利用CRISPR/Cas12a技术快速检测胸膜肺炎放线杆菌方法的建立

Establishment of a visual method for detection of Actinobacillus pleuropneumoniae based on CRISPR-Cas12a

为建立可用于临床检测猪胸膜肺炎放线杆菌(App)的方法,本研究根据App apxIVA基因3’末端保守区,设计CRISPR/Cas12a特异性gRNA,在gRNA序列与靶序列结合位点的上下游区域内,利用Primer Premier 5.0设计重组酶聚合酶扩增(RPA)引物,利用RPA引物扩增靶基因apxIVA,通过gRNA识别特异性靶基因序列,激活Cas12a蛋白单链DNA酶的切割活性,根据免疫层析试纸条检测原理设计单链DNA报告分子,使用免疫层析试纸条检测体系内报告分子是否被切割进而实现对靶基因的检测。并通过各反应条件的优化建立了一种针对APP的检测方法。反应条件优化结果显示,最佳单链DNA报告分子浓度及gRNA浓度分别为500 nmol/L、100 nmol/L;最佳孵育缓冲液为10%聚乙二醇,最佳反应时间为30 min。该方法仅对App的检测为阳性,而对副猪嗜血杆菌、猪链球菌、沙门氏菌、猪圆环病毒、猪伪狂犬病毒、猪流行性腹泻病毒的检测结果均为阴性,特异性较强;敏感性试验结果显示该方法对App重组质粒标准品的检测下限可达10拷贝/μL,比普通PCR方法敏感性高1 000倍;重复性试验结果显示,该方法对3个不同稀释度重组质粒标准品的批内和批间重复性试验检测结果均一致,重复性较好。对40份小鼠的肺脏病料样品进行检测,结果显示本研究建立的方法检出27份阳性样品,且通过试纸条即可直接观察结果。常规PCR方法检出25份阳性样品,二者的符合率为95%。本研究所建立的方法具有操作简单、快捷、结果直观,无需昂贵设备等优点,为现地快速检测该病原提供了一种切实可行的技术手段。

The aim of this study was to establish a method for detecting Actinobacillus pleuropneumoniae(App), gRNA was designed according to the conservative sequence of apx Ⅳ A gene which was specific for App. In the upstream and downstream regions of the binding site between the gRNA sequence and the target sequence. Primer Premier 5.0 was used to design recombinase polymerase amplification(RPA) primers, and the RPA primers were used to amplify the target gene apxIVA. The specific target gene sequence was recognized by g RNA, followed by the activation of the Cas12 a protein-mediated single-stranded DNA(ss DNA) enzyme activity, and then design a ss DNA reporter according to the principle of test strip, and use the test strip to detect whether the ss DNA reporter in the system is cleaved to detect the target gene. The optimization of various conditions established a detection method for APP. Results showed that the optimal ss DNA reporter concentration and g RNA concentration were 500 nmol/L and 100 nmol/L, respectively;the optimal incubation buffer was 10% polyethylene glycol;and the optimal reaction time was 30 min. The test results of this method for Haemophilus parasuis, Streptococcus suis, Salmonella, Porcine circovirus,Porcine pseudorabies virus and Porcine epidemic diarrhea virus showed that the specificity was high. The sensitivity test results showed that the detection limit of plasmid could reach 10 copies/μL, which is 1000 times more sensitive than ordinary PCR methods. The repeatability test results showed that the method was consistent intra-assays and inter-assays, and the results of3 different dilutions of plasmid standard products were consistent. The repeatability is well. Forty lung disease samples of mice were tested. The results showed that the method established in this study detected 27 positive samples and 13 negative samples;and conventional PCR methods detected 25 positive samples and 15 negative samples. The coincidence rate of the two methods is 95%,and the experimental results can be directly observed through the test strip. The method established in this study has the advantages of simple and fast operation, intuitive results, and no need for expensive equipments, and provides a practical and feasible technical means for rapid detection of pathogens on-site.