Multiplex TaqMan Real-Time PCR (qPCR) Assay Targeting prot6E and invA Genes for Fast and Accurate Detection of Salmonella Enteritidis

Salmonella is an important foodborne pathogen causing significant public health concern, both domestically and internationally (Tirado and Schmidt, 2001; Scallan et al., 2011). According to the latest CDC report Salmonella infections affect millions of people every year accounting for 11%, 35% and 28%, of illnesses, hospitalizations and deaths, respectively of the total U.S. foodborne diseases caused by all known foodborne pathogens (Scallan et al., 2011). Among those non-typhoid salmonellosis, S. Enteritidis (SE) has emerged as a major egg-associated pathogen. SE transmission to humans has been linked mainly to consumption of contaminated foods containing undercooked eggs (Rabsch et al., 2000). Fresh shell-eggs can be contaminated easily with SE through cracks in the shell by contact with chicken feces or by transovarian infection (Snoeyenbos et al., 1969). Consequently, the increase of consumption of shell eggs and egg products per capita in the United States to approximately 249 eggs per year (American Egg Board, 2008) may have contributed, in part, to increases in foodborne outbreaks (Altekruse et al., 1997), including a large multistate SE outbreak of SE outbreak associated with eggs in the US in 2010.

. qPCR is faster, is more sensitive than conventional PCR, and provides real-time data avoiding the use of gels (Valasek and Repa, 2005). In particular, the invA gene represents a good candidate for Salmonella detection as it is present in all pathogenic serovars described to date (Rahn et al., 1992;Boyd EF et al., 1997). The product of this gene is essential for the organism's ability to invade mammalian cells and subsequently cause disease (Galan and Curtiss, III, 1991;Galan JE et al., 1992). In the case of SE in specific, several PCR and isothermal methodologies has also been developed targeting different genes (Seo et al., 2004;Malorny et al., 2007a;O'Regan et al., 2008;Hadjinicolaou et al., 2009). Although isothermal amplification techniques has some advantages over qPCR, such as increased detection limit and lower cost; still has the disadvantage that a single target can be used at a time and lacks internal control for monitoring possible inhibitors of the reaction that might exist in the food matrix analyzed.
In the present study we developed a fast and accurate qPCR assay for the specific detection of SE in eggs. This qPCR contained primers and probes to detect three different targets: the invA gene (Salmonella genus specific), the prot6E gene (SE specific), and the internal amplification control (IAC). A foreign internal amplification control (IAC) was incorporated into the assay with the aim of detecting potential inhibitors present in the matrix analyzed (eggs). Salmonella spp. detection in foods is usually achieved after food samples preenrichment approaches using overnight incubation (Feder et al., 2001). Consequently, this method described herein is intended as an initial screening of 24 h pre-enrichments for the presence of Salmonella in eggs. In turn, this method will dramatically decrease the time and effort required during standard microbiological testing, since only positive pre-enrichment samples will be processed further.

Preparation of SE inocula
C u l t u r e s o f i n d i v i d u a l S E s t r a i n s w e r e p r epared by transferring a loopful for three consecutive 24-h intervals to 10 ml of tryptic soy broth (TSB, Difco, Becton Dickinson) at 35 C. SE cells from an overnight broth culture were centrifuged at 3,000 x g for 15 min at 4 C. The pellet was washed twice with sterile 0.1% peptone water and re-suspended in sterile 0.1% peptone water. Serial dilutions of the suspension were prepared in sterile 0.1% peptone water to obtain the desired cell populations. The cell number in the inoculum was determined by plating 100µl dilutions (in sterile 0.1% peptone water) on TSA and incubating at 35 ºC for 24 h.

Microbiological assay
All eggs were purchased from local grocery stores in College Park, MD. Analysis of liquid eggs was performed by following USDA procedure with some modifications. Shell eggs were broken by hands aseptically into sterile glass beakers. They were mixed well with a sterile stick by hands for about 2 minutes until it looked uniform. These liquid eggs were inoculated (day 1) at around 5 SE or at > 10 4 cells in 100 g. Each 100 g sample was placed into a 2-liter sterile glass beaker, mixed with 900 ml pre-enrichment broth. Five preenrichment broths were used for testing performance of pre-enrichments for SE recovery. They were TSB, TSB plus ferrous sulfate (TSB + Fe), universal pre-enrichment broth (UPB), nutrient broth (NB), and buffered peptone water (BPW). After 24 hr (day 2) preenrichment, 1 ml of each pre-enriched sample was transferred to 10 ml of selective enrichment media (Rappaport-Vassiliadis (RV) medium and Tetrathionate broth (TT) (Difco) and incubated for 24 h at 42 ºC and 43 °C, respectively. On day 3, tube contents were vortexed for 10 sec, and 10 µl portions of the TT and RV media were streaked on bismuth sulfite (BS) agar, xylose lysine desoxycholate (XLD) agar, and Hektoen enteric (HE) agar and incubated at 35 °C for 24 h. On day 4, the plates were examined for the presence of typical Salmonella colonies. Typical colonies were confirmed with Salmonella agglutination test kit from BD.

Design of primers and standards for qPCR
All primers and probes (Table 5) employed in this study were purchased from IDT (Coralville, IA, USA). The targets for qPCR were invA gene and prot6E gene of SE. Primers and probes for invA assay were designed previously (Gonzalez-Escalona et al., 2009). Primers and probes for prot6E were designed using Beacon designer v.7 (PREMIER Biosoft, Palo Alto, CA). DNA from strain CHS44 was used to determine the prot6E/invA qPCR detection limit. DNA extraction was performed with the DNeasy kit as recommended by the manufacturer (QIAGEN). DNA concentration was determined using Qubit® 2.0 Fluorometer and Qubit™ dsDNA HS Assay Kit following manufacturer's instructions (Invitrogen). The numbers of copies of the qPCR standards were calculated by assuming average molecular masses of 680 Da for 1 nucleotide of double stranded DNA. The calculation was done with the following equation: copies per nanogram = (NL x 10-9)/ (n x mw), where n is the length of the SE strain P125109 complete genome (4,685,848 bp), mw is the molecular weight per nucleotide, and NL is Avogadro constant (6.02 x 1023 molecules per mol).  Table 5. Primers and probes employed in this study to detect prot6E/invA by qPCR. TX -Texas Red.

qPCR and data analysis
The qPCR reactions were carried out using the Platinum® Quantitative PCR SuperMix-UDG kit according to the specifications of the manufacturer (Invitrogen). This kit is a ready to use cocktail consisting of a 2X Reaction Mix (Platinum® Taq polymerase, 40 mM Tris-HCl, 100 mM KCl, 6 mM MgCl 2 , 0.4 mM of each dNTP, 0.8 of dUTP, uracil DNA glycosilase (UDG) and stabilizers). Reactions were scaled down to a final volume of 20 µl. Additional MgCl 2 was added to the master mix to a final concentration per tube reaction of 5 mM. Also additional Platinum® Taq polymerase was supplied in order to have 2.5 final units per reaction. Final concentrations of primers in the qPCR mix were 200 nM for invA and prot6E, and 100 nM for IAC, respectively. Probes were added to a final concentration of 150 nM. qPCR and data analysis was performed on a Mx3005P QPCR System (Agilent Technologies, Inc., Santa Clara, CA) real-time PCR machine. qPCR conditions were as follows: an initial cycle of 2 min at 50°C for UDG incubation, a second cycle of 2 min at 95°C to activate the hot-start Taq polymerase and 35 cycles of denaturation at 95°C for 15 secs, primer annealing and extension at 60°C for 30 secs (the acquisition of dyes Cy5, FAM and Texas Red were performed at the end of this cycle). Two microliters of DNA IAC (10 pg -3,0 * 10 5 copies/2µl) was added to each qPCR reaction.

Evaluation of the prot6E/invA multiplex qPCR TaqMan assay
The detection limit of the prot6E/invA qPCR was determined using 10-fold dilutions of DNA extracted from S. enterica Enteritidis strain CHS44. PCR primers specific for prot6E gene (prot6E-NGE-f and prot6E-NGE-r) and invA gene (invA_176F and invA_291R) were used ( Table 5). Linear calibration curves with a correlation coefficient (R 2 ) of  0.99 and linear ranges of  5 orders of magnitude for both prot6E and invA were obtained ( Fig. 1A and B). This corresponds to detection limits of about 40 genome copies for both prot6E and invA genes. The efficiency of the qPCR was  0.99 for both SE targets. The robustness of DNA IAC was observed for all dilutions tested (Fig. 1C). The inclusion of the DNA IAC (internal amplification control) did not affect amplification of either Salmonella gene target (Fig. 1C).

Performance assessment of different pre-enrichment media for the recovery of SE from eggs using prot6E/invA qPCR and USDA microbial culture methods
The usefulness of the qPCR assay developed in this study for detecting SE in eggs was assessed by artificial contamination of eggs with SE. One hundred grams of pooled eggs were artificially contaminated with two different SE strains (CDC-2010K_1543 and 13-2) at high (10 6 CFU/100 g) and low (<10 CFU/100 g) levels (Table 6). We further tested the performance of 5 different pre-enrichment media for SE growth (BPW, TSB, TSB+Fe, NB, and UP). After 24 h, the pre-enrichments were used for detection of SE using both prot6E/invA qPCR and USDA Salmonella culture method (Chapter MLG 4.05 -"Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg and Catfish Products"; http://www.fsis.usda.gov/PDF/MLG_4_05.pdf). Un-inoculated egg samples were used as negative controls. One milliliter of pre-enrichment was boiled and used for qPCR amplification in triplicate. All artificially contaminated egg samples were positive for Salmonella using both prot6E/invA qPCR and the USDA methodologies (Table 6). We chose to show in the table only lower inoculation levels in order to highlight the sensitivity of this qPCR method. SE levels as low as 5 CFU/100 g were detected after 24 ± 2 h preenrichments. All pre-enrichment media showed fairly similar performances for SE recovery, save for NB which showed less growth after 24h, with SE levels 10-fold lower than other www.intechopen.com Please use Adobe Acrobat Reader to read this book chapter for free.
Just open this same document with Adobe Reader. If you do not have it, you can download it here. You can freely access the chapter at the Web Viewer here. media (Table 6). Absence of qPCR inhibitors was demonstrated by amplification of the IAC since IAC would not have been amplified had there been PCR inhibitors present in the samples analyzed (Table 6). Fig. 1. Detection limit of the multiplex qPCR assay. Calibration curves were generated using 10-fold dilutions of CHS44 DNA (three replicates). A) Average prot6E amplification from dilutions 4.0*e10 6 -40 CHS44 genome copies/reaction tube (FAM channel). B) Average invA amplification from dilutions 4.0*e10 6 -40 CHS44 genome copies/reaction tube (ROX channel). C) Concurrent IAC amplification for each dilution (Cy5 channel). The Cq values were plotted against the nucleic acid target concentration (as copies per reaction for both DNA). The reaction efficiency (E) and R 2 values are also shown. Cq -Cycle quantification threshold, where the fluorescent is higher than the background. + = Salmonella positive by the method. In the case of IAC stands for positive signal for IAC. Table 6. Pre-enrichment medium assessment for Salmonella Enteritidis (SE) recovery and detection using prot6E/invA multiplex qPCR and USDA culture method in pooled eggs artificially contaminated. Cq values are given in parentheses.

Application of the prot6E/invA qPCR assay for Salmonella Enteritidis detection in eggs artificially contaminated with different SE strains
After determining the effectiveness of the different pre-enrichment medias, we decided to employ TSB media as pre-enrichment for testing artificially contaminated eggs with 9 additional SE strains ( Table 7). One of these strains lacked prot6E gene (SE-10). Eighteen pooled egg samples (100 g each) were artificially contaminated with high (~ 10 6 CFU/100 g) and low (<10 CFU/100 g) levels of SE and were analyzed as mentioned previously. After 24h, all artificially contaminated egg pre-enrichments were used for detection of SE using both prot6E/invA qPCR and USDA Salmonella culture method. Un-inoculated egg samples resulted in negative results by both prot6E/invA qPCR and USDA Salmonella culture method. On the other hand, all artificially contaminated egg samples were positive for www.intechopen.com Please use Adobe Acrobat Reader to read this book chapter for free. Just open this same document with Adobe Reader. If you do not have it, you can download it here. You can freely access the chapter at the Web Viewer here.
Salmonella using both prot6E/invA qPCR and the USDA methodologies except for SE-10 strain which was negative for prot6E (Table 7). Contrary to what is shown in Table 6, we showed the results of both high and low inoculation levels Table 7. SE levels as low as 2 CFU/100 g were detected after 24 ± 2 h pre-enrichments. As mentioned previously for the pre-enrichment media, the absence of qPCR inhibitors was demonstrated by observing no inhibition of the amplification of the IAC in every sample (Table 7)

Discussion
This study reports the development of a multiplex qPCR TaqMan assay that allowed for the fast and accurate detection of SE cells from eggs. The assay performed comparably to the traditional SE culture methods described in Chapter MLG 4.05 (USDA) for the detection of SE from meat, poultry, pasteurized egg and catfish products. The overall analysis took roughly 24 h, in contrast to the 5 days to 2 weeks that traditional microbiological culture methods often take. It is noteworthy that agreement between the qPCR and the two microbial culture methods was 100% for all artificially spiked samples.
This novel quantitative real-time PCR (qPCR) assay uses specific primers for the detection of prot6E and invA genes of SE with TaqMan probes. This assay also includes an internal amplification control (IAC) to detect potential PCR inhibitors that may be present in egg samples. It has become increasingly evident that there is a need for internal controls for PCR reaction, to rule out the presence of PCR inhibitors that can cause false negative results for Salmonella-positive samples (Hartman et al., 2005;Hoorfar et al., 2004). The inclusion of this internal control did not affect either the amplification or the detection limit of the qPCR assay. The qPCR developed here as opposed to an invA single target qPCR method (Feder et al., 2001;Malorny et al., 2004;Bohaychuk et al., 2007;Gonzalez-Escalona et al., 2009) is able to detect specifically SE strains by the use of an SE specific marker (prot6E). Additionally it is capable to detect other SE that might lack the prot6E gene (Malorny et al., 2007a), such as the case for SE-10. The lack of prot6E in SE strains has been co-related with the absence of the SE virulence plasmid (~ 55 kb) (Malorny et al., 2007a). Due to the importance of that plasmid in SE virulence (Bakshi et al., 2003), without it SE has a diminish virulence, and such could be the case for SE-10 which was isolated from chicken. Moreover this assay has a further advantage in that it is an open formula assay, whereby no primers, no probes or IAC are patented or proprietary.
Among the most common gene targets used for SE detection by qPCR are: 1) Sdf1, a chromosomal fragment (Agron et al., 2001); 2) sefA, encoding for fimbrial antigen SEF14 (Seo et al., 2004); and 3) prot6E, encoding for a unique surface fimbriae (Malorny et al., 2007a;Clavijo et al., 2006). Sdf1 is highly specific for SE but is missing in SE phage types (PT) 6A, 9A, 11, 16, 20, and 27 and besides that were only tested on pure cultures (Malorny et al., 2007a). SefA gene is also present in all members of S. enterica serogroup D (Gallinarum, Pollorum, Dublin, Rostock, and Typhi, among others) which might lead to false positives results (Seo et al., 2004;Malorny et al., 2007a) and therefore it is not recommended for specific identification of SE. prot6E is present in the SE 60 kb virulence plasmid, which is present in most SE (>90%) (Chu et al., 1999;Helmuth and Schroeter, 1994;Clavijo et al., 2006) and therefore was our target of choice for SE specific detection by qPCR.
The detection limit of this qPCR assay was ~ 40 copies of genomic DNA. Usually 1 ml of pre-enrichment is boiled and 2 µl of supernatant is used for qPCR reaction. Thus, a population of approximately 4*10 4 CFU/ml needs to be reached in the pre-enrichment to render a positive result. Commonly SE levels reach ~ 10 8 CFU/ml in the pre-enriched cultures. Therefore, this assay could be used for identification and/or quantification of SE cells in foods directly after pre-enrichment. It is also important to note, however, that in non-host environments, Salmonella persists most likely in a starved and highly stressed state. However, the addition of a requisite pre-enrichment step in culture media substantially increases cell number. Thus, a pre-enrichment culture provides an essential preliminary step in the application of this assay to the reliable detection of SE from eggs.

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Rather than performing replicates of several inoculations with the same strain, we opted to spike the eggs in Table 7, with 9 different SE strains. This provided, in our opinion, a more powerful approach than simply repeating the experiment with the same strain multiple times as other investigators usually do. The ultimate goal of the assay is to detect different SE strains. Thus increasing the bio-complexity of testing provided a more thorough and rigorous challenge to the capability of the qPCR method to detect SE, in general. The IAC amplification was not affected in all the samples tested, however a possible failure in samples containing high levels of SE could be expected. That sort of possible failure is not un-expected given the competitive nature of this qPCR reaction, where primers and probes for Salmonella two targets are in excess. Thereby favoring SE targets instead of the DNA IAC. Nevertheless, it is important to emphasize that performance of the IAC in the presence of low DNA copy numbers or in the observed absence of SE was robust and reliable for each food sample analyzed, an imperative finding for any Salmonella detection qPCR assay (Malorny et al., 2007b).
In addition to being both effortless and reproducible, the use of ready to use mixtures, such as the one used in this study, facilitate performance of the assay. Likewise, conventional PCR methods are incapable of producing products with known identity (i.e. DNA sequence), subsequently failing to ensure proper specificity of PCR product(s) (Rahn et al., 1992;. Additionally, we employed TaqMan probes for our qPCR assay which had several advantages over the use of nonspecific (although cheaper) SYBR Green I assays, including greater sensitivity and a probe-based sequence-specific verification of PCR product identity (Wittwer et al., 1997;Fey et al., 2004;Jacobsen and Holben, 2007).
In conclusion, we have developed a method that has the potential to be used as an initial screen for pre-enrichment cultures for SE without precluding the USDA culture method which is deemed necessary to yield a physical isolate that is acceptable to the regulatory process. This assay showed a high selectivity, accuracy and detection capacity. In addition, we believe that this assay will reduce the amount of samples, overall time, and effort expended in the laboratory since only positive samples will be further processed after the initial pre-enrichment step. As an added benefit, this is also a quantitative assay which allows for SE quantification in pre-enrichments or other samples. Last but certainly not least, the inclusion of the IAC makes it useful for rapid diagnosis of SE in foods directly. Moreover, in order to be applied extensively, collaborative studies should be conducted to assess the inter-laboratory reproducibility of this assay.

Acknowledgement
This project was supported by the FDA Foods Program Intramural Funds.

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