Establish Real-Time PCR Method To Detect Human Parvovirus B19 In Serum

The parvovirus B19 is an erythrovirus included in the family of Parvoviridae. Transmission of B19 occurs via the respiratory route, through blood-derived products administered parenterally, or vertically from mother to fetus. The parvovirus B19 was known as the single pathogenic virus among Parvoviridae before the finding of PARV4, PARV5 and the human bocanvirus. In healthy immunocompetent children, B19 is the cause of erythema infectiosum (also called the fifth disease), an innocuous rash illness. When infection occurs in adults, it will appear with acute symmetric polyarthropathy and transient aplastic crisis. Likewise, the pregnant woman who was infected with B19 would subject to hydrops fetalis or fetal death in utero. In immunocompromised host, infection with B19 will manifest as chronic anemia, ephemeral aplastic crisis and acute lymphatic leukemia. So it's necessary to establish an efficient method for B19 detection.Examination methods for B19 mainly include serologic detection and PCR, identifying B19 antibody or virus DNA in serum, respectively. The PCR methods can be classified as single-step PCR, nest PCR, Real-time PCR, and so on. The Real-time PCR is especially used for checking out nucleic acid because of its sensitivity, specificity, accuracy, real-time detection and high-flux. For the competitive comparison in reaction system, competitive PCR can steer the discrepancies of amplifiable efficiency among reaction so it makes the detective consequence more creditable.In this study, the competitive Real-time PCR method was established for B19 detection, and utilized in the examination of B19 in human sera. The genome of B19 was also cloned from a positive sample for future virological research.Establishment of Real-time PCR method for B19 detectionObjective:to establish Taqman Real-time PCR method and apply it in serum B19 examination. Methods:Firstly, we chose a reliable virus nucleic acid extraction method through comparing experiments among 4 biological kits. A pair of PCR primers was then designed and synthesized. Two probes labeled with different fluoresceins were also synthesized for positive sample or internal control examination, respectively. The predicted PCR product was 113 bp in length. Due to lack of positive sample at the beginning, the 113 bp PCR product was synthesized with overlap-extension PCR method for constructing the standard curve. Another 113bp DNA was also synthesized similarly, which was different from the positive fragment in the probe binding site and used as the internal control. Results:The standard curves for positive reaction and the internal control were constructed respectively. The internal control DNA was quantified and added to the serum sample just before nucleic acid extraction. The Real-time PCR was established and applied in B19 detection in human serum samples. Two positive specimens were found and conformed by routine PCR method. Conclusion:Taqman PCR with internal control was established successfully and applied in B19 detection.Cloning of B19 genomeObjective:to clone B19 genome. Method:B19 genome sequence (NC₀00883) were analyzed with software pDraw32, and then 6 restriction enzyme single-cutting sites was labeled, which divided the whole genome into 7 fragments about 800 bp in length for each. Primers around these restriction sites were designed and synthesized to amplifying the 7 fragments. PCR products were purified after agarose gel electrophoresis and inserted the T vector (pMD-18T). Following sequencing, these B19 fragments were excised from T vector and ligated together sequentially to form a full length genome. Result:we cloned B19 genome fragments except LTR regions. Finally, a plasmid, pMD-B23456, were successfully constructed, which contained all B19 genes.Conclusion:Real-time PCR method for B19 detection has been established, which was reliable and sensitive. Two B19-postive samples were found from 160 serum specimens. Genome of one B19 strain was cloned from one of the two positive samples.