Kits Of RT-PCR And RT-Fluorescent Quantitive PCR For Universal Detection Of Bluetongue Virus

Bluetongue (BT), a viral acute disease of ruminant transmitted by some insects, is a World Organization for Animal Health reportable (formerly List A) and is infectious but noncontagious disease of considerable socioeconomic concern and of major importance in the international trade of animals and animal products. BT is a Ministry of Agriculture of the People`s Republic of China listⅠdisease. Bluetongue virus (BTV) is one of the viruses which need to be detected in animal products such as bovine source thrombin, platelet cofactorⅠ, and bovine hemoglobin with mPEG, thymic peptide, and biological product dressing, e.g. bovine serum, bovine source gelatin, and bovine source collagen and so on.BTV belongs to the family Reoviridae, genus Orbivirus. BTV has a global distribution, and there are at least 24 serotypes of BTV worldwide. As global air temperature become warmer, many countries broke out BT from 2006 through 2008, which broadens the distribution of BTV and is emerging the characteristic of caused by new serotype in some area.To monitor and control the disease and to detect it reliably, it is very urgent to develop universal viral nucleic acid detecting techniques and viral nucleic acid detecting kits for different serotypes of BTV.Bioinformatics methods such as computer software, network database and software on line were employed to obtain some conservative genes of BTV, which were compared and analyzed. The most conservative gene segment of BTV was found, to which a pair of primers of polymerase chain reaction (PCR) and a TaqMan fluorescent probe of fluorescent PCR were designed according. The features and the homologies of the primers and the probe were analyzed. And the results showed that the primers and the probe were special, then, had no homologies with other viruses.BTV-1 to 22 reference strains and Epizootic hemorrhagic disease virus (EHDV) serotype 5 were cultured in BHK-21 cells. BTV positive reference substances and EHDV negative reference substances were established after these viruses were identified through cytopathic effect (CPE), viral morph and enzyme linked immunosorbent assay (ELISA). Recombinant plasmid and in vitro transcription RNA were prepared and identified through molecular biology techniques. Then recombinant plasmid and in vitro transcription RNA positive reference substances were established respectively, and diethyl pyrocarbonate (DEPC) water was established for blank negative reference substances.The special pair of primers and these reference substances was used to reverse transcription (RT) PCR for BTV detection. The suitable detection system was determined through some tests and evaluated through specificity tests, sensitiveness tests, repeatability tests, coincidence tests, rise and fall regular tests, serum samples detection and stability tests etc. Results showed that: (1) The RT-PCR detection system for various serotypes of BTV was special, and had no cross reaction with EHDV and 6 common viruses in bovine and sheep. 15 negative serum samples were negative. (2) The RT-PCR detection system was sensitive. 20 mock BTV infected bovine serum samples were detected, and 20 were positive and the positive rate was 100%. 20 BTV antibodies serum samples were detected, and the positive rate was 90%. Limit of detection was 102 copies of RNA molecules, and 1 TCID50/mL BTV. (3) The repeatability of the RT-PCR detection protocol was good. Strong positive, limited positive and negative reference substances were detected repeatedly. The positive rates and negative rates in these tests were 100%. (4) In coincidence tests, a same kind of the duplex RT-PCR assay was developed firstly. BTV-1 to 22 reference strains and EHDV-5 reference strain were detected through the RT-PCR and duplex RT-PCR, results showed, that the coincidence was 95.65% and 22 serotypes of BTV were detected in the RT-PCR but 21 serotypes of BTV were detected in the duplex RT-PCR. EHDV was negative in these two assays. (5) In rise and fall regular tests, BTV reference strains cultured in 72 h were detected through RT-PCR methods. (6) 40 serum samples of sheep were tested through the RT-PCR system and Bluetongue Virus (BTV) Direct FA Conjugate, and the results were coincidence. (7) The BTV RT-PCR detection kit should be preserved in -20℃for 6 months according to the results of stability tests.On the basis of preclinical tests of BTV RT-PCR nucleic acid detection kit, we had made Bluetongue Virus RT-PCR Nucleic Acid Detection Kit Manufacture and Check Protocol, Quality Standard Protocol, Compiling Description, Mark Manuscript and so on. According to the these protocols et al, 5 lots productions of Bluetongue virus RT-PCR nucleic acid detection kits had been prepared, and up to standard. Then, we had contrived the clinical trial scheme, had signed clinical trial contracts with 2 institutions. On account of some regulations and commands of National Ministry of Agriculture, we had compiled Application Document for Clinical Trial of Bluetongue Virus RT-PCR Nucleic Acid Detection Kit, had submitted it to National Ministry of Agriculture, and the application had been approved.The TaqMan fluorescent probe, primers and the reference substances was used to RT fluorescent PCR (FPCR) for BTV gene detection. The RT-FPCR detection system was determined through some tests and evaluated through specificity tests, sensitiveness tests, repeatability tests, coincidence tests, rise and fall regular tests, serum samples detection and stability tests and so on. Results indicated that: (1) The RT-FPCR detection system for BTV groups was special, showing no cross reaction with EHDV and common viruses in bovine and sheep. 15 negative serum samples were detected negatively. (2) The RT-FPCR detection system was sensitive. 20 mock BTV infected bovine serum samples were detected positively, and the positive rate was 100%. 20 BTV antibodies serum samples were detected, and the positive rate was 95%. Limit of detection was 102 copies of RNA molecules, and 1 TCID50/mL BTV. (3) The repeatability of the RT-FPCR detection protocol was good. The CV of Ct values of strong positive were 2.17, 2.18, 3.66 intra-tests repeatedly, 3.23 inter-tests, and the CV of negative reference substances detected was 0. The CV of Ct of limited positive reference substances detected in RT-FPCR was 2.96, 4.96, 4.43 intra-tests, 4.04 inter-tests. (4) BTV-1 to 22 reference strains and EHDV-5 reference strain were detected through the RT-FPCR and the duplex RT-PCR in coincidence tests, and the coincidence was 95.65%. 22 serotypes of BTV were detected in the RT-FPCR but 21 serotypes of BTV were detected in the duplex RT-PCR. EHDV was negative in these two assays. (5) Results of rise and fall regular tests showed, BTV-8 reference strains cultured in 36 h and BTV-1 reference strains cultured in 48 h were detected positively through RT-FPCR methods. (6) Applying the RT-FPCR system and Bluetongue Virus (BTV) Direct FA Conjugate to detect 40 serum samples of sheep, the results were coincidence. (7) The results of stability tests indicated that the BTV RT-FPCR detection kit should be preserved in -20℃for 6 months.According to the preclinical tests of BTV RT-FPCR nucleic acid detection kit, we had made Bluetongue Virus RT-FPCR Nucleic Acid Detection Kit Manufacture and Check Protocol, Quality Standard Protocol, Compiling Description, Mark Manuscript and so on. Then, 5 lots productions of Bluetongue virus RT-FPCR nucleic acid detection kits had been prepared, which was up to standard. So, we had contrived the clinical trial scheme, had signed clinical trial contracts with 2 institutions. On account of some regulations and commands of National Ministry of Agriculture, we had written Application Document for Clinical Trial of Bluetongue Virus RT-FPCR Nucleic Acid Detection Kit, had submitted it to National Ministry of Agriculture, and the application had been approved.Conclusion: The preclinical studies on Bluetongue Virus RT-PCR Nucleic Acid Detection Kit and Bluetongue Virus RT-FPCR Nucleic Acid Detection Kit have finished successfully. The applications for clinical tests of these two products have approved, which drives the studies up effectively.