| To construct external standard for detection of rotavirus in water using the quantitative real-time polymerase chain reaction, rotavirus cDNA standard for real-time PCR assay was prepared by cell culture, PCR and T-A clone methods with primers specific for the viral structure protein VP7 gene, and this cDNA standard was confirmed by enzyme cleavage and DNA sequencing. Specificity, stability and reproducibility of the cDNA standard quantified were detected by common polymerase chain reaction (PCR) and quantitative real-time PCR. The results of standard curve showed a very good linear negative regression between threshold cycle (Ct) and Log starting quantity of copy number. The melting curve analysis of real-time PCR showed melting temperature at 81℃±0.5℃,indicating PCR products were that of the rotavirus VP7 sequence and thus the standard used in this study was specific for rotavirus. Moreover, the result of real-time PCR also indicated detection range was from 9×100 to 9×1011 copies per reaction, and the detection limit for this assay was 9 copies of rotavirus cDNA per reaction, and thus real-time PCR assay using the standard had a high sensitivity for detection of rotavirus. Furthermore, the results indicated a high stability and reproducibility of cDNA standard were assessed according to the CVs of three independent experiments in the range of 0.2%～0.9%. Taken together, in this study rotavirus cDNA standard prepared could be used as quantitative detection of rotavirus cDNA from water samples. |
