Chahorm Kanchana, Prakitchaiwattana Cheunjit
Department of Food Technology, Faculty of Science, Chulalongkorn University, Patumwan, Bangkok 10330, Thailand.
Department of Food Technology, Faculty of Science, Chulalongkorn University, Patumwan, Bangkok 10330, Thailand.
Int J Food Microbiol. 2018 Jan 2;264:46-52. doi: 10.1016/j.ijfoodmicro.2017.10.014. Epub 2017 Oct 12.
The aim of this research was to evaluate the feasibility of PCR-DGGE and Reverse Transcriptase-PCR-DGGE techniques for rapid detection of Vibrio species in foods. Primers GC567F and 680R were initially evaluated for amplifying DNA and cDNA of ten references Vibrio species by PCR method. The GC-clamp PCR amplicons were separated according to their sequences by the DGGE using 10% (w/v) polyacrylamide gel containing 45-70% urea and formamide denaturants. Two pair of Vibrio species, which could not be differentiated on the gel, was Vibrio fluvialis - Vibrio furnissii and Vibrio parahaemolyticus - Vibrio harveyi. To determine the detection limit, in the community of 10 reference strains containing the same viable population, distinct DNA bands of 3 species; Vibrio cholerae, Vibrio mimicus and Vibrio alginolyticus were consistently observed by PCR-DGGE technique. In fact, 5 species; Vibrio cholerae, Vibrio mimicus, Vibrio alginolyticus, Vibrio parahaemolyticus and Vibrio fluvialis consistently observed by Reverse Transcriptase-PCR-DGGE. In the community containing different viable population increasing from 10 to 10CFU/mL, PCR-DGGE analysis only detected the two most prevalent species, while RT-PCR-DGGE detected the five most prevalent species. Therefore, Reverse Transcriptase-PCR-DGGE was also selected for detection of various Vibrio cell conditions, including viable cell (VC), injured cells from frozen cultures (IVC) and injured cells from frozen cultures with pre-enrichment (PIVC). It was found that cDNA band of all cell conditions gave the same migratory patterns, except that multiple cDNA bands of Plesiomonas shigelloides under IVC and PIVC conditions were found. When Reverse Transcriptase-PCR-DGGE was used for detecting Vibrio parahaemolyticus in the pathogen-spiked food samples, Vibrio parahaemolyticus could be detected in the spiked samples containing at least 10CFU/g of this pathogen. The results obtained also corresponded to standard method (USFDA, 2004). In comparison with the detection of the Vibrio profiles in fourteen food samples using standard method, Reverse Transcriptase-PCR-DGGE resulted in 100%, 75% and 50% similarity in 3, 1 and 6 food samples, respectively.
本研究的目的是评估聚合酶链反应-变性梯度凝胶电泳（PCR-DGGE）和逆转录聚合酶链反应-变性梯度凝胶电泳（RT-PCR-DGGE）技术用于快速检测食品中弧菌属细菌的可行性。首先通过PCR方法评估引物GC567F和680R对十种参考弧菌属细菌的DNA和cDNA的扩增能力。使用含有45-70%尿素和甲酰胺变性剂的10%（w/v）聚丙烯酰胺凝胶，通过DGGE根据序列分离GC夹PCR扩增产物。在凝胶上无法区分的两对弧菌属细菌是河流弧菌-弗氏弧菌和副溶血性弧菌-哈维氏弧菌。为了确定检测限，在含有相同活菌数的10种参考菌株群落中，通过PCR-DGGE技术始终观察到霍乱弧菌、模仿弧菌和解蛋白弧菌这3种细菌的独特DNA条带。实际上，通过逆转录聚合酶链反应-变性梯度凝胶电泳（RT-PCR-DGGE）始终观察到霍乱弧菌、模仿弧菌、解蛋白弧菌、副溶血性弧菌和河流弧菌这5种细菌。在活菌数从10增加到10CFU/mL的不同群落中，PCR-DGGE分析仅检测到两种最常见的细菌，而RT-PCR-DGGE检测到5种最常见的细菌。因此，逆转录聚合酶链反应-变性梯度凝胶电泳（RT-PCR-DGGE）也被选择用于检测各种弧菌细胞状态，包括活细胞（VC）、来自冷冻培养物的损伤细胞（IVC）和经过预富集的来自冷冻培养物的损伤细胞（PIVC）。结果发现，除了在IVC和PIVC条件下志贺邻单胞菌出现多条cDNA条带外，所有细胞状态的cDNA条带都呈现相同的迁移模式。当使用逆转录聚合酶链反应-变性梯度凝胶电泳（RT-PCR-DGGE）检测接种病原体的食品样品中的副溶血性弧菌时，在每克至少含有10CFU这种病原体的加标样品中可以检测到副溶血性弧菌。获得的结果也与标准方法（美国食品药品监督管理局，2004年）相符。与使用标准方法检测14个食品样品中的弧菌谱相比，逆转录聚合酶链反应-变性梯度凝胶电泳（RT-PCR-DGGE）在3个、1个和6个食品样品中分别产生了100%、75%和50%的相似度。
