Chieffi Daniele, Bongiorno Dafne, Licitra Anna, Campanile Floriana, Fusco Vincenzina
Institute of Sciences of Food Production, National Research Council of Italy (CNR-ISPA), 70126 Bari, Italy.
Medical Molecular Microbiology and Antibiotic Resistance Laboratory (MMARLab), Department of Biomedical and Biotechnological Sciences (BIOMETEC), University of Catania, 95123 Catania, Italy.
Foods. 2024 Sep 22;13(18):3004. doi: 10.3390/foods13183004.
(.) is a pathogenic bacterium able to cause several diseases in humans and animals as well as foodborne intoxications. , being phenotypically and genotypically related to , is part of the so-called complex and recently recognized as an emerging pathogen able to cause, like , several diseases both in humans and animals, and foodborne poisoning outbreaks. However, it has been reported that the widely used conventional PCR of Brakstad et al. [, (7), 1654-1660, (1992)] targeting the thermostable nuclease gene may provide false-positive , as it is able to amplify also . Here, we developed a novel two-step approach that, following the PCR of Brakstad et al. (1992), discriminates from by a real-time PCR with high-resolution melting analysis (rt-PCR-HRM). In particular, targeting a polymorphic 137 bp region of the gene, our developed rt-PCR-HRM method clearly discriminated from , showing a remarkable difference in their amplification product melting temperatures (approximately 1.3 °C) as well as distinct melting curve shapes. The good sensitivity, reproducibility, user friendliness, and cost effectiveness of the developed method are advantageous attributes that will allow not only its easy employment to correctly identify misidentified isolates present in various collections of but also expand the still lacking knowledge on the prevalence and distribution of .
（.）是一种病原菌，能够在人类和动物中引发多种疾病以及食源性中毒。（.）在表型和基因型上与（.）相关，是所谓的（.）复合体的一部分，最近被确认为一种新兴病原体，能够像（.）一样在人类和动物中引发多种疾病以及食源性中毒事件。然而，据报道，广泛使用的针对热稳定核酸酶基因的Brakstad等人[（.），（7），1654 - 1660，（1992）]的传统PCR可能会产生假阳性结果，因为它也能够扩增（.）。在此，我们开发了一种新颖的两步法，在Brakstad等人（1992）的PCR之后，通过高分辨率熔解分析实时PCR（rt-PCR-HRM）从（.）中区分出（.）。特别是，针对（.）基因的一个137 bp多态性区域，我们开发的rt-PCR-HRM方法能够清晰地将（.）与（.）区分开来，显示出它们扩增产物熔解温度有显著差异（约1.3°C）以及不同的熔解曲线形状。所开发方法具有良好的灵敏度、重现性、用户友好性和成本效益，这些优势不仅使其能够轻松用于正确鉴定存在于各种（.）菌株库中的错误鉴定菌株，还能扩展关于（.）的流行情况和分布的仍很欠缺的知识。
