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当前位置: 首页 > 文章 > 仔猪粪便DNA中产气荚膜梭菌主要毒素基因检测及荧光定量PCR计数方法的建立 中国兽医学报 2019 (1) 57-62
Position: Home > Articles > Detection of major toxin genes and establishment of a real-time PCR assay with quantitative of Clostridium perfringens from the fecal DNA of piglets Chinese Journal of Veterinary Science 2019 (1) 57-62

仔猪粪便DNA中产气荚膜梭菌主要毒素基因检测及荧光定量PCR计数方法的建立

作  者：
韩春生;戴益民;王林康;王婧祺;周姣;张锦华
单  位：
江西农业大学动物科学技术学院
关键词：
仔猪;产气荚膜梭菌;毒素基因;β2毒素;荧光定量PCR
摘  要：
为探究产气荚膜梭菌(Cp)及其产生的β2毒素对7日龄内仔猪腹泻的影响,本试验利用PCR技术对江西不同地区96份仔猪粪样DNA进行分析。首先检测产气荚膜梭菌α、β、β2、ε、ι、CPE毒素基因来推断Cp存在和基因型,然后对不同地区的β2毒素基因全长进行测序比对、MEGA7构建进化树,最后采用荧光定量PCR对携带CpA(β2+)的健康、腹泻样品中的Cp菌数进行测定。结果显示:α毒素基因在健康、腹泻仔猪的检测率分别为85.4%(41/48),81.3%(39/48),β2毒素基因则分别为79.2%(38/48),77.1%(37/48);β、ε、CPE及ι毒素基因未能检测到,综合各分型毒素检测结果,阳性粪样存在的Cp均为A型。β2基因全长除九江地区得到的β2基因在第706～733bp缺失28个碱基,其他地区β2全长序列不多于3个碱基的差异,与GenBank登录号AJ537530.1对应序列同源性最高。携带CpA(β2+)的健康、腹泻粪样同浓度DNA中Cp菌数为104～105 CFU,经SPSS17分析,差异不显著(P>0.05)。结果表明,江西地区的猪源β2毒素基因保守性较高,但仔猪腹泻与β2毒素基因存在与否和Cp菌数没有明显的相关关系,是否与毒素表达有关有待进一步的研究,本试验结果为研究Cp致病机制提供了依据。
译  名：
Detection of major toxin genes and establishment of a real-time PCR assay with quantitative of Clostridium perfringens from the fecal DNA of piglets
作  者：
HAN Chun-sheng;DAI Yi-min;WANG Lin-kang;WANG Jing-qi;ZHOU Jiao;ZHANG Jin-hua;College of Animal Science and Technology,Jiangxi Agricultural University;Jiangxi Provincial Key Laboratory of Animal Disease Diagnosis and Prevention;
单  位：
HAN Chun-sheng%DAI Yi-min%WANG Lin-kang%WANG Jing-qi%ZHOU Jiao%ZHANG Jin-hua%College of Animal Science and Technology,Jiangxi Agricultural University%Jiangxi Provincial Key Laboratory of Animal Disease Diagnosis and Prevention
关键词：
piglet;;C.perfringens;;toxin genes;;β2 toxin;;q-PCR
摘  要：
Keywords:In order to study the impact of Clostridium perfringens and itsβ2 toxin on piglet diarrhea within 7-day old,96 piglets fecal DNA from different areas in Jiangxi were collected and amplified all those gene sequences from DNA by PCR.The existence of toxin genes(α,β,β2,ε,ι,CPE)and the genetyping of Cp were detected.Then,the completeβ2 toxin gene was obtained and analyzed by using program MEGA7.And the quantum of CpA in healthy or diarrhea piglet fecal DNA(only CpA carryingβ2+)was tested by quantitative real-time PCR(q-PCR).The results showed thatαtoxin gene was identified 85.4%(41/48)in the healthy piglets(H)and 81.3%(39/48)in the diarrhea piglets(D),β2 toxin gene was 79.2%(38/48,H)and 77.1%(37/48,D),respectively.All positive samples from 96 piglets were identified as CpA.Obviously,7 sequences of complete geneβ2 were slightly different about not more than 3 bases,except the sequence from Jiujiang,in which 28 bases were deleted in 706 th to 733 th bp.And they were less distant to AJ537530.1.The bacterial density of CpA in healthy or diarrhea piglet fecal DNA was about 10~4-10~5 CFU,which were not significant each other(P>0.05).Reasult suggested that theβ2 genes found in pig in Jiangxi were highly conservative,but piglet diarrhea did not correlate to the bacterial density of CpA in intestinal tract of piglet or the presence ofβ2 gene carrying by Cp.Further researchis to explore whether it is relate to the expression ofβ2 toxin genes.This experiment provides an important reference to study the pathogenesis of Cp.

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