目的 采用环介导等温扩增（LAMP）方法检测疟原虫DNA,并评价其检测效果。方法 采集2013-2018年从疟疾流行区回国人员中确诊为疟疾患者的滤纸血样。根据疟原虫的18S rRNA序列,使用PrimerExplorer V5软件设计恶性疟原虫与非恶性疟原虫LAMP引物,建立疟原虫LAMP检测方法,进行敏感性、特异性、灵敏度和扩增效率评价,并与巢式PCR比较。检测钙黄绿素指示剂对LAMP反应的影响。结果 LAMP和巢式PCR各检测96份疟疾患者血样（47份恶性疟血样和49份其他疟疾血样）,均检出93份疟原虫阳性。LAMP检测结果显示,恶性疟血样均被检出,非恶性疟假阴性率为2.1%,10份利什曼原虫玻片血、8份日本血吸虫感染兔血和38份健康人血均为阴性,与巢式PCR检测结果具有极高的一致性（Kappa值 = 0.956）。LAMP检测恶性疟原虫、间日疟原虫、卵形疟原虫、三日疟原虫的灵敏度分别为25、3、4、2个疟原虫/μl血。扩增效率检测结果显示,4种疟原虫LAMP反应出现浊度时间（Tt值）均小于60 min,速率曲线峰值（Df）达0.14以上,其中恶性疟原虫扩增效率较高,Tt值为20.4 min,Df达0.16。可视化LAMP检测结果显示,钙黄绿素指示剂对不同种疟原虫LAMP反应延时约8~22 min,Df下降约21%~35%。结论 LAMP技术检测疟原虫有高度敏感性、特异性和灵敏度,且可视,具有在现场及基层医疗机构应用前景。

Objective To detect the DNA of malaria parasites using the loop-mediated isothermal amplification (LAMP) method and evaluate the efficacy of this method.Methods Blood samples on filter papers were collected from confirmed malaria patients returning from endemic areas of other countries during 2013-2018. According to the 18S rRNA sequences of malaria parasites, LAMP primers for Plasmodium falciparum and other species of Plasmodium were designed using the Primer Explorer V5 software. The LAMP method for detecting malaria parasites was established and its sensitivity, specificity, detection limit and amplification efficiency were assessed and compared with the nested-PCR. The effect of calcein indicator on LAMP reaction was tested.Results The LAMP and nested-PCR were performed separately on 96 blood samples, including 47 with P. falciparum and 49 with other species of Plasmodium. Either method found 93 samples as malaria-positive. The LAMP method produced positive results for all the 47 blood samples with P. falciparum, while having a false negative rate of 2.1% among the samples with other Plasmodium spp.. The LAMP method provided negative results for 10 samples with Leishmania, 8 with Schistosomiasis japonicum, and 38 blood samples from healthy participants, highly consistent with the results of nested-PCR (Kappa value, 0.956). The sensitivity of LAMP for P. falciparum, P. vivax, P. ovale and P. malariae was 25, 3, 4, and 2 parasites/μl blood, respectively. Amplification efficiency test showed that the turbity time (Tt) and peak value of rate (Df) of LAMP for the four malaria species were less than 60 min and above 0.14, respectively, of them the amplification efficacy was particularly high for P. falciparum (Tt, 20.4 min; Df, 0.16). In visualized LAMP assay for the four malaria species, using calcein indicator delayed LAMP reaction Tt about 8-22 min, and lower the Df by about 21%-35%.Conclusion The LAMP method is highly sensitive, specific, of low detection limit and has potentials for application on-site in the field and in primary medical settings.