Maurischat Sven, Szabo Istvan, Baumann Beatrice, Malorny Burkhard
Federal Institute for Risk Assessment, Unit Molecular Microbiology and Genome Analysis, National Salmonella Reference Laboratory, Max-Dohrn Str. 8-10, D-10589 Berlin, Germany.
Federal Institute for Risk Assessment, Unit Molecular Microbiology and Genome Analysis, National Salmonella Reference Laboratory, Max-Dohrn Str. 8-10, D-10589 Berlin, Germany.
J Microbiol Methods. 2015 May;112:92-8. doi: 10.1016/j.mimet.2015.03.015. Epub 2015 Mar 18.
Salmonella enterica serovar Enteritidis is a major non-typhoid Salmonella serovar causing human salmonellosis mainly associated with the consumption of poultry and products thereof. To reduce infections in poultry, S. Enteritidis live vaccine strains AviPro SALMONELLA VAC E and Salmovac SE/Gallivac SE have been licensed and used in several countries worldwide. To definitively diagnose a S. Enteritidis contamination in vaccinated herds a reliable and fast method for the differentiation between vaccine and wildtype field isolates is required. In this study, we developed and validated real-time PCR (qPCR) assays to distinguish those variants genetically. Suitable target sequences were identified by whole genome sequencing (WGS) using the Illumina MiSeq system. SNP regions in kdpA and nhaA proved to be most useful for differentiation of AviPro SALMONELLA VAC E and Salmovac SE/Gallivac SE, respectively, from wildtype strains. For each vaccine strain one TaqMan-qPCR assay and one alternative approach using High Resolution Melting (HRM) analysis was designed. All 30 Salmovac SE and 7 AviPro SALMONELLA VAC E vaccine strain reisolates tested were correctly identified by both approaches (100% inclusivity). Furthermore, all 137 (TaqMan) and 97 (HRM) Salmonella non-vaccine and related Enterobacteriaceae strains tested were excluded (100% exclusivity). The analytical detection limits were determined to be approx. 10(2) genome copies/reaction for the TaqMan and 10(4) genome copies/reaction for the HRM approach. The real-time PCR assays proved to be a reliable and fast alternative to the cultural vaccine strain identification tests helping decision makers in control measurements to take action within a shorter period of time.
肠炎沙门氏菌肠炎血清型是一种主要的非伤寒沙门氏菌血清型，可引起人类沙门氏菌病，主要与食用家禽及其产品有关。为减少家禽感染，肠炎沙门氏菌活疫苗株AviPro SALMONELLA VAC E和Salmovac SE/Gallivac SE已获得许可并在全球多个国家使用。为明确诊断接种疫苗鸡群中的肠炎沙门氏菌污染情况，需要一种可靠且快速的方法来区分疫苗株和野生型田间分离株。在本研究中，我们开发并验证了实时PCR（qPCR）检测方法，以从基因上区分这些变体。使用Illumina MiSeq系统通过全基因组测序（WGS）鉴定了合适的靶序列。结果证明，kdpA和nhaA中的单核苷酸多态性（SNP）区域分别对于区分AviPro SALMONELLA VAC E和Salmovac SE/Gallivac SE与野生型菌株最为有用。针对每种疫苗株，设计了一种TaqMan-qPCR检测方法和一种使用高分辨率熔解（HRM）分析的替代方法。两种方法均正确鉴定了所有测试的30株Salmovac SE和7株AviPro SALMONELLA VAC E疫苗株重分离株（100%的包容性）。此外，所有测试的137株（TaqMan）和97株（HRM）非疫苗沙门氏菌及相关肠杆菌科菌株均被排除（100%的排他性）。TaqMan检测方法的分析检测限约为10²基因组拷贝/反应，HRM方法的分析检测限约为10⁴基因组拷贝/反应。实时PCR检测方法被证明是一种可靠且快速的替代方法，可用于替代培养疫苗株鉴定试验，有助于控制措施中的决策者在更短时间内采取行动。
