Isolation and Identification of Pathogenic <i>Yersinia enterocolitica</i> and Development of a Triple PCR Detection Method

Nanchi ZHANG; Xiaolan GOU; Li WANG

doi:10.13386/j.issn1002-0306.2020050227

Volume 42 Issue 7

Mar. 2021

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Science and Technology of Food Industry > 2021 > 42(7): 95-101. > DOI: 10.13386/j.issn1002-0306.2020050227

ZHANG Nanchi, GOU Xiaolan, WANG Li. Isolation and Identification of Pathogenic Yersinia enterocolitica and Development of a Triple PCR Detection Method[J]. Science and Technology of Food Industry, 2021, 42(7): 95−101. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2020050227.

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Isolation and Identification of Pathogenic Yersinia enterocolitica and Development of a Triple PCR Detection Method

Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Exploitation of Ministry of Education, Key Laboratory of Animal Science of State Ethnic Affairs Commission, Southwest Minzu University, Chengdu 610041,China

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Received Date: May 21, 2020
Available Online: January 27, 2021

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Abstract

Abstract

This paper aimed to identify the pathogenic bacterium that caused anal swelling and abdominal bleeding of Carassius auratus, and establish a rapid detection method for the pathogenic bacterium. In this study, the pathogenic bacterium was isolated from Carassius auratus, and identified by morphological, physical and chemical characteristics analysis and 16S rDNA sequence analysis. The virulence gene of the bacterium was detected by PCR method. The drug sensitivity of isolated strain was detected by disk agar diffusion method and its pathogenicity was discussed by artificial infection. Three specific primers of ail gene, inv gene and intB gene were designed for the isolated strain. A triple PCR method for detecting the isolated strain was established and this method was used to test samples of sick Carassius auratus. The result showed that a Yersinia enterocolitica strain named fsznc-10 was isolated from the heart of diseased Carassius auratus. Yersinia enterocolitica fsznc-10 had virulence genes of ail, ystB, virF and intB. Yersinia enterocolitica fsznc-10 was nonresistant to five antibiotics such as norfloxacin and gentamicin, and it had certain pathogenicity to Carassius auratus. Triple PCR method could accurately amplify three target genes of ail, inv and intB of Yersinia enterocolitica, while other strains had not amplified the target genes. The minimum template concentration for the detection of Yersinia enterocolitica was 1.704 × 10⁻⁶ ng/μL. The positive rate of the triple PCR method for detecting heart samples of sick fish was about 86.67%, and its detection coincidence rate with the 16S rDNA sequence analysis method was 100%. The triple PCR detection method has the advantages of strong specificity, high sensitivity, simple operation and low cost. The present paper research provides scientific data reference for the prevention, control and detection of Yersinia enterocolitica in clinical practice.
- Yersinia enterocolitica,
- virulence gene,
- rapid detection,
- multiplex PCR

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References

[1]	布坎南R E, 吉本斯N E. 伯杰细菌鉴定手册(9版)[M]. 北京: 科学出版社, 1989.
[2]	Venkitanarayanan K, Annamalai T. Expression of major cold shock proteins and genes by Yersinia enterocolitica in synthetic medium and foods[J]. J Food Prot,2005,68(11):2454. doi: 10.4315/0362-028X-68.11.2454
[3]	Stachelska M A. Determining the prevalence of inv-positive and ail-positive Yersinia enterocolitica in pig tonsils using PCR and culture methods[J]. Acta Sci Pol Technol Aliment,2017,16(3):303−310.
[4]	付宏杰. 乳糖酸抑菌性及其对小肠结肠炎耶尔森氏菌抑菌机理的研究[D]. 沈阳: 沈阳农业大学, 2018.
[5]	关乃瑜, 夏爽, 赵丽丽, 等. 断奶仔猪源小肠结肠炎耶尔森氏菌的分离鉴定及其致病性研究[J]. 中国预防兽医学报,2015,37(9):679−682. doi: 10.3969/j.issn.1008-0589.2015.09.07
[6]	张霞, 温华蔚, 倪松, 等. 新等温扩增技术检测小肠结肠炎耶尔森氏菌[J]. 食品研究与开发,2015,36(23):113−116. doi: 10.3969/j.issn.1005-6521.2015.23.030
[7]	胡惠娟, 吴清平, 张菊梅, 等. 食品中小肠结肠炎耶尔森氏菌污染调查和ERIC-PCR分型研究[J]. 现代食品科技,2014,30(6):294−300.
[8]	冯育芳, 邢进, 岳秉飞, 等. 小肠结肠炎耶尔森氏菌、假结核耶尔森氏菌、志贺氏菌和空肠弯曲菌多重PCR方法的建立[J]. 实验动物科学,2016,33(1):1−6. doi: 10.3969/j.issn.1006-6179.2016.01.001
[9]	章志超, 龙慧, 吴鑫, 等. 食品中小肠结肠炎耶尔森氏菌分离鉴定方法研究进展[J]. 食品安全质量检测学报,2020,11(2):455−461.
[10]	王利, 苟小兰. 鱼源小肠结肠炎耶尔森菌5种毒力基因的检测和分析[J]. 中国兽医杂志,2013,49(2):68−70. doi: 10.3969/j.issn.0529-6005.2013.02.026
[11]	陈宇明, 程天印, 王小君, 等. 藤黄微球菌PCR检测方法的建立[J]. 畜牧兽医科技信息,2007(1):47−48. doi: 10.3969/j.issn.1671-6027.2007.01.028
[12]	李天芝, 于新友, 莫玲. 基于16S rRNA基因的枯草芽孢杆菌PCR快速检测方法的建立[J]. 广东畜牧兽医科技,2016(2):53−55.
[13]	刘变芳, 王涛, 王蕊, 等. 生鲜肉中产毒素大肠杆菌多重PCR检测方法构建[J]. 中国食品学报,2018,18(12):225−231.
[14]	丁天翊, 潘阳阳, 何翃闳, 等. 快速检测奶牛乳房炎四种病原菌的多重PCR方法的建立及应用[J]. 中国兽医科学,2018,48(1):19−26.
[15]	布勒. 鱼类及其他水生动物细菌实用鉴定指南[M]. 北京: 海洋出版社, 2013.
[16]	伍晔晖, 孟庆玲, 乔军, 等. 奶牛源金黄色葡萄球菌新疆分离株生物被膜、肠毒素基因与毒力的检测及其相关性分析[J]. 西南农业学报,2019,32(11):2693−2698.
[17]	Tan L K, Ooi P T, Thong K L. Prevalence of Yersinia enterocolitica from food and pigs in selected states of malaysia[J]. Food Control,2014,35(1):94−100. doi: 10.1016/j.foodcont.2013.06.053
[18]	杨澜. 实时荧光环介导等温扩增技术检测食品中小肠结肠炎耶尔森氏菌的研究[D]. 保定: 河北农业大学, 2013.
[19]	刘翔, 刘阳波, 郭邦成, 等. 2008~2013年宁夏小肠结肠炎耶尔森菌分布特征分析[J]. 中国人兽共患病学报,2015,31(3):260−263, 271. doi: 10.3969/cjz.j.issn.1002-2694.2015.03.016
[20]	许文炯, 丁洁, 陈晓蔚, 等. 小肠结肠炎耶尔森氏菌主要毒力基因分析[J]. 中国人兽共患病学报,2007(7):675−677. doi: 10.3969/j.issn.1002-2694.2007.07.011
[21]	姜晓梅, 李刚山, 王意银, 等. 小肠结肠炎耶尔森菌的分离鉴定及毒力基因检测[J]. 西南国防医药,2015,25(8):844−846. doi: 10.3969/j.issn.1004-0188.2015.08.011
[22]	苟小兰, 王利. 小肠结肠炎耶尔森氏菌对黄颡鱼的致病性及毒力基因检测[J]. 水产科学,2013,32(5):293−296. doi: 10.3969/j.issn.1003-1111.2013.05.009
[23]	杨兴林, 熊金凤, 李丽, 等. 荧光聚合酶链式反应与酶联免疫吸附法筛查肠道病毒71型病原感染的比较研究[J]. 华西医学,2014,29(7):1309−1312. doi: 10.7507/1002-0179.20140399
[24]	张祥林, 李京, 罗明, 等. 基于16S rDNA基因的谷斑皮蠹PCR检测技术[J]. 生物安全学报,2017,26(1):75−79. doi: 10.3969/j.issn.2095-1787.2017.01.012
[25]	谭秋杉, 高利荣, 王晓杜, 等. 基于16S rRNA基因的不同血清型鸭疫里默氏杆菌PCR检测方法的建立[J]. 中国预防兽医学报,2014,36(1):50−53. doi: 10.3969/j.issn.1008-0589.2014.01.13
[26]	毕水莲. 动物性食品源弓形菌23S rRNA基因PCR检测方法的建立[J]. 湖北农业科学,2014,53(14):3419−3423. doi: 10.3969/j.issn.0439-8114.2014.14.054