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目的建立鉴别恶性疟和间日疟的SYBR Green实时PCR检测方法。方法针对恶性疟原虫和间日疟原虫18S rRNA基因设计引物,优化引物浓度与退火温度,建立可扩增出两种疟原虫基因片段的SYBR Green实时PCR,并进行54例临床标本检测,其中恶性疟32例、间日疟22例。以镜检法为金标准分析敏感度和特异度等指标。结果该方法可扩增出恶性疟原虫和间日疟原虫18S rRNA基因片段,并能检出混合感染,特异性好。该方法检测恶性疟原虫或间日疟原虫的敏感度为97.30%(36/37),特异度为5.88%(1/17);检测恶性疟原虫,敏感度为87.50%(21/24),特异度为63.33%(19/30);检测间日疟原虫,敏感度为69.23%(9/13),特异度为68.29%(28/41)。结论所建立的SYBR Green实时PCR方法能较准确地诊断疟疾并鉴别虫种,敏感度高,在混合感染的诊断方面具有优越性。
Objective To establish a SYBR Green real-time PCR assay for the identification of P. falciparum and P. vivax. Methods Primers were designed according to the 18S rRNA gene of Plasmodium falciparum and Plasmodium vivax, and the primer concentration and annealing temperature were optimized. SYBR Green real-time PCR was established to amplify two kinds of plasmodium gene fragments and 54 clinical samples were detected. Malignant 32 cases of malaria, vivax malaria in 22 cases. To microscopic examination method for the gold standard analysis of sensitivity and specificity and other indicators. Results The method could amplify the 18S rRNA gene fragment of Plasmodium falciparum and Plasmodium vivax and could detect the mixed infection with good specificity. The sensitivity and specificity of this method for detecting Plasmodium falciparum or Plasmodium vivax were 97.30% (36/37) and 5.88% (1/17), respectively. The detection rate of Plasmodium falciparum was 87.50% (21/24) The specificity was 63.33% (19/30). The detection of P. vivax was 69.23% (9/13) in sensitivity and 68.29% (28/41) in specificity. Conclusion The established SYBR Green real-time PCR method can accurately diagnose malaria and identify worms with high sensitivity and is superior in the diagnosis of mixed infection.