A novel integrated strategy for detection of human bocavirus based on a heminested PCR assay combined with boiling lysis method of samples in human specimens.

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.