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目的评估聚合酶链反应(PCR)加限制酶分析在婴儿巨细胞病毒(CMV)感染中的应用。方法在巨细胞病毒DNA多聚酶基因中设计一对引物,对CMV进行PCR扩增并选择BstUI进行酶切以鉴别扩增产物。怀疑CMV感染患儿的32份血标本和5份脑脊液标本进行PCR和ELISA法检测。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0·1fg,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37·5%,ELISA为15·7%(P<0·05)。结论PCR加限制酶分析具有特异敏感,快速准确的特点,能提高婴儿巨细胞病毒感染诊断的特异性和敏感性。
Objective To evaluate the use of polymerase chain reaction (PCR) plus restriction enzyme analysis in infants with cytomegalovirus (CMV) infection. Methods A pair of primers was designed in cytomegalovirus DNA polymerase. PCR was performed on CMV and BstUI was selected to identify the amplification products. Thirty-two blood samples and five cerebrospinal fluid samples suspected of CMV infection were tested by PCR and ELISA. Results The PCR product of cytomegalovirus was 592bp. After digested by BstUI, the product was proved to be CMVDNA. The minimum detectable amount of cytomegalovirus was 0.1fg. There was no cross reaction with HBV, fungi, bacteria and human genome. This method was used to detect CMV, 12 blood samples and 1 cerebrospinal fluid in 32 clinical samples and 5 cerebrospinal fluid samples. The blood samples were detected by ELISA, and 5 were positive. The positive rate of PCR was 37.5% and the ELISA was 15.7% (P <0.05). Conclusion PCR plus restriction enzyme analysis is sensitive, rapid and accurate, which can improve the specificity and sensitivity of diagnosis of infant cytomegalovirus infection.