基于内参的志贺氏菌实时荧光定量PCR快速检测
发布时间：2019-08-13 11:58
【摘要】：根据Genbank已公布的志贺氏菌基因组序列,筛选特异性靶基因ipa H,设计特异性引物和探针,优化反应体系,并在体系中加入内参(IAC),通过标记不同荧光基团的Taq Man探针来监测IAC,进而实时监控整个PCR反应过程。按照人工污染样品,以评价所建立反应体系的性能。以志贺氏菌基因组DNA为模板,最低检测限为1 pg/u L;以10倍梯度稀释的菌液用水煮法提取DNA为模板,最低检测限为9×103CFU/m L;以含有ipa H的质粒为模板,最低检测限可以达到103考贝/u L;建立ipa H和ipa H-IAC标准曲线,Ct值与模板拷贝数均呈良好的线性关系(R2=0.999);人工污染初始菌量为20 g中10 CFU的羊肉时,志贺氏菌在增菌6 h后即可检出(水洗加试剂盒法)。作者建立的ipa H-IAC实时荧光定量PCR方法,既能有效检测食品中志贺氏菌,又能实时监测PCR反应过程,有效防止"假阴性"的发生,进一步保证了结果的可靠性,有利于实现样品中志贺氏菌实时荧光定量PCR检测方法的标准化。
[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，
本文编号：2526109
[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，
本文编号：2526109
本文链接：https://www.wllwen.com/huliyixuelunwen/2526109.html
最近更新
教材专著
