蓝舌病病毒生物条形码及荧光定量型生物条形码检测体系的建立与评价

发布时间：2018-08-17 11:20

【摘要】： 蓝舌病(bluetongue, BT)是由蓝舌病病毒(blue tongue virus, BTV)引起的一种侵害反刍动物的虫媒性传染病,被世界动物卫生组织(World Organization for Animal Health, OIE)列为上报疾病,我国将其列为一类动物疫病,是国内外动物防疫和出入境检验检疫机构监测和防控的重点疫病。此外,蓝舌病病毒还是人用动物源生物制品(如人用牛源凝血酶、凝血因子Ⅷ、聚合牛血红蛋白、胸腺肽)和生物制品敷料(如牛源明胶、牛源胶原)病毒安全性检测的病毒之一。 BTV属于呼肠孤病毒科(Reoviridae)环状病毒属(Orbivirus)成员,至少存在24个血清型,为血清型多样性,呈全球性分布。随着全球气温变暖,2006年～2008年许多国家爆发蓝舌病,使其分布范围不断扩张,并出现了地域性新血清型,而现在的检测试剂存在着的灵敏度不高覆盖血清型范围窄的缺点阻碍了BTV的病原监控工作。因此,建立高灵敏度的适于各血清型BTV的特异性检测技术,以及研制相应的检测试剂盒已经迫在眉睫。生物条形码检测技术(bio-bar codes assay, BCA)是2003年美国科学家Mirkin领导的课题组首次报道的一种新型标记免疫测定技术,其突出特点是具有极高的灵敏度,其基本原理几乎等同于ELISA检测中双抗体夹心法高度特异地捕获抗原,通过检测特异条形码DNA链(bar code DNA)而实现对抗原物质的间接检测。对特异条形码DNA链的检测采用常规PCR扩增或基于金标银染的芯片检测,由于PCR扩增和芯片检测的放大效应,使得检测灵敏度得到极大提高,可达常规ELISA方法的106倍。荧光定量PCR技术(fluorescent quantitative PCR, FQ-PCR)是通过在PCR反应体系中加入荧光基团,利用荧光信号的变化实时检测PCR扩增反应中每一个循环扩增产物量的变化,通过Ct值和标准曲线的分析对起始模板进行定量分析,被认为是当今核酸定量的金标准。本研究针对蓝舌病毒的检测现状,建立了BTV的BCA检测体系和BTV的荧光定量型生物条形码检测体系(fluorescent quantitative bio-bar codes assay, FQ-BCA),并对检测体系进行了灵敏度、特异性、重复性等方法学评价。本研究主要包括以下几部分: 1. NP探针制备、纯化及鉴定利用柠檬酸三钠还原法制备金纳米颗粒,并对金纳米颗粒进行光学、TEM、UV-vis和浓度鉴定;利用BTV全病毒免疫新西兰大白兔制备BTV多抗,纯化后进行浓度、SDS-PAGE电泳和效价鉴定;利用金纳米颗粒、BTV多抗和人工合成的互补probe NP链、条形码DNA链制备NP探针,纯化后进行TEM、UV-vis和浓度鉴定。 2. MMP探针制备、纯化及鉴定利用BTV杂交瘤细胞株免疫BALB/c小鼠制备BTV单抗,纯化后进行浓度、效价和SDS-PAGE鉴定;利用制备的BTV单抗和磁性微球制备MMP探针,并对探针进行标记效率鉴定。 3.检测探针制备、纯化及鉴定利用金纳米颗粒和人工合成的NP标签链制备检测探针,纯化后对检测探针进行TEM、UV-vis和浓度鉴定。 4. BTV的BCA及FQ-BCA检测体系的建立及优化利用NP探针和MMP探针通过抗原抗体作用制备NP-VP7-MMP三明治复合物,在高温低盐条件下,释放条形码DNA链。对获得的条形码DNA链分别进行常规PCR检测、芯片检测和FQ-PCR检测,并对检测条件进行优化。 5. BCA及FQ-BCA检测体系的灵敏度评价 VP7蛋白检测灵敏度评价:ELISA方法检测灵敏度为1ng/mL;BCA检测体系的常规PCR检测灵敏度为1fg/mL,芯片检测灵敏度为10fg/mL;FQ-BCA检测体系的检测灵敏度为100ag/mL,灵敏度分别为常规ELISA方法的106、105、10~7倍。 BTV全病毒检测灵敏度评价:ELISA方法检测灵敏度为102TCID50;BCA检测体系的常规PCR检测灵敏度为10-4TCID50,芯片检测灵敏度为10-3TCID50;FQ-BCA检测体系的检测灵敏度为10-5TCID50,灵敏度分别为常规ELISA方法的106、105、107倍。结果表明BCA和FQ-BCA检测体系的检测灵敏度远远超过现在的蛋白检测首选方法——ELISA方法。 6. BCA及FQ-BCA检测体系的特异性评价用建立的BCA和FQ-BCA检测体系对BTV、EHDV、BVDV进行特异性交叉试验。结果为:BCA及FQ-BCA检测体系均具有良好的特异性。 7. BCA及FQ-BCA检测体系的重复性评价 BTV滴度为10-1TCID50时:BCA检测体系的芯片检测的批内CV值分别为4.2%、4.1%、3.4%,批间CV值为6.3%;FQ-BCA检测体系的批内CV值分别为2.06%、1.45%、1.43%,批间CV值为2.52%。 BTV滴度为10-2TCID50时:BCA检测体系芯片检测的批内CV值分别为4.2%、3.9%、4.4%,批间CV值为5.5%;FQ-BCA检测体系的批内CV值分别为1.95%、1.37%、1.56%,批间CV值为2.1%。 BTV滴度为10-3TCID50时:BCA检测体系的芯片检测的批内CV值分别为4.1%、4.4%、3.8%,批间CV值为6.2%;FQ-BCA检测体系的批内CV值分别为1.43%、1.30%、1.19%,批间CV值为1.91%。结果表明BCA及FQ-BCA检测体系都具有良好的批内和批间重复性,且FQ-BCA检测体系重复性更好。综上所述,本研究成功地建立了BTV的BCA检测体系和BTV的FQ-BCA检测体系,并对体系进行了方法学评价。其意义在于:1)本研究为研制BTV的BCA检测试剂和FQ-BCA检测试剂奠定了基础;2)本研究为其它类似的被检物尤其是那些不适于建立核酸检测体系的微量物质如毒素、生物战剂、类固醇、脂类、维生素、肿瘤特异性标志物等的检测提供了新方法,对被检物建立类似检测体系起到良好的示范作用。
[Abstract]:Bluetongue disease (BT) is an insect-borne infectious disease caused by blue tongue virus (BTV) which infects ruminants. It is listed as a reported disease by the World Organization for Animal Health (OIE). It is classified as a kind of animal epidemic disease in China and abroad, and it is also an animal quarantine and entry-exit inspection and quarantine. Bluetongue virus is also one of the viruses used to detect the safety of human animal-derived biological products (such as human bovine thrombin, coagulation factor_, polymerized bovine hemoglobin, thymosin) and biological dressings (such as bovine gelatin, bovine collagen).
BTV belongs to the Orbivirus family of Reoviridae. There are at least 24 serotypes of BTV. BTV is a globally distributed serotype with diverse serotypes. The presence of low sensitivity and narrow coverage of serotypes limits the monitoring of BTV pathogens.
Therefore, it is imminent to establish a highly sensitive and specific detection technique for BTV of various serotypes and develop corresponding detection kits.
Bio-bar codes assay (BCA) is a new labeled immunoassay technique first reported by a research group led by American scientist Mirkin in 2003. Its outstanding feature is its high sensitivity. Its basic principle is almost equal to the double antibody sandwich method in ELISA detection, which can capture antigens with high specificity. Specific bar code DNA strands (bar code DNA) can be used for indirect detection of antigen substances. Detection of specific bar code DNA strands using conventional PCR amplification or gold labeled silver staining chip detection, due to the amplification effect of PCR amplification and chip detection, the detection sensitivity has been greatly improved, up to 106 times the conventional ELISA method.
Fluorescent quantitative polymerase chain reaction (FQ-PCR) is a real-time detection of changes in the amount of each amplified product in a PCR amplification reaction by adding fluorescent groups to the PCR reaction system. The initial template is quantitatively analyzed by the analysis of CT value and standard curve. FQ-PCR is considered to be today's nucleic acid. Quantitative gold standard.
In this study, a BCA detection system for BTV and a fluorescent quantitative bio-bar codes assay (FQ-BCA) for BTV were established, and the sensitivity, specificity and repeatability of the detection system were evaluated.
This study mainly includes the following parts:
Preparation, purification and identification of 1. NP probe
Gold nanoparticles were prepared by trisodium citrate reduction method, and the gold nanoparticles were identified by optical, TEM, UV-vis and concentration analysis; BTV polyclonal antibodies were prepared by immunizing New Zealand white rabbits with BTV whole virus, then purified, SDS-PAGE electrophoresis and titer identification; gold nanoparticles, BTV polyclonal antibodies and synthetic complementary NP chains, bar code D were used. NA chain was used to prepare NP probe. After purification, TEM, UV-vis and concentration were identified.
Preparation, purification and identification of 2. MMP probe
BTV monoclonal antibody was prepared by immunizing BALB/c mice with BTV hybridoma cell line, then purified and identified by concentration, titer and SDS-PAGE. MMP probe was prepared by using BTV monoclonal antibody and magnetic microspheres, and the labeling efficiency of the probe was evaluated.
3. detection, preparation, purification and identification of probes
Gold nanoparticles and synthetic NP tag chains were used to prepare detection probes. After purification, the probe was identified by TEM, UV-vis and concentration.
Establishment and optimization of BCA and FQ-BCA detection system for 4. BTV
NP-VP7-MMP sandwich complex was prepared by antigen-antibody interaction of NP probe and MMP probe, and barcode DNA strands were released under high temperature and low salt conditions. The obtained barcode DNA strands were detected by conventional PCR, chip detection and FQ-PCR respectively, and the detection conditions were optimized.
Sensitivity evaluation of 5. BCA and FQ-BCA detection system
VP7 protein detection sensitivity evaluation: ELISA detection sensitivity is 1ng/mL; BCA detection system of conventional PCR detection sensitivity is 1fg/mL, chip detection sensitivity is 10fg/mL; FQ-BCA detection system detection sensitivity is 100ag/mL, sensitivity is 106,105,10-7 times that of conventional ELISA method.
BTV whole virus detection sensitivity evaluation: ELISA detection sensitivity of 102 TCID 50; BCA detection system of conventional PCR detection sensitivity of 10-4 TCID 50, chip detection sensitivity of 10-3 TCID 50; FQ-BCA detection system of 10-5 TCID 50, sensitivity of 106, 105, 107 times as conventional ELISA method.
The results showed that the sensitivity of BCA and FQ-BCA detection system was much higher than that of ELISA method, which is the preferred method for protein detection.
Specificity evaluation of 6. BCA and FQ-BCA detection system
BCA and FQ-BCA were used to test BTV, EHDV and BVDV. The results showed that BCA and FQ-BCA had good specificity.
Repeatability evaluation of 7. BCA and FQ-BCA detection system
When BTV titer was 10-1 TCID50, the CV values of BCA chip were 4.2%, 4.1%, 3.4% and 6.3% respectively, while that of FQ-BCA chip was 2.06%, 1.45%, 1.43% and 2.52% respectively.
When BTV titer was 10-2TCID50, the CV values of BCA chip were 4.2%, 3.9%, 4.4% and 5.5% respectively, while that of FQ-BCA chip was 1.95%, 1.37%, 1.56% and 2.1% respectively.
When BTV titer was 10-3 TCID50, the intra-batch CV was 4.1%, 4.4%, 3.8% and the inter-batch CV was 6.2% for BCA, 1.43%, 1.30%, 1.19% and 1.91% for FQ-BCA, respectively.
The results showed that both BCA and FQ-BCA detection systems had good intra-batch and inter-batch repeatability, and FQ-BCA detection system had better repeatability.
To sum up, the BCA detection system for BTV and the FQ-BCA detection system for BTV were successfully established, and the methodological evaluation of the system was carried out. The detection system of trace substances such as toxins, biological warfare agents, steroids, lipids, vitamins, tumor specific markers and so on provides a new method for the detection of the detection system, which plays a good role in establishing a similar detection system.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R373

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