婴儿血中四种疱疹类病毒DNA及特异性IgM抗体检测

目的 建立1种能快速区分4种疱疹类病毒DNA的特异性酶切图谱。方法 在疱疹类病毒高度同源序列DNA多聚酶基因中设计1对通用引物,该引物能同时扩增单纯疱疹病毒Ⅰ型与Ⅱ型(HSVⅠ／Ⅱ)、爱泼斯坦-巴尔病毒(EBV)、人巨细胞病毒(CMV)4种病毒。选择BamHI和BstUI2种内切酶对同一PCR产物进行酶切,建立了能区分4种疱疹类病毒的PCR限制性内切酶片段长度多态性分析(RFLP)法。采用PCR-RFLP和ELISA法,对75例怀疑病毒感染的患儿和38名健康儿童的血标本进行上述4种病毒DNA及IgM抗体检测。结果 4种标准病毒株PCR扩增后产物为510～592bp,经酶切后能区分病毒种类,其最小检出量为0．1fg,与乙型肝炎病毒、新型隐球菌、金黄色葡萄球菌、大肠埃希氏菌和人类基因组：DNA无交叉反应。用该方法检测临床75份血标本,阳性23份,其中CMV、EBV和HSVⅡ感染分别为13、4和5例,HSVⅠ为1例,同时用ELISA法检测该标本,10例阳性,分别为CMV5例、EBV3例和HSVⅡ2例。在38例健康体检儿童的血标本中上述4种病毒DNA及IgM抗体均为阴性。结论 本研究建立的4种疱疹类病毒特异性酶切图谱具有特异敏感,快速准确的特点,能检测到ELISA法不能检测到的疱疹病毒感染。

Detection of four human herpesviruses DNA and virus-specific IgM antibody in blood specimens of infants

OBJECTIVE: To establish a restriction endonuclease pattern which could detect and differentiate four major human herpesviruses by polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), DNA cloning and sequence analysis. METHODS: A pair of primer, which was designed according to sequences in well-conserved regions of the DNA polymerase gene in human herpesviruses, was designed to amplify herpes simplex virus type 1 and 2 (HSVI/II), Epstein-Barr virus (EBV) and cytomegalovirus (CMV). Sequences of the primers are as follows: P(1) (5'-CGACTTTGCCAGCCTGACC-3') and P(2) (5'-AGTCCGTGTCCCCGTAGATG-3'). DNA of four strains of standard herpesviruses were amplified by PCR, and further studied by DNA cloning, sequence analysis and RFLP. At last, the authors established the PCR-RFLP technique to differentiate the four different herpesviruses. Meanwhile, 75 clinical blood specimens from infants with suspected viral infection and 38 blood specimens from healthy children were evaluated for herpesviruses DNA or virus-specific IgM antibody by PCR-RFLP or by ELISA. RESULTS: The PCR amplified products of four human herpesviruses were from 510 bp to 592 bp in length and were analyzed for herpesvirus types with restriction endonuclease technique. The specificity and sensitivity of this PCR-RFLP were examined. There was no cross-reaction with Escherichia coli, Staphylococcus aureus, hepatitis B virus (HBV), Clostridium neoformans and human-genomic DNA and the lowest detection level was 0.1 fg DNA. Among 75 specimens, 23 were positive by PCR and the positive rate was 30.7%, including 13 for CMV, four for EBV, five for HSVII and one for HSVI after restriction enzyme digestion with BamHI and BstUI, while 10 were positive by ELISA and positive rate was 13.3%. All ELISA-positive specimens were likewise positive by PCR. Thirteen of 65 specimens that were ELISA-negative were tested positive by PCR. An infant with CMV infection was determined with viral DNA and virus-specific IgM antibody in blood at 3, 4 and 6 months after birth, respectively. The result showed that she was still CMV DNA-positive in blood whereas IgM antibody was positive only at month 3 after birth. None of the 38 control blood specimens was positive for herpesvirus by this PCR-RFLP or by ELISA. CONCLUSIONS: This PCR-RFLP technique was specific, sensitive, rapid and accurate in diagnosing herpesviruses infection in infants, and it could detect herpesviruses DNA in specimens which were negative for IgM antibody by ELISA.