Detection of Plasmodium vivax sporozoites-carrying mosquitoes using loop-mediated isothermal amplification (LAMP)
- VernacularTitle:环介导等温扩增技术检测蚊体内间日疟原虫子孢子的研究
- Author:
Hanwu ZHU
;
Jun CAO
;
Huayun ZHOU
;
Julin LI
;
Guoding ZHU
;
Yaping GU
;
Weiming WANG
;
Yaobao LIU
;
Zhiyong TAO
;
Qi GAO
- Publication Type:Journal Article
- Keywords:
Loop-mediated isothermal amplification (LAMP);
Plasmodium vivax;
Sporozoites;
Mosquito vector
- From:
Chinese Journal of Schistosomiasis Control
2010;22(2):158-163
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a simple,convenient,quick and high sensitive method of loop-mediated isothermal amplification (LAMP) for detection of Plasmodium vivax-carrying mosquitoes.Methods The species conservative regions of P.v CSP gene were selected to design 2 pairs of primers which recognized 6 distinct regions.To evaluate the specificity of detection by LAMP,infected Anopheles,An.sinensis (An.s),Plasmodium falciparum (P.f),and healthy human blood DNA were selected as templates.To assess the sensitivity of detection,1.3×10~6,1.3×10~5,1.3×10~4,1.3×10~3,1.3×10~2,1.3×10~1 and 1.3×10~0 copies of P.v CSP plasmid DNA mixed with 1.0 μl An.s DNA were used as the templates of LAMP.The infected An.s DNAs 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,and compared with the detection of microscopic examination and nested PCR in parallel.Results By using LAMP,the detection of infected An.s 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 1.3×10~2 copies.The limits of detection of different proportion dilutions of the mixture of infected An.s DNA with An.s DNA were 1:128.The positive rate of detecting the same batch of 67 artificial infected mosquitoes was 47.76% by LAMP,25.37% by the microscopic examination (X~2 = 7.24,P<0.01),40.30% by nested PCR (X~2 = 0.73,P>0.05).Compared with the test of the microscopic examination and then with a statistical analysis,the sensitivity of LAMP was 100%,which agreed well with the sensitivity of nested PCR (100%).Conclusion The method of LAMP is simple,convenient and high sensitive,and it is a potential method for detecting Plasmodium vivax-carrying mosquitoes in the field.
