CAJ | 학술논문

目的建立一种简便、快速和高敏感度的蚊体内间日疟原虫检测的环介导等温扩增方法(LAMP).方法针对间日疟原虫环子孢子蛋白(CSP)基因种属特异性保守区域6个位点设计2对引物,以感染性按蚊、阴性按蚊、恶性疟原虫及正常人血DNA为模板评价LAMP的特异性.将间日疟原虫CSP基因质粒DNA梯度稀释,并与阴性按蚊DNA按1.0μl加1.3×10~6、1.3×10~5、1.3×10~4、1.3×10~3、1.3×10~2、1.3×10~1、1.3×10~0拷贝混合后为模板进行LAMP反应,观察其检测敏感性;将感染性按蚊与阴性按蚊DNA作1:2、1:4、1:8、1:16、1:32、1:64、1:128、1:256稀释,然后以稀释样本为模板进行LAMP反应,观察批量检测的敏感性.再用此方法与镜检解剖、巢式PCR同时检测同批次人工感染的67只按蚊,评价其应用价值.结果此法检测感染性按蚊阳性,对照组均为阴性;检测不同比例稀释的间日疟原虫CSP基因质粒DNA与按蚊DNA混合物,最低可检测1.3×10~2拷贝的间日疟原虫CSP基因质粒DNA与按蚊DNA的混合物;检测不同比例感染按蚊与阴性按蚊DNA混合样本,最低可检测出在128个按蚊中有1个感染按蚊的混合样本;用此法检测67只同批次人工感染的按蚊,检出率为47.76%,解剖镜检检出率为25.37%(X~2=7.24,P<0.01),巢式PCR检出率为40.30%(X~2=0.73,P>0.05).以镜检解剖作为金标准,LAMP敏感性为100%,巢式PCR敏感性为100%.结论 LAMP检测蚊体内间日疟原虫简便、快速、敏感性高,具有广阔的应用前景.
목적 건립일충간편、쾌속화고민감도적문체내간일학원충검측적배개도등온확증방법(LAMP).방법 침대간일학원충배자포자단백(CSP)기인충속특이성보수구역6개위점설계2대인물,이감염성안문、음성안문、악성학원충급정상인혈DNA위모판평개LAMP적특이성.장간일학원충CSP기인질립DNA제도희석,병여음성안문DNA안1.0μl가1.3×10~6、1.3×10~5、1.3×10~4、1.3×10~3、1.3×10~2、1.3×10~1、1.3×10~0고패혼합후위모판진행LAMP반응,관찰기검측민감성;장감염성안문여음성안문DNA작1:2、1:4、1:8、1:16、1:32、1:64、1:128、1:256희석,연후이희석양본위모판진행LAMP반응,관찰비량검측적민감성.재용차방법여경검해부、소식PCR동시검측동비차인공감염적67지안문,평개기응용개치.결과 차법검측감염성안문양성,대조조균위음성;검측불동비례희석적간일학원충CSP기인질립DNA여안문DNA혼합물,최저가검측1.3×10~2고패적간일학원충CSP기인질립DNA여안문DNA적혼합물;검측불동비례감염안문여음성안문DNA혼합양본,최저가검측출재128개안문중유1개감염안문적혼합양본;용차법검측67지동비차인공감염적안문,검출솔위47.76%,해부경검검출솔위25.37%(X~2=7.24,P<0.01),소식PCR검출솔위40.30%(X~2=0.73,P>0.05).이경검해부작위금표준,LAMP민감성위100%,소식PCR민감성위100%.결론 LAMP검측문체내간일학원충간편、쾌속、민감성고,구유엄활적응용전경.
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.