多重荧光定量PCR测定疟原虫方法建立

发布时间：2019-04-18 06:23

【摘要】：目的建立可同时检测四种疟原虫的多重荧光定量PCR方法。方法 PCR扩增四种疟原虫目的片段,克隆并构建标准质粒,进行梯度稀释。体系按浓度加入五重引物探针,对临床四种疟原虫阳性血样及单一腺病毒和肺炎支原体阳性血样、人基因组进行特异性检测。将本地区优势虫种恶性疟及间日疟阳性血样混合并检测。每个反应做一式三份验证重复性。检测标准品梯度浓度,获得体系最低检测限。结果成功构建四种疟原虫标准质粒。对20份样本进行检测,其中15份恶性疟,3份间日疟,1份三日疟,1份卵形疟,与原有确证结果一致。对单一腺病毒及肺炎支原体阳性血样、人基因组检测结果均为阴性。恶性疟及间日疟阳性血样混合检测显示3条特异性扩增曲线。三个复孔的变异系数在0.17~4.96之间。恶性疟可检测到10~2copies/m L,间日疟、三日疟、卵形疟可检测到10~3copies/m L。结论建立五重荧光定量PCR检测四种疟原虫特异性及重复性均较好,灵敏度高,可用于疟原虫快速筛选及分型鉴定。
[Abstract]:Objective to establish a multiplex fluorescence quantitative PCR method for simultaneous detection of four species of Plasmodium malaria. Methods the fragments of four species of Plasmodium were amplified by PCR, and the standard plasmids were cloned and constructed for gradient dilution. In this system, four clinical samples of malaria parasite, single adenovirus and mycoplasma pneumoniae positive blood samples were detected by adding five-primer probe according to the concentration, and the human genome was detected specifically. The positive blood samples of Plasmodium falciparum and Plasmodium vivax were mixed and detected. Each reaction is done in triplicate to verify repeatability. The minimum detection limit of the system was obtained by detecting the gradient concentration of the standard sample. Results four standard plasmids of Plasmodium malaria were constructed successfully. There were 15 falciparum malaria, 3 vivax malaria, 1 egg malaria and 15 falciparum malaria, 3 vivax malaria, 1 egg malaria. The results of human genome detection were negative for single adenovirus and mycoplasma pneumoniae positive blood samples. The mixed detection of Plasmodium falciparum and Plasmodium vivax showed 3 specific amplification curves. The coefficient of variation of the three complex pores was between 0.17 and 4.96. Plasmodium falciparum can detect 10~2copies/m L, Plasmodium vivax, Plasmodium triviparum, Plasmodium ovale can detect 10~3copies/m L. Conclusion five-fold fluorescence quantitative PCR can be used for rapid screening and typing identification of Plasmodium malaria, which has good specificity and reproducibility and high sensitivity.
【作者单位】：丽水出入境检验检疫局;浙江省丽水市疾病预防控制中心检验科;
【基金】：浙江出入境检验检疫局重点科研计划项目计划(2014-ZKZ-006)
【分类号】：R531.3

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