A study on the single -tube multiplex RT -real time PCR assay in detecting rotavirus,astrovirus and hepatitis A virus / 预防医学

ABSTRACT

Objective To develop and evaluate a single -tube multiplex RT -real time PCR assay for the simultaneous detection of rotavirus,astrovirus and hepatitis A virus.Methods Gen -bank sequences of rotavirus,astrovirus and hepatitis A virus were included as reference sequences.Primers and probes were designed based on the reference sequences.A multiplex real -time RT -PCR assay was developed,and the reproducibility,specificity and sensitivity of the assay were evaluated.Fecal samples from 1 28 patients with viral diarrhea were detected and verified by gene sequencing.Results There was high reproducibility and specificity of the single -tube multiplex real -time RT -PCR assay for detecting rotavirus,astrovirus and hepatitis A virus.Detection limits of 1 01 copies were achieved in tests of rotavirus,1 02 copies for astrovirus and hepatitis A virus in one reaction.3.1 3% and 1 7.97% of 1 28 clinical specimens were detected positive for astrovirus RNA and rotavirus RNA respectively.And the positive samples were verified by gene sequencing.Conclusion Rotavirus,astrovirus and hepatitis A virus can be detected and identified by the single -tube multiplex RT -real time PCR assay with high specificity and sensitivity.The assay developed in this study can be applied to the clinical diagnosis and epidemiological investigation.