疟疾双重PCR检测方法的建立和初步应用研究

目的 建立一种适合于我国恶性疟、间日疟和两混合感染地区，一次性检测人体内或蚊体内的恶性疟原虫(P.f.)和间日疟原虫(P.v，)的双重聚合酶链反应(PCR)法。方法 根据疟原虫红内期SSUrRNA基因特定片段，设计合成3条寡核苷酸特异引物，在一个反应体系中，同时检测P.f．与P．v.的双重PCR技术，操作方法按PCR常规。结果 从P.f.和P．v．感染血样中，分别扩增出274bp和412bp的特异片段，检出水平达1-5个虫／μl血，而人基因组DNA、约氏疟原虫、伯氏疟原虫均未见扩增条带；但食蟹猴疟原虫(P．c，)与P.v．相似，在412bp可见扩增条带。经对采自不同疟区发热待查病人的滤纸干血滴和确诊疟疾病人的静脉血检测，344例血样中，检出阴性112例、P.f．109例、P.v.l114例、P．f. P.v.9例，阳性率67．4％；比常规镜检的阳性率66．3％稍高，尤以检出P．f． P．v．感染病例高。分别检测人工感染P.f．与P.v.各10只大劣按蚊的阳性唾腺，均被检出同种的阳性结果。结论一次性检测P．f.DNA与P.v.DNA的双重PCR法，敏感性高，特异性强，稳定性好，具有简化操作、提高工效、降低耗费的优点。对发热待查病人诊断和蚊媒感染疟原虫调查，以及疟疾病原学的检测质控方面，均有实用价值。

Studies on the Establishment and Preliminary Application of Double PCR Technique for Detection of Malaria.

Objective To establish a double plymerase chain reaction(PCR) technique suitable for the detection of Plasmodium falciparum(P.f.),plasmodium vivax(P.v.) and mixed infections with P.f. and P.v. In human and mosquitos in the areas prevalent with P.f.,P.v.,and mixed infections with P.f. and P.v. in China. Methods Oligonucleotide primers were, based on the specific fragments of SSUrRNA gene of plasmodium at blood stage, designed and synthesized, P. f. and P. v. could be simultaneously detected in an amplification with double PCR technique. Results Specific fragments of 274 bp and 412 bp wer amplified from P. f. and P. v. infected blood samples, respectively 1-5 parasites per ul blood could be detected. No amplified bands could be found from human genomic DNA, Plasmodium Yoelii(P. y.), Plasmodium berghei(P. b.). As Plasmdium cynomolgi(P. c.) is similar to P. v. ,thus an amplified bands of 412bp in length was noticed. 344 samples consisted of veinous blood samples from confirmed patients and dried filter paper samples from feverish patients in different malaria endemic areas were detected. The results show that 112 negatives, 109 P.f. positives, 114 P.v. positives and 9 positives with P. f. and P.v. mixed infection with a positive rate of 67.4% , a little higher than microscopy(66.3%) . One case was misdiagnosed by PCR and 5 casess were misdiagnosed by microscopy and the coincidence of PCR with microscopy was 95% . The same positive results were obtained in the detection of the positive Salivarg glands of 10 mosquitoes infected with P.f. and 10 with P. v. in the laboratory. Conclusion the sensitivity and specificity of double PCR technique capable of simultaneous detection of P. f. and P. v. in a single amplification is higher and with the advantages of simplicity, efficiency and tow cost in addition to practical value in the diagnosis of feverish patients, investigation of plasmodium infected mosquito vectors and quality control in detection of malaria parasites.