基于CRISPR-Cas13a技术的乙型肝炎病毒cccDNA检测方法临床应用评价

Clinical application evaluation of the detection method for hepatitis B virus cccDNA based on CRISPR-Cas13a technology

目的 评价基于CRISPR-Cas13a技术的乙型肝炎病毒(HBV)共价闭合环状DNA(cccDNA)检测方法的应用价值。方法 收集25例HBV感染患者和5例非HBV感染患者的肝组织标本,提取肝组织的总DNA,依次使用HindⅢ内切酶和质粒安全性ATP依赖DNA酶(PSAD)消化;设计HBV cccDNA的特异性引物,扩增消化后的产物;利用CRISPR/Cas13a技术建立CRISPR-HBV cccDNA检测方法,通过HBV cccDNA质粒和临床样本对该方法进行评价,并利用数字PCR和荧光定量PCR方法进行比较。结果 本研究建立的方法检测HBV cccDNA质粒的灵敏度为1 copies/μL,显著高于数字PCR(10 copies/μL,P<0.01)和荧光定量PCR(10^(3)copies/μL,P<0.01);建立的方法检测HBV cccDNA阳性样本的灵敏度为1 copies/μL,与数字PCR相同,高于荧光定量PCR(10 copies/μL,P<0.01);建立的方法对25例HBV感染的临床肝组织样本的检出阳性率为80%,均高于数字PCR(64%)和荧光定量PCR(48%)。结论 本研究建立的CRISPR-HBV cccDNA检测方法在乙肝患者肝组织标本HBV cccDNA检测中具有较高的灵敏度,为评价乙肝患者的临床治疗效果和监测停药复发等情况提供了有效帮助。

Objective To evaluate the application value of the detection method for hepatitis B virus(HBV) covalently closed circular DNA(cccDNA) based on CRISPR-Cas13a technology. Methods Liver tissue samples from 25 HBV-infected patients and 5 non-HBV-infected patients were collected, and the total DNA was extracted and digested with HindⅢ endonuclease and Plasmid-safeTMATP-dependent DNase(PSAD) in turn. The digested products were amplified with the designed specific primers of HBV cccDNA. Then, a method for detecting HBV cccDNA was established using the CRISPR/Cas13a technology. The established method was evaluated by the HBV cccDNA plasmid and clinical samples, and compared with digital PCR and fluorescence quantitative PCR. Results The sensitivity of the established method to detect HBV cccDNA plasmid was 1 copy/μL, which was higher than that of digital PCR(10 copies/μL, P<0.01) and fluorescence quantitative PCR(10^(3) copies/μL, P<0.01). The sensitivity of the established method to detect the clinical samples with positive HBV cccDNA was also 1 copy/μL, which was the same as that of digital PCR and higher than that of fluorescence quantitative PCR(10 copies/μL, P<0.01). The positive rate of 25 clinical liver tissue samples from HBV-infected patients detected by the established method was 80%, which was higher than that of digital PCR(64%) and fluorescence quantitative PCR(48%). Conclusion The established CRISPR-HBV cccDNA method for detecting HBV cccDNA in the liver tissue samples of HBV-infected patients in this study has good sensitivity, which provides effective help for evaluating the clinical treatment effect of HBV-infected patients and monitoring the relapse after drug withdrawal.