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目的建立一种快速、敏感、特异检测恶性疟原虫的交叉引物等温扩增(cross priming amplification,CPA)技术。方法根据疟原虫的18SrRNA基因保守序列,设计CPA引物和探针。分别用CPA法、吉氏染色镜检法检测78份发热患者血样,验证CPA法特异性和灵敏性。结果采用CPA法对不同病原体核酸的检测结果显示,38份含有恶性疟原虫血样中检出37例阳性,其他病原体检测结果均为阴性,证明其特异性良好;该方法检测的灵敏度可达102拷贝/μl;与镜检法比较,其检测的灵敏度为97.37%,特异性为100%,准确度为98.72%。结论用于检测恶性疟原虫的CPA检测体系具有简便、快速、灵敏、特异的优点,可用于临床、预防和出入境口岸现场疟疾的快速检测。
Objective To establish a rapid, sensitive and specific cross-priming amplification (CPA) technique for the detection of Plasmodium falciparum. Methods According to the conserved sequence of 18S rRNA gene of Plasmodium, CPA primers and probes were designed. 78 cases of fever patients were detected by CPA method and Kyrgyzstan stain, respectively, to verify the specificity and sensitivity of CPA method. Results The results of CPA assay on different pathogens showed that 37 samples were positive in blood samples of P. falciparum and all other pathogens were negative, which proved their specificity was good. The sensitivity of this method was 102 copies / μl; compared with the microscopic examination, the detection sensitivity was 97.37%, the specificity was 100% and the accuracy was 98.72%. Conclusion The CPA assay system for the detection of Plasmodium falciparum has the advantages of simple, rapid, sensitive and specific. It can be used in clinic, prevention and rapid detection of malaria at the port of entry and exit.