Methodology Development of a Locked Nucleic Acid-based Fluorescent Quantitation RT-PCR for Detecting Porcine Epidemic Diarrhea Virus
In this study,a LNA-based fluorescent quantitation PCR method was developed for the rapid,accurate and trace detection of porcine epidemic diarrhea virus(PEDV)by targeting the M gene.Special primers and LNA-TaqMan probe were designed according to the M gene,and the reaction conditions have been optimized.The LNA-based fluorescent quantitation PCR method was compared with the conventional TaqMan probe method.Results showed that the detection limit of the method was 3.2 copies/μL,improved by 10 times compared to conventional TaqMan probe method.The method was specific of PEDV and no cross reaction to TGEV,PoRV,and PDCoV.The method had a good repeatability with variation coefficient of intra-and inter-group less than 1%.The method was used to detect 103 clinical samples,the results showed that 47 samples were positive for PEDV,with a positive rate of 45.63%,and the positive coincidence rate with the conventional TaqMan probe PCR assay was 90.38%.This LNA-based fluorescent quantitation PCR method provides a new technical for accurate detection and epidemiological investigation of PEDV.
NETL
NSTL
万方数据
维普
