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Many critical aquatic habitats are in close proximity to human activity (i.e., adjacent to residences, docks, marinas, etc.), and it is vital to monitor biodiversity in these and similar areas that are subject to ongoing urbanization, pollution, and other environmental disruptions. Environmental DNA (eDNA) metabarcoding is an accessible, non-invasive genetic technique used to detect and monitor species diversity and is a particularly useful approach in areas where traditional biodiversity monitoring methods (e.g., visual surveys or video surveillance) are challenging to conduct. In this study, we implemented an eDNA approach that used a combination of three distinct PCR primer sets to detect marine vertebrates within a canal system of Biscayne Bay, Florida, an ecosystem representative of challenging sampling conditions and a myriad of impacts from urbanization. We detected fish species from aquarium, commercial, and recreational fisheries, as well as invasive, cryptobenthic, and endangered vertebrate species, including charismatic marine mammals such as the protected West Indian manatee, Trichechus manatus. Our results support the potential for eDNA analyses to supplement traditional biodiversity monitoring methods and ultimately serve as an important tool for ecosystem management. This approach minimizes stress or disturbance to organisms and removes the intrinsic risk and logical limitations of SCUBA diving, snorkeling, or deploying sensitive equipment in areas that are subject to high vessel traffic and/or low visibility. Overall, this work sets the framework to understand how biodiversity may change over different spatial and temporal scales in an aquatic ecosystem heavily influenced by urbanization and validates the use of eDNA as a complementary approach to traditional ecological monitoring methods., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: Viral hepatitis caused by hepatitis B virus (HBV) and hepatitis C virus (HCV) infections remain a major global public health challenge, particularly in low- and middle-income countries. It is crucial to utilize a pointof-care (POC) testing platform that is sensitive, specific, rapid, and user-friendly for screening and diagnosis of the two infections. Here, a novel molecular diagnostic assay, integrating multiplex loop-mediated isothermal amplification with a gold nanoparticle-based lateral flow biosensor (mLAMP-AuNPs-LFB) was developed and applied for one-step, visual, rapid, sensitive, and specific identification of HBV and HCV., Methods: The AuNPs-based LFB was devised and constructed for the simultaneous detection of HBV and HCV. The HBV-LAMP and HCV-LAMP primers were designed against the S and 5'-untranslated region (5'-UTR) genes from the major HBV genotypes (B, C, D, B/C recombinant, and C/D recombinant) and HCV subtypes (1b, 2a, 3a, 3b, and 6a) in China, respectively. Our assay conditions, both multiplex-LAMP amplification temperature and time were optimized. The sensitivity and specificity of our assay were tested, and the feasibility of our assay was verified through clinical samples., Results: The AuNPs-based LFB used here was successfully manufactured according to our devise manual. The two unique independent primer pairs were successfully designed based on the S and 5'-UTR genes, respectively. The optimal mLAMP-AuNPs-LFB detection process, involving rapid nucleic acid isolation (10 min), mLAMP (63 °C for 35 min), and visual AuNPs-LFB interpretation (less than 2 min), could be completed within 50 min. The HBV&HCV-mLAMP-AuNPs-LFB assay can detect the target genes (HBV-S and HCV-5'-UTR) with as low as 20 copies of plasmid template per test, and the specificity was 100% for the experimental pathogens., Conclusions: The preliminary results manifested that our mLAMP-AuNPs-LFB assay is a valuable tool and has tremendous potential as a POC testing approach for HBV and HCV identification, especially in undeveloped regions., (© 2024. The Author(s).)

Background: The currently used methods for the detection of methylene tetrahydrofolic acid reductase (MTHFR) C677T single nucleotide polymorphism (SNP) are either time-consuming or expensive. In this study, we devised an accurate, rapid and easy-to-use SNP detection system based on fluorescent primers amplification refractory mutation system qPCR, known as FP ARMS-qPCR., Methods: Fluorescent primers (FPs) modified by fluorescent dye or quencher near the 3' terminal thymine were designed. The reaction conditions were optimized and the performance was evaluated. Using commercial kits as controls, 242 samples were tested in parallel to verify the feasibility of the FP ARMS-qPCR assay., Results: We demonstrated the good sensitivity and specificity of the FP ARMS-qPCR with optimized conditions. The assay was able to accurately distinguish between different SNP sites of MTHFR C677T in less than 2 h using as low as 50 pg of template genomic DNA. Completely consistent genotyping results reveal that FP ARMS-qPCR is concordant with commercial kits., Conclusion: We established a specific, sensitive, and rapid FP ARMS-qPCR method for the detection of MTHFR C677T genotype. This could also serve as a potential diagnostic tool for a variety of diseases., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: Recombinase polymerase amplification (RPA) is a novel nucleic acid isothermal amplification technique that can achieve rapid detection of the target, under 37 to 42°C conditions, within 30 minutes. It has the advantage of extreme sensitivity, strong specificity, and low instrument dependency and is particularly suitable for real-time detection in the field. It can be widely used in fields such as in vitro diagnostics, biosafety, and agriculture. This study was based on RPA technology, targeting the gyrA gene of Neisseria gonorrhoeae (N. gonorrhoeae), to establish a quick, accurate, and easy to operate method for detecting N. gonorrhoeae and to evaluate its specificity, sensitivity, and clinical, practical value., Methods: Specific primers and probes suitable for RPA and qPCR methods based on the specific conserved region of the gyrA gene of N. gonorrhoeae on GenBank (no. U08817.1) were designed An RPA method was developed and N. gonorrhoeae ATCC49226 and a number of clinical isolates were used as study subjects to validate the specificity and sensitivity of the RPA method for the detection of N. gonorrhoeae. A real-time fluorescence quantitative polymerase chain reaction (qPCR) method, with N. gonorrhoeae ATCC49226 as the research object, was established to verify the sensitivity of qPCR method for detecting N. gonorrhoeae. Finally, clinical samples were tested by using RPA and qPCR methods as performance validation experiments to determine the clinical utility of the RPA technique in detecting N. gonorrhoeae., Results: The established RPA detection method showed excellent specificity, with a specific amplification curve for N. gonorrhoeae alone, no cross-reactivity with other bacteria, and excellent reproducibility. The detection results could be obtained within 30 minutes, under the condition of 39°C, which was significantly lower than the detection time of traditional methods. The sensitivity of the RPA method for detecting pathogenic bacteria samples was 4 × 102 CFU/mL, which is consistent with the detection limit of qPCR methods. RPA and qPCR methods were used to detect 121 clinical isolates, out of which 30 strains of N. gonorrhoeae showed a specific amplification curve, while the remaining 91 strains of non-N. gonorrhoeae did not. Both methods had 100% accuracy and specificity in detecting N. gonorrhoeae., Conclusions: The RPA method developed in this study has the characteristics of being quick, accurate, and easy to operate, which was of great value for the rapid detection of N. gonorrhoeae in clinical samples.

Background: Songling virus (SGLV) within the genus Orthonairovirus, family Nairoviridae, is an emerging tick-borne virus associated with human febrile illness. However, no rapid detection method for SGLV has been established., Methods: In this study, four primer sets targeting the nucleocapsid protein gene of SGLV were designed for use in the LAMP assay and evaluated to identify the optimal primer set. Recombinant plasmids were constructed and utilized for assessing the sensitivity of the assay. Tacheng tick virus 1 (TcTV-1)-, Beiji nairovirus (BJNV)-, Yezo virus (YEZV)-, severe fever with thrombocytopenia syndrome virus (SFTSV)-, and tick-borne encephalitis virus (TBEV)-positive tick samples were utilized to assess the specificity. Field-collected ticks were also evaluated as biological specimens to validate the assay., Results: A SGLV-specific LAMP assay was established with a detection limit of 1 × 10 -2  copies/μl and could be visually confirmed by a color change from purple to blue in SGLV-positive samples. No cross-reactivity was observed in the detection of TcTV-1, BJNV, YEZV, SFTSV, and TBEV using the LAMP assay. In addition to the detection of the same seven high-copy numbers of SGLV as the SYBR Green quantitative RT-PCR assay within a reduced timeframe, the developed LAMP method also effectively identified an additional sample with a low copy number in the field-collected tick samples., Conclusions: We successfully developed a sensitive, specific, and cost-effective visual method for the rapid detection of SGLV using the LAMP assay, which can be applied in pathogenesis and epidemiological surveillance studies of SGLV., (© 2024. The Author(s).)

Purpose: Loop-mediated isothermal amplification (LAMP) is a simple and rapid nucleic acid method for DNA amplification at a constant temperature. The "gold standard" culture method for yeast detection, has low sensitivity with severe consequences, increasing morbidity and mortality rates. Here, we report the development of a LAMP method for the specific detection of C. glabrata., Methodology: The specific LAMP primers for C. glabrata detection were designed and evaluated., Results: The LAMP assay accurately detected C. glabrata with no cross-reactivity with other Candida species., Conclusion: The developed molecular method would be a promising tool in the management of invasive candidiasis., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Isothermal PCR can be performed by imposing a static temperature gradient that continuously circulates reagents through denaturing, annealing, and extension conditions inside a PCR tube. But despite early promise, these systems have yet to demonstrate performance and repeatability sufficient for adoption in validated laboratory tests because the rate-limiting extension step is inherently short and cannot be increased independently of the other stages in a temperature cycle. Here, a discovery that enables isothermal PCR to be achieved with statistically robust repeatability that meets or exceeds diagnostic assay requirements (false positive/negative rate <8% at 95% confidence) by manipulating the interplay between the DNA replication biochemistry (via the amplicon GC content) and the microscale circulatory flow inside a PCR tube is reported. Surprisingly, optimal performance depends on selecting primer sequences that replicate high GC content amplicons, contradicting established PCR primer design rules. This innovative thermocycling approach accelerates PCR to speeds rivaling ultra-fast instruments, enabling rapid, repeatable isothermal DNA analysis across a range of targets relevant to diagnostics and pathogen detection., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Cotton root rot caused by Macrophomina phaseolina pose a significant threat to cotton production, leading to substantial yield and quality losses. Early and accurate diagnosis of this pathogen in soil is crucial for effective disease management. This study presents a pioneering investigation into the utilization of the nit gene encoding nitrilase for the development of a molecular diagnostic assay aimed at the rapid detection of M. phaseolina in field soils. The methodology involved the design and validation of primers targeting the Nit gene sequence, followed by the optimization of PCR conditions for efficient amplification. Leveraging state-of-the-art molecular techniques, the assay offers a novel protocol to accurately identify the presence of M. phaseolina in soil with high sensitivity and specificity. The specificity of the designed primers was confirmed through PCR amplification using DNA from M. phaseolina and other related fungi. Sensitivity tests demonstrated that the PCR assay reliably detected M. phaseolina DNA at concentrations as low as 1 ng. Furthermore, the performance of the diagnostic assay was rigorously evaluated using field soil samples with a known status of M. phaseolina infection, demonstrating its reliability and efficacy in real-world scenarios. This study introduces a novel molecular marker for the detection of M. phaseolina and offers a rapid and efficient means for screening M. phaseolina in large soil samples with minimal time and manpower., (© 2024 Wiley‐VCH GmbH.)

LAMP (Loop-mediated isothermal amplification) is a popular method for the molecular diagnostics of numerous pathogens, specifically useful for point-of-care testing. However, the efficacy and sensitivity of LAMP still need to be maximised for the best performance in clinical settings. Adding a novel fourth primer pair is a promising way to accelerate the LAMP speed. Here, we report PI primers that are part of inner primers and can be used in LAMP without a specific design. PI primers were tested in quantitative LAMP detecting SARS-CoV-2 and MS2. The new primers have increased the speed and sensitivity of quantitative LAMP, RT-LAMP, and duplex LAMP with artificial templates and RNA samples from nasal swabs. Adding PI primers could become a valuable option for LAMP optimisation, especially when a desirable LAMP target is a highly variable DNA sequence with a few conservative sites for primers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Oyster aquaculture is one of the fastest-growing food production industries worldwide; however, it faces a significant challenge from the protist Perkinsus marinus, particularly in the USA. Although several quantitative molecular diagnostic methodologies are available for identifying diseases caused by P. marinus, the primer pairs used therein led to non-specific identification of other Perkinsus spp. Hence, a quantitative real-time PCR (Pm-qPCR) assay specific for P. marinus was developed using a TaqMan-based probe with the internal quencher in this study. A primer pair and probe specific to P. marinus were designed from a hypothetical protein of P. marinus collected from the whole-genome shotgun sequence database of the National Center for Biotechnology Information (NCBI). In silico analysis using homologous sequences of P. olseni and P. chesapeaki confirmed the high specificity of primers designed in this study. The Pm-qPCR assay was performed using seven different strains of P. marinus, P. olseni, and P. chesapeaki, revealing high specificity and sensitivity for detecting only P. marinus strains. In conclusion, it was demonstrated that Pm-qPCR can effectively and accurately diagnose P. marinus with high specificity and sensitivity. This assay is promising for monitoring oyster health and disease management in ecosystems and aquaculture., (© 2024. The Author(s).)

Background: Senecavirus A (SVA) is a newly pathogenic virus correlated with the acute death of piglets and vesicular lesions in pigs. The further prevalence of SVA will cause considerable economic damage to the global pig farming industry. Therefore, rapid and accurate diagnostic tools for SVA are crucial for preventing and controlling the disease., Methods: We designed multiple primer pairs targeting the most conserved region of the SVA 3D gene and selected those with the highest specificity. Nfo-probes were subsequently developed based on the highest specificity primer pairs. Subsequently, the recombinase-assisted amplification (RAA) reaction was completed, and the reaction temperature and duration were optimized. The RAA amplicons were detected using a lateral flow device (LFD). Finally, a rapid and intuitive RAA-LFD assay was established against SVA., Results: The SVA RAA-LFD assay can be performed under reaction conditions of 35°C within 17 minutes, with results observable to the naked eye. We then evaluated the performance of this method. It exhibited high specificity and no cross-reaction with the other common swine pathogens. The lowest detectable limits of this method for the plasmid of pMD18-SVA-3D, DNA amplification product, and viral were 3.86×10 1 copies/µL, 8.76×10 -7 ng/µL, and 1×10 0.25 TCID 50 /mL, respectively. A total of 44 clinical samples were then tested using the RAA-LFD, PCR, and RT-qPCR methods. The results demonstrated a consistent detection rate between the RAA-LFD and RT-qPCR assays., Conclusion: The SVA RAA-LFD assay developed in our study exhibits excellent specificity, sensitivity, and time-saving attributes, making it ideally suited for utilization in lack-instrumented laboratory and field settings., Competing Interests: Authors ZL and LW were employed by the company Wen’s Foodstuffs Group Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Song, Fang, Zhu, Wang, Wang, Chen, He, Yi, Ding, Zhao, Fan, Li and Chen.)

Hulless barley sheath rot is a spike disease caused by Dactylobotrys graminicola. In recent years, it has generally occurred in hulless barley growing areas in China, resulting in reduced hulless barley yields. In this study, primers and probes were designed based on conserved genome sequences, and a method was established using recombinant enzyme polymerase amplification-lateral flow burette (RPA-LFD) technology for the rapid diagnosis of sheath rot in hulless barley. The method can be successfully established in five minutes at a constant temperature of 39℃, and the results are consistent with those of normal PCR analysis. The method demonstrated high sensitivity, with a detection limit of 10 fg/µL. Furthermore, the rapid method was able to successfully detect D. graminicola in hulless barley during field incubation, which highlighted the significant advantage of the method in practical applications. In conclusion, the RPA method established in this study exhibited several advantageous characteristics, including high efficiency, simplicity, rapidity and practicality, which provide a theoretical basis for the early detection and prevention of D. graminicola., (© 2024. The Author(s).)

Introduction: In clinical diagnosis of porcine diseases, co-infection with multiple viruses often leads to similar clinical symptoms. Postweaning multisystemic wasting syndrome (PMWS) can be caused by infections with TTSuV or PCV2, while PCV2, PRV, and PPV can cause respiratory and reproductive disorders in pigs. The overlapping clinical and pathological features of these infections necessitate the development of a rapid and specific method for differentiating and detecting these four DNA viruses., Methods: In this study, four pairs of primers and TaqMan probes were designed targeting the conserved sequence of TTSuV, the Rep gene of PCV2, the gE gene of PRV, and the VP2 gene of PPV. After optimizing reaction conditions, including annealing temperature, primer concentration, and probe concentration, a quadruplex real-time PCR method was developed., Results: This method can specifically detect TTSuV1, PCV2, PRV, and PPV simultaneously, with no cross-reactivity with ASFV, CSFV, PRRSV, PEDV, PSV, and TGEV. The minimum detection limit for each virus was 10 copies/μl, and the inter-assay and intra-assay coefficients of variation ranged from 0.33% to 1.43%. Subsequently, 150 clinical samples were tested to evaluate the practical applicability of this method. The positive rates for TTSuV1, PCV2, PRV, and PPV were 8.6% (13/150), 10.67% (16/150), 14% (21/150), and 11.33% (17/150), respectively., Discussion: The results indicate that the established quadruplex real-time PCR method can assist in the accurate and rapid diagnosis of TTSuV1, PCV2, PRV, and PPV in clinical settings, providing robust support for the prevention and control of these infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Quan, Geng, Wu, Jiang, Li and Yu.)

This study aimed to unveil the genetic diversity among 47 bacterial isolates from various species using start codon targeted (SCoT) markers. Six SCoT primers yielded 219 reproducible bands, with 89.04% exhibiting polymorphism. The amplification process generated 28 to 50 fragments per primer, with an average of 36.50. Genetic diversity was quantified using polymorphic information content (PIC) values ranging from 0.11 to 0.14, with SCoT32 showing the highest PIC (0.14) and SCoT23 the lowest (0.11). The resolving power (RP) index, used to assess primer discriminatory power, varied significantly, with SCoT23 demonstrating the highest RP (6.00) and SCoT29 the lowest (4.51). Comparative analysis with conventional markers like M13 and (GTG) 5 revealed that certain SCoT primers exhibited superior PIC values, which indicates enhanced utility for interspecies differentiation. The high discrimination level achieved by SCoT primers underscores their effectiveness in genetic differentiation and biodiversity assessment within bacterial populations. This research highlights SCoT markers as powerful tools for microbial genetic studies, which offers valuable insights into bacterial diversity and provides a robust methodological framework for future investigations aimed at elucidating genetic variation and improving species identification. The application of SCoT markers represents a significant advancement in molecular techniques for bacterial characterization and phylogenetic analysis, demonstrating their potential to enhance our understanding of microbial genetics and evolution., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The resurgence of two bed bug species, the common bed bug (Cimex lectularius Linnaeus, 1758) and tropical bed bug (Cimex hemipterus Fabricius, 1803), in the same geographical regions has been frequently reported recently. Consequently, the rapid identification of these species is crucial for implementing targeted capture traps and tailored pyrethroid resistance diagnosis, due to differences in genetic and physiological traits., Methods: To develop molecular diagnostic methods, distinct protocols were established for multiplex PCR and loop-mediated isothermal amplification (LAMP) using species-specific primers based on species-specific segments of internal transcribed spacer 2 sequences. These methods were optimized for rapid and accurate identification of the two bed bug species., Results: Both multiplex PCR and LAMP protocols were effective in simultaneously identifying the two bed bug species, even when utilizing DNA released from dead specimens. Notably, the straightforward procedure and minimal time commitment of LAMP suggest its potential for rapid and accurate diagnosis of bed bugs in the field. The diagnostic accuracy of these methods was validated through a blind test., Conclusions: The multiplex PCR and LAMP protocols lay the foundation for rapid and accurate field identification of bed bug species, enabling the use of appropriate traps and the detection of species-specific pyrethroid resistance mutations. This approach ensures effective management tailored to the unique characteristics of each bed bug species., (© 2024. The Author(s).)

Objectives: This study aims to develop a novel diagnostic approach using the recombinase aided amplification-lateral flow dipstick(RAA-LFD) assay for the distinction of Mycobacterium tuberculosis (MTB) and Mycobacterium avium complex (MAC), enabling rapid and convenient as well as accurate identification of them in clinical samples., Methods: Our study established a duplex RAA-LFD assay capable of discriminating between MTB and MAC. Based on the principles of RAA primer and probe design, specific primers and probes were developed targeting the MTB IS6110 and the MAC DT1 separately. Optimization of reaction time points and temperatures was conducted, followed by an evaluation of specificity, sensitivity, and reproducibility. The established detection method was then applied to clinical samples and compared with smear microscopy, liquid culture, LAMP, and Xpert/MTB RIF in terms of diagnostic performance., Results: The complete workflow allows for the effective amplification of the MTB IS6110 and MAC DT1 target sequences at constant 37°C within 20min, and the amplification products can be visually observed on the LFD test strip. This method exhibits high specificity, showing no cross-reactivity with nucleic acids from M. kansassi , M. abscessus, M. gordonae, M. chelonae, M. fortuitum, M. scrofulaceum, M. malmoense, M. chimaera, M. szulgai and common respiratory pathogens. It also demonstrates high sensitivity, with a detection limit as low as 10 2 CFU/mL. Additionally, the method's Coefficient of Variation (CV) is less than 5%, ensuring excellent repeatability and reliability. Furthermore, clinical performance evaluations, using Xpert/MTB RIF as the gold standard, demonstrated that the duplex RAA-LFD assay achieves a sensitivity of 92.86% and a specificity of 93.75%. It is also noteworthy that the assay exhibits considerable diagnostic efficacy in smear-negative patients., Conclusions: Our study introduces a rapid, specific, and sensitive duplex RAA-LFD assay for the discriminatory diagnosis of MTB and MAC. This method represents a significant advancement in the field of infectious disease diagnostics, offering a valuable tool for rapid detection and management of MTB and MAC infections. The implementation of this approach in point-of-care settings could greatly enhance TB control and prevention efforts, especially in resource-limited environments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Chen, Zhang, Wang, Yi and Fu.)

Objective: To explore the application of triplet-primer PCR (TP-PCR) for the genetic testing and prenatal diagnosis in patients with Myotonic dystrophy type 1 (DM1)., Methods: A total of 60 individuals from 48 pedigrees undergoing genetic testing at the Genetic and Prenatal Diagnosis Center of the First Affiliated Hospital of Zhengzhou University from May 2018 to October 2022 were selected as the study subjects. TP-PCR combined with capillary electrophoresis was applied to determine the number of CTG repeats of the DMPK gene, and prenatal testing was provided to four DM1 pedigrees. This study was approved by the First Affiliated Hospital of Zhengzhou University (Ethics No. KS-2018-KY-36)., Results: A total of 52 DM1 patients were detected, mostly with muscle weakness, muscular atrophy and myotonia as the initial symptoms, along with typical myotonic potentials. Some patients also had abnormalities of other systems. The number of abnormal CTG repeats of the DMPK gene was > 50, whilst the number of CTG repeats on the normal allele had ranged from 5 to 18. The number of the most common normal CAG repeats was 6 (30.77%, 16/52). Among the four DM1 pedigrees undergoing prenatal diagnosis, one fetus was healthy, whilst three fetuses were found to have abnormal CTG repeats (> 50 times) and diagnosed with DM1., Conclusion: TP-PCR can diagnose DM1 patients with speed and accuracy. However, this method cannot accurately determine the number of CTG repeats when it exceeds 50.

Loop-Mediated Isothermal Amplification (LAMP) represents a valuable technique for DNA/RNA detection, known for its exceptional sensitivity, specificity, speed, accuracy, and affordability. This study focused on optimizing a LAMP-based method to detect early signs of Plasmopara halstedii, the casual pathogen of sunflower downy mildew, a severe threat to sunflower crops. Specifically, a set of six LAMP primers (two outer, two inner, and two loop) were designed from P. halstedii genomic DNA, targeting the ribosomal Large Subunit (LSU). These primers were verified by in silico analysis and experimental validation using both target and non-target species' DNAs. Optimizations encompassing reaction conditions (temperature, time) and component concentrations (magnesium, Bst DNA polymerase, primers, and dNTP) were determined. Validation of these optimizations was performed by agarose gel electrophoresis. Furthermore, various colorimetric chemicals (Neutral Red, Hydroxynaphthol Blue, SYBR Safe, Thiazole Green) were evaluated to facilitate method analysis, and the real-time analysis has been optimized, presenting multiple approaches for detecting sunflower downy mildew using the LAMP technique. The analytical sensitivity of the method was confirmed by detecting P. halstedii DNA concentrations as low as 0.5 pg/μl. This pioneering study, establishing P. halstedii detection through the LAMP method, stands as unique in its field. The precision, robustness, and practicality of the LAMP protocol make it an ideal choice for studies focusing on sunflower mildew, emphasizing its recommended use due to its operational ease and reliability., (© 2024. The Author(s).)

The Staphylococcus genus comprises multiple pathogenic and opportunistic species that represent a risk to public health. Epidemiological studies require accurate taxonomic classification of isolates with enough resolution to distinguish clonal complexes. Unfortunately, 16 S rRNA molecular analysis and phenotypic characterization cannot distinguish all species and do not offer enough resolution to assess intraspecific diversity. Other approaches, such as Multilocus Sequence Tagging, provide higher resolution; however, they have been developed for Staphylococcus aureus and a few other species. Here, we developed a set of genus-targeted primers using five orthologous genes (pta, tuf, tpi, groEs, and sarA) to identify all Staphylococcus species within the genus. The primers were initially evaluated using 20 strains from the Collection of Microorganisms of Interest in Animal Health from AGROSAVIA (CMISA), and their amplified sequences were compared to a set of 33 Staphylococcus species. This allowed the taxonomic identification of the strains even on close species and the establishment of intraspecies diversity. To enhance the scope and cost-effectiveness of the proposed strategy, we customized the primer sets for an Illumina paired-end amplicon protocol, enabling gene multiplexing. We assessed five genes across 177 strains, generating 880 paired-end libraries from the CMISA. This approach significantly reduced sequencing costs, as all libraries can be efficiently sequenced in a single MiSeq run at a fraction (one-fourth or less) of the cost associated with Sanger sequencing. In summary, this method can be used for precise identification and diversity analysis of Staphylococcus species, offering an advancement over traditional techniques in both resolution and cost-effectiveness., (© 2024. The Author(s).)

Since the discovery of complete ammonia oxidizers (comammox) within the genus Nitrospira , their distribution and abundance across habitats have been intensively studied to better understand their ecological significance. Many primers targeting their ammonia monooxygenase subunit A gene ( amoA ) have been designed to detect and quantify comammox bacteria and to describe their community structure. We identified 38 published primers, but only few had high coverage and specificity for all known comammox Nitrospira or one of the two described subclades. For each target group, we comprehensively evaluated selected primer pairs using in silico analyses, endpoint PCRs, qPCRs, and amplicon sequencing on samples from various environments. Endpoint PCRs and qPCRs showed that the most commonly used primer pairs (comaA-244F/659R, comaB-244F/659R, and Ntsp-amoA162F/359R) produced several bands, which likely inflated quantifications via qPCR. In contrast, the recently published primer combinations CA377F/C576R, CB377F/C576R, and CA-CB377F/C576R resulted mostly in a single band. Furthermore, amplicon sequencing demonstrated that these primer combinations also captured the highest richness of comammox Nitrospira . Taken together, our results indicate that few existing comammox amoA primer combinations have both high specificity and coverage and that the choice of these high-specificity and high-coverage primer pairs substantially impacts the accurate detection, quantification, and community description of comammox bacteria. We, therefore, recommend using the CA377F/C576R, CB377F/C576R, and CA-CB377F/C576R primer pairs.IMPORTANCEBacteria that can fully convert ammonia via nitrite to nitrate, the complete ammonia oxidizers (comammox), were recently discovered and are found in many natural and engineered environments. PCR-based tools to study their abundance and diversity were rapidly developed, resulting in a plethora of primers available, many of which are widely used. The presence of comammox bacteria in an environment can, however, only be correctly determined if the used primers detect all members of this group while not detecting any other guilds. This study assesses the coverage and specificity of existing primers targeting comammox bacteria using both computational and standard molecular techniques, revealing large differences in their performance. The uniform usage of well-performing primers across studies could aid in generating comparable and generalizable data to better understand the importance of comammox bacteria in the environment., Competing Interests: The authors declare no conflict of interest.

This study aimed to design and evaluate a universal primer for Polymerase Chain Reaction (PCR)- based detection of mucormycosis-causing fungi by targeting their Internal Transcribed Spacer (ITS) sequences. In-silico analysis was conducted to assess primer suitability. Using Clustal Omega and Primer-BLAST, ITS sequences of 32 fungi species causing mucormycosis were aligned and subjected to primer design. Generated primers were sorted and in silico PCR simulations were performed to identify primers capable of amplifying all fungal species. Instead of identifying one pair of universal primer, in silico PCR analysis identified a panel of 14 primer pairs designed from full-length ITS sequences, and a panel of 12 primer pairs designed from conserved regions, that could detect 31 species. The study recommends a panel of 12 primers for multiplex-PCR to detect mucormycosis-causing fungi instead of a long list of PCR analyses for each fungus in diagnosing mucormycosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bacterial blight caused by Xanthomonas phaseoli pv. manihotis (Xpm) is considered the main bacterial disease that affects cassava, causing significant losses when not properly managed. In the present study, a fast, sensitive, and easy-to-apply method to detect Xpm via colorimetric loop-mediated isothermal amplification (LAMP) was developed. To ensure the use of a unique-to-the-target pathovar core region for primer design, 74 complete genomic sequences of Xpm together with different bacterial species and pathovars were used for comparative genomics. A total of 42 unique genes were used to design 27 LAMP primer sets, from which nine primers were synthesized, and only one (Xpm&#95;Lp1 primer set) showed sufficient efficiency in preliminary tests. The sensitivity, assessed by a serial dilution of the type strain (IBSBF 278) DNA, yielded high sensitivity, detecting up to 100 fg. The LAMP primers showed high specificity, did not cross-react with other bacterial species or other pathovars tested, and amplified only the Xpm isolates. Tests confirmed the high efficiency of the protocol using infected or inoculated macerated cassava leaves without the need for additional sample treatment. The LAMP test developed in this study was able to detect Xpm in a fast, simple, and sensitive way, and it can be used to monitor the disease under laboratory and field conditions., Competing Interests: The author(s) declare no conflict of interest.

Eucalyptus spp. in plantations are negatively affected by canker and wilt diseases caused by several species of Ceratocystis, particularly those in the Latin American Clade (LAC). Ceratocystis eucalypticola and Ceratocystis manginecans are of particular concern where disease epidemics are reported globally, with recent outbreaks emerging in South African and Indonesian Eucalyptus plantations. Consequently, a rapid screening protocol is required for these pathogens. In this study, a high-resolution melting curve analysis (HRMA) was developed to detect C. eucalypticola and C. manginecans that bypasses time-consuming isolation and post-PCR procedures. Primers targeting a 172 bp region of the cerato-platanin (CP) gene were designed. Using these primers, the accuracy of HRMA to detect and distinguish between these two LAC species was assessed using pure fungal DNA, and DNA extracted directly from Eucalyptus samples naturally infected with C. eucalypticola. The assay accurately detected the presence of C. eucalypticola and C. manginecans and quantifies their DNA, both from cultures, and directly from wood samples. HRMA further differentiated these two species from all other tested LAC individuals. This assay was also able to detect the presence of all the tested LAC species and distinguish seven of these, including C. fimbriata, to species level. Ceratocystis polyconidia was the only non-LAC off-target species detected. Based on these results, the developed assay can be used to rapidly identify C. eucalypticola and C. manginecans directly from infected plant material or fungal cultures, with the potential to also screen for several other LAC species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Dollar spot is a major fungal disease affecting turfgrass worldwide and can quickly destroy turfgrass swards. An assimilating probe-based loop-mediated isothermal amplification (LAMP) assay was developed to detect Clarireedia monteithiana and C. jacksonii , the causal agents of dollar spot within the continental United States. Five LAMP primers were designed to target the calmodulin gene with the addition of a 6-carboxyl-fluorescein florescent assimilating probe, and the temperature amplification was optimized for C. jacksonii and C. monteithiana identification. The minimum amount of purified DNA needed for detection was 0.05 ng μl -1 . Specificity assays against host DNA and other turfgrass pathogens were negative. Successful LAMP amplification was also observed for dollar spot-infected turfgrass field samples. Further, a DNA extraction technique via rapid heat-chill cycles and visualization of LAMP results via a florescent flashlight was developed and adapted for fast, simple, and reliable detection in 1.25 h. This assimilating probe-based LAMP assay has proved successful as a rapid, sensitive, and specific method for the detection of C. monteithiana and C. jacksonii in pure cultures and from symptomatic turfgrass leaves blades. The assay represents a promising technology to be used in the field for on-site, point-of-care pathogen detection., Competing Interests: The author(s) declare no conflict of interest.

Although several primers targeted to the internal transcribed-spacer 1 (ITS1) of the ribosomal DNA (rDNA) have been designed to improve the detection of African trypanosomes, no study tried to compare their agreement level and ability to amplify different trypanosome species in tsetse flies and mammals in various epidemiological settings. This study was designed to fill this gap, by targeting tsetse-infested areas of Cameroon. For this, archived DNA samples reporting at-least one trypanosome species with species-specific PCR primers were reviewed. Ten sets of primers targeting different ITS1 rDNA sequences of trypanosomes were selected for assessment using single-round and nested-PCR method. Amplification rates (sensitivity) and agreement level of different ITS1 assays were compared using Cohen's-Kappa and McNemar's x 2 statistic. Little agreement level (k = 0.05-0.52) were observed between different ITS1-primers PCRs detection of African trypanosome species despite significant (X 2 =54.3, p = 0.0001) high amplification rate 91.6 % (339/370). This sensitivity varied from quite low for T. simiae (11.9 %) and T. vivax (27.3 %) to fairly good for T. congolence (51.9 %), Trypanozoon (32.4 %) and T. theileri (40.3 %). Primers set targeting ITS1-A sequence of trypanosome species recorded the highest sensitivity (50.5 %) with fairly good agreement compared to 39.2 % for ITS1-C (k = 0.52), 32.4 % for ITS1-R (k = 0.47), 29.7 % for ITS1-N (k = 0.48) and 23.0 % for ITS1-KIN (k = 0.43) respectively. This study revealed a diversity in the sensitivity of different trypanosome species with different sets of ITS-primers enhancing the need to use the same sets of primers in different bio-ecological settings. The use of nested-PCR instead of single-round PCR enabled improvement of trypanosome infections detection in both tsetse and mammals. Among the sets of ITS1-primers tested, those designed by to amplify ITS1-A can be considered as the most appropriate for the detection of trypanosome infections in mammals and tsetse flies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing personal relationships or financial interests that could have appeared to influence the work reported in this paper. Authors also confirm that the manuscript has been read and approved by all named authors. Also that there are no other person who satisfied the criteria for authorship but are no listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us., (Copyright © 2024 Elsevier B.V. All rights reserved.)

COVID-19 has afflicted millions of lives worldwide. Although there are many rapid methods to detect it based on colorimetric loop-mediated isothermal amplification, there remains room for improvement. This study aims to 1) integrate multiple primers into a singleplex assay to enhance the diagnostic sensitivity, and 2) utilize a high-throughput smartphone-operatable AI-driven color reading tool to enable a rapid result analysis. This setup can improve the sensitivity by 10-100 times and can analyze approximately 6700 samples per minute. The assay is simpler than RT-qPCR, with a turnaround time of less than 75 min. It can detect various types of SARS-CoV-2 by targeting 3 genes, increasing the likelihood that it will remain effective even if the virus undergoes mutations in any single target gene. In summary, it affords potential for adaptation to detection of new/re-emerging diseases with the visual readout for maximum assay simplicity and AI-operated mode for large-scale testing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Mosquitoes take blood meals from a diverse range of host animals and their host associations vary by species. Characterizing these associations is an important element of the transmission dynamics of mosquito-vectored pathogens. To characterize mosquito host associations, various molecular techniques have been developed, which are collectively referred to as blood meal analysis. DNA barcoding has diverse biological applications and is well-suited to mosquito blood meal analysis. The standard DNA barcoding marker for animals is a 5' fragment of the cytochrome c oxidase I ( COI ) gene. A major advantage of this marker is its taxonomic coverage in DNA sequence reference databases, making it feasible to identify a wider range of mosquito host species than with any other gene. However, the COI gene contains high sequence variation at potential priming sites between vertebrate orders. Coupled with the need for primer sequences to be mismatched with mosquito priming sites so that annealing to mosquito DNA is inhibited, it can be difficult to design primers suitable for blood meal analysis applications. Several primers are available that perform well in mosquito blood meal analysis, annealing to priming sites for most vertebrate host taxa, but not to those of mosquitoes. Because priming site sequence variation among vertebrate taxa can cause amplification to fail, a hierarchical approach to DNA barcoding-based blood meal analysis can be applied. In such an approach, no single primer set is expected to be effective for 100% of potential host species. If amplification fails in the initial reaction, a subsequent reaction is attempted with primers that anneal to different priming sites, and so on, until amplification is successful., (© 2024 Cold Spring Harbor Laboratory Press.)

The microbiota of hydrothermal vents has been widely implicated in the dynamics of oceanic biogeochemical cycling. Lithotrophic organisms utilize reduced chemicals in the vent effluent for energy, which fuels carbon fixation, and their metabolic byproducts can then support higher trophic levels and high-biomass ecosystems. However, despite the important role these microorganisms play in our oceans, they are difficult to study. Most are resistant to culturing in a lab setting, so culture-independent methods are necessary to examine community composition. Targeted amplicon surveying has become the standard practice for assessing the structure and diversity of hydrothermal vent microbial communities. Here, the performance of primer pairs targeting the V3V4 and V4V5 variable regions of the SSU rRNA gene was assessed for use on environmental samples from microbial mats surrounding Kama'ehuakanaloa Seamount, an iron-dominated hydrothermal vent system. Using the amplicon sequence variant (ASV) approach to taxonomic identification, the structure and diversity of microbial communities were elucidated, and both primer pairs generated robust data and comparable alpha diversity profiles. However, several distinct differences in community composition were identified between primer sets, including differential relative abundances of both bacterial and archaeal phyla. The primer choice was determined to be a significant driver of variation among the taxonomic profiles generated. Based on the higher quality of the raw sequences generated and on the breadth of abundant taxa found using the V4V5 primer set, it is determined as the most efficacious primer pair for whole-community surveys of microbial mats at Kama'ehuakanaloa Seamount., Competing Interests: Craig Moyer is an Academic Editor for PeerJ., (© 2024 Smith et al.)

African swine fever virus (ASFV) has been spreading through Europe, Asia, and the Caribbean after its introduction in Georgia in 2007 and, due to its particularly high mortality rate, poses a continuous threat to the pig industry. The golden standard to trace back the ASFV is whole genome sequencing, but it is a cost and time-intensive methodology. A more efficient way of tracing the virus is to amplify only specific genomic regions relevant for genotyping. This is mainly accomplished by amplifying single amplicons by PCR followed by Sanger sequencing. To reduce costs and processivity time, we evaluated a multiplex PCR based on the four primer sets routinely used for ASFV genotyping ( B646L , E183L , B602L , and intergenic I73R-I329L ), which was followed by Nanopore ligation-based amplicon sequencing. We show that with this protocol, we can genotype ASFV DNA originating from different biological matrices and correctly classify multiple genotypes and strains using a single PCR reaction. Further optimization of this method can be accomplished by adding or swapping the primer sets used for amplification based on the needs of a specific country or region, making it a versatile tool that can speed up the processing time and lower the costs of genotyping during ASFV outbreaks.

Candida glabrata (Nakaseomyces glabratus) is an opportunistic fungal pathogen that has become a significant concern in clinical settings due to its increasing resistance to antifungal treatments. Understanding the genetic basis of its pathogenicity and resistance mechanisms is crucial for developing new therapeutic strategies. One powerful method of studying gene function is through targeted gene deletion. This paper outlines a comprehensive protocol for the deletion of genes in C. glabrata, encompassing primer design, preparation of electrocompetent cells, transformation, and finally confirmation of the gene deletion. The protocol begins with the identification and design of primers necessary for generating deletion constructs, involving the precise targeting of up- and downstream regions flanking the gene of interest to ensure high specificity and efficiency of homologous recombination. Followed is the preparation of electrocompetent cells, a critical step for successful transformation. Transformation of the competent cells is achieved through electroporation, facilitating the introduction of exogenous DNA into the cells. This is followed by the selection and confirmation of successfully transformed colonies. Confirmation involves the use of colony PCR to verify the correct integration of the NAT resistance cassette and deletion of the target gene. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Primer design for gene deletion in C. glabrata Basic Protocol 2: Preparing competent C. glabrata cells Basic Protocol 3: Transforming C. glabrata using electroporation Basic Protocol 4: Confirming deletion strains with colony PCR., (© 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.)

In vitro amplification of single-stranded oligonucleotide libraries presents a significant challenge due to the potential for excessive byproduct formation. This phenomenon largely affects the quality of the ssDNAs created using the most commonly used methods, e.g., asymmetric PCR, biotin-streptavidin separation, or lambda exonuclease digestion of dsDNA. Here, we describe an improved protocol that combines primer-blocked asymmetric PCR (PBA-PCR) with emulsion PCR and a cost-effective downstream process that altogether alleviates byproduct formation without distorting the sequence space of the ssDNA library. In PBA-PCR, the reaction mixture is complemented with a 3'-phosphate-blocked limiting primer that decreases mispriming, thus reducing polymerization of DNA byproducts. The downstream process includes mixing of the PBA-PCR product with excess reverse complement of the 3'-phosphate-blocked limiting primer and removal of dsDNA strands via biotin-streptavidin separation, yielding purified ssDNAs. In conclusion, we have devised a universally applicable approach for simple and cost-effective production of ssDNA libraries and unique ssDNA sequences with on-demand labeling. Our protocol could be beneficial for a variety of uses, such as generating aptamer libraries for SELEX, creating unique molecular identifiers for a wide range of sequencing applications, providing donor DNA for CRISPR-Cas9 systems, developing scaffold nanostructures, and enabling DNA-based data storage. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Amplification of ssDNA libraries using PBA-PCR Alternate Protocol 1: Amplification of ssDNA libraries using emulsion PBA-PCR with a simplified extraction of PBA-PCR products Basic Protocol 2: Purification of PBA-PCR products to remove dsDNA and conversion of 3'-blocked primer to double-stranded complexes Alternate Protocol 2: Purification of PBA-PCR products to remove both dsDNA and blocking primers from the reaction mixture Support Protocol: Analysis of PBA-PCR products by gel electrophoresis., (© 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC.)

One-step RT-qPCR TaqMan assays have been developed for six plant viruses with considerable economic impact in the growing of tulip and lily bulbs: lily mottle virus, lily symptomless virus, lily virus X, Plantago asiatica mosaic virus, tulip breaking virus and tulip virus X. To enhance efficacy and cost-efficiency these assays were combined into multiplex panels. Four different multiplex panels were designed, each consisting of three virus assays and an adapted assay for the housekeeping gene nad5 of lilies and tulips, that acts as an internal amplification control. To eliminate false negative results due to variation in the viral genome sequences, for each target virus two assays were developed on distinct conserved genomic regions. Specificity, PCR efficiency and compatibility of primers and probes were tested using gBlock constructions. Diagnostic samples were used to evaluate the strategy. High Throughput Sequencing of a set of the diagnostic samples, further verified the presence or absence of the viruses in the RNA samples and sequence variations in the target genes. This interchangeable multiplex panel strategy may be a valuable tool for the detection of viruses in certification, surveys and virus diagnostics., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Authentication of true (genuine) cow leathers is in high demand to promote merchandise and economic growth. The present study employs RT-PCR-based TaqMan assay to facilitate the identification. Species-specific primers and probes were designed utilizing the existing NCBI data on mitochondrial DNA (mtDNA) genes, particularly the cytochrome b region (Cyt b). Mitochondrial DNA extracted from leather samples of both Bos taurus and Bos indicus and analyzed following the appropriate procedures. The RT-PCR results showed the designed primers and probes are exceptionally precise for cow leather samples. The established detection limit for the assay is estimated as 0.1 ng of DNA. In summary, the amplifiable mtDNA extracted from finished leather enables the identification of authentic cow leathers using the RT-PCR TaqMan assay, representing a pioneering report in this field., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Food falsification is a pressing issue in today's food industry, with fraudulent substitution of costly ingredients with cheaper alternatives occurring globally. Consequently, developing straightforward and efficient diagnostic systems to detect such fraud is a top priority in scientific research. The aim of the work was to develop a test system and protocol for polymerase chain reaction (PCR) to detect in food products of animal origin the substitution of expensive meat raw materials for by-products of poultry processing. For this, real-time polymerase chain reaction (RT-PCR) was used, which allows determining the qualitative and quantitative substitution in raw and technologically prepared products. Other methods for detecting falsification - enzyme immunoassay (ELISA/ELISA) or express methods in the form of a lateral flow immunoassay are less informative. The extraction of nucleic acids for real-time polymerase chain reaction depends on the source matrix, with higher concentrations obtained from germ cells and parenchymal organs. Extraction from muscle and plant tissues is more challenging, but thorough grinding of these samples improves nucleic acid concentration by 1.5 times using DNA extraction kits. The selection of primers and fluorescent probes through GenBank and PCR Primer Design/DNASTAR software enables efficient amplification and identification of target chicken DNA fragments in various matrices.

Infectious hematopoietic necrosis virus (IHNV) severely and lethally infects salmonid fish, including Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) worldwide. Rapid and accurate viral detection is crucial for preventing pathogen spread and minimizing damage. Although several IHNV detection assays have been developed, their analytical and diagnostic performances have not been evaluated and field usability assessments have not been completely validated. Here, we developed a reverse-transcription cross-priming amplification-based lateral flow assay (RT-CPA-LFA) and validated its diagnostic performance. To detect the IHNV, primers were designed based on the consensus sequence of the nucleocapsid (N) gene. Notably, when combined with a lateral flow dipstick, it could visualize the IHNV amplification products within 5 min and the detection limit of the developed RT-CPA-LFA was 3.28×10 5 copies/μL. The diagnostic sensitivity and specificity in fish samples (n=140) were 98.88 % and 96.08 %, respectively. Moreover, the IHNV detection rate by RT-CPA-LFA in dead rainbow trout artificially injected with the virus was 100 %, consistent with to the results obtained from second conventional and real-time PCR, indicating its applicability for rapid IHNV detection and presumptive IHN diagnosis during the endemic period., Competing Interests: Declaration of Competing Interest The author declares no conflicts of interest, (Copyright © 2024 Elsevier B.V. All rights reserved.)

Molluscum contagiosum virus (MOCV) is an important human pathogen causing a high disease burden worldwide. It is the last exclusively human-infecting poxvirus still circulating in its natural reservoir-a valuable model of poxviral evolution. Unfortunately, MOCV remains neglected, and little is known about its evolutionary history and circulating genomic variants, especially in non-privileged countries. The design weaknesses of available MOCV detection/genotyping assays surfaced with recent accumulation of abundant sequence information: all existing MOCV assays fail at accurate genotyping and capturing sub-genotype level diversity. Because complete MOCV genome characterization is an expensive and labor-intensive task, it makes sense to prioritize samples for whole-genome sequencing by diversity triage screening. To meet this demand, we developed a novel assay for accurate MOCV detection and genotyping, and comprehensive sub-genotype qualification to the level of phylogenetic groups (PGs). The assay included a novel set of oligonucleotide primers and probes, and it was implemented using digital polymerase chain reaction (dPCR). It offers sensitive, specific, and accurate detection, genotyping (MOCV1-MOCV3), and PG qualification (PG1-6) of MOCV DNA from clinical samples. The novel dPCR assay is suitable for MOCV diversity triage screening and prioritization of samples for complete MOCV genome characterization., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Detection and quantification of pathogen propagules in the air or other environmental samples is facilitated by culture-independent assays. We developed a quantitative PCR assay for the hop powdery mildew fungus, Podosphaera macularis , for detection of the organism from air samples. The assay uses primers and a TaqMan probe designed to target species-specific sequences in the 28S large subunit of the nuclear ribosomal DNA. Analytical sensitivity was not affected by the presence of an exogenous internal control or potential PCR inhibitors associated with DNA extracted from soil. The level of quantification of the assay was between 200 and 350 conidia when DNA was extracted from a fixed number of conidia. The assay amplified all isolates of P. macularis tested and had minimal cross-reactivity with other Podosphaera species when assayed with biologically relevant quantities of DNA. Standard curves generated independently in two other laboratories indicated that assay sensitivity was qualitatively similar and reproducible. All laboratories successfully detected eight unknown isolates of P. macularis and correctly discriminated Pseudoperonospora humuli and a water control. The usefulness of the assay for air sampling for late-season inoculum of P. macularis was demonstrated in field studies in 2019 and 2020. In both years, airborne populations of P. macularis in hop yards were detected consistently and increased during bloom and cone development., Competing Interests: The author(s) declare no conflict of interest.

Efficient DNA assembly methods are an essential prerequisite in the field of synthetic biology. Modular cloning systems, which rely on Golden Gate cloning for DNA assembly, are designed to facilitate assembly of multigene constructs from libraries of standard parts through a series of streamlined one-pot assembly reactions. Standard parts consist of the DNA sequence of a genetic element of interest such as a promoter, coding sequence, or terminator, cloned in a plasmid vector. Standard parts for the modular cloning system MoClo, also called level 0 modules, must be flanked by two BsaI restriction sites in opposite orientations and should not contain internal sequences for two type IIS restriction sites, BsaI and BpiI, and optionally for a third type IIS enzyme, BsmBI. We provide here a detailed protocol for cloning of level 0 modules. This protocol requires the following steps: (1) defining the type of part that needs to be cloned, (2) designing primers for amplification, (3) performing polymerase chain reaction (PCR) amplification, (4) cloning of the fragments using Golden Gate cloning, and finally (5) sequencing of the part. For large standard parts, it is preferable to first clone sub-parts as intermediate level -1 constructs. These sub-parts are sequenced individually and are then further assembled to make the final level 0 module., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The global impact of the COVID-19 pandemic has emphasized the critical need for effective viral diagnostics. Although polymerase chain reaction (PCR) is a well-established nucleotide amplification technique, its limitations, such as the need for expensive equipment and skilled technicians, have led to the exploration of alternative methods, including loop-mediated isothermal amplification (LAMP). Bats, as a crucial natural reservoir of coronaviruses (CoVs), particularly Scotophilus bat coronavirus 512 (Scotophilus bat-CoV 512) prevalent among Taiwan's bat population, are the focus of this study. We aimed to detect Scotophilus bat-CoV 512 from bats in field conditions using loop-mediated isothermal amplification (LAMP) assay for on-site detection. Therefore, our study delves into the specificity of the LAMP reaction, emphasizing the careful design of primers to prevent false positive results. A cross reactivity and primer specificity test involving seven different microorganisms, including closely related bat CoVs and two bacterial species typically found in feces, revealed that the LAMP assay uniquely detected Scotophilus bat-CoV 512. The developed colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was optimized for the primers targeting nucleocapsid (N) gene, and the sensitivity test revealed a detection limit of 2.4 × 10 3 copies/µL. Our findings indicate the potential of the RT-LAMP assay for on-site detection in the field and subsequent laboratory analysis for comprehensive sampling and further research on bat CoV isolation. The surveillance and monitoring of bat CoVs contribute substantially to mitigating human threats, particularly concerning the emergence of new pandemic variants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Nuomin virus (NOMV), an emerging tick-borne virus (TBVs) identified in 2020, has been associated with fever, headache, and potential liver dysfunction in infected individuals. This study presents a novel TaqMan real-time quantitative PCR method designed for the rapid, sensitive, and specific detection of NOMV, facilitating early diagnosis. Utilizing Beacon Designer software 8.0, we optimized the PCR assay including the development of primers and probes to precisely target the conserved region of the NOMV genome, followed by optimization of primer and probe concentrations and annealing temperature. The resulting assay demonstrated robust performance, with standard curve represented by the equation y=-3.29x+39.42, a high correlation coefficient (R 2 = 0.995) and an efficiency 99.53 %. Importantly, the method exhibited exceptional specificity, which did not yield cross-reacting signals from other TBVs, including Songling virus (SGLV), Beiji virus (BJNV), tick-borne encephalitis virus (TBEV), Yezo virus (YEZV), Alongshan virus (ALSV), and severe fever with thrombocytopenia syndrome bunyavirus (SFTSV). The assay's detection limit was remarkably low, reaching 10 copies/μL, representing a 100-fold increase compared to semi-nested RT-PCR. Additionally, it demonstrated excellent repeatability, with coefficients of variation for intra- and inter-group tests consistently below 3 %. Clinical evaluations confirmed the assay's superior performance, highlighting its high specificity, sensitivity, and reproducibility for NOMV detection. In conclusion, the method developed in this study provides a valuable tool to support timely management of NOMV infections, with significant implications for clinical practice., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Due to shared routes of transmission, including sexual contact and vertical transmission, HIV-HBV co-infection is common, particularly in sub-Saharan Africa. Measurement of viral load (VL), for both HIV and HBV, plays a critical role for determining their infectious phase and monitoring response to antiviral therapy. Implementation of viral load testing in clinical settings is a significant challenge in resource-limited countries, notably because of cost and availability issues. We designed HIV and HBV primers for conserved regions of the HIV and HBV genomes that were specifically adapted to viral strains circulating in West Africa that are HIV-1 subtype CRF02AG and HBV genotype E. We first validated two monoplex qPCR assays for individual quantification and, then developed a multiplex qPCR for simultaneous quantification of both viruses. HIV RNA and HBV DNA amplification was performed in a single tube using a one-step reverse transcription-PCR reaction with primers and probes targeting both viruses. Performance characteristics such as the quantification range, sensitivity, and specificity of this multiplex qPCR assay were compared to reference qPCR tests for both HIV and HBV viral load quantification. The multiplex assay was validated using clinical samples from co- or mono-infected patients and gave comparable viral load quantification to the HIV and HBV reference test respectively. The multiplex qPCR demonstrated an overall sensitivity of 71.25 % [68.16-74.3] for HBV and 82 % [78.09-85.90] for HIV and an overall specificity of 100 % [94.95-100] for both viruses. Although the overall sensitivities of the HIV and HBV assays were lower than the commercial comparator assays, the sensitivity in the clinical decision range of >1000 copies/mL for HIV was 80 % [71.26-88.73] and >1000 IU/mL for HBV was 100 % [95.51-100] which indicates the test results can be used to guide treatment decisions. This in-house developed multiplex qPCR assay represents a useful diagnostic tool as it can be performed on affordable "open" real-time PCR platforms currently used for HIV or SARS-Cov-2 infection surveillance in Mali., Competing Interests: Declaration of Competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Five simplex and a multiplex-RT-PCR (m-RT-PCR) protocols were developed for detection and differentiation of bean pod mottle virus (BPMV), cherry leaf roll virus (CLRV), raspberry ringspot virus (RpRSV), soybean mosaic virus (SMV) and tomato ringspot virus (ToRSV) infecting soybean. The simplex RT-PCR protocols produced virus-specific amplicons of 538 bp for BPMV, 139 bp for CLRV, 298 bp for RpRSV, 403 bp for SMV, and 282 bp for ToRSV, with sensitivity down to 10 -4 diluted cDNA. Further, to detect all the five viruses simultaneously in a single tube a quintuplex RT-PCR protocol was optimized with as low as 10 -3 diluted cDNA and 0.05 µM primer. To validate the reliability of the simplex RT-PCR protocol, imported soybean samples were tested by ELISA as well as RT-PCR. The results revealed that the developed protocol could detect the viruses in imported soybean, and found to be efficient than ELISA in resolving ambiguity in detection of seed borne viruses. The developed simplex and quintuplex RT-PCR protocol will be quite helpful for the diagnosis of soybean germplasm co-infected with viruses during the quarantine processing for ensuring virus free long term seed conservation in the National Gene Bank as well as for quarantine certification., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

China has the largest aquaculture eel production in the world. High-density cultivation pattern often results in an outbreak of epidemic diseases. Since the 1990s, eel "mucus sloughing and hemorrhagic septicemia disease" was often broke out in China, and brought huge economic losses to eel breeders. Anguillid herpesvirus 1 (AngHV) was detected and isolated from the diseased eel, and proved to be the pathogen of the disease. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for rapid, sensitive, and specific detection of AngHV. A set of six primers targeting the ORF51 gene of AngHV was designed, which could effectively detect purified AngHV virions, AngHV-infected cells, or eel tissue samples. The suitable reaction temperature is 63℃, and the reaction time is 40 min. There was no cross-reaction with eel and other fish viruses, including Infectious pancreatic necrosis virus (IPNV), Marine birnavirus (MABV), Rana grylio virus (RGV), Cyprinid herpesvirus 3 (CyHV-3), and Eel iridovirus (EIV). The lower detection limit of the AngHV LAMP assay is 10 copies of AngHV genome DNA, which is at least 100 times more sensitive than conventional PCR in detecting AngHV. The assay could effectively detect AngHV from collected samples with typical clinical symptoms of AngHV infection. It suggested that the LAMP assay could be used in specific detection of AngHV and has great potential for early diagnosis of AngHV infection in the farm., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The tcdA gene codes for an important toxin produced by Clostridioides difficile ( C. difficile ), but there is currently no simple and cost-effective method of detecting it. This article establishes and validates a rapid and visual loop-mediated isothermal amplification (LAMP) assay for the detection of the tcdA gene., Methods: Three sets of primers were designed and optimized to amplify the tcdA gene in C. difficile using a LAMP assay. To evaluate the specificity of the LAMP assay, C. difficile VPI10463 was used as a positive control, while 26 pathogenic bacterial strains lacking the tcdA gene and distilled water were utilized as negative controls. For sensitivity analysis, the LAMP assay was compared to PCR using ten-fold serial dilutions of DNA from C. difficile VPI10463, ranging from 207 ng/µl to 0.000207 pg/µl. The tcdA gene of C.difficile was detected in 164 stool specimens using both LAMP and polymerase chain reaction (PCR). Positive and negative results were distinguished using real-time monitoring of turbidity and chromogenic reaction., Results: At a temperature of 66 °C, the target DNA was successfully amplified with a set of primers designated, and visualized within 60 min. Under the same conditions, the target DNA was not amplified with the tcdA12 primers for 26 pathogenic bacterial strains that do not carry the tcdA gene. The detection limit of LAMP was 20.700 pg/µl, which was 10 times more sensitive than that of conventional PCR. The detection rate of tcdA in 164 stool specimens using the LAMP method was 17% (28/164), significantly higher than the 10% (16/164) detection rate of the PCR method (X 2 = 47, p < 0.01)., Conclusion: LAMP method is an effective technique for the rapid and visual detection of the tcdA gene of C. difficile , and shows potential advantages over PCR in terms of speed, simplicity, and sensitivity. The tcdA -LAMP assay is particularly suitable for medical diagnostic environments with limited resources and is a promising diagnostic strategy for the screening and detection of C. difficile infection in populations at high risk., Competing Interests: The authors declare that they have no competing interests., (© 2024 Lin et al.)

PCR is tolerant to single nucleotide mismatches. Therefore, genotyping of point mutations by PCR requires special conditions for the amplification of allele-specific PCR fragments. MS-PCR (mutagenically separated PCR) is an improved version of ARMS (amplification refractory mutation system) in which additional nucleotide mismatches near the mutation site are used to separate the wt fragments from the mutant fragments in a single-tube PCR. In the originally described procedure, the resulting fragments are resolved on agarose gels according to differences in size introduced by different lengths of the allele-specific primers. In order to evaluate the PCR fragments by melting curve analysis, we enlarged the difference in the melting temperatures of the fragments of the two alleles by increasing the GC content of the longer allele-specific primer resulting in a higher melting temperature of the corresponding fragment. Using the murine retinal degeneration mutations rd1 and rd8 as an example, we show that such primers result in an easy to handle genotyping procedure: qPCR followed by melting curve analysis. In summary, MS-PCR is a simple and easy-to-use method for detecting single nucleotide variants., (© 2024. The Author(s).)

Single-cell RNA sequencing (scRNA-seq) has emerged as a versatile tool in biology, enabling comprehensive genomic-level characterization of individual cells. Currently, most scRNA-seq methods generate barcoded cDNAs by capturing the polyA tails of mRNAs, which exclude many non-coding RNAs (ncRNAs), especially those transcribed by RNA polymerase III (Pol III). Although previously thought to be expressed constitutively, Pol III-transcribed ncRNAs are expressed variably in healthy and disease states and play important roles therein, necessitating their profiling at the single-cell level. In this study, we developed a measurement protocol for nc886 as a model case and initial step for scRNA-seq for Pol III-transcribed ncRNAs. Specifically, we spiked in an oligo-tagged nc886-specific primer during the polyA tail capture process for the 5'scRNA-seq. We then produced sequencing libraries for standard 5' gene expression and oligo-tagged nc886 separately, to accommodate different cDNA sizes and ensure undisturbed transcriptome analysis. We applied this protocol in three cell lines that express high, low, and zero levels of nc886. Our results show that the identification of oligo tags exhibited limited target specificity, and sequencing reads of nc886 enabled the correction of non-specific priming. These findings suggest that gene-specific primers (GSPs) can be employed to capture RNAs lacking a polyA tail, with subsequent sequence verification ensuring accurate gene expression counting. Moreover, we embarked on an analysis of differentially expressed genes in cell line sub-clusters with differential nc886 expression, demonstrating variations in gene expression phenotypes. Collectively, the primer spike-in strategy allows combined analysis of ncRNAs and gene expression phenotype., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Shin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Globally, around 7 to 20 million people are believed to be suffering from coinfection with both hepatitis B virus (HBV) and hepatitis C virus (HCV). The loop-mediated isothermal amplification (LAMP) approach, introduced by Notomi and colleagues, has undergone substantial advancements as an effective molecular tool that enables the simultaneous analysis of multiple samples in a single tube., Methods: The present study examined the simultaneous detection of HBV and HCV in a single tube using melt curve analysis multiplex LAMP (mLAMP), which is based on the identification of unique melting peak temperatures. Selected regions for primer design including the S gene of HBV and the UTR gene of HCV. Primer optimization is initially performed through individual HBV and HCV LAMP analysis. Following the optimization process, the mLAMP assay was evaluated by optimizing the multiplex reaction mixture, determining the reaction time, and analyzing the limit of detection (LOD). The results are also analyzed using lateral flow dipsticks (LFD), which enable the visual detection of HBV and HCV by adding 20 pmol FITC-labeled LF primers into the reaction mixture prior the mLAMP., Results: The LOD for the mLAMP assay was determined as 10 copies/µl, and no cross-reactivity with other microorganisms was detected. The detection results obtained from patient plasma were also visually demonstrated using LFD, and displayed significant concordance with those obtained from Real-Time Polymerase Chain Assay. The mLAMP assay revealed a diagnostic sensitivity of 95% for detecting the HBV, and LOD is 90% for HCV. The overall diagnostic sensitivity of the mLAMP assay for both viruses was 85%. The assay confirmed a specificity of 100%., Conclusion: The mLAMP assay displays significant promise for analyzing coinfected samples by simultaneously detecting the dual targets HBV and HCV within a set temperature of 62 °C, all within a time frame of 1 h. Additionally, when paired with disposable LFD, the mLAMP assay enables rapid visual detection of assay results in a matter of minutes. The result contributes to the mLAMP assay being highly suitable for coinfection screening, particularly in field conditions., (© 2024. The Author(s).)

Mycoplasma pneumoniae is a significant pathogen responsible for community-acquired pneumonia, predominantly affecting children and adolescents. Here, we devised a rapid method for M. pneumoniae that combined multiple cross displacement amplification (MCDA) with real-time fluorescence technology. A set of ten primers, which were specifically designed for M. pneumoniae detection, were employed in a real-time fluorescence MCDA reaction. Of these, one primer incorporated a restriction endonuclease recognition sequence, a fluorophore, and a quencher, facilitating real-time fluorescence detection. The real-time (RT)-MCDA reactions were monitored in a simple real-time fluorescence instrument and conducted under optimised conditions (64°C for 40 min). The detection limit of the M. pneumoniae RT-MCDA assay for genomic DNA extracted from M. pneumoniae culture was down to 43 fg/µl. This assay accurately identified M. pneumoniae strains without cross-reacting with other bacteria. To validate its practical application, we tested the M. pneumoniae RT-MCDA assay using genomic DNA extracted from clinical samples. The assay's detection capability proved comparable with real-time PCR, MCDA-based biosensor detection, and visual inspection under blue light. The entire process, including rapid DNA extraction and real-time MCDA detection, was completed within 1 h. Overall, the M. pneumoniae RT-MCDA assay reported here is a simple and effective diagnostic tool for rapid M. pneumoniae detection, which holds significant potential for point-of-care testing and in resource-limited regions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Xiao, Zhang, Xu, Fu, Huang, Jia, Sun, Xu, Zheng, Zhou, Wang and Meng.)

When the polymerase chain reaction (PCR) is used to amplify complex templates such as metagenomic DNA using single or degenerate primers, preferential amplification of templates (PCR bias) leads to a distorted representation of the original templates in the final amplicon pool. This bias can be influenced by mismatches between primers and templates, the locations of mismatches, and the nucleotide pairing of mismatches. Many studies have examined primer-template interactions through interrogation of the final products of PCR amplification with controlled input templates. Direct measurement of primer-template interactions, however, has not been possible, leading to uncertainty when optimizing PCR reactions and degenerate primer pools. In this study, we employed a method developed to reduce PCR bias ( i.e. , Deconstructed PCR, or DePCR) that also provides empirical data regarding primer-template interactions during the first two cycles of PCR amplification. We systematically examined interactions between primers and templates using synthetic DNA templates and varying primer pools, amplified using standard PCR and DePCR protocols. We observed that in simple primer-template systems, perfect match primer-template interactions are favored, particularly when mismatches are close to the 3' end of the primer. In more complex primer-template systems that better represent natural samples, mismatch amplifications can dominate, and heavily degenerate primer pools can improve representation of input templates. When employing the DePCR methodology, mismatched primer-template annealing led to amplification of source templates with significantly lower distortion relative to standard PCR. We establish here a quantitative experimental system for interrogating primer-template interactions and demonstrate the efficacy of DePCR for amplification of complex template mixtures with complex primer pools., Competing Interests: The authors declare there are no competing interests., (©2024 Kahsen et al.)

Introduction: Goose astrovirus (GAstV) is a newly emerging pathogen that is currently widespread among geese, causing visceral gout and leading to substantial gosling mortalities, posing a severe threat to the waterfowl industry. GAstV II is the predominant epidemic strain, characterized by its high morbidity and mortality rate. Consequently, there is an urgent necessity to develop an effective diagnostic approach to control the dissemination of GAstV II, particularly in clinical farms with limited laboratory resources., Methods: In this study, a novel multi-enzyme isothermal rapid amplification (MIRA) and lateral flow dipstick (LFD) combined assay was developed. Different primers designed specific targeting a highly conserved region within the viral RdRp gene for the detection of GAstV II. Primers optimized and MIRA-LFD assay analyzed its performance regarding limits of detection, specificity, and efficiency of detection., Results: The developed MIRA amplification is conducted at a constant temperature and accomplished within 10 minutes. Subsequent naked-eye observation of the LFD strips merely takes 5 minutes. The established MIRA-LFD method exhibits high specificity, with no cross-reaction with other pathogens and attains a detection sensitivity of 1 copy/μl, which is consistent with the reverse transcription quantitative PCR (RT-qPCR) assay. Further evaluation with clinical samples indicates that the accuracy of this MIRA-LFD method correlates well with RT-qPCR for the detection of GAstV II., Conclusion: In summary, the convenience, sensitivity, and rapidity of this newly developed detection method offer a significant advantage for on-site diagnosis of GAstV II., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhu, Chen, Xu, Ye, Ni, Huo, Hua, Yun, Yao, Wang and Zhang.)