Further development of sample preparation and detection methods for O157 and the top 6 non-O157 STEC serogroups in cattle feces
Further development of sample preparation and detection methods for O157 and the top 6 non-O157 STEC serogroups in cattle feces
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DOI：
10.1016/j.mimet.2014.06.020
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发表时间：
2014-10-01
影响因子：
2.2
通讯作者：
Reuter, Tim
中科院分区：
文献类型：
--
作者：
Conrad, Cheyenne C.;Stanford, Kim;Reuter, Tim
产滋贺毒素大肠杆菌（STEC）是引起全球人类感染暴发的食源性病原体。反刍家畜在其肠道中携带STEC，并且通过粪便污染具有损害食物和水的安全的可能性。作为一种人类健康安全风险，需要USDA-FSIS强制要求的牛肉屠体和胴体上的STEC检测方法。为了在收获和人类食用之前监测STEC，我们的目标是评估和/或改进牛粪便中7种STEC血清群的检测。与传统方法相比，使用涉及冷冻或冻干粪便的多因子拉丁方设计评价牛粪便中的样品处理方法。在高压灭菌与非高压灭菌粪便中加标不同稀释度的O26：H11或O 157：H7血清型，并富集长达6 h。每小时，使用传统培养方法和定量聚合酶链反应（qPCR）比较富集的等分试样。建立了7群多重PCR（mPCR）方法，可同时检测O26、O 45、O 103、O 111、O 121、O 145和O 157血清群。与先前建立的PCR测定法（O26和O 103为10 CFU/g;所有其他血清组>= 10（4）CFU/g）相比，我们的mPCR测定法在6 h富集后的诊断灵敏度上级（所有血清组均为10 CFU/g）。获得活的分离株似乎受到当前免疫磁性分离（IMS）方法效率的限制，该方法根据血清型检索菌落的有效性范围为20%至100%。IMS后，通过mPCR筛选70个推定的STEC分离株的滋贺毒素和附着基因。65株分离株含有一种或两种滋贺毒素基因。(C)© 2014 Elsevier B. V.保留所有权利。
Shiga toxin-producing Escherichia coli (STEC) are food-borne pathogens responsible for outbreaks of human infections worldwide. Ruminant livestock harbor STEC in their intestinal tract, and through fecal contamination possess the potential to compromise the safety of food and water. As a human health safety risk, STEC detection methods on beef carcasses and trim are needed as mandated by the USDA-FSIS. In order to monitor STEC prior to harvest and human consumption, our goal was to evaluate and/or improve detection of seven STEC serogroups in cattle feces. In comparison to traditional approaches, sample processing methods in bovine feces were evaluated using a multi-factorial Latin square design which involved freezing or freeze drying feces. Autoclaved versus non-autoclaved feces were spiked with O26:H11 or O157:H7 serotypes in various dilutions and enriched for up to 6 h. Each hour, enriched aliquots were compared using traditional culture methods and quantitative polymerase chain reaction (qPCR). Furthermore, a 7-serogroup multiplex PCR (mPCR) was developed to detect O26, O45, O103, O111, O121, O145 and O157 serogroups simultaneously. The diagnostic sensitivity of our mPCR assay following 6 h enrichment was superior (10 CFU/g across all serogroups) compared to a previously established PCR assay (10 CFU/g for O26, and O103; >= 10(4) CFU/g for all other serogroups). Obtaining viable isolates appeared to be limited by the efficiency of current immunomagnetic separation (IMS) methods, which ranged from 20 to 100% effectiveness at retrieving colonies depending on serogroup. After IMS, 70 putative STEC isolates were screened for Shiga toxin and attachment genes by mPCR. Sixty-five isolates contained one or both Shiga toxin genes. (C) 2014 Elsevier B.V. All rights reserved.