Comparative Study Of Four Techniques For The Detection Of Bluetongue Virus

Bluetongue disease(BT) is a viral acute disease of ruminant transmitted by Culicoides. Bluetongue virus (BTV) belongs to the family Reoviridae, genus Orbivirus. Its outbreaks have serious socio-economic consequences in international trade of animals and animal products, but it does not have a highly effective vaccine and control measures at present. In this case, it is necessary to strengthen our monitoring of BT. Early and accurate detection can timely control and treat infected animals, and it is the key to reduce economic losses.Our laboratory had established Bluetongue Virus nucleic acid real-time fluorescent RT-PCR detection system, nucleic acid RT-PCR detection system and antigen-captured ELISA detection system, each of them was evaluated through specificity tests, sensitiveness tests, repeatability tests and coincidence tests etc, so the main content of this study was to detect BTV-5 using the above detection systems and OIE standard nested RT-PCR detection method, and to have a comparative analysis of results. The aim of study is to provide reliable experimental guide for the detection of clinical samples, and to make timely and effective diagnosis of BT.1. The culture of BTV-5BTV-5 reference strain stored in our laboratory was cultured in BHK-21 cells. These viruses were identified through cytopathic effect (CPE), virus morphology and RT-PCR. The positive BTV-5 culture harvest was measured by titer determination test, and TCID50 was 10-2.83/0.1mL. Used DMEM culture medium to dilution(1:22~1:221) as BTV positive test samples. The Epizootic hemorrhagic disease virus(EHDV)-5 standard reference strain was used as EHDV reference substances.2. Evaluation of Four BTV detection systems to BTV-5The nucleic acid real-time fluorescent RT-PCR detection system, nucleic acid RT-PCR detection system, OIE standard nested RT-PCR detection method and antigen-captured ELISA detection system were evaluated through specificity tests and sensitiveness tests. The results showed that: (1) The nucleic acid real-time fluorescent RT-PCR detection system was special, and had no cross reaction with EHDV-5. Limit of detection was 3.3TCID50/mL. (2) The nucleic acid RT-PCR detection system was special, and had no cross reaction with EHDV-5. Limit of detection was 3.3TCID50/mL. (3) OIE standard nested RT-PCR detection method was special, and had no cross reaction with EHDV-5. Limit of detection was 6.4×10-3TCID50/mL. (4) The antigen-captured ELISA detection system was special, and had no cross reaction with EHDV-5. Limit of detection was 211.3TCID50/mL.3. The comparative analysis of Four BTV detection systemsThe test data were collected to evaluate BTV detection systems through specificity, sensitivity, time-consuming and instrumentation. The results showed that: (1) All four detection systems were specificity with no cross-reaction with EHDV-5. (2) The nucleic acid detection systems (nucleic acid real-time fluorescent RT-PCR detection system, nucleic acid RT-PCR detection system, OIE standard nested RT-PCR detection method) were more sensitive than antigen-captured ELISA detection system. The detection limit gap was 2 orders of magnitude at least. (3) Compared with antigen-captured ELISA detection system, nucleic acid detection systems were time-consuming and complicated, required excellent equipment situation. (4) Compared with the other nucleic acid detection systems, nucleic acid real-time fluorescent RT-PCR detection system was simple, without complicated electrophoresis. It used detector to complete the entire process from PCR amplification to signal acquisition, so it reduced the chance of false positive. (5) OIE standard nested RT-PCR detection method used two pairs of PCR primers and two rounds of PCR amplification to improve the specificity and sensitivity, but it increased the complexity and testing time at the same time.