【摘要】：本研究旨在建立檢測(cè)豬流行性腹瀉病毒(PEDV)的恒溫隔絕式PCR(iiPCR)。采用靶向PEDV S基因的引物和探針,通過對(duì)引物、探針、Taq酶及反轉(zhuǎn)錄酶等進(jìn)行優(yōu)化,建立了檢測(cè)PEDV的iiPCR,對(duì)其特異性、敏感性和穩(wěn)定性進(jìn)行評(píng)價(jià),對(duì)15份PEDV陽性樣本和7份陰性樣本進(jìn)行檢測(cè),比較與現(xiàn)有的熒光定量RT-PCR方法的符合率,并對(duì)115份仔豬腹瀉樣本進(jìn)行檢測(cè)。結(jié)果顯示,iiPCR的最佳反應(yīng)體系為:上、下游引物各3.5μL、探針0.25μL、Taq酶1μL、反轉(zhuǎn)錄酶1.25μL、Buffer A 25μL、模板2μL,補(bǔ)足DEPC水至50L,從樣本處理到報(bào)告結(jié)果僅需1 h;該方法能檢出樣本中的PEDV,對(duì)TGEV、PPV、CSFV等無關(guān)病原不檢出,檢測(cè)下限為17.5 copiesμL,對(duì)5個(gè)稀釋度的陽性標(biāo)準(zhǔn)品進(jìn)行檢測(cè),3次重復(fù)結(jié)果一致,與現(xiàn)有熒光定量RT-PCR的總符合率為90.9%;本研究建立的iiPCR對(duì)仔豬腹瀉樣本中PEDV的檢出率為22.6%(26/115)。綜上可見,本研究成功建立了檢測(cè)PEDV的iiPCR,為PED的診斷提供了更為便捷并可現(xiàn)場(chǎng)使用的可靠方法。
[Abstract]:The purpose of this study was to establish a isothermal isolated PCR (iiPCR). For detection of porcine epidemic diarrhea virus (PEDV). By using primers and probes targeting PEDV S gene, the specificity, sensitivity and stability of iiPCR, for detecting PEDV were evaluated by optimizing the primers, probes, Taq enzymes and reverse transcriptase. 15 PEDV positive samples and 7 negative samples were detected, and the coincidence rate was compared with the existing fluorescence quantitative RT-PCR method, and 115 piglets diarrhea samples were detected. The results showed that the best reaction system of iPCR was: Downstream primer 3.5 渭 L, probe 0.25 渭 L Taq 1 渭 L, reverse transcriptase 1.25 渭 L buffer A 25 渭 L, template 2 渭 L, complement DEPC water to 50 L, from sample processing to report result only 1 hour, this method can detect PEDV, in the sample not to TGEV,PPV,CSFV and other unrelated pathogens. The detection limit was 17.5 copies 渭 L, and the results of three repeats of five dilution positive standard samples were consistent with the total coincidence rate of fluorescence quantitative RT-PCR was 90.90.The detection rate of PEDV in diarrhea samples of piglets was 22.6% (26 / 115). It can be seen that the iiPCR, for detecting PEDV provides a more convenient and reliable method for the diagnosis of PED.
【作者單位】： 西南民族大學(xué)生命科學(xué)與技術(shù)學(xué)院;金瑞鴻捷(廈門)生物科技有限公司;
【基金】：十三五國家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目(2016YFD0500705)
【分類號(hào)】：S852.651

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