目的应用套式聚合酶链式反应(PCR)扩增小亚单位核糖体核糖核酸(SSUrRNA)基因诊断三日疟原虫感染,以减少三日疟的漏诊和误诊。方法分别提取可疑三日疟患者抗凝血DNA和对照间日疟、恶性疟患者抗凝血DNA,以此为模板,用疟原虫属特异性引物进行第一轮扩增;然后以第一轮扩增产物为模板,用4种疟疾的种特异性引物进行第二轮扩增,比较扩增出的18 SSU rRNA基因片段的大小,并对目的片段进行测序鉴定。结果经属特异性引物PCR扩增后,3个样本均出现大小约为1 200bp的条带。经种特异性引物PCR扩增后,间日疟及恶性疟确诊样本均扩增出相应的120bp和205bp特异性条带;可疑患者样本仅在用三日疟原虫种特异性作引物时扩增出144bp特异条带,与理论值相符,与Genbank标准序列对比显示,扩增片段大小及测序结果均完全正确。证实患者感染三日疟原虫。结论利用小亚单位核糖体核糖核酸基因片段三日疟种特异引物进行扩增可以用于诊断三日疟原虫感染。 Objective To detect the small subunit ribosomal RNA (SSUrRNA) gene by nested polymerase chain reaction (PCR) to diagnose Plasmodium malariae infection and to reduce the missed diagnosis and misdiagnosis of Plasmodium malariae. Methods The anticoagulant DNA of Plasmodium falciparum and Plasmodium falciparum in patients with suspected three-day malaria was extracted and used as a template for the first round of amplification with Plasmodium-specific primers. Then, The amplified product was used as a template and the second round of amplification was carried out using four kinds of malaria species-specific primers. The size of the amplified 18 SSU rRNA gene fragment was compared, and the target fragment was sequenced and identified. Results After PCR amplification with specific primers, bands of about 1 200 bp appeared in all three samples. After PCR amplification of species-specific primers, specific bands of 120 bp and 205 bp were amplified from diagnosed samples of P. vivax and P. falciparum; suspicious patient samples were amplified only with P. vivax species-specific primers A 144 bp specific band was obtained, which was in good agreement with the theoretical value. Comparing with the Genbank standard sequence, the amplified fragment size and sequencing result were completely correct. Confirmed patients infected with P. malariae. Conclusion The amplification of small subunit ribosomal RNA gene fragment of Plasmodium malariae can be used to diagnose Plasmodium malariae infection.