Duplex Real-time RT-PCR Assay Research Articles

Development of a duplex real-time RT-PCR assay for the detection and identification of two subgroups of human metapneumovirus in a single tube

Human metapneumovirus (hMPV) is a common cause of respiratory infections in children. Many genetic diagnostic assays have been developed, but most detect hMPV regardless of the subgroup. In this study, we developed a real-time RT-PCR assay that can detect and identify the two major subgroups of hMPV (A and B) in one tube. Primers and probes were designed based on the sequences of recent clinical isolates in Japan. The assay showed comparable analytical sensitivity to a previously reported real-time RT-PCR assay and specific reactions to hMPV subgroups. The assay also showed no cross-reactivity to clinical isolates of 19 species of other respiratory viruses. In a validation assay using post-diagnosed clinical specimens, 98% (167/170) positivity was confirmed for the duplex assay, and the three specimens not detected were of low copy number. The duplex assay also successfully distinguished the two major subgroups for all 12 clinical specimens, for which the subgroup had already been determined by genomic sequencing analysis. The duplex assay described here will contribute to the rapid and accurate identification and surveillance of hMPV infections.

An Improved Duplex Real-Time Quantitative RT-PCR Assay with a Canine Endogenous Internal Positive Control for More Sensitive and Reliable Detection of Canine Parainfluenza Virus 5.

A duplex real-time quantitative reverse transcription-polymerase chain reaction (dqRT-PCR) assay was successfully developed to simultaneously detect canine parainfluenza virus 5 (CPIV5) and a canine endogenous internal positive control (EIPC) in canine clinical samples. Two sets of primers and probes for the CPIV5 L and canine 16S rRNA genes were included in the dqRT-PCR assay to detect CPIV and monitor invalid results throughout the qRT-PCR process. The developed dqRT-PCR assay specifically detected CPIV5 but no other canine pathogens. Furthermore, 16S rRNA was stably amplified by dqRT-PCR assay in all samples containing canine cellular materials. The assay's sensitivity was determined as below ten RNA copies per reaction, with CPIV5 L gene standard RNA and 1 TCID50/mL with the CPIV5 D008 vaccine strain, which was 10-fold higher than that of the previous HN gene-specific qRT-PCR (HN-qRT-PCR) assays and was equivalent to that of the previous N gene-specific qRT-PCR (N-qRT-PCR) assays, respectively. Moreover, the Ct values of the CPIV5-positive samples obtained using the dqRT-PCR assay were lower than those obtained using the previous HN- and N-qRT-PCR assays, indicating that the diagnostic performance of the dqRT-PCR assay was superior to those of previous HN- and N-qRT-PCR assays. The calculated Cohen's kappa coefficient values (95% confidence interval) between dqRT-PCR and the HN- or N-specific qRT-PCR assays were 0.97 (0.90-1.03) or 1.00 (1.00-1.00), respectively. In conclusion, the newly developed dqRT-PCR assay with high sensitivity, specificity, and reliability will be a promising diagnostic tool for the detection of CPIV5 in clinical samples and useful for etiological and epidemiological studies of CPIV5 infection in dogs.

Detection and Genomic Characterization of Bovine Rhinitis Virus in China.

Bovine rhinitis virus (BRV) is an etiological agent of bovine respiratory disease complex (BRDC) and can be divided into two genotypes-bovine rhinitis A virus (BRAV) and bovine rhinitis B virus (BRBV). However, knowledge about the prevalence and molecular information of BRV in China is still limited. In this study, 163 deep nasal swabs collected from bovines with BRDC syndrome on 16 farms across nine provinces of China were tested for BRAV and BRBV by a duplex real-time RT-PCR assay. The results showed that 28.22% (46/163) of the samples were BRV-positive, and the positive rates were 22.09% (36/163) for BRAV and 9.2% (15/163) for BRBV. The co-circulation of both BRV genotypes was observed on two farms. Furthermore, five near-complete BRV genomes, including three BRAVs and two BRBVs, were obtained. The phylogenetic analysis showed that the three obtained BRAVs were phylogenetically independent, while the two BRBVs exhibited significant genetic heterogeneity. Recombination analysis revealed that three BRAVs and one BRBV strain obtained in this study were recombinants. The present study confirmed the presence and prevalence of BRAV in China, and it found that both types of BRV are circulating in beef cattle, which contributes to a better understanding of the prevalence and molecular characteristics of BRV.

New RT-PCR Assay for the Detection of Current and Future SARS-CoV-2 Variants.

Multiple lineages of SARS-CoV-2 have been identified featuring distinct sets of genetic changes that confer to the virus higher transmissibility and ability to evade existing immunity. The continuous evolution of SARS-CoV-2 may pose challenges for current treatment options and diagnostic tools. In this study, we have first evaluated the performance of the 14 WHO-recommended real-time reverse transcription (RT)-PCR assays currently in use for the detection of SARS-CoV-2 and found that only one assay has reduced performance against Omicron. We then developed a new duplex real-time RT-PCR assay based on the amplification of two ultra-conserved elements present within the SARS-CoV-2 genome. The new duplex assay successfully detects all of the tested SARS-CoV-2 variants of concern (including Omicron sub-lineages BA.4 and BA.5) from both clinical and wastewater samples with high sensitivity and specificity. The assay also functions as a one-step droplet digital RT-PCR assay. This new assay, in addition to clinical testing, could be adopted in surveillance programs for the routine monitoring of SARS-CoV-2's presence in a population in wastewater samples. Positive results with our assay in conjunction with negative results from an Omicron-specific assay may provide timely indication of the emergence of a novel SARS-CoV-2 variant in a certain community and thereby aid public health interventions.

Rapid detection of human respiratory syncytial virus A and B by duplex real-time RT-PCR

Respiratory syncytial virus (RSV) is a common cause of acute respiratory disease worldwide, especially in young children. The World Health Organization (WHO) has initiated an RSV Surveillance Pilot program that aims to perform worldwide RSV surveillance, requiring the development of reliable and rapid molecular methods to detect and identify RSV. A duplex real-time RT-PCR assay developed for simultaneous detection of both A and B subtypes of RSV was included as part of this program. This duplex assay targeted a conserved region of the RSV polymerase gene and was validated for analytical sensitivity, specificity, reproducibility and clinical performance with a wide range of respiratory specimens. The assay was highly specific for RSV and did not react with non-RSV respiratory pathogens, including the SARS-CoV-2 virus.

Duplex real-time RT-PCR assay for detection and subgroup-specific identification of human respiratory syncytial virus

Human respiratory syncytial virus (HRSV) is a leading cause of acute respiratory illness in young children worldwide. Reliable detection and identification of HRSV subgroup A and B infections are essential for accurate disease burden estimates in anticipation of licensure of novel HRSV vaccines and immunotherapies. To ensure continued reliability, molecular assays must remain current with evolving virus strains. We have developed a HRSV subgroup-specific real-time RT-PCR (rRT-PCR) assay for detection and subgroup identification using primers and subgroup-specific probes targeting a conserved region of the nucleoprotein gene combined in a single duplex reaction using all genome sequence data currently available in GenBank. The assay was validated for analytical sensitivity, specificity, reproducibility, and clinical performance with a geographically diverse collection of viral isolates and respiratory specimens in direct comparison with an established pan-HRSV rRT-PCR reference test. The assay was sensitive, reproducibly detecting as few as 5–10 copies/reaction of target RNA. The assay was specific, showing no amplification with a panel of 16 other common respiratory pathogens or predicted by in silico primer/probe analysis. The duplex rRT-PCR assay based on the most current available genome sequence data permits rapid, sensitive and specific detection and subgroup identification of HRSV.

Development and validation of a real-time, reverse transcription PCR assay for rapid and low-cost genotyping of hepatitis C virus genotypes 1a, 1b, 2, and 3a

Hepatitis C virus (HCV) infection affects millions of people and leads to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Treatment regimen selection requires HCV genotype (Gt) and Gt 1 subtype determination. Use of a laboratory developed, reverse transcription (RT)-PCR assay was explored as a low-cost, high-throughput screening approach for the major HCV genotypes and subtypes in North America. A commercial line probe assay (LiPA) was used for comparison. Sequencing and/or an alternative PCR assay were used for discordant analyses. Testing of 155 clinical samples revealed that a paired, duplex real-time RT-PCR assay that targets Gts 1a and 3a in one reaction and Gts 1b and 2 in another had 95% overall sensitivity and individual Gt sensitivity and specificity of 98–100% and 85–98%, respectively. The RT-PCR assay detected mixed HCV Gts in clinical and spiked samples and no false-positive reactions occurred with rare Gts 3b, 4, 5, or 6. Implementation of the RT-PCR assay, with some reflex LiPA testing, would cost only a small portion of the cost of using LiPA alone, and can also save 1.5h of hands-on time. The use of a laboratory developed RT-PCR assay for HCV genotyping has the potential to reduce cost and labour burdens in high-volume testing settings.

Duplex realtime RT-PCR assay for the detection of Potato spindle tuber viroid (PSTVd) along with ef 1-α gene of potato

SYBR green based realtime RT-PCR assay coupled with melt curve analysis was developed for the detection of Potato spindle tuber viroid (PSTVd) along with and without internal control from potato. The amplification of the specific targets was identified by their melting points viz., 85.93±0.22, 85.62±0.34 and 82.07±0.23 for primer pairs PSTVd-1F/PSTVd-1R, PSTVd-NFP/PSTVd-NRP and PSTVd-QFP1/PSTVd-QRP1, respectively. The realtime RT-PCR assay was 1×104 times more sensitive than RT-PCR assay and the assay could detect up to 20 copies of the target using serially diluted plasmid and up to 0.025 fg of total RNA from infected tissues diluted in healthy plant RNA. Duplex realtime RT-PCR assay was also standardized to detect PSTVd along with elongation factor 1- α (ef-1α) gene, a stable housekeeping gene of potato. Duplex realtime RT-PCR assay showed two melt peaks indicating the successful amplification both the PSTVd RNA and internal control RNA. The developed assays could consistently detect PSTVd and proved to be highly sensitive and rapid for the detection of PSTVd in post entry quarantine testing.

Development and evaluation of a duplex real-time RT-PCR for detection and differentiation of virulent and variant strains of porcine epidemic diarrhea viruses from the United States

Porcine epidemic diarrhea virus (PEDV) has caused significant economic losses in the US swine industry since May 2013. A new variant strain of PEDV emerged in the US in the late December, 2013. This variant strain of PEDV differs from the virulent strain of PEDV currently circulating in the US in 1170nt of the 5’end of the S1 domain in the spike gene. Importantly, the variant PEDV caused significantly less mortality in piglets than the virulent PEDV, based on clinical observations. This suggests it may be a potential vaccine candidate for PED. Variant PEDV has been detected in samples from multiple states by our laboratory as well as other laboratories in the US. It is critical to detect and differentiate variant PEDV from the virulent PEDV during outbreaks to enhance control and to prevent PED associated disease. In this study, the development and validation of a duplex real-time RT-PCR assay for detection and differentiation of the variant and the virulent strains of PEDV currently circulating in the US was reported.

Development of duplex real‐time RT‐PCR based on Taqman technology for detecting simultaneously the genome of pan‐enterovirus and enterovirus 71

Human enterovirus (EV) 71 is the main etiological agent of hand, foot, and mouth disease (HFMD). It is associated with neurological complications, and caused fatalities during recent outbreaks in the Asia-Pacific region. Infections caused by EV71 could lead to many complications, ranging from brainstem encephalitis to pulmonary oedema, resulting in high mortality. In this study, a duplex real-time RT-PCR assay was developed in order to simultaneously detect pan-EV and EV71. EV71-specific primers and probes were designed based on the highly conserved VP1 region of EV71. Five EV71 strains were detected as positive, and no positive fluorescence signal was observed in the duplex real-time RT-PCR for other viral RNA, which showed 100% specificity for the selected panel, and no cross-reactions were observed in this duplex real-time RT-PCR. The EV71-specific duplex real-time RT-PCR was more sensitive than conventional RT-PCR, and detected viral titers that were 10-fold lower than those measured by the latter. Of the 381 HFMD clinical specimens, 196 (51.4%) cases were pan-EV-positive, of which 170 (86.7%) were EV71-positive when tested by pan-EV and EV71-specific duplex real-time RT-PCR. EV71-specific duplex real-time RT-PCR offers a rapid and sensitive method to detect EV71 from clinical specimens, and will allow quarantine measures to be taken more effectively during outbreaks.

Development of a New Duplex Real-Time Polymerase Chain Reaction Assay for Detection of Dicer in G. gallus

Recently, there has been a growing body of evidence showing that cellular microRNAs (miRNAs) are involved in virus-host interactions. Numerous studies have focused on analyses of the expression profiles of cellular miRNAs, but the expression patterns of Dicer, which is responsible for the generation of miRNAs, have only rarely been explored in Gallus gallus. We developed a duplex realtime reverse transcriptase polymerase chain reaction (RTPCR) assay for the relative quantification of the mRNAs of Dicer and beta-actin in G. gallus. To apply this method, the expression of Dicer in avian cells after infection with avian leukosis virus subgroup J (ALV-J) was detected using our established duplex real-time RT-PCR. The duplex realtime RT-PCR assay is sufficiently sensitive, specific, accurate, reproducible, and cost-effective for the detection of Dicer in G. gallus. Furthermore, this study, for the first time, demonstrated that ALV-J can induce differential expression of Dicer mRNA in the ALV-J-infected cells.

A novel duplex real-time PCR for HPIV-4 detects co-circulation of both viral subtypes among ill children during 2008

The two subtypes of the human parainfluenzavirus type 4 (HPIV-4) are rarely sought in testing for acute respiratory illness (ARI) and this may be confounding our understanding of its role. This study presents a novel duplex real-time RT-PCR assay targeting the P gene that can detect and differentiate the two subtypes in a single reaction. Subtype-specific synthetic RNA positive controls were prepared and used to determine an analytical sensitivity of 10 copies per reaction with an 8log10 dynamic range. The assays were validated using 1140 clinical specimens mostly nasopharyngeal aspirates collected from children during 2008. These included specimens previously determined to be positive for all commonly considered respiratory viruses. The novel assay did not cross-reaction with any other virus. Fourteen HPIV-4 positives, ten detected in the absence of any co-detections (four with rhinovirus), were identified in 2008 and their subtype confirmed by conventional RT-PCR and sequencing of P gene fragments. Most detections were in children two years of age or younger. Our assay proved suitably sensitive and specific for inclusion in future studies seeking to better understand the role HPIV-4 and other respiratory viruses in children with ARI.

A duplex real-time RT-PCR assay for the simultaneous genogroup-specific detection of noroviruses in both clinical and environmental specimens

Norovirus (NoV) is the major etiological agent causing foodborne and waterborne outbreaks worldwide. We developed a novel duplex real-time quantitative RT-PCR assay designed for the simultaneous detection of and discrimination between NoV genogroups GI and GII, by targeting the short junction region between ORF1 and ORF2, with sensitivity and efficiency comparable to those of each simplex RT-PCR assay. This new duplex assay was evaluated against clinical stool (n = 82) and environmental (groundwater or surface water, n = 60) specimens from South Korea, and the results were compared with those of conventional RT-PCR (cRT-PCR) assays. The duplex assay detected more positive samples than did the cRT-PCR for both clinical (74 vs. 71) and, more strikingly, environmental (24 vs. 10) specimens. No cross-reactivity against specimens containing other enteric viruses such as rotavirus, adenovirus, and poliovirus were observed. These results suggest that this newly developed duplex real-time RT-PCR assay can be used for the sensitive and simultaneous genogroup-specific detection of NoV in both clinical and environmental specimens.

Detection of human metapneumovirus and respiratory syncytial virus by duplex real-time RT-PCR assay in comparison with direct fluorescent assay

Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are important respiratory pathogens of small children and adults. The present study aimed to design a sensitive real-time RT-PCR assay for the detection of hRSV and hMPV in comparison with direct fluorescent assay (DFA) and to determine the incidence of hMPV and hRSV as causative agents of respiratory infections in a Finnish population. For DFA detection of hMPV antigen, four commercial antibodies were evaluated. The duplex real-time RT-PCR assay achieved a sensitivity of 103 copies/mL of specimen for hRSV and hMPV type A viruses and 104 copies/mL for type B hMPV. The detection rate of the RT-PCR assay was compared with those for DFA detection of hMPV and hRSV in analyses of 350 nasopharyngeal aspirates sent to HUSLAB, Helsinki University Hospital, for routine virus diagnostics during November 2007 to June 2008. Of the samples analyzed, 43 (12.3%) were positive for hRSV by DFA and an additional 13 specimens (3.7%) were positive for hRSV by RT-PCR. Only four samples (1.1 %) were found to be positive for hMPV RNA by RT-PCR, with two of them also positive by DFA. The duplex real-time RT-PCR assay described in the present study can therefore be applied for efficient identification of hMPV and hRSV in clinical specimens and collection of information on the epidemiology and clinical outcome of these viruses.

Rapid differential detection of classical and highly pathogenic North American Porcine Reproductive and Respiratory Syndrome virus in China by a duplex real-time RT-PCR

Since the emergence of highly pathogenic North American Porcine Reproductive and Respiratory Syndrome virus (H-US-PRRSV) in 2006, the classical North American PRRSV (C-US-PRRSV) and H-US-PRRSV isolates have coexisted in Chinese swine herds. A duplex real-time RT-PCR assay using minor groove binder (MGB) probes for differential detection of the two US PRRSV isolates was developed. The specificity, sensitivity, reproducibility, and interference test of this assay were validated. The sensitivity of the assay was 3.2 TCID 50/ml or 38 RNA copies/μl for C-US-PRRSV and 0.4 TCID 50/ml or 14 RNA copies/μl for H-US-PRRSV. Both assays were 10 times more sensitive than the current methods. A total of 302 clinical samples were tested by duplex real-time RT-PCR and conventional RT-PCR assays, and the results showed over 98.7% agreement. In addition, the new assay can be completed in less than 2 h. This duplex real-time RT-PCR assay is a promising tool for rapid differential detection and epidemiology of US PRRSV in China.

Development of a real-time RT-PCR assay for improved detection of Borna disease virus

Borna disease virus (BDV) is a non-segmented, negative-stranded RNA virus, which infects cells of the central nervous system (CNS) in many different species. BDV is the causative agent of the neurological disorders in horses and sheep termed classical Borna disease (BD), as well as staggering disease in cats. At present, the diagnosis staggering disease or feline BD is made by histopathology or immunohistochemistry of the CNS. In order to obtain a better clinical diagnostic tool, a duplex real-time RT-PCR assay (rRT-PCR) was developed. TaqMan ® probes and primers specific for the BDV P and BDV L genes were designed by aligning the sequences of known BDV strains. After optimisation, the sensitivity and specificity of the rRT-PCR were established. The detection limit was set to 10–100 viral genomic copies per reaction and the assay detects the BDV strains V and He/80, as well as the most divergent BDV strain known so far, No/98. Furthermore, the system detected feline BDV variants in five naturally infected cats and a feline isolate used in experimental infection of cats. This rRT-PCR assay will be a powerful tool in further studies of BDV, including epidemiological screening and diagnosis.