One-time Multiplex Pcr Method Detected Serum Hbv Dna, Hcv Rna And Hdv Rna

Polymerase Chain Reaction(PCR)technique has been widely used in detecting different hepatitis viruses in laboratory, because of its high specificity and sensitivity. Hepatitis B virus(HBV) , hepatitis C virus(HCV) and hepatitis D virus (HDV) are the major causative agents of Posttransfusion hepatitis (PTH) . They are all transmittedparenterally through human blood and blood products. Concurrent infections of all these three viruses are possible in patients and they can put patients into more critical conditions. It is difficult to distiguish these three hepatitis in clinical symbols and biochmical results . Using conventional HBV DNA PCR , HCV RNA RT-nested PCR or HDV RNA RT-PCR methods to detect HBV ,HCV and HDV separately in one serum sample is limited by their labor-intensity , time-cost and also increase the potential for contamination.In order to study HBV , HCV and HDV infections, occurring alone or in combination, we developed a combined two-step PCR method to detect HBV DNA , HCV RNA and HDV RNA in a single serum specimen from patients or blood donors. We designed four pairs of oligoprimers, one from the HBV preC/C region, one from the HDV ORF₅ region,the other two from the HCV 5' -UTR. The primers could amplified a 418bp,270bp or 121bp DNA fragment from HBV , HDV or HCV gene, respectively. We extracted the templates of these three viruses from the same serum sample simultaneously using the guanidinium isothiocyanate technique. The oligoprimers for HBV , HDV were added into the same reation tube together with an external set of oligoprimers for HCV,then the reverse transcription and the first-round PCR amplification were carried in that tube,and the products were subjected to a second round of PCR with the same primers for HBV and HDV,and an internal set of primers for HCV. The concentration of PCR reagents and the condition of PCR circulation were different between the two PCR rounds. The amplification reaction mixture could be detected and distinguished by a 2 % agrarose gel electrophoresis accordingto their molecular sizes. Specificity of HBV and HCV PCR products were confirmed on the basis of Southern Blot hybridization.To evaluate the sensitivity and specif ity of the combined PCR method,we collected 94 serum samples from Anhui province and Beijing No. 2 Hospital of Inf ecious Diseases. These samples were detected by conventional HBV DNA, HCV RNA RT-nes ted PCR > HDV RNA RT-PCR methods and our combined PCR method, the concordance between results of combined method and those of conventional PCR methods in patients with any hepatitis virus infections was 98.9%. It indicates that our combined PCR method is as sensitive and specific as the conventional PCR methods. The analysis of the results of these serum samples is also significant in the clinical epidemiology. Among the samples of anti-HCV positive, the positive ratio of HBV DNA was 2 6.3%, The ratio of HBV DNA , HCV RNA and HDV RNA all positive from these 94 samples was 3.2%, which indicated that the combined infections are not occassional but are representative in the clinical epidemiology.The combined HBV DNA , HCV RNA and HDV RNA Multi-PCR method is a easy-operating , sensitive , specific , rapid and cost-effective method,especially suited for epidemiological screening and clinical diagnosis of HBV ?? HCV and HDV infections occurring along or in combination. It is available for hospitals to assess the cure effect of combined infections of hepatitis viruses which are increasely concerned by the medical world and for laboratories to research them.