Methodological Approach Of Loop-mediated Isothermal Amplification (LAMP) For Malaria Detection

Malaria, one of the deadliest human infectious diseases with high incidence and fatality rate, is transmitted by Anopheles mosquito vectors. Along with Acquired Immunodeficiency Syndrome (AIDS) and tuberculosis, it has been considered as one of the top global public health issues. Exact diagnosis is the important tache during the malaria control at all times. Microscopic method which was considered as a golden standard for malaria parasite identification is the primary measure presently. Nevertheless, microscopic method needs professional technique and abundant experiences, but the situation nowdays is that extremely lack of salted technologists. Polymerase chain reaction (PCR) is another wildely using method. Although it has the high sensitivity, the test requires special equipments, time-consuming and high-cost. Hence, there is a need to develop a simple, convenient and quick method with high sensitivity and specificity for plasmodium.Objective To establish a quick, simple and convenient method of loop-mediated isothermal amplification (LAMP) for detection of plasmodium.Methods Two pair of distinct LAMP primers were synthesized according to the target gene of 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. The detection of Plasmodium yoelii (murine) in Anopheles stephensi by LAMP was pre-applied, the sensitivity and specificity of this method was evaluated.Results The results showed that we established the quick detection of Plasmodium Vivax 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 patients,43 Plasmnodium falciparum 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%, and the specificity of them were 86.15% and 100%. Positive predictive value (PPV) and negative predictive value (NPV) of LAMP were 88.16% and 98.25%, PPV and NPV of multiplex PCR were 100% and 97.01% respectively. The distinct LAMP primers were synthesized according to the target sequences of sporozoite microneme protein essential for cell traversal 2 (SPECT2) gene. The specificity of the method was good, and the sensitivity was that it was able to detect one positive Plasmodium yoelii(murine) in Anopheles stephensi out of eighty negative Anopheles stephensi.Conclusion The method of LAMP for detection of Plasmodium vivax whi established in this study is quick, simple, convenient, high sensitive, low-equipment demand and cost-effective, and is a potential method in the field. The detection of Plasmodium yoelii (murine) in Anopheles stephensi by LAMP was pre-applied, the sensitivity and specificity were both preferable. It will be helpful for further studying the method of LAMP for the detection of plasmodium sporozoites-carrying mosquitoes.