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

NOTE: A LAMP reaction mix contains DNA polymerase, buffer, MgSO₄, dNTPs, primers, DNA template, and water. The first four reagents are contained in an isothermal master mix (Table of Materials). Primers are premixed in-house to become a primer mix (10x). DNA templates can be prepared from enrichment broths of animal food samples for screening purpose or from cultures of presumptive Salmonella isolates for confirmation purpose. In addition, a positive control (DNA extracted from any Salmonella reference strains, e.g., Salmonella enterica serovar Typhimurium ATCC 19585 [LT2]) and a no template control (NTC; sterile molecular grade water) are included in every LAMP run.

1. Preparation of DNA templates

1. To prepare DNA templates from animal food enrichments, follow these steps.
   1.  Aseptically weigh 25 g of animal food sample (e.g., dry cat food, dry dog food, cattle feed, horse feed, poultry feed, and swine feed) into a sterile filter bag (Table of Materials), or equivalent. Place the bag into a large container or rack for support during incubation.
   2. Add 225 mL of sterile buffered peptone water (BPW). Mix well by swirling and brief hand-massage. Let stand at room temperature for 60 ± 5 min.
   3. Mix well by swirling and determine pH with a test paper. Adjust pH, if necessary, to 6.8 ± 0.2 with sterile 1 N NaOH or 1 N HCl. Incubate at 35 ± 2 °C for 24 ± 2 h.
   4. Mix well by swirling the bag containing animal food enrichment broths. Transfer 1 mL from the filtered side of the bag to a microcentrifuge tube. Vortex briefly.
   5. Extract DNA using a sample preparation reagent (Table of Materials) as follows.
      1. Centrifuge at 900 x g for 1 min to remove large particles and transfer supernatant to a new microcentrifuge tube.
      2. Centrifuge at 16,000 x g for 2 min and discard supernatant.
      3. Suspend the pellet in 100 µL of the sample preparation reagent and heat at 100 ± 1 °C for 10 min in a dry heat block.
      4. Cool to room temperature and store sample DNA extracts at -20 °C.
2. To prepare DNA templates from presumptive Salmonella cultures, follow these steps.
   1. Obtain presumptive Salmonella isolates from culture isolation in all foods following FDA’s BAM Chapter 5 Salmonella section D: Isolation of Salmonella²⁷.
   2. Inoculate presumptive Salmonella isolates on a nonselective agar plate (e.g., blood agar, nutrient agar, and trypticase soy agar) and incubate at 35 ± 2 °C for 24 ± 2 h.
   3. Transfer several single colonies to 5 mL of trypticase soy broth (TSB) or brain heart infusion (BHI) broth and incubate at 35 ± 2 °C for 16 ± 2 h.
      ​NOTE: This step can be optional if the presumptive Salmonella culture is pure. In that case, DNA templates can be prepared by suspending several single colonies in 5 mL of TSB and heat 500 μL of the suspension at 100 ± 1 °C for 10 min in a dry heat block. Continue with step 5 below.
   4. Transfer 500 µL of the overnight culture to a microcentrifuge tube and heat at 100 ± 1 °C for 10 min in a dry heat block.
   5. Cool to room temperature and store isolate DNA extracts at -20 °C.
3. To prepare positive control DNA, follow similar steps as above for preparing DNA templates from presumptive Salmonella cultures with one extra dilution step.
   1. Inoculate S. Typhimurium ATCC 19585 (LT2) or any Salmonella reference strains on a nonselective agar plate (e.g., blood agar, nutrient agar, and trypticase soy agar) and incubate at 35 ± 2 °C for 24 ± 2 h.
   2. Transfer several single colonies to 5 mL of TSB or BHI broth and incubate at 35 ± 2 °C for 16 ± 2 h to reach ~10⁹ CFU/mL.
   3. Serially dilute the overnight culture in 0.1% peptone water to obtain ~10⁷ CFU/mL.
   4. Transfer 500 µL of this dilution to a microcentrifuge tube and heat at 100 ± 1 °C for 10 min in a dry heat block.
   5. Cool to room temperature and store positive control DNA at -20 °C.

2. Preparation of primer mix (10x)

1. Obtain commercially synthesized LAMP primers (Sal4-F3, Sal4-B3, Sal4-FIP, Sal4-BIP, Sal4-LF, and Sal4-LB) with standard desalting purification (Table 1).

Table 1: LAMP primers for screening Salmonella in animal food and confirming Salmonella from culture isolation. The primers are designed based on the Salmonella invA sequence (GenBank accession number M90846).

2. Prepare stock solutions of each primer (100 µM) by rehydrating the primer with appropriate amount of sterile molecular grade water. Mix well by vortexing for 10 s and store at -20 °C (-80 °C for long-term storage).
3. Prepare the primer mix (10x) according to a worksheet (Table 2). Add appropriate volumes of primer stock solutions and sterile molecular grade water into a microcentrifuge tube. Mix all reagents well by vortexing for 10 s.

Table 2: Worksheet for preparing the LAMP primer mix (10x). The primers are listed in Table 1.

4. Aliquot the 10x primer mix to 500 µL per microcentrifuge tube and store at -20 °C.

3. Assembly of a LAMP reaction

NOTE: To prevent cross-contamination, it is highly recommended to physically separate the areas used for preparing the LAMP master mix and adding DNA templates. Figure 1 is a LAMP diagram.

Figure 1: A schematic diagram of the Salmonella LAMP workflow. Please click here to view a larger version of this figure.

1. Preparation and run setup
   1. Clean bench with isopropanol and a DNA- and DNase-degrading solution (Table of Materials). Clean pipettes and tube strip holders (Table of Materials) with the DNA- and DNase-degrading solution.
   2. Thaw the isothermal master mix, primer mix (10x), molecular grade water, positive control DNA, and DNA templates at room temperature.
   3. Turn on the LAMP instrument (Table of Materials) and tap the opening screen to access the home screen. Follow these steps to create a run.
      NOTE: One model of the LAMP instrument has 2 blocks (A and B) with 8 samples in each block and another model has a single block that accommodates 8 samples (Table of Materials).
      1. Tap LAMP+Anneal and select Edit to enter sample information.
         NOTE: The default LAMP run profile consists of amplification at 65 °C for 30 min and an anneal phase from 98 °C to 80 °C with 0.05 °C decrement per sec.
      2. Tap each sample row to activate the cursor and enter relevant sample information, using the AB block icon to switch between the two LAMP instrument blocks.
      3. Tap the Check icon when all sample information has been entered.
         NOTE: Optionally, the run setup (termed “Profile,” which contains sample information along with the default LAMP run profile) may be saved for later use. Tap the Save icon and give the profile a unique name. When testing this same set of samples next time, a new run can be initiated using the saved profile. Tap the Folder icon at the bottom left of the home screen and select Profile to load saved profiles.
2. LAMP reaction assembly
   NOTE: When using both LAMP instrument blocks (A and B, a total of 16 samples), prepare the LAMP master mix for 18 samples. If using only one LAMP instrument block (8 samples total), prepare the LAMP master mix for 10 samples. For other sample numbers, adjust the volume accordingly to accommodate pipetting loss. Always include a positive control and an NTC in every LAMP run. Duplicate testing of each sample in independent LAMP runs is recommended.
   1. Prepare the LAMP master mix according to a worksheet (Table 3). Add appropriate volumes of the isothermal master mix, primer mix, and molecular grade water into a microcentrifuge tube and vortex gently for 3 s. Centrifuge briefly.
   2. Place the tube strip in the strip holder and distribute 23 µL of the LAMP master mix to each well.
   3. Vortex all DNA templates and centrifuge briefly. Add 2 µL of DNA template to the appropriate well and cap tightly.
   4. Remove the tube strip from the holder and flick wrist to ensure all reagents have pooled at the bottom of the tube.
   5. Load the tube strip into the LAMP instrument block(s), ensuring caps are secure before closing the lid.

Table 3: Worksheet for preparing the LAMP reaction mix. The primer mix (10x) is prepared according to Table 2 using stock solutions of primers listed in Table 1.

4. LAMP Run

NOTE: During a LAMP run, fluorescence readings are acquired using the FAM channel. The time-to-peak values (T_max; min) are determined automatically by the instrument for the time point when fluorescence ratio reaches the maximum value of the amplification rate curve. The T_m (°C) is the melting/annealing temperature of the final amplified product.

1. Click on the Run icon at the upper right of the screen and select the block(s) containing tube strip(s) to start the LAMP run.
2. Optionally, while the reaction is in progress, tap the Temperature, Amplification, and Anneal tabs to see dynamic changes of various parameters during the LAMP run.
3. Once the run is complete, tap the Amplification and Anneal tabs to see complete amplification and anneal curves and tap the 結果 tab to view the results.
4. Optionally, for record keeping, record the run number located at the top left of the screen, using the format of “instrument serial number_run number,” e.g., “GEN2-2209_0030.”

5. Interpretation of LAMP Results

NOTE: LAMP results can be viewed on the LAMP instrument panel directly and/or using a LAMP software (Table of Materials).

1. To interpret LAMP results on the instrument panel, follow these steps.
   1. Tap the Folder icon at the bottom left of the home screen and select Log to navigate to the file location to load the LAMP run of interest.
      NOTE: The LAMP runs are organized by date, starting with year.
   2. Observe the five tabs associated with each run: Profile, Temperature, Amplification, Anneal, and 結果.
      NOTE: The Profile and Temperature tabs show programmed and actual temperatures, respectively, in the sample wells as the LAMP reaction proceeds. The Amplification and Anneal tabs show fluorescence readings and changes in fluorescence during the amplification and anneal phases, respectively. The 結果 tab shows a tabular view of the LAMP results.
   3. Tap the 結果 tab to observe LAMP results for each well.
      NOTE: There are three columns (Well, Amplification, and Anneal). The “Amplification” column shows the time-to-peak values (T_max; min:sec) for each sample (“Well”) and the “Anneal” column shows the melting/annealing temperatures (T_m; °C) for any amplified product in that well.
   4. Interpret the LAMP results and report final LAMP results as follows.
      1. Examine the control wells first. The NTC well should have blank T_max while T_m can be either blank (both LAMP instrument models) or < 83 °C (only for the LAMP instrument model with two blocks). The positive control well should have T_max between 5 and 10 min and T_m around 90 °C.
      2. Examine the sample wells. All samples with the correct T_m (approximately 90 °C) and T_max (between 5−30 min) are considered positive for Salmonella.
      3. Report final LAMP results based on results from duplicate runs. If the duplicate runs have consistent results, final LAMP results can be reported. If duplicate runs are inconsistent, repeat both runs independently. If results are still inconsistent, the sample should be considered presumptive positive for Salmonella and will need to go through culture confirmation.
2. To interpret LAMP results using the software, follow these steps.
   1. Click on the Computer icon on the left panel and navigate to the file location to load the LAMP run of interest.
      NOTE: The computer with the software installed does not need to be connected to the LAMP instrument to analyze LAMP results, i.e., remote access is available. The LAMP runs are organized by date.
   2. Observe the seven tabs associated with each run: Profile, Temperature, Amplification, Amplification Rate, Anneal, Anneal Derivative, and Result.
      NOTE: Similar to the instrument panel view, the Profile and Temperature tabs show programmed and actual temperatures, respectively, in the sample wells as the LAMP reaction proceeds. The Amplification/Amplification Rate and Anneal/Anneal Derivative tabs show fluorescence readings or changes in fluorescence during the amplification and anneal phases, respectively. The 結果 tab shows a tabular view of the LAMP results which differ slightly from the instrument panel view.
   3. Tap the Amplification Rate tab to view a graphic display of the fluorescence ratios by time. Click on the Setting icon at the top right of the screen and adjust the “Peak Detection Threshold Ratio” from 0.020 to 0.010.
      NOTE: The adjustment is necessary to ensure that all valid peaks are identified, and the results obtained using the software match with those displayed on the instrument panel.
   4. Tap the Result tab to observe LAMP results for each well.
      NOTE: There are four columns (Graph Name, Well Number, Well Name, and Peak Value). The top portion of the “Peak Value” column shows “Amp Time” (T_max; min:sec) for each sample (“Well Name”) while the bottom portion shows “Anneal Derivative” (T_m; °C) for any amplified product in that well.
   5. Interpret the LAMP results and report final LAMP results following similar steps as when using the instrument panel with one exception that the NTC well and other negative samples should have blank T_m as the LAMP software settings eliminate those T_m < 83 °C results. Similarly, all samples with the correct T_m (approximately 90 °C) and T_max (between 5−30 min) are considered positive for Salmonella.