检测猪圆环病毒2型PCR方法的建立

目的]为快速诊断猪圆环病毒病提供参考。方法]根据GenBank中猪圆环病毒2型(PCV-2)的核苷酸序列设计并合成1对特异性引物,建立诊断PCV-2地方分离株的PCR方法。结果]各物质在PCR反应中最佳浓度分别为:dNTP 220 pmol/ml,Taq酶0.1 U/μl,引物20 pmol/μl。从PCV-2阳性病料中提取DNA,在优化条件下进行PCR扩增,获得预期的494 bp特异性条带。用该PCR方法对PCV-2、PRRSV、HCVS、IV、PPV进行检测,PCV-2为阳性,而PRRSV、HCV、SIV、PPV检测均为阴性,未出现交叉反应,说明该PCR方法对PCV-2具有良好的特异性。该PCR方法可检出1.25 ng模板DNA,具备较高的敏感性。结论]使用该PCR方法检测PCV-2是切实可行的。

Establishment of PCR Method for Detecting Porcine Circovirus 2

Objective] The aim of the research was to provide reference for rapid diagnosis of porcine circovirus disease.Method] According to the nucleotide sequence of PCV-2 on GenBank website,one pair of primers were designed and synthesized.And PCR method for diagnosing the local isolates of PCV2 was established.Result] The optimum concn.of each substance in PCR reaction were as follows: dNTP 220 pmol/ml,Taq enzyme 0.1 U/μl,primers 20 pmol/μl. DNA was extracted from diseased materials with the positive PCV-2 to make PCR amplification under the optimized conditions,and the expected specific bands with the size of 494 bp was obtained.PCV-2,PRRSV,HCV,SIV and PPV were detected by this PCR method,PCV-2 was positive and PRRSV,HCV,SIV and PPV were negative,without cross reaction.This indicated that this PCR method had good specificity to PCV-2.1.25 template DNA could be detected by this PCR method which had higher sensitivity.Conclusion] It was feasible to detect PCV-2 by using this PCR method.