| Transfusion saved countless lives of patients , but also bring patients the potential risk of being infected with transfusion transmitted diseases, among which, the most serious consequences are infections of hepatitis viruses or HIV . So in our country, blood donor screening is an obligatory course which including: the detection of Hepatitis B Virus (HBVX Hepatitis C Virus (HCV) and Human Immunodeficiency Virus ( HIV) with enzyme immuno-assay. Yet even eligible blood tested by present method might still be infectious. 90% of the undetectable infectious blood is due to the existence of "window period" nowadays. In order to minimize the undetectable infectious blood units due to "window period", it is regard as a trend that the blood donor screening should be conducted with certain proper nucleic acid amplification techniques (NATs). As a high sensitive and specific nucleic acid amplification technique, Polymerase Chain Reaction (PCR) has been chosen as todetect nucleic acid of pathogens among blood donors. Unfortunately, the expensive cost and excessive demands to the lab and the operator restrict its application in practice greatly. In order to overcome the restriction rise from economic aspect, the nucleic acid detection of mixed plasma pool is developed, but it is difficult to dispose the blood units and the donors when self-contradictory results occurs in the course and also it might lengthen the time span of detection. An alternative PCR method is multiple PCR, which means that more than one pair of primers are added to a PCR reaction system, and multiple transfusion related pathogens could be detected simultaneously. The joint detection of HBV, HCV and HIV using this method has been reported, among which the templates are extracted from serum. Such kind of detection cannot ensure the safety of whole blood, since HBV DNA and HCV RNA may also exist in peripheral blood mononuclear cells(PBMCs) and the existence does not depends on the appearance of pathogen nucleic acid in serum. So a more reliable method is to make a joint detection of pathogen nucleic acid from whole blood. Our research is to explore and improve the disposal and preservation of whole blood, the extraction of template, the selection of enzyme and the optimization of the amplifying conditions so as to establish a rapid, economic, valid and reliable joint detection of HBV DNA and HCV RNA from whole blood, which is a base to make a final joint detection of transfusion related pathogens from whole blood infuture. METHODS:1. Blood samples are collected from liver disease outpatients of infectious disease department of the first teaching hospital, Zhengzhou University.2. The preparation of HCV RNA: Three different methods are used as bellows :(1) routine method provide by commercial kit; (2) precipitation with Catrimox-14 (3) PBMCs separation-lysis-precipitation. These methods are used in the preparation of template from serum, whole blood and PBMCs respectively.3 . Amplification of HCV RNA: Three different reaction systems are used to accomplish the Reverse Transcription-nested Polymerase Chain Reaction (RT-nested PCR): (1) reaction mix (provided by commercial kit) +RT mixed enzyme+ Taq DNA polymerase; (2) reaction mix (self confect) +RT mixed enzyme+Taq DNA polymerase; (3) reaction mix (self confect) +Tth DNA polymerase +Taq DNA polymerase;4. Amplification of the first round PCR product by rapid PCR.5. Preparation of HBV DNA template: lysis-boiling method is used to extract template from serum; Catrimox-14+co-agent is used to extract template from whole blood.6. Amplification of HBV DNA template: Two different reaction systems are used to amplify HBV DNA template: (1) reaction mix(provided by kit) + Taq DNA polymerase; (2) reaction mix (self-confect) +Taq DNA polymerase. Routine PCR and rapid PCR are used respectively.7. Simultaneous detection of HBV DNA and HCV RNA from whole blood: choose a HBV DNA positive sample and a HCV RNA positive sample, mix the two samples together with vo... |
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