猪附红细胞体重组酶介导等温扩增技术(RAA)荧光检测方法的建立

Establishment of a recombinase aided amplification for fluorescence detection of Eperythrozoon suis

为建立一种准确、快速的猪附红细胞体检测方法,根据猪附红细胞体α-烯醇化酶基因保守序列设计特异性引物和探针,经过筛选优化反应条件,建立了重组酶介导的等温扩增(RAA)荧光检测方法。结果显示,建立的方法在39℃恒温条件下20 min内即可特异性地检出猪附红细胞体,与猪肺炎支原体、猪链球菌、猪大肠杆菌、猪瘟病毒、猪圆环病毒2型、非洲猪瘟病毒、健康猪血液无交叉反应,方法的最低检出限为1 copy/μL。用该方法对15份临床样本进行检测,结果阳性率为60%,与荧光定量PCR方法检测结果一致。本研究建立的猪附红细胞体荧光RAA检测方法具有较好的敏感性和特异性,操作简便可应用于猪附红细胞体病的临床快速检测。

In order to establish a accurate and rapid method to detect Eperythrozoon suis,the specific primers and probes were designed based on the conserved sequence of a-enolase genome of Eperythrozoon suis,and a fluorescence detection method of recombinase aided amplification(RAA)was established through screening and optimization of reaction conditions.The results showed that the whole amplified fluorescence reaction of RAA was completed within 20 minutes under 39℃which can detect Eperythrozoon suis specifically and low detection limit 1 copy per microlite.There were no cross reaction for detecting Mycoplasma hyopneumoniae,Streptococcus suis,Escherichia coli,swine fever virus,porcine circovirus type 2,African swine fever virus and healthy pig blood.15 clinical samples were tested by this method,the positive rate was 60%,which was consistent with the result of fluorescent quantitative PCR.The amplified fluorescence reaction of RAA has good sensitivity and specificity,and it can be applied to the clinical rapid detection of Eperythrozoono suis.