多重荧光定量PCR测定疟原虫方法建立..docVIP

多重荧光定量PCR测定疟原虫方法建立 　　[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102 copies/mL，间日疟、三日疟、卵形疟可检测到103 copies/mL。 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 [关键词] 多重荧光定量PCR；疟原虫；蚊传染病；疟疾 [中图分类号] R440 [文献标识码] A [文章编号] 1673-9701（2016）23-0004-03 [Abstract] Objective To establish the multiple fluorescence quantitative PCR measure to detect four types of plasmodium at the same time. Methods Target fragments of four types of plasmodium were extended by PCR， the standard plasmid was cloned， constructed and diluted by gradient. Five primers and probes were added into the system according to the concentration and the clinical four plasmodium positive blood sample， single adenovirus and mycoplasma pneumonia positive blood sample as well as human gene was detected specifically. The falciparum malaria and tertian malaria positive blood sample caused by the local superior plasmodium were mixed and detected. Each experiment was performed for three times to validate the repetitiveness. The gradient concentration of the sample was detected and the lowest detection limitation was achieved. Results The standard plasmids of four plasmodiums were successfully constructed and 20 samples were detected. 15 samples were falciparum malaria and 3 samples were tertian malaria， 1 sample was quartan malaria and 1 sample was ovale malaria， which was in accordance with the original validated results. The results of detection of single adnovirus and mycoplasma pneumonia positive blood sample and human gene were negative. 3 specifically extended curves were presented by the mixed detection of falciparum malaria and tertian malaria positive blood sample. The variable coefficients of the three duplicati