本文選題：雞白痢沙門(mén)菌 + 套式PCR�。� 參考：《動(dòng)物醫(yī)學(xué)進(jìn)展》2017年08期

【摘要】：為了建立一種快速、高效和簡(jiǎn)便的雞白痢沙門(mén)菌套式PCR檢測(cè)方法,根據(jù)沙門(mén)菌侵襲蛋白基因invA的高度保守區(qū)域,利用Primmer 5.0軟件設(shè)計(jì)2對(duì)特異性的套式引物,以提取的沙門(mén)菌DNA為模板克隆invA基因,將其連入T載體并計(jì)算拷貝數(shù)作為套式PCR的模板,在對(duì)反應(yīng)條件進(jìn)行優(yōu)化的基礎(chǔ)上,建立最佳的套式PCR反應(yīng)條件,確定其上、下游引物用量分別為0.3μL,套式PCR的第1次退火溫度為57.4℃,第2次為53.5℃。應(yīng)用該方法對(duì)已構(gòu)建好的不同濃度的標(biāo)準(zhǔn)陽(yáng)性質(zhì)粒作為模板進(jìn)行檢測(cè),其靈敏度為102拷貝數(shù)/20μL,遠(yuǎn)高于常規(guī)PCR的106拷貝數(shù)/20μL。用建立的套式PCR方法對(duì)從新疆石河子某雞場(chǎng)采集的25樣品進(jìn)行檢測(cè),與傳統(tǒng)的診斷方法比較符合率達(dá)95.00%。建立的雞白痢沙門(mén)菌套式PCR檢測(cè)方法具有快速、可行、特異性強(qiáng)、準(zhǔn)確率高等特點(diǎn),為雞沙門(mén)菌病的早期診斷和研究提供了技術(shù)支撐。
[Abstract]:In order to establish a rapid, efficient and simple nested PCR method for detection of Salmonella pullorum, two pairs of specific nested primers were designed by using Primmer 5.0 software according to the highly conserved region of invA gene of Salmonella pullorum invasion protein.The extracted Salmonella DNA was used as template to clone the invA gene, and was inserted into T vector and the copy number was calculated as template of nested PCR. Based on the optimization of reaction conditions, the optimal reaction conditions of nested PCR were established, and the optimal reaction conditions were determined.The amount of downstream primer was 0.3 渭 L, the first annealing temperature of nested PCR was 57.4 鈩，

本文編號(hào)：1736603