Detection of respiratory syncytial virus by reverse transcription-PCR and hybridization with a DNA enzyme immunoassay

Abstract

Nasal aspirates from 238 infants hospitalized with acute respiratory infections during the winter of 1994 and 1995 were tested for respiratory syncytial virus (RSV) by immunofluorescence assay (IFA) and the viral isolation technique (VIT) and by two PCR and hybridization methods: reverse transcription PCR 1 (RT-PCR1), which amplifies the RNAs of all RSV strains, and RT-PCR-2, which allows subgroup classification of RSV. RT-PCR-1 and RT-PCR-2 detected viral sequences in 56.7% (135 of 238) and 48.3% (115 of 238) of the samples, respectively, while only 80 (33.6%) samples were found to be positive by IFA and VIT. Of the PCR-positive specimens, 57 were missed by these routine techniques in RT-PCR-1 and 45 were missed in RT-PCR-2. Although the RSV-PCR-1 and RSV-PCR-2 techniques amplified two different sequences of the RSV genome, they gave similar results for 218 (91.6%) nasal aspirates. Compared with conventional methods, the sensitivity, specificity, and agreement were 97.5, 63.9, and 75.2%, respectively, for RT-PCR-1 and 89.7, 71.9, and 77.7%, respectively, for RT-PCR-2, and for these two RT-PCR assays, the positive predictive value (PPV) and the index of agreement () were comparable and moderate, respectively: PPV was 57.8% and was 0.52 in RT-PCR-1, and PPV was 60.9% and was 0.54 in RT-PCR-2. However, there was a perfect correlation between the two RT-PCRs, with a PPV of 100% and an excellent index of agreement (‫؍‬ 0.88). Therefore, most RT-PCR results were really true positive, and VIT and IFA, which missed some of them, appeared to be less sensitive.

Detection of human Coronavirus 229E in nasal specimens in large scale studies using an RT-PCR hybridization assay

“…To test for HCoV-229E in the course of large scale epidemiological studies, we used a RT-PCR hybridization method for rapid, specific, and more sensitive detection of HCoV-229E DNA products. Hybridization is more specific and sensitive than agarose gel visualization [13]. The microplate enzyme immunoassay format allows the handling of large number of samples needed for epidemiological studies.…”

Detection of viral, Chlamydia pneumoniae and Mycoplasma pneumoniae infections in exacerbations of asthma in children

“…The polymerase chain reaction (PCR) and retrotranscription PCR (RT-PCR), followed by internal probe hybridization, were used to detect sequences for RS virus, adenovirus, Chlamydia pneumoniae and Mycoplasma pneumoniae. Primers, probes (Table 1) and molecular procedures previously defined and assessed in other studies, were used for the detection of RS virus (Freymuth et al, 1995), adenovirus (Hierholzer et al, 1993), Chlamydia pneumoniae (Petitjean et al, 1998), and Mycoplasma pneumoniae (de Barbeyrac et al, 1993). For the detection of coronavirus OC43 and 229E, the primers and probes were selected in our laboratory from reported sequences of M genes obtained from the gene bank.…”

Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses

“…Primers targeted specifically the haemagglutinin neuraminidase genes of PIV-1, -2 (Echevarria et al, 1998) and-3 (Karron et al, 1994), the phosphoprotein gene of VIP-4A and -4B (Aguilar et al, 2000), the nucleocapsid gene of hRSV sub-groups A and B (Cane and Pringle, 1991;Freymuth et al, 1995), the matrix protein genes of influenza viruses A and B (Donofrio et al, 1992), the matrix protein gene of hMPV (this study), the haemagglutinin-esterase gene of influenza C virus (Zhang and Evans, 1991), the M gene of OC43 and 229E (Vabret et al, 2001) and the VP4/VP2 and hypervariable region in the 5 -non-coding region of hRV (Savolainen et al, 2002). The sequences of the primers, as well as their annealing temperatures and amplicon sizes are given in Table 1.…”