摘要
为建立一种快速鉴别禽致病性大肠杆菌（avian pathogenic Escherichia coli,APEC）与禽非致病性大肠杆菌（avian non-pathogenic Escherichia coli,ANPEC）的多重PCR检测方法,本研究针对APEC的iss、cvaC、irp2、iucD、iroN和tsh共6个毒力基因的序列进行分析,分别设计合成6对特异性引物,通过正交组合法优化引物比例和梯度法确定最佳退火温度,并进一步完善方法的敏感性、特异性和判定标准,建立了一种快速灵敏检测APEC的多重PCR检测方法,并初步应用该方法对本实验室102株大肠杆菌样本进行鉴定和分群。结果显示,该多重PCR方法对菌液最低检测浓度为5×10^7CFU/m L;对其他种属的4株革兰阳性菌和5株革兰阴性菌无扩增。检测实验室保存的鸭源APEC和ANPEC各10株,通过比较两者所含毒力基因的分布情况及数量,建立了多重PCR判定标准即所含毒力基因数大于等于3者为APEC阳性菌株。初步应用该方法检测实验室分离保存的23株APEC和79株ANPEC,同时进一步验证了其准确性。结果表明,APEC检出率为100%,ANPEC的检出率亦为100%。以上结果充分说明本研究建立的多重PCR特异性强、灵敏度高、简便快捷,可进一步应用于禽类的APEC与ANPEC的快速鉴别诊断和APEC的分子流行病学调查。
In order to establish a rapid and sensitive detection method for avian pathogenic Escherichia coli（APEC） and avian non-pathogenic Escherichia coli（ANPEC）,primers were designed according to 6 genes sequences of APEC, including iss,cva C,irp2,iuc D,iro N and tsh genes from Gen Bank.We determined the proportion of primers and the annealing temperature by orthogonal mean and gradient method,after that the multiplex PCR method for APEC detection was established.The sensitivity,specificity and criterion of the method were further improved,and it was also applied to detect 102 Escherichia coli strains from our laboratory for phylogeny.In result,the minimum detectable concentration of bacterium was 5 × 10^7 CFU/m L.The specificity was proved by amplifying 4 gram-positive bacteria and 5 gram-negative bacteria strains,and they were detected negatively.By detecting duck source APEC and ANPEC preserved in our laboratory,we made the criterion was that the strain is APEC if it had 3 or more than 3 virulence genes,according to their virulence genes distribution and numbers.The preliminary application of detection for 23 APEC strains and 79 ANPEC strains saved by our laboratory further indicated that the detection rate was 100% both in APEC and ANPEC.In conclusion, we established a simple and quick multiplex PCR with strong specificity,high sensitivity,and it can be further applied in rapid differential diagnosis for APEC and ANPEC as well as providing a method for APEC molecular epidemiology survey.
作者
王怡平
孙畅
赵丽丽
陈洪岩
WANG Yi-ping;SUN Chang;ZHAO Li-li;CHEN Hong-yan(State Key Laboratory of Veterinary Biotechnology/Heilongjiang Provincial Key Laboratory of Laboratory Animal and Comparative Medicine/Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China;College of Life Science and Technology,Mudanjiang Normal University,Mudanjiang 157011,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2018年第11期1358-1364,共7页
Chinese Veterinary Science
基金
黑龙江省自然科学基金重点项目(ZD2016006)
上海市科委科研计划项目(16140900500)
中央级公益性科研院所基本科研业务费专项(1610302017013
1610302018013)
关键词
禽致病性大肠杆菌
禽非致病性大肠杆菌
多重PCR
avian pathogenic Escherichia coli （APEC）
avian non-pathogenic Escherichia coli（ANPEC）
multiplex PCR
