Department of Food Science and Technology, University of Tennessee, Knoxville, Tennessee 37996-4591, USA.
J Food Prot. 2010 Mar;73(3):507-14. doi: 10.4315/0362-028x-73.3.507.
Reverse transcriptase PCR (RT-PCR) detects the presence of mRNA and has a greater potential for detecting viable pathogens than do DNA-based PCR assays, with improved speed and sensitivity compared with traditional methods. Our objective was to rapidly and sensitively detect Salmonella Typhimurium from pork within two 8-h work shifts using a SYBR Green I real-time RT-PCR (rt-RT-PCR) assay. Pork chop and sausage samples (25 g) were inoculated with 10(8) to 10(0) CFU of Salmonella Typhimurium and stomached in 225 ml of tetrathionate broth. Serial dilutions were spread plated on xylose lysine Tergitol 4 agar either immediately or after 10 h of selective preenrichment or preenrichment followed by 12 h of selective enrichment (for stressed cells) at 37 degrees C for standard cultural enumeration. RNA was extracted using the TRIzol method. The rt-RT-PCR assay was carried out in a Bio-Rad iCycler using a SYBR Green I one-step RT-PCR kit and Salmonella specific invA gene primers with an internal amplification control (IAC). The PCR was followed by melting temperature (T(m)) analysis to determine specific Salmonella invA (T(m) = 87.5 degrees C) and IAC (T(m) = 82 degrees C) products. Improved Salmonella detection up to 10(1) CFU/25 g of pork and 10(0) CFU/25 g of sausages was obtained after 10 h of enrichment within approximately 24 h. Even without enrichment, Salmonella could be detected from both pork chop and sausage at 10(6) CFU/25 g within 1 day. This robust rt-RT-PCR detects and confirms Salmonella in pork within approximately 24 h and thus is significantly faster than traditional methods that take >/=1 week. This assay shows promise for routine testing and monitoring of Salmonella by the pork industry.
逆转录聚合酶链反应 (RT-PCR) 可检测 mRNA 的存在，并且比基于 DNA 的 PCR 检测方法更有潜力检测存活的病原体，与传统方法相比，具有更快的速度和更高的灵敏度。我们的目标是使用 SYBR Green I 实时 RT-PCR (rt-RT-PCR) 检测在两个 8 小时工作班次内从猪肉中快速灵敏地检测出肠炎沙门氏菌。猪排和香肠样品（25 克）用 10(8) 到 10(0) CFU 的肠炎沙门氏菌接种，并在 225 毫升四硫代盐肉汤中搅拌。将连续稀释液立即或在选择性预富集 10 小时后或预富集后 12 小时（对于应激细胞）在 37 度下进行选择性富集，在木糖赖氨酸 Tergitol 4 琼脂上进行平板划线，用于标准培养计数。使用 TRIzol 法提取 RNA。rt-RT-PCR 检测在 Bio-Rad iCycler 上使用 SYBR Green I 一步 RT-PCR 试剂盒和沙门氏菌特异性 invA 基因引物进行，带有内部扩增对照 (IAC)。PCR 后进行熔点 (T(m)) 分析，以确定特定的沙门氏菌 invA（T(m) = 87.5 摄氏度）和 IAC（T(m) = 82 摄氏度）产物。在富集 10 小时后，从猪肉和香肠中分别提高了高达 10(1) CFU/25 g 和 10(0) CFU/25 g 的沙门氏菌检测水平，大约在 24 小时内。即使没有富集，在 1 天内也可以从猪排和香肠中检测到 10(6) CFU/25 g 的沙门氏菌。这种强大的 rt-RT-PCR 在大约 24 小时内检测并确认猪肉中的沙门氏菌，因此比需要 >/=1 周的传统方法快得多。该检测方法有望用于猪肉行业的常规沙门氏菌检测和监测。
