Detection Of Plasmodium Vivax Sporozoites-carrying Mosquitoes Using Loop-mediated Isothermal Amplification (LAMP)

Malaria is one of the major parasitic diseases that pose serious health risk to humans. Along with acquired immunodeficiency syndrome (AIDS) and tuberculosis, it is one of the top three global public health issues. Vector control is an important component of malaria comprehensive prevention and control measures. The effective vector survey requests that the susceptibility, population density, bloodthirsty habits, life span, insecticide resistance and sporozoites infection of Anopheles should be grasped accurately. Anopheles is the unique vector for malaria transmission, so the detection of Plasmodium sporozoites in Anopheles is not only the important content of the vector survey , but also conductive to the awareness of malaria endemic situation and the evaluation of effect of malaria control, which do great significance to the generation of malaria control strategies. Traditionally, manual dissection of the salivary glands of Anopheles mosquitoes is done for microscopic detection of the sporozoites. However, this method proves not only to be time-consuming and labor-intensive but the success rate is also low. In recent years, polymerase chain reaction (PCR) has been used to detect presence of malaria parasites in mosquitoes. However, this test requires special equipments and entails high cost. Hence, there is a need to develop a cost-effective, simple, and rapid method with high sensitivity and specificity for malaria sporozoites detection in malaria control and vector survey. Objective To establish a simple, convenient,quick and high sensitive method of loop-mediated isothermal amplification (LAMP) for the detection of Plasmodium vivax sporozoites-carrying mosquitoes. Methods Find out the conserved sequence of circumsporozoite protein gene through the comparison of multiple sequences and comparison in blast. Then, the species conservative regions of P. v CSP gene were selected to design 2 pairs of primers which recognized 6 distinct regions. Set up the LAMP method for detection of Plasmodium vivax sporozoites-carrying mosquitoes by optimizing most of the components and conditions of reaction including the content of Mg2+, dNTPs, betaine, FIP/BIP and F3/B3 in LAMP buffer, and the condition of reaction temperature and reaction time. To evaluate the specificity of detection by LAMP, plasmid DNA, An.sinensis (An. s), Plasmodium falciparum (P. f), and healthy human blood DNA were selected as templates and double distilled water (ddH2O) as the blank control. To assess the sensitivity of detection, 1.3×106, 1.3×105, 1.3×104, 1.3×103, 1.3×102, 1.3×101 and 1.3×100 copies of P. v CSP plasmid DNA mixed with 1.0μL An. s DNA were used as the templates of LAMP. The positive infected An.s DNA were diluted with negative An. s DNA by 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128 and 1:256 and then detected by LAMP to show the sensitivity of batch quantity detection. The applied value of this method was evaluated by detecting the same batch of 67 artificial infected An. s mosquitoes through the comparison of the detecting results of LAMP, microscopical examination and nested PCR in parallel. And then, a chi-square analysis (χ2) of the results was performed using SPSS 16.0.Results The results showed that amplification at the condition of 8 mM Mg2+, 1.4 mM dNTPs, 0 M betaine, 1.6μM FIP/BIP, 0.2μM F3/B3, and at 65℃for 70 min was the optimal one. Using LAMP, the detection of plasmid DNA was positive, while the control samples were all negative. The limits of detection of different proportion dilutions of the mixture of P. v CSP plasmid DNA with An. s DNA were 1.3×102copies. The limits of detection of different proportion dilutions of the mixture of positive infected An.s DNA with An. s DNA were 1:128. The statistical analysis of the results of the dtection of the same batch of 67 artificial infected mosquitoes using SPSS16.0 was that the positive detection rate was 47.76% by LAMP, which was significant higher than the positive detection rate of microscopic examination (25.37%) with statistically significant difference (χ2=7.24, P<0.01), and higher than the positive detection rate of nested PCR (40.30%) without statistically significant difference (χ2=0.73, P>0.05).Conclusion The method of LAMP for detection of Plasmodium vivax sporozoites-carrying mosquitoes which was established in this study is simple, convenient, quick, high sensitive and cost-effective, and is a potential method for detecting Plasmodium vivax-carrying mosquitoes in the field after further optimizing, perfecting and promoting.