Takeuchi S, Itoh N, Uchio E, Aoki K, Ohno S
Department of Ophthalmology, Yokohama City University School of Medicine, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan.
J Clin Microbiol. 1999 Jun;37(6):1839-45. doi: 10.1128/JCM.37.6.1839-1845.1999.
To detect and identify adenovirus (Ad), we investigated hypervariable regions (HVRs) of Ad by using a combination of PCR and direct sequencing (PCR-sequence) method. Primers for nested PCR to amplify the conserved region in the hexon protein containing HVRs were designed based on hexon gene sequences derived from GenBank. These two primer sets amplified a DNA fragment of 7 HVRs from 16 prototypes of Ad, which were divided into five subgenera, including seven serotypes that are the predominant causative agents of acute conjunctivitis in Japan, and from 31 recent conjunctival scraping specimens from patients with adenoviral conjunctivitis. HVR DNA sequences were determined by means of universal sequence primers. Analysis of the predicted amino acid homology of HVRs among Ad prototypes suggested three regions, HVR4, -5, and -7, to be candidates for the neutralization epitopes. The clinical serotype of specimens was determined by the PCR-sequence method with reference to these three HVRs. The serotype determined according to this method was identical to that obtained by culture isolation and the neutralization test (NT) in all scraping samples, whereas the results of this method did not match PCR and restriction fragment length polymorphism (PCR-RFLP) analysis in five samples. It took only three days to detect Ad and to identify the serotype, in contrast to culture isolation-NT, which took at least 2 weeks. These findings indicate that our newly developed PCR-sequence method is applicable for the detection and serotyping of human Ads.
为了检测和鉴定腺病毒（Ad），我们采用聚合酶链反应（PCR）与直接测序相结合的方法（PCR测序法）对腺病毒的高变区（HVRs）进行了研究。基于从GenBank获得的六邻体基因序列，设计了用于巢式PCR扩增包含高变区的六邻体蛋白保守区的引物。这两组引物从16种腺病毒原型中扩增出了包含7个高变区的DNA片段，这些原型被分为五个亚属，其中包括在日本急性结膜炎的主要致病血清型中的7种血清型，以及来自31例腺病毒性结膜炎患者近期结膜刮片标本。通过通用序列引物测定高变区DNA序列。对腺病毒原型之间高变区预测氨基酸同源性的分析表明，高变区4、-5和-7这三个区域是中和表位的候选区域。参照这三个高变区，采用PCR测序法确定标本的临床血清型。根据该方法确定的血清型与所有刮片样本通过培养分离和中和试验（NT）获得的结果一致，而在五个样本中，该方法的结果与PCR和限制性片段长度多态性分析（PCR-RFLP）不匹配。检测腺病毒并鉴定血清型仅需三天，相比之下，培养分离-中和试验至少需要两周时间。这些结果表明，我们新开发的PCR测序法适用于人类腺病毒的检测和血清分型。
