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检测法可快速定量污染即食蔬菜和水果中的细菌总数。使用靶向编码细菌RNA聚合酶β亚基的rpoB基因（大多数细菌物种共有）的引物，而不是具有多个拷贝且在细菌物种之间变化的16S rRNA基因。利用从5种果蔬中分离的49株细菌，对rpoB特异性引物进行扩增，结果表明，该引物能在大范围内扩增rpoB。我们从零售店购买水果和蔬菜，并使用实时PCR计数与它们相关的细菌，并将其与培养方法发现的数量进行比较。我们发现与平板计数培养物数量相比，阈值PCR循环数之间具有高度相关性。此外，将通过培养方法检测到的优势细菌植物群与通过使用变性梯度凝胶电泳分析的实时PCR测定检测到的优势细菌群进行比较。两种方法的结果几乎相同。本研究建立的实时荧光定量PCR检测方法可以对即食水果和蔬菜中的优势细菌种类进行计数。

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.