婴儿巨细胞病毒感染的分子生物学诊断研究

Molecular Diagnostic Study on Human Cytomegalovirus Infection in Infants

目的评价婴儿巨细胞病毒感染中聚合酶链反应(PCR)加限制酶分析的应用价值。方法在巨细胞病毒(CMV)DNA多聚酶基因中设计一对引物,对CMV进行PCR扩增、分子克隆、序列分析后,选择BstUI进行酶切以鉴别扩增产物。结果巨细胞病毒PCR扩增后产物为592bp,经BstUI酶切后证实为CMVDNA,其最小检出量为01fg,与乙肝病毒、真菌、细菌和人类基因组无交叉反应。用该方法检测临床32份血和5份脑脊液标本中的CMV,12份血和1份脑脊液标本阳性;同时用ELISA法检测血标本,5例阳性。PCR阳性检出率为375%,ELISA为156%(P<005)。9例诊断为CMV感染患儿经更昔洛韦治疗10～14天后,复查外周血巨细胞病毒DNA,有1例仍为阳性。结论PCR加限制酶分析能提高婴儿巨细胞病毒感染的诊断率。更昔洛韦可作为抗CMV感染的有效药物。

Objective To evaluate the diagnostic value of polymerase chain reaction combined with restriction fragment length polymorphism (RFLP PCR) in the diagnosis of human cytomegalovirus (HCMV) in infants.Methods A pair of primers was designed located at the DNA polymerase gene position of HCMV. After the PCR amplification, molecular cloning and sequencing analysis, the product was discriminated by RFLP with the restriction enzyme BstUI.Results The PCR product was 592bp long and proved to be HCMV DNA by BstUI digestion. There was no cross reaction with HBV, fungus, bacterium and human genomic DNA, and the lowest detection level was 0 1fg. 12 among 35 blood specimens and 1 among 5 cerebral spinal fluid (CSF) specimens were positive using this method, while using ELISA to detect HCMV only in blood specimens, there were 5 blood specimens occurring positive reaction. The positive detection rates of these two methods were 37 5% and 15 6%, respectively. After treating with ganciclovir for 10～14 days, there was only 1 infant with HCMV positive among 9 definite patients.Conclusions Using RFLP PCR can raise the detection rate of HCMV diagnosis, and ganciclovir is an effective drug against HCMV infection.