Multiplex Real-time Reverse Transcription PCR Research Articles

Epidemiology and Clinical Presentations of the Four Human Coronaviruses 229E, HKU1, NL63, and OC43 Detected over 3 Years Using a Novel Multiplex Real-Time PCR Method

Four human coronaviruses (HCoV-229E, HCoV-HKU1, HCoV-NL63, and HCoV-OC43) are associated with a range of respiratory outcomes, including bronchiolitis and pneumonia. Their epidemiologies and clinical characteristics are poorly described and are often reliant on case reports. To address these problems, we conducted a large-scale comprehensive screening for all four coronaviruses by analysis of 11,661 diagnostic respiratory samples collected in Edinburgh, United Kingdom, over 3 years between July 2006 and June 2009 using a novel four-way multiplex real-time reverse transcription-PCR (RT-PCR) assay. Coronaviruses were detected in 0.3 to 0.85% of samples in all age groups. Generally, coronaviruses displayed marked winter seasonality between the months of December and April and were not detected in summer months, which is comparable to the pattern seen with influenza viruses. HCoV-229E was the exception; detection was confined to the winter of 2008 and was sporadic in the following year. There were additional longer-term differences in detection frequencies between seasons, with HCoV-OC43 predominant in the first and third seasons and HCoV-HKU1 dominating in the second (see Results for definitions of seasons). A total of 11 to 41% of coronaviruses detected were in samples testing positive for other respiratory viruses, although clinical presentations of coronavirus monoinfections were comparable to those of viruses which have an established role in respiratory disease, such as respiratory syncytial virus, influenza virus, and parainfluenza viruses. The novel multiplex assay for real-time pan-coronavirus detection enhances respiratory virus diagnosis, overcomes potential diagnostic problems arising through seasonal variation in coronavirus frequency, and provides novel insights into the epidemiology and clinical implications of coronaviruses.

Evolutionary Dynamics of GII.4 Noroviruses over a 34-Year Period

Noroviruses are a major cause of epidemic gastroenteritis in children and adults, and GII.4 has been the predominant genotype since its first documented occurrence in 1987. This study examined the evolutionary dynamics of GII.4 noroviruses over more than three decades to investigate possible mechanisms by which these viruses have emerged to become predominant. Stool samples (n = 5,424) from children hospitalized at the Children's Hospital in Washington, DC, between 1974 and 1991 were screened for the presence of noroviruses by a custom multiplex real-time reverse transcription-PCR. The complete genome sequences of five GII.4 noroviruses (three of which predate 1987 by more than a decade) in this archival collection were determined and compared to the sequences of contemporary strains. Evolutionary analysis determined that the GII.4 VP1 capsid gene evolved at a rate of 4.3 x 10(-3) nucleotide substitutions/site/year. Only six sites in the VP1 capsid protein were found to evolve under positive selection, most of them located in the shell domain. No unique mutations were observed in or around the two histoblood group antigen (HBGA) binding sites in the P region, indicating that this site has been conserved since the 1970s. The VP1 proteins from the 1974 to 1977 noroviruses contained a unique sequence of four consecutive amino acids in the P2 region, which formed an exposed protrusion on the modeled capsid structure. This protrusion and other observed sequence variations did not affect the HBGA binding profiles of recombinant virus-like particles derived from representative 1974 and 1977 noroviruses compared with more recent noroviruses. Our analysis of archival GII.4 norovirus strains suggests that this genotype has been circulating for more than three decades and provides new ancestral strain sequences for the analysis of GII.4 evolution.

Comparison of Viral Isolation and Multiplex Real-Time Reverse Transcription-PCR for Confirmation of Respiratory Syncytial Virus and Influenza Virus Detection by Antigen Immunoassays

We evaluated the Prodesse ProFlu-1 real-time reverse transcription-PCR multiplex assay with the SmartCycler instrument for the detection of human respiratory syncytial virus (RSV) and influenza A and B viruses in comparison to conventional cell culture and antigen immunoassays with the BD Directigen A+B and Binax NOW RSV assays over two successive respiratory virus seasons. Ninety-two percent of the 361 specimens tested were nasopharyngeal aspirates obtained from individual patients, of which 119 were positive for RSV and 59 were positive for influenza virus. The median age of the patients whose specimens were positive for RSV and influenza virus were 6.3 months and 42.4 years, respectively. The specificity of all of the methods tested was >or=99%, and the individual sensitivities of NOW RSV, RSV culture, Directigen A+B, influenza virus culture, and the Proflu-1 PCR for influenza/RSV were 82% (95% confidence interval [CI], 73 to 88), 57% (95% CI, 44 to 69), 59% (95% CI, 44 to 72), 54% (95% CI, 38 to 69), and 98% (95% CI, 93 to 100)/95% (95% CI, 85 to 99), respectively. In a clinical setting where viral isolation is performed to confirm rapid antigen immunoassay results for these common respiratory viruses, one-step real-time reverse transcriptase PCR testing can be a more sensitive and timely confirmatory method.

Detection and characterization of F+ RNA bacteriophages in water and shellfish: Application of a multiplex real-time reverse transcription PCR

Genotyping of F+ RNA bacteriophages has been used to distinguish between human and animal contributions to contaminated water and food. There are four genetically distinct genogroups of F+ RNA bacteriophages. Genogroups I and IV predominate in animal wastes and genogroups II and III in wastes of human origin. In this study, a multiplex real-time RT-PCR-based method was developed to detect and genotype F+ RNA bacteriophages. The assay was shown to be broadly reactive against a wide spectrum of F+ RNA bacteriophage strains, including MS2, GA, Qβ, MX1, SP and FI, and was able to detect and genotype F+ RNA bacteriophages in shellfish and river water. The assay is highly sensitive, with detection limits <10 PFU/reaction and <10 copies/reaction of the target sequences carried in plasmids, respectively. The applications of this assay include F+ RNA semi-quantitation and microbial source tracking.

Preparation of Armored RNA as a Control for Multiplex Real-Time Reverse Transcription-PCR Detection of Influenza Virus and Severe Acute Respiratory Syndrome Coronavirus

The common respiratory viruses, including influenza A, influenza B, and newly emerging severe acute respiratory syndrome (SARS) viruses, may cause similar clinical symptoms. Therefore, differential diagnosis of these virus pathogens is frequently required for single clinical samples. In addition, there is an urgent need for noninfectious and stable RNA standards and controls for multivirus detection. In this study, reverse transcription-PCR (RT-PCR) targeting of the RNAs of influenza A and influenza B viruses and SARS coronavirus was performed, and the resulting products were spliced into a fragment which was packaged into armored RNA for use as a noninfectious, quantifiable synthetic substitute. Furthermore, in the present study we developed a multiplex real-time RT-PCR assay in which the armored RNA was used as an external positive control and the three RNA viruses could be detected simultaneously in a single reaction mix. The detection limit of the multiplex real-time PCR was 10 copies/microl of armored RNA.

Evaluation of the One-Step Multiplex Real-Time Reverse Transcription-PCR ProFlu-1 Assay for Detection of Influenza A and Influenza B Viruses and Respiratory Syncytial Viruses in Children

We evaluated the one-step multiplex real-time reverse transcription-PCR ProFlu-1 assay for the detection of influenza A and influenza B viruses and respiratory syncytial viruses from 353 pediatric nasopharyngeal aspirates. As assessed by comparison with the results of immunofluorescence testing and cell culture, the specificity and the sensitivity of the ProFlu-1 assay ranged from 97% to 100%. In addition, the ProFlu-1 assay amplified 9% of samples not detected by conventional methods.

Sensitive Multiplex Real-Time Reverse Transcription-PCR Assay for the Detection of Human and Animal Noroviruses in Clinical and Environmental Samples

In this study, we developed a triplex real-time reverse transcription-PCR (RT-PCR)-based method that detects and distinguishes between noroviruses belonging to genogroups I, II, and III and that targets the junction between the regions of open reading frame 1 (ORF1) and ORF2. This is the first assay to include all three genogroups and the first real-time RT-PCR-based method developed for the detection of bovine noroviruses. The assay was shown to be broadly reactive against a wide spectrum of norovirus genotypes, including GI/1 through GI/7, GII/1 through GII/8, GII/10, GII/12, and GII/17, in different matrices (including fecal specimens, treated and raw sewage, source water, and treated drinking water). The assay is highly sensitive, detecting low copy numbers of plasmids that carry the target sequence. A new bovine norovirus, Bo/NLV/Norsewood/2006/NZL, was identified by this assay and was further genetically characterized. The results implicate a broad range of possible applications, including clinical diagnostics, tracing of fecal contaminants, and due to its sensitivity and broad reactivity, environmental studies.

Somatodendritic microRNAs identified by laser capture and multiplex RT-PCR

The catalog of RNAs present in dendrites represents the complete repertoire of local translation that contributes to synaptic plasticity. Most views hold that a pool of dendritic mRNAs is selectively transported to a dendritic destination. This view requires that some mRNAs in the dendrite are locally enriched relative to the cell body; however, quantitative comparisons that would support this assumption do not currently exist. These issues related to somatodendritic distribution of mRNAs also apply to the microRNAs, approximately 21 nucleotide noncoding transcripts that bind to target mRNAs and either inhibit their translation or destabilize them. We combined laser capture with multiplex real-time RT (reverse transcription) PCR to quantify microRNAs in the neuritic and somatic compartments separately. The samples were standardized by RT-PCR measurements of a set of mRNAs, including known dendritic mRNAs, in these two compartments. Most neuronal miRNAs were detected in dendrites. With a few notable exceptions, most miRNAs were distributed through the somatodendritic compartment across a nearly constant gradient. Thus for lower-abundance miRNAs, the total neuronal concentration of the miRNA can remain readily detectable in the cell body but vanish from the dendrite. A very small number of miRNAs deviate from the distribution gradient across the miRNA population as relatively enriched or depleted in the dendrite.

Detection of three porcine vesicular viruses using multiplex real-time primer-probe energy transfer

Rapid identification of the etiologic agent in infected animals is important for the control of an outbreak of vesicular disease in livestock. We have in the present study developed a multiplex real-time reverse transcription-PCR, based on primer-probe energy transfer (PriProET), for simultaneous detection and differentiation of three Office International des Epizooties (OIE) classified vesicular viruses: foot-and-mouth disease virus, vesicular stomatitis virus and swine vesicular disease, causing clinically indistinguishable vesicular diseases in swine. The multiplex assay consists of extraction of total RNA from clinical samples; reverse transcription to cDNA using random primers and one-tube real-time amplification of cDNA using multiplex PriProET with specific fluorescent-labelled primers and probes for detection of the three viruses from the vesicular disease complex. The probes are labelled with unique reporter fluorophores, which during amplification are excited by donor fluorophores incorporated in the 5′ end of specific amplicons by primer extension. The sensitivity of the multiplex assay was approximately 100 TCID 50, which is 10-fold lower compared to the individual PriProET assays for the three vesicular viruses.