Analysis of major band of Enterobacter sakazakii by ERIC-PCR and development of a species-specific PCR for detection of Ent. sakazakii in dry food samples |
| |
Affiliation: | 1. State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou 510070, China;2. Guangzhou Wondfo Biotech. Co., Ltd., Guangzhou Accurate and Correct Test Co., Ltd., Guangzhou, Guangdong 510643, China;3. School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China;1. Guangdong Institute of Microbiology, State Key Laboratory of Applied Microbiology Southern China, Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangzhou 510070, PR China;2. Department of Laboratory Medicine, First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120, PR China;3. Department of Cardiology, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, PR China;4. College of Food Science, South China Agricultural University, Guangzhou 510642, PR China;1. Guangdong Institute of Microbiology, State Key Laboratory of Applied Microbiology Southern China, Guangzhou 510070, PR China;2. Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, Guangzhou 510070, PR China |
| |
Abstract: | ERIC (Enterobacterial Repetitive Intergenic Consensus)-PCR was employed to generate stable and reproductive ERIC-PCR fingerprints of Ent. sakazakii ATCC51329. Moreover, this study also cloned and sequenced a major band of Ent. sakazakii (ATCC51329) ERIC-PCR fingerprints. The major band was amplified with primer ERIC2 and sequences extending primer ERIC 2 showed poor similarity with ERIC elements. A comparison of the nucleotide acid with other sequences available in the GenBank revealed 90% of identity with Ent. sakazakii ATCC BAA-894, and 73%–74% of identity with oligopeptiase gene or proteaseⅡgene of some species from the Enterobacteriaceae family. Two primers were synthesized to develop and optimize an Enterobacter sakazakii-specific PCR based on regions of major band unique to Ent. sakazakii. The expected fragment was amplified from all of Ent. sakazkaii but not from the negative controls. As few as 102 CFU/ml of Ent. sakazakii of PCR were directly detected in the infant formulas. This was the case even in the presence of other bacteria. A comparison of traditional methods and new developed PCR in commercial foods suggested that without using API20-E test, the DFI chromogenic medium and FDA method showed 46.15% and 50% false positive respectively. Moreover, one false negative was observed with FDA method. In contrast, PCR was highly sensitive and specific to Ent. sakazakii. A high heterogeneity between Ent. sakazakii and the other microorganisms was found on expected fragment sequence. In addition, Ent. sakazakii ATCC51329 formed a separate branch with > 5% divergence from the type strain ATCC BAA-894 and major strains. |
| |
Keywords: | |
本文献已被 ScienceDirect 等数据库收录! |
|