Sethulekshmi C, Latha C, Anu C J
Department of Veterinary Public Health, College of Veterinary and Animal Sciences, Mannuthy, Thrissur, Kerala, India.
Vet World. 2018 Feb;11(2):104-111. doi: 10.14202/vetworld.2018.104-111. Epub 2018 Feb 3.
The objective of the study was to detect Shiga toxin-producing (STEC) and develop a quantitative polymerase chain reaction (qPCR) assay to quantify the bacterial DNA present in different food matrices.
A total of 758 samples were collected during a period from January 2015 to December 2016 from Kozhikode, Thrissur, and Alappuzha districts of Kerala. The samples consisted of raw milk (135), pasteurized milk (100), beef (132), buffalo meat (130), chevon (104), beef kheema (115), and beef sausage (42). All the samples collected were subjected to isolation and identification of STEC by conventional culture technique. Confirmation of virulence genes was carried out using PCR. For the quantification of STEC in different food matrices, a qPCR was standardized against 1 gene of STEC by the construction of standard curve using SYBR green chemistry.
The overall occurrence of STEC in raw milk (n=135), beef (n=132), buffalo meat (n=130), chevon (n=104), and beef kheema (n=115) samples collected from Kozhikode, Thrissur, and Alappuzha districts of Kerala was 19.26%, 41.6%, 16.92%, 28.85%, and 41.74%, respectively. PCR revealed the presence of 1 and 2 genes in 88.46 and 83.64 and 30.77 and 40.00% of STEC isolates from raw milk and beef samples, respectively, while 100% of the STEC isolates from buffalo beef and beef kheema samples carried 1 gene. Real-time qPCR assay was used to quantify the bacterial cells present in different food matrices. The standard curve was developed, and the slopes, intercept, and R of linear regression curves were -3.10, 34.24, and 0.99, respectively.
The considerably high occurrence of STEC in the study confirms the importance of foods of animal origin as a vehicle of infection to humans. In the present study, on comparing the overall occurrence of STEC, the highest percentage of occurrence was reported in beef kheema samples. The study shows the need for rigid food safety measures to combat the potential pathogenic effects of harmful bacteria throughout the production chain from production to consumption.
本研究的目的是检测产志贺毒素大肠杆菌（STEC），并开发一种定量聚合酶链反应（qPCR）检测方法，以量化不同食品基质中存在的细菌DNA。
2015年1月至2016年12月期间，从喀拉拉邦的科泽科德、特里苏尔和阿拉普扎地区共采集了758份样本。样本包括生牛奶（135份）、巴氏杀菌牛奶（100份）、牛肉（132份）、水牛肉（130份）、山羊肉（104份）、牛肉末（115份）和牛肉香肠（42份）。所有采集的样本均通过传统培养技术进行STEC的分离和鉴定。使用PCR对毒力基因进行确认。为了量化不同食品基质中的STEC，通过使用SYBR绿色化学构建标准曲线，针对STEC的1个基因对qPCR进行了标准化。
从喀拉拉邦的科泽科德、特里苏尔和阿拉普扎地区采集的生牛奶（n = 135）、牛肉（n = 132）、水牛肉（n = 130）、山羊肉（n = 104）和牛肉末（n = 115）样本中，STEC的总体发生率分别为19.26%、41.6%、16.92%、28.85%和41.74%。PCR显示，分别有88.46%和83.64%以及30.77%和40.00%的生牛奶和牛肉样本中的STEC分离株存在1和2基因，而水牛肉和牛肉末样本中的STEC分离株100%携带1基因。使用实时qPCR检测方法对不同食品基质中存在的细菌细胞进行量化。绘制了标准曲线，线性回归曲线的斜率、截距和R分别为-3.10、34.24和0.99。
本研究中STEC的高发生率证实了动物源性食品作为人类感染源的重要性。在本研究中，比较STEC的总体发生率时，牛肉末样本的发生率最高。该研究表明需要采取严格的食品安全措施，以对抗从生产到消费的整个生产链中有害细菌的潜在致病影响。
