快速检测日本血吸虫感染性钉螺LAMP试剂盒的建立及应用

Creation and use of a LAMP kit to rapidly detect snails infected with Schistosoma japonicum

目的构建快速检测日本血吸虫感染性钉螺环介导同温DNA扩增(LAMP)试剂盒,用于血吸虫病流行区感染性钉螺调查。方法采用碱裂解法制备钉螺基因组DNA样品,以缩短检测过程中样品DNA制备时间;对LAMP试剂进行组合优化,采用染料显色方法判定LAMP结果,以减少LAMP检测的操作步骤,避免污染,减少假阳性结果产生;建立批量检测钉螺的LAMP方法,以适用于血吸虫病防治现场大规模钉螺筛查与有感染性钉螺地块的调查。将以上优化技术及试剂整合成快速检测日本血吸虫感染性钉螺LAMP试剂盒。结果制备的日本血吸虫感染性钉螺LAMP检测试剂盒可用于血吸虫流行区现场钉螺快速检测,检测时间可由原来的6h缩短为2h,方法的灵敏性与常规LAMP法相似。该试剂盒能检测出感染后1周钉螺,能对现场钉螺进行大批量检测。结论建立的LAMP试剂盒用于日本血吸虫感染性钉螺检测快速、特异、操作简便,适用于日本血吸虫病流行区感染性钉螺调查。

Objective To construct a LAMP kit to rapidly detect snails infected withSchistosoma japonicum.This kit will be used to survey the distribution of infected snails in areas with endemic schistosomiasis.Methods Alkaline lysis was used to prepare genomic DNA from snails,thus reducing the time for sample DNA preparation.LAMP reagents were optimized,and coloration was used to judge LAMP results.The steps in the LAMP test were reduced to avoid contamination and false positives were decreased.A batch method of detection was developed to allow mass screening of infected snails in the field and investigation of the distribution of snails infected withS.japonicumin areas with snails.A LAMP kit was constructed to rapidly detect snails infected withS.japonicumby optimizing the reagents and techniques as mentioned.Results The LAMP kit to rapidly detect snails infected withS.japonicumwas able to rapidly test for infected snails collected from areas with endemic schistosomiasis.The testing time was reduced from 6 to 2 hours and testing was effectively simplified.The sensitivity of this LAMP kit is similar to regular LAMP.The LAMP kit identified infected snails one week after infection and sampled large numbers of snails in the field.Conclusion This study has successfully constructed a rapid,specific,and simple kit to detect snails infected with schistosomes.