伪狂犬病病毒微滴数字PCR定量检测方法的建立

doi:10.11843/j.issn.0366-6964.2017.09.015

Abstract

Abstract:

A droplet digital polymerase chain (ddPCR) detection method, based on gB gene of pseudorabies virus (PRV) was developed to improve the detection and quantification of PRV. The concentration of primer, probe concentration and annealing temperature in ddPCR reaction were optimized. The specificity and sensitivity of the established ddPCR method were also determined and applied to the detection of field samples. In results,the optimal primer concentration was 0.9 μmol·L^-1, the probe concentration was 0.25 μmol·L^-1, and the annealing temperature was 60℃ for ddPCR.The R² value of ddPCR standard curve was 0.998,showed ddPCR had a good linear response. The Limit of Detection of ddPCR was 6.1 copies·μL^-1, lower than that of fluorescence quantitative PCR (qPCR). The coefficient of variation was 2.81%,the ddPCR was specific for detecting PRV. Field samples were detected by ddPCR, qPCR and virus isolation. Compared with the virus isolation, the sensitivity of ddPCR method was 87.5%, specificity was 96.2%, and the coincidence rate was 97%. The results indicate that the new ddPCR assay provides a specific, sensitive tool for quantitative detection of PRV.

CLC Number:

S852.659.1

CHEN Ya-na, WANG Jing, ZI Zhan-chao, KANG Wen-hua, WANG Bao-yue, NI Jian-qiang, YUAN Lin. Development of Droplet Digital PCR for the Detection of Pseudorabies Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(9): 1705-1710.

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Development of Droplet Digital PCR for the Detection of Pseudorabies Virus

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