Xiong Dan, Zhou Yi, Song Li, Liu Bowen, Matchawe Chelea, Chen Xiang, Pelle Roger, Jiao Xinan, Pan Zhiming
Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou 225009, China.
Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China.
Foods. 2022 Mar 3;11(5):742. doi: 10.3390/foods11050742.
Salmonella enteritidis is a major causative agent of foodborne illnesses worldwide. As the traditional serotyping and quantification methods are labor-intensive, time-consuming, and expensive, faster and more convenient molecular diagnostic methods are needed. In this study, we developed and validated a rapid duplex TaqMan real-time polymerase chain reaction (PCR) for the accurate identification and quantification of S. enteritidis. The primers and TaqMan probes were designed based on the S. enteritidis-specific gene lygD and the Salmonella genus-specific gene invA. The melt curve and gel electrophoresis analysis showed that the designed primers had potent specificity for the amplification of lygD and invA. The duplex real-time PCR specifically identified S. enteritidis from a panel of 40 Salmonella strains that represented 29 serovars and 12 non-Salmonella organisms. The duplex real-time PCR assay detected four copies of S. enteritidis DNA per reaction. The intra- and inter- assays indicated a high degree of reproducibility. The real-time PCR could accurately detect and quantify S. enteritidis in chicken organs after Salmonella infection. Furthermore, the assay identified 100% of the S. enteritidis and Salmonella genus isolates from chicken egg samples with superior sensitivity after 6 h of pre-enrichment compared to the traditional culture method. Additionally, the most-probable-number (MPN) combined with qPCR and a shortened incubation time (MPN-qPCR-SIT) method was developed for the population determination of S. enteritidis and compared with various enumeration methods. Thus, we have established and validated a new duplex real-time PCR assay and MPN-qPCR-SIT method for the accurate detection and quantification of S. enteritidis, which could contribute to meeting the need for fast detection and identification in prevention and control measures for food safety.
肠炎沙门氏菌是全球食源性疾病的主要病原体。由于传统的血清分型和定量方法 labor-intensive、耗时且昂贵，因此需要更快、更便捷的分子诊断方法。在本研究中，我们开发并验证了一种快速双重 TaqMan 实时聚合酶链反应（PCR），用于准确鉴定和定量肠炎沙门氏菌。引物和 TaqMan 探针是基于肠炎沙门氏菌特异性基因lygD和沙门氏菌属特异性基因invA设计的。熔解曲线和凝胶电泳分析表明，设计的引物对lygD和invA的扩增具有很强的特异性。双重实时 PCR 从一组代表 29 个血清型的 40 株沙门氏菌菌株和 12 种非沙门氏菌生物体中特异性鉴定出肠炎沙门氏菌。双重实时 PCR 检测每个反应中肠炎沙门氏菌 DNA 的四个拷贝。批内和批间分析表明具有高度的重现性。实时 PCR 能够在沙门氏菌感染后准确检测和定量鸡器官中的肠炎沙门氏菌。此外，与传统培养方法相比，该检测方法在预富集 6 小时后能以更高的灵敏度从鸡蛋样本中鉴定出 100%的肠炎沙门氏菌和沙门氏菌属分离株。此外，还开发了最可能数（MPN）与 qPCR 相结合以及缩短孵育时间的方法（MPN-qPCR-SIT）用于肠炎沙门氏菌的种群测定，并与各种计数方法进行了比较。因此，我们建立并验证了一种新的双重实时 PCR 检测方法和 MPN-qPCR-SIT 方法，用于准确检测和定量肠炎沙门氏菌，这有助于满足食品安全预防和控制措施中快速检测和鉴定的需求。
