Detection of non-emetic and emetic Bacillus cereus by propidium monoazide multiplex PCR (PMA-mPCR) with internal amplification control

详细信息    查看全文

• 作者：Zhihong Zhang ; Lijun Wang ; Hengyi Xu ; Zoraida P. Aguilar ; Chengwei Liu ; Bei Gan ; Yonghua Xiong ; Weihua Lai ; Feng Xu ; Hua Wei
• 关键词：Bacillus cereus ; Propidium monoazide ; Multiplex PCR ; IAC ; cesB gene ; Detection
• 刊名：Food Control
• 出版年：2014
• 出版时间：January, 2014
• 年：2014
• 卷：35
• 期：1
• 页码：401-406
• 全文大小：635 K

文摘

Bacillus cereus is an etiological agent of food-borne disease that can cause a type of emesis. To develop a sensitive and reliable diagnosis technique for detecting all the species of the B.?cereus group, specific primers were designed to target a recently discovered part of the cereulide synthetase gene (cesB) for emetic B.?cereus and 16S rRNA for non-emetic B.?cereus. To detect PCR signals only from viable cells, propidium monoazide (PMA) was selected to eliminate the false-positive results. In addition, an internal amplification control (IAC) was applied to meet diagnostic multiplex PCR requirements that will prevent the occurrence of false-negative results. The inclusivity and exclusivity of the mPCR assay were estimated using a panel of 100 strains, including 2 emetic B.?cereus, 77 non-emetic B.?cereus and 21 non-Bacillus strains. The limit of detection (LOD) for dead B.?cereus without PMA treatment in pure bacteria culture was 4.0?¡Á?10²?CFU/mL, as low as 7.5?¡Á?10⁰?CFU/mL for viable B.?cereus without PMA treatment, and 7.5?¡Á?10¹?CFU/mL for viable B.?cereus with PMA treatment. B.?cereus in spiked food produce was detected specifically and sensitively at 1.0?¡Á?10³?CFU/g which was the lowest concentration detected. This novel PMA-mPCR-IAC assay is rapid and reliable, providing an efficient diagnostic tool with promising application in monitoring food samples.