套式聚合酶链式反应诊断三日疟原虫感染的临床应用

Diagnosis of plasmodium malariae infection by nested PCR

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 diagnose the imported plasmodium malariae cases with nested-PCR amplification of 1 8SSUrRNA gene.Methods Plasmodium DNA was extracted from anticoagulant blood as template with the plasmodium gene-specific primers in the first round of amplification,then the products of the first round were amplified as the second round template.The results of different subgroups of plasmodium were acquired.The target fragment of PCR was used for species identification and sequencing,comparing with the control sample of P.vivax and P.falciarum.Results By using the plasmodium gene-specific primers amplification,the fragments were amplified from the three samples with 1 200 bp.By using the interspecific primers amplification,specific fragments were amplified from the blood samples of P.vivax and P.falciarum with 1 20 bp and 205 bp respectively,and 1 44 bp from the suspicious patient with P.malariae specific primers.The suspicious patients did not have the specific fragment of P.ovale.The product of the suspicious patients was sequenced and compared with standard sequence registered in Genbank by blast.The comparison results showed that the fragment size and product sequence were in accordance with the reference.The results showed that nested PCR was more objective than the traditional microscopic examination.Conclusions The results indicate that nested PCR can be used to detect plasmodium with high specificity and identify plasmodium at the species level when microscopy is equivocal.The specific primers to amplify 1 8 SSUrRNA gene fragment can be used to diagnose plasmodium malariae infection.