Hirasawa T, Kaneshige T, Mikazuki K
Aburahi Laboratories, Shionogi & Co., Ltd., Shiga, Japan.
Vet Microbiol. 1994 Jul;41(1-2):135-45. doi: 10.1016/0378-1135(94)90143-0.
A polymerase chain reaction (PCR) for the detection of canine parvovirus (CPV) was developed. To increase the sensitivity and specificity of the reaction, the nested PCR with a double-nested primer pair (inner primer pair) was designed. The sequences of the PCR primer pairs were selected from the conserved region in the CPV VP1/VP2 gene. The PCR with the outer or inner primer pair alone (single PCR) could detect 10 fg of viral replicative form (RF) DNA on agarose gel electrophoresis; whereas as little as 100 ag of the RF DNA was detected by the nested PCR, which was shown to be 100 times more sensitive than the single PCR. Samples prepared from feline panleukopenia virus and mink enteritis virus, both having a very close antigenic relationship to CPV, were also amplified by the nested PCR. The specificity of the reaction was confirmed by restriction enzyme analysis and Southern hybridization. Next, fecal samples were examined by the nested PCR. All 10 samples suspected of CPV infection were positive, and two restriction sites (HaeIII and HindIII sites) in the PCR product were conserved among them. On the other hand, specific amplification was not observed in the samples derived from normal dogs. The number of the genome copy in positive samples was estimated about 10(9)-10(11)/g by the single PCR and 10(11)-10(13)/g by the nested PCR. The assay can be completed in 1-1.5 days, and does not need radioisotopes. Thus, the nested PCR seems to be a sensitive, specific and practical method for the detection of CPV in fecal samples.
建立了一种用于检测犬细小病毒（CPV）的聚合酶链反应（PCR）。为提高反应的敏感性和特异性，设计了带有双嵌套引物对（内部引物对）的巢式PCR。PCR引物对的序列选自CPV VP1/VP2基因的保守区域。单独使用外部或内部引物对进行的PCR（单重PCR）在琼脂糖凝胶电泳上可检测到10 fg的病毒复制型（RF）DNA；而巢式PCR可检测到低至100 ag的RF DNA，其敏感性比单重PCR高100倍。从与CPV具有非常密切抗原关系的猫泛白细胞减少症病毒和水貂肠炎病毒制备的样品也通过巢式PCR进行了扩增。通过限制性内切酶分析和Southern杂交证实了反应的特异性。接下来，通过巢式PCR检测粪便样本。所有10份疑似CPV感染的样本均为阳性，且PCR产物中的两个限制性位点（HaeIII和HindIII位点）在这些样本中是保守的。另一方面，在来自正常犬的样本中未观察到特异性扩增。通过单重PCR估计阳性样本中的基因组拷贝数约为10(9)-10(11)/g，通过巢式PCR估计为10(11)-10(13)/g。该检测可在1-1.5天内完成，且不需要放射性同位素。因此，巢式PCR似乎是一种检测粪便样本中CPV的灵敏、特异且实用的方法。
