發(fā)布時間：2019-08-13 11:58

【摘要】：根據(jù)Genbank已公布的志賀氏菌基因組序列,篩選特異性靶基因ipa H,設(shè)計特異性引物和探針,優(yōu)化反應(yīng)體系,并在體系中加入內(nèi)參(IAC),通過標(biāo)記不同熒光基團(tuán)的Taq Man探針來監(jiān)測IAC,進(jìn)而實時監(jiān)控整個PCR反應(yīng)過程。按照人工污染樣品,以評價所建立反應(yīng)體系的性能。以志賀氏菌基因組DNA為模板,最低檢測限為1 pg/u L;以10倍梯度稀釋的菌液用水煮法提取DNA為模板,最低檢測限為9×103CFU/m L;以含有ipa H的質(zhì)粒為模板,最低檢測限可以達(dá)到103考貝/u L;建立ipa H和ipa H-IAC標(biāo)準(zhǔn)曲線,Ct值與模板拷貝數(shù)均呈良好的線性關(guān)系(R2=0.999);人工污染初始菌量為20 g中10 CFU的羊肉時,志賀氏菌在增菌6 h后即可檢出(水洗加試劑盒法)。作者建立的ipa H-IAC實時熒光定量PCR方法,既能有效檢測食品中志賀氏菌,又能實時監(jiān)測PCR反應(yīng)過程,有效防止"假陰性"的發(fā)生,進(jìn)一步保證了結(jié)果的可靠性,有利于實現(xiàn)樣品中志賀氏菌實時熒光定量PCR檢測方法的標(biāo)準(zhǔn)化。
[Abstract]:According to the genome sequence of Shigella published by Genbank, the specific target gene ipa H was screened, specific primers and probes were designed, and the reaction system was optimized. Internal reference (IAC), was added to the system to monitor IAC, by labeling Taq Man probes with different fluorescent groups to monitor the whole PCR reaction process in real time. The performance of the reaction system was evaluated according to the artificially polluted samples. Using Shigella genomic DNA as template, the minimum detection limit was 1 pg/u / L, the minimum detection limit was 9 脳 103CFU/m L using 10 times gradient diluted bacteria solution to extract DNA with boiling method, and the minimum detection limit was 10 3 pg/u / u L with plasmid containing ipa H as template. The standard curves of ipa H and ipa H-IAC were established, and there was a good linear relationship between KT value and copy number of template (R2 鈮，

本文編號：2526109