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In 2022, citrus fruits were the second most widely produced fruit globally, highlighting their significant role in the fruit industry. However, due to their clonal propagation, these fruits are highly susceptible to viral infections, posing challenges for growers. In response to the booming nursery market, the Korean plant quarantine station reported over 80 million sapling stocks, with 15% being discarded after rigorous inspection due to contamination or disease. As the global nursery trade continues to expand, there is an urgent need for a fast and accurate diagnostic tool to ensure the health of plant stocks. In this study, we developed a TaqMan-based real-time reverse transcription-quantitative PCR assay specifically designed to detect two critical citrus viruses: citrus psorosis virus and citrus leprosis virus C. Our assay demonstrated the capability to detect virus sequences with as few as 30 copies, maintaining high PCR efficiency with RNA extracted from both twig and leaf tissues. Additionally, we incorporated an artificial sequence into the positive controls, which effectively served as a marker for detecting potential sample contamination. This comprehensive diagnostic system promises to enhance plant quarantine measures and phytosanitation practices, providing a reliable and efficient solution to safeguard citrus crops from viral threats.

IntroductionSpecies of Botryosphaeriaceae fungi are relevant pathogens of almond causing trunk cankers, extensive gumming, necrosis of internal tissues and plant dieback and dead, threatening almond productivity. A novel triplex quantitative real-time PCR (qPCR) assay was designed for the simultaneous detection and quantification of Neofusicoccum parvum, Botryosphaeria dothidea and the Botryosphaeriaceae family.Material and methodsThe method was validated in symptomatic and asymptomatic almond, avocado, blueberry and grapevine plants and in environmental samples, such as cropping soil and rainwater and in artificially inoculated trapped spores, demonstrating the same performance on several matrices.Results and discussionThe limit of detection of the triplex qPCR was 10 fg of genomic DNA for the three fungal targets, with high correlation coefficients (R2) and amplification efficiencies between 90 and 120%. Although the triplex qPCR demonstrated to be more sensitive and accurate than the traditional plate culturing and further sequencing method, a substantial agreement (kappa index = 0.8052 ± 0.0512) was found between the two detection methods. The highly sensitive qPCR assay allows for accurate diagnosis of symptomatic plants and early detection of Botryosphaeriaceae fungi in asymptomatic plants (rootstocks and grafting scions from almond nurseries). Furthermore, the triplex qPCR successfully detected Botryosphaeriaceae fungi in environmental samples, such as cropping soils and rainwater. It was also capable of detecting as few as 10 conidia in artificially inoculated tapes. Therefore, the triplex qPCR is a valuable tool for accurate diagnosis, aiding in the implementation of suitable control measures. It enables preventive detection in asymptomatic samples, helping to avoid the introduction and spread of these pathogens in production fields. Moreover, it assists in identifying inoculum sources and quantifying inoculum levels in crop environments, contributing to a precise phytosanitary application schedule, thereby reducing production costs and preserving the environment.

Objectives: The accuracy of malaria rapid diagnostic tests is threatened by Plasmodium falciparum with pfhrp2/3 deletions. This study compares gene deletion prevalence determined by multiplex real time polymerase chain reaction (qPCR) and conventional polymerase chain reaction (cPCR) using existing samples with clonality previously determined by microsatellite genotyping. Methods: Multiplex qPCR was used to estimate prevalence of pfhrp2/3 deletions in three sets of previously collected patient samples from Eritrea and Peru. The qPCR was validated by multiplex digital polymerase chain reaction. Sample classification was compared with cPCR, and receiver operating characteristic curve analysis was used to determine the optimal ΔCq threshold that aligned the results of the two assays. Results: qPCR classified 75% (637 of 849) of samples as single, and 212 as mixed-pfhrp2/3 genotypes, with a positive association between clonality and proportion of mixed-pfhrp2/3 genotype samples. The sample classification agreement between cPCR and qPCR was 75.1% (95% confidence interval [CI] 68.6-80.7%) and 47.8% (95% CI 38.9-56.9%) for monoclonal and polyclonal infections. The qPCR prevalence estimates of pfhrp2/3 deletions showed almost perfect (κ = 0.804, 95% CI 0.714-0.895) and substantial agreement (κ = 0.717, 95% CI 0.562-0.872) with cPCR for Peru and 2016 Eritrean samples, respectively. For 2019 Eritrean samples, the prevalence of double pfhrp2/3 deletions was approximately two-fold higher using qPCR. The optimal threshold for matching the assay results was ΔCq = 3. Conclusions: Multiplex qPCR and cPCR produce comparable estimates of gene deletion prevalence when monoclonal infections dominate; however, qPCR provides higher estimates where multi-clonal infections are common.

Downy mildews are major constraints on spinach and lettuce production globally. Disease management can be achieved with fungicides, but routine applications are costly and can lead to pathogen resistance. Detection of airborne spores could guide sustainable fungicide application, and, considering that spinach and lettuce are often grown in the same cycles, simultaneous detection of both pathogens is practical. Here, a multiplex hydrolysis probe quantitative PCR assay was designed using single copy mitochondrial DNA targets for spinach downy mildew (Peronospora effusa) and lettuce downy mildew (Bremia lactucae). To quantify P. effusa and B. lactucae sporangia, a standard curve was developed for each pathogen using the multiplex qPCR to amplify DNA obtained from known dilutions of sporangia. Analysis of these curves revealed a greater sensitivity for B. lactucae, indicating that the sporangia of B. lactucae may harbor more mitochondria than those of P. effusa, providing insight into the biology of these pathogens. The multiplex qPCR assay was partnered with two different spore trap types: a cyclone spore trap and an impaction spore trap. Results from air sampling revealed that the cyclone spore traps collect significantly more sporangia compared with impaction traps. Exposure of P. effusa sporangia to desiccation was performed to assess the environmental impact on the assay, and although detection levels were reduced, they were still apparent. This detection and quantification tool will be useful in efforts to improve the accuracy of downy mildew forecasting, which in return may reduce fungicide usage or improve its efficiency. [Figure: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

[Display omitted] • We developed a quantitative PCR (qPCR) able to simultaneously detect and quantify the main species of canine lungworms. • This multiplex qPCR displayed high analytical and diagnostic sensitivity and specificity. • This qPCR identified 42.9% additional mixed lungworm species infections compared with microscopy. • This qPCR will aid clinicians evaluating disease progression and treatment outcomes. • This novel tool will assist parasitologists in monitoring the spread of canine lungworms, worldwide. In recent years, Angiostrongylus vasorum , Crenosoma vulpis , Eucoleus aerophilus (syn. Capillaria aerophila) and Eucoleus boehmi (syn. Capillaria boehmi), commonly referred to as canine lungworms, have gained a growing interest worldwide as the result of their geographical expansion. Each of these nematode species differs considerably in its biology and pathogenicity. Despite their impact on dogs' health, these parasites are often underdiagnosed owing to diagnostic challenges. Here, we describe the development and validation of a Taq-Man-based multiplex quantitative PCR (qPCR) for the simultaneous detection of the main species of canine lungworms in faeces of infected dogs. Using 10-fold serial dilutions of synthetic gene block fragments containing individual sequence targets of each lungworm species, the analytical sensitivity of the assay ascertained was 1.84 ng/μl for A. vasorum , 3.08 ng/μl for C. vulpis and 0.79 ng/μl for Eucoleus spp. The sensitivity of the assays and their ability to detect mixed species infections were compared with microscopy-based techniques (faecal floatation and Baermann technique) applied to faecal samples submitted for lungworm testing through an accredited diagnostic laboratory at the Institute of Parasitology, University of Zurich, Switzerland, and from community dogs as part of a research project on canine endoparasites in Cambodia. The multiplex qPCR displayed high diagnostic sensitivity (42/46, 91.3%; 95% Confidence Interval (CI): 79.1–97.1%) and a diagnostic specificity of 100% (45/45, 95% CI: 90.6–100%), and was able to detect 42.9% additional mixed lungworm species infections compared with microscopy-based methods. Kappa statistics showed substantial agreement between the qPCRs and microscopy for mixed infections (κ = 0.72, 95% CI: 0.4–1) and Eucoleus spp. (κ = 0.65, 95% CI: 0.45–0.85) and almost perfect agreement for C. vulpis (κ = 0.85, 95% CI: 0.63–1) and A. vasorum (κ = 0.92, 95% CI: 0.84–1). This multiplex qPCR enables timely, accurate, and sensitive diagnosis of canine lungworm species in faecal samples and can be used to monitor the geographical distribution and emergence of these parasitic species, globally. [ABSTRACT FROM AUTHOR]

To elaborate the utility of multiplex quantitative polymerase chain reaction (multiplex qPCR) for the accurate diagnosis of severe respiratory tract infections (RTIs) in hospitalized children. In two separate periods during 2022, 76 respiratory specimens (combined throat/nasopharyngeal swabs) were submitted for multiplex qPCR regarding 26 respiratory pathogens. The specimens were obtained from children with severe RTIs hospitalized in the Institute for Respiratory Diseases in Children, Skopje. Multiplex qPCR detected at least one respiratory pathogen in all examined specimens (76/76), with 83% (63/76) rate of co-infections. Considering that positive results are only the ones with Ct value below 28, the rates of detected pathogens and co-infections decrease to 75% and 22%, respectively. The most commonly detected pathogens during the spring period were Parainfluenza type 3 (PIV3) followed by Adenovirus (AdV) and Respiratory syncytial virus type B (RSVB) with frequency rate of 23%, 19% and 19%, respectively. During the autumn period, the most common were RSVB and Streptococcus pneumoniae with frequency rate of 31% and 17%, respectively. Multiplex qPCR is a powerful tool for diagnosing RTIs. Semi-quantification of the viral load by reporting Ct values added higher level of evidence for accurate diagnosis. Seasonal detection of the examined viruses was notable with higher prevalence of PIV3 in spring and RSVB in autumn period. [ABSTRACT FROM AUTHOR]

In newborn screening (NBS), it is important to consider the availability of multiplex assays or other tests that can be integrated into existing systems when attempting to implement NBS for new target diseases. Recent developments in innovative testing technology have made it possible to simultaneously screen for severe primary immunodeficiency (PID) and spinal muscular atrophy (SMA) using quantitative real-time polymerase chain reaction (qPCR) assays. We describe our experience of optional NBS for severe PID and SMA in Osaka, Japan. A multiplex TaqMan qPCR assay was used for the optional NBS program. The assay was able to quantify the levels of T-cell receptor excision circles and kappa-deleting recombination excision circles, which is useful for severe combined immunodeficiency and B-cell deficiency screening, and can simultaneously detect the homozygous deletion of SMN1 exon 7, which is useful for NBS for SMA. In total, 105,419 newborns were eligible for the optional NBS program between 1 August 2020 and 31 August 2023. A case each of X-linked agammaglobulinemia and SMA were diagnosed through the optional NBS and treated at early stages (before symptoms appeared). Our results show how multiplex PCR-based NBS can benefit large-scale NBS implementation projects for new target diseases. [ABSTRACT FROM AUTHOR]

Multiplex assays implementing DNA-based methods have been demonstrated as suitable alternatives to culture-based microbiological methods; however, in most cases, they still require a suitable enrichment step. Finding suitable enrichment conditions for different bacteria may result in challenges. In the present study, a novel selective broth named MSB (mTA10 selective broth) was formulated for the simultaneous recovery of Salmonella spp., E. coli O157:H7 and L. monocytogenes. Attention was paid to ensure the optimal enrichment of L. monocytogenes as its enrichment is more challenging. To this end, cellobiose was added to increase the growth of L. monocytogenes, and sodium pyruvate was also added to improve the recovery of stressed bacteria. Four selective agents were added, namely nalidixic acid, sodium cholate, lithium chloride and potassium tellurite, to control the growth of interfering microorganisms. It was concluded that the novel broth was suitable for the simultaneous enrichment of the target pathogens, allowing them to reach concentrations higher than 7 log CFU/mL for each bacterium in pure culture. Furthermore, all heavily contaminated ready-to-eat salad samples reached concentrations higher than 5 log CFU/g. Finally, after 24 h of enrichment of spiked salad, it was possible to detect concentrations below 10 CFU/25 g. [ABSTRACT FROM AUTHOR]

Illicit administration of transgene into horses is a form of gene doping that has been a key concern in equine sports. The large number of potential performance‐enhancing transgenes has demanded a cost‐effective and reliable detection method. Multiplex qPCR is a relevant technique, but the cross‐talking between fluorophores and high background noise limits the method sensitivity and specificity. This study reports a simpler multiplexing approach by using the same fluorophore for four hydrolysis probes each targeting one of the four transgenes: human growth hormone, insulin‐like growth factor 1, equine erythropoietin and interleukin‐10. Any positive findings from this multiplex qPCR assay can then be confirmed by individual qPCR assays to identify potential transgene(s). This has effectively eliminated the cross‐talking issue and allowed an improved signal‐to‐noise than conventional multiplex qPCR assay. It has also removed the limitation imposed by the available choice of fluorophores and optical channels of qPCR instruments on the number of transgenes that can be analysed in a multiplex qPCR assay. This novel multiplex qPCR has been successfully validated. The estimated limits of detection were ~1500–2500 copies/mL of blood, thus demonstrating comparable sensitivity with the corresponding duplex qPCR assays. Concurring results were obtained by analysing hundreds of official blood samples provided by racehorses with this multiplex qPCR assay and the accredited individual duplex qPCR assays. This novel multiplex qPCR assay for detecting multiple transgenes is a cost‐effective screening method using a conventional laboratory setup and has opened up the potential to include the testing of additional transgenes in a single assay. [ABSTRACT FROM AUTHOR]

Woody canker diseases caused by fungi of the Botryosphaeriaceae family are producing increasing losses in many economically important woody crops, including almond. To develop a molecular tool for the detection and quantification of the most aggressive and threatening species is of main importance. This will help to prevent the introduction of these pathogens in new orchards and to conveniently apply the appropriate control measures. Three reliable, sensitive and specific duplex qPCR assays using TaqMan probes have been designed for the detection and quantification of (a) Neofusicoccum parvum and the Neofusicoccum genus, (b) N. parvum and the Botryosphaeriaceae family and (c) Botryosphaeria dothidea and the Botryosphaeriaceae family. The multiplex qPCR protocols have been validated on artificially and naturally infected plants. Direct systems to process plant materials, without DNA purification, allowed high-throughput detection of Botryosphaeriaceae targets even in asymptomatic tissues. These results validate the qPCR using the direct sample preparation method as a valuable tool for Botryosphaeria dieback diagnosis allowing a large-scale analysis and the preventive detection of latent infection. [ABSTRACT FROM AUTHOR]

Food contaminated with bacterial pathogens is a great threat to human health and food spoilage, having an impact on public health and the food industry. Research in food safety seeks to develop a practical, rapid, and sensitive detection technique for food-borne pathogens. In the past few decades, real-time quantitative polymerase chain reaction (qPCR) has been developed, and multiplex qPCR is a preferred feature. Multiplex qPCR enables the simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers. In this study, we have developed and evaluated a hydrolysis (TaqMan) probe-based system for simultaneous detection of eight of the most common food-borne pathogens in a single-step procedure by multiplex qPCR. A multicolor combinational probe coding (MCPC) strategy was utilized that allows multiple fluorophores to label different probes in combinatorial manner. This strategy enabled simultaneous detection, identification, and quantification of targeted genes. The efficiency of the individual qPCR reactions for each target gene had values comparable to those established for multiplex qPCR, with detection limits of approximately < 10 copies of DNA per reaction. Pathogen load helps to predict bacteriological quality status in food products and serves to validate the efficiency of procedures to minimize or eliminate their presence, so newly developed multiplex qPCR was quantitative for each pathogen. During sample preparation, a step to concentrate the target organism from a relatively large sample size, remove all potential PCR inhibitors, and yield samples in a volume suitable for qPCR was incorporated.

BACKGROUND:Most genetically modified (GM) plants contain a promoter, P35S, from the plant virus, Cauliflower mosaic virus (CaMV), and many have a terminator, TNOS, derived from the bacterium, Agrobacterium tumefaciens. Assays designed to detect GM plants often target the P35S and/or TNOS DNA sequences. However, because the P35S promoter is derived from CaMV, these detection assays can yield false-positives from non-GM plants infected by this naturally-occurring virus. RESULTS:Here we report the development of an assay designed to distinguish CaMV-infected plants from GM plants in a single multiplexed quantitative PCR (qPCR) reaction. Following initial testing and optimization via PCR and singleplex-to-multiplex qPCR on both plasmid and plant DNA, TaqMan qPCR probes with different fluorescence wavelengths were designed to target actin (a positive-control plant gene), P35S, P3 (a CaMV-specific gene), and TNOS. We tested the specificity of our quadruplex qPCR assay using different DNA extracts from organic watercress and both organic and GM canola, all with and without CaMV infection, and by using commercial and industrial samples. The limit of detection (LOD) of each target was determined to be 1% for actin, 0.001% for P35S, and 0.01% for both P3 and TNOS. CONCLUSIONS:This assay was able to distinguish CaMV-infected plants from GM plants in a single multiplexed qPCR reaction for all samples tested in this study, suggesting that this protocol is broadly applicable and readily transferrable to any interested parties with a qPCR platform.

Abstract Glaesserella parasuis, Mycoplasma hyorhinis, and Mycoplasma hyosynoviae are important porcine pathogens responsible for polyserositis, polyarthritis, meningitis, pneumonia, and septicemia causing significant economic losses in the swine industry. A new multiplex quantitative polymerase chain reaction (qPCR) was designed on one hand for the detection of G. parasuis and the virulence marker vtaA to distinguish between highly virulent and non‐virulent strains. On the other hand, fluorescent probes were established for the detection and identification of both M. hyorhinis and M. hyosynoviae targeting 16S ribosomal RNA genes. The development of the qPCR was based on reference strains of 15 known serovars of G. parasuis, as well as on the type strains M. hyorhinis ATCC 17981T and M. hyosynoviae NCTC 10167T. The new qPCR was further evaluated using 21 G. parasuis, 26 M. hyorhinis, and 3 M. hyosynoviae field isolates. Moreover, a pilot study including different clinical specimens of 42 diseased pigs was performed. The specificity of the assay was 100% without cross‐reactivity or detection of other bacterial swine pathogens. The sensitivity of the new qPCR was demonstrated to be between 11–180 genome equivalents (GE) of DNA for M. hyosynoviae and M. hyorhinis, and 140–1200 GE for G. parasuis and vtaA. The cut‐off threshold cycle was found to be at 35. The developed sensitive and specific qPCR assay has the potential to become a useful molecular tool, which could be implemented in veterinary diagnostic laboratories for the detection and identification of G. parasuis, its virulence marker vtaA, M. hyorhinis, and M. hyosynoviae.

Largemouth bass virus (LMBV) and infectious spleen and kidney necrosis virus (ISKNV) are both belong to Iridoviridae that cause considerable economic losses in the fish industry. There is no reported literature that can detect these two viruses simultaneously. In this study, we established a multiplex quantitative polymerase chain reaction (qPCR) assay that can specifically and simultaneously detect both LMBV and ISKNV in fish samples. The specificity experiment showed that the method only amplified LMBV and ISKNV but not the other 10 common fish viruses. The slope (m), efficiency (E) and linearity (R 2 ) determined from the generated standard curve were all within the optimal range of qPCR values. The detection limit of the multiplex qPCR assay was as low as 4 copies/μL for LMBV DNA and 7 copies/μL for ISKNV DNA, respectively. The established method exhibited adequate repeatability and reproducibility, and the intra- and inter-assay coefficients of variation were both less than 3 %. The accuracy of the multiplex qPCR method was validated using 229 fish samples and was more precise than that of the conventional PCR assay. In summary, the established multiplex qPCR assay can simultaneously detect LMBV and ISKNV to monitor the risk of infection LMBV and ISKNV and control the disease early. • A one-step duplex RT-qPCR assay testing for largemouth bass virus\ infectious spleen and kidney necrosis virus was developed. • No cross-reactivity were present. • The duplex assay was highly sensitive and had low limits of detections. • The established method exhibited adequate repeatability and reproducibility. [ABSTRACT FROM AUTHOR]

• An assay called BFML-qMC was developed for multiplex methylation detection. • The BFML-qMC assay combines blocking FRET probes with machine learning. • The assay blocks unmethylated amplification and produces melting curves with probes. • Validation with 80 clinical samples showed 83.33% sensitivity and 84.62% specificity. Breast cancer (BC) is the second most prevalent form of cancer, and poses a significant threat to public health. DNA methylation is an ideal marker for the early detection of BC. Fluorescence quantitative polymerase chain reaction (PCR)-based DNA methylation detection is simpler and faster but is constrained by its multiplexing capability and specificity. To address this, we developed a multiplex quantitative methylation PCR assay for the simultaneous analysis of methylation status at multiple sites specific to BC (cg11754974, cg13828440, cg18637238, and cg16652347). The machine learning model was trained using 1200 cases of multipeak data to enhance the melting curve resolution. Performance testing demonstrated the method's ability to selectively amplify methylated genes at a DNA concentration of 1 × 105 copies μL−1, with high replicability (coefficient of variation <5 %) and mutation detection capabilities as low as 10 %. When applied to 80 clinical BC samples, the assay effectively distinguished patients with early-stage BC from normal controls, achieving an area under the curve of 0.8938, sensitivity of 83.33 %, and specificity of 84.62 %. Our essay exhibits superior clinical performance when compared to the quantitative methylation-specific PCR assays for noninvasive detection of early-stage BC, which is poised to become a favorable clinical diagnostic method for early-stage BC owing to its simplicity, speed, and capacity to improve diagnostic accuracy. [ABSTRACT FROM AUTHOR]

Binmaeil H, Hanafiah A, Mohamed Rose I, Raja Ali RA. Infect Drug Resist. 2021;14:4129–4145. The authors have advised that Table 1 on page 4132 is incorrect. There is an error in the clarithromycin resistant gene reverse primer and UreA probe sequences. The authors apologize for these errors. The correct Table 1 is as follows. Table 1 Oligonucleotides Sequence Used in This Study

Multiplex assays implementing DNA-based methods have been demonstrated as suitable alternatives to culture-based microbiological methods; however, in most cases, they still require a suitable enrichment step. Finding suitable enrichment conditions for different bacteria may result in challenges. In the present study, a novel selective broth named MSB (mTA10 selective broth) was formulated for the simultaneous recovery of Salmonella spp., E. coli O157:H7 and L. monocytogenes. Attention was paid to ensure the optimal enrichment of L. monocytogenes as its enrichment is more challenging. To this end, cellobiose was added to increase the growth of L. monocytogenes, and sodium pyruvate was also added to improve the recovery of stressed bacteria. Four selective agents were added, namely nalidixic acid, sodium cholate, lithium chloride and potassium tellurite, to control the growth of interfering microorganisms. It was concluded that the novel broth was suitable for the simultaneous enrichment of the target pathogens, allowing them to reach concentrations higher than 7 log CFU/mL for each bacterium in pure culture. Furthermore, all heavily contaminated ready-to-eat salad samples reached concentrations higher than 5 log CFU/g. Finally, after 24 h of enrichment of spiked salad, it was possible to detect concentrations below 10 CFU/25 g.

IntroductionThe imminent risk of zoonoses of non-human malaria parasites is not far from reality in India, as has been observed in the case of Plasmodium knowlesi (Pk), and so is possible with P. cynomolgi (Pc), already reported from South East Asian countries. Therefore, a novel multiplex qPCR assay was developed and evaluated for detection of non-human malaria parasites- Pk and Pc in populations at risk.MethodsThe qPCR primers were designed in-house with fluorescence labeled probes (HEX for Pk and FAM for Pc). DNA samples of Pk and Pc were used as templates and further the qPCR assay was evaluated in 250 symptomatic and asymptomatic suspected human blood samples from malaria endemic areas of North Eastern states of India.ResultsThe qPCR assay successfully amplified the target 18S rRNA gene segment from Pk and Pc and was highly specific for Pk and Pc parasites only, as no cross reactivity was observed with P. falciparum (Pf), P. vivax (Pv), P. malariae (Pm), and P. ovale (Po). Standard curves were generated to estimate the limit of detection (LOD) of Pk and Pc parasites DNA (0.00275 & 0.075 ng/μl, respectively). Due to COVID-19 pandemic situation during 2020–21, the sample accessibility was difficult, however, we managed to collect 250 samples. The samples were tested for Pf and Pv using conventional PCR- 14 Pf and 11 Pv infections were observed, but no Pk and Pc infections were detected. For Pk infections, previously reported conventional PCR was also performed, but no Pk infection was detected.DiscussionThe multiplex qPCR assay was observed to be robust, quick, cost-effective and highly sensitive as compared to the currently available conventional PCR methods. Further validation of the multiplex qPCR assay in field setting is desirable, especially from the high-risk populations. We anticipate that the multiplex qPCR assay would prove to be a useful tool in mass screening and surveillance programs for detection of non-human malaria parasites toward the control and elimination of malaria from India by 2030.

Simple Summary: β-lactam antibiotics are commonly used for the treatment of severe infection in both animals and humans, and the resistance of E. coli to third-generation cephalosporins is becoming a worldwide problem. Our results revealed 407 E. coli strains isolated from central China exhibited strong resistance to first- to fourth-generation cephalosporins and monobactam antibiotics, and piperacillin/tazobactam was the most effective drug. Phenotypically, 63.88% of the isolates were positive for ESBL production and the isolation rates kept growing. Genetic characterization identified CTX-M as the most prevalent type and blaTEM + blaCTX-M as the most common ESBL genotype combination. Furthermore, a novel multiplex real-time PCR method for detecting the three most common ESBL genes was developed as a complementary rapid-screening test for antimicrobial resistance genes. These results confirm the cephalosporins resistance urgency in the central Chinese poultry sector and suggest the continuous monitoring and timely detection of ESBL-producing isolates is vitally important in establishing appropriate antimicrobial therapies and blocking their transmission. The emergence and dissemination of Escherichia coli (E. coli) strains that produce extended-spectrum beta-lactamases (ESBLs) represents a major public health threat. The present study was designed to evaluate the prevalence and characteristics of ESBL-producing Escherichia coli isolates from chickens in central China during 2016–2019. A total of 407 E. coli strains isolated from 581 chicken swabs were identified conventionally and analyzed for various cephalosporin susceptibility by disk-diffusion assay. ESBL-producing strains were screened using the double=disk synergy test and ESBL-encoding genes were carried out by PCR/sequencing. A total of 402 E. coli isolates exhibited strong resistance to first- to fourth-generation cephalosporins and monobactam antibiotics, especially cefazolin (60.69%), cefuroxime (54.05%), cefepime (35.14%), ceftriaxone (54.30%), and aztreonam (40.29%). Piperacillin/tazobactam (1.72%) was the most effective drug against the strains, but the resistance rates increased each year. Among the isolates, 262 were identified as ESBL producers and the isolation rates for the ESBL producers increased from 63.37% to 67.35% over the four years. CTX-M (97.33%) was the most prevalent type, followed by TEM (76.72%) and SHV (3.05%). The most common ESBL genotype combination was blaTEM + blaCTX-M (74.46%), in which the frequency of carriers increased steadily, followed by blaCTX-M + blaSHV (3.05%). In addition, the most predominant specific CTX-M subtypes were CTX-M-55 (48.47%) and CTX-M-1 (17.94%), followed by CTX-M-14 (11.01%), CTX-M-15 (8.02%), CTX-M-9 (6.11%), CTX-M-65 (4.58%), and CTX-M-3 (1.15%). Moreover, a novel multiplex qPCR assay was developed to detect blaCTX-M, blaTEM, and blaSHV, with limits of detection of 2.06 × 101 copies/μL, 1.10 × 101 copies/μL, and 1.86 × 101 copies/μL, respectively, and no cross-reactivity with other ESBL genes and avian pathogens. The assays exhibited 100% sensitivity and specificities of 85%, 100%, and 100% for blaCTX-M, blaTEM, and blaSHV, respectively. In conclusion, our findings indicated that ESBL-producing E.coli strains isolated from chickens in central China were highly resistant to cephalosporins and frequently harbored diversity in ESBL-encoding genes. These isolates can pose a significant public health risk. The novel multiplex qPCR method developed in this study may be a useful tool for molecular epidemiology and surveillance studies of ESBL genes. [ABSTRACT FROM AUTHOR]

Background: The extent to which infections with Ixodes ricinus-borne pathogens (TBPs), other than Borrelia burgdorferi s. l. and tick-borne encephalitis virus (TBEV), cause disease in humans remains unclear. One of the reasons is that adequate diagnostic modalities are lacking in routine or research settings. Methods: We evaluated the analytical specificity, sensitivity and robustness of qPCR assays for the detection of Anaplasma phagocytophilum, Neoehrlichia mikurensis, Spiroplasma ixodetis, several Babesia species and Spotted Fever Rickettsia species as well as Bartonella species in human samples. Results: The qPCRs were found to perform well, given the difficulties of dealing with microorganisms for which confirmed patient materials are scarce or non-existent, a hurdle that was partially overcome by using synthetic controls. Spiking blood samples with the tested microorganisms showed that the detection of the TBPs was not inhibited by the presence of blood. The acceptable sensitivity when multiplexing the different pathogens, the good inter-assay variability and the absence of cross-reactivity make them potentially suitable as human diagnostics. Conclusions: The qPCRs evaluated in this study are technically suitable for the laboratory diagnostic assessment of clinical samples for infection with tick-borne pathogens. However, clinical validation and independent confirmation are still needed, pending the availability of sufficient human samples for testing in different laboratories. [ABSTRACT FROM AUTHOR]

Aims: Diarrhoea is a common health problem in calves and a main reason for use of antimicrobials. It is associated with several bacterial, viral and parasitic pathogens, most of which are commonly present in healthy animals. Methods, which quantify the causative agents, may therefore improve confidence in associating a pathogen to the disease. This study evaluated a novel commercially available, multiplex quantitative polymerase chain reaction (qPCR) assay (Enterit4Calves) for detection and quantification of pathogens associated with calf‐diarrhoea. Methods and Results: Performance of the method was first evaluated under laboratory conditions. Then it was compared with current routine methods for detection of pathogens in faecal samples from 65 calves with diarrhoea and in 30 spiked faecal samples. The qPCR efficiencies were between 84%–103% and detection limits of 100–1000 copies of nucleic acids per sample were observed. Correct identification was obtained on 42 strains of cultured target bacteria, with only one false positive reaction from 135 nontarget bacteria. Kappa values for agreement between the novel assay and current routine methods varied between 0.38 and 0.83. Conclusion: The novel qPCR method showed good performance under laboratory conditions and a fair to good agreement with current routine methods when used for testing of field samples. Significance and impact of study: In addition to having fair to good detection abilities, the novel qPCR method allowed quantification of pathogens. In the future, use of quantification may improve diagnosis and hence treatment of calf diarrhoea. [ABSTRACT FROM AUTHOR]

In 2022, citrus fruits were the second most widely produced fruit globally, highlighting their significant role in the fruit industry. However, due to their clonal propagation, these fruits are highly susceptible to viral infections, posing challenges for growers. In response to the booming nursery market, the Korean plant quarantine station reported over 80 million sapling stocks, with 15% being discarded after rigorous inspection due to contamination or disease. As the global nursery trade continues to expand, there is an urgent need for a fast and accurate diagnostic tool to ensure the health of plant stocks. In this study, we developed a TaqMan-based real-time reverse transcription-quantitative PCR assay specifically designed to detect two critical citrus viruses: citrus psorosis virus and citrus leprosis virus C. Our assay demonstrated the capability to detect virus sequences with as few as 30 copies, maintaining high PCR efficiency with RNA extracted from both twig and leaf tissues. Additionally, we incorporated an artificial sequence into the positive controls, which effectively served as a marker for detecting potential sample contamination. This comprehensive diagnostic system promises to enhance plant quarantine measures and phytosanitation practices, providing a reliable and efficient solution to safeguard citrus crops from viral threats.

Many wildlife conservation efforts focus on the effects of one pathogen, but for many conservation efforts to be successful, researchers require an understanding of ecological processes that may include multiple co-occurring pathogens. We developed a multiplex quantitative PCR (qPCR) assay to detect four pathogens in eastern box turtles (Terrapene carolina carolina), including frog virus 3 (FV3), Terrapene herpesvirus 1 (TerHV1), box turtle Mycoplasma sp. (BTMyco), and Terrapene adenovirus (TerAdv). TaqMan™ primer probes were designed using previously published assays with four different fluorophores. Multiplex Cq values plotted against singleplex Cq values demonstrated slopes of 0.967, 1.00, 0.980, and 0.973 for TerHV1, TerAdv, FV3, and BTMyco, respectively, and R 2 values of 0.999 for all four pathogens. The assay was highly consistent with the intra-assay variation of all four pathogen targets, ranging from 0.05-1.826 % across all concentrations, while inter-assay variation ranged from 0.031-4.569 % among all four targets at all concentrations. Clinical samples were tested using previously collected samples from eastern box turtles and red-eared sliders (Trachemys scripta elegans) and performed similarly to singleplex assays. This multiplex assay is an effective, time-efficient diagnostic tool to quickly monitor chelonian pathogens by detecting FV3, TerHV1, BTMyco, and TerAdv within a single reaction. A validated and clinically utilized multiplex assay will be beneficial to characterizing a more complex pathogen profile for future chelonian epidemiological studies to better describe pathogen dynamics and their impacts on individual and population health., 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.)

The endemic status of goose parvovirus (GPV), H5 subtype avian influenza virus (AIV), and goose astrovirus (GoAstV) infections continues to devastate the poultry industry in China. Despite this, there exists a notable gap in the application of molecular diagnostic techniques. This investigation described the development of a multiplex qualitative polymerase chain reaction (qPCR) assay capable of concurrently detecting GPV, H5 AIV, and GoAstV, with no cross-reactivity observed with other avian viral pathogens. The assay exhibited a detection threshold of 10 copies/μL for both GPV and GoAstV, and 1 copy/μL for H5 AIV. The intra- and inter-assay coefficients of variation were < 3.0%, signifying high repeatability within and across assay batches. Utilizing this multiplex qPCR assay, a batch of 60 clinical samples was analyzed to assess its practical utility. The detected prevalence rates for GoAstV, GPV, and H5 AIV were 35.0% (21/60), 21.7% (13/60), and 15.0% (9/60), respectively. Concurrent infections were also identified, with rates for GPV + GoAstV, GPV + H5 AIV, GoAstV + H5 AIV, and GPV + GoAstV + H5 AIV being 6.7% (4/60), 3.3% (2/60), 3.3% (2/60), and 3.3% (2/60), respectively. The developed multiplex qPCR assay exhibited a diagnostic concordance rate equivalent to that of traditional PCR techniques. This novel assay serves as a rapid, efficient, specific, and sensitive tool for the detection of prevalent goose viruses, thereby enhancing disease management strategies and epidemiological monitoring efforts., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Introduction: Species of Botryosphaeriaceae fungi are relevant pathogens of almond causing trunk cankers, extensive gumming, necrosis of internal tissues and plant dieback and dead, threatening almond productivity. A novel triplex quantitative real-time PCR (qPCR) assay was designed for the simultaneous detection and quantification of Neofusicoccum parvum , Botryosphaeria dothidea and the Botryosphaeriaceae family., Material and Methods: The method was validated in symptomatic and asymptomatic almond, avocado, blueberry and grapevine plants and in environmental samples, such as cropping soil and rainwater and in artificially inoculated trapped spores, demonstrating the same performance on several matrices., Results and Discussion: The limit of detection of the triplex qPCR was 10 fg of genomic DNA for the three fungal targets, with high correlation coefficients (R2) and amplification efficiencies between 90 and 120%. Although the triplex qPCR demonstrated to be more sensitive and accurate than the traditional plate culturing and further sequencing method, a substantial agreement (kappa index = 0.8052 ± 0.0512) was found between the two detection methods. The highly sensitive qPCR assay allows for accurate diagnosis of symptomatic plants and early detection of Botryosphaeriaceae fungi in asymptomatic plants (rootstocks and grafting scions from almond nurseries). Furthermore, the triplex qPCR successfully detected Botryosphaeriaceae fungi in environmental samples, such as cropping soils and rainwater. It was also capable of detecting as few as 10 conidia in artificially inoculated tapes. Therefore, the triplex qPCR is a valuable tool for accurate diagnosis, aiding in the implementation of suitable control measures. It enables preventive detection in asymptomatic samples, helping to avoid the introduction and spread of these pathogens in production fields. Moreover, it assists in identifying inoculum sources and quantifying inoculum levels in crop environments, contributing to a precise phytosanitary application schedule, thereby reducing production costs and preserving the environment., 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 Romero-Cuadrado, Aguado, Ruano-Rosa and Capote.)

Hasyanee Binmaeil,1 Alfizah Hanafiah,1,2 Isa Mohamed Rose,3 Raja Affendi Raja Ali2,4 1Department of Medical Microbiology and Immunology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, 56000, Malaysia; 2GUT Research Group, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, 56000, Malaysia; 3Department of Pathology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, 56000, Malaysia; 4Department of Medicine, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, 56000, MalaysiaCorrespondence: Alfizah HanafiahDepartment of Medical Microbiology and Immunology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Cheras, Kuala Lumpur, 56000, MalaysiaEmail alfizah@ppukm.ukm.edu.my; alfizah@ukm.edu.myAims and Objectives: More than half of the world’s population is infected with Helicobacter pylori, which can cause chronic gastritis. WHO has regarded clarithromycin-resistant H. pylori as a high priority pathogen. Hence, accurate diagnosis and detection of clarithromycin- and levofloxacin-resistant H. pylori strains is essential for proper management of infection. The objective of this study was to develop and optimize multiplex quantitative PCR assay for detection of mutations associated with clarithromycin and levofloxacin resistance in H. pylori directly from the gastric biopsies.Materials and Methods: Specific primers and probes were designed to amplify ureA and mutations in 23S rRNA and gyrA genes. Singleplex and triplex qPCR assays were optimized and the assay’s sensitivities and specificities were determined. The optimized multiplex qPCR assay was performed on 571 gastric biopsies.Results: In this study, 14.7% (84/571) of the gastric biopsies were positive for H. pylori by conventional methods and 23.8% (136/571) were positive by the ureA-qPCR with 96.4% sensitivity and 88.5% specificity, while the +LR and −LR were 8.72 and 0.04, respectively. The ureA-positive samples (n=136) were subjected to multiplex qPCR which detected A2142G and A2143G mutations in the 23S rRNA gene (20.6%, 28/136) conferring clarithromycin resistance and gyrA mutations N87K, N87I, D91N, and D91Y (11.8%, 16/136) leading to levofloxacin resistance. The sensitivity and specificity of qPCR of 23S rRNA gene were 100% and 98.7%, respectively, while 100% and 99.8% for qPCR of gyrA, respectively.Conclusion: The effectiveness of this qPCR is that it is sensitive in detecting low bacterial load and will help in timely detection of clarithromycin- and levofloxacin-resistant strains, especially in case of mixed infections. Since it is culture independent, it can inform clinicians about antibiotics to be included in the first-line therapy, thereby improving the management of H. pylori infection at a much greater pace.Keywords: Helicobacter pylori, multiplex qPCR, resistance, mutation, clarithromycin, levofloxacin

Spinal muscular atrophy (SMA) is a progressive neuromuscular disease caused by biallelic pathogenic/likely pathogenic variants of the survival motor neuron 1 (SMN1) gene. Early diagnosis via newborn screening (NBS) and pre-symptomatic treatment are essential to optimize health outcomes for affected individuals. We developed a multiplex quantitative polymerase chain reaction (qPCR) assay using dried blood spot (DBS) samples for the detection of homozygous absence of exon 7 of the SMN1 gene. Newborns who screened positive were seen urgently for clinical evaluation. Confirmatory testing by multiplex ligation-dependent probe amplification (MLPA) revealed SMN1 and SMN2 gene copy numbers. Six newborns had abnormal screen results among 47,005 newborns screened during the first year and five were subsequently confirmed to have SMA. Four of the infants received SMN1 gene replacement therapy under 30 days of age. One infant received an SMN2 splicing modulator due to high maternally transferred AAV9 neutralizing antibodies (NAb), followed by gene therapy at 3 months of age when the NAb returned negative in the infant. Early data show that all five infants made excellent developmental progress. Based on one year of data, the incidence of SMA in Alberta was estimated to be 1 per 9401 live births.

Many wildlife conservation efforts focus on the effects of one pathogen, but for many conservation efforts to be successful, researchers require an understanding of ecological processes that may include multiple co-occurring pathogens. We developed a multiplex quantitative PCR (qPCR) assay to detect four pathogens in eastern box turtles (Terrapene carolina carolina), including frog virus 3 (FV3), Terrapene herpesvirus 1 (TerHV1), box turtle Mycoplasma sp. (BTMyco), and Terrapene adenovirus (TerAdv). TaqMan™ primer probes were designed using previously published assays with four different fluorophores. Multiplex Cq values plotted against singleplex Cq values demonstrated slopes of 0.967, 1.00, 0.980, and 0.973 for TerHV1, TerAdv, FV3, and BTMyco, respectively, and R2 values of 0.999 for all four pathogens. The assay was highly consistent with the intra-assay variation of all four pathogen targets, ranging from 0.05–1.826 % across all concentrations, while inter-assay variation ranged from 0.031–4.569 % among all four targets at all concentrations. Clinical samples were tested using previously collected samples from eastern box turtles and red-eared sliders (Trachemys scripta elegans) and performed similarly to singleplex assays. This multiplex assay is an effective, time-efficient diagnostic tool to quickly monitor chelonian pathogens by detecting FV3, TerHV1, BTMyco, and TerAdv within a single reaction. A validated and clinically utilized multiplex assay will be beneficial to characterizing a more complex pathogen profile for future chelonian epidemiological studies to better describe pathogen dynamics and their impacts on individual and population health. • Development of TaqMan™ multiplex qPCR assay for four chelonian pathogens. • Multiplex assay performs with low inter- and intra- assay variability. • Multiplex assay detects as few as 10 copies per reaction of each pathogen. • Presence of clinical DNA does not hinder multiplex assay efficiency. [ABSTRACT FROM AUTHOR]

Woody canker diseases caused by fungi of the Botryosphaeriaceae family are producing increasing losses in many economically important woody crops, including almond. To develop a molecular tool for the detection and quantification of the most aggressive and threatening species is of main importance. This will help to prevent the introduction of these pathogens in new orchards and to conveniently apply the appropriate control measures. Three reliable, sensitive and specific duplex qPCR assays using TaqMan probes have been designed for the detection and quantification of (a) Neofusicoccum parvum and the Neofusicoccum genus, (b) N. parvum and the Botryosphaeriaceae family and (c) Botryosphaeria dothidea and the Botryosphaeriaceae family. The multiplex qPCR protocols have been validated on artificially and naturally infected plants. Direct systems to process plant materials, without DNA purification, allowed high-throughput detection of Botryosphaeriaceae targets even in asymptomatic tissues. These results validate the qPCR using the direct sample preparation method as a valuable tool for Botryosphaeria dieback diagnosis allowing a large-scale analysis and the preventive detection of latent infection.

• The pfhrp2/3 deletion status was compared between real time polymerase chain reaction (qPCR) and conventional polymerase chain reaction. • There are similar prevalence estimates for pfhrp2/3 deletions in monoclonal infections. • qPCR yields higher pfhrp2/3 deletion prevalence in polyclonal infections. • The optimal threshold for qPCR to match the conventional polymerase chain reaction results is ΔCq = 3. The accuracy of malaria rapid diagnostic tests is threatened by Plasmodium falciparum with pfhrp2/3 deletions. This study compares gene deletion prevalence determined by multiplex real time polymerase chain reaction (qPCR) and conventional polymerase chain reaction (cPCR) using existing samples with clonality previously determined by microsatellite genotyping. Multiplex qPCR was used to estimate prevalence of pfhrp2/3 deletions in three sets of previously collected patient samples from Eritrea and Peru. The qPCR was validated by multiplex digital polymerase chain reaction. Sample classification was compared with cPCR, and receiver operating characteristic curve analysis was used to determine the optimal ΔCq threshold that aligned the results of the two assays. qPCR classified 75% (637 of 849) of samples as single, and 212 as mixed- pfhrp2/3 genotypes, with a positive association between clonality and proportion of mixed- pfhrp2/3 genotype samples. The sample classification agreement between cPCR and qPCR was 75.1% (95% confidence interval [CI] 68.6-80.7%) and 47.8% (95% CI 38.9-56.9%) for monoclonal and polyclonal infections. The qPCR prevalence estimates of pfhrp2/3 deletions showed almost perfect (κ = 0.804, 95% CI 0.714-0.895) and substantial agreement (κ = 0.717, 95% CI 0.562-0.872) with cPCR for Peru and 2016 Eritrean samples, respectively. For 2019 Eritrean samples, the prevalence of double pfhrp2/3 deletions was approximately two-fold higher using qPCR. The optimal threshold for matching the assay results was ΔCq = 3. Multiplex qPCR and cPCR produce comparable estimates of gene deletion prevalence when monoclonal infections dominate; however, qPCR provides higher estimates where multi-clonal infections are common. [ABSTRACT FROM AUTHOR]

The study aimed to develop a multiplex qPCR to detect Leishmania infantum load in different sandfly sample settings using Leishmania kDNA and sandfly vacuolar ATPase (VATP) subunit C as internal control gene. The amplification of Lutzomyia longipalpis VATP gene was evaluated together with Leishmania infantum kDNA in a multiplex reaction. The concentration of VATP gene oligonucleotides was adjusted until no statistically significant difference was observed between all multiplex standard curves and singleplex curves, that is, only kDNA amplification. Limit of detection (LoD) was measured using a probit model and a cut‐off defined by receiver operating characteristic analysis. Limit of quantification (LoQ) was assessed by a linear model using the coefficient of variation threshold of 25%. After assuring VATP gene amplification, its primer–probe concentrations were best at 100 nM/10 nM, respectively. The cut‐off Cq value for L. infantum kDNA was defined as 35.46 with 100% of sensitivity and specificity. A total of 95% LoD was determined to be of 0.162 parasites while LoQ was 5.858. Our VATP/kDNA multiplex qPCR assay shows that it can be used to evaluate both DNA integrity and determine L. infantum load in L. longipalpis even for low yielded samples, that is, individual midguts. [ABSTRACT FROM AUTHOR]

BACKGROUND Dasineura oleae (Angelini 1831) (Diptera: Cecidomyiidae) was considered a minor pest in olive orchards, but in recent years severe outbreaks have been registered in several Mediterranean countries. Damage is caused by the feeding activity of larvae that induce gall formations and alters the physiological activity of the leaves. In Italy, this pest may be controlled by four Hymenoptera parasitoid species belonging to Platygaster and Mesopolobus genera such as Platygaster demades Walker 1835, Platygaster oleae Szelenyi 1940 (Hymenoptera: Platygastridae), Mesopolobus aspilus (Walker 1835) and Mesopolobus mediterraneus (Mayr 1903) (Hymenoptera: Pteromalidae), but parasitization becomes evident only after gall dissection. RESULTS: In this study, we aim to: (i) design a primer for the detection of specimens belonging to Platygaster and Mesopolobus genera; (ii) develop a multiplex quantitative polymerase chain reaction (qPCR) protocol combined to a fast samples DNA extraction method; (iii) apply the developed protocol to field‐collected specimens and compare this method with traditional techniques based on visual estimation of parasitism rate on larvae. Primers were designed to anneal with cytochrome oxidase subunit I (COI) sequences of Platygaster and Mesopolobus genera while protocols were developed to be fast and capable to process several samples at the same time. Molecular analyses demonstrated to provide almost double of the parasitism rate assessed by visual inspection. Furthermore, on second instar larvae the PCR‐based method was able to detect ten‐fold times the parasitization rate estimated by visual inspection. CONCLUSION: The application on a greater scale of this newly developed method could be fundamental in the determination of the biological control potential in olive orchards. [ABSTRACT FROM AUTHOR]

1. The aim of this study was to develop a multiplex quantitative polymerase chain reaction (qPCR) based molecular diagnostic kit for rapid diagnosis of Salmonella enterica serovar Enteritidis and S. enterica serovar Typhimurium serotypes, which are frequently isolated worldwide from poultry samples. 2. Detection and discrimination of S. Enteritidis and S. Typhimurium were performed by targeting the sdf and the STM4492 (putative cytoplasmic protein) gene, respectively. The invA (invasion protein) gene was used to detect Salmonella spp. as a target gene, since it is considered a standard. In this study, a total of 200 bacterial strains (178 Salmonella spp. strains and 22 other genera) were used to test the specificity and sensitivity of the developed kit. The limit of detection (LOD) of the assays was determined to be 100–101 cfu/25 g from chicken meat samples artificially contaminated by litter and 100–101 cfu/ml for cloacal swab samples. 3. The multiplex qPCR results were 100% compatible with conventional serotyping results while the specificity and sensitivity values were 100%. These findings indicated that the newly developed multiplex qPCR technique can provide an alternative method to conventional serotyping of S. Enteritidis and S. Typhimurium in laboratories lacking adequate infrastructure. [ABSTRACT FROM AUTHOR]

Brazil currently has the highest number of individuals infected with human T-lymphotropic virus 1- and 2- (HTLV-1 and HTLV-2) globally. At present, neither molecular protocols nor commercial assays are available for HTLV-1/-2 diagnosis or validated by the Brazilian Ministry of Health regulatory agency (ANVISA). We developed and validated two in-house multiplex quantitative real-time PCR for HTLV-1/-2 (mqPCR_HTLV) assays, targeting the pol and tax genes, for the simultaneous identification of HTLV-1, HTLV-2, and the albumin reference gene. The robustness of the assays was evaluated on two platforms using seven commercial master mix formulations. The reactions employed double plasmids (pHTLV1-Alb and pHTLV2-Alb) for the standard curve's construction and for expressing the detection limit of the assays. They were able to detect 10 and 10 copies of HTLV-1 and 10 and 70 copies of HTLV-2 for the tax and pol targets, respectively. High efficiency was obtained using both the platforms and all the reagents evaluated and were successfully reproduced by other analysts. DNA samples from HTLV-1/-2-infected and non-infected patients and from HIV/HTLV-coinfected patients were evaluated to determine the feasibility of their use in routine diagnosis. The mqPCR_HTLV (pol and tax) assays demonstrated an overall specificity of 100% and a sensitivity of 97.4% when testing samples from patients without HIV infection, and sensitivities of 77.1% (pol) and 74.6% (tax) in samples from HIV/HTLV-coinfected patients. In addition, they resolved the issue of HTLV western blotting (WB) indeterminate and WB-untyped results in 45.5 and 66.7% of cases, respectively. The developed mqPCR_HTLV (pol and tax) assays indicated their feasibility for efficient and reliable HTLV diagnosis in various core facility laboratories under different conditions and supplies. [ABSTRACT FROM AUTHOR]

Brazil currently has the highest number of individuals infected with human T-lymphotropic virus 1- and 2- (HTLV-1 and HTLV-2) globally. At present, neither molecular protocols nor commercial assays are available for HTLV-1/-2 diagnosis or validated by the Brazilian Ministry of Health regulatory agency (ANVISA). We developed and validated two in-house multiplex quantitative real-time PCR for HTLV-1/-2 (mqPCR_HTLV) assays, targeting the pol and tax genes, for the simultaneous identification of HTLV-1, HTLV-2, and the albumin reference gene. The robustness of the assays was evaluated on two platforms using seven commercial master mix formulations. The reactions employed double plasmids (pHTLV1-Alb and pHTLV2-Alb) for the standard curve’s construction and for expressing the detection limit of the assays. They were able to detect 10 and 10 copies of HTLV-1 and 10 and 70 copies of HTLV-2 for the tax and pol targets, respectively. High efficiency was obtained using both the platforms and all the reagents evaluated and were successfully reproduced by other analysts. DNA samples from HTLV-1/-2-infected and non-infected patients and from HIV/HTLV-coinfected patients were evaluated to determine the feasibility of their use in routine diagnosis. The mqPCR_HTLV (pol and tax) assays demonstrated an overall specificity of 100% and a sensitivity of 97.4% when testing samples from patients without HIV infection, and sensitivities of 77.1% (pol) and 74.6% (tax) in samples from HIV/HTLV-coinfected patients. In addition, they resolved the issue of HTLV western blotting (WB) indeterminate and WB-untyped results in 45.5 and 66.7% of cases, respectively. The developed mqPCR_HTLV (pol and tax) assays indicated their feasibility for efficient and reliable HTLV diagnosis in various core facility laboratories under different conditions and supplies.

The emergence and dissemination of Escherichia coli (E. coli) strains that produce extended-spectrum beta-lactamases (ESBLs) represents a major public health threat. The present study was designed to evaluate the prevalence and characteristics of ESBL-producing Escherichia coli isolates from chickens in central China during 2016–2019. A total of 407 E. coli strains isolated from 581 chicken swabs were identified conventionally and analyzed for various cephalosporin susceptibility by disk-diffusion assay. ESBL-producing strains were screened using the double=disk synergy test and ESBL-encoding genes were carried out by PCR/sequencing. A total of 402 E. coli isolates exhibited strong resistance to first- to fourth-generation cephalosporins and monobactam antibiotics, especially cefazolin (60.69%), cefuroxime (54.05%), cefepime (35.14%), ceftriaxone (54.30%), and aztreonam (40.29%). Piperacillin/tazobactam (1.72%) was the most effective drug against the strains, but the resistance rates increased each year. Among the isolates, 262 were identified as ESBL producers and the isolation rates for the ESBL producers increased from 63.37% to 67.35% over the four years. CTX-M (97.33%) was the most prevalent type, followed by TEM (76.72%) and SHV (3.05%). The most common ESBL genotype combination was blaTEM + blaCTX-M (74.46%), in which the frequency of carriers increased steadily, followed by blaCTX-M + blaSHV (3.05%). In addition, the most predominant specific CTX-M subtypes were CTX-M-55 (48.47%) and CTX-M-1 (17.94%), followed by CTX-M-14 (11.01%), CTX-M-15 (8.02%), CTX-M-9 (6.11%), CTX-M-65 (4.58%), and CTX-M-3 (1.15%). Moreover, a novel multiplex qPCR assay was developed to detect blaCTX-M, blaTEM, and blaSHV, with limits of detection of 2.06 × 101 copies/μL, 1.10 × 101 copies/μL, and 1.86 × 101 copies/μL, respectively, and no cross-reactivity with other ESBL genes and avian pathogens. The assays exhibited 100% sensitivity and specificities of 85%, 100%, and 100% for blaCTX-M, blaTEM, and blaSHV, respectively. In conclusion, our findings indicated that ESBL-producing E.coli strains isolated from chickens in central China were highly resistant to cephalosporins and frequently harbored diversity in ESBL-encoding genes. These isolates can pose a significant public health risk. The novel multiplex qPCR method developed in this study may be a useful tool for molecular epidemiology and surveillance studies of ESBL genes.

Aim: Clavibacter michiganensis (Cm) is a seed‐borne plant pathogen that significantly reduces tomato production worldwide. Due to repeated outbreaks and rapid spread of the disease, seeds/transplants need to be certified free of the pathogen before planting. To this end, we developed a multiplex TaqMan qPCR assay that can accurately detect Cm in infected samples. Methods and Results: A specific region of Cm (clvG gene) was selected for primer design using comparative genomics approach. A fully synthetic universal internal control (UIC) was also designed to detect PCR inhibitors and false‐negative results in qPCRs. The Cm primers can be used alone or in a triplex TaqMan qPCR assay with UIC and previously described Clavibacter primers. The assay was specific for Cm and detected up to 10 fg of Cm DNA in sensitivity and spiked assays. Addition of the UIC did not change the specificity or sensitivity of the multiplex TaqMan qPCR assay. Conclusion: The triplex TaqMan qPCR provides a specific and sensitive diagnostic assay for Cm. Significance and Impact of the Study: This assay can be used for biosecurity surveillance, routine diagnostics, estimating bacterial titres in infected material and for epidemiological studies. The UIC is fully synthetic, efficiently amplified and multiplex compatible with any other qPCR assay. [ABSTRACT FROM AUTHOR]

Abstract Background A question of epidemiological relevance in Chagas disease studies is to understand Trypanosoma cruzi transmission cycles and trace the origins of (re)emerging cases in areas under vector or disease surveillance. Conventional parasitological methods lack sensitivity whereas molecular approaches can fill in this gap, provided that an adequate sample can be collected and processed and a nucleic acid amplification method can be developed and standardized. We developed a duplex qPCR assay for accurate detection and quantification of T. cruzi satellite DNA (satDNA) sequence in samples from domestic and sylvatic mammalian reservoirs. The method incorporates amplification of the gene encoding for the interphotoreceptor retinoid-binding protein (IRBP), highly conserved among mammalian species, as endogenous internal amplification control (eIAC), allowing distinction of false negative PCR findings due to inadequate sample conditions, DNA degradation and/or PCR interfering substances. Results The novel TaqMan probe and corresponding primers employed in this study improved the analytical sensitivity of the assay to 0.01 par.eq/ml, greater than that attained by previous assays for Tc I and Tc IV strains. The assay was tested in 152 specimens, 35 from 15 different wild reservoir species and 117 from 7 domestic reservoir species, captured in endemic regions of Argentina, Colombia and Mexico and thus potentially infected with different parasite discrete typing units. The eIACs amplified in all samples from domestic reservoirs from Argentina and Mexico, such as Canis familiaris, Felis catus, Sus scrofa, Ovis aries, Equus caballus, Bos taurus and Capra hircus with quantification cycles (Cq’s) between 23 and 25. Additionally, the eIACs amplified from samples obtained from wild mammals, such as small rodents Akodon toba, Galea leucoblephara, Rattus rattus, the opossums Didelphis virginiana, D. marsupialis and Marmosa murina, the bats Tadarida brasiliensis, Promops nasutus and Desmodus rotundus, as well as in Conepatus chinga, Lagostomus maximus, Leopardus geoffroyi, Lepus europaeus, Mazama gouazoubira and Lycalopex gymnocercus, rendering Cq’s between 24 and 33. Conclusions This duplex qPCR assay provides an accurate laboratory tool for screening and quantification of T. cruzi infection in a vast repertoire of domestic and wild mammalian reservoir species, contributing to improve molecular epidemiology studies of T. cruzi transmission cycles.

Abstract Background Most genetically modified (GM) plants contain a promoter, P35S, from the plant virus, Cauliflower mosaic virus (CaMV), and many have a terminator, TNOS, derived from the bacterium, Agrobacterium tumefaciens. Assays designed to detect GM plants often target the P35S and/or TNOS DNA sequences. However, because the P35S promoter is derived from CaMV, these detection assays can yield false-positives from non-GM plants infected by this naturally-occurring virus. Results Here we report the development of an assay designed to distinguish CaMV-infected plants from GM plants in a single multiplexed quantitative PCR (qPCR) reaction. Following initial testing and optimization via PCR and singleplex-to-multiplex qPCR on both plasmid and plant DNA, TaqMan qPCR probes with different fluorescence wavelengths were designed to target actin (a positive-control plant gene), P35S, P3 (a CaMV-specific gene), and TNOS. We tested the specificity of our quadruplex qPCR assay using different DNA extracts from organic watercress and both organic and GM canola, all with and without CaMV infection, and by using commercial and industrial samples. The limit of detection (LOD) of each target was determined to be 1% for actin, 0.001% for P35S, and 0.01% for both P3 and TNOS. Conclusions This assay was able to distinguish CaMV-infected plants from GM plants in a single multiplexed qPCR reaction for all samples tested in this study, suggesting that this protocol is broadly applicable and readily transferrable to any interested parties with a qPCR platform.

Listeria in food, are a serious risk to consumer's health that may even have a fatal outcome. To analyze Listeria in food, the methods used must provide reliable results and detect all strains of Listeria. Several qPCR systems have been published for the identification of Listeria monocytogenes, innocua, ivanovii, welshimeri and seeligeri. PCR systems for Listeria spp. have also been published. However, they do not detect all known Listeria. To achieve this, we have developed a novel multiplex real-time PCR method. This multiplex qPCR system was able to determine DNA specifically from the five most common Listeria as well as 15 other known Listeria strains simultaneously after cultivation on selective plates. [ABSTRACT FROM AUTHOR]

Vegan food claims to be free of all animal material. This must be verified by the food control authority and the production control laboratory. The method must be sensitive and able to detect animal material after processing such as pasteurization and storage. We have, therefore, developed a multiplex qPCR to simultaneously detect animal, fish and plant DNA in food samples. The system is cost-efficient and exhibited a high sensitivity. The specificity tests confirmed that all relevant species are detected. The plant-specific system served as a DNA isolation control and was used to generate valid results. A large selection of market samples had been analyzed and showed the expected results and the robustness of this analytical tool in routine analysis. [ABSTRACT FROM AUTHOR]

Periodontitis is a widespread chronic inflammatory disease caused by interactions between periodontal bacteria and homeostasis in the host. We aimed to investigate the performance and reliability of machine learning models in predicting the severity of chronic periodontitis. Mouthwash samples from 692 subjects (144 healthy controls and 548 generalized chronic periodontitis patients) were collected, the genomic DNA was isolated, and the copy numbers of nine pathogens were measured using multiplex qPCR. The nine pathogens are as follows: Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), Prevotella intermedia (Pi), Fusobacterium nucleatum (Fn), Campylobacter rectus (Cr), Aggregatibacter actinomycetemcomitans (Aa), Peptostreptococcus anaerobius (Pa), and Eikenella corrodens (Ec). By adding the species one by one in order of high accuracy to find the optimal combination of input features, we developed an algorithm that predicts the severity of periodontitis using four machine learning techniques. The accuracy was the highest when the models classified “healthy” and “moderate or severe” periodontitis (H vs. M-S, average accuracy of four models: 0.93, AUC = 0.96, sensitivity of 0.96, specificity of 0.81, and diagnostic odds ratio = 112.75). One or two red complex pathogens were used in three models to distinguish slight chronic periodontitis patients from healthy controls (average accuracy of 0.78, AUC = 0.82, sensitivity of 0.71, and specificity of 0.84, diagnostic odds ratio = 12.85). Although the overall accuracy was slightly reduced, the models showed reliability in predicting the severity of chronic periodontitis from 45 newly obtained samples. Our results suggest that a well-designed combination of salivary bacteria can be used as a biomarker for classifying between a periodontally healthy group and a chronic periodontitis group.

Periodontitis is a widespread chronic inflammatory disease caused by interactions between periodontal bacteria and homeostasis in the host. We aimed to investigate the performance and reliability of machine learning models in predicting the severity of chronic periodontitis. Mouthwash samples from 692 subjects (144 healthy controls and 548 generalized chronic periodontitis patients) were collected, the genomic DNA was isolated, and the copy numbers of nine pathogens were measured using multiplex qPCR. The nine pathogens are as follows: Porphyromonas gingivalis (Pg), Tannerella forsythia (Tf), Treponema denticola (Td), Prevotella intermedia (Pi), Fusobacterium nucleatum (Fn), Campylobacter rectus (Cr), Aggregatibacter actinomycetemcomitans (Aa), Peptostreptococcus anaerobius (Pa), and Eikenella corrodens (Ec). By adding the species one by one in order of high accuracy to find the optimal combination of input features, we developed an algorithm that predicts the severity of periodontitis using four machine learning techniques. The accuracy was the highest when the models classified "healthy" and "moderate or severe" periodontitis (H vs. M-S, average accuracy of four models: 0.93, AUC = 0.96, sensitivity of 0.96, specificity of 0.81, and diagnostic odds ratio = 112.75). One or two red complex pathogens were used in three models to distinguish slight chronic periodontitis patients from healthy controls (average accuracy of 0.78, AUC = 0.82, sensitivity of 0.71, and specificity of 0.84, diagnostic odds ratio = 12.85). Although the overall accuracy was slightly reduced, the models showed reliability in predicting the severity of chronic periodontitis from 45 newly obtained samples. Our results suggest that a well-designed combination of salivary bacteria can be used as a biomarker for classifying between a periodontally healthy group and a chronic periodontitis group. [ABSTRACT FROM AUTHOR]

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Shiga toxin-producing E. coli (STEC), are among the most frequently reported foodborne pathogens worldwide. They produce two powerful cytotoxins, called Shiga toxins (Stx1 and Stx2). Most DNA-based methods target these genes, and rely on lengthy enrichment protocols which delay early identification of potential threats. Vegetable samples are particularly problematic, as they are rich in components that may inhibit these techniques. In the present study, a short enrichment and improved matrix lysis protocols were combined and tested with qPCR implementing two different detection chemistries, a hydrolysis probe, and an intercalating dye-based one taking advantage of SYBR Green, to determine which performed best. The methodology presented a turnaround time of ∼6 h including the enrichment, sample treatment followed by DNA extraction, and STEC detection with either detection chemistry. Minor differences were identified among both detection chemistries, as the LOD95 were 6.9 and 8.6 CFU/25 g for the probe-based and intercalating dye respectively; both got relative sensitivities, specificities, and accuracies of 100 % and a Cohen's kappa, of 1.00, being this an "almost complete concordance" with the expected result. Overall, same-day detection of STEC in ready-to-eat salad samples, was achieved, and both qPCR detection chemistries demonstrated suitable for the intended application. • Evaluation of the growth of different E. coli serogroups in mTSB with different concentrations of novobiocin was performed. • Decreasing the concentration of novobiocin down to 1–2 mg/L allowed to recover a wider number of serogroups. • An LOD50 of <10 CFU/25 g was reached retaining a relative sensitivity, specificity and accuracy of 100 %. • A Cohen's kappa result of "almost complete concordance", among the multiplex qPCR and culture-based method was obtained. • The method presented a turnaround time of ∼6 h including culture, DNA extraction and multiplex qPCR detection. [ABSTRACT FROM AUTHOR]

Edwardsiellosis causes severe economic losses in the aquaculture industry worldwide. Accurate identification of the fish pathogenic Edwardsiella species is crucial for preventing disease spread and providing effective treatment strategies in the aquaculture industry. These species have high phenotypic and genetic similarities (> 99% in 16S rRNA sequences), which makes their differentiation challenging. In this study, we developed a multiplex real-time PCR diagnostic assay based on the single-copy DNA gyrase subunit B (gyr B) gene using a peptide nucleic acid (PNA) probe to discriminate among four fish pathogenic Edwardsiella species, E. tarda, E. piscicida, E. anguillarum, and E. ictaluri. Through phylogenetic analysis based on gyrB sequences (1462 bp), 57 Korean Edwardsiella isolates were affiliated to E. piscicida, E. anguillarum, and E. tarda , accounting for host and geographical origin specificity. The assay targeted a conserved region in gyr B, and designed four probes, each capable of distinguishing among the four Edwardsiella species, even in cases of 1–3 nucleotide mismatch, based on differences in melting temperature. The assay exhibited high specificity and sensitivity, with reaction efficiency in the range 85–100% and a reliable quantifiable limit of 102 copies per reaction for all fluorescence signals. In addition, it could detect and quantify the four species, even in mixed infections, allowing accurate identification within 2 h in a single reaction. This PNA probe-based multiplex qPCR assay could be a practical and reliable diagnostic tool in the field and provide correct taxonomic assignment for further research on the widened host range and niche, pathogenicity, and susceptibility of each Edwardsiella species, to help establish appropriate treatment and vaccine strategies. • A PNA probe-based multiplex qPCR assay developed in our study allows to rapid and accurate identification and quantification of four fish-pathogenic Edwardsiella species, E. tarda , E. anguillarum , E. piscicida , and E. ictaluri. • This assay exhibits high specificity and sensitivity (E = 85–100%, R2 > 0.99, and quantifiable limit = 102 copies/reaction) with highly repeatable and reproducible (CV < 2.0). • Perfectly hybridized PNA probe enables specific discrimination among the four Edwardsiella species even in 1–3 nucleotide variation and in a mixed infections by their melting temperature (T m). [ABSTRACT FROM AUTHOR]

Listeria monocytogenes is an important foodborne pathogen worldwide, which could cause listeriosis with a 20–30 % fatality rate in immunocompromised individuals. Listeria monocytogenes MLST clonal complex (CC) 8 strain is a common clone in food and clinical cases. The aim of this study was to develop multiplex PCR (mPCR) and high-resolution melting (HRM) qPCR to simultaneously detect L. monocytogenes CC8 and the other L. monocytogenes strains based on pan-genome analysis. A novel multiplex PCR and HRM qPCR targeted for the genes LM5578_1180 (specific for CC8) and LM5578_2262 (for L. monocytogenes) were developed. The specificity of this multiplex PCR and HRM qPCR were verified with other CCs of L. monocytogenes and other species strains. The detection limit of this multiplex PCR and HRM qPCR is 2.1 × 103 CFU/mL and 2.1 × 100 CFU/mL, respectively. This multiplex PCR and HRM qPCR could accurately detect CC8 strains with the interference of different ratios of L. monocytogenes CC9, CC87, CC121, CC155, and L. innocua strains. Subsequently, the detection ability of mPCR and HRM qPCR were also evaluated in spiked samples. The mPCR method could successfully detect 6.2 × 103 CFU/mL of CC8 L. monocytogenes after 6 h enrichment while the multiplex HRM qPCR method could successfully detect 6.2 × 104 CFU/mL of CC8 L. monocytogenes after 3 h enrichment. The feasibility of these methods were satisfactory in terms of sensitivity, specificity, and efficiency after evaluating 12 mushroom samples and was consistent with that of the National Standard Detection Method (GB4789.30–2016). In conclusion, the developed assays could be applied for rapid screening and detection of L. monocytogenes CC8 strains both in food and food production environments, providing accurate results to adopt monitoring measures to improve microbiological safety. • Mining of specific gene in Listeria monocytogenes and L. monocytogenes CC8 strains based on comparative genome analysis. • A multiplex PCR and HRM qPCR were developed for simultaneous identification Listeria monocytogenes CC8 strains. • This novel method can be applied to the detection of Listeria monocytogenes CC8 in food samples. • This novel method is specific, sensitive, economical, and had a run-time within 9 h. [ABSTRACT FROM AUTHOR]