摘要
猪瘟是由猪瘟病毒引起猪的一种高度接触性和致死性传染病。病猪的主要临床特征是高热和出血常呈急性、慢性和亚临床经过。猪瘟病毒同边界病毒、牛病毒性腹泻病毒1型、牛病毒性腹泻病毒2型、长颈鹿瘟病毒一个暂定种同属于黄病毒科瘟病毒属的成员。猪瘟的暴发可以导致养猪业的巨大经济损失且影响国际贸易,被世界动物卫生组织列为报告疾病名录。对于猪瘟的预防尽管弱毒疫苗很有效,但是由于缺乏血清学标志鉴别诊断的方法,通过抗体检测很难区分免疫弱毒和野毒感染。现有的检测猪瘟病毒的方法主要包括病毒分离、抗原捕捉ELISA、聚合酶链式反应等。这些方法需要几小时到几天才能完成,且有的需要熟练的操作者和特殊的仪器。因此对于猪瘟病毒的检测急需要一种快速、可靠、廉价的检测方法。本研究使用了抗猪瘟病毒E2蛋白的单克隆抗体,研发了一个可以鉴别诊断猪瘟病毒野毒和兔化弱毒的免疫层析试纸条。本研究的内容及结果如下:
1.为建立一种快速、简便检测猪瘟病毒野毒的胶体金免疫层析方法,采用柠檬酸三钠还原法制备胶体金颗粒,标记纯化的抗猪瘟病毒E2蛋白的单克隆抗体6E10作为捕捉抗体,将纯化的抗猪瘟病毒E2蛋白的单克隆抗体HQ06和兔抗鼠IgG抗体包被在硝酸纤维素膜上,分别作为检测线和质控线,进行条件优化,然后组装成胶体金免疫层析试纸条。结果表明,所制备的试纸条用于检测猪瘟病毒野毒感染的PK15细胞培养物,在检测线和质控线处均出现红色条带,未感染病毒的细胞培养物仅在质控线出现红色条带;试纸条检出病毒培养物的最低限为103.5 TCID50;用不同批次的试纸条重复检测,结果无差异;该试纸条不与猪瘟兔化弱毒、牛病毒性腹泻病毒、猪繁殖与呼吸综合征病毒、传染性胃肠炎病毒、猪流行性腹泻病毒、猪轮状病毒、伪狂犬病病毒、猪细小病毒和猪圆环病毒2型反应。所制备的试纸条具有一定的特异性和敏感性,重复性良好,初步达到了区分检测猪瘟野毒和弱毒的目的。
2.应用病毒分离、胶体金免疫层析试纸条、抗原捕捉ELISA、反转录-聚合酶链式反应、TaqMan荧光定量RT-PCR和反转录-环介导等温扩增方法等6种方法,分别对50份疑似猪瘟病料中的猪瘟病毒进行检测。结果表明:TaqMan荧光定量RT-PCR方法和反转录-环介导等温扩增方法检出阳性样品数为13份,反转录-聚合酶链式反应为11份,病毒分离为10份,抗原捕捉ELISA为9份,胶体金试纸条为8份;6种方法均检测为阳性8份,均为阴性37份。结果提示,在对猪瘟病毒进行检测时,TaqMan荧光定量RT-PCR方法、反转录-环介导等温扩增方法和反转录-聚合酶链式反应由于其灵敏性高,可作为首选检测方法,但操作时需要避免假阳性的出现;病毒分离方法虽然操作繁琐,但结果准确,是确诊猪瘟必不可少的检测方法;抗原捕捉ELISA和胶体金试纸条检测时间较短,操作简单,结果易判断,不足之处是敏感性较低。
本研究使用了特异性识别猪瘟病毒野毒的单克隆抗体,研发了可以鉴别诊断猪瘟病毒野毒和兔化弱毒的胶体金免疫层析试纸条,对于猪瘟的诊断具有一定的指导意义。
Classical swine fever virus (CSFV) is the causative agent of classical swine fever, a highly contagious and often fatal disease of domestic pigs. It is characterized by fever and hemorrhages and can run an acute, chronic, or even subclinical course. CSFV, together with Border disease virus (BDV), Bovine viral diarrhea virus 1 (BVDV-1), Bovine viral diarrhea virus 2 (BVDV-2) and a tentative species Pestivirus of giraffe, is classified within the genus Pestivirus in the family Flaviviridae .CSF outbreaks can result in large economic losses in pig production and affect international trade and therefore CSF is a notifiable disease to The World Organization for Animal Health (OIE). Although effective live-attenuated vaccines are available, vaccinated and infected pigs are serologically indistinguishable. Current methods for detection CSFV include virus isolation, antigen-capture ELISA, reverse transcription polymerase chain reaction and others. These methods, however, take a few hours to several days to accomplish, and require skilled personnel and specialized equipment. There is, therefore, an urgent need for rapid, reliable, and inexpensive tests for detection of CSFV. In this study, an immunochromatographic test strip using anti-E2 monoclonal antibodies for rapid diagnosis of CSFV was developed. This strip makes discrimination between wild-type virus and C-strain vaccine of classical swine fever virus. The results were as follows:
1. A colloidal gold immunochromatographic strip was developed for the rapid detection of the wild-type classical swine fever virus (CSFV) antigen using two monoclonal antibodies (mAbs), 6E10 and HQ06, against the E2 protein. The wild-type CSFV-specific 6E10 mAb was labeled with colloidal gold prepared by reduction of sodium citrate as capture antibody and the HQ06 mAb was immobilized on the test line as detection antibody, while a rabbit anti-mouse IgG antibody was blotted on the control line of the nitrocellulose membrane. PK15 cell cultures infected with CSFV and non-infected were detected by the strips. The results showed that two red lines appeared in the test line and control line region only when detecting the CSFV-infected cells, while only a red line appeared in the control line region when detecting the non-infected cells. The detection limit of the immunochromatographic strip was 103.5 TCID50 CSFV, and the results of different batches were reproducible. The test strip did not react with hog cholera lapinized virus (HCLV), bovine viral diarrhea virus (BVDV), porcine reproductive and respiratory syndrome virus (PRRSV), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PRV), pseudorabies virus (PrV), porcine parvovirus (PPV) and porcine circovirus type 2 (PCV2). Taken together, the test strip has good specificity, sensitivity and reproducibility, and can be used to differentiate cells infected with wild-type CSFV from those infected with HCLV.
2. Six methods, including the virus isolation, colloidal gold immunochromatographic assay (CGIA), antigen-capture ELISA (AC-ELISA), reverse transcription polymerase chain reaction(RT-PCR),TaqMan real-time RT-PCR(RT-qPCR) and reverse transcription loop mediated isothermal amplification assay (RT-LAMP), were used to detect CSFV in 50 samples in parallel. The results showed that 13 samples were detected positive by RT-qPCR and RT-LAMP, 11 by PCR, 10 by virus isolation, 9 by AC-ELISA and 8 by CGIA, and 8 samples were detected positive and 37 samples negative by the six methods. These results indicated that the 3 RNA-amplification assays could be used as the first choice for detection of CSFV due to the high sensitivity, while they were vulnerable to false positive results arising from sample to sample contaminations or from other contaminated sources. Although the virus isolation was time-consuming, it was still considered the“gold standard”and was indispensable for confirming CSF outbreaks. The rest two methods, AC-ELISA and CGIA, yielded the results in a short time yet their performance was hampered by a low sensitivity. Therefore, they were mainly used for herd diagnosis.
In this study, the wild-type CSFV-specific mAb was used to develop a colloidal gold immunochromatographic strip. This strip, which makes discrimination between wild-type virus and C-strain vaccine of classical swine fever virus, might contribute to diagnosis of CSFV.
引文
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