Multiplex Real-time Reverse Transcription PCR Research Articles

Validation of a direct multiplex real-time reverse transcription PCR assay for rapid detection of African swine fever virus using swine field samples in Vietnam

ObjectiveThis study validates a direct multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay which was previously established for enabling rapid and simultaneous detection of African swine fever (ASF) virus (ASFV) and classical swine fever virus. The assay eliminates the need for viral nucleic acid purification using a buffer system for crude extraction and an impurity-tolerant enzyme. However, the assay had not yet been validated using field samples of ASFV-infected pigs. Therefore, to address this gap, we tested 101 samples collected from pigs in Vietnam during 2018 and 2021 for validation.ResultsThe rRT-PCR assay demonstrated a diagnostic sensitivity of 98.8% and a specificity of 100%. Remarkably, crude samples yielded results comparable to those of purified samples, indicating the feasibility of using crude samples without compromising accuracy in ASFV detection. Our findings emphasize the effectiveness of the rRT-PCR assay for the prompt and accurate diagnosis of both swine fever viruses, which is essential for effective disease prevention and control in swine populations.

Development and Clinical Application of a Molecular Assay for Four Common Porcine Enteroviruses.

Porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus-A (PoRVA) are the four main pathogens that cause viral diarrhea in pigs, and they often occur in mixed infections, which are difficult to distinguish only according to clinical symptoms. Here, we developed a multiplex TaqMan-probe-based real-time RT-PCR method for the simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA for the first time. The specific primers and probes were designed for the M protein gene of PEDV, N protein gene of TGEV, N protein gene of PDCoV, and VP7 protein gene of PoRVA, and corresponding recombinant plasmids were constructed. The method showed extreme specificity, high sensitivity, and excellent repeatability; the limit of detection (LOD) can reach as low as 2.18 × 102 copies/μL in multiplex real-time RT-PCR assay. A total of 97 clinical samples were used to compare the results of the conventional reverse transcription PCR (RT-PCR) and this multiplex real-time RT-PCR for PEDV, TGEV, PDCoV, and PoRVA detection, and the results were 100% consistent. Subsequently, five randomly selected clinical samples that tested positive were sent for DNA sequencing verification, and the sequencing results showed consistency with the detection results of the conventional RT-PCR and our developed method in this study. In summary, this study developed a multiplex real-time RT-PCR method for simultaneous detection of PEDV, TGEV, PDCoV, and PoRVA, and the results of this study can provide technical means for the differential diagnosis and epidemiological investigation of these four porcine viral diarrheic diseases.

Evaluating the efficiency of ELISA, monoplex and multiplex probe-based real-time reverse-transcription PCR assays in the detection of SARS-CoV-2 (COVID-19) and influenza A and B viruses: A cross-sectional study.

The current study aimed to evaluate the efficiency of Enzyme-linked immunosorbent assay (ELISA) assay and monoplex and multiplex real-time reverse-transcription PCR (rRT-PCR) in the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A and B viruses (Flu A and Flu B). The SARS-CoV-2 -specific IgG and IgM antibodies, as well as, Flu A (H1N1 and H3N2 serotypes) and Flu B virus antibodies were determined by ELISA assay. The one-step qRT-PCR method was used to detect the SARS-CoV-2 in nasopharyngeal swab samples. Furthermore, the presence of Flu A and B viruses was evaluated using probe-based RT-PCR. Simultaneous detection of SARS-CoV-2, Flu A and B viruses was performed by multiplex rRT-PCR assay. SARS CoV-2 IgM and IgG antibodies were detected in 33.3% and 58.3% of patients, respectively. In contrast, the SARS CoV-2 genome was detected in 50% of patients using the one-step monoplex RT-PCR assay. Flu A serotypes H1N1 and H3N2 were found in 16.7% and 8.3% of patients. Probe-based RT-PCR revealed that 39.3% of patients were positive for the Flu A virus. Multiplex rRT-PCR detect the SARS-CoV-2, Flu A, and Flu B in 50%, 39.3%, and 19% of samples, respectively. The sensitivity and specificity of multiplex rRT-PCR assay in comparison to monoplex RT-PCR were 100% and 55%, respectively. Coinfection with SARS-CoV-2, Flu A, and Flu B viruses was found in 9.5% of patients. Multiplex rRT-PCR can be used as a repaid, cost-effective and suitable tool for molecular surveillance of SARS-CoV-2 and Flu A/B viruses.

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Detection of Borrelia miyamotoi and Powassan Virus Lineage II (Deer Tick Virus) from Odocoileus virginianus Harvested Ixodes scapularis in Oklahoma

Odocoileus virginianus (white-tailed deer) is the primary host of adult Ixodes scapularis (deer tick). Most of the research into I. scapularis has been geographically restricted to the northeastern United States, with limited interest in Oklahoma until recently as the I. scapularis populations spread due to climate change. Ticks serve as a vector for pathogenic bacteria, protozoans, and viruses that pose a significant human health risk. To date, there has been limited research to determine what potential tick-borne pathogens are present in I. scapularis in central Oklahoma. Using a one-step multiplex real-time reverse transcription-PCR, I. scapularis collected from white-tailed deer was screened for Anaplasma phagocytophilum, Borrelia burgdorferi, Borrelia miyamotoi, Babesia microti, and deer tick virus (DTV). Ticks (n = 394) were pooled by gender and life stage into 117 samples. Three pooled samples were positive for B. miyamotoi and five pooled samples were positive for DTV. This represents a minimum infection rate of 0.8% and 1.2%, respectively. A. phagocytophilum, B. burgdorferi, and B. microti were not detected in any samples. This is the first report of B. miyamotoi and DTV detection in Oklahoma I. scapularis ticks. This demonstrates that I. scapularis pathogens are present in Oklahoma and that further surveillance of I. scapularis is warranted.

POCT Detection of 14 Respiratory Viruses Using Multiplex RT-PCR

Over the past 6 years, acute respiratory infections have constituted an average of more than 70,000 cases in South Korea. It results in a high mortality rate in infants and the elderly with weak immunity. There are several types of respiratory viruses that invade the human respiratory tract and cause infectious disease. Reverse transcription PCR (RT-PCR) is mainly used for respiratory virus detection owing to its high sensitivity and reproducibility. In response, a multiplex real-time RT-PCR (rRT-PCR) assay was developed for the detection of influenza A and B viruses, parainfluenza viruses 1–4 (PIV1-4), human metapneumovirus, adenovirus, human rhinovirus, respiratory syncytial virus (RSV), and SARS-CoV-2. Detection ability of RT-PCR assay was confirmed by applying it to a portable device capable of point-of-care testing (POCT). Amplicons were synthesized using primer pairs and probe sets designed for each target virus, and a standard curve was constructed to confirm the limit of detection. An experiment using nasopharyngeal swab samples was conducted to understand the field applicability of the rRT-PCR assay. Detection was confirmed in most samples. This study demonstrated that rapid and accurate detection results can be obtained using the multiplex rRT-PCR based POC test, and that it is possible to detect 14 types of respiratory viruses that are generally difficult to distinguish at the same time, enabling timely treatment. Furthermore, we expect that the portable PCR device can significantly reduce the processing procedure of clinical samples before testing, which is the main disadvantage of common RT-PCR tests and can help reduce costs.

Single-Amplicon Multiplex Real-Time Reverse Transcription-PCR with Tiled Probes To Detect SARS-CoV-2 spike Mutations Associated with Variants of Concern.

ABSTRACTTo provide an accessible and inexpensive method to surveil for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutations, we developed a multiplex real-time reverse transcription-PCR (rRT-PCR) assay, the Spike single-nucleotide polymorphism (SNP) assay, to detect specific mutations in the spike receptor binding domain. A single primer pair was designed to amplify a 348-bp region of spike, and probes were initially designed to detect K417, E484K, and N501Y. The assay was evaluated using characterized variant sample pools and residual nasopharyngeal samples. Variant calls were confirmed by SARS-CoV-2 genome sequencing in a subset of samples. Subsequently, a fourth probe was designed to detect L452R. The lower limit of 95% detection was 2.46 to 2.48 log10 genome equivalents (GE)/ml for the three initial targets (∼1 to 2 GE/reaction). Among 253 residual nasopharyngeal swabs with detectable SARS-CoV-2 RNA, the Spike SNP assay was positive in 238 (94.1%) samples. All 220 samples with threshold cycle (CT) values of <30 for the SARS-CoV-2 N2 target were detected, whereas 18/33 samples with N2 CT values of ≥30 were detected. Spike SNP results were confirmed by sequencing in 50/50 samples (100%). Addition of the 452R probe did not affect performance for the original targets. The Spike SNP assay accurately identifies SARS-CoV-2 mutations in the receptor binding domain, and it can be quickly modified to detect new mutations that emerge.

A Clinical Epidemiology and Molecular Attribution Evaluation of Adenoviruses in Pediatric Acute Gastroenteritis: a Case-Control Study

The objective of this study was to characterize the etiological role of human adenovirus (HAdV) serotypes in pediatric gastroenteritis. Using a case-control design, we compared the frequencies of HAdV serotypes between children with ≥3 episodes of vomiting or diarrhea within 24 h and <7 days of symptoms (i.e., cases) and those with no infectious symptoms (i.e., controls). Stool samples and/or rectal swabs underwent molecular serotyping with cycle threshold (Ct) values provided by multiplex real-time reverse transcription-PCR testing. Cases without respiratory symptoms were analyzed to calculate the proportion of disease attributed to individual HAdV serotypes (i.e., attributable fraction). Between December 2014 and August 2018, adenoviruses were detected in 18.8% (629/3,347) of cases and 7.2% (97/1,355) of controls, a difference of 11.6% (95% confidence interval [CI], 9.6%, 13.5%). In 96% (95% CI, 92 to 98%) of HAdV F40/41 detections, the symptoms could be attributed to the identified serotype; when serotypes C1, C2, C5, and C6 were detected, they were responsible for symptoms in 52% (95% CI, 12 to 73%). Ct values were lower among cases than among controls (P < 0.001). HAdV F40/41, C2, and C1 accounted for 59.7% (279/467), 17.6% (82/467), and 12.0% (56/467) of all typed cases, respectively. Among cases, Ct values were lower for F40/41 serotypes than for non-F40/41 serotypes (P < 0.001). HAdV F40/41 serotypes account for the majority of HAdV-positive gastroenteritis cases, and when detected, disease is almost always attributed to infection with these pathogens. Non-F40/41 HAdV species have a higher frequency of asymptomatic infection and may not necessarily explain gastroenteritis symptoms. Real-time quantitative PCR may be useful in differentiating asymptomatic shedding from active infection.

Simultaneous Detection and Differentiation between Wild-Type and Vaccine Measles Viruses by a Multiplex Real-Time Reverse Transcription-PCR Assay.

Measles is one of the most contagious viral respiratory infections and was declared to be eliminated from Canada in 1998; however, measles cases and outbreaks still occur every year through reintroduction from other parts of the world. Laboratory confirmation of measles virus (MV) RNA by real-time PCR provides a definitive diagnosis, and molecular analysis to determine the genotype is the only way to distinguish between wild-type and vaccine strains. This distinction is important since live attenuated vaccine strains are able to replicate in the patient and can be associated with rash and fever but are poorly transmissible, if at all. Prompt reporting of measles cases to local authorities, including differentiation between wild-type and vaccine strains, allows for optimal management and contact tracing. The development and validation of a multiplex real-time reverse transcription-PCR (rtRT-PCR) assay for the simultaneous detection and differentiation of the Moraten and Schwarz vaccine strains from presumptive wild-type MV in a format that can be easily implemented for high-throughput testing of patient samples are reported here. This assay is sensitive, specific, reproducible, and 100% accurate in comparison with the gold standard comparator assay.

Molecular-Genetic Characterization of Human Rotavirus A Strains Circulating in Moscow, Russia (2009-2014).

Enteric viruses are the most common cause of acute gastroenteritis (AGE) in young children and a significant public health problem globally. Hospital admissions of children under 5years of age with diarrhea are primarily associated with group A rotavirus (RVA) infection. In this retrospective study, the population structure of viruses linked to AGE etiology in young children hospitalized with AGE in Moscow was evaluated, and molecular characterization of RVA strains was performed. Fecal specimens were collected from children under 5years old hospitalized with AGE between 2009 and 2014 in Moscow, Russia. Multiplex real-time reverse transcription PCR was used to detect enteric viruses and for G/[P]-genotyping of isolated RVAs. Sequencing of RVA VP7 and VP4 cDNA fragments was used to validate the data obtained by PCR-genotyping. The main causes for hospitalization of children with AGE were RVA (40.1%), followed by noroviruses (11.4%), while adenoviruses, astroviruses, sapoviruses, enteroviruses, and orthoreoviruses were detected in 4.7%, 1.9%, 1.4%, 1.2%, and 0.2% of samples tested, respectively. Nosocomial infections, predominantly associated with RVAs and noroviruses, were detected in 24.8% of cases and occurred significantly more frequently in younger infants. The predominant RVA genotype was G4P[8], detected in 38.7% of RVA-positive cases, whereas genotypes G1P[8], G9P[8], G3P[8], and G2P[4] were found in 11.8%, 6.6%, 4.2%, and 3.3% of cases, respectively. Together, the presence of circulating RVA strains with rare VP7 and VP4 gene variants (G6 and P[9]) highlights the need to conduct continuous epidemiological monitoring of RVA infection.

Human Coronavirus NL63 Molecular Epidemiology and Evolutionary Patterns in Rural Coastal Kenya.

BackgroundHuman coronavirus NL63 (HCoV-NL63) is a globally endemic pathogen causing mild and severe respiratory tract infections with reinfections occurring repeatedly throughout a lifetime.MethodsNasal samples were collected in coastal Kenya through community-based and hospital-based surveillance. HCoV-NL63 was detected with multiplex real-time reverse transcription PCR, and positive samples were targeted for nucleotide sequencing of the spike (S) protein. Additionally, paired samples from 25 individuals with evidence of repeat HCoV-NL63 infection were selected for whole-genome virus sequencing.ResultsHCoV-NL63 was detected in 1.3% (75/5573) of child pneumonia admissions. Two HCoV-NL63 genotypes circulated in Kilifi between 2008 and 2014. Full genome sequences formed a monophyletic clade closely related to contemporary HCoV-NL63 from other global locations. An unexpected pattern of repeat infections was observed with some individuals showing higher viral titers during their second infection. Similar patterns for 2 other endemic coronaviruses, HCoV-229E and HCoV-OC43, were observed. Repeat infections by HCoV-NL63 were not accompanied by detectable genotype switching.ConclusionsIn this coastal Kenya setting, HCoV-NL63 exhibited low prevalence in hospital pediatric pneumonia admissions. Clade persistence with low genetic diversity suggest limited immune selection, and absence of detectable clade switching in reinfections indicates initial exposure was insufficient to elicit a protective immune response.

Avian Metapneumovirus circulation in Italian broiler farms

With increasing frequency, avian Metapneumovirus (aMPV) is reported to induce respiratory signs in chickens. An adequate knowledge of current aMPV prevalence among Italian broilers is lacking, with little information available on its economical and health impact on the poultry industry. In order to collect preliminary data on the epidemiological context of aMPV in broiler flocks, a survey was performed in areas of Northern Italy with high poultry density from 2014 to 2016. Upper respiratory tract swabs were collected and processed by A and B subtype-specific multiplex real-time reverse transcription PCR (RT-PCR). Samples were also screened for infectious bronchitis virus (IBV) by generic RT-PCR and sequencing. Productive data and respiratory signs were detailed where possible. The high prevalence of aMPV was confirmed in broilers older than 26 d and also attested in IBV-negative farms. All aMPV detections belonged to subtype B. Italian strain genetic variability was evaluated by the partial attachment (G) gene sequencing of selected strains and compared with contemporary turkey strains and previously published aMPV references, revealing no host specificity and the progressive evolution of this virus in Italy.

A multiplex real-time RT-PCR for simultaneous detection of four most common avian respiratory viruses

A one-step multiplex real-time reverse transcription-PCR (rRT-PCR) assay was developed for simultaneous detection and quantification of four avian respiratory viruses: avian influenza virus (AIV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV) and infectious laryngotracheitis virus (ILTV). In comparison with the singleplex rRT-PCR, the specificity, the sensitivity and the reproducibility of the new assay were evaluated and validated using 70 clinical samples. The optimal cutoff point, the corresponding limit of quantification (LoQ) and the limit of detection (LoD) were statistical established based on receiver operating characteristic (ROC) curve analysis. The results showed that the multiplex assay presents higher sensitivity and specificity. Correlation coefficients (R2) and amplification efficiencies (E) of all singleplex and multiplex rRT-PCR reactions are within the acceptable range. The 95% LoDs of multiplex assay were in the range [3–19] copies genomic/ µl, and its corresponding cutoff cycles were in the range [34.16–36.59]. No competitive inhibition for the detection of the four targets and no specific amplification or cross reactivity with other tested viruses was observed. Excellent results were attained in the inter-assay and intra-assay reproducibility evaluation. All identified samples by the multiplex rRT-PCR assay proved to be 100% concordant with the results of the singleplex assays. The results achieved showed that the multiplex assay is very suitable as a routine laboratory test for rapid and specific detection and quantification of co-infections in field samples.

Evaluation of 793/B-like and Mass-like vaccine strain kinetics in experimental and field conditions by real-time RT-PCR quantification.

Infectious bronchitis virus (IBV) is a great economic burden both for productive losses and costs of the control strategies. Many different vaccination protocols are applied in the same region and even in consecutive cycles on the same farm in order to find the perfect balance between costs and benefits. In Northern Italy, the usual second vaccination is more and more often moved up to the chick's first d of life. The second strain administration together with the common Mass priming by spray at the hatchery allows saving money and time and reducing animal stress. The present work compared the different vaccine strains (Mass-like or B48, and 1/96) kinetics both in field conditions and in a 21-day-long experimental trial in broilers, monitoring the viral replication by upper respiratory tract swabbing and vaccine specific real time reverse transcription PCR (RT-PCR) quantification. In both field and experimental conditions, titers for all the vaccines showed an increasing trend in the first 2 wk and then a decrease, though still remaining detectable during the whole monitored period. IBV field strain and avian Metapneumovirus (aMPV) presence also was also investigated by RT-PCR and sequencing, and by multiplex real-time RT-PCR, respectively, revealing a consistency in the pathogen introduction timing at around 30 d, in correspondence with the vaccine titer's main decrease. These findings suggest the need for an accurate knowledge of live vaccine kinetics, whose replication can compete with the other pathogen one, providing additional protection to be added to what is conferred by the adaptive immune response.

Development of a Multiplex Real-Time RT-PCR Assay for Detection of Human Parainfluenza Viruses in Patients With Respiratory Symptoms

Aim: The human parainfluenza viruses (HPIVs) are common viral causes of both upper and lower respiratory tract infections in infants and children. The purpose of this study was to develop a real-time reverse transcription PCR assay to detect HPIVs in clinical specimens in a single test. Specimens: Specific primers and probes matched to conserved region of hemagglutinin–neuraminidase genes were designed. Clinical specimens were collected from hospitalized children of ≤5 years old. Results &amp; conclusion: Of 230 specimens, 48 samples contained one of HPIV types 1–4. The most percentage of positive HPIVs belonged to HPIV2. HPIVs were found in fall, winter and spring but not in summer months. In conclusion, our in-house multiplex real-time reverse transcription PCR is a suitable technique for simultaneous detection of HPIVs in clinical samples.

Single-Reaction Multiplex Reverse Transcription PCR for Detection of Zika, Chikungunya, and Dengue Viruses.

Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses.

Comparison of the FDA-Approved CDC DENV-1-4 Real-Time Reverse Transcription-PCR with a Laboratory-Developed Assay for Dengue Virus Detection and Serotyping

Dengue virus (DENV) is the agent of the most common vector-borne disease worldwide. Using 199 clinical samples collected from Nicaragua and Sri Lanka, a laboratory-developed DENV multiplex real-time reverse transcription-PCR (rRT-PCR) proved more clinically sensitive than the FDA-approved CDC assay for DENV serotypes 1 to 4 when measured against a composite reference standard, with sensitivities of 97.4% versus 87.1%, respectively.

A one-step multiplex real-time RT-PCR assay for rapid and simultaneous detection of human norovirus genogroup I, II and IV

A one-step multiplex real-time reverse transcription-PCR (RT-PCR) assay was developed for one-tube and simultaneous detection of three genogroups of human norovirus, genogroup I, II and IV (GI, GII and GIV). The specificity and sensitivity of the assay were evaluated and 50 samples were tested by using this assay. The results showed that the multiplex assay had high sensitivity and specificity. The amplification efficiencies of the assay were 91.3%, 90.1%, 88.9% and the detection limits were up to 16.9, 6.3, 43.0 copies/reaction respectively for norovirus GI, GII and GIV detection. No cross-reaction with the other examined RNA viruses was observed, and the qualitative analysis of samples showed that the multiplex assay had a good consistency with its corresponding monoplex assays for the detection of norovirus GI, GII and GIV (Kappa values were 0.848, 0.876 and 0.812 respectively).

Comparison of a laboratory-developed RT-PCR and the CDC RT-PCR protocol with rapid immunodiagnostic testing during the 2009 H1N1 influenza A pandemic

We evaluated the performance of a laboratory-developed multiplex real-time reverse transcription-PCR assay (LDT rRT-PCR), the Centers for Disease Control and Prevention (CDC) 2009 H1N1 rRT-PCR protocol using the LightCycler 480 II, the multiplex xTAG Respiratory Virus Panel (xTAG RVP), and rapid immunodiagnostic testing (RIDT) using the BinaxNOW Influenza A & B to detect 2009 H1N1 with 426 nasopharyngeal swab specimens during the 2009 H1N1 pandemic. The specificity of the methods tested was ≥98%, and the individual test sensitivities were RIDT at 42.3% [95% confidence interval (CI), 31.4–54.0], LDT rRT-PCR at 98.9% (95% CI, 92.9–99.9), CDC 2009 H1N1 rRT-PCR at 78.2% (95% CI, 67.8–86.0), and xTAG RVP at 93.1% (95% CI, 85.0–97.2). A negative RIDT result should not be used to make decisions with respect to treatment or infection prevention. rRT-PCR is the preferred first-line diagnostic test for detecting 2009 H1N1 influenza A.

Laboratory Diagnosis, Epidemiology, and Clinical Outcomes of Pandemic Influenza A and Community Respiratory Viral Infections in Southern Brazil

Community respiratory viruses (CRVs) are commonly associated with seasonal infections. They have been associated with higher morbidity and mortality among children, elderly individuals, and immunosuppressed patients. In April 2009, the circulation of a new influenza A virus (FLUA H1N1v) was responsible for the first influenza pandemic of this century. We report the clinical and epidemiological profiles of inpatients infected with CRVs or with FLUA H1N1v at a tertiary care hospital in southern Brazil. In addition, we used these profiles to evaluate survivor and nonsurvivor patients infected with FLUA H1N1v. Multiplex reverse transcription-PCR (RT-PCR) and real time RT-PCR were used to detect viruses in inpatients with respiratory infections. Record data from all patients were reviewed. A total of 171 patients were examined over a period of 16 weeks. Of these, 39% were positive for FLUA H1N1v, 36% were positive for CRVs, and 25% were negative. For the FLUA H1N1v- and CRV-infected patients, epidemiological data regarding median age (30 and 1.5 years), myalgia (44% and 13%), need for mechanical ventilation (44% and 9%), and mortality (35% and 9%) were statistically different. In a multivariate analysis comparing survivor and nonsurvivor patients infected with influenza A virus H1N1, median age and creatine phosphokinase levels were significantly associated with a severe outcome. Seasonal respiratory infections are a continuing concern. Our results highlight the importance of studies on the prevalence and severity of these infections and that investments in programs of clinical and laboratory monitoring are essential to detect the appearance of new infective agents.