Development and evaluation of a novel TaqMan fluorescence probe-based real-time reverse transcriptase polymerase chain reaction assay for detection and quantification of West Nile virus

摘要

In order to improve and accelerate the detection of West Nile virus (WNV), a rapid and specific real-time reverse transcription polymerase chain reaction (rtRT-PCR) was established. Primers and probe were designed according to the conservative sequence of capsid protein gene of WNV. Tenfold successive dilutions of positive WNV DNA were used to measure the sensitivity of rtRT-PCR. The amplifying curve showed that this method could successfully amplify 101?copies/μl WNV gene, while reference to Japanese encephalitis virus (JEV) and blank control were all negative. The assay system showed high reproducibility with coefficient of variation (CV) < 2%. The detection of WNV can be completed within 2 to 3 h. By detecting cDNA samples (n = 55) with rtRT-PCR and the conventional PCR assay, the established rtRT-PCR showed 96.36% (37 + 16/55) coincidence rate with the conventional PCR. All the results showed that the newly established rtRT-PCR assay was shown to be a rapid, sensitive and specific test for detecting WNV.