Chen Long, Yao Qing, Ma Jing, Li Jianning, Zhang Qian, Yang Yi, Li Fang, Sun Yuning
Department of Biochemistry and Molecular Biology, Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, Yinchuan, Ningxia, China.
Department of Respiratory and Critical Care Medicine, General Hospital of Ningxia Medical University, Yinchuan, Ningxia, China.
J Virol Methods. 2014 Jul;203:48-53. doi: 10.1016/j.jviromet.2014.03.009. Epub 2014 Mar 27.
Human bocavirus (HBoV) has been shown to be associated with acute respiratory tract infection in children. The aim of the work was to develop a novel integrated strategy for human bocavirus detection: heminested PCR assay combined with boiling lysis method of samples. The detection limit of the heminested PCR assay was 1.2 copies of a recombinant DNA plasmid, and no cross-reaction with other respiratory viruses or bacteria was observed. By using the integrated strategy, a total of 202 secretions of the lower respiratory tract of children with acute respiratory diseases were collected and tested. The samples were treated and lysed in boiling lysis buffer rather than extracting viral DNA from secretions, then these sample lysates could be templates and tested by heminested PCR assay, and the amplification of HBoV DNA was detected by using agarose gel electrophoresis. The results showed that, only 7 samples were found to be positive by conventional single-round PCR; importantly, the other new 41 samples were positive by heminested PCR assay. Additionally, the genomic viral DNA was extracted from all positive and some negative specimens, amplified, and sequenced. The results were perfectly consistent with those of the integrated strategy. Taken together, these results suggest that the novel integrated strategy (heminested PCR assay combined with boiling lysis method of samples) is a convenient, sensitive, cost-effective and reliable detective method for HBoV detection and will have broad application prospects in clinical diagnosis.
人博卡病毒（HBoV）已被证明与儿童急性呼吸道感染有关。这项工作的目的是开发一种用于人博卡病毒检测的新型综合策略：半巢式PCR检测法结合样本煮沸裂解方法。半巢式PCR检测法的检测限为1.2拷贝的重组DNA质粒，未观察到与其他呼吸道病毒或细菌的交叉反应。通过使用该综合策略，共收集并检测了202份患有急性呼吸道疾病儿童的下呼吸道分泌物。样本在煮沸裂解缓冲液中进行处理和裂解，而不是从分泌物中提取病毒DNA，然后这些样本裂解物可作为模板，通过半巢式PCR检测法进行检测，并使用琼脂糖凝胶电泳检测HBoV DNA的扩增。结果显示，仅7份样本通过常规单轮PCR检测为阳性；重要的是，另外41份新样本通过半巢式PCR检测法为阳性。此外，从所有阳性和部分阴性标本中提取基因组病毒DNA，进行扩增和测序。结果与综合策略的结果完全一致。综上所述，这些结果表明，新型综合策略（半巢式PCR检测法结合样本煮沸裂解方法）是一种用于HBoV检测的方便、灵敏、经济高效且可靠的检测方法，在临床诊断中将具有广阔的应用前景。
