本文選題：多重RT-PCR　切入點(diǎn)：豬流行性腹瀉病毒　出處：《中國(guó)獸醫(yī)科學(xué)》2016年06期

【摘要】：為建立一種同時(shí)檢測(cè)豬流行性腹瀉病毒(PEDV)、豬傳染性胃腸炎病毒(TGEV)、豬Delta冠狀病毒(PDCo V)的多重RT-PCR方法,根據(jù)Gen Bank中PEDV和TGEV基因序列保守區(qū)設(shè)計(jì)2對(duì)引物,參照文獻(xiàn)合成1對(duì)PDCo V引物,優(yōu)化三對(duì)引物在同一RT-PCR擴(kuò)增體系下的濃度、退火溫度等反應(yīng)條件,同時(shí)優(yōu)化方法的靈敏度、特異性。將初步建立的多重RT-PCR方法用于檢測(cè)采自四川省及重慶市14個(gè)豬場(chǎng)的222份樣品。結(jié)果表明,本試驗(yàn)建立的多重RT-PCR在引物量分別為PEDV 0.2 L,PDCo V 0.2 L和TGEV 0.4L,退火溫度為53℃時(shí)的擴(kuò)增效果最佳;最低檢測(cè)量分別為PEDV:60.96 pg、PDCo V:58.85 pg、TGEV:102.69 pg;用該法對(duì)多種豬傳染病病原DNA或c DNA進(jìn)行擴(kuò)增時(shí)均無(wú)非特異性擴(kuò)增條帶出現(xiàn)。對(duì)222份腹瀉樣品的檢測(cè)結(jié)果表明,PEDV陽(yáng)性檢出率為52.2%,PDCo V為2.7%,TGEV為2.3%。同時(shí)多重RT-PCR檢測(cè)方法和單項(xiàng)RT-PCR檢測(cè)方法符合率分別為PEDV 92.9%、PDCo V 100%、TGEV 100%。表明該法具較高可信度。本試驗(yàn)建立了一種高效、特異地同時(shí)檢測(cè)PEDV、TGEV和PDCo V的多重RT-PCR方法,為病原的實(shí)驗(yàn)室診斷和分子流行病學(xué)調(diào)查提供了技術(shù)支持。對(duì)四川省及重慶市部分豬場(chǎng)三種病原流行情況進(jìn)行調(diào)查和統(tǒng)計(jì)分析,為地區(qū)豬腹瀉病的防控提供了依據(jù)。
[Abstract]:In order to establish a multiplex RT-PCR method for simultaneous detection of porcine epidemic diarrhea virus (PEDV), porcine transmissible gastroenteritis virus (TGEV) and porcine Delta coronavirus (PDCo V), two pairs of primers were designed according to the conserved region of PEDV and TGEV gene in Gen Bank. A pair of PDCo V primers were synthesized with reference to the literature. The reaction conditions such as the concentration of three pairs of primers under the same RT-PCR amplification system and annealing temperature were optimized, and the sensitivity of the method was optimized. The multiplex RT-PCR method was used to detect 222 samples from 14 pig farms in Sichuan province and Chongqing city. In this experiment, the amplification effect of multiple RT-PCR was the best when the number of primers was 0.2L PEDV 0.2L PDCo V 0.2L and TGEV 0.4L, and the annealing temperature was 53 鈩，

本文編號(hào)：1695531