The authors thank members of the FDA&#8217;s Microbiology Methods Validation Subcommittee (MMVS) and Bacteriological Analytical Manual (BAM) Council for critically reviewing Salmonella LAMP method validation studies.

<ol>
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C., Cepica, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+for+diagnosis+of+18+World+Organization+for+Animal+Health+(OIE)+notifiable+viral+diseases+of+ruminants,+swine+and+poultry.">Loop-mediated isothermal amplification for diagnosis of 18 World Organization for Animal Health (OIE) notifiable viral diseases of ruminants, swine and poultry.</a> Animal Health Research Reviews. 16 (2), 89-106 (2015).</li><li>Kumar, Y., Bansal, S., Jaiswal, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+(LAMP):+A+rapid+and+sensitive+tool+for+quality+assessment+of+meat+products.">Loop-mediated isothermal amplification (LAMP): A rapid and sensitive tool for quality assessment of meat products.</a> Comprehensive Reviews in Food Science and Food Safety. 16 (6), 1359-1378 (2017).</li><li>Kundapur, R. R., Nema, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification:+Beyond+microbial+identification.">Loop-mediated isothermal amplification: Beyond microbial identification.</a> Cogent Biology. 2, 1137110 (2016).</li><li>. Compliance Policy Guide Sec. 690.800 Salmonella in Food for Animals Available from: <a target="_blank" href="https://www.fda.gov/downloads/iceci/compliancemanuals/compliancepolicyguidancemanual/ucm361105.pdf">https://www.fda.gov/downloads/iceci/compliancemanuals/compliancepolicyguidancemanual/ucm361105.pdf</a> (2013)</li><li>Bird, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+3M+molecular+detection+assay+(MDA)+2+-+Salmonella+for+the+detection+of+Salmonella+spp.+in+select+foods+and+environmental+surfaces:+collaborative+study,+first+action+2016.01.">Evaluation of the 3M molecular detection assay (MDA) 2 - Salmonella for the detection of Salmonella spp. in select foods and environmental surfaces: collaborative study, first action 2016.01.</a> Journal of AOAC International. 99 (4), 980-997 (2016).</li><li>D'Agostino, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+a+loop-mediated+amplification/ISO+6579-based+method+for+analysing+soya+meal+for+the+presence+of+Salmonella+enterica.">Validation of a loop-mediated amplification/ISO 6579-based method for analysing soya meal for the presence of Salmonella enterica.</a> Food Analytical Methods. 9 (11), 2979-2985 (2016).</li><li>Ge, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-laboratory+validation+of+a+loop-mediated+isothermal+amplification+method+for+screening+Salmonella+in+animal+food.">Multi-laboratory validation of a loop-mediated isothermal amplification method for screening Salmonella in animal food.</a> Frontiers in Microbiology. 10, 562 (2019).</li><li>D'Agostino, M., Diez-Valcarce, M., Robles, S., Losilla-Garcia, B., Cook, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+loop-mediated+isothermal+amplification-based+method+for+analysing+animal+feed+for+the+presence+of+Salmonella.">A loop-mediated isothermal amplification-based method for analysing animal feed for the presence of Salmonella.</a> Food Analytical Methods. 8 (10), 2409-2416 (2015).</li><li>Galan, J. E., Ginocchio, C., Costeas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+functional+characterization+of+the+Salmonella+invasion+gene+invA:+homology+of+InvA+to+members+of+a+new+protein+family.">Molecular and functional characterization of the Salmonella invasion gene invA: homology of InvA to members of a new protein family.</a> Journal of Bacteriology. 174 (13), 4338-4349 (1992).</li><li>Chen, S., Wang, F., Beaulieu, J. C., Stein, R. E., Ge, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+viable+salmonellae+in+produce+by+coupling+propidium+monoazide+with+loop-mediated+isothermal+amplification.">Rapid detection of viable salmonellae in produce by coupling propidium monoazide with loop-mediated isothermal amplification.</a> Applied and Environmental Microbiology. 77 (12), 4008-4016 (2011).</li><li>Yang, Q., Chen, S., Ge, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+Salmonella+serovars+in+shell+eggs+by+loop-mediated+isothermal+amplification.">Detecting Salmonella serovars in shell eggs by loop-mediated isothermal amplification.</a> Journal of Food Protection. 76 (10), 1790-1796 (2013).</li><li>Yang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+loop-mediated+isothermal+amplification+for+the+rapid,+reliable,+and+robust+detection+of+Salmonella+in+produce.">Evaluation of loop-mediated isothermal amplification for the rapid, reliable, and robust detection of Salmonella in produce.</a> Food Microbiology. 46, 485-493 (2015).</li><li>Yang, Q., Domesle, K. J., Wang, F., Ge, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+of+Salmonella+in+food+and+feed+by+coupling+loop-mediated+isothermal+amplification+with+bioluminescent+assay+in+real-time.">Rapid detection of Salmonella in food and feed by coupling loop-mediated isothermal amplification with bioluminescent assay in real-time.</a> BMC Microbiology. 16 (1), 112 (2016).</li><li>Domesle, K. J., Yang, Q., Hammack, T. S., Ge, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+a+Salmonella+loop-mediated+isothermal+amplification+assay+in+animal+food.">Validation of a Salmonella loop-mediated isothermal amplification assay in animal food.</a> International Journal of Food Microbiology. 264, 63-76 (2018).</li><li>Bustin, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MIQE+guidelines:+minimum+information+for+publication+of+quantitative+real-time+PCR+experiments.">The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments.</a> Clinical Chemistry. 55 (4), 611-622 (2009).</li></ol>

The authors declare that they have no competing financial interests. The views expressed in this manuscript are those of the authors and do not necessarily reflect the official policy of the Department of Health and Human Services, the U.S. Food and Drug Administration, or the U.S. Government. Reference to any commercial materials, equipment, or process does not in any way constitute approval, endorsement, or recommendation by the Food and Drug Administration.

We have presented here a simple, rapid, specific, and sensitive LAMP method for screening and confirming Salmonella in animal food and pure culture, respectively. With the convenience of an isothermal master mix that contains four key reagents, and a ready-to-use, in-house prepared primer mix, assembling a LAMP reaction requires only a few pipetting steps (Figure 1). The total run time including amplification and anneal phases is less than 38 min (Figure 2A,B and Figure 3A,B). Positive results are monitored via real-time fluorescence (Figure 2C and Figure 3C,D) and can be detected as early as 5 min26. The anneal phase serves as an extra confirmation of LAMP specificity since only samples with correct Tm (around 90 &#176;C) are reported as positive (Figure 2D,E and Figure 3E&#8722;G). Sensitivities of 1 Salmonella cell in pure culture and &#60; 1 CFU/25 g in animal food have been reported previously26.
As LAMP is quite effective and generates a large quantity of DNA1, it is critical that best laboratory practices are used to prevent cross-contamination, which may include physically separating the areas for preparing the LAMP master mix and adding DNA templates, avoiding generating aerosols, using filter pipette tips, changing gloves often, and refraining from opening LAMP reaction tubes post-amplification.
The specificity of this Salmonella LAMP method was previously tested using 300 bacterial strains (247 Salmonella of 185 serovars and 53 non-Salmonella) and demonstrated to be 100% specific26. Notably, significant differences in Tmax were observed between the two Salmonella species, S. enterica and Salmonella bongori, and among S. enterica subspecies, especially subsp. arizonae (IIIa)26. Nonetheless, these were still valid positive results per the rules for interpreting LAMP results. In our multi-laboratory collaborative study in dry dog food which involved 14 analysts19, samples having inconsistent results in duplicate LAMP runs were occasionally observed. These usually involved samples with delayed positive results (Tmax &#62; 15 min). Repeating both runs independently usually resolved the issue. More rarely, we observed samples with correct Tm but no or irregular Tmax values (&#60; 5 min). This was usually caused by air bubbles in the reaction tube.
Throughout the lifecycle of LAMP method development, evaluation, precollaborative study, and multi-laboratory validation, we have observed high tolerance of LAMP to inhibitors in various animal food or food matrices and culture media4,19,22,23,24, highlighting the robustness of the method and collaborating numerous other studies on a global scale8. This is superior compared to PCR or real-time PCR, which usually requires an internal amplification control to ensure that negative results are not due to matrix inhibition28. Further, LAMP demonstrated similar (or superior) specificity and sensitivity compared to PCR or real-time PCR in the vast majority of studies8. The cost of LAMP reagents is at about $1 per reaction. The LAMP instruments used in this protocol are small, low-maintenance, and portable. They can handle any isothermal amplification method that employs target detection by fluorescence measurement, LAMP included. Using the LAMP software, comprehensive reports can be generated in multiple format (pdf, text, and image).
Method validation is a critical step before a new method can be adopted for routine use. It is noteworthy that the LAMP protocol reported here has successfully completed multi-laboratory validation19. With the recent incorporation of this LAMP protocol into the U.S. FDA&#8217;s BAM Chapter 5 Salmonella27, it is expected that the method will gain much wider use, both as a rapid screening method in animal food and as a reliable confirmation method for presumptive Salmonella isolates from all food categories.

Loop-mediated isothermal amplification (LAMP) is a novel isothermal nucleic acid amplification test (iNAAT) invented in 2000 by a group of Japanese scientists1. Through the formation of a target-specific stem-loop DNA structure during initial steps, LAMP uses a strand-displacing DNA polymerase to efficiently amplify this starting material quasi-exponentially, resulting in 109 copies of target in less than 1 h1. Compared to polymerase chain reaction (PCR), a widely used NAAT, LAMP possesses several advantages. First, LAMP reactions are carried out under isothermal conditions. This obviates the need for a sophisticated thermal cycling instrument. Second, LAMP is highly tolerant to culture media and biological substances2 with robustness demonstrated for both clinical and food applications3,4. This simplifies sample preparation and minimizes false negative results5. Third, LAMP is amenable to multiple detection platforms, such as turbidity, colorimetry, bioluminescence, fluorescence, and microfluidics6. Fourth, LAMP is highly specific as it uses four to six specially designed primers to target six to eight specific regions1,7. Fifth, LAMP is ultrasensitive and numerous studies have reported its superior sensitivity to PCR or real-time PCR8. Finally, LAMP is faster with many assays now adopting a 30 min standard run time while PCR-type assays usually take 1&#8722;2 h8.
These attractive features fueled the application of LAMP in broad pathogen detection areas, including in vitro diagnostics9, animal disease diagnostics10, and food and environmental testing11. Notably, a TB-LAMP (LAMP for Mycobacterium tuberculosis) has been recommended by WHO as a valid replacement test for sputum-smear microscopy for pulmonary tuberculosis diagnoses in peripheral settings12. LAMP application also expands beyond microbial identification to include the detection of allergens, animal species, drug resistance, genetically modified organisms, and pesticides13.
Nontyphoidal Salmonella is a zoonotic pathogen of substantial food safety and public health concern worldwide14. It has also been identified as an important microbial hazard in food for animals (i.e., animal food)15,16. To prevent Salmonella illnesses/outbreaks from contaminated human food and animal food, it is imperative to have rapid, reliable, and robust methods for testing Salmonella in a variety of matrices. In the past decade, considerable efforts have been made internationally on the development and application of Salmonella LAMP assays in a wide array of food matrices, as recently summarized in an extensive review8. Several Salmonella LAMP assays, including the one presented here, have successfully completed multi-laboratory validation following well-established international guidelines17,18,19,20.
Our Salmonella LAMP assay specifically targets the Salmonella invasion gene invA (GenBank accession number M90846)21 and is rapid, reliable, and robust in multiple food matrices4,22,23,24,25,26. The method has been validated in six animal food matrices in a precollaborative study26 and in dry dog food in a multi-laboratory collaborative study19. As a result, the Salmonella LAMP method presented here has recently been incorporated into the U.S. Food and Drug Administration (FDA)&#8217;s Bacteriological Analytical Manual (BAM) Chapter 5 Salmonella27 to serve two purposes, one as a rapid screening method for the presence of Salmonella in animal food and two as a reliable confirmation method for presumptive Salmonella isolated from all foods.

<table><tbody><tr><td>Brain heart infusion (BHI) broth</td><td>BD Diagnostic Systems, Sparks, MD</td><td>299070</td><td>Liquid growth medium used in the cultivation of Salmonella.</td></tr><tr><td>Buffered peptone water (BPW)</td><td>BD Diagnostic Systems, Sparks, MD</td><td>218105</td><td>Preenrichment medium for the recovery of Salmonella from animal food samples.</td></tr><tr><td>DNA AWAY</td><td>Thermo Fisher Scientific, Waltham, MA</td><td>7010</td><td>Eliminates unwanted DNA and DNase from laboratory bench, glassware, and plasticware without affecting subsequent DNA samples.</td></tr><tr><td>Genie Explorer software</td><td>OptiGene Ltd., West Sussex, United Kingdom</td><td>Version 2.0.6.3</td><td>Supports remote operation of Genie instruments including LAMP runs and data analysis.</td></tr><tr><td>Genie II or Genie III (LAMP instrument)</td><td>OptiGene Ltd., West Sussex, United Kingdom</td><td>GEN2-02 or GEN3-02</td><td>A small instrument capable of temperature control up to 100 &amp;deg;C with &amp;plusmn; 0.1 &amp;deg;C accuracy and simultaneous fluorescence detection via the FAM channel. Genie II has 2 blocks (A and B) with 8 samples in each block. Genie III has a single block that accommodates 8 samples.</td></tr><tr><td>Genie strip</td><td>OptiGene Ltd., West Sussex, United Kingdom</td><td>OP-0008</td><td>8-well microtube strips with integral locking caps and a working volume of 10 to 150 &amp;micro;l.</td></tr><tr><td>Genie strip holder</td><td>OptiGene Ltd., West Sussex, United Kingdom</td><td>GBLOCK</td><td>Used to hold Genie strips when setting up a LAMP reaction, the aluminum holder can also be used as a cool block.</td></tr><tr><td>Hydrochloric acid (HCl) solution, 1 N</td><td>Thermo Fisher Scientific, Waltham, MA</td><td>SA48-500</td><td>Adjusts pH of animal food samples after adding BPW and prior to overnight enrichment.</td></tr><tr><td>Heat block</td><td>Thermo Fisher Scientific, Waltham, MA</td><td>88-860-022</td><td>Heats samples at 100 &amp;plusmn; 1 oC for DNA extraction.</td></tr><tr><td>Incubator</td><td>Thermo Fisher Scientific, Waltham, MA</td><td>3960</td><td>Standard laboratory incubator.</td></tr><tr><td>ISO-001 isothermal master mix</td><td>OptiGene Ltd., West Sussex, United Kingdom</td><td>ISO-001</td><td>An optimized master mix to simplify the assembly of a LAMP reaction, containing a strand-displacing GspSSD DNA polymerase large fragment from Geobacillus spp., thermostable inorganic pyrophosphatase, reaction buffer, MgSO4, dNTPs, and a double-stranded DNA binding dye (FAM detection channel).</td></tr><tr><td>Isopropanol</td><td>Thermo Fisher Scientific, Waltham, MA</td><td>A416</td><td>Disinfects work surfaces.</td></tr><tr><td>LAMP primers</td><td>Integrated DNA Technologies Inc., Coralville, IA</td><td>Custom</td><td>LAMP primers with detailed information in Table 1.</td></tr><tr><td>Microcentrifuge</td><td>Eppendorf North America, Hauppauge, NY</td><td>22620207</td><td>MiniSpin plus personal microcentrifuge.</td></tr><tr><td>Microcentrifuge tubes</td><td>Thermo Fisher Scientific, Waltham, MA</td><td>05-408-129</td><td>Standard microcentrifuge tubes.</td></tr><tr><td>Molecular grade water</td><td>Thermo Fisher Scientific, Waltham, MA</td><td>AM9938</td><td>Used in making primer stocks, primer mix, and LAMP reaction mix.</td></tr><tr><td>Sodium hydroxide (NaOH) solution, 1 N</td><td>Thermo Fisher Scientific, Waltham, MA</td><td>SS266-1</td><td>Adjusts pH of animal food samples after adding BPW and prior to overnight enrichment.</td></tr><tr><td>Nonselective agar (e.g., blood agar, nutrient agar, and trypticase soy agar)</td><td>Thermo Fisher Scientific, Waltham, MA</td><td>R01202</td><td>Solid growth medium used in the cultivation of Salmonella.</td></tr><tr><td>Peptone water</td><td>BD Diagnostic Systems, Sparks, MD</td><td>218071</td><td>Dilutes overnight Salmonella cultures to make positive control DNA.</td></tr><tr><td>Pipettes and tips</td><td>Mettler-Toledo Rainin LLC, Oakland CA</td><td>Pipet Lite LTS series</td><td>Standard laboratory pipettes and tips.</td></tr><tr><td>PrepMan Ultra sample preparation reagent</td><td>Thermo Fisher Scientific, Waltham, MA</td><td>4318930</td><td>A simple kit used for the rapid preparation of DNA templates for use in a LAMP reaction.</td></tr><tr><td>Salmonella reference strain LT2</td><td>ATCC, Manassas, VA</td><td>700720</td><td>Salmonella reference strain used as positive control.</td></tr><tr><td>Trypticase soy broth (TSB)</td><td>BD Diagnostic Systems, Sparks, MD</td><td>211768</td><td>Liquid growth medium used in the cultivation of Salmonella.</td></tr><tr><td>Vortex mixer</td><td>Scientific Industries, Inc., Bohemia, NY</td><td>SI-0236</td><td>Standard laboratory vortex mixer.</td></tr><tr><td>Whirl-pak filter bag</td><td>Nasco Sampling Brand, Fort Atkinson, WI</td><td>B01318</td><td>Filter bags to hold animal food samples for preenrichment.</td></tr></tbody></table>

loop-mediated isothermal amplification for screening salmonella in animal food and confirming salmonella from culture isolation

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, fungal, parasitic, and viral agents. Salmonella is a bacterial pathogen of worldwide food safety concern, including food for animals. Presented here is a multi-laboratory-validated Salmonella LAMP protocol that can be used to rapidly screen animal food for the presence of Salmonella contamination and can also be used to confirm presumptive Salmonella isolates recovered from all food categories. The LAMP assay specifically targets the Salmonella invasion gene (invA) and is rapid, sensitive, and highly specific. Template DNAs are prepared from enrichment broths of animal food or pure cultures of presumptive Salmonella isolates. The LAMP reagent mixture is prepared by combining an isothermal master mix, primers, DNA template, and water. The LAMP assay runs at a constant temperature of 65 &#176;C for 30 min. Positive results are monitored via real-time fluorescence and can be detected as early as 5 min. The LAMP assay exhibits high tolerance to inhibitors in animal food or culture medium, serving as a rapid, reliable, robust, cost-effective, and user-friendly method for screening and confirming Salmonella. The LAMP method has recently been incorporated into the U.S. Food and Drug Administration&#8217;s Bacteriological Analytical Manual (BAM) Chapter 5.

Figure 2 and Figure 3 show representative LAMP graphs/tables displayed on both platforms. In this LAMP run, samples S1 to S6 are 10-fold serial dilutions of S. enterica serovar Infantis ATCC 51741 ranging from 1.1 x 106 CFU to 11 CFU per reaction. Positive control is S. enterica serovar Typhimurium ATCC 19585 (LT2) at 1.7 x 104 CFU per reaction and NTC is molecular grade water.
As shown in Figure 2E and Figure 3G, both NTC and PC wells are valid controls. The NTC well has blank Tmax while Tm is &#60; 83 &#176;C on the LAMP instrument panel and blank in the LAMP software, suggesting a negative result. The PC well has Tmax of 7 min 45 sec and Tm of ~ 90 &#176;C on both platforms, suggesting a positive result. Samples S1 to S6 have Tmax between 6 min 30 sec&#160;and 12 min 15 sec, all being Salmonella-positive.&#160;
Following duplicate runs of the same set of samples, the final LAMP results are reported for these samples. This representative LAMP run shows that LAMP successfully detects Salmonella with a wide range of concentrations in the samples.&#160;
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61239/61239fig2v2.jpg" /> 
Figure 2: Representative LAMP results displayed on the LAMP instrument panel.&#160;(A) The Profile tab shows the programmed temperature profile. (B) The Temperature tab shows actual temperatures in the sample wells as LAMP reaction proceeds. (C) The Amplification tab shows fluorescence readings during LAMP amplification. (D) The Anneal tab shows changes in fluorescence (derivative) during the anneal phase. (E) The Results tab shows a tabular view of the LAMP results. <a href="https://www.jove.com/files/ftp_upload/61239/61239fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61239/61239fig3v2.jpg" /> 
Figure 3: Representative LAMP results viewed in the LAMP software.&#160;(A) The Profile tab shows the programmed temperature profile. (B) The Temperature tab shows actual temperatures in the sample wells as LAMP reaction proceeds. (C) The Amplification tab shows fluorescence readings during LAMP amplification. (D) The Amplification Rate tab shows changes in fluorescence (fluorescence ratio) during LAMP amplification. (E) The Anneal tab shows fluorescence readings during the anneal phase. (F) The Anneal Derivative tab shows changes in fluorescence (derivative) during the anneal phase. (G) The Result tab shows a tabular view of the LAMP results. <a href="https://www.jove.com/files/ftp_upload/61239/61239fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation at JoVE.com

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification test (iNAAT) that has attracted broad interest in the pathogen detection field. Here, we present a multi-laboratory-validated Salmonella LAMP protocol as a rapid, reliable, and robust method for screening Salmonella in animal food and confirming presumptive Salmonella from culture isolation.

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, ...

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JoVE Journal

Biology

8.0K Views.  U.S. Food and Drug Administration. Loopgemedieerde isothermale versterking (LAMP) is een isothermische nucleïnezuurversterkingstest (iNAAT) die brede belangstelling heeft getrokken voor het detectieveld van ziekteverwekkers. Hier presenteren we een multilaboratorium gevalideerd Salmonella LAMP-protocol als een snelle, betrouwbare en robuuste methode voor het screenen van Salmonella in dierlijk voedsel en het bevestigen van vermoedelijke Salmonella uit cultuurisolatie.

Video: Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.

A PCR-based protocol was adapted to detect Cronobacter spp., Salmonella enterica, and Listeria monocytogenes from body surfaces and alimentary canals of individual wild-caught flies. The goal of this protocol is to detect and isolate bacterial pathogens from individual insects collected as part of an environmental sampling program during foodborne outbreak investigations.

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a ...

Environment

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Immunology and Infection

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.
We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.
Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

We describe a multiplex PCR for the rapid detection of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. Specific Salmonella serovars can be identified by targeting a multiplex PCR to genes and sequences unique to the O-antigen biosynthesis cluster and flagellin of a given serovar. Serovar is assigned then to a Salmonella isolate based on the appearance of specific, size amplicons (PCR product) corresponding to the target allele.

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus.  However, there are two species, six subspecies, and over ...

Application of the Intelligent High-Throughput Antimicrobial Sensitivity Testing/Phage Screening System and Lar Index of Antimicrobial Resistance

To improve the efficiency of antimicrobial susceptibility testing (AST) and phage high-throughput screening for resistant bacteria and to reduce the detection cost, an intelligent high-throughput AST/phage screening system, including a 96-dot matrix inoculator, image acquisition converter, and corresponding software, was developed according to AST criteria and the breakpoints of resistance (R) formulated by the Clinical &amp; Laboratory Standards Institute (CLSI). AST and statistics of minimum inhibitory concentration (MIC) distributions (from R/8 to 8R) of 1,500 Salmonella strains isolated from poultry in Shandong, China, against 10 antimicrobial agents were carried out by the intelligent high-throughput AST/phage screening system. The Lar index, meaning "less antibiosis, less resistance and residual until little antibiosis", was obtained by calculating the weighted average of each MIC and dividing by R. This approach improves accuracy in comparison with using the prevalence of resistance to characterize the antimicrobial resistance (AMR) degree of highly resistant strains. For the strains of Salmonella with high AMR, lytic phages were efficiently screened from the phage library by this system, and the lysis spectrum was computed and analyzed. The results showed that the intelligent high-throughput AST/phage screening system was operable, accurate, highly efficient, inexpensive, and easy to maintain. Combined with the Shandong veterinary antimicrobial resistance monitoring system, the system was suitable for scientific research and clinical detection related to AMR.

Here we introduce the principle, structure, and instruction of the intelligent high-throughput antimicrobial sensitivity testing/phage screening system. Its application is illustrated by using Salmonella isolated from poultry in Shandong, China, as an example. The Lar index is calculated, and its significance in evaluating antimicrobial resistance is discussed comprehensively.

To improve the efficiency of antimicrobial susceptibility testing (AST) and phage high-throughput screening for resistant bacteria and to reduce the ...

Chronic Salmonella Infected Mouse Model

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and tumorigenesis. Many researchers have taken advantage of the colitis induced by Salmonella typhimurium for the studies on the early phase of inflammation and infection. However, only few reports are on the chronic infection in vivo. Mice with Salmonella persistent existence in the gastrointestinal tract allow us to explore the long-term host-bacterial interaction, signal transduction, and tumorigenesis. We have established a chronic bacterial infected mouse model with Salmonella typhimurium colonization in the mouse intestine over 6 months. To use this system, it is necessary for the researcher to learn how to prepare the bacterial culture and gavage the animals. We detail a methodology for prepare bacterial culture and gavage mice. We also show how to detect the Salmonella persistence in the gastrointestinal tract. Overall, this protocol will aid researchers using the bacterial infected mouse model to address fundamentally important biological and microbiological questions.

Chronic Salmonella Infected Mouse Model

Establish a chronic bacterial infected mouse model with persistent Salmonella typhimurium colonization in intestine for 27 weeks.

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Specific and Rapid Detection of Tilapia Lake Virus

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the aquaculture industry that has resulted in the mass morbidity and mortality of tilapia in many parts of the world. An effective, rapid, and accurate diagnostic assay for TiLV infection is therefore necessary to detect the initial infection and to prevent the spread of the disease in aquaculture farming. In this study, a highly sensitive and practical reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is presented to detect tilapia lake virus in fish tissue. A comparison of the RT-qPCR and RT-LAMP assays of infected samples revealed positive results in 63 (100%) and 51 (80.95%) samples, respectively. Moreover, an analysis of uninfected samples showed that all 63 uninfected tissues yielded negative results for both the RT-qPCR and RT-LAMP assays. The cross-reactivity with five pathogens in tilapia was evaluated using RT-LAMP, and all the tests showed negative results. Both the liver and mucus samples obtained from infected fish showed comparable results using the RT-LAMP method, suggesting that mucus can be used in RT-LAMP as a nonlethal assay to avoid killing fish. In conclusion, the results demonstrated that the presented RT-LAMP assay provides an effective method for TiLV detection in tilapia tissue within 1 h. The method is therefore recommended as a screening tool on farms for the rapid diagnosis of TiLV.

Reverse Transcription Loop-Mediated Isothermal Amplification RT-LAMP Assay for the Specific and Rapid Detection of Tilapia Lake Virus

We present an RT-LAMP assay for the detection of TiLV in tilapia fish using simple instruments over a relatively short period of time compared to conventional RT-PCR techniques. This protocol may help control the epidemic spread of TiLVD, especially in developing countries.

Tilapia lake virus disease (TiLVD), an emerging viral disease in tilapia caused by the tilapia lake virus (TiLV), is a persistent challenge in the ...

Human Respiratory Bacterial Infection Diagnosis Using Isothermal Amplification

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

Author Spotlight: Advancing Rapid Detection of Respiratory Pathogens Using Microfluidic Chip 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, microbiome modification is designed to concentrate genomic DNA of a target foodborne pathogen contaminant to facilitate the detection and subtyping of the pathogen in a single workflow. Here, we explain and demonstrate the sample preparation steps for the quasi-metagenomics analysis of Salmonella enterica from representative food and environmental samples including alfalfa sprouts, ground black pepper, ground beef, chicken breast and environmental swabs. Samples are first subjected to the culture enrichment of Salmonella for a shortened and adjustable duration (4&#8211;24 h). Salmonella cells are then selectively captured from the enrichment culture by immunomagnetic separation (IMS). Finally, multiple displacement amplification (MDA) is performed to amplify DNA from IMS-captured cells. The DNA output of this protocol can be sequenced by high throughput sequencing platforms. An optional quantitative PCR analysis can be performed to replace sequencing for Salmonella detection or assess the concentration of Salmonella DNA before sequencing.

Quasi-metagenomic Analysis of Salmonella from Food and Environmental Samples

Here, we present a protocol to prepare DNA samples from food and environmental microbiomes for concerted detection and subtyping of Salmonella through quasimetagenomic sequencing. The combined use of culture enrichment, immunomagnetic separation (IMS), and multiple displacement amplification (MDA) allows effective concentration of Salmonella genomic DNA from food and environmental samples.&#160;

Quasi-metagenomics sequencing refers to the sequencing-based analysis of modified microbiomes of food and environmental samples. In this protocol, ...

Modified Most Probable Number Assay to Quantify Salmonella in Raw and Ready-to-Cook Chicken Products

Salmonella is a leading cause of foodborne illness in the United States, particularly in poultry products. Traditional methods for detecting Salmonella focus on prevalence rather than quantification, which limits their utility in assessing contamination levels and risks. This study introduces a novel most probable number (MPN) assay designed to quantify Salmonella in ready-to-cook poultry products, such as chicken cordon bleu. The method involves washing the poultry sample, concentrating the rinse through centrifugation, and serially diluting it in a 48-well block. The MPN assay is integrated with the loop-mediated isothermal amplification (LAMP) method to provide a sensitive, accurate, and rapid quantification of Salmonella contamination within the same timeframe as existing Food Safety and Inspection Service (FSIS) protocols. Results show a strong linear correlation between the MPN-LAMP measurements and theoretical inoculation levels (R&#178; = 0.933). However, variability at lower concentrations highlights challenges in accurately detecting Salmonella at these levels, with the practical lower limit of detection estimated at approximately 300 CFU/g. Potential refinements to improve the protocol's applicability include increasing the quantity sampled to further improve the limit of detection, optimizing enrichment media formulations, and expanding molecular detection to target multiple Salmonella serovars. Overall, this study presents a practical tool for the food industry, enabling reliable quantification of Salmonella contamination in poultry products, contributing to improved food safety and public health.

Accurately quantifying Salmonella in poultry at low levels is a current industrial and regulatory challenge. This protocol describes an MPN assay that enables quantification of Salmonella in raw and ready-to-cook poultry products. This method is fast, sensitive, and aligns with FSIS guidelines, enhancing food safety and supporting public health efforts.

Salmonella is a leading cause of foodborne illness in the United States, particularly in poultry products. Traditional methods for detecting Salmonella ...

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

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