Rotavirus Detection Technology

Objectives: To develop a more sensitive and specific diagnostic test for rotavirus (RV) by applying real-time quantitative RT-PCR technology.Methods: By multiple alignment analysis of the sequences downloaded from GenBank with the MegAlign software, one region of high interspecies homology of segment 6 of group A RV genome was selected as PCR target. A fluorescent probe labeled with a FAM reporter at 5'-end while a TAMRA quencher at 14bp from the 5'-end as well as primer pairs were designed according to the general principles of real-time quantitative PCR. A DNA standard derived from recombinant plasmid pcDNA-II-220 was prepared and serially diluted to set up the standard curve, and the real-time quantitative RT-PCR specific for RV was developed. The sensitivity of the developed real-time quantitative RT-PCR was validated by using serial diluted nucleic acid extracts from standard RV SA-11 stocks which were propagated on MA104 cells. Commercial monoclonal antibody-based RV antigen ELISA kits were used to detect group A rotaviruses. The reliability of the assay was verified by detecting RV in 113 stool specimens collected from children under 3 years with diarrhea and the results were compared with a commercial RV detection ELISA kit. Results: The detection limit of the real-time quantitative RT-PCR was 1TCID₅₀/ml (median tissue culture-infective dose), which is 1 log more sensitive than ELISA. The relationship between threshold cycle (Ct) and amount of virus was linear (r = 0.998) over a range of 1 to 10,000 TCID₅₀/ml. The assay is specific to RV and its efficiency was confirmed by the detection of viral RNA in clinical samples. The results showed that out of the 113 specimens, 80 (70%) were RV positive by the real-time RT-PCR assay and 76 (67%) positive by ELISA. Sixty seven were positive in both real-time RT-PCR and ELISA assay, whereas 13 samples were only found to be positive in the real-time RT-PCR assay. Twenty four were negative in both assays. The overall detection rate of the real-time RT-PCR assay is not statistically different from that of ELISA (Chi-square test, x2 =0.41, P>0.5)Conclusion: The real-time RT-PCR assay to group A rotavirus described here allows a large number of samples to be screened rapidly, sensitively and specifically. It will potentially be a suitable tool for the detection of rotavirus in various situations including clinical specimen detection, molecular epidemiological study and food safety control. Significance: Rotaviruses are the major pathogenic agents of acute diarrhea disease of human and animals worldwide. Apart from causing severe clinical disease, rotaviruses also lead to subclinical infections in human and animals. Further more, rotaviruses have shown to be transmitted from the original host to a heterologous one. Thus, it is necessary to detect rotaviruses rapidly and accurately from clinical and environmental samples. Though EIA has been recommended by World Health Organization (WHO) as a rapid diagnosis assay for rotavirus, the sensitivity and specificity of which are limited once used for field samples such as food specimens, waste waters, etc. It is indeed important to develop a more sensitive and specific diagnostic test. The polymerase chain reaction (PCR) has achieved tremendous developments in the past decade and been accepted as the gold standard in molecular diagnostic techniques, however, the majority of which reported to date have been used in a quanlitative format. Real-time PCR has proved itself a valuable tool in true quantitation of different target nucleic acids at present, and it has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. However, development of such assays for RV is still in its early stage and needs to be further investigated, evaluated and improved, despite a real-time RT-PCR protocol for the detection of RV using SYBR Green I dye as fluorimetry and an assay relying on the detection for the NSP3 gene of RV has been described very recently. In the present work, we developed and evaluated a real-time RT-PCR system specific for the segment 6 of RV genome using an improved highly specific TaqMan probe.