Development of a Quantitative Real-time Polymerase Chain Reaction Method to Enumerate Total Bacterial Count in Ready-to- eat Fruits and Vegetables

与标准平板计数法相比，新开发的实时PCR检测可快速定量污染即食蔬菜和水果的细菌总数。引物针对大多数细菌常见的编码细菌RNA聚合酶β-亚基的rpoB基因，而不是具有多个拷贝且在细菌物种中存在差异的16S rRNA基因。利用从5种果蔬中分离的49株菌株，确定了rpoB特异引物对，可在多种细菌中扩增rpoB。我们从零售商店购买水果和蔬菜，并使用实时聚合酶链反应（real-time PCR）枚举与其相关的细菌数量，并将其与培养方法发现的细菌数量进行比较。我们发现阈值PCR循环数与平板计数培养数之间存在高度相关性。此外，利用变性梯度凝胶电泳分析，将培养法检测到的优势菌群与实时PCR检测到的优势菌群进行了比较。两种方法的结果几乎相同。本研究建立的实时PCR检测方法可以枚举即食水果和蔬菜中的优势菌种。

A newly developed real-time PCR assay rapidly quantifies the total bacterial numbers in contaminating ready-to-eat vegetables and fruits as compared to the standard plate count method. Primers targeting the rpoB gene that encodes for the β-subunit of the bacterial RNA polymerase, common to most bacterial species was used instead of the 16S rRNA gene that has multiple copies and varies among bacterial species. A primer pair specific for rpoB was confirmed to amplify rpoB in wide-range of bacterial species by using 49 strains isolated from five kinds of fruits and vegetables. We purchased fruits and vegetables from retail shops and enumerated the bacteria associated with them using the real-time PCR and compared this to the number found by the culture method. We found a high correlation between the threshold PCR cycle numbers when compared to the plate count culture number. Further, the predominant bacterial flora detected by the culture method was compared with those detected by the real-time PCR assay using a denaturing gradient gel electrophoresis analysis. The results from the two methods were almost identical. The real-time PCR assay developed in this study can enumerate the dominant bacterial species in ready-to-eat fruits and vegetables.