Development and verification of a qPCR method based on TaqMan probe targeting E301R gene of African swine fever virus
Objective To develop and verify a fluorescent quantitative polymerase chain reaction(qPCR)method based on TaqMan probe for the detection of E301R gene of African swine fever virus(ASFV),so as to apply the method to the detection of ASFV in clinical samples.Methods By analyzing the E301R gene sequence of ASFV,specific primers and TaqMan probes were designed for the conserved region of the gene.The fragment of E301R gene amplified by PCR was cloned into pCAGGS vector,and the standard plasmid was constructed.The qPCR detection method based on TaqMan probe was developed and verified for the linear range,precision,specificity,sensitivity and accuracy.The developed method was used to detect 92 clinical samples,and the results were compared with those detected by commercial real-time PCR diagnostic kit.In addition,the transcriptional dynamics of E301R gene was analyzed by using the developed method.Results In the range of 1.6 ×(101-108)copies/μL,the standard plasmid showed a good linear relationship with Ct values,and the lin-ear regression equation was:y=-3.239 x+40.774,with the correlation coefficient of 0.994.The CVs of repeatability and intermediate precision verification were both less than 2％.Except for ASFV,there was no amplification curve of classi-cal swine fever virus(CSFV),porcine reproductive and respiratory syndrome virus(PRRSV),pseudorabies virus(PRV),and porcine circovirus type 2(PCV2).The minimum detection limit was 1.6 × 101 copies/μL of standard plasmid.The spike recoveries of 1.6 × 102 and 1.6 × 101 copies/μL standard plasmid were between 90％and 110％.The coincidence rate of the detection results between the developed method and commercial real-time PCR diagnostic kit for 92 clinical samples was 96.7％(Kappa=0.932,P=1.000).E301R gene may be the transcription gene in the middle stage of ASFV infec-tion.Conclusion The developed detection method of qPCR based on TaqMan probe has good precision,specificity,sensi-tivity and accuracy which can be used for the detection of ASFV in clinical samples.
NETL
NSTL
万方数据
