Establishment Of Simultaneous Detection Of HBV, HCV And HIV-1 With Multiplex Nested PCR

BackgroundWith the development of molecular biology techniques and the further understanding to the virus which can cause human diseases, the transfusion transmitted virus occupies a pivotal position, and HBV, HCV and HIV become the main factors threatening the safety of blood. In order to ensure the safety of blood transfusion, the appropriate test items and methods of blood screening are determined by every country in accordance with its national condition.In China, "donor health check-standard" provides that hepatitis B virus surface antigen (HBsAg), hepatitis C virus antibody (HCV antibody) and HIV antibodies (HIV antibodies) can be detected by ELISA (enzyme-linked immunosorbent assay). However, because of the long "window period", virus mutation, immunosilent infection, manual errors and other factors, the transfusion transmitted virus infection still exists.PCR-detection technology, on behalf of NAT (nucleic acid test), is a new detection method of transfusion-transmitted diseases. With the high sensitivity, the trace amounts of nucleic acid can be detected by PCR, and the "window period" is reduced greatly. In addition, the infected blood which was undetected by ELISA can detected by NAT. Although the "window period" can not eliminated completely by NAT in theory, the possibility of the infectious blood is very low before the virus nucleic acid become positive, so you can effectively control the spread of viral diseases by blood transfusion.As a routine blood screening method, there are a number of issues in PCR. To reduce the cost, while ensuring the sensitivity, the plasma pool strategy (MP-NAT) is often taken. However, the more of the number of the plasma pool samples, the lower of the sensitivity of nucleic acid amplification detection. Another better approach is multiplex PCR. With multiplex PCR, a variety of DNA in the same reaction tube can be detected simultaneously, which can not only save precious experimental samples, but the operation can save time and effort, and many conventional PCR could be achieved through the multi-PCR. Compared with conventional PCR, the number of the affecting factors is more. Therefore, it's needed to optimize the reaction conditions repeatedly.ObjectiveThe design of this study was based on the whole, to build a fast, economical, practical, reliable and synchronous detection of HBV, HCV and HIV-1 approach, both to effectively ensure the safety of blood transfusion, but also reduce costs.Material and Methods1 Specimen Source:They were mainly from the First Affiliated Hospital of Henan Province, Zhengzhou University, Division of Infectious Diseases laboratory and the HIV-infected persons who were from a village of blood-borne dissemination in Henan Province.2 Choice of viral nucleic acid extraction method:First, with the viral DNA/ RNA Purification Kit, HBV DNA/HCV RNA were extracted, then the separate amplification reaction conditions were determined. Second, with three different nucleic acid extraction methods, HBV DNA/HCV RNA were extracted, by comparing the results, a fast, economical and practical method of viral nucleic acid extraction were determined.3 To determine simultaneously amplified HBV DNA and HCV RNA conditions: With the identified nucleic acid extraction method, HBV DNA and HCV RNA were extracted, and then, the synchronization amplification conditions were determined.4 To determine simultaneously amplified HBV DNA, HCV RNA and HIV-1 RNA conditions:On the base of determined reaction conditions of the amplification of HIV-1 RNA, simultaneous detection of HBV, HCV and HIV-1 multiple nested PCR reaction conditions were determined.Results1 Compared with four different nucleic acid extraction methods, TRIzol was found the best, the kit and the lysis buffer were quite effective, Catrimox-14 was poorer than the other. But TRIzol needs low-temperature centrifugal, and the time is long; kit is expensive; Catrimox-14 still need to be improved; the lysis buffer can be used as the viral DNA and RNA extraction method.2 When amplifying HBV DNA and HCV RNA simultaneously with multiplex nested PCR, the first time, the amplification of HBV was dominant, so the proportion of the primers of HCV and HBV was 2:1; The second time, HCV accounted for a advantage, so the proportion of the primers of HCV and HBV was 1:2.3 When amplifying HCV RNA, HBV DNA and HIV-1 RNA simultaneously with multiplex nested PCR, the primer concentration ratio of HCV, HBV and HIV-1 in the first amplification was 2:1:4; in the second amplification, the primer concentration ratio was 1:2:4, thus the HIV-1 fragments could be effectively amplified.Conclusions1 Extracting HBV DNA, HCV RNA and HIV-1 RNA simultaneously with the lysis buffer is feasible.2 With Multi-PCR, the effect of reducing the primers of the higher efficiency of the fragments is better than that of increasing the primers of the lower efficiency of the fragments.3 HBV, HCV and HIV-1 can be detected by the method of this study simultaneously, but its sensitivity and specificity need to be further identified.