基于ipaH基因的志贺菌LAMP检测方法的建立及应用

为建立一种快速、特异、敏感的志贺菌环介导等温扩增(LAMP)检测方法,试验针对志贺菌属侵袭性质粒抗原H基因(invasive plasmid, ipaH)设计一套特异性引物,条件优化及特异性和敏感性验证后建立了该菌的LAMP检测方法,并应用于临床样本的检测。结果显示,62℃恒温扩增1 h能得到明显的扩增产物,除志贺菌标准菌株扩增结果为阳性外,沙门菌、大肠埃希菌、金黄色葡萄球菌、枯草芽孢杆菌和阴性对照均为阴性,检测限值达到120 fg/μL。利用建立的方法检测了20株疑似菌株和38份粪样样品,结果显示20株疑似菌和27份粪样样品为阳性,阳性率分别为100%和71.05%,说明建立的LAMP检测法具有良好的特异性和敏感性,在粪便等临床样本中志贺菌的检测与鉴定方面具有优势和应用前景。

Shigella LAMP Detection Method Based on ipaH Gene: Establishment and Application

To establish a LAMP detection method which is rapid, specific and sensitive for Shigella, the sequence of invasion plasmid antigen H gene (invasion plasmid, ipaH) of Shigella was used as target sequence to design a set of specific primers, the LAMP method was established after condition optimization, specificity and sensitivity verification, and applied to detect clinical samples. The results showed that the amplification products could be obtained within 1 h at 62℃. The standard Shigella strains showed the positive result, while Salmonella, Escherichia coli, Staphylococcus aureus, Bacillus subtilis and the negative control showed the negative result. The detection limit of the LAMP method for Shigella was 120 fg/μL. 20 suspected strains and 38 fecal samples were tested by the established method. The results showed that 20 suspected bacteria and 27 fecal samples were positive, and the positive rate was 100% and 71.05%, respectively, indicating that this LAMP assay was a specific and sensitive method, and it had advantages and application prospects in the detection and identification of Shigella in clinical samples such as feces.