An Experimental Method Was Established For Detection Of Huaman Hookworm Eggs By Loop-mediated Isothermal Amplification

Objective:Hookworm diseases is epidemic in worldwide,especially in developing countries in tropical and subtropical regions.Due to the senitary conditions are poor,the hookworm infection rate is high,which is most important public healthy problems in these regions even in the whole world.In China,hookworm infection is the first infection of soil-transmitted nematode,and one of the five parasites damaging national health.Currently,the detection of hookworn infection is Kato-Katz recommended by WTO,which is ecomomic and and convenient,and can be diagnosed by observing the hookworm eggs under the microscope.Other detection methods include PCR and immunology.However,these methods exist differrent degree of defects,for instance,time consuming,low sensitivity,complex operation,expensive equipments,higher requestments on operators,and easy to cause miss detection,making it difficult to popularize and apply in those economic backward areas.It bring difficulties for prevention and control of hookworm diseases.The purpose of this study is to construct a new,rapid and visual detection method.Methods:This study designed primers according to ITS gene of Necator americanus,using Loop-mediated Isothermal Amplification to amplify the DNA of Necator americanus eggs,and plasmid DNA was constructed as standar positive control.The amplification system established in this study contained the following components:10×Reaction Buffer,Primr Mix,dNTPs,ddH2O,Bst DNA polymrase,plasmid tempelate,Cresol red(dye),Betaine.The standar rection system after optimizing was as follow:10×Reaction Buffer(100 mM(NH4)2SO4,500 mM KCl,80 mM MgSO4,1%Tween 20,without Tris-HCl,adjusting PH to 8.8),1.6?M FIP/BIP,0.2?M F3/B3,0.4?M LF/LB,1.2mM dNTPs,0.8M Betaine,50?M Cresol red,8U Bst polymrase,1 ?l DNA tempelate,ddH20,and the amplification time and temperature was 50min and 65? respectively.In this study,crsesol red used as color indicator.It was purple before reaction,brilliant yellow after amplification,and purple remain unchanged after negative amplification.Setting PCR as a parrallel control experiment for LAMP sensitivity.Results:(1)hookworm positive plasmid was constructed:The cloning vector was PMD-18T,the clone fragment size was 250bp,the plasmid concentration was 83.315ng/?l,and the calculated copy numbers were 3×1011 copies/?l.Positive plasmid entrusted for synthesis:the cloning vector was pUC57,the clone fragment size was 600bp,the original concentration was 100ng/?l,and the calculated plasmid copy numbers were 3×1010 copies/?l.(2)The detection limit of LAMP were 3×102 copies/?l,and The detection limit of PCR in parallel control experiment were 3×104 copies/?l.The sensitivity of LAMP was 100 times higher than that of PCR.(3)On-site sample verification:sensitivity was 73.68%(14/19).(4)The methods of fecal DNA extraction was improved to enhance the PCR detection effect(15 positive samples of human hookworm eggs were successfully amplified),which have a good application value.Conclusions:An experimental system was established for LAMP detection of human hookworm:a suitable primers were selected,the reaction system was optimized,and constructed a visual and rapid detection method(results can be obtained within 50 minutes).