PCR-RFLP检测疟原虫方法的建立和应用效果

Development and Application of PCR-RFLP to Plasmodium detection

目的建立PCR-RFLP方法检测疟原虫,并评价其应用效果。方法收集2010-2018年武汉市输入性疟疾患者血样。根据4种人疟原虫线粒体细胞色素b(Cytb)基因属、种特异位点,设计1组疟原虫属特异性公共引物。提取患者血样中的疟原虫DNA,巢式PCR扩增得到产物后,用限制性内切酶AluⅠ进行酶切,不同种疟原虫将产生特异性的酶切条带,建立PCR-RFLP检测方法。分析该方法的灵敏度和特异度,并与巢式PCR相比较,评价其应用价值。结果PCR-RFLP检测恶性疟、三日疟、间日疟和卵形疟血样(包括curtisi亚种和wallikeri亚种),最低检出阈值分别为2.5、2、3.6、4、1.5个虫/μL血。共检测121份疟疾血样和45份健康人血样,巢式PCR检出阳性115份,PCR-RFLP方法检出阳性118份,灵敏度分别为95.0%和97.5%,结果一致性较好(Kappa值=0.93),差异无统计学意义(χ2=0.8,P>0.05)。PCR-RFLP分别检测日本血吸虫、利氏曼原虫、隐孢子虫样品无任何扩增条带产生,均为阴性。结论建立的PCR-RFLP方法实验流程相对简单,且检出阈值低,敏感度、特异度高,检测结果易于判断,是一种新的疟原虫核酸检测方法。

Objective To establish a PCR-RFLP method for detecting Plasmodium,and evaluate the application effect of this technique.Methods The blood samples were collected from patients with imported malaria between 2010 and 2018.Group-specific primers for Plasmodium was designed according to the genus and species specific sites of 4 Plasmodium mitochondrial cytochrome b(cytb)genes.The DNA was extracted from the blood samples,and amplified for the gene fragments using nested PCR.The amplified products were digested by restriction enzyme Alu I.PCR-RFLP detection procedures were established on the specific bands generated by different species of Plasmodium.Then the sensitivity and specificity of this newly developed method were analyzed and compared with the detection findings by nested PCR,together with application of the value of this method.Results The minimal threshold was 2.5,2,3.6,4 and 1.5 Plasmoda/μL blood sample for P.falciparum,P.malariae,P.vivax,P.ovale(P.oc and P.ow),respectively,by PCR-RFLP.Totally,121 blood samples of malaria patients and 45 blood samples of healthy subjects were detected.Nested-PCR identified 115 positive samples,and PCR-RFLP exposed 118 positive samples.The sensitivity was 95.0%and 97.5%,respectively,which indicated better consistency between the two techniques(Kappa value=0.93),yet no statistical difference(χ2=0.8,P>0.05).No specific bands were seen for Schistosoma japonicum,Leishmania and Cryptosporidium.Conclusion PCR-RFLP can be served as a new approach to detection of nucleic acid in Plasmodium,because this technique possesses higher sensitivity and specificity,with minimal threshold and easy judgment of the findings.