Long-read sequencing data from pure cultures of <i>Escherichia coli</i> O157:H7 and ground beef inoculated with <i>E. coli</i> O157:H7

Foodborne pathogens are a significant cause of illness and infection with Shiga toxin-producing <i>Escherichia coli</i> (STEC) has the potential to produce life-threatening complications. The current methods to identify STEC in meat involve culture-based, molecular, and proteomic assays and take at least four days to complete. This time could be reduced by using long-read whole genome sequencing to identify foodborne pathogens. Therefore, the goal of this project was to evaluate using long-read sequencing to detect STEC in ground beef. The objectives of the project included: establishing optimal sequencing parameters, determining the limit of detection of all STEC virulence genes of interest in pure cultures and spiked ground beef, and evaluating selective sequencing to enhance STEC detection in ground beef. Sequencing libraries were run on Oxford Nanopore Technologies’ MinION sequencer. Optimal sequencing output was obtained using the default parameters in MinKNOW, except for setting the minimum read length to 1 kb. All genes of interest (<i>eae</i>, <i>stx1</i>, <i>stx2</i>, <i>fliC</i>, <i>wzx</i>, <i>wzy</i>, <i>rrsC</i>) were detected in DNA extracted from STEC pure cultures within 1 hour of sequencing, and 30X coverage was obtained within 2 hours. All virulence genes were confidently detected in STEC DNA quantities as low as 12.5 ng. In STEC inoculated ground beef, software-controlled selective sequencing improved virulence gene detection; however, several virulence genes were not detected due to high bovine DNA concentrations in the samples. Growth enrichment of inoculated meat samples in mTSB resulted in a 100-fold increase in virulence gene detection as compared to the unenriched samples. The results of this project suggest that further development of long-read sequencing protocols may result in a faster, less labor-intensive method to detect STEC in ground beef. The sequencing data from this project has been uploaded.食源性致病菌是引发疾病与感染的重要诱因，产志贺毒素大肠杆菌（Shiga toxin-producing Escherichia coli, STEC）感染则可能引发危及生命的并发症。 当前用于肉类样本中STEC检测的方法包括基于培养、分子生物学及蛋白质组学的检测手段，且至少需要四天方可完成全部检测流程。采用长读长全基因组测序（long-read whole genome sequencing）技术识别食源性致病菌，则有望大幅缩短这一检测周期。因此本研究的目标为评估长读长测序技术在绞碎牛肉样本中检测STEC的可行性与效果。 本项目的研究目标包括：确立最优测序参数；确定纯培养物与加标绞碎牛肉样本中所有目标STEC毒力基因的检测限；并评估选择性测序技术以提升绞碎牛肉中STEC的检出能力。 测序文库在牛津纳米孔技术公司（Oxford Nanopore Technologies）的MinION测序仪上完成上机运行。通过使用MinKNOW软件的默认参数（仅将最小读长设置为1 kb），获得了最优的测序产出数据。 在测序启动后1小时内，即可从STEC纯培养物提取的DNA样本中检出所有目标基因（<i>eae</i>、<i>stx1</i>、<i>stx2</i>、<i>fliC</i>、<i>wzx</i>、<i>wzy</i>、<i>rrsC</i>），并在2小时内达到30倍测序覆盖度。 当STEC DNA含量低至12.5 ng时，仍可可靠检出全部目标毒力基因。 在接种了STEC的绞碎牛肉样本中，软件控制的选择性测序技术提升了毒力基因的检出效果，但由于样本中牛源DNA浓度过高，仍有部分毒力基因未能被成功检出。 将接种了STEC的绞碎牛肉样本在mTSB培养基中进行增菌培养后，与未增菌的对照组样本相比，毒力基因的检出率提升了100倍。 本研究结果表明，进一步优化长读长测序流程，有望开发出一种更快速、劳动强度更低的绞碎牛肉STEC检测方法。 本项目产生的测序数据已完成上传存档。