摘要
目的对多重PCR、巢式PCR、实时PCR检测结果进行比较,筛选适合低密度恶性疟原虫血症的检测方法。方法实验室培养恶性疟原虫,同步化处理至环状体期占99%以上时涂制薄血膜,计数其原始密度。实验分3组,原虫血均按1︰40、1︰80、1︰160、......1︰40 960等11个梯度进行稀释,涂制厚血膜,并用显微镜检查确定低密度恶性疟原虫血症密度;再作倍比系列稀释,用3种PCR方法进行检测,确定各种检测方法的检测限,并进行比较。结果经显微镜检查确定配制的待测血样恶性疟原虫密度为21.92个/μl,多重PCR检测限为0.171 3个疟原虫/μl血,实时PCR为0.171 3个疟原虫/μl血,巢式PCR为0.085 6个疟原虫/μl血。检出率巢式PCR≥实时PCR>多重PCR。结论 3种PCR方法均可用于检测低密度恶性疟原虫血症。巢式PCR检出率和检测限均优于其他两种方法。
Objective To explore suitable methods of testing for submicroscopic Plasrnodium falciparum infection by comparing detection with multiple PCR, nested PCR, and real time PCR. Methods P. falciparurn （FCCI/HN） was cultured and synchronized to the ring stage （〉 99 % ） in vitro, and parasites in Giemsa-stained thin smears were counted. The parasites were divided into three groups and each was then diluted to 1 ： 40, 1 ： 80, 1 ： 160, 1 ： 320 1 ： 40 960. Giemsa-stained thick smears of all of the diluted samples were tested microscopically and the threshold for submi croscopic P. falciparurn was determined. Samples with submicroscopic parasites were then diluted 2-fold （1 ： 2, 1 ： 22, 1 ： 23, 1 ： 24 ）. All of the samples were tested using multiple PCR, nested PCR, and real-time PCR and the limit of each method was determined. Results The threshold for submicroscopic P. falciparurn was 21.92/μl. The LOD was 0. 171 3/μl for multiple PCR, 0. 171 3/μl for real time PCR, and 0. 085 6 /μl for nested PCR. Nested PCR had the highest rate of detection, followed by real-time PCR and then multiple PCR. Conclusion All three methods are suitable for detecting submicroscopic P. falciparum. Nested PCR had much better detection than the other two meth- ods.
出处
《中国病原生物学杂志》
CSCD
北大核心
2013年第4期331-335,共5页
Journal of Pathogen Biology
基金
亚洲消除疟疾合作网络(APMEN)资助项目(No.108-06)
