Department of Food Science and Technology, Pukyong National University, Busan, 48513, South Korea.
Department of Chemical and Biological Engineering, College of Engineering, University of Saskatchewan, 57 Campus Dr, Saskatoon, SK, S7N 5A9, Canada.
Arch Microbiol. 2021 Dec 6;204(1):5. doi: 10.1007/s00203-021-02613-y.
Vibrio cholerae and Vibrio vulnificus are critical foodborne pathogens that need to be intensively controlled for their infection due to the intake and distribution of seafood, especially raw oysters. For this reason, various methods have already been developed for the detection and enumeration of these bacteria. The most probable number (MPN)-PCR (polymerase chain reaction) method is commonly used with the selective-differential medium for the efficiency and convenience of cell enumeration. One of the most frequently used for detecting Vibrio spp. is thiosulfate-citrate-bile salts-sucrose (TCBS) agar. But this selective-differential medium can fail to distinguish between V. cholerae, V. vulnificus, and Vibrio alginolyticus. For this reason, the conventional MPN-PCR method with TCBS medium for the detection of Vibrio spp. has a problem with processing PCR two times. This study suggests a simple and minimized detection method using one-time PCR and non-NaCl Luria-Bertani (LB-0) medium culture. This detection method is based on the difference in salt requirement between V. cholerae and V. vulnificus. Employing the developed methodology, the simultaneous cell enumeration of V. cholerae and V. vulnificus can be possible at a low cost. Furthermore, this study proposes a new specific primer to detect virulence-related genes from V. cholerae and V. vulnificus. This advanced MPN-PCR method was verified using bioaccumulated pacific oysters (Crassostrea gigas) by V. cholerae and V. vulnificus.
霍乱弧菌和创伤弧菌是两种重要的食源性致病菌，由于海鲜（尤其是生牡蛎）的摄入和分布，需要对其进行严格的控制以防止感染。出于这个原因，已经开发出了各种用于检测和计数这些细菌的方法。最可能数（MPN）-PCR（聚合酶链式反应）方法通常与选择性差异培养基一起用于细胞计数的效率和便利性。最常用于检测弧菌属的方法之一是硫代硫酸盐-柠檬酸盐-胆汁盐-蔗糖（TCBS）琼脂。但是，这种选择性差异培养基可能无法区分霍乱弧菌、创伤弧菌和溶藻弧菌。出于这个原因，使用 TCBS 培养基的传统 MPN-PCR 方法在处理 PCR 两次方面存在问题。本研究提出了一种使用一次 PCR 和非 NaCl 的简化检测方法，即 Luria-Bertani（LB-0）培养基培养。这种检测方法基于霍乱弧菌和创伤弧菌对盐的需求差异。采用所开发的方法，可以以低成本同时对霍乱弧菌和创伤弧菌进行细胞计数。此外，本研究提出了一种新的特异性引物，用于检测霍乱弧菌和创伤弧菌的毒力相关基因。这种先进的 MPN-PCR 方法已通过生物蓄积的太平洋牡蛎（Crassostrea gigas）被霍乱弧菌和创伤弧菌验证。
