【摘要】：為建立快速、準(zhǔn)確的豬瘟病毒(CSFV)血清抗體檢測(cè)方法,根據(jù)Gen Bank上豬瘟兔化弱毒全基因序列,設(shè)計(jì)1對(duì)引物,擴(kuò)增E2蛋白主要抗原區(qū)基因片段;然后,將其克隆入載體p ET-28a(+),采用大腸桿菌Rosetta(DE3)表達(dá)出含E2蛋白主要抗原區(qū)的重組蛋白。以純化的截短E2蛋白為包被抗原,優(yōu)化間接ELISA反應(yīng)條件,建立了檢測(cè)CSFV血清抗體的間接ELISA。經(jīng)過(guò)優(yōu)化,確定最佳抗原包被濃度為8 g/m L,待檢血清稀釋倍數(shù)為1∶160,酶標(biāo)二抗稀釋倍數(shù)為1∶2000。該方法的陽(yáng)性臨界值為0.37,陰性臨界值為0.32。批內(nèi)變異系數(shù)在1.58%~3.33%之間,批間變異系數(shù)在2.67%~5.35%之間,說(shuō)明該方法重復(fù)性良好。特異性檢驗(yàn)中,該方法與豬繁殖與呼吸綜合征病毒、豬圓環(huán)病毒2型、豬流行性腹瀉病毒、豬流感病毒、傳染性胃腸炎病毒、乙型腦炎病毒、偽狂犬病病毒、豬細(xì)小病毒陽(yáng)性血清均沒(méi)有交叉反應(yīng)。用該方法與IDEXX CSFV抗體檢測(cè)試劑盒對(duì)1 101份臨床豬血清樣品進(jìn)行符合性檢測(cè),結(jié)果,該方法的敏感性、特異性、符合率分別為89.07%(603/677)、77.59%(329/424)和84.65%(932/1101)。上述結(jié)果表明,該方法可用于臨床CSFV血清抗體的檢測(cè),為CSFV血清抗體檢測(cè)試劑盒的研制奠定了基礎(chǔ)。
[Abstract]:In order to establish a rapid and accurate method for detection of (CSFV) serum antibody of CSFV, a pair of primers were designed according to the whole gene sequence of Gen Bank to amplify the main antigen region of E2 protein. Then, the recombinant protein containing the main antigen region of E2 protein was expressed by Escherichia coli Rosetta (DE3). Using the purified truncated E2 protein as the coating antigen and optimizing the reaction conditions of indirect ELISA, an indirect ELISA. for the detection of serum antibodies of CSFV was established. After optimization, the best antigen coating concentration was 8 g / m L, the dilution multiple of serum to be tested was 1: 160, and the dilution multiple of enzyme labeled second antibody was 1: 2000. The positive critical value and negative critical value of this method are 0. 37 and 0. 32 respectively. The coefficient of variation is between 1.58% and 3.33%, and the coefficient of variation between batches is between 2.67% and 5.35%, which indicates that the method has good reproducibility. Specific tests were performed with porcine reproductive and respiratory syndrome virus, porcine circovirus type 2, porcine epidemic diarrhea virus, swine influenza virus, infectious gastroenteritis virus, Japanese encephalitis virus, pseudorabies virus. There was no cross-reaction in serum of porcine parvovirus positive. The method and IDEXX CSFV antibody kit were used to detect the conformance of 1 101 clinical pig serum samples. The results showed that the sensitivity, specificity and coincidence rate of this method were 89.07% (603 / 677), respectively. 77.59% (329 / 424) and 84.65% (932 / 1101). These results indicate that this method can be used for the detection of serum antibodies in clinical CSFV, which lays a foundation for the development of a kit for detection of serum antibodies of CSFV.
【作者單位】： 南京農(nóng)業(yè)大學(xué)動(dòng)物醫(yī)學(xué)院;江蘇高校動(dòng)物重要疫病與人獸共患病防控協(xié)同創(chuàng)新中心;
【基金】：江蘇省農(nóng)業(yè)科技自主創(chuàng)新資金項(xiàng)目(CX(15)1056;CX(16)1028)
【分類號(hào)】：S852.651

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