Establishment and application of multiplex fluorescent quantitative PCR method for enterotoxigenic Escherichia coli and Salmonella
-
摘要: 为了建立能同时检测产肠毒素大肠杆菌和沙门氏菌的多重荧光定量PCR方法，试验根据GenBank中产肠毒素大肠杆菌ST和LT基因及沙门氏菌invA基因序列设计引物和探针，通过优化扩增体系中引物和探针剂量建立检测产肠毒素大肠杆菌和沙门氏菌的多重荧光定量PCR方法，分析该方法的特异性、敏感性和重复性，用建立的方法对85份临床样品进行检测，并与实验室传统血清学分离鉴定方法进行比较。结果表明：在ST基因的最佳引物和探针剂量为0.4μL、0.8μL,LT基因的最佳引物和探针剂量为0.6μL、1.0μL,invA基因的最佳引物和探针剂量为1.0μL、1.2μL时，扩增曲线良好，ST基因、LT基因、invA基因的标准曲线回归方程相关系数（R2）分别为0.975,0.968,0.915,扩增曲线具有良好线性关系；建立的多重荧光定量方法对铜绿假单胞菌、金黄色葡萄球菌等细菌核酸无交叉反应；组内重复变异系数均低于2%,且产肠毒素大肠杆菌和沙门氏菌的最低检出浓度均为1×10～1 copies/μL;从85份临床样品中检出产肠毒素大肠杆菌20份，沙门氏菌阳性样品10份，双阳性样品2份，与实验室传统血清学分离鉴定方法的符合率为94.12%。说明试验建立的多重荧光定量PCR方法的特异性好、敏感性强，稳定性高，具有良好的使用前景。Abstract: In order to establish a multiplex fluorescent quantitative PCR method for the simultaneous detection of enterotoxigenic Escherichia coli and Salmonella, in this experiment, primers and probes were designed by using enterotoxigenic Escherichia coli ST gene and LT gene and Salmonella invA gene in GenBank and the amounts of primers and probes in the amplification system were optimized. Thus, a multiplex fluorescent quantitative PCR method for the detection of enterotoxigenic Escherichia coli and Salmonella was established. Its specificity, sensitivity and repeatability were determined. Eighty-five clinical samples were tested using the established method, which was compared with traditional laboratory serological isolation and identification methods. The results showed that when the optimal primer and probe amounts for the ST gene of 0.4 μL and 0.8 μL respectively, the optimal primer and probe amounts for the LT gene of 0.6 μL and 1 μL respectively, and the optimal primer and probe amounts for the invA gene of 1.0 μL and 1.2 μL respectively, good amplification curves were obtained. The correlation coefficients（R~2） of the standard curve regression equations for ST gene, LT gene and invA gene were 0.975, 0.968 and 0.915, respectively, and the amplification curves had good linear relationships. The established multiplex fluorescent quantitative PCR method had no cross-reaction against bacterial nucleotides such as Pseudomonas aeruginosa and Staphylococcus aureus. The coefficient of intra-batch repeat variation was less than 2%, and the lowest detection concentrations of enterotoxigenic Escherichia coli and Salmonella were 1×10~1 copies/μL. From 85 clinical samples, 20 samples of enterotoxigenic Escherichia coli, 10 positive samples of Salmonella and 2 double positive samples were detected. The consistency rate with the traditional laboratory serological separation and identification methods was 94.12%. The results suggested multiplex fluorescent quantitative PCR method had good specificity, strong sensitivity, high stability, and good practical prospects.
-
-
[1] 陈真强.犊牛腹泻的病因及防治措施探讨[J].江西畜牧兽医杂志,2005(5):35. [2] 陆承平.兽医微生物学[M].北京:中国农业出版社,2012. [3] BARKLEY J A,PEMPEK J A,BOWMAN A S,et al.Longitudinal health outcomes for enteric pathogens in preweaned calves on Ohio dairy farms[J].Prev Vet Med,2021,190:105323.
[4] NAGY B,FEKETE P Z.Enterotoxigenic Escherichia coli in veterinary medicine[J].Int J Med Microbiol,2005,295(6):443-454.
[5] ZHANG Y C,TAN P,ZHAO Y,et al.Enterotoxigenic Escherichia coli:intestinal pathogenesis mechanisms and colonization resistance by gut microbiota[J].Gut Microbes,2022,14(1):2055943.
[6] KOPIC S,GEIBEL J P.Toxin mediated diarrhea in the 21 century:the pathophysiology of intestinal ion transport in the course of ETEC,Ⅴ.cholerae and rotavirus infection[J].Toxins,2010,2(8):2132-2157.
[7] YIN Y,ZHOU D.Organoid and enteroid modeling of Salmonella infection[J].Front Cell Infect Microbiol,2018,8:102.
[8] 严惠馨.动物沙门氏菌病的防治[J].现代畜牧兽医,2018(3):28-31. [9] 杨帆,王红宁,张安云,等.多重PCR检测病死鸡中沙门氏菌方法的研究[J].四川大学学报(自然科学版),2015,52(1):163-169. [10] BRANCHU P,BAWN M,KINGSLEY R A.Genome variation and molecular epidemiology of Salmonella enterica serovar typhimurium pathovariants[J].Infect Immun,2018,86(8):e00079-18.
[11] DUBREUIL J D.Pig vaccination strategies based on enterotoxigenic Escherichia coli toxins[J].Braz J Microbiol,2021,52(4):2499-2509.
[12] BELL R L,ZHENG J,BURROWS E,et al.Ecological prevalence,genetic diversity,and epidemiological aspects of Salmonella isolated from tomato agricultural regions of the Virginia Eastern Shore[J].Frontiers Microbiol,2015,6:415.
[13] 王芳.ETEC肠毒素SYBR Green Ⅰ荧光定量PCR检测方法的建立[D].杨凌:西北农林科技大学,2011. [14] 王华健,张宁,杨威,等.3种食源性致病菌TaqMan多重荧光定量PCR检测方法的建立[J].畜牧兽医学报,2022,53(4):1201-1209. [15] 杨德鸿.苏北地区猪产肠毒素大肠杆菌(ETEC)流行病学调查及灭活疫苗初步研究[D].南京:南京农业大学,2018. [16] 宋美英.致病性牛源大肠杆菌毒力基因分子流行病学调查和耐药突变选择窗的应用[D].哈尔滨:东北农业大学,2017. [17] 王丽扬.苏皖部分地区动物源沙门菌流行病学调查及复方白头翁散对其生物被膜形成的抑制作用[D].扬州:扬州大学,2021. [18] 孙璐,王娟,黄秀梅,等.山东地区肉鸡屠宰环节沙门菌流行病学调查与毒力分析[J].中国家禽,2015,37(7):67-70. -
期刊类型引用(2)
1. 于有利，王建东，郭亚男，何薇，王攀. 宁夏地区羔羊源大肠杆菌分离鉴定及病理组织学分析. 中国草食动物科学. 2025(01): 50-56 . 
百度学术
2. 张红，田秋丰，董佳强，杨坤，苗艳，张蕾，陈志峰. 鹅沙门氏菌病的诊断与防治. 家禽科学. 2024(08): 71-73 . 百度学术
其他类型引用(0)
计量
- 文章访问数: 1
- HTML全文浏览量: 0
- PDF下载量: 0
- 被引次数: 2
下载:
