Loop-Mediated Isothermal Amplification-Based Workflow for the Detection and Serotyping of Salmonella spp. in Environmental Poultry Flock Samples

Citation: Regal, P.; Doval, A.; García-Ramos, I.; Cepeda, A.; Garrido-Maestu, A.; Lamas, A. Loop-Mediated Isothermal Amplification-Based Workflow for the Detection and Serotyping of Salmonella spp. in Environmental Poultry Flock Samples. Foods 2024,13, 4069. https://doi.org/10.3390/ foods13244069 Academic Editor: Ewen C. D. Todd Received: 18 November 2024 Revised: 4 December 2024 Accepted: 13 December 2024 Published: 17 December 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Article Loop-Mediated Isothermal Amplification-Based Workflow for the Detection and Serotyping of Salmonella spp. in Environmental Poultry Flock Samples Patricia Regal 1, Anne Doval 1, Iria García-Ramos 1, Alberto Cepeda 1, Alejandro Garrido-Maestu 2,* and Alexandre Lamas 1,* 1Food Hygiene, Inspection and Control Laboratory (LHICA-USC), Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Veterinary Science, Campus Terra, Universidade de Santiago de Compostela (USC), 27002 Lugo, Spain; [email protected] (P.R.); [email protected] (A.D.); [email protected] (I.G.-R.); [email protected] (A.C.) 2Laboratory of Microbiology and Technology of Marine Products (MicroTEC), Instituto de Investigaciones Marinas (IIM—CSIC), Eduardo Cabello, 6, 36208 Vigo, Spain *Correspondence: [email protected] (A.G.-M.); [email protected] (A.L.) Abstract: Salmonella spp. is one of the most important foodborne pathogens worldwide. Given the fact that poultry and poultry products are the main source of human infection, Salmonella control in these farms is of utmost importance. To better control this pathogen in farms, boot swabs are used to sample farm environments but the analysis of these swabs is mainly based on culture-dependent methods. In the present study, a novel loop-mediated isothermal amplification (LAMP) method was developed for the rapid screening of Salmonella spp. in boot swab samples from broiler flock environments. Four different DNA extraction protocols were evaluated in depth, including a simple thermal lysis, a chelex-based protocol and two thermal lysis protocols followed by the purification of magnetic beads made of silica (“glass milk”) in order to determine the most suitable alternative for potential on-site, farm analyses. The methodology evaluation included a blind interlaboratory assay and as a proof-of-concept, a naked-eye colorimetric assay was also included. Following the final methodology, it was possible to reach an LoD 50 of 1.8 CFU/25 g of the samples, with a high relative sensitivity (95.7%), specificity (100%) and accuracy (96.6%) along with Cohen’s kappa of concordance with respect to the ISO standard 6579-1:2017 of 0.9, with an RLOD of 1.3. In addition to this, due to the relevance of certain serotypes with the genus Salmonella spp., a serotype LAMP panel for the specific identification of S. Typhimurium, S. Enteritidis, S. Infantis, S. Hadar and S. Virchow was also developed. Even though some degree of cross-reactivity among the primers developed was observed, all the serotypes could be accurately identified based on their melt curve analysis profile. Taken together, in the present study, a rapid Salmonella spp. screening method, suitable for farm applications, was developed, along with a serotyping panel that could be used in a laboratory setup for the identification of the most relevant serotypes of the genus, taking advantage of real-time amplification followed by melt curve analysis. Keywords: loop-mediated isothermal amplification; Salmonella spp.; S. Typhimurium; S. Enteritidis; S. Infantis; S. Hadar; S. Virchow; poultry; environment; farm 1. Introduction The poultry market, through the production of meat and eggs, is one of the most important sectors worldwide due to its influence in global markets [ 1 ]. Additionally, this industry plays a crucial role in maintaining food safety. Poultry products are one of the main sources of Salmonella spp. in the food chain. The control of this pathogen is carried out from primary production to retail markets. Based on the European Union Regulation (EC) No 2160/2003 [ 2 ], member states have developed their national plans for Salmonella control Foods 2024,13, 4069. https://doi.org/10.3390/foods13244069 https://www.mdpi.com/journal/foods Foods 2024,13, 4069 2 of 17 in poultry production. These plans include environmental self-monitoring sampling by the operator to determine the presence of Salmonella spp. in farms. One sampling method, exclusively used in the case of broilers, involves walking inside the breeding facility with boot swabs, which are later analyzed for the presence of Salmonella spp. [ 3 ]. This procedure has been reported to be the most sensitive and cost-effective approach for the detection of Salmonella spp. in farms [ 4 , 5 ]. In addition to the genus, certain serotypes are considered particularly relevant for human health, namely S. Typhimurium, S. Enteritidis, S. Infantis, S. Virchow, and S. Hadar [6]. The ISO 6579-1:2017 is the reference method for Salmonella detection in the food chain [ 7 ]. This standard applies to food and environmental control in the food industry, as well as environmental samples from primary production. The method is based on classical microbiology, involving two enrichment steps, selective media seeding, and confirmation through biochemical methods. A minimum of three days is needed to classify a sample as negative and a minimum of five days to determine if a sample is positive. These turnaround times are further extended due to serotyping using antisera. This delays the production system, and may pose a challenge for an industry operating on narrow economic margins. Alternative methods for the detection of Salmonella spp., such as immunoassays like the VIDAS or molecular methods, have been developed in recent decades [ 8 – 10 ]. Molecular methods, particularly real-time PCR (qPCR), have gained importance in pathogen detection in the food chain as they allowed us to overcome the limitations of classical, culture-based methods [ 11 , 12 ]. While these methods still require a prior enrichment step, the times are significantly shortened, and results can be obtained within 24 h. Although qPCR is the gold standard among molecular amplification methods, it has some limitations, primarily requiring relatively complex equipment capable of temperature ramping and fluorescence detection [13,14]. In recent years, a series of molecular isothermal amplification techniques, such as Recombinase Polymerase Amplification (RPA) or loop-mediated isothermal amplification (LAMP), have emerged [ 15 , 16 ]. In these techniques, amplification occurs at a constant temperature in a simple thermal block or water bath. Moreover, these techniques support the introduction of chemicals that enable visual detection without the need for fluorescence measurement [17,18]. The current study presented two main goals. The first was to develop a LAMP-based method to detect the presence of Salmonella in environmental samples from poultry farms in less than 24 h, along with a proof of concept of a low-cost, colorimetric format for future laboratory assay decentralization. The second goal consisted of a subsequent step to be performed on Salmonella-positive samples, and was focused on a panel of independent LAMP assays for rapid identification of the presence of some of the most relevant serotypes for the poultry industry, namely S. Typhimurium, S. Enteritidis, S. Infantis, S. Virchow, and S. Hadar. 2. Materials and Methods 2.1. Bacterial Strains and Culture Preparation All bacterial strains used in the present work are listed in Table 1. Specifically, Salmonella Enteritidis WDCM 00030 was selected as the reference strain for the development and optimization of the LAMP assays for the detection of Salmonella spp., as well as the S. Enteritidis serotype-specific LAMP. S. Virchow LHICA C11/22, S. Infantis LHICA C12/20, S. Hadar LHICA C11/21, and S. Typhimurium WDCM 00031 were used to develop the corresponding serotype-specific LAMP assays. Additional Salmonella serotypes, as well as non-Salmonella bacterial species, were used for inclusivity/exclusivity assays. All strains were conserved in cryovials at − 20 ◦ C. The preparation of fresh cultures was performed by transferring one cryoball to a 25 mL flask with 10 mL of Brain Heart Infusion (BHI, Merck Millipore, Burlington, MA, USA) and incubated with agitation (150 rpm) at 37 ◦ C during 18 h. Upon completion, the strains were plated in nutrient agar (NA, VWR, Barcelona, Spain) and incubated at 37 ◦ C for 24 h. Finally, the plates were conserved and Foods 2024,13, 4069 3 of 17 4 ◦ C until use. For spiking assays, Salmonella strains were grown as described and the culture was serially diluted and samples inoculated with different volumes of different dilutions. Also, dilutions were plated in NA to determine bacterial concentration. Buffered Peptone Water (BPW, Merck Millipore, Darmstadt, Germany) was used for sample enrichment. Semisolid Rappaport Vassiliadis medium (MRVS, DifcoTM, BD, Madrid, Spain) was used for Salmonella selective enrichment. Xylose Lysine Deoxycholate agar (XLD, Thermo Scientific, Oxoid) and RAPID’Salmonella agar (Bio-Rad, Hercules, CA, USA) were used for Salmonella isolation. Media were used according to ISO 6579-1:2017 [19]. Table 1. Bacterial strains for inclusivity and exclusivity tests. Strain Strain Target ttr STM4497 safA Group_29846 Group_21126 Group_27174 S. Typhimurium WDCM 00031 + + - - - - C13/22 + + - - - - C3/21 + + - - - - C4/20 + + - - - - C4/22 + + - - - - C7/21 + + - - - - C9/20 + + - - - - S. Typhimurium monophasi C15/22 + + - - - - C9/21 + + - - - - C9/22 + + - - - - C7/20 + + - - - - S. Enteritidis C1/20 + - + - - - C1/21 + - + - - - C10/20 + - + - - - C10/21 + - + - - - C2/22 + - + - - - C7/22 + - + - - - S. Hadar C11/21 + - - + - - C2/20 + - - + - - C3/22 + - - + - - S. Infantis C12/20 + - - - + - C2/21 + - - - + - S. Virchow C11/22 + - - - - + C15/20 + - - - - + C6/21 + - - - - + S. Fresno C13/20 + - - - - - S. Lawndale C6/20 + - - - - - S. Abony C11/20 + - - - - - S. Agama C15/21 + - - - - - S. Agbeni C13/21 + - - - - - S. Augustenborg C12/22 + - - - - - S. Berta C14/21 + - - - - - C8/22 + - - - - - S. Coeln C8/20 + - - - - - S. Dublin C1/22 + - - - - - C5/21 + - - - - - S. Give C5/20 + - - - - - S. Gloucester C4/21 + - - - - - S. Lagos C10/22 + - - - - - S. Stanley C12/21 + - - - - - S. Stanley C5/22 + - - - - - S. Teddington C3/20 + - - - - - Foods 2024,13, 4069 4 of 17 Table 1. Cont. Strain Strain Target ttr STM4497 safA Group_29846 Group_21126 Group_27174 S. Wernigerode C14/22 + - - - - - C8/21 + - - - - - S. Yoruba C14/20 + - - - - - B. cereus WDCM 00151 - NT NT NT NT NT E. faecalis 20825 - NT NT NT NT NT CECT 481 - NT NT NT NT NT WDCM 00009 - NT NT NT NT NT A. baumannii CECT 452 - NT NT NT NT NT Klebsiella pneumoniae CECT 8453 - NT NT NT NT NT S. uberis CECT 994 - NT NT NT NT NT S. agalactiae CECT 183 - NT NT NT NT NT S. dysgalactiae CECT 758 - NT NT NT NT NT L. monocytogenes WDCM 00110 - NT NT NT NT NT WDCM 00021 - NT NT NT NT NT L1AM0 - NT NT NT NT NT L. innocua CUP 1375 - NT NT NT NT NT P. aeruginosa WDCM 00024 - NT NT NT NT NT P. fluorescens WDCM 00115 - NT NT NT NT NT P. fragi WDCM 00116 - NT NT NT NT NT E. coli CECT 99 - NT NT NT NT NT AMC 76 - NT NT NT NT NT CECT 5947 - NT NT NT NT NT C179-12 - NT NT NT NT NT C. difficile CECT 531 - NT NT NT NT NT S. aureus WDCM 00034 - NT NT NT NT NT CECT 54 - NT NT NT NT NT Y. enterocolitica WDCM 00038 - NT NT NT NT NT WDCM 00039 - NT NT NT NT NT C. jejuni AMC - NT NT NT NT NT C. coli UM - NT NT NT NT NT AMC - NT NT NT NT NT CECT: Spanish Type Culture Collection. WDCM: World Data Center for Microorganisms. AMC: collection from the Institute of Applied Microbiology, ASMECRUZ. UM: University of Minho. CUP: Catholic University of Porto. NT: Not tested. 2.2. Primer Design For the detection of S. Virchow, S. Hadar, S. Infantis and Salmonella spp., new sets of primers were designed with Primer Explorer V5 (https://primerexplorer.jp/e/index. html, 16 December 2024). For the detection of Salmonella spp., the primers reported by Costa-Ribeiro et al., targeting the ttr gene, were selected [ 20 ], while for the serotypes Typhimuriums and Enteritidis, the primers described by Azinheiro et al. were selected and targeted the genes STM4497 and safA, respectively [ 21 ]. In Table 2, a complete list of all the primers used in the present study is provided. For the design of the new sets of primers, the reference genomes retrieved from RefSeq were used, including NZ_CP025094.1 (group_27174), NZ_CP121068.1 (group_21126), and NZ_CP022069.2 (group_29846) for S. Virchow, S. Hadar, and S. Infantis, respectively. Foods 2024,13, 4069 5 of 17 Table 2. Bacteria, genetic targets and primer sequences. Bacteria Target Primer Name Sequence (5′→3′) Reference Salmonella spp. ttr FIP_ttr GCA TCA GCC AAC ATA GCG CCA tttt CTA CGC CAT CCG TTA TCA CA [20] BIP_ttr TCA GGT ACA AAC CGT CCC CAA G tttt CAT CCG TTC CGC CTG GTA F3_ttr ACA CTG CTG TTC TGT AGC CT B3_ttr AGG TGC CGA GAA TAG CCA LF_ttr CCA GCA GGA CGC GTC TT LB_ttr CGC GCA ATT TAA CCC TTA CTC G S. Typhimurium STM4497 FIP_STM ACC TGC AGC TCA TTC TGA GCA G tttt TCA AAA ACA ACG GCT CCG G [21] BIP_STM GAA AAG GAC CAC AAG TTC GCG C tttt TCA GTG AGC ATG TCG ACG AT F3_STM AGC CGC ATT AGC GAA GAG B3_STM GCG GTC AAA TAA CCC ACG T S. Enteritidis safA FIP_SEN AGC CCA CAG TGA GTA TCG TG tttt CGC TGC TGG TAG TGC ATG G [21] BIP_SEN CAG AGG TCA TGG CGC GCA AAT tttt GGC ATT GGT ATC AAA GGT GA F3_SEN GTT GCT AAC ACG ACA CTG GAC B3_SEN GTG GGA TAT TCT GAG CCC CTA T S. Infantis group_21126 FIP_INF ATA GCC CAC CCC GCA ATT TCG tttt GAC TAC ATA CCG TAG CCC CA Current study BIP_INF CCA GGCG AAT TAG TAT ACG ACC CAT tttt TTG AGC CAA GCT TCG AGG A F3_INF GCA GAT ATC CCA TTA AAA ACT GAG C B3_INF CGG TAC CAA TAG TAT CCC TAC CT LF_INF GGG CGC ATC TTC CCA ATG LB_INF TTT GTG GTT CTG GTA CTG TGC S. Virchow group_27174 FIP_VIR TGG GCC AGC ACA AAT GAA TAC TGT G tttt CCA TGA TGG CAA CGG GAT Current study BIP_VIR TTA GGT GGC ACC CAT CCA GTG tttt TAA GGC AGC TCA CAA CGC F3_VIR TGT ACC TGG TGT TTG ATA TTT CGT B3_VIR CTG CAA TTG ACC AGT CGG T LF_VIR TGG ATC TTA AAT AGT CAT CAA ACG A LB_VIR CTG AAA CTT TTA TTT ATG CTT GGG T S. Hadar group_29846 FIP_HAD GCC GTG ATT TTC TTG ACT AAT TGA T tttt CAT GTG GCA ACA TTA GAA CG Current study BIP_HAD TCT TTG GCG AGA AAA CAG CAA tttt TCC TTC ATA AAC GGA ACC G F3_HAD AGA AGT CCG AGA GGA TGA B3_HAD ACA GAT TAA GTT CCC TTC CAA LF_HAD GCA TAC TGA AGC TCT TTT TCT GC LB_HAD ATT TGC ATT GCT GGC GT tttt represents a polyT linker between F2 and F1c, and B2 and B1c. 2.3. Nucleic Acid Extraction In the present work, four different DNA extraction methods were evaluated from simple thermal lysis to other methods that include different types of purification. In all cases, 1 mL of enriched sample was transferred to a 1.5 mL microtube and centrifuged at 900 × gfor 1 min to eliminate sample debris (step omitted for pure bacterial culture). The supernatant was transferred to a new microtube and centrifuged at 16,000 × gfor 1 min. The supernatant was discarded, and the pellet was used for DNA isolation. All the DNA samples were conserved at −20 ◦C until use. Foods 2024,13, 4069 6 of 17 2.3.1. Thermal Lysis (TL) The pellet was resuspended in 100 µ L of nuclease-free water and heated at 99 ◦ C for 5 min at 1400 rpm in a heater block (Thermomixer, Eppendorf AG, Wesseling-Berzdorf, Germany). Then, the sample was centrifuged again at 16,000 × gfor 1 min to eliminate bacterial and sample debris and the supernatant was transferred to a new microtube. 2.3.2. Chelex The pellet was resuspended in 100 µ L of 6% Chelex ® 100 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The sample was incubated for 15 min at 56 ◦ C and 1400 rpm, and then heated at 99 ◦ C for 8 min at 1400 rpm. Then, the samples were centrifuged at 16,000 × gfor 1 min and the supernatant was transferred to a new tube. 2.3.3. Thermal Lysis with Magnetic Bead Purification DNA was isolated as described in Section 2.3.1, but, in this case, the final supernatant was mixed with 100 µ L of magnetic beads (Mag-Bind ® TotalPure NGS, Omega-Biotek, Norcross, GA, USA). The mixture was incubated for 5 min at room temperature. The beads were recovered with a magnetic particle concentrator (Dynal ® MPC, Invitrogen, Carlsbad, CA, USA) until the liquid was clear. The supernatant was removed, and the pellet was washed two times with 200 µ L of 70% ethanol. Finally, the pellet was air-dried to eliminate the rest of the ethanol leaving the caps open and the tubes in the magnetic rack. The microtubes were retrieved from the magnetic rack and the magnetic pellet was resuspended in 100 µ L of nuclease-free water. Sample was incubated for 2 min at room temperature and then the tubes were placed again in the magnetic rack. When the liquid containing the released DNA was clear, it was transferred to a new microtube. 2.3.4. Thermal Lysis with “Glass Milk” Purification (GM) This DNA extraction was based on the method described by Page, Robert, et al. with some modifications. In this case, the pellet was resuspended in 100 µ L of nuclease-free water and 100 µ L of a 4% SDS solution and incubated for 5 min at 99 ◦ C and 1400 rpm in a heater block. Then, 400 µ L of 100% isopropanol, 200 µ L of 1.25 M NaCl and 10 µ L of “glass milk” were added and the sample was incubated for 5 min at room temperature. After that, it was centrifuged for 15 s in minicentrifuge at 2000 × g. The supernatant was discarded, and the pellet was washed two times with ethanol at 70%. Then, the pellet was air dried in a heater block at 65 ◦ C for 5 min with lead. After that, the pellet was resuspended in 100 µ L nuclease-free water to release the DNA from the silica. The sample was centrifuged at 2000×gand the supernatant was transferred to a new tube. 2.4. DNA Concentration and Quality The comparison among the four DNA extraction protocols was performed by quantifying the DNA concentration in all the samples spiked with Salmonella. The quantification was performed with the dsDNA Broad Range (BR) assay kit (Invitrogen™, ThermoFisher Scientific, Waltham, MA, USA) in combination with the commercial fluorometer Qubit (Invitrogen™, ThermoFisher Scientific, Waltham, MA, USA) and DNA purity was determined with NanoDrop Lite Plus (Thermo Scientific, Waltham, MA, USA). 2.5. ttr-LAMP 2.5.1. Real-Time ttr-LAMP Fluorescent LAMP assays were performed in a QuantStudio 12k Flex Real Time PCR system (Applied Biosystems, ThermoFisher Scientific, Waltham, MA, USA). Primers were designed by targeting the ttr gene. Reactions were carried using 12 µ L Fast Master Mix (ISO-004, OptiGene, UK), 800 nM of FIP/BIP primers, 400 nM of LB/LF primers and 200 nM of F3/B3 primers, 50 nM of CXR Reference Dye (Promega, Madison, WI, USA) and 2 µ L of template DNA and the remaining volume was completed with nuclease-free water. Technical duplicates were performed for all samples, and the experiments were run Foods 2024,13, 4069 7 of 17 at 65 ◦ C for 30 min, with fluorescence acquisition every 30 s. Then, melt curve analysis was performed as follows: samples were heated at 95 ◦ C for 1 s, 80 ◦ C for 20 s, and heated up to 95 ◦ C with temperature increments of 0.05 ◦ C/s and fluorescence acquisition after each temperature increment. Only samples with both positive technical replicates and Tm values falling within the calculated average temperature ± its standard deviation were considered positive. 2.5.2. Colorimetric ttr-LAMP Colorimetric LAMP assays were performed in 1.5 mL microtubes in a heater block (Thermomixer) at 65 ◦ C for 30 min. The reaction composition and volume were the same as the fluorescent LAMP but without ROX. After filling 1.5 mL microtubes, the top of the tube was covered with Parafilm ® leaving an opening of 1–2 mm on the hinge side of the tube. First, 1 µ L of SYBR Green I 1000X (Invitrogen ™ , ThermoFisher Scientific, Waltham, MA, USA) was deposited in the center of Parafilm ® . Then, the lid was carefully closed, and the tubes were incubated. After that, tubes were shaken to mix the sample with SYBR Green I and centrifuged for 10 s in a minicentrifuge (mySPIN 6, ThermoScientific, Waltham, MA, USA). Positive samples were greenish while negative samples remained orange. Furthermore, under UV light, positive samples emitted fluorescence, while the negative ones did not. 2.5.3. Serotyping LAMP Once the presence of Salmonella was confirmed, i.e., ttr-LAMP positive, the serotyping LAMP was applied. A real-time, fluorescence-based LAMP to determine the five serotypes of importance in poultry production (S. Typhimurium, S. Enteritidis, S. Infantis, SVirchow, S. Hadar) was developed. The list of LAMP primers designed for this purpose are included in Table 2. The reaction conditions were the same as that of the ttr-LAMP. The acceptance criteria were the same defined as those for the ttr-LAMP in Section 2.5.1. 2.6. LAMP Validation 2.6.1. Evaluation of the Inclusivity and Exclusivity For inclusivity assays, 54 Salmonella strains belonging to 31 different serotypes and 3 different subspecies were included. For exclusivity assays, strains from 28 other bacterial strains, belonging to 18 different species, were included. In Table 1, a detailed list of the microorganisms included in the present study is provided. Pure cultures were prepared as described in Section 2.1, and the DNA of this pure culture was extracted with the method described in Section 2.3.1. 2.6.2. Dynamic Range The dynamic ranges covered with the different protocols described in Section 2.2 were evaluated with pure DNA extracted from the strain WDCM 00030, as well as with a feces-spiked sample. After preparing the pure culture, and the spiked sample, the DNA was extracted with all the DNA extraction protocols, it was quantified, ten-fold serially diluted in nuclease-free water (Promega, Madison, WI, USA) and analyzed in technical triplicates by ttr-LAMP. Regarding the chicken feces, 100 µ L of Salmonella overnight pure culture was diluted in 900 µ L of feces in BPW. DNA was isolated with the four methods described in Section 2.2. Then, isolated DNA was serially diluted as for the pure culture. 2.6.3. Determination of the Limit of Detection (LoD) and Relative Limit of Detection (RLOD) The Limit of Detection (LoD) with confidence of 50% (LoD 50 ) and 95% (LoD 95 ) was determined for the four DNA extraction methods tested as described by Wilrich and Wilrich [ 22 ]. Regarding the Relative Limit of Detection (RLOD), the model described by Mărgăritescu and Wilrich was used [ 23 , 24 ]. To determine these limits, chicken bedding Foods 2024,13, 4069 8 of 17 was collected from a chicken farm and the absence of Salmonella was determined by ISO 6579-1:2017. Two pairs of boot swabs were placed in a stomacher bag with 25 g of chicken bedding collected and spiked with different concentrations of Salmonella Enteritidis WDCM 00030. Then, samples were homogenized with 225 mL of pre-warmed BPW and incubated for 18 h at 37 ◦ C. After that, 1 mL of the sample was collected and processed as indicated in Section 2.2 for DNA isolation with the four different methods tested. In addition, the samples were analyzed by the reference method ISO 6579-1:2017. Briefly, after BPW incubation, 0.1 mL was transferred to modified MRVS. Plates were incubated at 41.5 ◦ C for 48 h. Suspected samples were streaked in XLD and RAPID’ Salmonella plates and incubated for 24 h at 37 ◦ C. Samples with presumptive Salmonella colonies were confirmed with the latex agglutination test (Microgen Bioproducts Ltd., Surrey, UK). 2.6.4. Fitness-for-Purpose Once the LoD 50 with each different DNA extraction method was determined, all the samples above the corresponding value were considered and classified as being in Positive or Negative Agreement (PA/NA) if the ttr-LAMP result matched that obtained by the ISO reference method and were considered to be Positive or Negative Deviations (PD/ND) if the results did not match the reference method. Once classified, these values were used to determine the relative sensitivity, specificity and accuracy (SE, SP and AC, respectively) along with Cohen’s kappa of concordance (k) as previously described by Anderson et al. and Tomás et al. [8,25]. The developed LAMP method was also tested with interlaboratory tests carried out annually by the Central Veterinary Laboratory in Spain. This test includes ten samples of chicken feces inoculated with 100 CFU, 10 CFU or not inoculated. The samples were analyzed following the protocol previously described and evaluating the four DNA isolation methods described. The final workflow was also used to analyze the routine samples analyzed in the laboratory as part of the national Salmonella control plan. 2.7. Graphical Representation and Statistical Analysis The statistical analyses and the representation of the data obtained in the present study were performed with Graphpad Prism 10 (Boston, MA, USA). One-way ANOVA analysis with Dunn’s test was used to determine the existence of differences between groups (p< 0.05). 3. Results 3.1. LAMP Assay Evaluation 3.1.1. Inclusivity/Exclusivity The evaluation of the inclusivity indicated that all the 31 serotypes and 54 strains tested in the present study reported positive results for the ttr gene with an average melting temperature (Tm) of 88.94 ± 0.18 ◦ C. Regarding the evaluation of the exclusivity, a panel of 28 strains covering 18 different species typically encountered in food and environmental samples were tested. None of the exclusivity panel strains reported positive results, thus demonstrating the specificity of the assay. When focusing on the serotype-specific assays, it was possible to amplify the different serotypes when using the corresponding serotype-specific LAMP assay. In this sense, the STM4497 (Typhimurium) gene reported an average Tm value of 86.98 ±0.28 ◦C (10 strains including monophasic variants), safA (Enteritidis) reported values of 86.19 ±0.37 ◦C (6 strains), and for group_29846 (Hadar, 3 strains), group_21126 (Infantis, 2 strains) and group_27174 (Virchow, 3 strains), the Tm values were 83.46 ± 0.35 ◦ C, 86.64 ± 0.21 ◦ C and 84.08 ± 0.28 ◦ C, respectively, as shown in Figure 1. When focusing on the exclusivity, 20 non-target strains were tested covering 16 different serotypes, which were all positive for ttr but all negative for the serotype-specific LAMP assays (note that all assays were intended to be run in simplex format to avoid Tm misidentification). Foods 2024,13, 4069 9 of 17 Foods 2024, 13, x FOR PEER REVIEW 9 of 18 environmental samples were tested. None of the exclusivity panel strains reported positive results, thus demonstrating the specificity of the assay. When focusing on the serotype-specific assays, it was possible to amplify the different serotypes when using the corresponding serotype-specific LAMP assay. In this sense, the STM4497 (Typhimurium) gene reported an average Tm value of 86.98 ± 0.28 °C (10 strains including monophasic variants), safA (Enteritidis) reported values of 86.19 ± 0.37 °C (6 strains), and for group_29846 (Hadar, 3 strains), group_21126 (Infantis, 2 strains) and group_27174 (Virchow, 3 strains), the Tm values were 83.46 ± 0.35 °C, 86.64 ± 0.21 °C and 84.08 ± 0.28 °C, respectively, as shown in Figure 1. When focusing on the exclusivity, 20 non-target strains were tested covering 16 different serotypes, which were all positive for ttr but all negative for the serotype-specific LAMP assays (note that all assays were intended to be run in simplex format to avoid Tm misidentification). Figure 1. Graphical summary of the typical Tm values obtained for the ttr and serotyping LAMP assays. Each assay was run in simplex format. 3.1.2. DNA Extraction Protocol Comparison and ttr-LAMP Dynamic Range The dynamic range of LAMP assay was determined in pure DNA and feces, inoculated with S. Enteritidis WDCM 00030. In both cases, the DNA was isolated with the four methods described. When analyzing the pure bacterial DNA, all four protocols reached the range of the picograms. In this sense, with TL and GM, the lowest concentration was 0.4 pg/µL, while with the beads, the value slightly decreased down to 0.2 pg/µL and with chelex, a value of 0.1 pg/µL was reached, as shown in Figure 2A. In the case of chicken feces inoculated with Salmonella, differences were observed among the different DNA extraction methods. It was determined that the sample was spiked with 8.3 log CFU of Salmonella. Three protocols, namely chelex, TL and magnetic beads, were able to carry out detection until 3.3 log CFU/mL, while the GM method carried out detection until 4.3 log CFU/mL, as shown in Figure 2B. Figure 1. Graphical summary of the typical Tm values obtained for the ttr and serotyping LAMP assays. Each assay was run in simplex format. 3.1.2. DNA Extraction Protocol Comparison and ttr-LAMP Dynamic Range The dynamic range of LAMP assay was determined in pure DNA and feces, inoculated with S. Enteritidis WDCM 00030. In both cases, the DNA was isolated with the four methods described. When analyzing the pure bacterial DNA, all four protocols reached the range of the picograms. In this sense, with TL and GM, the lowest concentration was 0.4 pg/ µ L, while with the beads, the value slightly decreased down to 0.2 pg/ µ L and with chelex, a value of 0.1 pg/ µ L was reached, as shown in Figure 2A. In the case of chicken feces inoculated with Salmonella, differences were observed among the different DNA extraction methods. It was determined that the sample was spiked with 8.3 log CFU of Salmonella. Three protocols, namely chelex, TL and magnetic beads, were able to carry out detection until 3.3 log CFU/mL, while the GM method carried out detection until 4.3 log CFU/mL, as shown in Figure 2B. Foods 2024, 13, x FOR PEER REVIEW 10 of 18 Figure 2. Dynamic range covered with the different DNA extraction protocols with the ttr-LAMP assay with pure DNA (A) and with bacteria inoculated in boot swabs (B). The amplification time is provided as Tt, Time to Threshold. 3.2. DNA Extraction Protocol Comparison In order to better determine the performance of each extraction protocol, spiked samples were used. There were no significant differences in the quantity of DNA isolated between the different extraction protocols tested (see Figure 3A). Contrary to the DNA concentration, when the purity of the extracts was measured, significant differences were observed, i.e., the A260/A280 ratio of magnetic beads (1.935 ± 0.084) and GM (1.899 ± 0.080) was significantly higher (p < 0.05) than the ratio of chelex (1.685 ± 0.351) and TL (1.612 ± 0.441). The same results were observed with the ratio A260/A230, as shown in Figure 3B,C. (A) (B) Figure 2. Dynamic range covered with the different DNA extraction protocols with the ttr-LAMP assay with pure DNA (A) and with bacteria inoculated in boot swabs (B). The amplification time is provided as Tt, Time to Threshold. 3.2. DNA Extraction Protocol Comparison In order to better determine the performance of each extraction protocol, spiked samples were used. There were no significant differences in the quantity of DNA isolated between the different extraction protocols tested (see Figure 3A). Contrary to the Foods 2024,13, 4069 16 of 17 16. Notomi, T.; Okayama, H.; Masubuchi, H.; Yonekawa, T.; Watanabe, K.; Amino, N.; Hase, T. Loop-Mediated Isothermal Amplification of DNA. Nucleic Acids Res. 2000,28, e63. [CrossRef] [PubMed] 17. Garrido-Maestu, A.; Prado, M. Naked-Eye Detection Strategies Coupled with Isothermal Nucleic Acid Amplification Techniques for the Detection of Human Pathogens. Compr. Rev. Food Sci. Food Saf. 2022,21, 1913–1939. [CrossRef] 18. Selva Sharma, A.; Lee, N.Y. Advancements in Visualizing Loop-Mediated Isothermal Amplification (LAMP) Reactions: A Comprehensive Review of Colorimetric and Fluorometric Detection Strategies for Precise Diagnosis of Infectious Diseases. Coord. Chem. Rev. 2024,509, 215769. [CrossRef] 19. ISO 6579-1:2017; Microbiology of the Food Chain—Horizontal Method for the Detection, Enumeration and Serotyping of Salmonella—Part 1: Detection of Salmonella spp. ISO: Geneva, Switzerland, 2017. 20. Costa-Ribeiro, A.; Lamas, A.; Garrido-Maestu, A. Evaluating Commercial Loop-Mediated Isothermal Amplification Master Mixes for Enhanced Detection of Foodborne Pathogens. Foods 2024,13, 1635. [CrossRef] [PubMed] 21. Garrido-Maestu, A.; Fuciños, P.; Azinheiro, S.; Carvalho, J.; Prado, M. Systematic Loop-Mediated Isothermal Amplification Assays for Rapid Detection and Characterization of Salmonella spp., Enteritidis and Typhimurium in Food Samples. Food Control 2017,80, 297–306. [CrossRef] 22. Wilrich, C.; Wilrich, P.T. Estimation of the Pod Function and the LOD of a Qualitative Microbiological Measurement Method. J. AOAC Int. 2009,92, 1763–1772. [CrossRef] [PubMed] 23. Mărgăritescu, I.; Wilrich, P.-T. Determination of the Relative Level of Detection of a Qualitative Microbiological Measurement Method with Respect to a Reference Measurement Method. J. AOAC Int. 2013,96, 1086–1091. [CrossRef] 24. NordVal International Protocol for the Validation of Microbiological Alternative (Proprietary) Methods Against a Reference Method. 2016. Available online: https://www.semanticscholar.org/paper/NordVal-International-Protocol-for-the-validationa/3e4849b0dbd841d01af148feb66d21c19fb596d7 (accessed on 16 December 2024). 25. Anderson, A.; Pietsch, K.; Zucker, R.; Mayr, A.; Müller-Hohe, E.; Messelhäusser, U.; Sing, A.; Busch, U.; Huber, I.; Muller-Hohe, E.; et al. Validation of a Duplex Real-Time PCR for the Detection of Salmonella spp. in Different Food Products. Food Anal. Methods 2011,4, 259–267. [CrossRef] 26. Valderrama, W.B.; Dudley, E.G.; Doores, S.; Cutter, C.N. Commercially Available Rapid Methods for Detection of Selected Food-Borne Pathogens. Crit. Rev. Food Sci. Nutr. 2016,56, 1519–1531. [CrossRef] [PubMed] 27. Report, S. The European Union One Health 2022 Zoonoses Report. EFSA J. 2023,21, e8442. [CrossRef] 28. ISO 6579-1:2017/Amd 1:2020; Microbiology of the Food Chain—Horizontal Method for the Detection, Enumeration and Serotyping of Salmonella—Part 1: Detection of Salmonella spp.—Amendment 1: Broader Range of Incubation Temperatures, Amendment to the Stat 2020. ISO: Geneva, Switzerland, 2020; pp. 1–11. 29. Ferone, M.; Gowen, A.; Fanning, S.; Scannell, A.G.M. Microbial Detection and Identification Methods: Bench Top Assays to Omics Approaches. Compr. Rev. Food Sci. Food Saf. 2020,19, 3106–3129. [CrossRef] [PubMed] 30. Chapela, M.-J.; Garrido-Maestu, A.; Cabado, A.G. Detection of Foodborne Pathogens by QPCR: A Practical Approach for Food Industry Applications. Cogent Food Agric. 2015,1, 1013771. [CrossRef] 31. Niessen, L.; Luo, J.; Denschlag, C.; Vogel, R.F. The Application of Loop-Mediated Isothermal Amplification (LAMP) in Food Testing for Bacterial Pathogens and Fungal Contaminants. Food Microbiol. 2013,36, 191–206. [CrossRef] [PubMed] 32. Kreitlow, A.; Becker, A.; Schotte, U.; Malorny, B.; Plötz, M.; Abdulmawjood, A. Evaluation of Different Target Genes for the Detection of Salmonella sp. by Loop-Mediated Isothermal Amplification. Lett. Appl. Microbiol. 2021,72, 420–426. [CrossRef] [PubMed] 33. González-Escalona, N.; Brown, E.W.; Zhang, G. Development and Evaluation of a Multiplex Real-Time PCR (qPCR) Assay Targeting ttrRSBCA Locus and invA Gene for Accurate Detection of Salmonella spp. in Fresh Produce and Eggs. Food Res. Int. 2012,48, 202–208. [CrossRef] 34. Malorny, B.; Paccassoni, E.; Fach, P.; Bunge, C.; Martin, A.; Helmuth, R. Diagnostic Real-Time PCR for Detection of Salmonella in Food. Appl. Environ. Microbiol. 2004,70, 7046–7052. [CrossRef] [PubMed] 35. Barrett, E.L.; Clark, M.A. Tetrathionate Reduction and Production of Hydrogen Sulfide from Thiosulfate. Microbiol. Rev. 1987, 51, 192–205. [CrossRef] [PubMed] 36. Pacheco, J.I.M.; dos Anjos, K.B.A.; Silva, I.V.; Okar, R.G.; Rodrigues, S.M.B.D.; Francabandiera, A.I.; Rodriguez, M.C. Comparison of Two Affordable DNA Extraction Methods for Molecular Detection of Salmonella Isolates from Broiler Farm’s Boot Swabs. Res. Soc. Dev. 2023,12, e28312139618. [CrossRef] 37. Geuther, R. 260/280 and 260/230 Ratios Introduction. Z. Allg. Mikrobiol. 2007,17, 86–87. [CrossRef] 38. Costa-Ribeiro, A.; Lamas, A.; Mora, A.; Prado, M.; Garrido-Maestu, A. Moving towards On-Site Detection of Shiga ToxinProducing Escherichia coli in Ready-to-Eat Leafy Greens. Curr. Res. Food Sci. 2024,8, 100716. [CrossRef] 39. Kim, J.H.; Jung, S.; Oh, S.W. Combination of Bacteria Concentration and DNA Concentration for Rapid Detection of E. coli O157:H7, L. monocytogenes, and S. Typhimurium without Microbial Enrichment. LWT 2020,117, 108609. [CrossRef] 40. Fachmann, M.S.R.; Josefsen, M.H.; Hoorfar, J.; Nielsen, M.T.; Löfström, C. Cost-Effective Optimization of Real-Time PCR-Based Detection of Campylobacter and Salmonella with Inhibitor Tolerant DNA Polymerases. J. Appl. Microbiol. 2015,119, 1391–1402. [CrossRef] Foods 2024,13, 4069 17 of 17 41. Ong, C.T.; Boe-Hansen, G.; Ross, E.M.; Blackall, P.J.; Turni, C.; Hayes, B.J.; Tabor, A.E. Evaluation of Host Depletion and Extraction Methods for Shotgun Metagenomic Analysis of Bovine Vaginal Samples. Microbiol. Spectr. 2022,10, e00412-21. [CrossRef] [PubMed] 42. Garrido-Maestu, A.; Lamas, A.; Fornés, D.T.; Rodríguez, M.P. The Use of Multiplex Real-Time PCR for the Simultaneous Detection of Foodborne Bacterial Pathogens. In Foodborne Bacterial Pathogens: Methods and Protocols; Bridier, A., Ed.; Springer: New York, NY, USA, 2025; pp. 19–31, ISBN 978-1-0716-4100-2. 43. Altman, D.G. Practical Statistics for Medical Research; Hall, C., Ed.; Chapman and Hall/CRC: New York, NY, USA, 1991. 44. Zheng, J.; Reed, E.; Maounounen-Laasri, A.; Deng, X.; Wang, S.S.; Ramachandran, P.; Ferreira, C.; Bell, R.; Brown, E.W.; Hammack, T.S.; et al. Evaluation of Universal Preenrichment Broth and Comparison of Rapid Molecular Methods for the Detection of Salmonella from Spent Sprout Irrigation Water (SSIW). Int. J. Food Microbiol. 2024,411, 110527. [CrossRef] [PubMed] 45. Fujiyoshi, S.; Yarimizu, K.; Miyashita, Y.; Rilling, J.; Acuña, J.J.; Ueki, S.; Gajardo, G.; Espinoza-González, O.; Guzmán, L.; Jorquera, M.A.; et al. Suitcase Lab: New, Portable, and Deployable Equipment for Rapid Detection of Specific Harmful Algae in Chilean Coastal Waters. Environ. Sci. Pollut. Res. 2021,28, 14144–14155. [CrossRef] 46. Higgins, O.; Chueiri, A.; Connor, L.O.; Lahiff, S.; Burke, L.; Morris, D.; Pfeifer, M. Portable Differential Detection of CTX-M ESBL Gene Variants, bla CTX-M-1 and bla CTX-M-15 , from Escherichia coli Isolates and Animal Fecal Samples Using Loop-Primer Endonuclease Cleavage Loop-Mediated Isothermal Amplification. Microbiol. Spectr. 2023,11, e0331622. 47. Fachmann, M.S.R.; Löfström, C.; Hoorfar, J.; Hansen, F.; Christensen, J.; Mansdal, S.; Josefsen, M.H.M.H.; Mandal, S.; Josefsen, M.H.M.H. Detection of Salmonella Enterica in Meat in Less than 5 Hours by a Low-Cost and Noncomplex Sample Preparation Method. Appl. Environ. Microbiol. 2017,83, e03151-16. [CrossRef] [PubMed] 48. Costa-Ribeiro, A.; Azinheiro, S.; Fernandes, S.P.S.; Lamas, A.; Prado, M.; Salonen, L.M.; Garrido-Maestu, A. Evaluation of Covalent Organic Frameworks for the Low-Cost, Rapid Detection of Shiga Toxin-Producing Escherichia coli in Ready-to-Eat Salads. Anal. Chim. Acta 2023,1267, 341357. [CrossRef] 49. Lamas, A.; Santos, S.B.; Prado, M.; Garrido-Maestu, A. Phage Amplification Coupled with Loop-Mediated Isothermal Amplification (PA-LAMP) for Same-Day Detection of Viable Salmonella Enteritidis in Raw Poultry Meat. Food Microbiol. 2023,115, 104341. [CrossRef] [PubMed] 50. Rossmanith, P.; Suss, B.; Wagner, M.; Hein, I. Development of Matrix Lysis for Concentration of Gram Positive Bacteria from Food and Blood. J. Microbiol. Methods 2007,69, 504–511. [CrossRef] [PubMed] 51. Mann, E.; Pommer, K.; Mester, P.; Wagner, M.; Rossmanith, P. Quantification of Gram-Positive Bacteria: Adaptation and Evaluation of a Preparation Strategy Using High Amounts of Clinical Tissue. BMC Vet. Res. 2014,10, 53. [CrossRef] [PubMed] 52. Mester, P.; Wagner, M.; Rossmanith, P. Molecular Enrichment for Qualitative Molecular Pathogen Detection in Food. Food Anal. Methods 2018,11, 1251–1256. [CrossRef] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.