应用CRISPR/Cas13a快速鉴定布鲁氏菌

Rapid identification of Brucella with CRISPR/Cas13a

目的建立一种基于CRISPR/Cas13a的布鲁氏菌鉴定方法。方法以布鲁氏菌BCSP31基因的保守区为靶标区域设计特异的引物和crRNA,建立鉴定布鲁氏菌的CRISPR/Cas13a方法。通过对纯菌株以及临床需氧血培养阳性菌液检测评价该方法的特异性和敏感性;通过梯度稀释法评估其检测下限。结果建立了基于CRISPR/Cas13a的布鲁氏菌快速鉴定方法,其检测下限可达10 fg/μL。通过鉴定64株布鲁氏菌、56株非布鲁氏菌、人DNA以及57例临床需氧血培养阳性菌液,计算该方法的敏感性、特异性可达100%。结论建立了一种基于CRISPR/Cas13a的布鲁氏菌鉴定方法,可用于临床需氧血培养阳性后快速鉴定布鲁氏菌。

This study aimed to establish a method based on CRISPR/Cas13a to identify Brucella.We designed specific primers for RPA amplification targeting the conserved region of the Brucella BCSP31 gene to develop a CRISPR/Cas13a method to identify Brucella.The specificity and sensitivity of the method were evaluated by testing pure strains and positive clinical aerobic blood cultures;the lower limit of this method was evaluated with the gradient dilution method.A rapid identification method for Brucella through CRISPR/Cas13a was successfully established,and its detection limit reached 10 fg/μL.On the basis of detection of 64 Brucella strains,56 non-Brucella strains,human DNA,and 57 cases of clinical aerobic blood culture positive bacteria,the sensitivity and specificity of this method reached 100%.In conclusion,a Brucella identification method based on CRISPR/Cas13a was established and can be applied for rapid identification of Brucella after a positive clinical aerobic blood culture.