Department of Human, Nutrition, Food and Animal Science, University of Hawaii at Manoa, 1955 East West Road, Agricultural Sciences Building 216, Honolulu, HI, 96822, USA.
Department of Plant and Environmental Protection Sciences, University of Hawaii at Manoa, 3190 Maile Way, St. John 305, Honolulu, HI, 96822, USA.
Food Microbiol. 2022 Sep;106:104052. doi: 10.1016/j.fm.2022.104052. Epub 2022 Apr 27.
Traditional culture-based detection methods for Campylobacteri jejuni, a leading cause of human bacterial gastroenteritis worldwide, are time-consuming, cumbersome, and lacking in reliability. While polymerase chain reaction (PCR) has been frequently used for pathogen testing, it might generate false-negative results due to inadequate sensitivity. This study was the first to explore novel single-tube nested PCR (STN-PCR) to detect pathogens in food. Two pairs of nested PCR primers were designed based on the hippuricase gene of C. jejuni. The annealing temperatures and concentrations of nested primers were optimized to ensure the sequential use of outer and inner pairs of primers during amplification. Efficacy of the developed STN-PCR assay was compared with standard culture-based methods and conventional PCR in artificially contaminated ground chicken homogenate. Limit of detection of the STN-PCR assay was determined to be 3.6 × 10 CFU/ml of C. jejuni in the homogenate without enrichment, which was 100 times lower than conventional PCR. Moreover, 0.1 CFU/g of C. jejuni in ground chicken homogenate was identified by STN-real time PCR (rtPCR) after 24 h of enrichment, while a 48-h enrichment was required for culture-based methods and conventional rtPCR. This developed assay provides a powerful tool for rapid, highly specific, and ultra-sensitive detection of C. jejuni and may potentially be used to identify contaminated chicken and other foods.
传统的基于文化的检测方法用于检测弯曲杆菌，一种导致全球人类细菌性胃肠炎的主要病原体，耗时、繁琐且缺乏可靠性。聚合酶链反应（PCR）已被频繁用于病原体检测，但由于灵敏度不足，可能会产生假阴性结果。本研究首次探索了新型单管嵌套 PCR（STN-PCR）用于检测食品中的病原体。根据弯曲杆菌的 hippuricase 基因设计了两对嵌套 PCR 引物。优化了嵌套引物的退火温度和浓度，以确保在扩增过程中顺序使用外引物和内引物。将开发的 STN-PCR 与基于标准培养的方法和常规 PCR 进行比较，在人工污染的鸡肉匀浆中进行了检测。STN-PCR 检测方法的检测限为未经富集的匀浆中 3.6×10 CFU/ml 的弯曲杆菌，比常规 PCR 低 100 倍。此外，STN-实时 PCR（rtPCR）在 24 小时富集后可检测到鸡肉匀浆中 0.1 CFU/g 的弯曲杆菌，而基于培养的方法和常规 rtPCR 需要 48 小时的富集。该方法为快速、高度特异性和超灵敏检测弯曲杆菌提供了有力工具，可能用于鉴定污染的鸡肉和其他食品。
