In this study we propose to develop a novel real-time multiplex-PCR (mRT-PCR) assay for the rapid and accurate serotype-specific detection of Salmonella Enteritidis (SE) from culture, environmental drag swabs and eggs. <P>We propose following specific aims: 1) Development of a mRT-PCR assay for the serotype-specific detection of SE and determination of selectivity and detection limit of mRT-PCR. 2) Determination of sensitivity and specificity of mRT-PCR assay in artificially and potentially naturally contaminated samples.
Salmonella infections are among the leading bacterial cause of foodborne illness in the United States, with shell eggs being the primary source in human infections. Salmonella Enteritidis (SE) is the most common serotype isolated from humans. Because of the public health concerns, the US Food and Drug Administration (FDA) require all poultry producers to test for SE infection in the flocks. Traditional detection methods for SE include isolation in culture and detection by biochemical testing and serotyping which is done manually by tube agglutination. Conventional culture methods are time consuming, and serotyping is only available at a few laboratories. Serotyping of SE may be particularly troublesome for some isolates that may loose the ability to express the antigens used for the. Few PCR assays have been developed and validated for the serogroup-specific identification of Salmonella isolates for routine diagnostics applications. While these PCR assays are considered as a good screening tool, they lack diagnostic specificity due to non-SE-specific targets used for detection. Therefore the positive results obtained by these PCR assays require further confirmation by conventional testing which is time consuming and labor intensive. Few PCR assays for serotype-specific detection of SE have also been developed; however none of these methods are approved by the federal agencies for the routine definitive identification of the SE isolates from culture and/or environmental and food specimen. In this study we propose to develop a novel real-time multiplex-PCR (mRT-PCR) assay for the rapid and accurate serotype-specific detection of Salmonella Enteritidis (SE) from culture, environmental drag swabs and eggs. We propose to 1) develop a mRT-PCR assay for the serotype-specific detection of SE and determination of selectivity and detection limit of mRT-PCR, and 2) determine the sensitivity and specificity of mRT-PCR assay in artificially and potentially naturally contaminated samples. The PCR we propose is unique because we will target three genetic markers; invA (Salmonella genus-specific), sdfI (SE-specific chromosomal gene) and prot6e (SE-specific gene located on virulence plasmid pSEV) which will allow us to unambiguously and rapidly detect all the field strains SE from routinely submitted samples. We expect that this would provide simple and rapid alternative serotype-specific detection method that could be used by most microbiologic and diagnostic laboratories. While this study is aimed towards development and preliminary testing, our long term goal is to conduct a multi-laboratory validation of this test and seek approval from FDA from routine use. In addition five other national laboratories have agreed to collaborate for validation studies. Funding for such trials will be sought from the USDA or USPEA. If successful there is also potential to include a Salmonella group D- specific gene target, sefA, which would increase the scope of this test for not only layer operations under the FDA regulation, but also in breeder operations under the National Poultry Improvement Plan.
Aim 1: For selectivity tests, Salmonella or non-Salmonella strains will be grown aerobically with gentle shaking at 37C for 18-20 h in LB medium. A 1 ml aliquot will be centrifuged, the pellet will be resuspended and suspension will be incubated at 56C for 15 min. An aliqot from this suspension will be diluted 1:10 in Tris HCl and an aliquot of this dilution will be used as a template in each reaction. The limit of detection of the mRT-PCR will be determined using gDNA of the reference strains UK (plasmid bearing) and G45 (non-plasmid bearing) purified using DNeasy Tissue kit (Qiagen). Salmonella copy numbers will be calculated based on the SE genome size (4.67 Mb) using a copy number calculator. A 10-fold dilution of gDNA from each strain will be prepared and tested in a range of approximately 1 to 106 genome equivalents per PCR. Three independent experiments will be performed for each target gene and for a combination of all targets. The line for calculating CT values for each channel will be assigned to a fixed value intersecting the amplification curves. The lowest dilution of gDNA that results in positive reaction for all the replicates tested will be considered as the detection sensitivity of the assay.
Aim 2: Environmental samples and pooled egg contents will be used. These samples will be artificially inoculated with two strains indicated above (UK and G45). For method comparison, each sample will be tested according to the FDA approved methods and in parallel to our proposed model. For determination of diagnostic sensitivity, five drag swabs and five egg samples will be artificially inoculated at five levels (0, 1-3, 4-10, 11-100 and 101-500 cfu/25g) per drag swab. For the mRT-PCR, the samples will be immersed into tetrathionate broth (TT) at 1:10 ratio and processed. At each time point, 1 ml of primary enrichment (TT) and secondary enrichment (MSRV) broth will be collected and processed for gDNA extraction using QIAamp DNA Mini Kit (Qiagen). Serial 10-fold dilutions of the TT broth and MSRV media will also be inoculated on XLT4 and BGN agar to determine the CFU and the results will be noted as positive (with CFU/ml) or negative (if no growth is reported). The gDNA extracted from broth will be subject to mRT-PCR as described in specific aim 1. Each sample will be tested in triplicates. CT values of the each sample will be determined and the results (positive or negative) will be compared with the results of culture isolation (positive or negative). Diagnostic specificity of the assay will be tested using similar methods with the exception that each sample will be artificially inoculated with a mixture of non-SE strains belonging to family Enterobacteriaceae with or without SE. These samples will serve as potentially naturally contaminated samples and our expectation is that the mRT-PCR assays developed in this study will specifically detect SE from such samples. In order to determine the diagnostic sensitivity and specificity, the results of the standard culture method of isolated reference strains will be compared with those of the mRT-PCR using previously described 2x2 box analysis.