Multiplex Real-time PCR For Detection Research Articles

Development of a multiplex real-time PCR assay for the simultaneous detection of mpox virus and orthopoxvirus infections

Since May 2022, the multi-country outbreak of monkeypox (mpox) has raised a great concern worldwide. Early detection of mpox virus infection is recognized as an efficient way to prevent mpox transmission. Mpox specific detection methods reported up to now are based on the SNPs among mpox virus and other orthopoxviruses. We have therefore developed a real-time PCR based mpox detection method targeting mpox virus specific sequences (N3R and B18Rplus). We have also optimized an orthopoxvirus detection system which targets the highly conserved E9L and D6R genes. The mpox and orthopoxvirus real-time PCR assays have a high sensitivity (1 copy/reaction) and specificity. Mpox viral DNA and clinical samples from mpox patients are detected with the mpox detection system. Furthermore, we have established a multiplex real-time PCR detection system allowing simultaneous and efficient detection of mpox and orthopoxvirus infections.

Development and application of one-step multiplex Real-Time PCR for detection of three main pathogens associated with bovine neonatal diarrhea.

Neonatal calf diarrhea (NCD) is one of the most common diseases in calves, causing huge economic and productivity losses to the bovine industry worldwide. The main pathogens include bovine rotavirus (BRV), bovine coronavirus (BCoV), and Enterotoxigenic Escherichia coli (ETEC) K99. Since multiple infectious agents can be involved in calf diarrhea, detecting each causative agent by traditional methods is laborious and expensive. In this study, we developed a one-step multiplex Real-Time PCR assay to simultaneously detect BRV, BCoV, and E. coli K99+. The assay performance on field samples was evaluated on 1100 rectal swabs of diseased cattle with diarrhea symptoms and compared with the conventional gel-based RT-PCR assay detect BRV, BCoV, and E. coli K99+. The established assay could specifically detect the target pathogens without cross-reactivity with other pathogens. A single real-time PCR can detect ~1 copy/µL for each pathogen, and multiplex real-time PCR has a detection limit of 10 copies/µL. Reproducibility as measured by standard deviation and coefficient of variation were desirable. The triple real-time PCR method established in this study was compared with gel-based PT-PCR. Both methods are reasonably consistent, while the real-time PCR assay was more sensitive and could rapidly distinguish these three pathogens in one tube. Analysis of surveillance data showed that BRV and BCoV are major enteric viral pathogens accounting for calves' diarrhea in China. The established assay has excellent specificity and sensitivity and was suitable for clinical application. The robustness and high-throughput performance of the developed assay make it a powerful tool in diagnostic applications and calf diarrhea research. ​.

Comparison of bacteriological culture method and multiplex real-time PCR for detection of mastitis

This study includes the evaluation of multiplex real-time PCR (rPCR) kit, which was developed to provide rapid diagnosis of mastitis infections, by working with milk samples of 2 different sources of mastitis and comparing the results with the classical bacteriological culture method (BC). A total of 273 bacteria were isolated in 226 samples (47.88%) out of 472 samples by BC. These were 139 (50.91%) Staphylococcus spp., 61 (22.34%) Streptococcus spp., 15 (5.49%) E. coli, 8 (2.93%) Enterococcus spp., 50 (18.31%) other bacteria. When we look at the multiplex rPCR results; 1052 positive were obtained for the gene regions of 14 different bacteria, 1 yeast, and 1 β-lactamase gene examined in 472 samples. While no searched gene region was found by rPCR in 78 (16.5%) of the 472 samples studied, at least 1 gene was detected in 394 (83.5%) samples. These 1052 positive samples by rPCR were; 263 (28.43%) Staphylococcus spp., 51 (5.51%) S. aureus, 57 (6.16%) Enterococcus spp., 49 (5.29%) C. bovis, 16 (1.73%) S. dysgalactiae, 84 (9.08%) S. agalactiae, 71 (7.67%) S. uberis, 73 (7.89%) E. coli, 14 (1.51%) Prototheca spp., 39 (4.21%) T. pyogenes/P. indolicus, 5 (0.54%) S. marcescens, 15 (1.62%) K. oxytoca/pneumonia, 117 (12.64%) Mycoplasma spp., 31 (3.35%) M. bovis, 40 (4.32%) yeast, and 127 samples (26.90%) were β-lactamase positive. When the antibiotic resistance of the isolates was evaluated, 78 (31.96%) tetracycline, 72 (29.5%) penicillin, and 60 (24.59%) clindamycin resistance were observed predominantly in Gram-positive isolates, while 6 (23.07%) tigecycline, 6 (23.07%) netilmicin, 6 (23.07%) pipercillin resistance was found in gram-negative isolates.While a bacteria and/or yeast gene was found by rPCR in 187 of 246 (76.01%) samples with no bacterial growth, a bacterium was isolated with BC in only 20 (8.84%) samples whose gene region was not found by rPCR. As a result, the multiplex rPCR system used in the diagnosis of mastitis has been found to be quite reliable as it can detect a large number of bacteria in a very short time compared to classical methods. Therefore, we advise the use of rPCR and/or culture for confirmation of clinical signs in mastitis and at routine mastitis surveillance.

Development of an ARMS multiplex real-time PCR assay for the detection of HLA-B*13:01 genotype by detecting highly specific SNPs.

HLA-B*13:01 was strongly associated with Dapsone Hypersensitivity Syndrome (DHS). This study aimed to develop and validate a rapid and economical method for HLA-B*13:01 genotyping. Two tubes multiplex real-time PCR detection system comprising amplification refractory mutation system primers and TaqMan probes was established for HLA-B*13:01 genotyping. Sequence-based typing was applied to validate the accuracy of the assay. The accuracy of the assay was 100% for HLA-B*13:01 genotyping. The detection limit of the new method was 0.025 ng DNA. The positive rate of HLA-B*13:01 in the Bouyei (20%, n = 50) populations was significantly higher than that in the Uighur population (4%, n = 100), Han (4.5%, n = 200), and Tibetan (1%, n = 100) ( P < 0.05). The proposed method is rapid and reliable for HLA-B*13:01 screening in a clinical setting.

Application of multiplex realtime PCR detection for hemorrhagic fever syndrome viruses

BackgroundMultiplex real-time PCR is a quick and cost effective method for detection of various gene simultaneously. HFSV (Hemorrhagic Fever Syndrome Virus) is a newly emerging infectious disease because of globalization and climate change. We tried to develop a molecular diagnostic technique for various causative viruses and evaluate its usefulness for improving public health. MethodsMolecular diagnostic test method that qualitatively detects viruses causing viral hemorrhagic fevers hired Taq-Man Real-time RT-PCR technique. The Ct value was experimentally observed three or more times at the RNA concentration before and after the detection limit. After designing a multiplex real-time RT-PCR test for target gene of selected 17 viruses, the detection limit for each target and the presence or absence of cross-reaction and interference reaction were evaluated to determine its availability. ResultsSix kinds of viruses, including Crimean-Congo hemorrhagic fever virus, Omsk hemorrhagic fever virus, Sabia virus, Chapare virus, Yellow fever virus, and Variola virus (A4L gene, B12R gene), were able to confirm the detection limit of 0.5 copies/μl, and other Ebola virus, Marburg virus, Rift Valley fever virus, Kyasanur Forest disease virus, Junin virus, Guanarito virus, Machupo virus, Chikungunya virus, Hantavirus, Dengue virus types 1–4, and Lassa virus (L gene, GPC gene), and 11 kinds of viruses, the detection limit was confirmed at 5 copies/μl. No cross-reaction or interference between detected genes was observed. ConclusionThe virus test method developed through this study using multiplex is expected to be used for public health and quarantine as a test method that can be used when a hemorrhagic fever virus of unknown cause is introduced.

Development of multiplex real-time PCR for detection of clarithromycin resistance genes for the Mycobacterium abscessus group.

Introduction. The M. abscessus molecular identification and its drug-resistance profile are important to choose the correct therapy.Aim. This work developed a multiplex real-time PCR (mqPCR) for detection of clarithromycin resistance genes for the Mycobacterium abscessus group.Methodology. Isolates received by Adolfo Lutz Institute from 2010 to 2012, identified by PCR restriction enzyme analysis of a fragment of the hsp65 gene (PRA-hsp65) as M. abscessus type 1 (n=135) and 2 (n=71) were used. Drug susceptibility test (DST) for CLA were performed with reading on days 3 and 14. Subespecies identification by hsp65 and rpoB genes sequencing and erm(41) and rrl genes for mutation detection and primer design were performed. erm(41) gene deletion was detected by conventional PCR. Primers and probes were designed for five detections: erm(41) gene full size and with deletion; erm(41) gene T28 and C28; rrl gene A2058.Results. In total, 191/206 (92.7 %) isolates were concordant by all methods and 13/206 (6.3 %) were concordant only between molecular methods. Two isolates (1.0 %) were discordant by mqPCR compared to rrl gene sequencing. The mqPCR obtained 204/206 (99.0 %) isolates in agreement with the gold standard, with sensitivity and specificity of 98 and 100 %, respectively, considering the gold standard method and 92 and 93 % regarding DST.Conclusion. The mqPCR developed by us proved to be an easy-to-apply tool, minimizing time, errors and contamination.

A single-tube multiplex real-time PCR for HLA-B*38:02 genotype by detecting highly specific SNPs.

Purpose: HLA-B*38:02 is closely related to carbimazole/methimazole-induced agranulocytosis. This study aimed to develop and validate a rapid and economical method for HLA-B*38:02 genotyping. Methods: A single-tube multiplex real-time PCR detection system comprising amplification refractory mutation system primers and TaqMan probes was established for HLA-B*38:02 genotyping. Sequence-based typing was applied to validate the accuracy of the assay. Results: The accuracy of the assay was 100% for HLA-B*38:02 genotyping. The detection limit of the new method was 0.05ng DNA. The positive rate of HLA-B*38:02 in the Han (8%, n=100), Bouyei (17.8%, n=90)and Tibetan (12.7%, n=110) populations was significantly higher than that in the Uighur population (1%, n=100) (p<0.05). Conclusion: The proposed method is rapid and reliable for HLA-B*38:02 screening in a clinical setting.

ALL-206 Management and Outcome of Coronavirus Disease 2019 (COVID-19) in Pediatric Cancer Patients: A Single Centre Experience from a Developing Country

Context: Few developing countries have described the impact of coronavirus disease 2019 (COVID-19) on pediatric cancer patients. Most patients had a favorable outcome. The potential benefits of remdesivir (RDV) in pediatric oncology patients require further studies. Objective: Describe the management and outcome of COVID-19 in pediatric oncology patients. Design: Conducted from May to November 2020, this study included pediatric oncology patients with confirmed COVID-19. RDV was the antiviral therapy used. Setting: Pediatric oncology patients were treated at Children's Cancer Hospital Egypt, a specialized cancer center for pediatric oncology. Patients or Other Participants: This prospective study recruited 76 pediatric oncology patients with confirmed COVID-19 infection. Interventions: Allplex2019-nCoV Assay (Seegene, Seoul, South Korea) was used for Multiplex real-time PCR detection of SARS-CoV-2 according to manufacturer instructions. Main Outcome Measures: This study described the clinical course and management of SARS-CoV-2 infections in 76 pediatric oncology patients, detailing disease severity, duration to achieve a negative RT-PCR test, modifications made to protocols, and survival outcomes in patients who had been treated with RDV and those treated without it. Results: The median age of patients was 9 years. Sixty patients were on first-line treatment. Hematological malignancies constituted 86.8% of patients. Severe to critical infections affected 35.4% of patients. The most common symptom was fever (93.4%). Chemotherapy was delayed in 59.2% of patients and doses were modified in 30.2%. The 60-day overall survival (OS) stood at 86.8%, with mortalities occurring only among critical patients. Of 16 acute leukemia patients in the first induction therapy, 13 survived and 10 achieved complete remission. A negative RT-PCR within 2 weeks and improvement of radiological findings were statistically related to disease severity (P=.008 and .002, respectively). Better OS was associated with regression of radiological findings 30 days after infection (P=.002). Forty-five patients received RDV, and 42.1% had severe and critical forms of infection compared to 25.7% in the no-RDV group, yet OS was comparable in both groups. Conclusions: Most pediatric cancer patients with COVID-19 should have good clinical outcomes, except for patients with critical infections. Cancer patients can tolerate chemotherapy, including induction phase, alongside COVID-19 treatment. In severe and critical COVID-19, RDV might have a potential benefit.

A multiplex real-time PCR assay for simultaneous detection of classical swine fever virus, African swine fever virus, and atypical porcine pestivirus

With the implementation of the C-strain vaccine, classical swine fever (CSF) has been under control in China, which is currently in a chronic atypical epidemic situation. African swine fever (ASF) emerged in China in 2018 and spread quickly across the country. It is presently occurring sporadically due to the lack of commercial vaccines and farmers’ increased awareness of biosafety. Atypical porcine pestivirus (APPV) was first detected in Guangdong Province, China, in 2016, which mainly harms piglets and has a local epidemic situation in southern China. These three diseases have similar clinical symptoms in pig herds, which cause considerable losses to the pig industry. They are difficult to be distinguished only by clinical diagnosis. Therefore, developing an early and accurate simultaneous detection and differential diagnosis of the diseases induced by these viruses is essential. In this study, three pairs of specific primers and Taq-man probes were designed from highly conserved genomic regions of CSFV (5′ UTR), African swine fever virus (ASFV) (B646L), and APPV (5′ UTR), followed by the optimization of reaction conditions to establish a multiplex real-time PCR detection assay. The results showed that the method did not cross-react with other swine pathogens (porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease virus (FMDV), pseudorabies virus (PRV), porcine parvovirus (PPV), and bovine viral diarrhea virus BVDV). The sensitivity results showed that CSFV, ASFV, and APPV could be detected as low as 1 copy mL−1; the repeatability results showed that the intra-assay and inter-assay coefficient of variation of ASFV, CSFV, and APPV was less than 1%. Twenty-two virus samples were detected by the multiplex real-time PCR, compared with national standard diagnostic and patented method assay for CSF (GB/T 27540–2011), ASF (GB/T 18648–2020), and APPV (CN108611442A), respectively. The sensitivity of this triple real-time PCR for CSFV, ASFV, and APPV was almost the same, and the compliance results were the same (100%). A total of 451 clinical samples were detected, and the results showed that the positive rates of CSFV, ASFV, and APPV were 0.22% (1/451), 1.3% (6/451), and 0% (0/451), respectively. This assay provides a valuale tool for rapid detection and accurate diagnosis of CSFV, ASFV, and APPV.

Viral aetiology in paediatric age group patients admitted with acute febrile encephalopathy in Western Rajasthan

PurposeIn this study, we are trying to find out viral aetiology in paediatric age group patients from 1 month to 15 years of age in Western Rajasthan region. MethodsA total of 105 patients from 1 month to 15 years were recruited into this study. CSF samples were collected and were processed by multiplex real-time PCR for detection of various predefined panels of viral agents. ELISA was also done for all samples for detection of dengue, JE, measles and mumps. ResultsA total of 32 samples out of 105 were tested positive for viral agents. Viral aetiology detected in this study were Adenovirus (n ​= ​2), EBV (n ​= ​1), HHV-1 (n ​= ​10), HHV-6 (N ​= ​5), Parechovirus (n ​= ​1), Parvovirus B19 (n ​= ​7), Dengue (n ​= ​2) and Measles (n ​= ​1). Mixed infections were also detected, HHV-1 and HHV-6 (n ​= ​2), HHV-1 and Parvovirus B19 (n ​= ​1). In 73 patients no viral aetiology could be detected. ConclusionsParvovirus B19 is sporadically prevalent in this geographical region. In this study, HHV-6 was also found which has not been reported earlier from India.

Improved diagnosis of gastrointestinal infections using a semi-automated multiplex real-time PCR for detection of enteropathogens.

Introduction. The identification of enteropathogens is critical for the clinical management of patients with suspected gastrointestinal infection. The FLOW multiplex PCR system (FMPS) is a semi-automated platform (FLOW System, Roche) for multiplex real-time PCR analysis.Hypothesis/Gap Statement. FMPS has greater sensitivity for the detection of enteric pathogens than standard methods such as culture, biochemical identification, immunochromatography or microscopic examination.Aim.The diagnostic performance of the FMPS was evaluated and compared to that of traditional microbiological procedures.Methodology. A total of 10 659 samples were collected and analysed over a period of 7 years. From 2013 to 2018 (every July to September), samples were processed using standard microbiological culture methods. In 2019, the FMPS was implemented using real-time PCR to detect the following enteropathogens: Shigella spp., Salmonella spp., Campylobacter spp., Giardia intestinalis, Entamoeba histolytica, Blastocystis hominis, Cryptosporidum spp., Dientamoeba fragilis, adenovirus, norovirus and rotavirus. Standard microbiological culture methods (2013-2018) included stool culture, microscopy and immunochromatography.Results. A total of 1078 stool samples were analysed prospectively using the FMPS from July to September (2019): bacterial, parasitic and viral pathogens were identified in 15.3, 9.71 and 5.29 % of cases, respectively. During the same period of 6 years (2013-2018), the proportion of positive identifications using standard microbiological methods from 2013 to 2018 was significantly lower. A major significant recovery improvement was observed for all bacteria species tested: Shigella spp./enteroinvasive Escherichia coli (EIEC) (P <0.05), Salmonella spp. (P <0.05) and Campylobacter spp. (P <0.05). Marked differences were also observed for the parasites G. intestinalis, Cryptosporidium spp. and D. fragilis.Conclusion. These results support the value of multiplex real-time PCR analysis for the detection of enteric pathogens in laboratory diagnosis with outstanding performance in identifying labile micro-organisms. The identification of unsuspected micro-organisms for less specific clinical presentations may also impact on clinical practice and help optimize patient management.

Multiplex real-time PCR for detection and quantification of Colletotrichum abscissum and C. gloeosporioides on Citrus leaves

Colletotrichum abscissum and C. gloeosporioides are the causal agents of citrus Postbloom Fruit Drop (PFD), a major disease in several countries of the American continent. These pathogens infect only floral structures; however, in the absence of floral tissue, they survive asymptomatically on the leaf tissue. Thus, detecting and quantifying pathogens on leaves are critical for epidemiological studies. Colletotrichum species are detected through isolation, serological methods and conventional PCR, but these techniques are not effective for accurate detection of PFD agents. This study aimed to develop specific, more sensitive, faster and less laborious techniques than the traditional ones routinely used. We developed, standardized and validated a multiplex real-time PCR with high sensitivity (98–99.3%) and specificity (94.3–97.1%) for the detection and quantification of both pathogens. This technique was 1000 and 10,000 times more sensitive than Nested-PCR and PCR, respectively for the detection of C. abscissum. Similarly, the multiplex qPCR was 100 and 1000 times more sensitive than Nested-PCR and PCR, respectively for the detection of C. gloeosporioides. The diagnostic parameters used to validate the multiplex qPCR showed a high positive (17–35) and low negative (0.006–0.95) likelihood ratios, low percentage of false positives (2.8–5.6%) and false negatives (0.6–1.9%), good agreement between the results generated between qPCR and multiplex qPCR, revealed by the Kappa index (0.91–0.98) and coincidental results (95–99%). All parameters indicated that the multiplex qPCR was effectively validated to detect and quantify the causal agents of PFD.

Multiplex Real-Time PCR Detection of Respiratory Viruses in Lower Respiratory Tract Infections in Children

Acute respiratory infections cause significant morbidity and mortality in children. Several new respiratory viruses have been identified and co-detection of multiple viruses is commonly reported. This is part of a prospective study which aims to detect respiratory viruses by multiplex molecular method and conventional methods. Nasopharyngeal aspirate specimens were taken from hospitalised children aged less than 5 years with lower respiratory tract infections. These were tested using viral culture, immunofluorescence and Seegene Anyplex™ II RV16 real-time polymerase chain reaction. From 102 samples, 69 (67.6%) were positive by PCR, 12 (11.8%) positive by culture method and 13 (12.7%) positive by IF. A single viral pathogen was detected in 48 samples (47.1%), while 21 samples (20.6%) had co-detection of 2 to 4 viral pathogens. Respiratory syncytial virus (RSV) was most common, detected in 17 samples (16.7% of all samples), followed by adenovirus and rhinovirus in 16 (15.7%), respectively. Bocavirus was detected in 15, enterovirus in 15, influenza A in 8 and parainfluenza-4 in 4 samples, with highest occurrences in co-detection (12/15, 10/15, 5/8 and 3/4, respectively). RSV was the least likely detected in co-detection (3/17). In PCR-positive samples, 54/69 (78.3%) were patients aged up to 24 months. Molecular methods detect more viral aetiologies than conventional methods, with simultaneous detection of multiple respiratory viruses. More sensitive, specific and rapid tools to determine aetiological agents could be incorporated into diagnostic algorithms of respiratory tract infections. Interpretations, significance, and applicability in clinical practice could be further explored, particularly for patients up to 2 years old.

Development of Molecular Diagnosis Using Multiplex Real-Time PCR and T4 Phage Internal Control to Simultaneously Detect Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis from Human Stool Samples.

This study aimed to develop a new multiplex real-time PCR detection method for 3 species of waterborne protozoan parasites (Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis) identified as major causes of traveler’s diarrhea. Three target genes were specifically and simultaneously detected by the TaqMan probe method for multiple parasitic infection cases, including Cryptosporidium oocyst wall protein for C. parvum, glutamate dehydrogenase for G. lamblia, and internal transcribed spacer 1 for C. cayetanensis. Gene product 21 for bacteriophage T4 was used as an internal control DNA target for monitoring human stool DNA amplification. TaqMan probes were prepared using 4 fluorescent dyes, FAM™, HEX™, Cy5™, and CAL Fluor Red® 610 on C. parvum, G. lamblia, C. cayetanensis, and bacteriophage T4, respectively. We developed a novel primer-probe set for each parasite, a primer-probe cocktail (a mixture of primers and probes for the parasites and the internal control) for multiplex real-time PCR analysis, and a protocol for this detection method. Multiplex real-time PCR with the primer-probe cocktail successfully and specifically detected the target genes of C. parvum, G. lamblia, and C. cayetanensis in the mixed spiked human stool sample. The limit of detection for our assay was 2×10 copies for C. parvum and for C. cayetanensis, while it was 2×103 copies for G. lamblia. We propose that the multiplex real-time PCR detection method developed here is a useful method for simultaneously diagnosing the most common causative protozoa in traveler’s diarrhea.

Multiplex Real-time PCR Detection of Intestinal Protozoa in HIV-infected Children in Malawi: Enterocytozoon Bieneusi Is Common and Associated With Gastrointestinal Complaints and May Delay BMI (Nutritional Status) Recovery.

Intestinal protozoa are common opportunistic infections in HIV patients. Longitudinal studies on either the clinical relevance or the effect of immune reconstitution by antiretroviral therapy on intestinal protozoan infections in children are lacking however. This study investigates prevalence and clinical relevance of intestinal protozoa in HIV-infected Malawian children before and during their first year of antiretroviral treatment (ART). Stool samples collected at enrolment and during follow-up were tested for nonopportunistic (Giardia lamblia, Dientamoeba fragilis, Entamoeba histolytica) and opportunistic protozoa (Enterocytozoon bieneusi, Encephalitozoon spp., Cryptosporidium spp. and Cystoisospora belli) using multiplex real-time polymerase chain reaction. Associations between infections and clinical symptoms were evaluated using univariate methods. Nonopportunistic and opportunistic protozoa were detected in 40% (14/35) and 46% (16/35) of children at baseline, respectively. E. bieneusi was the most prevalent protozoa (37%, 13/35) and associated with gastrointestinal complaints (43% in positive (10/13) versus 18% (4/22) in E. bieneusi-negative children, P = 0.001. Body mass index recovery during 12 months of ART was more commonly delayed in E. bieneusi-positive children (+0.29 +standard deviation 0.83) than E. bieneusi-negative children (+1.03 +standard deviation 1.25; P = 0.05). E. bieneusi was not detected after 12 months of ART. E. bieneusi was the most prevalent opportunistic intestinal protozoa, present in over a third of study participants before initiation of ART. Although all children cleared E. bieneusi after 12 months of ART, E. bieneusi was associated with gastrointestinal complaints and may delay body mass index recovery. Trials to assess effect of treatment of E. bieneusi on nutritional status should be considered in HIV-infected African children.

Evaluation of a multiplex real-time PCR for detection of four bacterial agents commonly associated with bovine respiratory disease in bronchoalveolar lavage fluid

BackgroundPasteurella multocida, Mannheimia haemolytica, Histophilus somni and Trueperella pyogenes are four bacterial agents commonly associated with bovine respiratory disease (BRD). In this study a bacterial multiplex real-time PCR (the RespoCheck PCR) was evaluated for the detection in bronchoalveolar lavage fluid (BALF) of these four bacterial agents.ResultsThe analytical sensitivity of the multiplex real-time PCR assay determined on purified DNA and on bacterial cells of the four target pathogens was one to ten fg DNA/assay and 4 × 10−1 to 2 × 100 CFU/assay. The analytical specificity of the test was, as evaluated on a collection of 118 bacterial isolates, 98.3% for M. haemolytica and 100% for the other three target bacteria. A set of 160 BALF samples of calves originating from ten different herds with health problems related to BRD was examined with bacteriological methods and with the RespoCheck PCR. Using bacteriological examination as the gold standard, the diagnostic sensitivities and specificities of the four bacterial agents were respectively between 0.72 and 1.00 and between 0.70 and 0.99. Kappa values for agreement between results of bacteriological examination and PCRs were low for H. somni (0.17), moderate for P. multocida (0.52) and M. haemolytica (0.57), and good for T. pyogenes (0.79). The low and moderate kappa values seemed to be related to limitations of the bacteriological examination, this was especially the case for H. somni.ConclusionIt was concluded that the RespoCheck PCR assay is a valuable diagnostic tool for the simultaneous detection of the four bacterial agents in BALF of calves.

Evaluation of a fully automated extended RAS-BRAF test on prospectively collected plasma samples from patients with metastatic colorectal cancer.

e15127 Background: Extended analysis of RAS genes in metastatic colorectal cancer (mCRC) is not available in many settings and the need for an easy plasma test is high. Methods: Pre-treatment plasma samples from 198 mCRC patients enrolled in the prospective multicenter RASANC study (NCT02502656) were retrospectively assessed for the presence of ctDNA mutations in KRAS, NRAS and BRAF by the fully automated Idylla platform.Proper informed consent and statistical analysis were included. The study is ongoing. We used Idylla platform which allows analysis of 21 KRAS, 18 NRAS and 4 BRAF ctDNA mutations from mCRC using 2 ml of plasma. The tests were performed using prototype versions of the Idylla ctKRAS and ctNRAS-BRAF-EGFR S492R Mutation Assays (RUO), which contain all reagents for integrated sample preparation and multiplex real time PCR detection. The tests each require 1 ml of plasma, less than 1 minute hands-on time, and have a turnaround time of less than 130 minutes.As a comparator test, NGS analysis with a sensitivity of 0.2% was performed on extracted plasma ctDNA according to Pécuchet et al. (2016). Results: The Idylla ctDNA assays identified 84 KRAS mutated (42.4%), 6 NRAS mutated (3%) and 13 BRAF mutated (6.6%) cases. The Idylla assays detected 62 KRAS codon 12 (31%), 16 codon 13 (6.5%), 3 codon 61 (1.5%), and 8 codon 146 (4%), and 1 NRAS codon 13 (0.5%) and 5 (2.5%) codon 61 mutated samples. All BRAF mutations detected were V600E. Overall agreement between Idylla and NGS for KRAS, NRAS, and BRAF was 96%, 100%, and 99.5% respectively. Overall RASconcordance with the NGS reference method was 96.5% with a positive agreement of 98% and a negative agreement of 95% (Table 1). Discordancy analysis by means of ddPCR on plasma and/or tissue will be presented. Conclusions: Plasma-based analysis of 41 extended RAS and BRAF mutations in ctDNA on the Idylla platform enables rapid and reliable genotyping in patients with advanced colorectal cancers. Clinical trial information: NCT02502656. [Table: see text]

Діагностика вірусів скручування листя винограду і коротковузля в процесі виробництва сертифікованого садивного матеріалу винограду

Діагностика вірусів скручування листя винограду і коротковузля в процесі виробництва сертифікованого садивного матеріалу винограду

A comparison of flocked swabs and traditional swabs, using multiplex real-time PCR for detection of common gastroenteritis pathogens in Botswana

We compared the performance of flocked and matched traditional rectal swabs collected from 236 children admitted with gastroenteritis in Botswana. All samples were tested using real time multiplex-PCR assays for nine enteric pathogens. There was a 20% higher detection of Shigella from flocked swabs, but most other pathogens had similar detection rates.

Multiplex real-time PCR for detection of Staphylococcus aureus, mecA and Panton-Valentine Leukocidin (PVL) genes from selective enrichments from animals and retail meat.

The aim of this study was to compare a real-time PCR assay, with a conventional culture/PCR method, to detect S. aureus, mecA and Panton-Valentine Leukocidin (PVL) genes in animals and retail meat, using a two-step selective enrichment protocol. A total of 234 samples were examined (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat). The multiplex real-time PCR targeted the genes: nuc (identification of S. aureus), mecA (associated with methicillin resistance) and PVL (virulence factor), and the primary and secondary enrichment samples were assessed. The conventional culture/PCR method included the two-step selective enrichment, selective plating, biochemical testing, and multiplex PCR for confirmation. The conventional culture/PCR method recovered 95/234 positive S. aureus samples. Application of real-time PCR on samples following primary and secondary enrichment detected S. aureus in 111/234 and 120/234 samples respectively. For detection of S. aureus, the kappa statistic was 0.68–0.88 (from substantial to almost perfect agreement) and 0.29–0.77 (from fair to substantial agreement) for primary and secondary enrichments, using real-time PCR. For detection of mecA gene, the kappa statistic was 0–0.49 (from no agreement beyond that expected by chance to moderate agreement) for primary and secondary enrichment samples. Two pork samples were mecA gene positive by all methods. The real-time PCR assay detected the mecA gene in samples that were negative for S. aureus, but positive for Staphylococcus spp. The PVL gene was not detected in any sample by the conventional culture/PCR method or the real-time PCR assay. Among S. aureus isolated by conventional culture/PCR method, the sequence type ST398, and multi-drug resistant strains were found in animals and raw meat samples. The real-time PCR assay may be recommended as a rapid method for detection of S. aureus and the mecA gene, with further confirmation of methicillin-resistant S. aureus (MRSA) using the standard culture method.