利用实时定量PCR快速检测冷鲜猪肉新鲜度指标的方法研究

摘要

The current detection techniques for freshness of chilled pork are complex, so exploring fast detection techniques becomes hotspot. In this study Total Volatile Basic Nitrogen(TVB-N)was detected by the method defined in national standard, and Plate Culture Method(PCM)and real-time quantitative PCR were used to count the total amount of microorganisms causing spoilage of chilled pork. With the extension of storage time of the chilled pork at 4℃, the total number of microorganism colony and TVB-N had the linear correlation, and the level of spoilage of chilled pork behaved the positive correlation. The extracted bacterial genome by acetone-chloroform had the clear bands and showed the fine extraction result. Single-factor analysis of variance revealed that there was no significant difference of total number of colony (P=0. 7190)between by real-time quantitative PCR based on SYBER Green I and by counting in PCM, i. e. , consistency was quite solid. The detection time was reduced by real-time quantitative PCR with higher detection sensitivity. The amount of microorganisms is a crucial index to evaluate the freshness of chilled pork, and real-time quantitative PCR can be a new technology for quickly checking the freshness of chilled pork.%目前冷鲜猪肉新鲜度指标检测繁琐，探索快速检测冷鲜猪肉新鲜度指标新技术成为研究热点。利用国标法检测发性盐基氮（Total volatile basic nitrogen，TVB-N）含量，平板培养法和实时定量PCR技术分别检测腐败微生物数量。结果显示，冷鲜猪肉在4℃条件下，随储藏时间延长，微生物的菌落总数和TVB-N逐渐增加且呈线性相关，与猪肉品的腐败程度呈正相关；丙酮-氯仿法提取的细菌基因组条带清晰，提取效果好；基于SYBER GreenⅠ的实时定量PCR技术检测的菌落数量与平板培养测得菌落总数利用单因素方差分析结果没有显著性差异（P=0.7190），一致性较好；实时定量PCR方法缩短了检测时间，提高了检测灵敏度。微生物是冷鲜猪肉新鲜度评价的重要指标，实时定量PCR可以作为一种快速高效检测冷鲜猪肉新鲜度指标的新技术。