LAMP Screening of Salmonella from Animal Food

Animal food may be screened for Salmonella using the loop-mediated isothermal amplification
(LAMP) method on the Genie II or Genie III platform (1). Samples are enriched in buffered
peptone water prior to DNA extraction for LAMP. Only LAMP-positive samples should
continue with the isolation of Salmonella as described in the FDA’s Bacteriological Analytical
Manual Chapter 5, Section D (2). The LAMP assay specifically targets the Salmonella invasion
gene invA (GenBank accession number M90846) (3) and is rapid, reliable, and robust in multiple
food matrices (1, 4-8). The method is capable of detecting < 1 CFU/25 g in animal food (1). The
LAMP assay has been validated in six animal food matrices via a single laboratory validation
(SLV) study (1) and in dry dog food via a multi-laboratory validation (MLV) study (9), with the
latter being performed by seven FDA, state, and academic laboratories. The FDA’s
Microbiological Methods Validation Subcommittee has approved both SLV and MLV reports.

A. Equipment and Materials

1. Genie II or Genie III (OptiGene, Ltd., West Sussex, United Kingdom; also available
through Pro-Lab Diagnostics Inc., Round Rock, TX) or other instrument capable of
temperature control up to 100°C with ± 0.1°C accuracy and simultaneous fluorescence
detection via the FAM channel
2. Genie strips (8-well microtube strips with integral locking caps, working volume of 10 to
150 µl; OptiGene Ltd.) or other equivalent LAMP reaction tubes for selected instrument
3. Genie strip holder (OptiGene Ltd.) or equivalent
4. Vortex mixer
5. Microcentrifuge (capable of spinning at 16,000 × g)
6. Microcentrifuge tubes (0.5 to 2 ml)
7. Pipettes (0.5 to 10 µl, 2-20 µl, 20-200 µl, and 200-1000 µl) and tips (aerosol resistant)
8. Heat block capable of maintaining 100°C
9. Whirl-Pak sterile filter bags with resealable tape and wire (19 × 30 cm and 38 × 38 cm)

B. Media and Reagents

1. Peptone water (0.1%)
2. Isopropanol (70%)
3. DNA AWAY (Thermo Fisher Scientific, Waltham, MA) or equivalent
4. PrepMan Ultra sample preparation reagent (Thermo Fisher Scientific)
5. Sterile molecular grade water
6. Positive control Salmonella DNA (PC)
Any Salmonella reference strains, e.g., Salmonella enterica serovar Typhimurium ATCC
19585 (LT2), may be used as the positive control. Grow the bacteria on a nonselective
agar plate, transfer several single colonies to 5 ml of trypticase soy broth or brain heart
infusion broth, and incubate 16 ± 2 h at 35°C with shaking to reach ca. 109 CFU/ml.

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 Serially dilute the overnight culture in 0.1% peptone water to obtain ca.107 CFU/ml (10-2
dilution). Transfer 500 µl of this dilution to a new microcentrifuge tube, heat 10 min at
100oC in a dry heating block, cool to room temperature, and store at -20°C.
7. ISO-001 isothermal master mix (400 reactions; OptiGene Ltd.)
8. Salmonella LAMP primers (Table 1) and worksheet for making 5× primer mix (Table 2)

Table 1. LAMP primers for use on the Genie II or Genie III platform based on the
Salmonella invA sequence (GenBank accession number M90846)
Primer Description Sequence (5′-3′) Length
name (bp)
Sal4-F3 Forward outer primer GAACGTGTCGCGGAAGTC 18
Sal4-B3 Backward outer CGGCAATAGCGTCACCTT 18
primer
Sal4-FIP Forward inner primer GCGCGGCATCCGCATCAATA- 38
TCTGGATGGTATGCCCGG
Sal4-BIP Backward inner GCGAACGGCGAAGCGTACTG- 38
primer TCGCACCGTCAAAGGAAC
Sal4-LF Loop forward primer TCAAATCGGCATCAATACTCATCTG 25
Sal4-LB Loop backward AAAGGGAAAGCCAGCTTTACG 21
primer

Stock solutions of primers in Table 1 (F3 and B3 at 40 µM each and FIP, BIP, LF, and
LB at 100 µM each) are prepared from commercially synthesized primers with standard
desalting purification (Integrated DNA Technologies, Coralville, IA or equivalent) by
rehydrating with appropriate amount of sterile molecular grade water, and store at -20°C.

Table 2. Worksheet for making 1 ml of primer mix (5×)

Component Stock concentration Concentration in 5× Volume
(µM) primer mix (µM) (µl)
Sal4-F3 primer 40 0.5 12.5
Sal4-B3 primer 40 0.5 12.5
Sal4-FIP primer 100 9 90
Sal4-BIP primer 100 9 90
Sal4-LF primer 100 5 50
Sal4-LB primer 100 5 50
Molecular grade water N/A N/A 695
Total N/A N/A 1,000

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 Primer mix (5×) is prepared by diluting the stock solutions above in sterile molecular
grade water. Mix all reagents well by vortexing for 10 s, aliquot to 200 µl per
microcentrifuge tube, and store at -20°C.

C. Sample Preparation, Enrichment, and DNA Extraction

1. Aseptically weigh 25 g sample into a sterile Whirl-Pak filter bag. Add 225 ml sterile
buffered peptone water and mix 2 min by hand or in a stomacher.
2. Incubate the sample mixture 24 ± 2 h at 35°C.
3. Gently shake incubated sample. Take 1 ml liquid from the filtered side of the bag and
transfer to a microcentrifuge tube. Vortex briefly.
4. Extract DNA using the PrepMan Ultra sample preparation reagent:
Centrifuge 1 min at 900 × g to remove large particles and transfer supernatant to a new
microcentrifuge tube. Centrifuge 2 min at 16,000 × g and discard supernatant. Suspend
the pellet in 100 μl of PrepMan Ultra sample preparation reagent, heat 10 min at 100°C,
cool to room temperature, and centrifuge again 2 min at 16,000 × g. Transfer supernatant
to a new microcentrifuge tube and store sample DNA extracts at -20°C.

D. LAMP Assembly and Data Analysis

1. Overview
Go to https://youtu.be/zJwnfyikWRo for a quick demonstration of the LAMP assembly.

2. Preparation
a. Clean bench with isopropanol and a DNA and DNase degrading solution such as
DNA AWAY and clean pipettes and Genie strip holder also with DNA AWAY.
b. Thaw LAMP ISO-001 isothermal master mix, primer mix (5×), molecular grade
water, positive control Salmonella DNA (PC), and sample DNA extracts at room
temperature.

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 c. Turn on the Genie II or Genie III instrument and tap on the opening screen to access
the home screen.
Create a run by tapping on [LAMP+Anneal] (a default profile consisting of 30 min at
65°C and an annealing step from 98°C to 80°C with 0.05°C decrement per second)
and selecting [Edit]. Enter sample information (8 samples in each block) by tapping
on each sample row to activate the cursor and using the [AB] icon to switch between
the blocks. Tap on the [√] icon when all sample information has been entered.
Optional: You can find all run profiles by tapping on the folder icon at the bottom left
side of the home screen and selecting [Profile].
3. LAMP reaction assembly
a. Always include a positive control Salmonella DNA (PC) and a no template control
(NTC, molecular grade water) in every LAMP run. It is recommended to perform
duplicate testing in independent LAMP runs for each sample.
Note: It is highly recommended to physically separate the areas for preparing the
LAMP master mix and adding DNA templates to prevent cross-contamination.
b. Prepare LAMP master mix (Table 3) by adding ISO-001 isothermal master mix,
primer mix, and sterile molecular grade water into a microcentrifuge tube and vortex
for 10 s. Centrifuge briefly.
Note: When using both blocks A and B (a total of 16 samples), prepare the master
mix for 18 samples. If using only one block (8 samples), prepare the master mix for 9
samples. For other sample numbers, adjust the volume accordingly to accommodate
pipetting loss.

Table 3. Components in a LAMP reaction

Component Working Final Volume Volume (µl) Volume (µl)
concentration concentration per in master in master
sample mix for 18 mix for 9
(µl) samples samples
ISO-001 1.67× 1× 15 270 135
isothermal
master mix
Primer mix 5× 1× 5 90 45
Molecular N/A N/A 3 54 27
grade water
Master mix N/A N/A 23 414 207
subtotal
DNA N/A N/A 2 N/A N/A
template

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 c. Place the Genie strip in the Genie strip holder and distribute 23 µl of the master mix
to each well.
d. Add 2 µl of sample DNA extracts or control template and cap tightly. Flick wrist
while holding the Genie strip to ensure all reagents have pooled at the tip of the tube.
e. Load the Genie strip into block A and/or B of Genie II or the single block in Genie
III.
4. LAMP run on Genie II or Genie III
a. After the Genie strip(s) have been loaded, close the lid and click the [►] icon in the
upper right corner of the screen, select the block or blocks containing Genie strips to
start the LAMP run.
Fluorescence readings are acquired using the FAM channel. The time-to-peak values
(Tmax; min) are determined automatically by the instrument for the time point when
fluorescence ratio reaches the maximum value of the amplification rate curve. The
annealing temperature values (Tm; °C) are obtained automatically for the temperature
point when fluorescence derivative reaches the maximum value of the anneal
derivative curve.
b. While the reaction is in progress, you may tap on the [Temperature], [Amplification],
and [Anneal] tabs to see dynamic changes of these parameters during the LAMP run.
c. Once the run is complete, tap on the [Results] tab to view the final results and tap on
[Amplification] and [Anneal] tabs to see complete amplification and anneal curves.
d. 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 on the instrument panel
a. Open the LAMP run of interest on the instrument panel. There are five tabs
associated with each run: [Profile], [Temperature], [Amplification], [Anneal], and
[Results] (Figure 1).
Note: To open previous runs, tap on the folder icon at the bottom left of the home
screen and select [log]. The runs are organized by date.
b. Tap on the [Results] tab for a tabular view of the results (Figure 1e). There are three
columns (Wells, Amplification, and Anneal). The column entitled “Amplification”
shows the time-to-peak values (Tmax) in minutes and seconds, and the column entitled
“Anneal” shows the annealing temperature values (Tm) in °C.
The NTC well should have no Tmax values while Tm can be either blank (both Genie II
and Genie III) or ca. 81°C (Genie II only). The PC well should have Tmax between 5
and 10 min and Tm around 90°C.
All sample DNA extracts with the correct Tm (approximately 90°C) and Tmax values
between 5-30 min are positive for Salmonella.
c. As duplicate runs have been performed for each sample, when the sample has
consistent results in both runs, final LAMP results can be reported. If two repeats

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 generate inconsistent results, one more time repeat (duplicate runs independently) is
recommended.
6. Interpretation of LAMP results using the Genie Explorer software (version 2.0.6.3 or
higher)
a. Open the Genie Explorer software and the file containing the LAMP run of interest.
There are two additional tabs, [Amplification Rate] and [Anneal Derivative], for a
total of 7 tabs (Figure 2).
Note: A Genie experimental report can be generated from the run by clicking on the
blue notebook icon at the bottom right of the software screen.
b. Tap on the [Result] tab for a tabular view of the results (Figure 2g).
The NTC well should have no Tmax values and blank Tm as the Genie Explorer
software has a refinement step that removes noise in detecting annealing peaks
(approximately 81°C), which may result from low threshold setting on the Genie II
instrument. The PC well should have Tmax between 5 and 10 min and Tm around 90oC.
All sample DNA extracts with the correct Tm (approximately 90°C) and Tmax values
between 5-30 min are positive for Salmonella.
c. As duplicate runs have been performed for each sample, when the sample has
consistent results in both runs, final LAMP results can be reported. If two repeats
generate inconsistent results, one more time repeat (duplicate runs independently) is
recommended.

Figure 1. Examples of LAMP run information and results shown on the Genie II
instrument panel when testing 10-fold serial dilutions of S. enterica serovar Infantis
ATCC 51741 ranging from 1.1 × 10 6 to 11 CFU per reaction. PC is S. Typhimurium
ATCC 19585 (LT2) at 1.7 × 10 4 CFU per reaction and NTC is molecular grade water.
a. The [Profile] tab

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b. The [Temperature] tab

c. The [Amplification] tab

d. The [Anneal] tab

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 e. The [Results] tab

Figure 2. Examples of LAMP run information and results shown in the Genie Explorer
software (for the same run as in Figure 1)
a. The [Profile] tab

b. The [Temperature] tab

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c. The [Amplification] tab

d. The [Amplification Rate] tab

e. The [Anneal] tab

f. The [Anneal Derivative] tab

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 g. The [Result] tab

References
1. Domesle KJ, Yang Q, Hammack TS, Ge B. 2018. Validation of a Salmonella loop-
mediated isothermal amplification assay in animal food. Int J Food Microbiol 264:63-76.
2. Andrews WH, Jacobson A, Hammack TS. 2018. Bacteriological Analytical Manual.
Chapter 5: Salmonella.
http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm070149.htm.
Accessed October 16, 2018.
3. Galan JE, Ginocchio C, Costeas P. 1992. Molecular and functional characterization of the
Salmonella invasion gene invA: homology of InvA to members of a new protein family. J
Bacteriol 174:4338-49.
4. Chen S, Wang F, Beaulieu JC, Stein RE, Ge B. 2011. Rapid detection of viable
salmonellae in produce by coupling propidium monoazide with loop-mediated isothermal
amplification. Appl Environ Microbiol 77:4008-16.
5. Yang Q, Chen S, Ge B. 2013. Detecting Salmonella serovars in shell eggs by loop-
mediated isothermal amplification. J Food Prot 76:1790-6.
6. Yang Q, Wang F, Prinyawiwatkul W, Ge B. 2014. Robustness of Salmonella loop-
mediated isothermal amplification assays for food applications. J Appl Microbiol 116:81-
8.
7. Yang Q, Wang F, Jones KL, Meng J, Prinyawiwatkul W, Ge B. 2015. Evaluation of loop-
mediated isothermal amplification for the rapid, reliable, and robust detection of
Salmonella in produce. Food Microbiol 46:485-93.
8. Yang Q, Domesle KJ, Wang F, Ge B. 2016. Rapid detection of Salmonella in food and
feed by coupling loop-mediated isothermal amplification with bioluminescent assay in
real-time. BMC Microbiol 16:112.
9. Ge B, Domesle KJ, Yang Q, Hammack TS, Wang SS, Deng X, Hu L, Zhang G, Hu Y,
Lai X, Chou KX, Dollete JR, Hirneisen KA, La SP, Richter RS, Rai DR, Yousefvand
AA, Park PK, Wu CH, Eames T, Kiang D, Sheng J, Wu D, Hahn L, Ledger L, Logie C,
You Q, Slavic D, Cai H, Ayers SL, Young SR, Pamboukian R. 2019. Multi-laboratory
validation of a loop-mediated isothermal amplification method for screening Salmonella
in animal food. Front Microbiol 10:562.

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