Simultaneous Detection, Subgrouping, and Quantitation of Respiratory Syncytial Virus A and B by Real-Time PCR

Abstract

Timely diagnosis of respiratory syncytial virus (RSV) infection is critical for appropriate treatment of lower respiratory infection in young children. To facilitate diagnosis, we developed a rapid, specific, and sensitive TaqMan Respiratory syncytial virus (RSV) is one of the major respiratory pathogens causing lower respiratory tract diseases in children (10,18,23). Based on genetic and antigenic variations in the structural proteins, RSV is classified into two subgroups, A and B (3, 24). At present, there is no vaccine available for prevention of the viral infection, although antiviral therapy is known to be effective in some patients (1, 2, 11). For appropriate treatment of RSV infection, it is crucial to have an accurate and timely diagnostic method for detection of the virus.A number of techniques are available for detection and identification of RSV, including cell culture, enzyme immunoassay (EIA), immunofluorescence (IF), and conventional reverse transcription (RT)-PCR (6,7,8,9,12,16,17,21,22,25). The classical cell culture method requires prompt inoculation of the labile virus and is a time-consuming process. In addition, the test has low sensitivity, detecting only 50 to 60% of RSV infections (17). More rapid immunoassays for the detection of RSV, such as EIA and IF, also have limitations in sensitivity and specificity. The accuracy of EIA, for example, is in the range of 57 to 98%, and that of IF varies from 65 to 92% (12). This wide variability in assay sensitivity and specificity may lead to inaccurate diagnosis of RSV infection, and consequently, the assays may be of limited value in patient care. Furthermore, such rapid immunoassays are rarely capable of differentiating RSV subgroups A and B, which may be associated with different disease severities (28).Conventional RT-PCR, which has recently been developed for the detection of RSV, is a more sensitive and specific diagnostic method that also allows for the subgrouping of the virus in a single reaction (6,7,8,9,17,25). However, it still requires time-consuming post-PCR analysis. Real-time RT-PCR, on the other hand, eliminates post-PCR processing (13). This is achieved by combining conventional RT-PCR with advanced fluorescence detection technology, which allows the fluorescence signals to be analyzed and recorded during PCR cycling. The advanced method also performs automatic sample analysis with enhanced sensitivity and specificity. In this report, we describe the development and application of a TaqManbased RT-PCR assay for simultaneous detection, subgrouping, and quantification of RSV A and B in clinical respiratory specimens. MATERIALS AND METHODSVirus stocks, plaque titration, and virus particle counts. RSV A2 (subgroup A) and 2B (subgroup B) were propagated in Vero cells. About 100 ml each of subgroup A and B virus stocks were prepared, and the stocks were stored in 0.5-ml aliquots for future use. For plaque titration of RSV stocks, confluent Vero cells in 24-well plates were inoculated with 100 l of a serial dilution of each virus at 37°...

Electrochemical genosensor based on disc and screen printed gold electrodes for detection of specific DNA and RNA sequences derived from Avian Influenza Virus H5N1

“…This step of genosensor preparation using SH-NC3 probe is widely applied [24][25][26][27][28][29][30]. The immobilization of SH-NC3/MCH SAM was confirmed by CV and OSWV in the presence of Fe(CN) 6 3-/4-as a redox marker. As expected, in the CV, the redox marker shows reversible behaviour on bare SPGE, with a peak-to-peak separation ΔE p = 0.071 ± 0.003 V (Figure 2A).…”

Development of a 1-Step Enzyme-Linked Immunosorbent Assay for the Rapid Diagnosis of Bovine Respiratory Syncytial Virus in Postmortem Specimens

“…3,8 Given the detection limit established with the RB94 BRSV strain, the real-time RT-PCR was the most sensitive method among the 4 tests evaluated in this paper. The higher sensitivity of realtime RT-PCR could explain the positive results obtained in the 6 samples that were negative with the other 3 tests.…”

“…1,3,7,8 However, at present, some veterinary diagnostic laboratories are not yet equipped to perform real-time PCR routinely. Thus, there is a need for sensitive and rapid BRSV diagnostic methods for routine use on postmortem specimens.…”

A new real-time RT-qPCR assay for the detection, subtyping and quantification of human respiratory syncytial viruses positive- and negative-sense RNAs

“…Different RSV genes have been targeted in these assays, such as the matrix, the polymerase (Kuypers et al, 2004), or the fusion protein genes (Mentel et al, 2003;van Elden et al, 2003), but the N gene is the most widely used due to its high conservation and the number of N sequences available in Genbank. Several assays were designed to subtype RSV, but most were validated more than 10 years ago (Dewhurst-Maridor et al, 2004;Do et al, 2012;Gueudin et al, 2003;Hu et al, 2003). In order to include Table 5 Validation of subtyping efficiency.…”

“…Real-time quantitative PCR (RT-qPCR) is the gold standard for both accurate RSV diagnosis and quantification and several assays have been developed during the last decades (Chi et al, 2007;Choudhary et al, 2013;Dewhurst-Maridor et al, 2004;Do et al, 2012;Gueudin et al, 2003;Hu et al, 2003;Kuypers, http://dx.doi.org/10.1016/j.jviromet.2016.05.004 0166-0934/© 2016 Elsevier B.V. All rights reserved. Mentel et al, 2003;van Elden et al, 2003), although these have to be adapted constantly to take into account genetic variations among circulating strains.…”