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In infections of the Central Nervous System (iCNS), rapid identification of causing pathogens is crucial for survival and to avoid long-term sequelae. Targeted therapy may reduce side effects and development of antibiotic resistance. New molecular-based syndromic tests such as the "meningitis/encephalitis panel" (MEP) allow accelerated pathogen identification from cerebrospinal fluid. We conducted a clinical study to evaluate the MEP's efficacy in paediatric patients.Cohort study in a unique clinical setting by comparing the outcome data of two neighbouring Children's Hospitals in Germany which are comparable in size, catchment area and equipment but differ regarding availability of the MEP: study centre 1 (SC1): yes; SC2: no. The study population included 213 paediatric patients with a suspected iCNS (SC1: 106; SC2: 107), with comparable age, CRP at admission and frequency of intensive care. The primary outcome was total use of antibiotics.Total antibiotic use per patient was numerically lower in SC1 than in SC2 (SC1: median 2.83 days; SC2 3.67 days; p = 0.671). Multiple linear regression analysis did not show a relevant association between MEP-availability and total antibiotic use (ß = 0.1, 95% confidence interval [-1.46; +1.67], p = 0.897). In the subcohort with suspected meningoencephalitis (SC1: 18, SC2: 17), duration of acyclovir treatment was shorter in SC1 than in SC2 (median 1.3 days vs. 2.7 days, descriptive p = 0.0397).The add-on use of the MEP in paediatric patients with suspected iCNS was associated with a non-significant reduction in total antibiotic use, and with a reduced exposure to acyclovir in treated patients.

We investigated the concordance between the Unyvero Hospitalized Pneumonia (HPN) application and quantitative culture for detection of bacterial pathogens from serial lower respiratory tract (LRT) specimens collected from the same subject. Comparison of results from HPN application and culture was evaluated using 69 LRT samples from 27 subjects, using two evaluation approaches. False positive detections by the HPN application was 29% (20/69) in Evaluation I vs 10% (7/68) in Evaluation II. Additional pathogens detected by the HPN application could be confirmed in many instances by culture positivity for the same organism from previous or subsequent samples from the same subject.

Detection of mutations by multiplex real-time RT-PCR is a widely used method for the screening of SARS-CoV-2 variants, but this method has several limitations. We describe three cases in which a Mu strain containing the mutation K417N was initially misclassified as the Beta variant. We recommend the detection of P681H to distinguish between these two variants. Our experience highlights the importance of keeping track of new variants and mutations in order to adapt the current workflows.

Short tandem repeats (STRs) are the preferred genetic markers in forensic DNA analysis, routinely measured by capillary electrophoresis (CE) method based on the fragment length features. While, the massive parallel sequencing (MPS) technology could simultaneously target a large number of intriguing forensic STRs, bypassing the intrinsic limitations of amplicon size separation and accessible fluorophores in CE, which is efficient and promising for enabling the identification of forensic biological evidence. Here, we developed a novel MPS-based Forensic Analysis System Multiplecues SetB Kit of 133-plex forensic STR markers (52 STRs and 81 Y-STRs) and one Y-InDel (M175) based on multiplex PCR and single-end 400 bp sequencing strategy. This panel was subjected to developmental validation studies according to the SWGDAM Validation Guidelines. Approximately 2185 MPS-based reactions using 6 human DNA standards and 8 male donors were conducted for substrate studies (filter paper, gauze, cotton swab, four different types of FTA cards, peripheral venous blood, saliva, and exfoliated cells), sensitivity studies (from 2 ng down to 0.0625 ng), mixture studies (two-person DNA mixtures), PCR inhibitor studies (seven commonly encountered PCR inhibitors), species specificity studies (11 non-human species), and repeatability studies. Results of concordance studies (413 Han males and 6 human DNA standards) generated by STRait Razor and in-house Python scripts indicated 99.98% concordance rate in STR calling relative to CE for STRs between 41,900 genotypes at 100 STR markers. Moreover, the limitations of present studies, the nomenclature rules and forensic MPS applications were also described. In conclusion, the validation studies based on ~ 2200 MPS-based and ~ 2500 CE-based DNA profiles demonstrated that the novel MPS-based panel meets forensic DNA quality assurance guidelines with robust, reliable, and reproducible performance on samples of various quantities and qualities, and the STR nomenclature rules should be further regulated to integrate the inconformity between MPS-based and CE-based methods.

Dried blood spots (DBS) are the best sampling for large and population-based genetic studies. The study aimed to develop a DNA extraction protocol for DBS samples in a 96-well microtiter plate format using DNA salting-out principle. The developed protocol was named High-Throughput Salting-Out (HTSO). The DBS samples were collected from 20 suspected Glutaric Acidemia Type I (GA-I) patients from unrelated families and 50 controls without any metabolic disorders. The DNA was isolated from DBS samples using HTSO protocol in a 96-well microtiter plate. The assessment of yield and quality of DNA after isolation by HTSO method was done using NanoDrop-8000. The DNA isolated by the HTSO was used in the genotyping of multiple mutations, R402W and W225X in the Glutaryl-CoA Dehydrogenase gene in GA-I patients by multiplex PCR and RFLP. The HTSO showed at least a 2- to 34-fold increased DNA yield against manual in-house, commercial, and automation-based protocols. The per sample DNA extraction cost of the HTSO method was four times lesser than that of the commercial kits. Without sophisticated instruments or automation, HTSO method considerably reduced the turnaround time of DNA extraction compared to manual in-house and commercial kit protocols. The HTSO method is efficient, inexpensive, and time-saving for large-scale and potential high-throughput DNA extraction for DBS samples. This method is adequate for high-end diagnostic and institution facilities, in addition to institutions having financial constraints or lacks sophisticated instruments.

A multiplex PCR assay was developed to simultaneously identify 22 mammalian species (alpaca, Asiatic black bear, Bactrian camel, brown rat, cat, cattle, common raccoon, dog, European rabbit, goat, horse, house mouse, human, Japanese badger, Japanese wild boar, masked palm civet, pig, raccoon dog, red fox, sheep, Siberian weasel, and sika deer) and four poultry species (chicken, domestic turkey, Japanese quail, and mallard), even from a biological sample containing a DNA mixture of multiple species. The assay was designed to identify species through multiplex PCR and capillary electrophoresis, with a combination of amplification of a partial region of the mitochondrial D-loop by universal primer sets and a partial region of the cytochrome b (cyt b) gene by species-specific primer sets. The assay was highly sensitive, with a detection limit of 100 copies of mitochondrial DNA. The assay's ability to identify species from complex DNA mixtures was demonstrated using an experimental sample consisting of 10 species. Efficacy, accuracy, and reliability of the assay were validated for use in forensic analysis with the guidelines of Scientific Working Group on DNA Analysis Methods (SWGDAM). The multiplex PCR assay developed in this study enables cost-effective, highly sensitive, and simultaneous species identification without massively parallel sequencing (MPS) platforms. Thus, the technique described is straightforward and suitable for routine forensic investigations.

In this study, we developed and optimized a multiplex polymerase chain reaction (PCR) for simultaneous detection of two important leaf rust resistance genes Lr24 and Lr68. Seedling resistance gene Lr24 and slow rusting gene Lr68 encoding resistance to leaf rust were identified in forty-one advanced salt-tolerant wheat genotypes using molecular markers viz. Sr24#50 and csGS, respectively. For re-validation of PCR reaction results, a seedling resistance test (SRT) was performed in controlled glasshouse conditions. Genotypes were evaluated under epiphytotic conditions for SRT by artificial inoculation with a spectrum of Puccinia triticina pathotypes. Results of SRT were supplemented by PCR results, indicated that the gene Lr24/Sr24 conferred leaf rust resistance in two wheat genotypes namely HD 2851 and KRL2029. In advanced line KRL2029, along with Lr24/Sr24 other rust resistance genes (Lr26/Sr31/ Yr9) were also present. Molecular marker csGS for a known slow rusting gene Lr68 was amplified in fifteen salt-tolerant wheat genotypes indicated the presence of Lr68 in these lines. These lines could also be utilized as donors for slow rusting characteristics. Combination of both the genes could provide resistance to leaf rust and stem rust. Presences of respective genes in salt-tolerant lines were confirmed by SRT as well as molecular markers separately and multiplex PCR assay was designed. Multiplex PCR method allowed to reduce the cost and time of analyses as well as reducing the workload.

Mycobacterium tuberculosis (M. tuberculosis) genotyping provides valuable information related to the origin and the evolution of the isolates. This study aimed to evaluate the applicability of single-nucleotide polymorphisms (SNPs) technique for lineages identification of M. tuberculosis and compare it with mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) method. The lineages of 162 clinically isolates were evaluated using six pair primers by Multiplex-PCR based on SNPs. Among 162 isolates, 70 (43.2%) isolates were lineage 4, following that 62 (38.3%) and 22 (13.6%) isolates were lineage 3 and 2, respectively. The method could not type 8 (4.9%) isolates. Moreover, we could identify 71 out of 79 unknown isolates resulted from the MIRU-VNTR method. The results showed that the SNP typing method has the potential to determine the lineages of M. tuberculosis as a rapid laboratory screening test.

A total of 220 birds of age ranging from 3 to 14 weeks old were collected from several backyards and different farms in Sharkia Governorate, Egypt, and surveyed for the presence of fowl cholera. Twenty Pasteurella multocida from chickens (15/145, 10%) and ducks (5/75, 6%) were bacteriologically isolated, and it was shown that the infection was significantly related to age and breed. Capsular typing, using multiplex polymerase chain reaction (PCR), demonstrated that all strains were type A (100%). Disk diffusion assay towards ten antimicrobials revealed high susceptibilities to amikacin, doxycycline, chloramphenicol, and neomycin with varying degrees. Doxycycline was effective at the lowest concentration (MIC 0.125–1 µg/ml). Multidrug resistance was detected with a percentage of 25%. Multidrug-resistant isolates (five isolates) were subjected to study their pathogenicity in embryonated chicken eggs (ECE). The results showed a variation in indices between different dilutions of the tested strains. The resulting pathogenicity indices showed significant differences (P

This study aimed to identify Mycoplasma bovis, Myc. dispar, and Myc. bovirhinis, which are involved in bovine respiratory disease through a multiplex PCR as an alternative to culture's features that hamper Mycoplasma isolation. Nasal swabs were taken from 335 cattle with and without respiratory disease background (RDB) from dairy herds in the central region of Mexico. Each sample was divided in two; the first part was processed for the direct DNA extraction of the nasal swab and the second for Mycoplasma isolation, culture, and then the multiplex PCR was performed. In the nasal swabs, Myc. bovis was identified in 21.1%; Myc. dispar, in 11.8%; and Myc. bovirhinis, in 10.8% in cattle with RDB. Isolates were identified as Myc. bovis, 20.1%; Myc. dispar, 11.8%; and Myc. bovirhinis, 6.1%. There is a strong correlation between the presence of Mycoplasma identified by PCR and the clinical history of the disease (ρ 0.0000). In animals without RDB, Myc. bovirhinis was the only species detected in 6.1% of the samples processed directly for multiplex PCR, and in 2% of the isolates. There is an excellent correlation (kappa 0.803) between the isolation and the 16S PCR and a high correlation (kappa 0.75) between the isolation and the multiplex PCR. Therefore, we conclude that the PCR multiplex test is highly sensitive and may be used for the diagnosis and surveillance of the three species in biological samples and mycoplasma isolates.

Shiga toxin-producing E. coli (STEC) are important foodborne pathogens that causing serious public health consequences worldwide. The present study aimed to estimate the prevalence ratio and to identify the zoonotic potential of E. coli O157 isolates in slaughtered adult sheep, goats, cows and buffaloes. A total of 400 Recto-anal samples were collected from two targeted sites Rawalpindi and Islamabad. Among them, 200 samples were collected from the slaughterhouse of Rawalpindi included sheep (n = 75) and goats (n = 125). While, 200 samples were collected from the slaughterhouse of Islamabad included cows (n = 120) and buffalos (n = 80). All samples were initially processed in buffered peptone water and then amplified by conventional PCR. Samples positive for E. coli O157 were then streaked onto SMAC media plates. From each positive sample, six different Sorbitol fermented pink-colored colonies were isolated and analyzed again via conventional PCR to confirm the presence of rfbE O157 gene. Isolates positive for rfbE O157 gene were then further analyzed by multiplex PCR for the presence of STEC other virulent genes (sxt1, stx2, eae and ehlyA) simultaneously. Of 400 RAJ samples only 2 (0.5%) showed positive results for E. coli O157 gene, included sheep 1/75 (1.33%) and buffalo 1/80 (1.25%). However, goats (n = 125) and cows (n = 120) found negative for E. coli O157. Only 2 isolates from each positive sample of sheep (1/6) and buffalo (1/6) harbored rfbE O157 genes, while five isolates could not. The rfbE O157 isolate (01) of sheep sample did not carry any of STEC genes, while the rfbE O157 isolate (01) of buffalo sample carried sxt1, stx2, eae and ehlyA genes simultaneously. It was concluded that healthy adult sheep and buffalo are possibly essential carriers of STEC O157. However, rfbE O157 isolate of buffalo RAJ sample carried 4 STEC virulent genes, hence considered an important source of STEC infection to humans and environment which should need to devise proper control systems.

The aim of this study was to identify and validate a sensitive, high-throughput, and cost-effective SARS-CoV-2 real-time RT-PCR assay to be used as a surveillance and diagnostic tool for SARS-CoV-2 in a university surveillance program. We conducted a side-by-side clinical evaluation of a newly developed SARS-CoV-2 multiplex assay (EZ-SARS-CoV-2 Real-Time RT-PCR) with the commercial TaqPath COVID-19 Combo Kit, which has an Emergency Use Authorization from the FDA. The EZ-SARS-CoV-2 RT-PCR incorporates two assays targeting the SARS-CoV-2 N gene, an internal control targeting the human RNase P gene, and a PCR inhibition control in a single reaction. Nasopharyngeal (NP) and anterior nares (AN) swabs were tested as individuals and pools with both assays and in the ABI 7500 Fast and the QuantStudio 5 detection platforms. The analytical sensitivity of the EZ-SARS-CoV-2 RT-PCR assay was 250 copies/ml or approximately 1.75 genome copy equivalents per reaction. The clinical performance of the EZ-SARS-CoV-2 assay was evaluated using NP and AN samples tested in other laboratories. The diagnostic sensitivity of the assay ranged between 94 and 96% across the detection platforms, and the diagnostic specificity was 94.06%. The positive predictive value was 94%, and the negative predictive value ranged from 94 to 96%. Pooling five NP or AN specimens yielded 93% diagnostic sensitivity. The overall agreement between these SARS-CoV-2 RT-PCR assays was high, supported by a Cohen’s kappa value of 0.93. The EZ-SARS-CoV-2 RT-PCR assay performance attributes of high sensitivity and specificity with AN sample matrix and pooled upper respiratory samples support its use in a high-throughput surveillance testing program. Supplementary Information The online version contains supplementary material available at 10.1007/s00705-021-05148-1.

Plasmid-mediated colistin-resistance genes have been reported in human origin clinical samples worldwide which raises its threats to human infections. Notably, mcr-1, mcr-3, mcr-8, and mcr-10 have been reported isolated directly from clinical samples which creates more seriously threaten to human health than other mcr gene types. A multiplex polymerase chain reaction (Multi-PCR) protocol was developed to detect and genotype mobile colistin-resistance genes (mcr-1, mcr-3, mcr-8, mcr-10) in Enterobacteria for clinical laboratory purposes. We first designed four pairs of new primers for the amplification of mcr-1, mcr-3, mcr-8, and mcr-10 gene respectively to achieve stepwise separation of amplicons between 216 and 241 bp, and complete this Multi-PCR system with the assistance of another pair of universal primer. Among which the forward primers for mcr-8 and mcr-10 amplicons were identical. The protocol was validated by testing 11 clinical isolates of Escherichia coli and 3 clinical isolates of Klebsiella from human origin, each well characterized and prospectively validated. The Multi-PCR assay showed full concordance with whole-genome sequence data and displayed higher sensitivity and 100% specificity. The assay could detect all variants of the various mcr alleles described. The Multi-PCR assay successfully genotyped of mcr alleles described in one test.

The target leaf spot, an end-of-cycle disease that can cause soybean losses of 40%, is caused by the fungus Corynespora cassiicola. The aim of this study was to devise and validate a multiplex PCR protocol for the molecular identification of C. cassiicola, in both pure cultures of isolates and plants. For the analysis, a total of 75 samples was evaluated been 57 possible fungal isolates of C. cassiicola and 18 soybean plants displaying symptoms associated with target leaf spot. Total DNA was extracted from the pure cultures of the isolates and of four symptomatic leaf discs taken from each of the plants soybean evaluated and submitted to the conventional multiplex PCR technique using two primer pairs: ITS-1/ITS-4 and GA4-F/GA4-R. The ideal conditions for multiplex PCR were adjusted. The 57 isolates identified as C. cassiicola and among the evaluated plants, 15 samples were infected. Conventional multiplex PCR is efficient at identifying Corynespora cassiicola isolates and for rapid diagnosis of infected plants.

Viral respiratory rapid multiplex PCR assays FilmArray® (FA) and ePlex® (eP) provide qualitative results which may not reflect clinical relevance. In a pilot study, we report retrospectively whether the semi-quantitative PCR assay R-GENE® would have facilitated clinical interpretation. Forty-four patients were hospitalized for various respiratory manifestations; all of them have benefited from a respiratory sample during acute symptoms. Among the 44 patients, FA detected 23 positive samples including 31 viruses, 26 of them gave high or moderate R-GENE® scores (cycle threshold

Helicobacter pylori cagA + genotype is a leading risk factor for gastric cancer development making accurate identification and timely eradication of H. pylori critical to deadly gastric cancer prevention. Traditional clinical diagnostic methods, including conventional in vitro culture, histological examination, and (13/14)C-urea breath test methods, could only identify the presence of H. pylori, but these means are not capable of identification of cagA + strains. Herein, we firstly built a multiplex detection system based on novel accelerated PCR that could realize one-step detection of as low as 20 copies of H. pylori 16S rDNA and cagA genes within 30 min. In addition, this novel system performed strong anti-jamming capacity, and exhibited that it could specifically differentiate H. pylori cagA- and cagA + genotypes co-existence with other 4 kinds of gastrointestinal pathogens. Furthermore, this one-step system showed remarkable performance on rapid H. pylori infection diagnosis and cagA + genotypes identification in clinical gastric mucosa samples. Specifically, it outperformed histological examination in terms of accuracy and was superior to conventional PCR and DNA sequencing in terms of efficiency. This rapid, sensitive, and reliable H. pylori detection and identification system would break the limitation of traditional methods and realize H. pylori infection diagnosis and cagA + genotypes identification.

The modern and rapid avenue for detecting pathogens provided by molecular genetic techniques including polymerase chain reaction (PCR) was explored in the present study to identify prevalent disease pathogens, from six aquaculture farms and in two commonly cultured fish in Ghana. The specific detection was carried out directly on clinical samples of naturally infected fish (O. niloticus and C. gariepinus) based on syber-mix reaction protocol in traditional PCR. Molecular diagnostic techniques allowed the detection of the six most common and important bacterial pathogens in aquaculture farms in Ghana. Also, three of the pathogens (Streptococcus agalactiae, Streptococcus iniae, and Staphylococcus aureus) were simultaneously isolated in a multiplex reaction. The results indicated 90-100% sensitivity and specificity for each of the six bacterial pathogens tested. Streptococcosis and motile aeromonad septicemia were found to be highly prevalent in most aquaculture farms in Ghana with severity in infections traced to the 85.7% and 14.9% co-infections with all six target pathogens in catfish and tilapia respectively. The prevalence rate of infections significantly correlated with variations in salinity, conductivity, and dissolved oxygen concentrations in the thermal stressed condition of the culture water. Multiplex techniques employed in this study represent one of the first to be used by a fish health laboratory in Ghana for rapid detection of pathogens in diseased fish and could be a useful alternative to the culture-based method for routine diagnosis of fish diseases in Ghana.

Background Brucella suis is a zoonotic pathogen with a serious impact on public health and the pig industry worldwide. Information regarding B. suis in pigs in Egypt is scarce. This study aimed to investigate the prevalence of B. suis in slaughtered domestic pigs at El-Basatin abattoir in Cairo, Egypt. A total of 1,116 domestic pigs slaughtered in 2020 were sampled for Brucella isolation and identification. Identified Brucella isolates were molecularly confirmed at species, and biovar levels using Bruce ladder PCR and Suis ladder multiplex PCR. Additionally, high-risk practices of 16 abattoir workers (4 veterinarians, 10 butchering and evisceration workers, and 2 scalding workers) were investigated using a pre-piloted structured questionnaire. Results Brucella isolates were recovered from 1.3% of examined pigs (n = 14) at consistently low rates (1.1—2.9%) across the year of sampling from February to December 2020. All isolates were confirmed as B. suis biovar (bv) 2. Remarkably, 92.9% (13/14) of isolates showed atypical ability to produce H2S and hence were considered as B. suis bv2 atypical phenotype. The prevalence was higher in males (1.8%) than in females (0.9). However, this difference was not significant (Odds ratio = 1.9; CI 95% 0.7 – 5.7; P = 0.2). No detectable pathological lesions were associated with B. suis bv2 infection in examined pigs. All strains were isolated from cervical lymph nodes, highlighting a potential oral transmission. High-risk practices were recorded among swine abattoir workers in this study: 75% do not wear gloves or disinfect their knives daily, and 18.8% were willing to work with open wound injuries. Conclusions To the best of our knowledge, this is the first isolation of B. suis bv2 in Egypt. Detection of H2S producing B. suis bv2 atypical phenotype is alarming as it may result in misinterpretation of these isolates as highly human pathogenic B. suis bv1 in Egypt and possibly elsewhere. Further epidemiological tracing studies are crucial for the detection of the origin of this biovar. Including pigs in the national surveillance program of brucellosis, and an education program for swine abattoir workers about occupational risk of B. suis is a need in Egypt.

Background This study investigated the diagnostic utility of the BioFire FilmArray Pneumonia Panel (PN panel), an automated and multiplexed nucleic acid detection system that rapidly detects 26 pathogens (18 bacteria and eight viruses) and seven antimicrobial resistance markers in a single assay. Methods We analyzed the targets in lower respiratory tract specimens using the PN panel and compared the detection results with those of bacterial culture methods and antimicrobial susceptibility testing. Results Of the 57 samples analyzed, the PN panel detected 97 targets (84 bacteria, four viruses, and nine antimicrobial resistance markers). Detection of bacteria and antimicrobial resistance was three times greater than that of the bacterial culture (25 bacteria and two resistant isolates) against the targets available in the panel. The overall positive and negative percent agreements between the PN panel and culture methods for bacterial detection were 100.0% and 92.9%, respectively. Multiple pathogens were detected by the PN panel in 24 samples (42.1%), ranging from two pathogens in 11 samples (19.3%) to six pathogens in one sample (1.8%). The PN panel semiquantitatively detected higher copies (≥ 106 copies/mL) of bacterial targets if the bacteria were positive by the culture method. In contrast, the semiquantitative values obtained by the panel varied (104 to 107 ≤ copies/mL) among bacteria that were negative by the culture method. Conclusions The PN panel enhanced the detection of pathogens and antimicrobial resistance markers in lower respiratory tract specimens.

Precise molecular diagnosis of pathogenic bacterial infections decreases the spread of the disease and facilitates to make suitable disease management strategies. Advanced molecular diagnostics has taken a noticeable place and has revealed advantageous in diagnostic laboratory for routine detection, fingerprinting and epidemiologic analysis of infectious microorganisms. Molecular tests reduce the exposure to infectious agents and decreases the health risk for the consumers. This review aims to discuss our most recent of state-of-the-art molecular diagnostic techniques enabling fast and accurate detection and identification of plant and food borne pathogens based on well-developed genotyping techniques like polymerase chain reaction, Multiplex PCR, RT PCR, LAMP, LSSP PCR and SSCP PCR assay.

Sensitive, simple, and rapid pathogen separation and detection methods are essential for the early diagnosis and immediate medical treatment of bacterial infections. In this study, a novel strategy based on vancomycin (Van)-modified ovalbumin (OVA) mediated magnetic nanoprobes (MBs-OVA-Van) combined with multiplex polymerase chain reaction (mPCR) was developed for efficient isolation and detection of Staphylococcus aureus (S. aureus) and Bacillus cereus (B. cereus). Van conjugated to ovalbumin coated magnetic microcarrier via strong covalent interaction. In addition, Van binds through hydrogen bonds with d-alanyl-d-alanine moieties on the surface of gram-positive bacteria cell wall. Under optimum conditions, the MBs-OVA-Van showed that the capture efficiency (CE) for S. aureus and B. cereus in PBS and juice samples were around 91%, 92% and 89%, 85%, respectively. In addition, with a mixture containing both target bacteria in PBS and juice samples, the CE for S. aureus and B. cereus were recorded at about 90%, 91% and 80%, 84%, respectively. The MBs-OVA-Van plus mPCR method applied in PBS and juice samples for both bacteria showed limits of detection at 102 CFU mL-1 in a < 3 h total assay time. The results indicated that the method holds promise for isolation and enrichment of gram-positive bacteria with S. aureus and B. cereus as models.

The identification of mixed stains has always been a difficult problem in personal identification in the forensic field. In recent years, tissue-specific methylation sites have proven to be very stable biomarkers for distinguishing tissue origin. However, it is still challenging to perform tissue source identification and individual identification simultaneously. In this study, we developed a method that uses tissue-specific methylation markers combined with single-nucleotide polymorphism (SNP) markers to detect semen from mixed biofluids and to identify individuals simultaneously. Semen-specific CpG markers were chosen from the literature and further validated utilizing methylation-sensitive restriction endonuclease (MSRE) combined with PCR technology. The neighboring SNP markers were searched in the flanking sequence of the target CpG within 400 bp, and SNP typing was then carried out through a single-base extension reaction followed by capillary electrophoresis. Eventually, a method of MSRE combined with SNaPshot that could detect 12 compound CpG-SNP markers was developed. Using this system, 10 ng of total DNA and DNA mixture with semen content up to 25% could be typed successfully. Moreover, the cumulative discrimination power of the system in the northern Chinese Han population is 0.9998. This study provides a valuable strategy for forensic practice to perform tissue origin and individual identification from mixed stains simultaneously.

The present review deals with the serological and molecular techniques available for detection of fungal pathogens in plants, followed by management practices using bioinoculants which can induce resistance in the hosts. Specific detection of pathogens in the plant tissues even before the appearance of symptoms are done either through protein based immunodiagnostic techniques or DNA/RNA based molecular techniques. Immunotechniques depend on specific interaction between antigen of the pathogen or infected tissues and antibodies prepared against the specific antigen. Positive reactions are detected through either qualitative or quantitative techniques. Among the immunodiagnostic techniques, the most widely and commonly used is the enzyme linked immunosorbent assay (ELISA), Dot blot, Western blot and Immunofluorescence. The molecular techniques are predominantly PCR based and include different variations with increasing sensitivity such as nested PCR, multiplex PCR, reverse transcriptase PCR, real time PCR, loop-mediated isothermal amplification (LAMP) and nano based PCR. Thus, by use of one or more sophisticated techniques it is possible to detect pathogens rapidly and specifically. Pathogen detection is generally followed by management techniques, and in the current scenario ecologically safe bioinoculants are used for induction of immunity in the plants. There are several bioinoculants based on beneficial microorganisms such as plant growth promoting bacteria, biocontrol agents and arbuscular mycorrhizal fungi. Application of these microorganisms, singly or as a consortium, has been reported to induce immunity in plants. Induced systemic resistance (ISR) operates through signal molecules and pathways involving jasmonate and ethylene, and in very few cases, salicylate. Upregulation of these pathways leading to enhanced accumulation of antimicrobial compounds (phytoalexin) help in warding off the pathogen during subsequent attack.

Because the diagnosis of co/superinfection in COVID-19 patients is challenging, empirical antibiotic therapy is frequently initiated until microbiological analysis results. We evaluated the performance and the impact of the BioFire® FilmArray® Pneumonia plus Panel on 112 respiratory samples from 67 COVID-19 ICU patients suspected of co/superinfections. Globally, the sensitivity and specificity of the test were 89.3% and 99.1%, respectively. Positive tests led to antibiotic initiation or adaptation in 15% of episodes and de-escalation in 4%. When negative, 28% of episodes remained antibiotic-free (14% no initiation, 14% withdrawal). Rapid multiplex PCRs can help to improve antibiotic stewardship by administering appropriate antibiotics earlier and avoiding unnecessary prescriptions. Supplementary Information The online version contains supplementary material available at 10.1007/s10096-021-04213-6.

As of 2020, 11 GM canola events have been authorized as food for humans in Korea. However, there are no simultaneous multiplex detection methods for 3 GM canola events (DP-073496-4, MON88302, and MS11). Thus, we established the multiplex polymerase chain reaction (PCR) method coupled with capillary electrophoresis to detect 3 GM canola events. To verify the specificity of event-specific primers, various GM crops of 3 GM soybean events, 6 GM maize events, 2 GM cotton events and 11 GM canola events were prepared. The limit of detection of the developed multiplex PCR was approximately 0.0125% for 3 GM canola events. Certified GM canola and stacked events were analyzed to validate the developed multiplex PCR. This study focuses on establishing multiplex PCR coupled with capillary electrophoresis for newly approved GM canola events and contributes to efficient monitoring GM canola samples in Korea.

Single nucleotide polymorphisms (SNPs) are well-established for forensic applications. Although they are not compatible with existing criminal databases, they offer some advantages over short tandem repeat (STR) markers including smaller amplicons, no stutter artifacts, and biogeographic ancestry and phenotype predictions. The Precision ID NGS System, a commercial workflow by Thermo Fisher Scientific, offers a streamlined solution for genotyping forensically relevant SNPs using next-generation sequencing. The Precision ID Ancestry and Identity Panels combined target 289 SNPs, and their sensitivity, reproducibility, and accuracy have been evaluated by the forensic community. The aim of this study was to develop an alternative workflow to genotype these SNP panels using Illumina chemistry. Commercial genomic DNAs (gDNAs) (n, 3) were amplified using three uracil-tolerant polymerase master mixes. Resulting amplicons were prepared into libraries using the KAPA Hyper Prep Kit (KAPA Biosystems) and sequenced via Illumina's MiniSeq. Reads were analyzed using a published analysis pipeline to compile final genotypes with read depth information. Phusion U Multiplex PCR Master Mix (Thermo Fisher Scientific) statistically outperformed the other master mixes tested (P

The study was undertaken to evaluate the performance of Unyvero Hospitalized Pneumonia (HPN) panel application, a multiplex PCR-based method for the detection of bacterial pathogens from lower respiratory tract (LRT) samples, obtained from COVID-19 patients with suspected secondary hospital-acquired pneumonia. Residual LRT samples obtained from critically ill COVID-19 patients with predetermined microbiological culture results were tested using the Unyvero HPN Application. Performance evaluation of the HPN Application was carried out using the standard-of-care (SoC) microbiological culture findings as the reference method. Eighty-three LRT samples were used in the evaluation. The HPN Application had a full concordance with SoC findings in 59/83 (71%) samples. The new method detected additional bacterial species in 21 (25%) and failed at detecting a bacterial species present in lower respiratory culture in 3 (3.6%) samples. Overall the sensitivity, specificity, positive, and negative predictive values of the HPN Application were 95.1% (95%CI 96.5–98.3%), 98.3% (95% CI 97.5–98.9%), 71.6% (95% CI 61.0–80.3%), and 99.8% (95% CI 99.3–99.9%), respectively. In conclusion, the HPN Application demonstrated higher diagnostic yield in comparison with the culture and generated results within 5 h. Supplementary Information The online version contains supplementary material available at 10.1007/s10096-021-04194-6.

Bloodstream infections (BSIs) require an accurate and fast identification of causative pathogens. Molecular diagnostics, in particular polymerase chain reaction (PCR)-based approaches for BSI diagnostics directly from whole blood, suffer from limitations such as inhibition leading to invalid results. In this retrospective study, we analyzed 23 parameters for their potential interference with LightCycler SeptiFast PCR tests (n = 2167) routinely performed at our institution. The overall inhibition rate was 9.1%. Test date, type of ward, procalcitonin levels, high leukocyte counts, and absolute neutrophil count were significantly associated with inhibition. For a subset (n = 448), cut-off values for leukocyte counts of

We retrospectively examined the yield of a cerebrospinal fluid (CSF) multiplex real-time PCR assay of febrile young infants undergoing a full sepsis work-up. Eighty infants were included in the study: Forty-nine (61%) neonates and 31 (39%) 29–90 day-old patients were included in the study. A viral pathogen was detected in 59% (47/80) of the samples, human enterovirus in 53% (42/80) and Human parechovirus in 6% (5/80). The CSF of nearly half of the subjects with CNS infection was without pleocytosis; all CSF cultures were negative. Multiplex PCR CSF testing enhances the diagnosis of pathogen-specific viral CNS infection among febrile young infants.

Diseases can play a role in species decline. Among them, haemosporidian parasites, vector-transmitted protozoan parasites, are known to constitute a risk for different avian species. However, the magnitude of haemosporidian infection in wild columbiform birds, including strongly decreasing European turtle doves, is largely unknown. We examined the prevalence and diversity of haemosporidian parasites Plasmodium, Leucocytozoon and subgenera Haemoproteus and Parahaemoproteus in six species of the order Columbiformes during breeding season and migration by applying nested PCR, one-step multiplex PCR assay and microscopy. We detected infections in 109 of the 259 screened individuals (42%), including 15 distinct haemosporidian mitochondrial cytochrome b lineages, representing five H. (Haemoproteus), two H. (Parahaemoproteus), five Leucocytozoon and three Plasmodium lineages. Five of these lineages have never been described before. We discriminated between single and mixed infections and determined host species-specific prevalence for each parasite genus. Observed differences among sampled host species are discussed with reference to behavioural characteristics, including nesting and migration strategy. Our results support previous suggestions that migratory birds have a higher prevalence and diversity of blood parasites than resident or short-distance migratory species. A phylogenetic reconstruction provided evidence for H. (Haemoproteus) as well as H. (Parahaemoproteus) infections in columbiform birds. Based on microscopic examination, we quantified parasitemia, indicating the probability of negative effects on the host. This study provides a large-scale baseline description of haemosporidian infections of wild birds belonging to the order Columbiformes sampled in the northern hemisphere. The results enable the monitoring of future changes in parasite transmission areas, distribution and diversity associated with global change, posing a potential risk for declining avian species as the European turtle dove.

Bacterial leaf spot and bacterial leaf blight are global threats to the cultivation of cruciferous vegetables, and it is necessary to develop methods to easily detect, identify, and distinguish the causative pathogens Pseudomonas syringae pv. maculicola (Psm) and P. cannabina pv. alisalensis (Pca). Here, we used the sequence specificity of the exchangeable effector loci flanking the hrp gene cluster to design primers that can help detect and discriminate between Psm and Pca. Primers common to both bacteria (hrpK_fw1 and hrpK_fw2) were designed within hrpK at the end of the hrp gene cluster. Psm-specific primers (MAC_rv1 and MAC_rv2) were designed in hopPtoB1 and Pca-specific primers (ALS_rv1 and ALS_rv2) were designed in hopX1 adjacent to hrpK. PCR using hrpK_fw1 and MAC_rv1 or hrpK_fw2 and MAC_rv2 amplified DNA fragments of only Psm, P. syringae pv. tomato (causal agent of tomato bacterial speck), and P. syringae pv. spinaciae (causal agent of spinach bacterial leaf spot), among 76 strains of phytopathogenic bacteria. PCR using hrpK_fw1 and ALS_rv1 or hrpK_2 and ALS_rv2 amplified DNA fragments of only Pca. Multiplex PCR with these primers could easily distinguish Psm and Pca from bacterial colonies isolated on growth media and detect the pathogen in symptomatic leaves. Multiplex nested PCR with the primers detected contamination in one Psm- and/or one Pca-infected seeds in 1000 seeds. These results suggest that these PCR primers could help detect and discriminate Psm and Pca. KEY POINTS: • We investigated Pseudomonas syringae pv. maculicola and P. cannabina pv. alisalensis. • Novel primers common to both bacteria were designed following genome comparison. • Multiplex PCR with new primers could discriminate Psm and Pca.

Human Papillomaviruses (HPV) have some associations with that of the beta and gamma genera with non melanoma skin cancers (NMSC). The detection of HPV in clinical specimens is molecular-based since the isolation of the virus in in vitro cell lines has not been achieved. In this contest, we provide information about the choices of molecular methods applicable in epidemiological surveys. A limited number of assays have been developed to detect and genotype cutaneous HPVs (associated with NMSC). Consensus PCR followed by hybridization, consensus PCR followed by sequencing, multiplex PCR, and new genotyping are some methods to detect a proportion of the HPV types implicated in skin lesions with differing sensitivities and specificities. The current review discusses about various choices on different tests in the detection of cutaneous HPVs. Most available methods rely on the amplification of DNA by consensus PCR followed by sequencing. Amplification of DNA by consensus PCR followed by sequencing assays is a powerful highthroughput tool that 24 could be used to detect multiple infections and to learn more about the natural history of genus alpha and beta skin HPV types. 25 This method is applicable for large epidemiological studies that require high-throughput testing to analyze the role of these HPVs 26 in human disease.

Autosomal short tandem repeats (asSTR) serve as genetic markers for discriminating individuals and have been extensively used for criminal investigations as well as the establishment of genetic relationships. Tri-allelic pattern usually occurs due to chromosomal duplication, trisomy, and chimerism during mitotic division, but a false tri-allelic pattern at the D7S820 locus was encountered in our laboratory during the analysis of a case exhibit. DNA isolation from exhibit for profiling was done as per manufacturer's protocol. This is the first report which observed false tri-allelic pattern (10, 11, 14.1 allele) on D7S820 locus by analysis with GlobalFiler™ PCR Amplification Kit in Indian population. Findings were re-confirmed using other available asSTR kits in the laboratory, viz., AmpFLSTR™ Identifiler™ Plus PCR Amplification Kit and PowerPlex® Fusion 6C System. Two alleles (10, 11) found at D7S820, apart from SE33 marker, showed homozygous condition, but one Off Marker (OMR) peak was observed before start of SE33 marker region with the analysis using PowerPlex® Fusion 6C System. As it has been confirmed that the OMR allele belongs to the SE33 locus, this could be possible because of the adjacent locations of the D7S820 and the SE33 in the GlobalFiler® PCR amplification kit. 14.1 allele appeared within the allelic window of D7S820. The false tri-allelic pattern was due to the overlapping of SE33 marker allele (1.2 repeat) with bin window of D7S820 Marker. This finding might create confusion for the establishment of genetic relationships. We, therefore, conclude that such uncommon observations with rare events should be carefully investigated and interpreted.

Enteropathogenic Escherichia coli (EPEC) is one of the most important diarrheagenic agents among infants under 5 years in developing countries. This study aimed to investigate the relationship of integron genes and class A extended-spectrum ESBLs genes in MDR E. coli strains isolated from children with diarrhea in Southwestern Iran. Totally, 321 fecal samples were collected from diarrheal children under 5 years admitted to teaching hospitals of Abadan and Khorramshahr, southwest Iran. Routine bacteriological tests were performed for the identification of E. coli isolates. Multiplex PCR was used for the presence of eae, bfp, stx1, and stx2 genes to detected EPEC strains. Serogrouping was performed for EPEC strains. The EPEC isolates’ antibiotic resistance pattern was determined by the disk-diffusion technique. All EPEC isolates were screened for integron and class A β-lactamase genes. Of the 14 EPEC isolates, 12 (85.7%) were found to be ESBL-positive by double disk synergy test (DDST) and PCR. In addition, blaCTX-M and blaTEM genes were detected in 83.3% (n = 10) and 58.3% (n = 7) of EPEC isolates, respectively. None of the isolates had the blaKPC gene. On the other hand, 64.2% (n = 9) and 7.1% (n = 1) were positive only for intlI and intlII genes, respectively. The results demonstrated that EPEC is one of the major causes of childhood diarrhea in our region and that the distribution of class 1 integrons and ESBLs in EPEC strains is highly prevalent. Moreover, the results revealed that continuous monitoring of the emergence and expansion of MDR in EPEC strains is necessary.

Background Bacterial identification at the strain level is a much-needed, but arduous and challenging task. This study aimed to develop a method for identifying and differentiating individual strains among multiple strains of the same bacterial species. The set used for testing the method consisted of 17 Escherichia coli strains picked from a collection of strains isolated in Germany, Spain, the United Kingdom and Vietnam from humans, cattle, swine, wild boars, and chickens. We targeted unique or rare ORFan genes to address the problem of selective and specific strain identification. These ORFan genes, exclusive to each strain, served as templates for developing strain-specific primers. Results Most of the experimental strains (14 out of 17) possessed unique ORFan genes that were used to develop strain-specific primers. The remaining three strains were identified by combining a PCR for a rare gene with a selection step for isolating the experimental strains. Multiplex PCR allowed the successful identification of the strains both in vitro in spiked faecal material in addition to in vivo after experimental infections of pigs and recovery of bacteria from faecal material. In addition, primers for qPCR were also developed and quantitative readout from faecal samples after experimental infection was also possible. Conclusions The method described in this manuscript using strain-specific unique genes to identify single strains in a mixture of strains proved itself efficient and reliable in detecting and following individual strains both in vitro and in vivo, representing a fast and inexpensive alternative to more costly methods.

Background The detection of coinfections is important to initiate appropriate antimicrobial therapy. Molecular diagnostic testing identifies pathogens at a greater rate than conventional microbiology. We assessed both bacterial coinfections identified via culture or the BioFire® FilmArray® Pneumonia Panel (FA-PNEU) in patients infected with SARS-CoV-2 in the ICU and the concordance between these techniques. Methods This was a prospective study of patients with SARS-CoV-2 who were hospitalized for no more than 48 h and on mechanical ventilation for no longer than 24 h in 8 ICUs in Medellín, Colombia. We studied mini-bronchoalveolar lavage or endotracheal aspirate samples processed via conventional culture and the FA-PNEU. Coinfection was defined as the identification of a respiratory pathogen using the FA-PNEU or cultures. Serum samples of leukocytes, C-reactive protein, and procalcitonin were taken on the first day of intubation. We analyzed the empirical antibiotics and the changes in antibiotic management according to the results of the FA-PNEUM and cultures. Results Of 110 patients whose samples underwent both methods, FA-PNEU- and culture-positive samples comprised 24.54% versus 17.27%, respectively. Eighteen samples were positive in both techniques, 82 were negative, 1 was culture-positive with a negative FA-PNEU result, and 9 were FA-PNEU-positive with negative culture. The two bacteria most frequently detected by the FA-PNEU were Staphylococcus aureus (37.5%) and Streptococcus agalactiae (20%), and those detected by culture were Staphylococcus aureus (34.78%) and Klebsiella pneumoniae (26.08%). The overall concordance was 90.1%, and when stratified by microorganism, it was between 92.7 and 100%. The positive predictive value (PPV) was between 50 and 100% and were lower for Enterobacter cloacae and Staphylococcus aureus. The negative predictive value (NPV) was high (between 99.1 and 100%); MecA/C/MREJ had a specificity of 94.55% and an NPV of 100%. The inflammatory response tests showed no significant differences between patients whose samples were positive and negative for both techniques. Sixty-one patients (55.45%) received at least one dose of empirical antibiotics. Conclusions The overall concordance was 90.1%, and it was between 92.7% and 100% when stratified by microorganisms. The positive predictive value was between 50 and 100%, with a very high NPV.

Objective Main malaria diagnosis is based on microscopic examination combined with rapid diagnostic tests. Both methods have low sensitivity and specificity. Loop-mediated isothermal amplification techniques have shown a sensitivity similar to PCR but with lower times of performance. This study aimed to assess a commercial LAMP for the diagnosis of malaria (Alethia® Malaria) against the Nested-Multiplex-Malaria PCR, including the analytical sensitivity and the operational characteristics. Results One hundred five samples out of 114 rendered valid results, obtaining 85 positive samples and 18 negative samples with an agreement of 98% compared to the reference method with a sensitivity, specificity and kappa coefficient of 98.84%, 94.74% and 0.94 respectively, with only two discrepant samples. The turnaround time was estimated in 1 h and 30 min, with a cost of 32.67€ per determination. The results showed several advantages of the Alethia® Malaria, as it was easy to perform, minimal training requirement and 40 min run. Moreover, it includes an internal control to avoid false negatives. However, it also showed some limitations such as the need for a specific amplification and detection device, the detection of only Plasmodium spp. and a very high price.

One major challenge in the design of highly multiplexed PCR primer sets is the large number of potential primer dimer species that grows quadratically with the number of primers to be designed. Simultaneously, there are exponentially many choices for multiplex primer sequence selection, resulting in systematic evaluation approaches being computationally intractable. Here, we present and experimentally validate Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE), a stochastic algorithm for design of multiplex PCR primer sets that minimize primer dimer formation. In a 96-plex PCR primer set (192 primers), the fraction of primer dimers decreases from 90.7% in a naively designed primer set to 4.9% in our optimized primer set. Even when scaling to 384-plex (768 primers), the optimized primer set maintains low dimer fraction. In addition to NGS, SADDLE-designed primer sets can also be used in qPCR settings to allow highly multiplexed detection of gene fusions in cDNA, with a single-tube assay comprising 60 primers detecting 56 distinct gene fusions recurrently observed in lung cancer.

The COVID-19 pandemic has resulted in significant diversion of human and material resources to COVID-19 diagnostics, to the extent that influenza viruses and co-infection in COVID-19 patients remains undocumented and pose serious public-health consequences. We optimized and validated a highly sensitive RT-PCR based multiplex-assay for the detection of SARS-CoV-2, influenza A and B viruses in a single-test. This study evaluated clinical specimens (n = 1411), 1019 saliva and 392 nasopharyngeal swab (NPS), tested using two-assays: FDA-EUA approved SARS-CoV-2 assay that targets N and ORF1ab gene, and the PKamp-RT-PCR based assay that targets SARS-CoV-2, influenza viruses A and B. Of the 1019 saliva samples, 17.0% (174/1019) tested positive for SARS-CoV-2 using either assay. The detection rate for SARS-CoV-2 was higher with the multiplex assay compared to SARS-specific assay [91.9% (160/174) vs. 87.9% (153/174)], respectively. Of the 392 NPS samples, 10.4% (41/392) tested positive for SARS-CoV-2 using either assay. The detection rate for SARS-CoV-2 was higher with the multiplex assay compared to SARS-specific assay [97.5% (40/41) vs. 92.1% (39/41)], respectively. This study presents clinical validation of a multiplex-PCR assay for testing SARS-CoV-2, influenza A and B viruses, using NPS and saliva samples, and demonstrates the feasibility of implementing the assay without disrupting the existing laboratory workflow.

Objective Cholera is an intestinal infection caused by Vibrio cholerae, it is usually occurs in developing countries that lack of sanitation. In developing country including Indonesia, awareness importance of sanitation is still low. Unfortunately, research related to the detection of V. cholerae from fruit and vegetables in Indonesia is still rare. In this study, MPN method was used to determine the prevalence of V. cholerae followed by single and multiplex PCR to detect virulence genes, including toxR, ctxA, tcpA, hlyA, ace, ompU, and zot. Results We found 3 fruits and 2 vegetables positive for toxR gene. Fruit samples which were showed toxR positive found from East Jakarta while for vegetables, it was recovered from West Jakarta and Central Jakarta. Twenty-three isolates were recovered from toxR positive samples. The result of antibiotic resistance analysis showed that 4.35% of the isolates resistant to gentamicin, streptomycin (17.39%), trimethoprim (52.17%), ciprofloxacin (30.43%), ampicillin (13.04%), nalidixic acid (82.61%), and polymyxin B (91.30%). None of these isolates were resistant to kanamycin. Combination of MPN and Multiplex PCR method can be used to detect the prevalence and characterize the virulence properties of V. cholerae.

The study describes the development of a new multiplex PCR system that simultaneously amplifies 16 X chromosome short tandem repeats (X-STRs) loci in a single PCR reaction and its applicability on a sample of 200 from the Sinhalese population in Sri Lanka. 13 X-STR loci located in four clusters are selected for the assay (DXS10148-DXS10135-DXS8378, DXS7132-DXS10079-DXS10074-DXS10075, DXS6801-DXS6809-DXS6789 and DXS7424-DXS101-DXS7133). In addition, three single loci were also selected (DXS9902, HPRTB and DXS7423). Genomic DNA extracted using the Chelex-100 method was amplified with modified published primers and subjected to capillary gel electrophoresis. Complete DNA profiles were obtained with 0.20 ng 9947A DNA and the band sizes ranged between 100 and 320 bp with 10 loci having sizes below 237 bp. A total of 160 alleles were observed among the sample with 5-23 alleles for each locus. The forensic efficiency evaluation showed high values for the combined power of discrimination in males (1 in 1 × 1010) and females (1 in 1 × 1017). Combined mean exclusion chance (MEC) indices calculated for deficiency, normal trio and duo cases were equally high (> 0.99999). Application of the new multiplex system to two actual kinship cases of full sibling and deficient paternity suggested that these 16 short tandem repeat loci are highly appropriate for forensic and kinship testing among the Sinhalese population.

Donkey meat is a rich source of protein excellent in indispensable amino acid and low in fat. Its quality has received great attention due to its high nutritional and medicinal value to human beings. Due to donkey resource scarcity and gradually increasing market demand for donkey meat products, adulterated donkey meat products with other low cost animal meat, especially sheep, cattle, pig and horse, were often found in market and had raised widespread concern in recent years. In this study, total 300 mg samples of binary mixtures, containing 90%, 50%, 25%, 10%, 5%, 1% (w/w) of donkey in horse meat respectively, were used for DNA extraction. Both conventional multiplex and real-time PCR techniques were developed for qualitatively and quantitatively detecting the adulteration in donkey meat. The multiplex PCR allowed rapid and simultaneous qualitative detection of donkey and four common adulterated species including sheep, cattle, pig and horse. Moreover, a normalized real-time PCR assay was further developed to detect and quantify the donkey mitochondrial DNA, and the results of simulate adulteration suggested this method could quantitatively detect donkey meat in range of 1–90% with high trueness. The results illustrated that both methods might be applied to detect commercial foodstuff adulteration. Therefore, both improved conventional multiplex and real-time PCR methods were developed to achieve qualitative and quantitative detection of the adulteration in donkey meat products.

The aim of this study was to isolate Aspergillus section Nigri from onion samples bought in supermarkets and to analyze the fungal isolates by means of molecular data in order to differentiate A. niger and A. welwitschiae species from the other non-toxigenic species of black aspergilli, and detect genes involved in the biosynthesis of ochratoxin A and fumonisin B2. Aspergillus section Nigri were found in 98% (94/96) of the onion samples. Based on the results of multiplex PCR (performed on 500 randomly selected strains), 97.4% of the Aspergillus section Nigri strains were recognized as A. niger/A. welwitschiae. Around half of them were subjected to partial sequencing of the CaM gene to distinguish one from the other.Atotal of 97.9% of the isolates were identified as A. welwitschiae and only 2.1% as A. niger. The fum8 gene, involved in fumonisin B2 biosynthesis, was found in 36% of A. welwitschiae isolates, but radH and pks genes, involved in ochratoxin A biosynthesis, were found in only 2.8%. The presence/absence of fum8 gene in the A. welwitschiae genome is closely associated with ability/inability of the isolates to produce fumonisin in vitro. Based on these results,we suggest that in-depth studies are conducted to investigate the presence of fumonisins in onion bulbs. FAPESP / CAPES / CNPq

Phyllosticta citricarpa, Elsinoe fawcettii, Elsinoe australis, and Pseudocercospora angolensis are major pathogens of citrus crops worldwide and can cause non-characteristic symptoms that may lead to confusion regarding the causative agent. These fungi are subject to international phytosanitary regulations, and testing on fruits or leaves requires accurate and easy-to-use tools. New multiplex conventional PCR and real-time PCR assays were developed here to achieve highly accurate simultaneous detection of all four fungal pathogens in fruit tissues. We designed new oligonucleotide combinations for P. citricarpa, E. fawcettii, and E. australis and combined them with already available primers and hydrolysis probes to be used in either PCR assay. The limit of detection for multiplex conventional PCR was as low as 100 pg μL−1 for P. citricarpa, E. fawcettii, and E. australis and 10 pg μL−1 of target DNA per reaction tube for P. angolensis. The quadruplex real-time PCR assay successfully yielded repeatable positive results with as low as 242, 243, 241, and 242 plasmidic copies of target DNA of P. citricarpa, E. fawcettii, E. australis, and P. angolensis, respectively. Moreover, analysis of 60 naturally infected citrus samples yielded 100% concordant results by both assays. Our validation experiment revealed that the multiplex real-time PCR assay showed high specificity except a cross-reaction with P. paracitricarpa DNA. Sensitivity, repeatability, reproducibility, and robustness were verified, and the assay could be used following different DNA extraction procedures, supporting fitness for routine analysis. These new multiplex tools should be of great interest as cost-effective solutions for regulatory authorities and diagnostic laboratories, enabling testing for four important pathogens in single-tube reactions. • Development of new conventional PCR and qPCR assays for four citrus pathogens. • Very low limits of detection were found for multiplex conventional PCR. • qPCR had high specificity, sensitivity, repeatability, reproducibility, and robustness.

In the food supply chain, quality control has a very important role in maintaining customer confidence. In the EU, food safety aspects are strictly regulated; however, composition requirements and standard control methods are generally undefined. The rapidly increasing wild boar population has a growing market share in venison or game meat production. Several methods have been described for species identification and control of composition in food products, but only some of these are suitable for routine measurements. The aim of our research was to design a rapid, reliable and simple PCR insertion/deletion (InDel)-based genetic tool suitable for species identification in food quality control laboratories. In total, 59 different swine (Sus scrofa) whole genomes were tested with bioinformatic tools to identify wild boar-specific insertions or deletions. Three independent InDels were suitable for marker development, multiplex PCR amplification and separation in agarose gel. Altogether, 209 samples of wild boar and ten other domestic pig breeds were taken for DNA extraction and validation of the three multiplexed InDel markers. Statistical analysis showed a very high combined predictive value (0.996), indicating the capability of the newly developed markers to detect wild boars with a probability over 99%. Breed assignment tests confirm that the InDel markers developed are suitable for rapid, sensitive and reliable identification of the wild boar meat content of food products. The use of the reported method in food quality control can mean a simple and cost-effective way to maintain consumer confidence and to support the competitiveness of fair producers.

The MiSeq® FGX Forensic system and the HID-Ion AmpliSeq Panel were previously developed for massively parallel sequencing (MPS) for forensic casework. Among the three major sequencing platforms, BGISEQ-500TM, which is based on multiple PCRs, is still lacking in forensics. Here, a novel forensic panel was constructed to detect 186 single-nucleotide polymorphisms (SNPs) and 123 short tandem repeats (STRs) with MPS technology on the BGISEQ-500™ platform. First, the library preparation, sequencing process, and data analysis were performed, focusing on the average depth of coverage and heterozygote balance. We calculated the allelic frequencies and forensic parameters of STR and SNP loci in 73 unrelated Chinese Han individuals. In addition, performance was evaluated with accuracy, uniformity, sensitivity, PCR inhibitor, repeatability and reproducibility, mixtures, degraded samples, case-type samples, and pedigree analyses. The results showed that 100% accurate and concordant genotypes can be obtained, and the loci with an abundance in the interquartile range accounted for 92.90% of the total, suggesting reliable uniformity in this panel. We obtained a locus detection rate that was higher than 98.78% from 78 pg of input DNA, and the optimal amount was 1.25-10 ng. The maximum concentrations of hematin and humic acid were 200 and 100 μM, respectively (the ratios of detected loci were 96.52% and 92.41%), in this panel. As a mixture, compared with those of SNPs, minor-contributor alleles of STRs could be detected at higher levels. For the degraded sample, the ratio of detected loci was 98.41%, and most profiles from case-type samples were not significantly different in abundance in our studies. As a whole, this panel showed high-performance, reliable, robust, repeatable, and reproducible results, which are sufficient for paternity testing, individual identification, and use for potentially degraded samples in forensic science.

Sweet corn has emerged as a popular vegetable worldwide. Commercial shrunken2 (sh2)-based sweet corn lacks lysine, tryptophan and provitamin-A, deficiency of which cause various health problems. To overcome these problems, marker-assisted stacking of sh2, opaque2 (o2), β-carotene hydroxylase (crtRB1) and lycopene epsilon cyclase (lcyE) was attempted to combine nutritional quality traits in maize endosperm. Gene-based simple sequence marker (SSR) specific to o2, InDel-based marker for crtRB1 and lcyE, and SSR linked to sh2 were used in marker-assisted selection. Four backcross populations segregating for the alleles of crtRB1, lcyE, o2 and sh2 were developed. The favourable alleles of sh2 and o2 produced 103 and 150 bp amplicons, while the unfavourable alleles were 96 and 160 bp, respectively. CrtRB1 produced 543 bp favourable and 296 bp unfavourable allele, while the same for lcyE was 650 and 300 bp, respectively. To detect desirable alleles of the target genes simultaneously, multiplex-PCR (Mx-PCR) assay was developed and validated in BC2F2 populations. Mx-PCR assay successfully identified the favourable alleles of four genes simultaneously, and validated the results of individual uniplex-PCR assays. Mx-PCR assay saved 68% of cost and approximately 63% of time. This is the first report of multiplexing sh2, o2, crtRB1 and lcyE genes in maize. The information generated here would be immensely useful in molecular breeding programme.

Bacillus cereus is a human pathogenic bacterium that produces emetic and diarrheal foodborne diseases. This study evaluated the genetic and toxigenic diversity in B. cereus group isolates from powdered foods collected in public educational institutions, bakeries and powdered food companies located in Medellin, Colombia. B. cereus was detected in 35 of 305 (11%) powdered food samples and 52 B. cereus were isolated. The presence of ten toxin genes, hblCDAB, nheABC, cytK2, entFM and cesB, was evaluated in the isolates by multiplex PCR. The nheABC operon was found in all isolates (100%), hblCDAB in 22 (42%), hblCDA in 8 (15%) and hblCD in 3 (6%); the cytK2 gene was detected in 32 isolates (62%) and entFM in 32 (62%). Notably, the cesB gene was not detected. According to the presence of toxin genes, fifteen profiles were identified. The predominant toxigenic profile contained all toxin genes but cesB. A large genetic diversity was observed by GTG5 fingerprinting with 46 isolates grouped in seven clusters and the remaining six clustering individually. There was no relationship between toxigenic profiles and genetic clusters, but some genetic clusters seemed to be related to particular powdered food types. In general, the results evidenced high genetic and enterotoxigenic diversity among the B. cereus group isolates.

Sex is an important biological variable as many physiological as well as disease processes differ between females and males. The fundamental biological distinction between females and males starts with chromosomal sex, and the establishment of XX and XY cells and tissues. Polymerase Chain Reaction (PCR) is a simple and effective method to easily determine chromosomal or genetic sex of cells and tissues. The goal of this study was to develop a simple multiplex PCR genotyping assay to distinguish XX and XY tissues in sheep. Primers were designed to amplify a fragment of the autosomal gene myogenin (MYOG) and sex determining region on the Y chromosome (SRY). PCR analysis was performed on a variety of genomic DNA samples isolated from fetal sheep skeletal muscle, brain, liver, and placenta, and revealed a single 259 bp band for MYOG in XX females, and a 259 bp band for MYOG and a 167 bp band for SRY in XY males. Amplicons were clearly distinguishable by gel electrophoresis, and their sequences revealed 100% identity to the known ovine MYOG and SRY sequence. The reported multiplex PCR genotyping assay provides a rapid means to distinguish XX and XY sheep tissues using low volume samples.

Banana is a staple food and cash crop grown in East and Central Africa (ECA). The main banana varieties grown in ECA are the East African highland bananas (EAHB), although dessert/beer bananas such as Sukari Ndizi, Kayinja (Pisang Awak) and Gros Michel are also produced due to their high value at local markets. The Fusarium wilt fungus Fusarium oxysporum f. sp. cubense (Foc) causes disease of susceptible dessert/beer bananas, which significantly reduces yields. Banana Fusarium wilt is managed by excluding the pathogen from disease-free areas and by planting disease-resistant varieties in infested fields. Six phylogenetically closely-related vegetative compatibility groups (VCGs) of Foc, VCGs 0124, 0125, 0128, 01212, 01220 and 01222 are present in ECA, which all group together in Foc Lineage VI. Rapid and accurate detection of Foc Lineage VI strains is thus important to prevent its spread to disease-free areas. In this study, molecular markers specific to Foc Lineage VI were therefore developed. Primer sets were then combined in a multiplex PCR assay, and validated on a worldwide population of 623 known Foc isolates, other formae speciales and non-pathogenic Fusarium oxysporum isolates. The Foc Lineage VI multiplex PCR was used to identify Foc isolates collected in banana fields at five locations in Uganda and Tanzania. Foc Lineage VI DNA was detected at a concentration as low as 0.1 ng/μl, both in the absence and presence of banana DNA, and can therefore be used as an accurate diagnostic tool for Foc Lineage VI strains.