套式聚合酶链反应及限制酶分析对孕早期孕妇巨细胞病毒感染的研究

A STUDY ON HUMAN CYTOMEGALO VIRUS (HCMV) INFECTION IN PREGNANT WOMEN OF THE FIRST TRIMESTER BY NESTED POLYMERASE CHAIN REACTION(PCR)

在人巨细胞病毒（ＨＣＭＶ）－ＡＤ＿（１６９）株直接早期基因的ＥｃｏＲＩＪ片段内，自行设计二对引物，建立套式聚合酶链反应（ＰＣＲ）加限制酶分析检测ＨＣＭＶＤＮＡ，经实验证明有高度的特异性与敏感性，对其它疽疹类病毒及正常人基因组ＤＮＡ无交叉反应。一次性ＰＣＲ检测ＨＣＭＶ－ＡＤ１６９株基因组的最小检出量为１００ｆｇ，而套式ＰＣＲ可达１０ｆｇ。经ＰｓｔＩ酶切后，得到的片段与设计相符。对５８名孕早期孕妇外周静脉血进行ＨＣＭＶＤＮＡ、病毒分离、特异性ＩｇＭ及ＩｇＡ检测，结果阳性率分别８．６％、６．９％、１．７％及３。４％。２８例死胎组织中，发现一死胎肺组织ＨＣＭＶＤＮＡ阳性，与病毒分离相比，套式ＰＣＲ的特异性，敏感性及符合率分别达到１００％，９８．１％及９８．３％。提示：套式ＰＣＲ能提高诊断ＨＣＭＶ感染的特异性与敏感性，适合于孕早期ＮＣＭＶ感染的监测。

ested PCR was developed to detect conserved sequence from the immediate early geneof HCMV. The two primers were from EcoRI J fragment of AD169.The experimentsshowed that nested PCR has highly specificity and sensitivity. There was no cross-reactionwith human DNA or with DNA from other herpes viruses. With single PCR,it was possibleto detect 100fg HCMV DNA,but with nested PCR,10fg were detectable,By Pst I endonu-clease analysis,the fragment was in accordance with the designed one, 58 peripheral venousblood samples of gravida in the first trimester were analysed by nested PCR,virus isolationand seriological tests. The results showed that the positive rate of HCMV DNA, virus isola-tion,anti-HCMV-IgM and anti-HCMV-IgA were 8.6%,6.9%,1.7%and 3.4%,re-spectively. HCMV DNA was found in the lung tissue of one among 28 stillbirths.The speci-ficity、sensitivity and agreement of nested PCR were 100%, 98.1%, and 98.3%,respective-ly, when virus isolation was used as a reference method. All these suggest that the diagnos-tic specificity and sensitivity can be raised by nested PCR, which is helpful in finding HCMVinfection in pregnant women of the first trimester.