Yamakawa Y, Oka H, Hori S, Arai T, Izumi R
Department of Obstetrics and Gynecology, Toyama Medical and Pharmaceutical University, Japan.
Obstet Gynecol. 1995 Jul;86(1):126-9. doi: 10.1016/0029-7844(95)00092-6.
We describe the development of a nested polymerase chain reaction (PCR) technique used to detect human parvovirus B19 DNA. It was performed in a two-step reaction, first with a pair of outer primers, then with a pair of inner (ie, nested) primers. Primer sequences were selected in the VP1 gene, corresponding to the capsid protein, of human parvovirus B19. To establish the nested PCR assay, the plasmid pGEM-1 containing almost the entire coding sequence of a human parvovirus B19 isolate was used. The nested PCR could detect up to 1.8 x 10(-3) ag of B19 DNA, equivalent to 10(-4) genomes, by electrophoresis. No amplification product was detected by gel electrophoresis when the reaction mixture contained human placental DNA, cytomegalovirus DNA, and sterile distilled water as templates. We used this assay to evaluate four cases of maternal B19 infection that were diagnosed by determination of the presence of anti-B19 immunoglobulin-M in maternal serum. The advantages of our nested PCR for detecting B19 DNA are plating simplicity, safety, sensitivity, and specificity. Our results suggest that this method may have general applicability in the evaluation of nonimmune hydrops fetalis and in the documentation of the natural history of fetal infection with B19 when applied to specimens of amniotic fluid or fetal blood.
我们描述了一种用于检测人细小病毒B19 DNA的巢式聚合酶链反应（PCR）技术的开发。该反应分两步进行，首先使用一对外部引物，然后使用一对内部（即巢式）引物。引物序列是在人细小病毒B19的衣壳蛋白VP1基因中选择的。为了建立巢式PCR检测方法，使用了含有几乎整个人细小病毒B19分离株编码序列的质粒pGEM-1。通过电泳，巢式PCR能够检测低至1.8×10^(-3) 皮克的B19 DNA，相当于10^(-4) 个基因组。当反应混合物以人胎盘DNA、巨细胞病毒DNA和无菌蒸馏水作为模板时，凝胶电泳未检测到扩增产物。我们使用该检测方法评估了4例通过检测孕妇血清中抗B19免疫球蛋白M确诊的孕妇B19感染病例。我们的巢式PCR检测B19 DNA的优点是操作简单、安全、灵敏且特异。我们的结果表明，当应用于羊水或胎儿血液样本时，该方法可能在评估非免疫性胎儿水肿以及记录胎儿感染B19的自然史方面具有普遍适用性。
