基于重组酶介导等温扩增技术的鸡滑液囊支原体检测方法建立和初步应用

Establishment and Preliminary Application of a Detection Method for Mycoplasma synoviae Based on Recombinase-aided Isothermal Amplification

为建立一种鸡滑液囊支原体(MS)快速简便的检测方法,研究根据MS vlhA基因保守序列设计3对引物,通过引物筛选和条件优化,建立了基于重组酶介导等温扩增技术(RAA)的MS检测方法,并对其特异性和灵敏度进行评估。应用该方法对68份鸡咽喉拭子临床样品进行检测,并和农业行业标准中的PCR方法进行对比。结果显示:该方法最佳引物为vlhA F3/vlhA R3,在38℃孵育25 min即可完成靶基因扩增,并且特异性强,与鸡毒支原体(MG)、禽多杀性巴氏杆菌、鸡沙门菌、鸡大肠杆菌、鸡新城疫病毒、鸡传染性支气管炎病毒(AIBV)、鸡传染性法氏囊病病毒等其他鸡病病原体无交叉反应;灵敏度高,对MS基因组DNA的最低检出限度为15.5 pg;应用该方法从68份临床样品中检出阳性样品29份,与PCR方法检测结果一致。研究表明,建立的RAA方法快速简便,不依赖昂贵的仪器设备,有望用于基层实验室或MS的临床快速诊断。

To develop a rapid and simple method for Mycoplasma synoviae(MS)detection,three pairs of primers targeting the conserved sequence of vlhA gene of MS were designed,and a recombinase-aided isothermal amplification(RAA)assay was established with the optimal primers after optimizing the reaction conditions.The specificity and sensitivity of the RAA method were evaluated.A total of 68 clinical samples of chicken throat swabs were detected by the RAA assay and PCR method from the agricultural industry standard.The results showed that the target gene of the established RAA assay could be amplified at 38℃for 25 minutes with the optimal primer vlhA F3/vlhA R3.The RAA assay demonstrated strong specificity that no cross-reaction was identified with other chicken pathogens such as Mycoplasma gallisepticum,avian Pasteurella multocida,chicken Salmonella enterica,chicken Escherichia coli,Newcastle disease virus,avian infectious bronchitis virus and infectious bursal disease virus.Furthermore,this method exhibited high sensitivity with the detection limit of 15.5 pg for MS genomic DNA.From the 68 clinical samples,twenty-nine were detected positive by the RAA assay,which was consistent with PCR result.It’s indicated that the RAA assay for MS established in the present study was fast,simple and independent of expensive instruments or equipment,it could be potentially applied for base laboratory or rapid diagnosis of MS.