HMA-SSCP方法研究人类巨细胞病毒UL144基因在临床低传代分离株中的多态性

Variability of human cytomegalovirus UL144 gene in low-passage clinical isolates analyzed by HMA-SSCP

目的探讨人类巨细胞病毒(HCMV)UL144序列在临床患儿低传代分离株中的多态性及与临床疾病的关系。方法对65株HCMV临床低传代分离株及7例同年龄组HCMV-DNA定量PCR方法检测阳性无症状感染儿尿液进行HCMV-UL144PCR扩增及HMA-SSCP分析,并对其中32份阳性标本进行测序。结果65株分离株中有55株UL144全序列引物PCR扩增阳性,7份QPCR检测HCMV-DNA阳性无症状感染儿尿液中5份UL144全序列引物PCR扩增阳性。60份UL144扩增阳性标本HMA-SSCP(异源双链泳动及单链构象多态分析)呈现3种典型带形,巨结肠患儿分离株序列、小头畸形患儿的序列分布以1型为主,巨结肠患儿分离株序列没有2型,黄疸患儿以3型为主。结论HCMV-UL144广泛存在于临床低传代分离株中,用HMA-SSCP检测HCMV-UL144基因在临床低传代分离株中的多态性是一种可行的方法。HCMV不同疾病类型的HCMV-UL144序列不同,提示UL144基因可能对HCMV致病性起一定作用。

Objective Human eytemegalovirus （HCMV） infection is an important infectious agent that results in neonatal disease and congenital deformity. HCMV infection may affect in many organs. The different symptoms and tissue tropism of HCMV infection perhaps resulted from the genetic polymorphism of HCMV. HCMV UL144 open reading frames encode a homoiogue of the tumor necrosis factor receptor. It seems important to study the strain-specific variability of UL144 sequence in low-passage clinical isolates and to discuss if the variability related to the clinical HCMV infection. Methods HCMV-UL144 gene was amplified by PCR assay in 65 low- passage clinical isolates and urine from 7 healthy children who were HCMV-DNA positive by quantitative PCR. All the positive PCR products were analyzed by Hetereduplex mobility assay and single-strandad conformation polymorphism （HMA-SSCP） and 32 of them were sequenced. Results Fifty-five isolates and 5 urine specimens were HCMV-UL144 positive by PCR. Sequencing and HMA-SSCP analysis showed that significant strain-specific variability was present in the UL144 ORFs. Comparing UL144 sequences and the corresponding symptoms showed that genotype 2 did not exist in megacolon isolates. And genotype 1 and 3 were the major types among micreeephaly and jaundice isolates respectively, Conclusion HCMV-UL144 existed in almost all the low passage isolates. HMA-SSCP assay is an easy and effective method to detect the genetype of HCMV-UL144 sequence, The characteristic of sequences in different isolates showed that UL144 gene may play an important role in HCMV infection.