人呼吸道腺病毒通用特异检测试剂的研究及应用

Development and application of universal and specific diagnostic reagents for human respiratory adenovirus

目的建立一种特异性强、灵敏度高、操作简便的呼吸道腺病毒检测方法，为临床呼吸道腺病毒通用特异诊断试剂的研发奠定实验基础。方法用生物信息学软件DNAMAN5．2．2、GeneRunner3．05、BLAST对GenBank中已收录的10个型别人呼吸道腺病毒核酸全序列进行比对分析，找m高度保守序列，设计5对腺病毒通用特异引物，同时确保PCR产物具有型别特异性。用NP-40裂解法制备模板，对引物的有效性、特异性及灵敏忡进行验证．并应用于64例急性呼吸道感染患儿咽拭子标本的检测，阳性标本的PCR产物直接测序鉴定血清型．将阳性标本接种HeLa细胞，光镜下观察细胞病变，电镜下观察病毒形态。结果BLAST结果表明，5对引物与其他病毒、细菌及人类基因组等无高度同源性，是腺病毒特异性引物。用5对引物扩增人3型腺病毒DNA，均出现阳性目的条带，但不能检测呼吸道合胞病毒和柯萨奇病毒，验证了引物的有效性和腺病毒特异性。模板稀释、病毒稀释均可检测到10。梯度，说明引物具有较高的灵敏性，同时验证了NP-40裂解法的稳定性。64例临床咽拭子标本巾检测出2例阳性标本，阳性率为3．13％（2／64）。阳性标本接种HeLa细胞，光镜下可见细胞变圆、聚集成葡萄串状、脱落等腺病毒所致细胞病变，电镜下可见细胞核内有典型的品格状排列的腺病毒颗粒。PCR产物测序结果表明，2株病毒均为B组腺病毒。结论成功设计了腺病毒通用的、特异的，同时扩增产物又具有腺病毒型别特异性的引物。用该引物建立的PCR方法检测呼吸道标本中的腺病毒DNA具有灵敏和特异的特点，适用于临床常规诊断腺病毒感染。

Objective To establish a specific, sensitive, simple human respiratory adenovirus detection method to put a good experimental foundation for developing universal and specific diagnostie reagents of human respiratory, adenovirus. Methods The nucleotide sequences of 10 serotype of human respiratory adenovirus were obtained from GenBank. Highly conserved five pairs of universal and specific adenovirus primers were designed on the evaluation of multiple sequence alignment of the 10 full genomic sequences with the software DNAMAN 5.2.2, Geue Runner 3.05, BLAST, and to ensure that the polymerase chain reaction（PCR） products were type-specific. NP-40 sample lytic method was employed to prepare the template. The effectiveness, specificity and sensitivity of primers were evaluated. And PCR was carried out to test 64 samples of throat swabs of acute respiratory infection children. The positive PCR products were sequenced directly to identify the adenovirus serotypes. The positive specimens was inoculated on HeLa cells to observe cytopathic effect（CPE） under light microscope, and virus morphology were observed under electron microscope. Results BLAST results indicated that the five pairs of primers were specific adenovirus primers with low homology to the others. The primers were identified as PCR positive fragments obtained by using five pairs of primers to amplify the human adenovirus type 3 DNA, which did not react to the respiratory syncytial virus and Coxsackie virus, the effectiveness and specificity of the primer were thus indicated. PCR sensitive results showed 10^-5 dilutin of adenovirus culture and DNA sample could be detected which meant the method was sensitive and stable. Two PCR positive specimens were detected in 64 clinical samples, the positive rate was 3.13%（2/64）. Using the two PCR positive specimens to inoculate the HeLa cells, the typical adenovirus CPEs of rounded and aggregated, detatched cells were observed under light microscope. And a large number of adenovirus typical particles with characteristic lattice arrangement were observed in the infected cells under electron microscope. PCR product sequencing results showed that these two isolated adenoviruses were typed in human adenovirus group B. Conclusions Universal and specific adenovirus PCR primers with the serotype specificity of the PCR products were designed successfully. The PCR primers was sensitive and specific and could be routinely applied in clinical adenovirus diagnosis for respiratory specimens.