CRISPR-Cas13a辅助RAA快速检测金黄色葡萄球菌的研究

Rapid detection of Staphylococcus aureus by CRISPR-Cas13a combined with recombinase aided amplification(RAA)

目的建立一种金黄色葡萄球菌快速分子检测技术。方法根据金黄色葡萄球菌耐热核酸酶基因(nuc)保守区序列设计合成特异性引物,通过对反应条件进行优化,建立金黄色葡萄球菌重组酶介导的等温扩增技术(Recombi-naseaidedamplification,RAA);表达纯化CRISPR-Cas13a蛋白,设计特异的crRNA(CRISPR RNA),以crRNA引导CRISPR-Cas13a蛋白对RAA产物进行检测;对优化的方法进行灵敏性和特异性评价,同时采用该方法与real-timePCR法对食品标本中的金黄色葡萄球菌进行检测,评价方法的一致性。结果CRISPR-Cas13a辅助RAA检测金黄色葡萄球菌的灵敏度为101 CFU/ml,高于real-timePCR,约102 CFU/ml,检测时间仅需30min,与其他食源性致病菌无交叉反应;该方法和real-timePCR检测80份食品样品的阳性率均为8.75%,具有高度一致性(Kappa=1,P>0.05)。结论建立的CRISPR-Cas13a辅助RAA方法具有简便、快速、灵敏、特异等优点,为金黄色葡萄球菌的检测提供了新的技术手段。

Objective To establish a technique for rapid molecular detection of Staphylococcus aureus.Methods Specific primers were designed and synthesized according to the sequence of the conserved region of the S.aureus thermonuclease gene(nuc).Recombinase-aided amplification(RAA)was performed by optimizing reaction conditions.CRISPR-Cas13a was expressed and purified.After specific crRNA(CRISPR RNA)was designed and synthesized,RAA products were detected with CRISPR-Cas13a,which was guided by crRNA.The sensitivity and specificity of the optimized method were also evaluated.At the same time,the technique and real-time PCR were both used to detect S.aureusin food samples,and their consistency was evaluated.Results The minimum detection limit of CRISPR-Cas13a combined with RAA with respect to S.aureus was 10~1 CFU/mL,which was more sensitive than real-time PCR(10~2 CFU/mL).The bacterium at a concentration of 101 CFU/mL was detected within 30 min,and templates at a higher concentration took less time.There was no cross-reaction with other food-borne pathogens.In 80 food samples,CRISPR-Cas13a combined with RAA and real-time PCR both detected the bacterium at a rate of 8.75%(Kappa=1,P>0.05),which indicated that the technique is highly consistent with real-time PCR.Conclusion As established in this study,CRISPR-Cas13a combined with RAA has the advantages of simplicity,rapidity,sensitivity,and specificity and provides a new method for detecting S.aureus.