猪圆环病毒1型与2型双重PCR检测方法的建立及应用

Development and application of a duplex PCR method for porcine circovirus type 1 and type 2

【目的】建立一种可同时检测猪圆环病毒1型(PCV1)和2型(PCV2)的双重PCR检测方法并进行临床应用。【方法】对PCV1和PCV2的单项PCR方法分别进行条件优化后对两种病毒的双重PCR进行条件优化并进行特异性与敏感性试验,然后用单项PCR方法和双重PCR对临床样品进行比较检测。【结果】双重PCR检测方法对PCV1和PCV2的检测灵敏度分别为1.7×10~3、4×10~3 copies/μL,该方法对当前常见猪源DNA病毒的检测结果都是阴性。该方法对2013—2017年15个猪场的145份断奶仔猪样品的检测结果显示,PCV1和PCV2的个体阳性率分别为11.7%和77.2%,与单项PCR方法检测结果无显著(P>0.05)差异,且两种方法检出的PCV1或PCV2的阳性猪场完全一致;临床样品中PCV2的猪场阳性率与个体阳性率都极显著(P<0.01)高于PCV1。【结论】建立了一种有较高敏感性与特异性的PCV1和PCV2的双重PCR检测方法,当前猪场中PCV2感染较PCV1感染明显占优势。

【Objective】This study is aimed to establish a duplex PCR method for simultaneous detection of porcine circovirus type 1(PCV1)and type 2(PCV2)in clinical samples.【Methods】The conditions of the single PCR method of PCV1and PCV2 were optimized respectively,and then the duplex PCR method of these 2viruses was optimized including its specificity and sensitivity.Thereafter,several clinical samples were comparatively detected with both the single PCR method and the duplex PCR method.【Results】The detection sensitivity of the duplex PCR method for PCV1and PCV2was 1.7×10^3 and 4.0×10^3 copies/μL,respectively,and the detection results of the other current epidemic swine DNA viruses by this method were all negative.The detection results of 145weaning piglets samples from 15pig farms from 2013to 2017showed that the positive rate of PCV1and PCV2was 11.7%and 77.2%,respectively;and showed no significant difference(P>0.05)from those with the single PCR method.The positive rate of PCV2for both pig farms and individuals in the clinical samples was all very significant(P<0.01)higher than that of PCV1.【Conclusion】A duplex PCR assay with relatively high specificity and sensitivity for PCV1 and PCV2was developed,and the PCV2infection was superior to the PCV1infection in pig farms.