An integrated cell absorption process and quantitative PCR assay for the detection of the infectious virus in water.

ABSTRACT

Here we developed an integrated cell absorption process and quantitative (reverse transcription) polymerase chain reaction (ICAP-q(RT)PCR) assay to detect infectious viruses, which based on the detection of the viral nucleic acid (RNA or DNA) in the early stage of viral attachment and entry towards cells. The results showed that the poliovirus or adenovirus whose concentration was as low as 0.2 TCID50/mL could be detected by ICAP-q(RT)PCR after 4 h incubation. The ICAP-q(RT)PCR exhibited much higher sensitivity than the plaque assay. In parallel, it took shorter time to detect the viruses towards field samples compared with the integrated cell culture (ICC)-qPCR, but could still get the consistent detecting results with ICC-qPCR. This method is verified by detecting four different kinds of viruses including poliovirus, adenovirus, rotavirus, and astrovirus, which existed in the actual water samples. Among all the 24 Jinhe river samples, 50% (12/24) of river water samples were positive for poliovirus when detected by ICAP-q(RT)PCR, which was in accordance with the results detected by ICC-qPCR. However, 21% (5/24) and 68% (18/24) of the samples were detected to be positive for poliovirus by the plaque counting and the direct qPCR method, respectively. Compared with ICAP-q(PT)PCR and ICC-qPCR, the detecting results of qPCR or plaque assay displayed a marked expansion or decline, respectively, which lead to the evident deviations in the accuracy. The results demonstrated that our developed ICAP-q(RT)PCR method could dramatically reduce the test duration and quite improve the sensitivity towards infectious viruses. Therefore, the ICAP-q(RT)PCR method could be an effective and quantitative tool for detecting infectious viruses in water environments.