Development of a Universal Droplet Digital RT-PCR for Absolute Quantitative Detection of Swine Influenza Virus
In order to develop a sensitive droplet digital RT-PCR(ddRT-PCR)for absolute quantitative detection of swine influenza virus(SIV),targeting at the conserved sequence of SIV M gene,a pair of specific primers and TaqMan probe were designed and synthesized to establish a ddRT-PCR method for the main prevalent subtypes of SIV,including H1N1,H1N2 and H3N2.Subsequently,the method was evaluated for its sensitivity,specificity and reproducibility,then tested and verified using clinical porcine nasopharyngeal swab samples.The results showed that the established ddRT-PCR was highly sensitive with a detection limit of 1.6 copies/µL;it could specifically detect H1N1,H1N2 and H3N2 subtype SIV,but failed for swine fever virus and other common pathogens;the coefficients of variation(CVs)of the intra and inter-batch results of reproducibility tests were 0.75%and 3.17%,respectively,with a good reproducibility;as verified by testing clinical samples,72 and 66 positive samples were detected by the established ddRT-PCR and fluorescent RT-qPCR,respectively,indicating that the former was superior to the latter in detecting samples with low viral load.In conclusion,the method established in this study,with high sensitivity,good specificity and reproducibility,was more advantageous in detecting clinical samples with low viral load,and could be effectively used for early diagnosis and absolute quantitative detection of SIV.
国家科技期刊平台
NSTL
万方数据
维普
