复式PCR检测间日疟原虫和恶性疟原虫的现场应用

目的探讨复式PCR方法在疟疾诊断中的现场应用价值。方法根据疟原虫18 S rRNA基因序列,合成疟原虫属特异性上游引物和间日疟原虫、恶性疟原虫种特异性下游引物,优化PCR反应体系,建立在同一PCR反应体系中同时检测间日疟原虫、恶性疟原虫基因组特异性DNA片段的疟疾诊断方法,并评价其现场应用价值。结果间日疟原虫和恶性疟原虫基因组DNA经过复式PCR后,分别扩增出1 451 bp和833 bp的特异性条带,而伯氏疟原虫、食蟹猴疟原虫及健康人血样均无扩增带出现,用该反应体系可检出原虫血症为1.1×10-6和5.6×10-7的间日疟原虫和恶性疟原虫感染。与镜检法相比,复式PCR检测119份现场样本,112份与镜检结果相同,阳性率为54%,漏诊率为0.8%,误诊率为0,而镜检法依次分别为53%、1.7%和3.4%。结论复式PCR方法检测疟原虫具有简便、快速、特异、敏感等优点,在疑似病例的鉴别诊断和分子流行病学调查中具有良好的应用价值。

Field application of multiplex PCR to distinguish Plasmodium vivax and Plasmodium falciparum

Objective To explore the application value of multiplex PCR in the diagnosis of malaria in field.Methods The plasmodium genus-specific primer,Plasmodium vivax,P.falciparum species-specific primers were synthesis based on the specific target segments of small subunit of 18 S rRNA ribosomal.The multiplex PCR system was optimized,and a PCR diagnostic method of malaria was established based on the genomic specific DNA fragment of P.vivax,and P.falciparum was amplified in the same PCR reaction system.The sensitivity,specificity,and the value of field application of the multiplex PCR were investigated.Results The sizes of amplification products of multiplex PCR amplifying genomic DNA of P.vivax and P.falciparum were 833 bp and 1 451 bp,respectively,and the amplification did not take place with the samples DNA of P.berghei,P.cynomolgus and healthy human blood.The sensitivities of multiplex PCR to detect P.vivax and P.falciparum were 1.1 × 10-6 and 5.6 × 10-7 parasitemia,respectively.Compared with the microscopic examination,the positive rate of multiplex PCR to detect 119 cases of field samples was 54%,missed diagnosis rate was 0.8%,and the misdiagnosis rate was naught,while the positive rate of the microscopic examination was 53%,its misdiagnosis rate and missed diagnosis rate were 1.7% and 3.4%,respectively.The compliance rate between the multiplex PCR and microscopic examination was 94%.Conclusion The multiplex PCR for detecting malaria is simple,rapid,specific,sensitive,etc.,which is suitable for the differential diagnosis of suspected cases,and molecular epidemiology investigation.