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.
This Salmonella LAMP method has been incorporated within the FDA's Bacteriological Analytical Manual, both as a screening method in animal food and a confirmation method for any presumptive Salmonella isolates. The method is rapid, reliable, and robust. Compared to PCR, its main advantages include rapid testing, simple instrumentation, a low false-negative rate, and flexible workflow.
Rapid screening for pathogens such as salmonella plays an important role in quickly detecting a potentially contaminated product and can prevent illnesses or outbreaks in humans and animals. Begin by preparing DNA templates for the LAMP assay. There are two types of samples used to make DNA templates.
To prepare templates from animal food samples, aseptically weigh 25 grams of food sample into a sterile filter bag. Add 225 milliliters of sterile buffered Peptone water to the bag and incubate it at 35 degrees Celsius for 24 hours. After the incubation, transfer one milliliter from the filtered side of the bag to a micro centrifuge tube.
Centrifuge the tube at 900 times G for one minute. Then transfer the supernatant to a new micro centrifuge tube. Centrifuge the sample at 16, 000 times G for two minutes.
And to discard the supernatant, resuspend the pellet in 100 microliters of the sample preparation reagent and heat it at 100 degrees Celsius for 10 minutes in a dry heat block. Then, cool the sample to room temperature and store it at negative 20 degrees Celsius. The second type of sample for this LAMP assay comes directly from presumptive salmonella cultures.
To prepare DNA templates, transfer 500 microliters of an overnight salmonella culture to a micro centrifuge tube, and heat it in a 100 degrees Celsius heat block for 10 minutes. Then, cool the sample to room temperature and store it at negative 20 degrees Celsius. To prevent cross-contamination, physically separate the areas used for preparing the LAMP Master Mix and adding the DNA templates.
Clean the work surfaces with isopropanol and a DNA and DNA's degrading solution. Then, clean the pipettes and tube strip holders. Thaw the isothermal Master Mix, 10X Primer Mix, molecular grade water, positive control DNA, and DNA templates at room temperature.
Turn on the LAMP instrument, and enter the relevant sample information. Prepare the LAMP Master Mix according to manuscript directions, vortex and centrifuge it briefly, and distribute 23 microliters of the Master Mix into each well of a tube strip. Vortex all DNA templates and centrifuge them briefly.
Then, add two microliters of DNA template to the appropriate well, and cap the well tightly. Remove the tube strip from the holder, and flick it to ensure all reagents have pooled at the bottom of the tube. Load the tube strip into the LAMP instrument, and start the LAMP run.
While the LAMP reaction is in progress, tap the Temperature, Amplification, and Anneal tabs to see the dynamic changes of various parameters during the LAMP run. The LAMP results can be viewed on the LAMP instrument panel in real time, or using LAMP software. To interpret the results on the instrument panel, open the LAMP run of interest.
Observe the five tabs associated with each run. The Profile and Temperature tabs show programmed and actual temperatures 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 Results tab shows the tabular view of the LAMP results that can be interpreted according to manuscripts directions. The second way to view LAMP results is using the LAMP software. To interpret the results with the software, click on the Computer icon on the left panel and navigate to the file location to load the LAMP run of interest.
Observe the seven tabs associated with each run. The Profile and Temperature tabs show programmed and actual temperatures in the sample wells as the LAMP reaction proceeds. The Amplification and Amplification Rate tabs show fluorescence readings and changes in fluorescence during the Amplification phase.
The Anneal and Anneal Derivative tabs show fluorescence readings and changes in fluorescence during the Anneal phase. The Results tab shows a tabular view of the LAMP results. There are four columns titled Graph Name, Well Number, Well Name, and Peak Value.
The Amp Time and Anneal Derivatives of all eight samples are shown. The results can be interpreted according to manuscripts directions. The LAMP instrument panel and LAMP software can be used to display results of the assay.
In this LAMP run, samples S1 through S6 are tenfold serial dilutions of Salmonella enterica serovar Infantis ATCC 51741, ranging from 1.1 million to 11 colony forming units per reaction. The positive control, or PC, is Salmonella enterica Serovar Typhimurium LT2 at 17, 000 colony forming units per reaction, and the no template control, or NTC, is molecular grade water. The NTC well has a blank Tmax and an Aneal temperature of approximately 81 degrees Celsius on the LAMP instrument panel, and a blank Tmax and a blank Anneal temperature in the LAMP software.
The positive control well has a Tmax of seven minutes and 45 seconds and Annealing temperature of approximately 90 degrees Celsius on both platforms. Samples S1 to S6 have Tmax between six minutes and 30 seconds and 12 minutes and 15 seconds, and Annealing temperature of approximately 90 degrees Celsius, all indicating a positive detection. LAMP is very effective and generates a large quantity of DNA.
So it's critical that best laboratory practices are used to prevent cross-contamination. Use similar practices when making your DNA templates, as enrichments from contaminated animal food can have high levels of salmonella. Animal food sample screening positive with LAMP should be confirmed by culture isolation following the procedures in the FDA's Bacteriological Analytical Manual.
Negative samples can be reported as such. The incorporation of this LAMP method into the FDA's Bacteriological Analytical Manual paves away for wider application of this rapid, robust and user-friendly technology in food safety testing.
