The Value Of Multiplex Real-time Fluorescence Quantitative PCR Detect Human Bocavirus

ObjectiveEstablishing human bocavirusl-3multiplex real-time fluorescence quantitative PCR (multiple Real-time PCR) method, and evaluating the specificity and sensitivity through ordinary PCR comparison and sequencing. Nasopharyngeal aspirate specimens of acute lower respiratory tract infection in hospital of the children from2009to2010is to HBoV detection, sequenced points type by this method. The clinical data of related summary and analysis in order to understand the HBoVl-3type in changsha area acute lower respiratory infection in children popular characteristics; for prevention and control of the respiratory tract infection and further research. lay the foundation.Subjects and Methods1. According to the literature and synthetic HBoVl-3type primers, probes of the gene sequence, determine the reaction system..2. Select3cases HBoVl, HBoV2, HBoV3known positive one copy of the specimen by ordinary PCR amplification, amplification product identified sequencing, purpose gene and carrier connection, plasmid into normal cells and feel the bacteria, extraction plasmid, containing purpose gene sequencing sure the plasmid, evaluation after plasmid quality ten times diluted gradient spare, standard curve production.3、Plasmid were diluted into101-108standard product through the multiple Real-time PCR for amplification, evaluation sensitivity; Select the known one different respectively contain DNA and RNA virus infection10specimens, double or multiple virus mixed specimens, does not contain the virus samples and sterilization deionized water, through the multiple Real-time PCR detected, evaluation specificity; On the same HBoV positive samples simultaneously5repeated measurements for5days of the same sample of repeated determination, the evaluation repeatability.4、Hunan people’s hospital pediatric randomly selected from acute lower respiratory tract infections NPAs hospitalized children samples from multiple real-time PCR and common PCR detection, and to evaluate their detection sensitivity and specificity of clinical specimens.5. NPAs specimens with acute lower respiratory tact infection hospitalized in the hospital of hunan Province, between september1,2009-october31,2010, were enrolled in the study. All of the children were<14years old. Nasopharyngeal aspirate specimens were collected and sent to the Chinese Center for Viral Disease Control and Prevention, China CDC.6. Respiratory specimen after extracting the nucleic acid were detected HBoV by establishing the multiple real-time PCR. At the same time each specimen using RT-PCR and half nested PCR to test the RSV, HRV, IFVA/B, PIV1/2/3, HMPV, HCoV-HKU1, HCoV-NL63, ADV,determination and the sequence.7. HBoV hospitalized children acute lower respiratory infection for analysis in2009-2010in this area.Results1、Multiple real-time fluorescence quantitative PCR different gradient templates to copy Numbers of quantitative numerical and Ct value is good linear relationship between a good relationship, the correlation coefficient were0.999,0.999and0.998, repeatability, batch CV value within0.95%, batch CV value in between102-106copies within the range is0.9%-2%, all less than5%, the method of sensitivity can be up to10/reaction (minimum copy of3.38×10/reaction).Use multiplex real-time fluorescence quantitative PCR to other respiratory virus and that common mycoplasma, chlamydia samples for testing, there were no amplification curve.2、Multiple real-time fluorescence quantitative PCR and traditional PCR parallelly detect HBoV of171NPAs. Among them, the real-time quantitative fluorescence detection HBoV PCR positive27(for four a false positive), ordinary PCR detection of positive16. All the general PCR positive samples are included in real-time PCR positive quantitative fluorescence samples, two methods of testing positive product are sequenced appraisal, multiple real-time PCR can detect fluorescent quantitative HBoVl,2, ordinary PCR detection HBoV1only.3. Nasopharyngeal aspirate specimens from september1,2009-october31,2010, were detected HBoV positive samples in110cases, total detection rate of14.19%. In the110cases were positive, HBoVl107cases were found, the detection rate was13.80%; HBoV23cases, the detection rate was0.39%,; HBoV3not detect. Among them, the males of the children of the detection rate was14.26%, and the detection rate of children of the females was14.07%, the male and female of the infection rate is1.01:1, The detected rate was no significant difference between male and female (X2=0.005, P=0.944). The median ages of the viruses infected children was18months (range from22days to13years old2months). Most of children infected with HBoV diagnosis of bronchopneumonia, bronchiolitis and bronchial asthma with lung infection. Of the110HBoV positive samples,34.5%samples were fond monoinfected with HBOV,65.5%HBoV positive samples were detected coinfected with other respiratory viruses, dual infection of54, triple infection in14cases, four heavy infection4cases, a single HBoV and mixed other virus detection of clinical cases positive is not significant.Conclusions1. The research has successfully constructed the multiple real-time fluorescence quantitative PCR detection HBoV1-3methods.2. In the clinical samples, multiple fluorescence quantitative real-time PCR than ordinary PCR fast, sensitivity.3. Using multiple fluorescence quantitative real-time PCR can detect ALRIT NPAs hospitalized children of the specimen HBoVl and HBoV2.4. This study period, HBoVl detection rate is higher, may is an important virus pathogenic in ALRIT children this area.