Srisawat Wimvipa, Saengthongpinit Chalermkiat, Nuchchanart Wirawan
Department of Animal Science, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Nakhon Pathom 73140, Thailand.
Department of Veterinary Public Health, Faculty of Veterinary Medicine, Kasetsart University, Nakhon Pathom 73140, Thailand.
Vet World. 2022 Mar;15(3):590-601. doi: 10.14202/vetworld.2022.590-601. Epub 2022 Mar 12.
is a critical foodborne pathogen that infects pregnant females and their newborns and older adults and individuals with comorbidities. It contaminates fresh vegetables, fruits, ready-to-eat foods, and frozen food products consumed by individuals. The culture conventional detection methods for are time-consuming, taking 4 days. This study aimed to describe the development and comparison of loop-mediated isothermal amplification (LAMP)- lateral flow dipstick (LFD), LAMP assay to PCR, and conventional culture for detecting in frozen food products.
Five LAMP primer sets, including F3, B3, forward inner primer, and backward inner primer, were designed from a specific region on ferrous iron transport protein B gene ( gene) to amplify LAMP products. The DNA probe was created, and the detection limit was determined in pure culture and purified DNA, as well as the detection in 20 frozen food product samples.
The LAMP primer sets and DNA probe were LAMP products amplified at 60°C for 50 min. The specificity of the assay revealed no cross-reactivity with other pathogenic bacteria. The limit of detection (LOD) of the LAMP-LFD and LAMP assays using purified genomic DNA was 219 fg/μL both in LAMP and LAMP-LFD assays. The LOD of LAMP and LAMP-LFD assays in pure culture was 4.3×10 colony-forming unit (CFU)/mL and 43 CFU/mL, respectively. The LOD of the LAMP-LFD assay using artificially inoculated chicken in frozen food samples with pre-enrichment was 3.2×10 CFU/mL. The LAMP-LFD was also more sensitive than the LAMP assay and polymerase chain reaction. Finally, LAMP-LFD revealed no false positives in any of the 20 frozen food product samples.
LAMP-LFD assay using a specific region on the gene to detect was highly specific, sensitive, faster, and convenient, making it a valuable tool for the monitoring and rapid screening of in frozen food products. This technique is applicable to the development of detection technologies for other pathogens in food products.
[病原体名称]是一种重要的食源性病原体，可感染孕妇及其新生儿、老年人以及患有合并症的个体。它会污染人们食用的新鲜蔬菜、水果、即食食品和冷冻食品。针对[病原体名称]的传统培养检测方法耗时较长，需要4天。本研究旨在描述环介导等温扩增（LAMP）-侧向流动试纸条（LFD）、LAMP检测法与聚合酶链反应（PCR）以及传统培养法在冷冻食品中检测[病原体名称]的方法开发与比较。
从亚铁转运蛋白B基因（[基因名称]）的特定区域设计了五组LAMP引物，包括F3、B3、正向内引物和反向内引物，用于扩增LAMP产物。制备了DNA探针，并在纯培养物和纯化DNA中确定了检测限，以及在20份冷冻食品样品中的检测情况。
五组LAMP引物和DNA探针在60°C下扩增50分钟得到LAMP产物。该检测方法的特异性显示与其他病原菌无交叉反应。使用纯化基因组DNA时，LAMP-LFD和LAMP检测法的检测限在LAMP和LAMP-LFD检测中均为219 fg/μL。在纯培养物中，LAMP和LAMP-LFD检测法的检测限分别为4.3×10菌落形成单位（CFU）/mL和43 CFU/mL。在预富集的冷冻食品样品中，使用人工接种鸡肉的LAMP-LFD检测法的检测限为3.2×10 CFU/mL。LAMP-LFD也比LAMP检测法和聚合酶链反应更灵敏。最后，LAMP-LFD在20份冷冻食品样品中均未出现假阳性。
利用[基因名称]特定区域的LAMP-LFD检测法检测[病原体名称]具有高度特异性、灵敏性、快速且便捷，使其成为冷冻食品中[病原体名称]监测和快速筛查的有价值工具。该技术适用于食品中其他病原体检测技术的开发。
