鸭腺病毒A型TaqMan实时荧光定量PCR检测方法的建立

doi:10.16431/j.cnki.1671-7236.2018.05.027

Abstract

Abstract:

This study was aimed to establish TaqMan-based Real-time PCR method of duck adenovirus A (DAdV-A).According to the sequence of DAdV-A Hexon gene,the specific primers and probe were designed to develop the TaqMan-based Real-time PCR method,and the specificity,sensitivity and repeatability of the established method were detected.85 clinic samples were tested by the established TaqMan-based Real-time PCR method,then the coincidence rate were calculated which compared with the conventional PCR.The results showed that a TaqMan-based Real-time PCR method was successfully established,the correlation (R²) was 0.996,and the efficiency was 99.9%.This method had strong specificity,high sensitivity and good repeatability.No amplification was detected from common duck origin pathogens,such as,duck virus enteritis,goose parvovirus,Muscovy duck virus,duck circovirus,Escherichia coli,Rimerella anatipstifer and Pasteurella multocida.The limit of detection concentration was 8.37 copies/μL.The intra-and inter-assay were ranged from 0.54% to 1.28% and 0.61% to 2.39%,respectively.85 clinical samples were tested by the established TaqMan-based Real-time PCR method and the conventional PCR method,the positive rate were 7.06%(6/85)and 5.88%(5/85),respectively.All PCR positive samples were also tested positive by TaqMan-based Real-time PCR method,the coincidence rate was 100%.The TaqMan-based Real-time PCR method provided a useful method for molecular epidemiological survey of DAdV-A.

Key words: duck adenovirus A (DAdV-A); Hexon gene; TaqMan probe; Real-time PCR method

CLC Number:

S852.65⁺9.1

WAN Chunhe, LIU Rongchang, CHENG Longfei, SHI Shaohua, FU Guanghua, CHEN Hongmei, FU Qiuling, HUANG Yu. Establishment of a TaqMan-based Real-time PCR Method of Duck Adenovirus A[J]. , 2018, 45(5): 1341-1348.

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Establishment of a TaqMan-based Real-time PCR Method of Duck Adenovirus A

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