Carrière C, Boulanger P, Delsert C
Laboratoire de Virologie & Pathogénèse Moléculaires (CNRS URA 1487), Faculté de Médecine, Montpellier, France.
J Virol Methods. 1993 Oct;44(2-3):221-34. doi: 10.1016/0166-0934(93)90057-x.
The sensitivity of detection of B19 virus DNA in clinical specimens was evaluated by comparing the results of single PCR and nested PCR assays, with or without subsequent Southern blot hybridization to a radiolabelled B19 DNA probe. The inhibitory activity of human serum components on polymerase reaction was also determined. The sensitivity of B19 virus DNA detection decreased by a factor of 10(7) in the presence of 10% serum in the single PCR reaction mixture, and of 10(3) for nested PCR. When nested PCR products were analysed by Southern blot hybridization to a B19 radioactive DNA probe, the sensitivity of the assay increased to such a level of B19 DNA detection that the reaction was no longer influenced by the presence of serum inhibitors in the original sample. Less than ten B19 genome copies could thus be detected in a 10-microliters sample. A panel of 38 clinical samples, originating from patients with possibility of B19 virus infection, were assayed by this method. Only one sample was found to be positive after single PCR, whereas seven samples (including the former) gave a positive signal after nested PCR. The specificity of the nested PCR products was controlled by hybridization to the B19 DNA probe and DNA sequencing. No discrepancy in the results was observed between nested PCR alone and nested PCR followed by Southern blot analysis.
通过比较单重PCR和巢式PCR检测结果，评估临床标本中B19病毒DNA的检测灵敏度，无论是否随后用放射性标记的B19 DNA探针进行Southern印迹杂交。还测定了人血清成分对聚合酶反应的抑制活性。在单重PCR反应混合物中存在10%血清时，B19病毒DNA检测的灵敏度降低了10的7次方倍，而在巢式PCR中降低了10的3次方倍。当通过与B19放射性DNA探针的Southern印迹杂交分析巢式PCR产物时，检测的灵敏度提高到了这样的B19 DNA检测水平，即反应不再受原始样品中血清抑制剂存在的影响。因此，在10微升样品中可以检测到少于十个B19基因组拷贝。用这种方法检测了一组38份临床样品，这些样品来自可能感染B19病毒的患者。单重PCR后仅发现一个样品呈阳性，而巢式PCR后七个样品（包括前者）给出了阳性信号。巢式PCR产物的特异性通过与B19 DNA探针杂交和DNA测序来控制。单独的巢式PCR与巢式PCR后进行Southern印迹分析之间未观察到结果差异。
