Establishment And Evaluation Of Bio-bar Codes Assay System And Fluorescent Quantitative Bio-bar Codes Assay System For BTV

Bluetongue (BT), an acute contagious disease of ruminant transmitted by some insects, is a World Organization for Animal Health reportable and is listed as I disease by China. Bluetongue virus (BTV) is one of the viruses which need to be detected in animal products such as bovine source thrombin, platelet cofactor I, bovine hemoglobin and thymic peptide, and biological product dressing including 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 becomes warmer, BT has broken out in many countries from 2006 to 2008, which broadens the distribution of BTV and new serotypes are emerging in some area.To monitor and control the disease and to detect it reliably, it is very urgent to develop universal high sensitive detecting techniques and corresponding detecting kits for different serotypes of BTV.BCA (bio-bar codes assay) is a kind of new labeling immunologic assay technique. its conspicuous feature is the exellent detection sensitivity. The principle of this technique is similar to double antibody sandwich ELSIA, through which we can capture the target. Then by detecting special bar code DNA, we can detect the target indirectly. Since the magnifying effect of PCR and chip-based detection, the detection sensibility is six orders of magnitude more sensitive than the ELISA method.FQ-PCR(fluorescent quantitative PCR) is based on detection of a fluorescent signal produced proportionally during the amplification of a PCR product, and we can quantitate the onset target by analyzing Ct value and standard curve.After studying the BTV detection status quo, we have established the ordinary BCA system and FQ-BCA system of BTV, and have evaluated the sensitivity, specificity and reproducibility of the two detection system.The study is described in 7 parts:1. Preparation, purification and identification of NP probe Au nanoparticle was manufactured through reduction method with trisodium citrate, and identified through colar, TEM, UV-vis and concentration. BTV polyclonal antibody was manufactured by immunizing the rabbits with BTV, and identified through concentration, SDS-PAGE and valency. NP probe was manufactured with Au nanoparticle, BTV polyclonal antibody, complementary probe NP and bar code DNA, and identified through TEM, UV-vis and concentration.2. Preparation, purification and identification of MMP probeBTV Mab was manufactured by immunizing BALB/c mouse with BTV hybridoma cell, and identified through concentration, valency and SDS-PAGE. MMP probe was manufactured with BTV Mab and magnetic microsphere, and identified by labeling efficiency.3. Preparation, purification and identification of detection probeDetection probe was manufactured with Au nanoparticle and NP label, and identified through TEM, UV-vis and concentration.4. Establishment and optimization of ordinary BCA and FQ-BCANP-VP7-MMP sandwich compounds were formed with NP probe and MMP probe through antigen-antibody interaction, then at the condition of high temperature and low salt, the bar code DNA is released. The bar code DNA was detected by routine PCR, chip detection and FQ-PCR, and the detection condition is optimized.5. The sensitivity evaluation of Common BCA and FQ-BCAThe sensitivity evaluation of VP7 protein: The detection sensitivity of ELISA is 1ng/mL; The detection sensitivity of the common BCA system is as follows: the routine PCR is 1fg/mL, and the chip detection is 10fg/mL; The detection sensitivity of FQ-BCA is 100ag/mL. Compared with ELISA method, the sensitivity is 6, 5, 7 orders of magnitude more sensitive.The sensitivity evaluation of BTV: The detection sensitivity of ELISA is 102TCID50; The detection sensitivity of the common BCA system is as follows: the routine PCR is 10-4TCID50, and the chip detection is 10-3TCID50; The detection sensitivity of FQ-BCA is 10-5TCID50. Compared with ELISA method, the sensitivity is 6, 5, 7 orders of magnitude more sensitive.6. The specificity evaluation of common BCA system and FQ-BCA systemThe specificity crossover trial has been made of BTV, EHDV, BVDV with established common BCA system and FQ-BCA system. The result is that the specificity of common BCA method and FQ-BCA system is satisfactory.7. The reproducibility evaluation of common BCA system and FQ-BCA system When the BTV titer is 10-1TCID50: The intra-assay CV value of common BCA method is 4.2%, 4.1%, 3.4%; the inter-assay CV value is 6.3%. The intra-assay CV value of FQ-BCA method is 2.06%, 1.45%, 1.43%; the inter-assay CV value is 2.52%.When the BTV titer is 10-2TCID50: The intra-assay CV value of common BCA method is 4.2%, 3.9%, 4.4%; the inter-assay CV value is 5.5%. The intra-assay CV value of FQ-BCA method is 1.95%, 1.37%, 1.56%; the inter-assay CV value is 2.1%.When the BTV titer is 10-3TCID50: The intra-assay CV value of common BCA method is 4.1%, 4.4%, 3.8%; the inter-assay CV value is 6.2%. The intra-assay CV value of FQ-BCA method is 1.43%, 1.30%, 1.19%; The inter-assay CV value is 1.91 %.The results show that the intra-assay CV value and the inter-assay CV value of common BCA and FQ-BCA are satisfactory.To sum up, the study has established the detection system of BCA system and FQ-BCA system for BTV. The significances are as follows: 1) The study has lied a foundation for developing common BCA detection kit and FQ-BCA detection kit; 2) The study provides a new method for the detection of some microamount matter such as toxin, biological warfare agent, steroid, lipoid, vitamin, tumor special marker and so on, and it shows an example for the establishment of similar detection system of the target.