Establishment and Application of Universal Taqman-based Real-time Fluorescence Quantitative RT-PCR Method for the Detection of Goose Astrovirus
In order to establish one real-time fluorescence quantitative RT-PCR to detect goose astrovirus 1(GAstV1)and goose astrovirus 2(GAstV2)infection,a pair of specific primers and one TaqMan probe were designed and synthesized according to the sequence between partial ORF2 gene and partial 3'UTR of the two type of goose astrovirus(GAstV).The optimal concentration of primers and probe were determined by matrix method.And based on optimized annealing temperature,the standard amplification curves of GAstV 1 and GAstV 2 were established,respectively.The specificity,sensitivity and repeatibility of the quantitative RT-PCR were further verified,and the established method was applied to the detection of clinical samples compared with the common RT-PCR.The results showed that the optimal concentrations of the forward primer and reward primer were 0.40 or 0.50 μmol/L,respectively.The optimal concentration of probe was 0.50 μmol/L.The linear range was 3.26×103 to 3.26×108 copies/μL and 7.09×103 to 7.09×108 copies/μL,and the correlation coefficient was 0.998,respectively.The method could specifically amplify for two types of GAstV,but there was no amplification signal for the nucleic acid of Newcastle disease virus,H9 subtype avian influenza virus,duck Tembusu virus,new duck reovirus,goose parvovirus and fowl adenovirus serotype 4.The detection limits was 3.26×102 copies/μL and 7.09×101 copies/μL.The intra-group and inter-group coefficients of variation of this method were both below 3%.The results of clinical samples detected by the quantitative RT-PCR assay was 90.57%(96/106),while the mixed positive rate of GAstV1 and GAstV2 was 62.26%that from the same tissue samples detected by common RT-PCR methods reported in the reference.The results indicated that the established quantitative RT-PCR could provide a rapid,sensitive and specific detection method for the simultaneous detection of GAstV 1 and GAstV 2.
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