仿刺参腐皮综合征病原灿烂弧菌RPA-Cas13a检测方法的建立

Establishment of a Cas13a-RPA Detection Method for the Causative Pathogen Vibrio splendidus of Sea Cucumber Apostichopus japonicus Skin Ulcerative Syndrome

为预防和控制灿烂弧菌引发的仿刺参腐皮综合征,促进仿刺参养殖业的健康可持续发展,利用LwCas13a中特异性CRISPR RNA(crRNA)与靶RNA结合后可激活Cas13a蛋白对外源RNA分子附属切割活性的特征,结合重组酶介导的聚合酶扩增技术(RPA),建立1种针对灿烂弧菌gyrB基因的快速、灵敏、特异的定量检测方法——RPA-Cas13a。RPA-Cas13a可实现在37℃恒温下、60 min内对灿烂弧菌的快速实时检测。灵敏度测试结果显示,RPA-Cas13a检测限为550拷贝/μL(gyrB),与荧光定量PCR的检测灵敏度水平一致;特异性测试结果表明,RPA-Cas13a对创伤弧菌、哈维氏弧菌、鳗弧菌等其他弧菌及产黑假交替单胞菌、嗜水气单胞菌、迟缓爱德华氏菌等常见水产动物病原菌均无交叉反应,特异性较强。RPA-Cas13a方法对仿刺参体表黏液(32份)、养殖池塘水体(15份)和表层沉积物(10份)等57份环境样本的阳性检出率为8.77%,与传统荧光定量PCR方法的检出一致性高(Kappa=1.00),无统计学差异(P>0.05)。试验结果表明,灿烂弧菌的RPA-Cas13a新型定量检测方法,快速简便、灵敏特异,可为预防和控制仿刺参腐皮综合征的发生提供有力的分子工具。

In order to prevent and control the skin ulcerative syndrome(SUS)of sea cucumber Apostichopus japonicus caused by Vibrio splendidus and maintain the healthy and sustainable development of the sea cucumber aquaculture industry,this study used the characteristic of LwCas13a protein when specific crRNA in LwCas13a binds to the target RNA,its collateral cleavage activity can be activated to degrade non-targeted exogenous RNA.Combined this characteristic with the recombinase polymerase amplification(RPA)technique,a rapid,sensitive and specific quantitative detection method for gyrB gene of V.splendidus-RPA-Cas13a was established.This method can realize rapid real-time detection of V.splendidus at constant 37℃within 60 mins.Sensitivity test showed that the detection limit of this method is 550 copies/μL(gyrB),which is consistent with the sensitivity level of fluorescence quantitative PCR(qPCR);Specificity test demonstrated a relatively high specificity with the fact that it had no cross-reactions with other Vibrio species,e.g.V.vulnificus,V.harveyi and V.anguillarum,and other common aquatic animal bacterial pathogens,including Pseudoalteromonas nigrifacien s,Aeromonas hydrophlia and Edwardsiella tarda.The detection rate of 57 environmental samples including skin mucus of A.japonicus(32 samples),culture pond seawater(15 samples)and surface sediments(10 samples)was 8.77%,highly consistent with that of traditional fluorescence qPCR method(Kappa=1.00)with no statistical difference(P>0.05).In conclusion,this study established a novel rapid,simple,sensitive and specific RPA-Cas13a quantitative detection method for V.splendidus,which provides a powerful molecular tool for preventing and controlling the occurrence of sea cucumber SUS.