Van Giau Vo, Nguyen Thuy Trang, Nguyen Thi Kim Oanh, Le Thi Thuy Hang, Nguyen Tien Dung
Deparment of Faculty of Food Technology, Ho Chi Minh City University of Food Industry (HUFI), 140 Le Trong Tan, Tan Phu District, Ho Chi Minh City, Vietnam.
Gachon Medical Research Institute, Gachon University, Seongnam, South Korea.
3 Biotech. 2016 Jun;6(1):5. doi: 10.1007/s13205-015-0319-0. Epub 2015 Dec 31.
Shiga toxin-producing Escherichia coli O157:H7 (E. coli O157:H7) strains are foodborne infectious agents that cause a number of life-threatening diseases, including hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Shiga toxin 1 (stx1), shiga toxin 2 (stx2), or a combination of both are responsible for most clinical symptoms of these diseases. Hence, various diagnostic methods have been developed so far to detect shiga toxins such as cell culture, ELISA, Rapid Latex Agglutination (RPLA) and hybridization, but due to high costs and labor time in addition to low sensitivity, they have not received much attention. The aim of this study was to develop a complete, rapid and reliable multiplex PCR (mPCR) method by using two pairs of specific primers to detect either the stx1 or the stx2 gene confirms the presence of E.coli O157:H7. The study results show that stx1F/stx1R primers are specific for stx1 and primers stx2F/stx2R are specific for stx2 genes in E. coli O157:H7. The mPCR method with two pairs of primers for amplifying the stx1, stx2 target genes to detect E. coli O157:H7 in food has been set up successfully. Complete method performed well in both types of food matrices with a detection limit of 3 CFU/25 g or mL of food samples. Tests on 180 food samples have shown a specificity value of 93.75 % (95 % confidence interval [CI], 82.83-100), a sensitivity of 100 % (95 % CI, 83.79-99.85 %), and an accuracy of 96.66 % (CI 95 %, 83.41-99.91 %). Interestingly, results indicate that the mPCR performed as well as the traditional culture methods and can reduce the diagnosis time to 2 days. Finally, complete mPCR method was applied to natural samples covering a wide variety of food types proving that the mPCR method was a rapid and reliable screening method for detection of E. coli O157:H7 in food and environmental samples.
产志贺毒素大肠杆菌O157:H7菌株是食源性感染因子，可引发多种危及生命的疾病，包括出血性结肠炎（HC）和溶血尿毒综合征（HUS）。志贺毒素1（stx1）、志贺毒素2（stx2）或两者的组合是这些疾病大多数临床症状的病因。因此，到目前为止已经开发了各种诊断方法来检测志贺毒素，如细胞培养、酶联免疫吸附测定（ELISA）、快速乳胶凝集试验（RPLA）和杂交，但由于成本高、耗时且灵敏度低，它们并未受到太多关注。本研究的目的是通过使用两对特异性引物开发一种完整、快速且可靠的多重聚合酶链反应（mPCR）方法，以检测stx1或stx2基因，从而确认大肠杆菌O157:H7的存在。研究结果表明，stx1F/stx1R引物对stx1具有特异性，stx2F/stx2R引物对大肠杆菌O157:H7中的stx2基因具有特异性。已成功建立了用于扩增stx1、stx2靶基因以检测食品中大肠杆菌O157:H7的两对引物的mPCR方法。完整方法在两种类型的食品基质中均表现良好，食品样品的检测限为3 CFU/25 g或mL。对180份食品样品的检测显示，特异性值为93.75%（95%置信区间[CI]，82.83 - 100），灵敏度为100%（95% CI，83.79 - 99.85%），准确度为96.66%（95% CI，83.41 - 99.91%）。有趣的是，结果表明mPCR的性能与传统培养方法相当，并且可以将诊断时间缩短至2天。最后，完整的mPCR方法应用于涵盖多种食品类型的天然样品，证明mPCR方法是一种快速且可靠的用于检测食品和环境样品中大肠杆菌O157:H7的筛选方法。
