本文關(guān)鍵詞： 馬立克氏病病毒 傳染性喉氣管炎病毒 雞痘病毒 DNA病毒 復(fù)合PCR　出處：《河南農(nóng)業(yè)科學(xué)》2017年07期 　論文類型：期刊論文

【摘要】：依據(jù)Gen Bank中注冊的雞馬立克氏病病毒(MDV)、雞傳染性喉氣管炎病毒(ILTV)、雞痘病毒(FPV)基因組核苷酸序列中的保守序列設(shè)計(jì)引物,3種病毒各設(shè)計(jì)3對引物,共計(jì)9對引物,經(jīng)生物學(xué)軟件篩選后,得到3對相互匹配較佳的引物。3對匹配引物經(jīng)單一PCR驗(yàn)證后,優(yōu)化復(fù)合PCR反應(yīng)條件,確定最佳的退火溫度、引物濃度和Taq DNA聚合酶濃度;經(jīng)特異性、敏感性試驗(yàn)及簡化PCR試驗(yàn),建立簡易復(fù)合PCR檢測方法。復(fù)合PCR擴(kuò)增出的3條帶大小與預(yù)期一致,即228 bp(MDV)、400 bp(ILTV)、499 bp(FPV);建立的復(fù)合PCR方法能同時(shí)檢測出100 fg MDV、ILTV、FPV的DNA。同時(shí)依據(jù)PCR技術(shù)原理,將經(jīng)典的三溫?zé)嵫h(huán)改進(jìn)為二溫?zé)嵫h(huán)試驗(yàn),即將退火和延伸合并為一步(62℃),縮短了反應(yīng)時(shí)間。建立的3種病毒的復(fù)合PCR檢測方法具有較高的特異性和敏感性,可以用于臨床病料的快速診斷。
[Abstract]:According to the Gen Bank registered chicken Marek's disease virus (MDV), chicken infectious laryngotracheitis virus (ILTV). Primer design for the conserved nucleotide sequence of fowlpox virus (FPVV) genome 3 pairs of primers (9 pairs of primers) were designed for each of the three viruses. The primers were screened by biological software. Three pairs of matching primer pairs were obtained, which were verified by a single PCR. The optimal conditions of PCR reaction were optimized, and the optimal annealing temperature, primer concentration and Taq DNA polymerase concentration were determined. Through specificity, sensitivity test and simplified PCR test, a simple complex PCR detection method was established. The size of the 3 bands amplified by the compound PCR was in accordance with the expected, that is, 228bpnmdv). 400 BP ILTVN 499 BP FPVA; The established composite PCR method can simultaneously detect the DNAs of 100fg MDV / ILTVV FPV, and according to the principle of PCR technology. The classical three temperature thermal cycle is improved to two temperature thermal cycle test, that is, annealing and extension are combined into one step (62 鈩，

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