Zhou Min, Yang Jielin, Zhou Xiujuan, Liu Bin, Liu Daixin, Yuan Chengang, He Yuping, Pan Liangwen, Shi Xianming
1 MOST-USDA Joint Research Center for Food Safety, School of Agriculture and Biology, and State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University , Shanghai, China .
Foodborne Pathog Dis. 2014 Jul;11(7):537-44. doi: 10.1089/fpd.2013.1701. Epub 2014 Jun 13.
Salmonella is the most common cause of bacterial food poisoning in humans worldwide. Thus, rapid and reliable methods for the detection of this pathogen are required. Real-time reverse-transcriptase polymerase chain reaction (rt-RT-PCR), which detects the presence of mRNA (shorter half-life than DNA) has shown great potential for detecting viable pathogens. We recently identified a few new potential specific DNA sequences for Salmonella enterica using a comparative genomics method (Chen et al., 2010). In the present study, we examined the expression of the Salmonella-specific sigDE operon (encoding invasion proteins within the pathogenicity island 5) under typical growth conditions to determine whether sigDE could be a useful viability marker for the bacterium. We then assayed sigDE mRNA from cells heat-treated at 60°C, 100°C, and 121°C (autoclaved), and found that mRNA was degraded in autoclaved bacterial samples. These results showed that the sigDE transcript is a suitable mRNA target for rt-RT-PCR with samples pretreated at 121°C. Thus, an rt-RT-PCR using sigDE primers was developed for the detection of viable Salmonella. An RNA internal amplification control was constructed by overlap extension PCR, synthesized using in vitro transcription with a T7 RNA polymerase promoter, and incorporated into the rt-RT-PCR system to eliminate false-negative results. The rt-RT-PCR system has the capability of specifically detecting all the tested S. enterica serovars, and the detection limit of this assay with cultures of Salmonella Typhimurium ATCC 13311 was 10(1) colony-forming units (CFU)/mL. After 18-h enrichment, sigDE-based rt-RT-PCR could detect as low as 10(0) CFU/mL of Salmonella from egg broth and milk.
沙门氏菌是全球人类细菌性食物中毒最常见的病因。因此，需要快速可靠的方法来检测这种病原体。实时逆转录聚合酶链反应（rt-RT-PCR）可检测mRNA（半衰期比DNA短）的存在，在检测活的病原体方面显示出巨大潜力。我们最近使用比较基因组学方法鉴定了一些新的肠炎沙门氏菌潜在特异性DNA序列（Chen等人，2010年）。在本研究中，我们检测了典型生长条件下沙门氏菌特异性sigDE操纵子（编码致病岛5内的侵袭蛋白）的表达，以确定sigDE是否可作为该细菌有用的生存力标记。然后，我们检测了在60°C、100°C和121°C（高压灭菌）下热处理的细胞中的sigDE mRNA，发现高压灭菌的细菌样本中的mRNA被降解。这些结果表明，sigDE转录本是对经121°C预处理的样本进行rt-RT-PCR的合适mRNA靶标。因此，开发了一种使用sigDE引物的rt-RT-PCR来检测活的沙门氏菌。通过重叠延伸PCR构建RNA内部扩增对照，使用具有T7 RNA聚合酶启动子的体外转录合成，并将其纳入rt-RT-PCR系统以消除假阴性结果。该rt-RT-PCR系统能够特异性检测所有测试的肠炎沙门氏菌血清型，该检测方法对鼠伤寒沙门氏菌ATCC 13311培养物的检测限为10(1) 菌落形成单位（CFU）/mL。经过18小时富集后，基于sigDE的rt-RT-PCR能够检测到蛋汤和牛奶中低至10(0) CFU/mL的沙门氏菌。
