基于核酸序列依赖性扩增反应比色法检测沙门氏菌

Improved nucleic acid sequence-based amplification method for colorimetric detection of Salmonella typhimurium

本文以食源性致病菌中危害居于前列的沙门氏菌侵袭蛋白A基因作为检测靶标,通过改进传统的核酸序列依赖性扩增反应,开发了针对长链DNA的纳米金比色检测方法。本方法利用限制性内切酶和连接酶将T7启动子和终止子序列连接至靶标序列上,形成小型环状双链DNA,在T7 RNA聚合酶的作用下,转录出大量单链RNA序列进行NASBA反应,反应终产物能够促发纳米金探针发生交联聚集,溶液颜色从红色变成蓝色,从而实现对沙门氏菌的定量分析。

In this work,we chose DNA sequence of invasive protein A(invA) gene of popular food borne pathogen Salmonella typhimurium,as target to develop a colorimetric detection assay by improving the traditional nucleic acid sequence-based amplification(NASBA).In terms of the issues of NASBA is not suitable for long double-stranded DNA(dsDNA) detection,we employed restriction endonuclease and ligases to introduce T7 promoter and terminator sequences onto the target sequence to produce a mini plasmid-like circle dsDNA.T7 RNA polymerase recognizes the T7 promoter and transcribes a great amount of RNA strands,which could further initiate NASBA.The production of a great amount of RNA strands from NASBA would trigger probe-modified AuNPs aggregation and a concomitant color change from red to blue.The absorbance ratio at A650/A520 is directly proportional to the concentration of invA present,allowing quantitative determination of Salmonella typhimurium.