Establishment and application of a TaqMan-based multiplex real-time PCR for simultaneous detection of three porcine diarrhea viruses.

Introduction: Porcine viral diarrhea is a common clinical disease, which results in high mortality and economic losses in the pig industry. Porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), and porcine deltacoronavirus (PDCoV) are important diarrhea viruses in pig herds. The similarities of their clinical symptoms and pathological changes make it difficult to distinguish these three viruses clinically. Therefore, there is a need for a highly sensitive and specific method to simultaneously detect and differentiate these viruses. Methods: A multiplex real-time PCR assay using TaqMan probes was developed to simultaneously detect PEDV, PoRV, and PDCoV. To assess the efficacy of the established assay, 30 clinical samples with diarrhea symptoms were used to compare the results obtained from the multiplex real-time PCR assay with those obtained from commercial singleplex real-time PCR kit. Importantly, a total of 4,800 diarrhea samples were tested and analyzed to validate the utility of the assay. Results: This multiplex real-time PCR assay showed high sensitivity, specificity, and excellent repeatability with a detection limit of 1 × 102 copies/µL. Comparing the results of the commercial singleplex real-time PCR kit and the multiplex real-time PCR method for detecting PEDV, PoRV, and PDCoV, there was complete agreement between the two approaches. Clinical data revealed single infection rates of 6.56% for PEDV, 21.69% for PoRV, and 6.65% for PDCoV. The co-infection rates were 11.83% for PEDV + PoRV, 0.29% for PEDV + PDCoV, 5.71% for PoRV + PDCoV, and 1.29% for PEDV + PDCoV + PoRV, respectively. Discussion: The multiplex real-time PCR method established in this study is a valuable diagnostic tool for simultaneously differentiating PEDV, PoRV, and PDCoV. This method is expected to significantly contribute to prevent and control the spread of infectious diseases, as well as aid in conducting epidemiological investigations.

Development and application of a TaqMan-probe-based multiplex real-time PCR assay for simultaneous detection of porcine circovirus 2, 3, and 4 in Guangdong province of China.

Porcine circoviruses disease (PCVD), caused by porcine circovirus (PCVs), is an important swine disease characterized by porcine dermatitis, nephrotic syndrome and reproductive disorders in sows. However, diseases caused by PCV2, PCV3, or PCV4 are difficult to distinguish, so a simple, rapid, accurate and high-throughput diagnostic and identification method is urgently needed to differentiate these three types. In this study, specific primers and probes were designed based on the conserved region sequences of the Rep gene of PCV2, and the Cap gene of PCV3 and PCV4. A multiplex qPCR assay was developed and optimized that the limit of detection concentration could reach as low as 3.8 copies/µL, with all correlation coefficients (R2) exceeding 0.999. Furthermore, the method showed no cross-reaction with other crucial porcine viral pathogens, and both intra-repeatability and inter-reproducibility coefficients of variation were below 2%. The assay was applied to the detection of 738 pig samples collected from 2020 to 2021 in Guangdong Province, China. This revealed positive infection rates of 65.18% for PCV2, 29.27% for PCV3, and 0% for PCV4, with a PCV2/PCV3 co-infection rate of 23.17%. Subsequently, complete genome sequences of 17 PCV2 and 4 PCV3 strains were obtained from the above positive samples and pre-preserved positive circovirus samples. Nucleotide sequence analysis revealed that the 17 PCV2 strains shared 96.7-100% complete nucleotide identity, with 6 strains being PCV2b and 11 strains being PCV2d; the 4 PCV3 strains shared 98.9-99.4% complete nucleotide identity, with 2 strains being PCV3a-1 and 2 strains being PCV3b. This research provides a reliable tool for rapid PCVs identification and detection. Molecular epidemiological investigation of PCVs in pigs in Guangdong Province will help us to understand PCV2 and PCV3 epidemiological characteristics and evolutionary trends.

Development and preliminary application of a quadruplex real-time PCR assay for differential detection of porcine circovirus 1-4 in Chengdu, China.

Porcine circovirus (PCV) typically causes severe immune suppression in pigs, leading to mixed clinical infections with various pathogens that can cause significant harm to the pig industry. PCV has four subgenotypes, with PCV4 being an emerging virus that requires investigation due to its potential for epidemic outbreaks. Therefore, there is a need to develop a method that can detect all four PCV strains simultaneously. In this study, four pairs of specific primers and TaqMan probes were designed based on the conserved sequence of the PCV1-4 ORF2 gene to establish a PCV1-4 TaqMan multiplex real-time quantitative PCR method. The novel method was compared to six commercial testing kits for its efficacy. Then, a total of 595 mixed samples of spleen and lymph node collected from 12 districts in Chengdu from July to December 2021 were tested using the novel method. The results showed that the novel PCV1-4 TaqMan multiplex real-time quantitative PCR detection method has satisfied specificity, sensitivity, and repeatability. The positive rates of PCV1, PCV2, and PCV3 in Chengdu were 2.18%, 31.60%, and 15.29%, respectively, while no positive PCV4 was detected. The mixed infection rate of PCV2 and PCV3 was 5.21%. Our novel method may be as a potential method for PCV1-4 detection. Currently, PCV2 is the main epidemic PCV subtype in Chengdu, while the potential threat of PCV4 should also be considered.

Development and Application of Four Foodborne Pathogens by TaqMan Multiplex Real-Time PCR.

A TaqMan multiplex real-time PCR (mRT-PCR) was developed to detect simultaneously Salmonella spp., Escherichia coli O157, Staphylococcus aureus, and Listeria monocytogenes in food samples. The method involves four sets of primers and probes tailored to the unique DNA sequences found in the invA, nuc, rfbE, and hly genes of each pathogen. The generated standard curves, correlating gene copy numbers with Ct values, demonstrated high accuracy (R2 > 0.99) and efficiency (92%-104%). Meanwhile, the limit of detection was 100 CFU/mL for the four target bacteria in artificially contaminated food samples after 6-8 h of enrichment. The assay's effectiveness was further verified by testing 80 naturally contaminated food samples, showing results largely in agreement with traditional culture methods. Overall, this newly developed TaqMan mRT-PCR, inclusive of a pre-enrichment step, proves to be a dependable and effective tool for detecting single or multiple pathogens in diverse food items, offering significant potential for in vitro diagnostics.

Paediatric pulmonary disease-are we diagnosing it right?

Background: It has been reported that differential diagnosis of bacterial or viral pneumonia and tuberculosis (TB) in infants and young children is complex. This could be due to the difficulty in microbiological confirmation in this age group. In this study, we aimed to assess the utility of a real-time multiplex PCR for diagnosis of respiratory pathogens in children with pulmonary TB. Methods: A total of 185 respiratory samples [bronchoalveolar lavage (15), gastric aspirates (98), induced sputum (21), and sputum (51)] from children aged 3-12 years, attending tertiary care hospitals, Chennai, India, were included in the study. The samples were processed by N acetyl L cysteine (NALC) NAOH treatment and subjected to microbiological investigations for Mycobacterium tuberculosis (MTB) diagnosis that involved smear microscopy, Xpert® MTB/RIF testing, and liquid culture. In addition, DNA extraction from the processed sputum was carried out and was subjected to a multiplex real-time PCR comprising a panel of bacterial and fungal pathogens. Results: Out of the 185 samples tested, a total of 20 samples were positive for MTB by either one or more identification methods (smear, culture, and GeneXpert). Out of these 20 MTB-positive samples, 15 were positive for one or more bacterial or fungal pathogens, with different cycle threshold values. Among patients with negative MTB test results (n = 165), 145 (87%) tested positive for one or more than one bacterial or fungal pathogens. Conclusion: The results suggest that tuberculosis could coexist with other respiratory pathogens causing pneumonia. However, a large-scale prospective study from different geographical settings that uses such simultaneous detection methods for diagnosis of childhood tuberculosis and pneumonia will help in assessing the utility of these tests in rapid diagnosis of respiratory infections.

The prevalence of 17 common respiratory viruses in patients with respiratory illness but negative for COVID-19: A cross-sectional study.

Background and Aims: Second to COVID-19 pandemic, other viral respiratory infections are still important causes of human diseases or co-infections. Hence, the present study was carried out to investigate the common respiratory viruses in patients with respiratory illness diagnosed negative for severe acute respiratory syndrome coronavirus-2 in primary screening. Methods: In a cross-sectional study, a real-time PCR was carried out using HiTeq. 17 Viro Respiratory pathogen One Step RT-PCR Kit (Genova, Bonda Faravar, Bioluence, Tehran, Iran). Results: A total of 311 individuals (mean age ± SD: 48.2 ± 21.7 years, range: 1-97 years) underwent second PCR. Among these, 161 (51.7%) were female. In total, 55 (17.6%) cases (mean age ± SD: 45.7 ± 18.1 years) were found positive for respiratory viruses panel in the second PCR. The HCoV-OC43/HKU1 was in 5.4% (17/311), Flu A in 4.5% (14/311), HCoV-229E/NL63 in 2.8% (9/311), HMPV in 1.9% (6/311), HPiV 1, 2, 3 in 1.2% (4/311), HRSV in 0.9% (3/311), and HAdV in 0.6% (2/311) of the cases studies. Also, co-infection was detected in 4 samples (1.2%). In addition, sore throat (0.028), headache (p = 0.016), and body pain (p = 0.0001) were statistically the most significant symptoms in studied cases. Conclusion: According to the findings of our study, respiratory virus infections and co-infections were 17.6% and 1.2% frequent, respectively. Interestingly, nearly half of our positive cases (47.2%) were identified by coronaviruses (ОС43, Е229, NL63, and HKUI), followed by influenza A virus (25.4%). However, for more comprehensive results, we recommend using greater sample size.

A novel multiplex real-time PCR assay for the detection of cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1/2 and strategies for application to blood screening.

A multiplex real-time PCR has been developed to simultaneously detect transfusion-transmissible pathogens cytomegalovirus, Epstein-Barr virus and herpes simplex virus, as well as to provide sample quality testing, for the conserved regions of the cytomegalovirus UL123 gene, the Epstein-Barr virus BKRF1 gene, and the herpes simplex virus 1/2 UL30 gene, tested on 500 blood donors and 320 transfusion recipients. The laboratory sensitivities for all 3 pathogens were 100 copies/µL. Compared to the commercial real-time PCR reference kit, the multiplex real-time PCR assay for the detection of CMV, EBV and HSV presented 100% consistency. In 820 whole blood samples, the multiplex real-time PCR assay identified 34 (4.15%) positive for CMV DNA, 15 (1.83%) positive for EBV DNA, and 6 (0.73%) positive for HSV DNA. For blood transfusions in high-risk groups, whole blood herpes virus test should be included in the spectrum of pathogen testing for blood donors and recipients.

Changes in Prevalence and Seasonality of Pathogens Identified in Acute Respiratory Tract Infections in Hospitalised Individuals in Rural and Urban Settings in South Africa; 2018-2022.

Severe acute respiratory tract infections (SARIs) has been well described in South Africa with seasonal patterns described for influenza and respiratory syncytial virus (RSV), while others occur year-round (rhinovirus and adenovirus). This prospective syndromic hospital-based surveillance study describes the prevalence and impact of public interventions on the seasonality of other respiratory pathogens during the coronavirus disease-19 (COVID-19) pandemic. This occurred from August 2018 to April 2022, with 2595 patients who met the SARS case definition and 442 controls, from three sentinel urban and rural hospital sites in South Africa. Naso/oro-pharyngeal (NP/OP) swabs were tested using the FastTrack Diagnostics® Respiratory pathogens 33 (RUO) kit. Descriptive statistics, odds ratios, and univariate/multivariate analyses were used. Rhinovirus (14.80%, 228/1540) and Streptococcus pneumoniae (28.50%, 439/1540) were most frequently detected in NP/OP swabs and in children <1 years old (35%, 648/1876). Among others, pathogens associated with SARI cases causing disease were influenza A&B, HRV, RSV, hCoV 229e, Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae. Pre-COVID-19, seasonal trends of these pathogens correlated with previous years, with RSV and influenza A seasons only resuming after the national lockdown (2021). It is evident that stringent lockdown conditions have severe impacts on the prevalence of respiratory tract infections.

Severe Pneumonia Caused by Respiratory Syncytial Virus and Adenovirus in Children from 2 to 24 Months at Children's Hospital 1 in Ho Chi Minh City, Vietnam.

In Vietnam, due to the lack of facilities to detect respiratory viruses from patients' specimens, there are only a few studies on the detection of viral pathogens causing pneumonia in children, especially respiratory syncytial virus (RSV) and adenovirus (Adv). Here, we performed a cross-sectional descriptive prospective study on 138 children patients from 2 to 24 months old diagnosed with severe pneumonia hospitalized at the Respiratory Department of Children's Hospital 1 from November 2021 to August 2022. The number of patients selected in this study was based on the formula n = ([Z(1 - α/2)]2 × P [1 - P])/d2, with α = 0.05, p = 0.5, and d = 9%, and the sampling technique was convenient sampling until the sample size was met. A rapid test was used to detect RSV and Adv from the nasopharyngeal swabs and was conducted immediately after the patient's hospitalization. Laboratory tests were performed, medical history interviews were conducted, and nasotracheal aspirates were collected for multiplex real-time PCR (MPL-rPCR) to detect viral and bacterial pathogens. The results of the rapid test and the MPL-rPCR in the detection of both pathogens were the same at 31.9% (44/138) for RSV and 8.7% (7/138) for Adv, respectively. Using MPL-rPCR, the detection rate was 21% (29/138) for bacterial pathogens, 68.8% (95/138) for bacterial-viral co-infections, and 6.5% (9/138) for viral pathogens. The results showed few distinctive traits between RSV-associated and Adv-associated groups, and the Adv group children were more prone to bacterial infection than those in the RSV group. In addition, the Adv group experienced a longer duration of treatment and a higher frequency of re-hospitalizations compared to the RSV group. A total of 100% of Adv infections were co-infected with bacteria, while 81.82% of RSV co-infected with bacterial pathogens (p = 0.000009). This study might be one of the few conducted in Vietnam aimed at identifying viral pathogens causing severe pneumonia in children.

Rapid multiplex real-time PCR assay using a portable device for the detection of oral pathogens.

Colonization by several oral pathogens and the onset of oral diseases, such as dental caries and periodontal diseases, are closely related. Therefore, the analysis of pathogens in oral specimens would be helpful for the risk assessment of oral diseases. We developed a rapid multiplex real-time polymerase chain reaction (PCR) method using a portable device and newly designed probe/primer sets to detect the oral pathogens Streptococcus mutans, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia. The theoretical minimum detectable cell numbers of S. mutans, P. gingivalis, T. denticola, and T. forsythia were 1, 1, 4, and 3, respectively. The multiplex real-time PCR system simultaneously detected the colonization of S. mutans and P. gingivalis in human saliva. These results suggest that the multiplex real-time PCR system may be useful for the risk assessment of oral diseases.

Multiplex real-time PCR FilmArray performance in the diagnosis of meningoencephalitis: lights and shadows.

PURPOSE: We aimed to evaluate the performance of the FilmArray (FA) meningitis/encephalitis (ME) panel. Secondarily, we analyzed the false positive (FP) and false negative (FN) results, as well as the predictive values of the technique, regarding the cerebrospinal fluid (CSF) characteristics. METHODS: FA is a multiplex real-time PCR detecting 14 of the most common ME pathogens in CSF. All FA performed at our hospital (2018-2022) were retrospectively reviewed. FA was compared to conventional techniques and its performance was assessed based on the final diagnosis of the episode. RESULTS: FA was performed in 313 patients with suspicion of ME. Most patients had altered mental status (65.2%) and fever (61%). Regarding CSF characteristics, 49.8% and 53.7% presented high CSF proteins and pleocytosis, respectively. There were 84 (26.8%) positive FA results, mainly for HSV-1 (10.9%), VZV (5.1%), Enterovirus (2.6%), and S. pneumoniae (1.9%). In the 136 cases where both FA and routine methods were performed, there was a 25.7% lack of agreement. We identified 6.6% FN results, but 28.6% FP, mainly due to HSV-1. This resulted in a high negative predictive value (NPV) of 93.4%, but a positive predictive value (PPV) of 73%. Remarkably, PPV as low as 36.9%, and 70.2%, were found in cases without pleocytosis, or lack of high CSF protein levels, respectively. CONCLUSION: FA was associated with high NPV, but frequent FP results and low PPV, particularly for HSV-1, and especially in patients without high CSF protein levels or pleocytosis.

Clinical characteristics of preterm and term infants with Ureaplasma in gastric fluid.

BACKGROUND: Ureaplasma spp. is an endemic microorganism that causes placental chorioamnionitis or preterm delivery in pregnant women, and the occurrence of bronchopulmonary dysplasia or intraventricular hemorrhaging in preterm infants after birth, although the pathogenicity of Ureaplasma remains controversial. The association between Ureaplasma exposure and the symptoms or outcomes of infected mothers or their infants born at term remains poorly understood. We investigated the clinical characteristics of preterm and term infants with or without Ureaplasma in their gastric fluid. METHODS: Gastric fluid samples were collected from 47 newborns in the neonatal intensive-care unit immediately after birth and tested using multiplex polymerase chain reaction (PCR) assays targeting Ureaplasma spp., Ureaplasma parvum, and Ureaplasma urealyticum. The clinical findings and outcomes of the neonates and their mothers were retrospectively evaluated. RESULTS: Ureaplasma spp. were detected in 9/47 samples (19%) by multiplex PCR assays. In all cases, the subspecies was U. parvum. The Ureaplasma-positive group had a significantly higher incidence of chorioamnionitis in utero than the Ureaplasma-negative group. Regarding preterm infants, the IgM levels in the Ureaplasma-positive group were significantly higher than in the Ureaplasma-negative group. In contrast, in term infants, the rates of a non-reassuring fetal status, a maternal fever, and maternal leukocyte counts and maternal C-reactive protein levels within five days before delivery in the Ureaplasma-positive group were significantly higher than those in the Ureaplasma-negative group. All three extremely-low-birth-weight infants with Ureaplasma developed bronchopulmonary dysplasia. The length of hospitalization in the Ureaplasma-positive group was almost same as that in the Ureaplasma-negative group for term infants. CONCLUSION: Mothers or their fetuses with exposure to Ureaplasma expressed characteristic clinical features during pregnancy and after birth.

The impact of the COVID-19 pandemic on the circulation, seasonal distribution, and research of other respiratory pathogens in Turkey.

INTRODUCTION: Respiratory infections are one of the world's most common infectious diseases. Following the species, numbers, and seasonal distribution of acute respiratory agents is important for the protection of public health. Our study aimed to determine the effect of the COVID-19 pandemic on the circulation and seasonal distribution of non-SARS-CoV-2 respiratory tract agents and research on non-SARS-CoV-2 agents. METHODS: The results of the Multiplex PCR respiratory panel of 3702 nasopharyngeal swab samples sent between January 2018 and December 2021 were evaluated retrospectively. Scientific articles on acute respiratory infections between 2010 and 2021 from Turkey were analyzed in Scopus for bibliometric analysis. RESULTS: 1.382 pathogens were detected. During the pandemic, the number of non-SARS-CoV-2 pathogens was found to be statistically significantly lower than before the pandemic. It was determined that while the most frequent agent before the pandemic was the Adenovirus, the most frequent agent was the RSV-A during the pandemic. Our network analysis of keywords indicated that academic interest in 2020-21 was directed toward COVID-19, which coincides with the pandemic period. CONCLUSIONS: Our study determined the fact that the incidence, species, and seasonal distribution of non-SARS-COV-2 respiratory agents changed after the onset of the pandemic. Increasing the identification and following-up of these pathogens in health organizations and also presenting these data to literature and sharing with academics is important. We are of the opinion that the results of our study shall shed light on the epidemiology of changing respiratory infections and the prevention and following-up of future health problems.

Investigation of the prevalence of carbapenem resistance genes in faecal carriage of carbapenem resistant Klebsiella spp. isolates by multiplex real-time PCR method.

INTRODUCTION: Increased carbapenem resistance in Klebsiella spp. strains causes high morbidity and mortality. The genes encoded for carbapenemaseare transferrable between different bacterial species. In the present study, we aimed to investigate carbapenem resistance genes in Klebsiella spp. strains. METHODOLOGY: Fifty Klebsiella spp. strains were isolated from rectal swabs of patients hospitalized in the neonatal intensive care unit (NICU). All strains were identified with API20E. The minimum inhibitory concentrations (MICs) of carbapenems were determined by the broth dilution method. The major five carbapenem genes (OXA-48, NDM, VIM, KPC, and IMP) were detected by the multiplex real-time PCR method. RESULTS: It was found that 49 (98%) of the strains were resistant to ertapenem (MIC ≥ 2µg/mL) and imipenem(MIC ≥ 4 µg/mL), and 47 (94%) of the strains were resistant to doripenem (MIC ≥ 4 µg/mL)and meropenem(MIC ≥ 4 µg/mL).NDM was detected in 42%, OXA-48 in 16%, and VIM in one (2%) isolate, and NDM + OXA-48 co-existed in 36% of the isolates. The KPC and IMP genes were not detected. CONCLUSIONS: NDM and NDM co-existing with OXA-48 were prevalent in the NICU of Istanbul Medical Faculty Hospital. Paying attention to the hand hygiene of healthcare workers, screening of rectal swabs of hospitalized patients for the presence of carbapenem resistance strains, and isolation of infected patients can effectively control the spread of carbapenem-resistant strains.

The Fascinating Cross-Paths of Pathogenic Bacteria, Human and Animal Faecal Sources in Water-Stressed Communities of Vhembe District, South Africa.

Access to clean and safe drinking water still remains a major challenge in the developing world, causing public health risks in terms of waterborne infections, especially in rural areas of sub-Saharan Africa. This study aimed to track and detect enteric pathogens (Salmonella enterica subsp. enterica serovar Typhimurium str. LT2, Shigella flexneri, and Campylobacter jejuni subsp. jejuni) in rural water sources. It also sought to establish a correlation between these pathogens and the sources of faecal pollution. Multiplex qPCR and specific primers and probes were used for detection and tracking. The study successfully correlated the occurrence of target pathogens with sources of human and animal faecal contamination using host-specific genetic markers (BacHum and HF183 for humans, BacCow for cows, Pig-2-Bac for pigs, Cytb for chickens, and BacCan for dogs). The study revealed that enteric pathogens were found in 47.69% and 32.80% of samples during the wet and dry seasons, respectively. These pathogens were associated with human or animal faecal contamination. Correlations between pathogens and contamination sources were significant (p ≤ 0.05), with varying strengths during the wet and dry seasons. The findings emphasize the importance of identifying faecal contamination sources to protect rural communities from waterborne infections.

Human Parainfluenza Virus (HPIV) Detection in Hospitalized Children with Acute Respiratory Tract Infection in the Western Cape, South Africa during 2014-2022 Reveals a Shift in Dominance of HPIV 3 and 4 Infections.

The epidemiology of human parainfluenza viruses (HPIV), particularly its role as a cause of acute respiratory infection (ARI) in infants, has not been formally studied in South Africa. We evaluated HPIV prevalence in diagnostic samples from hospitalized children from public sector hospitals in the Western Cape between 2014 and 2022. HPIV infection was detected in 2-10% of patients, with the majority of infections detected in children less than 1 year of age. Prior to 2020, HPIV 4 (40%) and HPIV 3 (34%) were the most prevalent types, with seasonal peaks in late winter/spring for HPIV 3 and autumn/winter for HPIV 4. HPIV 4A and 4B co-circulated during the seasonal activity between 2014 and 2017. Pandemic restrictions in 2020 had a profound effect on HPIV circulation and the rebound was dominated by waves of HPIV 3, accounting for 66% of detections and a sustained decline in the circulation of HPIV 1, 2 and 4. An immunity gap could account for the surge in HPIV 3 infections, but the decline in prior HPIV 4 dominance is unexplained and requires further study.

Development of detection methods by multiplex real-time PCR for highly pathogenic Yersinia enterocolitica, low pathogenic Yersinia enterocolitica, and Yersinia pseudotuberculosis based on SYBR Green and TaqMan probes.

This study aimed to develop multiplex real-time PCR methods using SYBR Green and TaqMan probes for rapid and sensitive diagnosis, differentiating three pathogenic Yersinia groups such as highly pathogenic Y. enterocolitica, low pathogenic Y. enterocolitica, and Y. pseudotuberculosis. Specific primer and probe combinations for differentiating three pathogenic Yersinia groups were designed from three chromosomally encoded genes (ail, fyuA, and inv). Twenty-six stains of pathogenic Yersinia species including 6 strains of low pathogenic Y. enterocolitica serotypes, 7 strains of highly pathogenic Y. enterocolitica serotypes, and 13 strains of pathogenic Y. pseudotuberculosis were used for specificity testing. Specific patterns of real-time amplification signals distinguished three pathogenic Yersinia groups. A detection limit of approximately 101 colony forming units (CFU) /reaction of genomic DNA was determined based on plate counts. Furthermore, the multiplex real-time PCR methods also detected Y. enterocolitica O:8 from the DNA extracted from spiked rabbit blood samples and potentially infected wild rodent fecal samples. These results demonstrated that the multiplex real-time PCR methods developed in this study are useful for rapid detection and differentiation of three pathogenic Yersinia groups. Therefore, these methods provide a new monitoring and detection capability to understand the epidemiology of pathogenic Yersinia and to diagnose three pathogenic Yersinia groups.

Construction of a one-step multiplex real-time PCR assay for the detection of serogroups A, B, and E of Pasteurella multocida associated with bovine pasteurellosis.

Bovine pasteurellosis, caused by serogroups A, B, and E of Pasteurella multocida (Pm), is mainly manifested as bovine respiratory disease (BRD) and hemorrhagic septicemia (HS). The disease has caused a great economic loss for the cattle industry globally. Therefore, identifying the Pm serogroups is critical for optimal diagnosis and subsequent clinical treatment and even epidemiological studies. In this study, a one-step multiplex real-time PCR assay was established. Three pairs of specific primers were prepared to detect the highly conserved genomic regions of serogroups A (HyaD), B (bcbD), and E (ecbJ) of Pm, respectively. The results depicted that the method had no cross-reaction with other bovine pathogens (Mannheimia hemolytica, Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Salmonella Dublin, Mycobacterium paratuberculosis, infectious bovine rhinotracheitis virus, and Mycoplasma bovis). The linear range (107 to 102 copies/µL) showed the R2 values for serogroups A, B, and E of Pm as 0.9975, 0.9964, and 0.996, respectively. The multiplex real-time PCR efficiency was 90.30%, 90.72%, and 90.57% for CartA, CartB, and CartE, respectively. The sensitivity result showed that the serogroups A, B, and E of Pm could be detected to be as low as 10 copies/µL. The repeatability result clarified that an intra-assay and an inter-assay coefficient of variation of serogroups A, B, and E of Pm was < 2%. For the clinical samples, the detection rate was higher than the OIE-recommended ordinary PCR. Overall, the established one-step multiplex real-time PCR assay may be a valuable tool for the rapid and early detection of the serogroups A, B, and E of Pm with high specificity and sensitivity.

Identification by High-Throughput Real-Time PCR of 30 Major Circulating Listeria monocytogenes Clonal Complexes in Europe.

Listeria monocytogenes is a ubiquitous bacterium that causes a foodborne illness, listeriosis. Most strains can be classified into major clonal complexes (CCs) that account for the majority of outbreaks and sporadic cases in Europe. In addition to the 20 CCs known to account for the majority of human and animal clinical cases, 10 CCs are frequently reported in food production, thereby posing a serious challenge for the agrifood industry. Therefore, there is a need for a rapid and reliable method to identify these 30 major CCs. The high-throughput real-time PCR assay presented here provides accurate identification of these 30 CCs and eight genetic subdivisions within four CCs, splitting each CC into two distinct subpopulations, along with the molecular serogroup of a strain. Based on the BioMark high-throughput real-time PCR system, our assay analyzes 46 strains against 40 real-time PCR arrays in a single experiment. This European study (i) designed the assay from a broad panel of 3,342 L. monocytogenes genomes, (ii) tested its sensitivity and specificity on 597 sequenced strains collected from 24 European countries, and (iii) evaluated its performance in the typing of 526 strains collected during surveillance activities. The assay was then optimized for conventional multiplex real-time PCR for easy implementation in food laboratories. It has already been used for outbreak investigations. It represents a key tool for assisting food laboratories to establish strain relatedness with human clinical strains during outbreak investigations and for helping food business operators by improving their microbiological management plans. IMPORTANCE Multilocus sequence typing (MLST) is the reference method for Listeria monocytogenes typing but is expensive and takes time to perform, from 3 to 5 days for laboratories that outsource sequencing. Thirty major MLST clonal complexes (CCs) are circulating in the food chain and are currently identifiable only by sequencing. Therefore, there is a need for a rapid and reliable method to identify these CCs. The method presented here enables the rapid identification, by real-time PCR, of 30 CCs and eight genetic subdivisions within four CCs, splitting each CC into two distinct subpopulations. The assay was then optimized on different conventional multiplex real-time PCR systems for easy implementation in food laboratories. The two assays will be used for frontline identification of L. monocytogenes isolates prior to whole-genome sequencing. Such assays are of great interest for all food industry stakeholders and public agencies for tracking L. monocytogenes food contamination.

Establishment and application of multiplex real-time PCR for simultaneous detection of four viruses associated with porcine reproductive failure.

Many pathogens cause reproductive failure in sows suffering a broad spectrum of sequelae, including abortions, stillbirth, mummification, embryonic death, and infertility. Although various detection methods, such as polymerase chain reaction (PCR) and real-time PCR, have been widely used for molecular diagnosis, mainly for a single pathogen. In this study, we developed a multiplex real-time PCR method for the simultaneous detection of porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), porcine parvovirus (PPV) and pseudorabies virus (PRV) associated with porcine reproductive failure. The R 2 values for the standard curve of multiplex real-time PCR of PCV2, PCV3, PPV, and PRV reached to 0.996, 0.997, 0.996, and 0.998, respectively. Importantly, the limit of detection (LoD) of PCV2, PCV3, PPV, and PRV, were 1, 10, 10, 10 copies/reaction, respectively. Meanwhile, specificity test results indicated that multiplex real-time PCR for simultaneous detection is specific for these four target pathogens and does not react with other pathogens, such as classical swine fever virus, porcine reproductive and respiratory syndrome virus, and porcine epidemic diarrhea virus. Besides, this method had good repeatability with coefficients of variation of intra- and inter-assay less than 2%. Finally, this approach was further evaluated by 315 clinical samples for its practicality in the field. The positive rates of PCV2, PCV3, PPV, and PRV were 66.67% (210/315), 8.57% (27/315), 8.89% (28/315), and 4.13% (13/315), respectively. The overall co-infection rates of two or more pathogens were 13.65% (43/315). Therefore, this multiplex real-time PCR provides an accurate and sensitive method for the identification of those four underlying DNA viruses among potential pathogenic agents, allowing it to be applied in diagnostics, surveillance, and epidemiology.