利用MTT法检测活体线虫的主要影响因素及可行性研究  

Analysis of possibility and influencing factors on the nematode counting with the MTT method

作　　者：王瑞 祝晓蕊 梁萌 王新芳 王文蕊 陈林军 钱英红 陈湘江 杨晓野 WANG Rui;ZHU Xiao-rui;LIANG Meng;WANG Xin-fang;WANG Wen-rui;CHEN Lin-jun;QIAN Ying-hong;CHEN Xiang-jianga;YANG Xiao-ye(Key Laboratory of Clinical Diag-nosis and Treatment Technology in Animal Disease,College of Veterinary Medicine,Inner Mon-golia Agricultural University,Hohhot 010018,China;Inner Mongolia of Agricultural and An-imal Husbandry Science,Hohhot 010031,China;Zhucheng Animal Husbandry and Veterinary Administration Bureau of Shandong Province,Zhucheng,Shandong 262200,China)

机构地区：[1]内蒙古农业大学兽医学院农业部动物疾病临床诊疗技术重点实验室,内蒙古呼和浩特010018 [2]内蒙古自治区农牧业科学院,内蒙古呼和浩特010031 [3]山东省诸城市畜牧兽医管理局,山东诸城262200

出　　处：《中国兽医学报》2018年第9期1711-1715,共5页Chinese Journal of Veterinary Science

基　　金：国家重点研发计划基金资助项目(2017YFD0501200);公益性行业(农业)科研专项基金资助项目(201303037);国家自然科学基金资助项目(31460656);内蒙古自然科学基金(2014MS0339);内蒙古自然科学基金资助项目(2015MS0308);家畜疫病病原生物学国家重点实验室开放基金项目(SKLVEB2015KFKT013)

摘　　要：MTT法是一种灵敏、快速和便捷的计数生物活细胞的方法。为探讨这一方法用于计数活体线虫的主要影响因素和可行性,本试验以秀丽新杆线虫为研究对象,对MTT法检测秀丽新杆线虫的检测波长、MTT的用量、溶解用DMSO用量和与药物作用时间等主要影响因素进行分析,并将MTT法检测结果与显微镜直接镜检计数法结果比较。结果表明：甲瓒（Formazan）生成量与线虫活虫体数呈正相关;在试验反应体系中,MTT为10g/L,反应3h时,Formazan的最大吸收波长为550nm,溶解用DMSO最佳用量为150μL,与药物作用时间为24h;MTT法检测和直接镜检结果相似。结果表明,MTT法可用于秀丽新杆线虫虫体等的计数,且可以大大缩短检测的时间,有效减少人力的消耗,结果准确性也较高,对大批量的进行药物或其他物质对虫体活力作用的研究具有重要积极意义。MTT method is a sensitive,rapid,and simple living cell counting technique.In order to ascertain wherther it can be used to count living nematodes and other parasites,and its optimal reaction conditions,the influencing factors,including MTT dosage and detection wavelength,DMSO dosage,incubation time with ivermectin and so on,Caenorhabditis elegans was used as the tested nematode.At the same time,the comparison between the MTT method and direct counting with microscope was also done.The results indicated that the production of formazan showed positive pertinence to the quantity of living nematodes.The biggest absorbancy value was got when the reaction time was 3 hours with 10 g/L MTT;the maximum absorption wavelength was 550 nm;the optimal quantity for DMSO was 150μL,while the incubation time with ivermectin was 24 h.The statistical results showed that there was no significant difference in the results between MTT method and direct counting by microscope.The result assumed that the MTT method developed in the report could be used to count the living nematodes,which was helpful to the assay related to living nematode counting in isolation,incubation and anthelmintics effects on the nematodes.

关 键 词：秀丽新杆线虫 线虫计数 MTT 甲瓒 

分 类 号：S855.9[农业科学—临床兽医学]