环介导等温扩增技术检测间日疟原虫方法的建立及应用分析

目的建立一种快速、简便的检测间日疟原虫的环介导等温扩增方法（LAMP）并与常规PCR方法作比较。方法根据间日疟原虫环子孢子蛋白（CSP）基因序列合成2对特异性LAMP引物,优化Mg2＋浓度、dNTPs浓度、BstDNA聚合酶添加量、反应温度、时间以及设计引物缺省试验。评估优化后的LAMP反应的特异性和灵敏性。检测133份患者血样,以显微镜检方法为金标准,比较LAMP和多重PCR法检测间日疟原虫的敏感性和特异性。结果 LAMP法检测重组质粒DNA（Pv-rDNA）的灵敏度达到10-10,为传统PCR方法的100倍。镜检确诊的68例间日疟、43例恶性疟和22例非疟疾患者中,LAMP法和多重PCR检测间日疟原虫的敏感性为98.53%和97.06%,两法基本相当（χ2=0.34,P〉0.05）;特异性为86.15%和100%,LAMP法低于多重PCR法,差异有统计学意义（χ2=9.67,P〈0.05）。LAMP法的阳性预测值和阴性预测值分别为88.16%和98.25%,多重PCR的阳性预测值和阴性预测值分别为100%和97.01%。结论 LAMP法检测间日疟原虫具有快速简便、敏感性高、设备要求低的特点,具有较好的应用前景。

Analysis and establishment of loop-mediated isothermal amplification for the diagnosis of Plasmodium vivax

Objective To establish a quick, simple and convenient method of loop-mediated isothermal amplification （LAMP） for detection of Plasmodium vivax and compare with the method of PCR. Methods Two pairs of distinct LAMP primers were synthesized according to the sequences of P. vivax circumsporozoite protein （CSP） gene. The concentrations of Mg2＋, dNTPs and Bst, the reaction temperature and the reaction time of LAMP were optimized. And design the experiment which default different primers of LAMP. The optimized LAMP was evaluated by sensitivity and specificity. The method of LAMP was tested with 133 patients′ blood samples. The results were compared with the microscopic method, which was considered as a golden standard for malaria parasite identification. The sensitivity and specificity of LAMP and multiplex PCR were compared and evaluated. Results By using LAMP, the sensitivity detection of recombinant plasmid Pv-rDNA （1：10 multiple dilution） was 10-10, it showed a sensitivity 100-fold higher than traditional PCR. Sample analyzing results from 68 Plasmodium vivax infected patients, 43 Plasmodium falciparum infected patients and 22 non-malaria patients with microscopic examination as the golden standard showed that the sensitivity of LAMP and multiplex PCR were 98.53% and 97.06% , respectively and the specificity of them were 86.15% and 100% , respectively. Positive predictive value （PPV） and negative predictive value （NPV） of LAMP were 88.16% and 98.25%,respectively, while the PPV and NPV of multiplex PCR were 100% and 97.01%, respectively. Conclusion The method is characterized by rapidity, ease of operation, high sensitivity and low equipment requirements, with preferable application prospects.