多重PCR种特异性检测恶性疟原虫和间日疟原虫方法的建立

摘要

目的建立恶性疟原虫和间日疟原虫种特异性检测的多蕈PCR方法,用于疟疾的检测和诊断.方法根据疟原虫18S核糖体小亚基ssRNA的基因序列设计合成8对11条引物,通过对恶性疟、间日疟患者及健康对照者血样的DNA进行扩增,选择出敏感性和特异性最佳的引物用于建立多重PCR方法,并用梯度变化的方法分别对引物浓度、复性温度、延伸温度和循环次数等反应参数进行比较分析,优化PCR反应条件.利用优化后的多重PCR埘采自云南和上海的139份疟疾患者血样和32份非疟疾患者血样进行检测,以镜检方法为金标准,分析多重PCR方法检测患者血样的敏感性和特异性.结果从8对11条引物中优选出2对共3条引物用于建立多重PCR.利用这3条引物进行多重PCR,一次反应即可完成对恶性疟原虫和间日疟原虫的种特异性鉴定.对疟疾和非疟疾患者血样检测结果显示,该方法检测患者血样的敏感性为97.8%,特异性为100%.结论多重PCR方法敏感、特异、可进行批量检测,适用于对人群的疟疾监测和疑似疟疾病例的诊断,并能鉴定恶性疟原虫和间日疟原虫虫种.%Objective To establish a multiplex polymerase chain reaction(PCR)system for species specific detection of Plasmodium vivax and Plasmodium falciparum.Methods Eight pairs of primer(11 strips in total)were designed according to the sequences of the small subunit ribosomal RNA(ssRNA)of Plas modium species.A group of blood samples from patients infected with Plasmodium vivax and Plasmodium falciparum was used in simple PCR amplification.The primers which had the best sensitivity and specificity were selected for establishing the multiplex PCR.The reacting system was optimized by analysis on the different primer concentration,annealing temperature,extension temperature and cycle times.The optimized multiplex PCR was tested with 139 patients'blood samples collected from Yunnan Province and Shanghai area and 32 samples from non-malaria patients.The results were compared with the microscopic method,which was considered as a golden standard for malaria parasite identification.The sensitivity and specificity of the multiplex PCR were evaluated.Results Multiplex PCR Was established using 2 pairs of primer(3 strips in total)selected from 11 strips of primer.Plasmodium vivax and Plasmodium falciparum could be identified in one PCR reaction.Sample analyzing results from malaria patients and non-malaria patients showed that the sensitivity was 97.8% and the specificity was 100%.Conclusion This multiplex PCR system had high sensitivity and specifieity,could be used for the detection of large samples,and was suitable for the field malaria surveillance,and for diagnosis of doubtful malaria cases and species identification.