流产衣原体RAA快速检测方法的建立

Establishment of a Rapid RAA for Chlamydia abortus

流产衣原体(Chlamydia abortus)是一种可造成羊、牛、猪等多种动物和人感染的原核微生物,广泛分布于世界各地,是危害畜牧业最严重的衣原体病原之一。为实现流产衣原体的快速准确鉴别,提高检测效率,提升应急响应速度,根据流产衣原体ompA基因保守区域,设计特异性引物和探针,建立了流产衣原体重组酶介导等温核酸扩增检测技术(recombinase aided amplification,RAA)。该方法仅需在39℃条件下扩增20 min,即可完成检测;最低检测限可达1.13 copies/μL,且与常见细菌核酸无交叉反应;8次重复试验组内变异系数(CV)约为5%;临床样品检测结果显示,建立的RAA方法与荧光PCR符合率为100%。结果表明,本研究建立的RAA快速检测方法灵敏度高、特异性强、重复性好,操作简便,为流产衣原体的现场快速检测提供了新的技术手段,具有良好的应用前景。

Chlamydia abortus is a prokaryotic microorganism infecting sheep,cattle,pigs and other animals and humans,and widely distributes all over the world,seriously endangering livestock industry as one of chlamydial pathogens.In order to identify chlamydia abortus rapidly,and to improve the detection efficiency and emergency response,a recombinase aided amplification(RAA)assay for chlamydia abortus was established after designing specific primers and probe based on the conserved region of ompA gene.Detection could be finished by the method within 20 min of amplification at 39℃,the lowest detection limit was 1.13 copies/μL,and no cross reaction was observed with common bacterial nucleic acids;and the intra-group coefficient of variation(CV)was about 5% for 8 repeated tests.Clinical sample detection results showed that the established method was 100% compatible with fluorescent PCR.In conclusion,the established method could be used for rapid on-site detection of Chlamydia abortus as a new tool with the advantages of high sensitivity,strong specificity,good repeatability and accessibility,indicating a good application prospect.