Simultaneous detection of Zika, chikungunya, dengue, yellow fever, West Nile, and Japanese encephalitis viruses by a two-tube multiplex real-time RT-PCR assay.

Due to the concurrent prevalence and increasing risk of coinfection of the clinically important Arboviruses, timely and accurate differential diagnosis is important for clinical management and the epidemiological investigation. A two-tube multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the simultaneous detection of Zika virus (ZIKV), chikungunya virus (CHIKV), dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) was developed and optimized with high specificity and sensitivity. The detection limit for all the six viruses could reach as low as five genome equivalent copies and 2.8 × 10-3 tissue culture infectious doses (TCID50 ) for ZIKV, YFV, CHIKV and 2.8 × 10-2 TCID50  for JEV per reaction, with high accuracy and precision (R2 > 0.99). The coefficient of variation of intra-assay and inter-assay for our quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was low, and the obtained positive rates ad Ct values of this assay were comparable with singleplex commercial kits. Moreover, the multiplex qRT-PCR assay was able to detect possible co-infections without competitive inhibition of target viral genomes. In conclusion, our rapid, sensitive, cost-effective multiplex qRT-PCR will be of great use for differential diagnosis in a clinical setting and epidemiological investigation during surveillance.

A minimum variance method for genome-wide data-driven normalization of quantitative real-time polymerase chain reaction expression data.

Advances in multiplex qRT-PCR have enabled increasingly accurate and robust quantification of RNA, even at lower concentrations, facilitating RNA expression profiling in clinical and environmental samples. Here we describe a data-driven qRT-PCR normalization method, the minimum variance method, and evaluate it on clinically derived Mycobacterium tuberculosis samples with variable transcript detection percentages. For moderate to significant amounts of nondetection (∼50%), our minimum variance method consistently produces the lowest false discovery rates compared to commonly used data-driven normalization methods.

Development of a quadruple qRT-PCR assay for simultaneous identification of hypervirulent and carbapenem-resistant Klebsiella pneumoniae.

IMPORTANCE: Globally, the increasing number of hypervirulent Klebsiella pneumoniae (hvKp) and carbapenem-resistant Kp (CR-Kp) infections poses a huge public health challenge with high morbidity and mortality. Worrisomely, due to the mobility of elements carrying virulence and drug-resistance genes, the increasing prevalence of CR-hvKp has also been found with an overwhelming mortality rate in recent years. However, the current detection methods for hvKp and CR-Kp have many disadvantages, such as long turnaround time, complex operation, low sensitivity, and specificity. Herein, a more sensitive, rapid, single-reaction, and multiplex quantitative real-time PCR was developed and validated to differentiate the circulating lineages of Kp with excellent performance in sensitivity and specificity, providing a useful tool for the differential diagnosis and the surveillance of the circulating Kp.

Development of a multiplex qRT-PCR assay for detection of classical swine fever virus, African swine fever virus, and Erysipelothrix rhusiopathiae.

Classical swine fever virus (CSFV), African swine fever virus (ASFV), and Erysipelothrix rhusiopathiae (E. rhusiopathiae) remain endemic in many parts of China. Co-infections make distinguishing their clinical symptoms and pathological changes difficult. This study developed a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) that can simultaneously detect CSFV, ASFV, and E. rhusiopathiae. Three sets of primers and probes were designed to target the CSFV 5΄ untranslated region, ASFV p72 gene, and E. rhusiopathiae 16sRNA gene. Multiplex qRT-PCR for simultaneous differential detection of these three pathogens was developed after optimizing reaction parameters such as annealing temperature, primer and probe concentrations, amplification cycles, etc. The multiplex qRT-PCR could detect CSFV, ASFV, and E. rhusiopathiae simultaneously but could not amplify other porcine pathogens. The assay's limit of detection (LOD) was 2.89 × 102 copies/µL for CSFV, ASFV, and E. rhusiopathiae. All correlation coefficients (R2) at higher than 0.99, and the amplification efficiency was 98, 90, and 84%, respectively. All correlation coefficients (R2) were higher than 0.99, and the efficacy of amplification was 84%. In a repeatability test utilizing standard recombinant plasmids, the intra- and inter-assay coefficients of variation (CVs) were less than 2.27 and 3.79 percent, respectively. Lastly, 150 clinical samples were used to evaluate the assay's applicability in the field. The positive rates of CSFV, ASFV, and E. rhusiopathiae were 1.33%, 0, and 3.33%, respectively. And no co-infection among the three pathogens was found. The concordance rate between the multiplex qRT-PCR and single-plex commercial PCR kits reached 100%. This study's multiplex qRT-PCR could provide a rapid, sensitive, and specific method for the simultaneous and differential detection of CSFV, ASFV, and E. rhusiopathiae.

Development of a quadruplex real-time quantitative RT-PCR for detection and differentiation of PHEV, PRV, CSFV, and JEV.

Porcine hemagglutinating encephalomyelitis virus (PHEV), porcine pseudorabies virus (PRV), classical swine fever virus (CSFV), and Japanese encephalitis virus (JEV) cause similar neurological symptoms in the infected pigs, and their differential diagnosis depends on laboratory testing. Four pairs of specific primers and probes were designed targeting the PHEV N gene, PRV gB gene, CSFV 5' untranslated region (5'UTR), and JEV NS1 gene, respectively, and a quadruplex real-time quantitative RT-PCR (qRT-PCR) was developed to detect and differentiate PHEV, PRV, CSFV, and JEV. The assay showed high sensitivity, with the limit of detection (LOD) of 1.5 × 101 copies/µL for each pathogen. The assay specifically detected only PHEV, PRV, CSFV, and JEV, without cross-reaction with other swine viruses. The coefficients of variation (CVs) of the intra-assay and the inter-assay were less than 1.84%, with great repeatability. A total of 1,977 clinical samples, including tissue samples, and whole blood samples collected from Guangxi province in China, were tested by the developed quadruplex qRT-PCR, and the positivity rates of PHEV, PRV, CSFV, and JEV were 1.57% (31/1,977), 0.35% (7/1,977), 1.06% (21/1,977), and 0.10% (2/1,977), respectively. These 1,977 samples were also tested by the previously reported qRT-PCR assays, and the coincidence rates of these methods were more than 99.90%. The developed assay is demonstrated to be rapid, sensitive, and accurate for detection and differentiation of PHEV, PRV, CSFV, and JEV.

Development of a Multiplex Crystal Digital RT-PCR for Differential Detection of Classical, Highly Pathogenic, and NADC30-like Porcine Reproductive and Respiratory Syndrome Virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) type 1 (European genotype) and PRRSV type 2 (North American genotype) are prevalent all over the world. Nowadays, the North American genotype PRRSV (NA-PRRSV) has been widely circulating in China and has caused huge economic losses to the pig industry. In recent years, classical PRRSV (C-PRRSV), highly pathogenic PRRSV (HP-PRRSV), and NADC30-like PRRSV (NL-PRRSV) have been the most common circulating strains in China. In order to accurately differentiate the circulating strains of NA-PRRSV, three pairs of specific primers and corresponding probes were designed for the Nsp2 region of C-PRRSV, HP-PRRSV, and NL-PRRSV. After optimizing the annealing temperature, primer concentration, and probe concentration, a multiplex real-time quantitative RT-PCR (qRT-PCR) and a multiplex Crystal digital RT-PCR (cdRT-PCR) for the differential detection of C-PRRSV, HP-PRRSV, and NL-PRRSV were developed. The results showed that the two assays illustrated high sensitivity, with a limit of detection (LOD) of 3.20 × 100 copies/µL for the multiplex qRT-PCR and 3.20 × 10-1 copies/µL for the multiplex cdRT-PCR. Both assays specifically detected the targeted viruses, without cross-reaction with other swine viruses, and indicated excellent repeatability, with coefficients of variation (CVs) of less than 1.26% for the multiplex qRT-PCR and 2.68% for the multiplex cdRT-PCR. Then, a total of 320 clinical samples were used to evaluate the application of these assays, and the positive rates of C-PRRSV, HP-PRRSV, and NL-PRRSV by the multiplex qRT-PCR were 1.88%, 21.56%, and 9.69%, respectively, while the positive rates by the multiplex cdRT-PCR were 2.19%, 25.31%, and 11.56%, respectively. The high sensitivity, strong specificity, excellent repeatability, and reliability of these assays indicate that they could provide useful tools for the simultaneous and differential detection of the circulating strains of C-PRRSV, HP-PRRSV, and NL-PRRSV in the field.

Clinical Comparative Evaluation of the LabTurboTM AIO® Reverse Transcription-Polymerase Chain Reaction and World Health Organization-Recommended Assays for the Detection of Emerging SARS-CoV-2 Variants of Concern.

PURPOSE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent behind coronavirus disease-2019 (COVID-19). Single-plex reverse transcription-polymerase chain reaction (RT-PCR)-based assays are widely used for COVID-19 detection but exhibit decreased sensitivity and specificity in detecting the rapidly spreading SARS-CoV-2 variants; in contrast, multiplex RT-PCR reportedly yields better results. Here, we aimed at comparatively analyzing the clinical performance of the LabTurboTM AIO COVID-19 RNA testing kit, a multiplex quantitative RT-PCR kit, including a three-target (E, N1, and RNase P), single-reaction, triplex assay used for SARS-CoV-2 detection, with that of the WHO-recommended RT-PCR assay. MATERIALS AND METHODS: Residual, natural, nasopharyngeal swabs obtained from universal transport medium specimens at SARS-CoV-2 testing centers (n = 414) were collected from May to October 2021. For SARS-CoV-2 qRT-PCR, total viral nucleic acid was extracted. The limit of detection (LOD) and the comparative clinical performances of the LabTurboTM AIO COVID-19 RNA kit and the WHO-recommended RT-PCR assay were assessed. Statistical analysis of the correlation was performed and results with R2 values >0.9 were considered to be highly correlated. RESULTS: The LOD of the LabTurboTM AIO COVID-19 RNA kit was 9.4 copies/reaction for the target genes N1 and E. The results obtained from 102 SARS-CoV-2-positive and 312 SARS-CoV-2-negative samples showed 100% correlation with previous WHO-recommended RT-PCR assay results. CONCLUSION: Multiplex qRT-PCR is a critical tool for detecting unknown pathogens and employs multiple target genes. The LabTurboTM AIO COVID-19 RNA testing kit provides an effective and efficient assay for SARS-CoV-2 detection and is highly compatible with SARS-CoV-2 variants.

A Quadruplex qRT-PCR for Differential Detection of Four Porcine Enteric Coronaviruses.

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and swine acute diarrhea syndrome coronavirus (SADS-CoV) are four identified porcine enteric coronaviruses. Pigs infected with these viruses show similar manifestations of diarrhea, vomiting, and dehydration. Here, a quadruplex real-time quantitative PCR (qRT-PCR) assay was established for the differential detection of PEDV, TGEV, PDCoV, and SADS-CoV from swine fecal samples. The assay showed extreme specificity, high sensitivity, and excellent reproducibility, with the limit of detection (LOD) of 121 copies/µL (final reaction concentration of 12.1 copies/µL) for each virus. The 3236 clinical fecal samples from Guangxi province in China collected between October 2020 and October 2022 were evaluated by the quadruplex qRT-PCR, and the positive rates of PEDV, TGEV, PDCoV, and SADS-CoV were 18.26% (591/3236), 0.46% (15/3236), 13.16% (426/3236), and 0.15% (5/3236), respectively. The samples were also evaluated by the multiplex qRT-PCR reported previously by other scientists, and the compliance rate between the two methods was more than 99%. This illustrated that the developed quadruplex qRT-PCR assay can provide an accurate method for the differential detection of four porcine enteric coronaviruses.

Development of a multiplex qRT-PCR assay for detection of African swine fever virus, classical swine fever virus and porcine reproductive and respiratory syndrome virus.

BACKGROUND: African swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV) are still prevalent in many regions of China. Co-infections make it difficult to distinguish their clinical symptoms and pathological changes. Therefore, a rapid and specific method is needed for the differential detection of these pathogens. OBJECTIVES: The aim of this study was to develop a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) for the simultaneous differential detection of ASFV, CSFV, and PRRSV. METHODS: Three pairs of primers and TaqMan probes targeting the ASFV p72 gene, CSFV 5' untranslated region, and PRRSV ORF7 gene were designed. After optimizing the reaction conditions, including the annealing temperature, primer concentration, and probe concentration, multiplex qRT-PCR for simultaneous and differential detection of ASFV, CSFV, and PRRSV was developed. Subsequently, 1,143 clinical samples were detected to verify the practicality of the assay. RESULTS: The multiplex qRT-PCR assay could specifically and simultaneously detect the ASFV, CSFV, and PRRSV with a detection limit of 1.78 × 100 copies for the ASFV, CSFV, and PRRSV, but could not amplify the other major porcine viruses, such as pseudorabies virus, porcine circovirus type 1 (PCV1), PCV2, PCV3, foot-and-mouth disease virus, porcine parvovirus, atypical porcine pestivirus, and Senecavirus A. The assay had good repeatability with coefficients of variation of intra- and inter-assay of less than 1.2%. Finally, the assay was used to detect 1,143 clinical samples to evaluate its practicality in the field. The positive rates of ASFV, CSFV, and PRRSV were 25.63%, 9.36%, and 17.50%, respectively. The co-infection rates of ASFV+CSFV, ASFV+PRRSV, CSFV+PRRSV, and ASFV+CSFV+PRRSV were 2.45%, 2.36%, 1.57%, and 0.17%, respectively. CONCLUSIONS: The multiplex qRT-PCR developed in this study could provide a rapid, sensitive, specific diagnostic tool for the simultaneous and differential detection of ASFV, CSFV, and PRRSV.

A Novel Multiplex qRT-PCR Assay to Detect SARS-CoV-2 Infection: High Sensitivity and Increased Testing Capacity.

Rapid and sensitive screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to limit the spread of the global pandemic we are facing. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is currently used for the clinical diagnosis of SARS-CoV-2 infection using nasopharyngeal swabs, tracheal aspirates, or bronchoalveolar lavage (BAL) samples. Despite the high sensitivity of the qRT-PCR method, false negative outcomes might occur, especially in patients with a low viral load. Here, we developed a multiplex qRT-PCR methodology for the simultaneous detection of SARS-CoV-2 genome (N gene) and of the human RNAse P gene as internal control. We found that multiplex qRT-PCR was effective in detecting SARS-Cov-2 infection in human specimens with 100% sensitivity. Notably, patients with few copies of SARS-CoV-2 RNA (<5 copies/reaction) were successfully detected by the novel multiplex qRT-PCR method. Finally, we assessed the efficacy of multiplex qRT-PCR on human nasopharyngeal swabs without RNA extraction. Collectively, our results provide evidence of a novel and reliable tool for SARS-CoV-2 RNA detection in human specimens, which allows the testing capacity to be expanded and the RNA extraction step to be bypassed.

Combination of microdissection and single cell quantitative real-time PCR revealed intercellular mitochondrial DNA heterogeneities in fibroblasts of Kearns-Sayre syndrome patients.

Kearns-Sayre syndrome (KSS) is a multisystemic disorder marked by aerobic cell metabolism dysfunction. Fibroblasts derived from KSS patient skin biopsy exhibit heterogeneous occurrence of mitochondrial genomes as those circular DNA molecules partially carry the common deletion. In our approach, we aim to evaluate the intercellular alterations in respect to mitochondrial DNA integrity by laser capture microdissection and multiplex quantitative real-time PCR in single cells. The obtained results give new insights into the understanding of mitochondrial genetics, e.g. postulated sorting of damaged mitochondria, and heterogeneity of cells. Further, we discuss the relevance of intercellular heterogeneities for human mitochondrial disorders in general.

Periodontopathogen levels following the use of an Er:YAG laser in the treatment of chronic periodontitis.

BACKGROUND: Inflammatory periodontal diseases are initiated by microbial biofilms. The reduction of the biofilm is important in the management of the disease. This study compares periodontopathogen levels following the treatment of chronic periodontitis using Er:YAG laser (ERL) debridement and mechanical scaling and root planing (SRP). METHODS: Using a split-mouth design, two quadrants were randomly allocated for treatment. Two hundred and fifty-two subgingival plaque samples were collected from 21 patients, before treatment (baseline) and at 6 and 12 weeks post-therapy. Multiplex qPCR was used to determine relative levels of Porphyromonas gingivalis (Pg), Treponema denticola (Td), Tannerella forsythensis (Tf), and Aggregatibacter actinomycetemcomitans (Aa). RESULTS: Tf and Pg were significantly reduced post-treatment for both ERL and SRP. ERL treatment resulted in a reduction of Td at 12 weeks. Following SRP treatment Aa was significantly reduced at 12 weeks. No statistically significant difference was seen when treatments were compared at 6 and 12 weeks. CONCLUSIONS: A comparable reduction in the level of the four periodontal pathogens assayed was achieved with Er:YAG laser debridement and mechanical scaling and root planing.

One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples.

Background. Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time. Methods. In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostable rTth polymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments. Results. The VP7 qRT-PCRs exhibited 98.8-100% sensitivity, 99.7-100% specificity, 85-95% efficiency and a limit of detection of 4-60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81-92% efficiency and limit of detection of 150-600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8-100% sensitivity, 100% specificity, 86-89% efficiency and a limit of detection of 12-400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82-90% efficiency and limit of detection of 120-4000 copies in multiplex reaction. Discussion. The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains.