关键词: 疟原虫, 细胞色素 b 基因, 聚合酶链式反应-限制性片段长度多态性, 检测方法

Abstract: 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. ocand 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 an 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.

Key words: Plasmodium, Cytb gene, PCR-RFLP, Detection methold