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

【摘要】：為建立一種同時檢測豬流行性腹瀉病毒(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2對引物,參照文獻合成1對PDCo V引物,優(yōu)化三對引物在同一RT-PCR擴增體系下的濃度、退火溫度等反應(yīng)條件,同時優(yōu)化方法的靈敏度、特異性。將初步建立的多重RT-PCR方法用于檢測采自四川省及重慶市14個豬場的222份樣品。結(jié)果表明,本試驗建立的多重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℃時的擴增效果最佳;最低檢測量分別為PEDV:60.96 pg、PDCo V:58.85 pg、TGEV:102.69 pg;用該法對多種豬傳染病病原DNA或c DNA進行擴增時均無非特異性擴增條帶出現(xiàn)。對222份腹瀉樣品的檢測結(jié)果表明,PEDV陽性檢出率為52.2%,PDCo V為2.7%,TGEV為2.3%。同時多重RT-PCR檢測方法和單項RT-PCR檢測方法符合率分別為PEDV 92.9%、PDCo V 100%、TGEV 100%。表明該法具較高可信度。本試驗建立了一種高效、特異地同時檢測PEDV、TGEV和PDCo V的多重RT-PCR方法,為病原的實驗室診斷和分子流行病學調(diào)查提供了技術(shù)支持。對四川省及重慶市部分豬場三種病原流行情況進行調(diào)查和統(tǒng)計分析,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：1695531