肽核酸荧光原位杂交技术检测生肉中沙门氏菌方法的建立及应用  

Peptide nucleic acid fluorescence in situ hybridization for identification of salmonella in meat products

作　　者：杨彤 吴姗 李可 帅江冰 叶子弘 张晓峰 YANG Tong;WU Shan;LI Ke;SHUAI Jiang-bing;YE Zi-hong;ZHANG Xiao-feng(Zhejiang Provincial Key Laboratory of Biometrology and Inspection&Quarantine,College of Life Sciences,China Jiliang University,Hangzhou 310018,China;Zhejing Academy of Science and Technology for Inspection and Quarantine,Hangzhou 310016,China)

机构地区：[1]中国计量大学生命科学学院,浙江省生物计量与检验检疫技术重点实验室,浙江杭州310018 [2]浙江省检验检疫科学技术研究院,浙江杭州310016

出　　处：《中国预防兽医学报》2020年第6期584-589,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划(2017YFF0210203);浙江省重点研发计划项目(2018C02041);海关总署科研项目(2019 HK097)。

摘　　要：本研究设计了一种基于沙门氏菌毒力基因侵袭蛋白A(invA)的特异性肽核酸(PNA)探针,在前期优化条件的基础上,建立并评估了沙门氏菌肽核酸荧光原位杂交(PNA-FISH)的检测方法。特异性试验结果显示,该方法的Sal-invA探针除对目标菌杂交呈阳性外,与12株常见的非目标菌杂交均为阴性,特异性强;灵敏度试验结果显示,该方法对沙门氏菌的检测限为105 cfu/mL,而荧光定量PCR方法和细菌分离培养法对沙门氏菌的最低检测限分别为102 cfu/mL和105 cfu/mL,PNA-FISH方法的灵敏度与分离培养方法相近。人工污染沙门氏菌的生肉制品,增菌培养后经上述3种方法检测的最低检测限均可达到100 cfu/mL。对83份生鲜肉经增菌培养后,用上述3种方法检测。结果显示,该方法与细菌分离培养方法和荧光定量PCR方法的符合率均达到98.8%。表明,增菌培养是提高该PNA-FISH检测方法灵敏性的重要步骤。本研究建立的检测沙门氏菌的PNA-FISH方法快速、准确、灵敏,适合用于临床肉制品的检测。Based on the previous optimization,a peptide nucleic acids(PNA)probe was designed to detect the virulence gene invasion protein A(invA)of Salmonella,and a peptide nucleic acids fluorescence in situ hybridization(PNA-FISH)method was established and evaluated to detect Salmonella.The assay showed high specificity,the hybridization of the Sal-invA probe with 12 ccommon non-Salmonella strains was negative,while it was positive with the testing Salmonella strains.The sensitivity assay showed that the detection limit of this method was 105 cfu/mL,while the detection limits of the qPCR and culture method were 102 cfu/mL and 105 cfu/mL,respectively.The sensitivity of PNA-FISH was similar to that of isolation and culture method.In raw meat samples artificially contaminated with Salmonella,all three methods showed the detection limit of 100 cfu/mL after enrichment.When the above three methods were applied to 83 raw meats samples after enrichment,the coincidence rate of PNA-FISH method with the bacterial isolation culture method and qPCR method was 98.8%.It revealed that enrichment is an important step to enhance the sensitivity of the PNA-FISH method.The method established in this research is rapid,accurate,sensitive and suitable for detection of the clinical samples.

关 键 词：肽核酸 荧光原位杂交 沙门氏菌 最低检测限 

分 类 号：S852.61[农业科学—基础兽医学]