Dept. of Food Science and Technology, Univ. of Tennessee, Knoxville, TN 37996, USA.
J Food Sci. 2010 Apr;75(3):M165-72. doi: 10.1111/j.1750-3841.2010.01554.x.
Loop-mediated isothermal amplification (LAMP) is a novel molecular detection method that is more rapid and simpler than PCR. Products can be detected by turbidity using one temperature without the need for expensive PCR equipment. Our objective was to sensitively detect Salmonella Typhimurium from pork products within 1 d using the LAMP assay. Pork chop and pork sausage samples (25 g) were inoculated with high (10(8) to 10(6) CFU) and low (10(5) to 10(0) CFU) inocula of S. Typhimurium. Serial dilutions in phosphate buffered saline were plated on XLT4 agar either immediately or after selective preenrichment in tetrathionate broth (225 mL) for 10-h at 37 degrees C. Nucleic acid was extracted using the TRIzol method from 1-mL samples. The LAMP assay using 6 specific invA gene primers and Bst DNA polymerase reaction mix was carried out at 62 degrees C for 90 min in a waterbath. Turbid products were detected visually and by agarose gel electrophoresis. Improved Salmonella detection at 10(2) CFU/25 g for both pork chop and sausage was obtained after 10-h enrichment and 10(6) CFU/25 g without enrichment for both products. This assay can detect Salmonella from pork within 1 d, significantly faster than traditional methods that take >5 d. This method shows tremendous potential for routine diagnostics and monitoring of Salmonella by the pork industry.
The novel loop-mediated isothermal amplification (LAMP) assay is a rapid, specific, and sensitive method that has potential application for routine diagnostics of Salmonella from pork products. The isothermal method does not require expensive equipment such as a PCR thermocycler but only a simple waterbath for amplification within 90 min. Detection is even simpler by visual eye or turbidimeters that are less expensive than fluorescent spectrophotometers or real-time PCR machines. All these advantages make it a practical approach for routine use by processing industries to rapidly detect Salmonella in their environment and to implement appropriate control strategies. To improve detection sensitivities, preenrichment followed by selective enrichment may be necessary. Even so, the entire assay can be completed at the most within two 8-h working shifts.
环介导等温扩增(LAMP)是一种新型的分子检测方法,比 PCR 更快、更简单。产物可以通过浊度检测,在一个温度下进行,无需昂贵的 PCR 设备。我们的目的是使用 LAMP 法在 1 天内从猪肉产品中灵敏地检测出肠炎沙门氏菌。猪排和猪肉香肠样品(25 g)用高(10(8)至 10(6)CFU)和低(10(5)至 10(0)CFU)接种肠炎沙门氏菌。在磷酸盐缓冲盐水(PBS)中进行系列稀释,立即或在 37°C 下选择性预富集 10 小时后,在 XLT4 琼脂上平板。使用 TRIzol 法从 1 mL 样品中提取核酸。使用 6 个特定的 invA 基因引物和 Bst DNA 聚合酶反应混合物的 LAMP 检测在水浴中于 62°C 下进行 90 分钟。浊度产物通过肉眼和琼脂糖凝胶电泳检测。经过 10 小时富集,猪排和香肠的沙门氏菌检测灵敏度均提高到 10(2)CFU/25 g,而无需富集,两种产品均为 10(6)CFU/25 g。该方法可在 1 天内从猪肉中检测到沙门氏菌,比传统方法快 5 天以上。这种方法为猪肉行业常规诊断和监测沙门氏菌提供了巨大的潜力。
新型环介导等温扩增(LAMP)检测方法快速、特异、敏感,有潜力用于常规诊断猪肉产品中的沙门氏菌。等温方法不需要昂贵的设备,如 PCR 热循环仪,只需一个简单的水浴即可在 90 分钟内扩增。通过比荧光分光光度计或实时 PCR 机更便宜的浊度计或肉眼检测,检测甚至更简单。所有这些优势使其成为加工行业的一种实用方法,可以快速检测其环境中的沙门氏菌,并实施适当的控制策略。为了提高检测灵敏度,可能需要预富集,然后进行选择性富集。即使如此,整个检测过程最多可在两个 8 小时的工作班次内完成。