Establishment and Comparison of Two Taq Man Real-time PCR Methods for PCV2

作     者：周忠涛 王小敏 汪伟 茅爱华 温立斌 倪艳秀 何孔旺 

作者机构：江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室国家兽用生物制品工程技术研究中心江苏南京210014 南京农业大学动物医学院江苏南京210095 

基　　金：Supported by National Natural Science Foundation of China(31302071) Special Fund for Agro-scientific Research in the Public Interest(201303046) Jiangsu Agricultural Science and Technology Innovation Fund(CX(14)2045) "333 High-level Personnel Training Project"of Jiangsu Province(BRA2012194) 

出 版 物：《Agricultural Science & Technology》 (农业科学与技术（英文版）)

年 卷 期：2015年第16卷第1期

页      码：3-8页

摘      要：[Objective] In this study, the quantitive detection of PCV2 （porcine circovirus type 2） in vitro was achieved. We aimed to establish two kinds of TaqMan real-time PCR methods based on PCV20RF1 and ORF2 respectively and compare them. [Method] According to the relatively＇conserved sequences of PCV20RF1 and ORF2 registered in GenBank, two pairs of specific primers and TaqMan probes were designed and synthesized. Then the recombinant plasmids containing the whole sequences of PCV20RF1 and ORF2 were constructed to draw the standard curves through optimizing the reaction system and conditions. And thus two kinds of TaqMan real-time PCR detection methods based on the whole sequences of ORF1 and ORF2 respectively were constructed for PCV2. [Result] For the two established standard curves, the Ct values showed a good linear relationship with the loga- rithms of copy numbers of templates （F2〉0.99）. The amplification efficiency ranged from 90% to 110%. The amplifications all had a good repeatability with variation coefficients within groups all less than 5%. Moreover, the amplifications all had a good specificity. When the sequences of porcine parvovirus （PPV）, porcine circovirus type 1 （PCV1）, swine pseudorabies virus （PRV）, porcine reproductive and respiratory syndrome virus （PRRSV） were used as templates, the target sequence was not amplified. The amplifications also had a high sensitivity. The ORF1 detection method could reach 1.0x10T copies/;ul, and the ORF2 detection method could reach 1.0×10^2 copies/μl. The two established real-time PCR detection methods were used to detect the 80 clinical samples respectively. The results showed the magnitudes of 72 amplified samples were basically consistent between the 2 detection methods, while the magnitudes of the other 8 amplified samples were inconsistent. Then the 8 samples were detected with SYBR Green I real-time PCR method established based on the sequence of PCV2-1ike factor P1 by Wen et aL The PCV2-1ike factor P1 was ampl

主 题 词：PCV2 TaaMan real-time PCR ORF1 ORF2 PCV2-1ike factor P1 

学科分类：090601[090601] 09[农学] 0906[农学-水产类] 

D　O　I：10.16175/j.cnki.1009-4229.2015.01.002

馆 藏 号：203233047...