A one-step rapid screening test of Listeria monocytogenes in food samples using a real-time loop-mediated isothermal amplification turbidity assay

A rapid and specific, hly-based, loop-mediated isothermal amplification (LAMP) was applied for the detection of Listeria monocytogenes in food and food products, using a real-time turbidimeter platform (LAMP-turbidity). The principle behind this method relies on an increase in a DNA yield, which correlates with the production of magnesium pyrophosphate, and the results can be determined via an amplification curve within 1 h. The specificity test revealed that L. monocytogenes (DMST 17303) was observed from 34.1 to 38.3 min, while thirty strains of non-L. monocytogenes demonstrated no crossreactions. The limits of detection for purified genomic DNA and pure culture were 800 pg mL 1 and 2.82 10 CFU mL , respectively. Investigation on 200 raw chicken meat samples indicated that the specificity, sensitivity, and accuracy of LAMP-turbidity were 100%, 62.75%, and 90.50%, respectively. These data suggest that an hly-based, real-time, quantitative LAMP-turbidity assay can be an applicable tool for the epidemiological screening of L. monocytogenes in food and food products.


Introduction
Listeria monocytogenes (L. monocytogenes) is an important foodborne pathogen that is found in raw and processed foods. Currently, there is increasing demand for ready-to-eat (RTE) foods, which are possible sources of Listeria infection. 1 Thus far, at least seven different species of the genus Listeria have been reported in the food industry. 2 Among them, three Listeria species, including L. monocytogenes, Listeria ivanovii, and Listeria innocua, are pathogenic. L. monocytogenes, which is a major Grampositive food-borne pathogen in both human beings and animals, has already raised great concerns in several countries as a cause of meningitis, neonatal listeriosis, septicemia, and abortion of infected fetuses. 3 Although the occurrence of listeriosis is quite low, the hospitalization and fatality rates are high, at 94% and 12.8-17%, respectively. 4 Traditional culture-based methods for isolating and enumerating L. monocytogenes from raw food samples generally involve the most probable number (MPN) technique. Although widely used, these methods involve laborintensive steps and are very time-consuming (4-7 days). Some rapid detection methods for L. monocytogenes, 5,6 such as multiplex polymerase chain reaction (PCR) assay, 7 involve gel electrophoresis for analysis of PCR amplicons, which is tedious and timeconsuming. Real-time PCR assays have been developed for the detection of L. monocytogenes, allowing for increased speed and sensitivity. 8 Nonetheless, these assays require a dedicated realtime PCR machine, which is rather expensive and complicated. Alternatively, a lateral ow, dipstick-based immunoassay has been developed and is available but results in a cross-reaction that may yield false-positive results. 1,9 These methods can achieve a high specicity and a low minimum detection limit for the detection of L. monocytogenes, but some of them may still produce false-positive results. 1 Therefore, the development of fast and specic detection approaches that enable the identication of food-borne pathogens is necessary.
Loop-mediated isothermal amplication (LAMP) has been applied for the detection and identication of many bacterial and viral agents. 10,11 The main advantages of the LAMP assay are the use of isothermal amplication and that no special apparatus is needed. A regular laboratory water bath or heating block is adequate for generating the single temperature needed for the synthesis of a large amount of DNA. In the LAMP reaction, a large amount of pyrophosphate ion by-product binds to magnesium ions, forming a white precipitate of magnesium pyrophosphate. This results in turbidity that increases in correlation with the DNA yield and can be quantied by a realtime measurement of turbidity. 12 For gene-based detection, hly gene encoding for listeriolysin O (LLO) toxin is unique and is present as a single copy gene in genome of pathogenic for L. monocytogenes. The LLO toxin is necessary for virulence that shows LLO activity on blood agar and is used for species identication. 13 Herein, the unique hly gene has been employed in the primer design to detect the presence of L. monocytogenes, using a real-time LAMP-turbidity platform. The limit of detection, sensitivity, specicity, and accuracy were investigated in comparison to standard culture and PCR methods.

Bacterial isolates and culture conditions
A total of 35 bacterial isolates (Table 1) were obtained from the Department of Medical Science, the Ministry of Public Health, Thailand (DMST), the Department of Pathology, the Faculty of Medicine, Srinakharinwirot University, HRH Princess Maha Chakri Sirindhorn Medical Center, Thailand (DPSWU), and the Department of Biology, the Faculty of Science, Srinakharinwirot University, Thailand (DBSWU). These bacterial isolates were used for LAMP assay specicity tests. L. monocytogenes (DMST 17303) was used as the positive control in each test involving the sensitivity of LAMP and PCR.
The cultures of the aforementioned isolates were grown overnight in brain heart infusion (BHI) broth (BBL, Becton Dickinson Microbiology Systems, Cockeysville, MD, USA) at 37 C.

DNA extraction
DNA was extracted from an overnight culture of the organism. Briey, bacterial cells were pelleted by centrifugation at 13 000 rpm for 5 min and were re-suspended in 100 mL of sterile, deionized, nanopure water. Then, 100 mL of the suspension was heated at 100 C for 10 min to extract DNA. Aer centrifugation at 13 000 rpm for 1 min, the supernatant was saved for use as the DNA template.
One positive control and one negative control were included in each LAMP run. The LAMP reaction mixtures were heated at 60 AE 2 C for 60 min in a real-time LAMP turbidimeter (Mobilis Automata Co., Ltd., Thailand). Turbidity readings at 650 nm were obtained every second, and the time threshold (T t ; in min) was determined when turbidity measurements increased  (differential values of moving averages of turbidity). Aer 60 min, aliquots (3 mL) of the LAMP products were electrophoresed on a 2% agarose gel (AGE) for 60 min at 80 volts. A negative control reaction was performed using sterile, deionized, nanopure water instead of the DNA template.

PCR reaction
To compare the specicity of the LAMP assay, a conventional PCR assay was performed using LAMP outer primers. The PCR reagent mixture (25 mL) contained 1Â supplied buffer, 1.0 mM MgCl 2 (Vivantis, Malaysia), 0.2 mM dNTPs, 0.3 mM LAMP outer primers, 5U of Taq DNA polymerase (Vivantis, Malaysia), and 2 mL of template DNA. The assay was conducted through an initial denaturation at 95 C for 5 min, followed by 35 cycles of denaturation at 95 C for 30 s, primer annealing at 50 C for 30 s, extension at 72 C for 30 s, and post-extension at 72 C for 10 min in a C1000™ Thermal Cycler (Bio-Rad, USA). The PCR products were analyzed via 2% agarose gel electrophoresis.

LAMP specicity and sensitivity
DNA templates of the 30 bacterial isolates (Table 1) were prepared by heating at 100 C for 10 min, as described previously. Aliquots (2 mL) of each template were subjected to the LAMP assay. LAMP sensitivity (the limit of detection) was determined by performing 10-fold serial dilutions of pure culture and puried genomic DNA of L. monocytogenes (DMST 17303). Aliquots (2 mL) of each template were tested via both LAMP and PCR assays, and repeated three times each.

Real-time turbidity detection in raw food samples
200 raw chicken meat samples were randomly purchased from local delicatessens in Bangkok, Thailand. Twenty-ve gram test portions of the raw chicken meat samples were placed in sterile lter bags (BagFilter®, Interscience, France) containing 225 mL of deionized, ultrapure water (sterile). The bagged samples were crushed and homogenized. The solution was then collected, followed by a 5 min centrifugation to pellet bacterial cells. Aer removing the supernatants, pellets were resuspended in 2 mL of deionized, ultrapure water (sterile). Aliquots (1 mL) of the mixtures were heated at 100 C for 10 min and centrifuged again at 13 000 rpm for 1 min. Two microliters of the sample DNA extracts were subjected to both LAMP and PCR assays, which were repeated in triplicate. The suspended samples (1 mL) were identied as L. monocytogenes using the method outlined by the Bacteriological Analytical Manual (BAM) of the Food and Drug Administration (FDA). 14 Briey, the Listeria enriched sample in selective medium broth was plated on Chromogenic agar. The positive colony was selected, isolated and identied by using Gram staining test, CAMP test on sheep blood agar, catalase test, motility test and biochemical test.

Data analysis
Turbidity proles were recorded by using the LAMP-turbidity Plotter-MEMS LAB-NECTEC program on the monitor of a real-AMP turbidimeter. Means and standard deviations of T t (time threshold for the real-time turbidimeter platform) for LAMP were calculated in Microso Excel (Microso, Seattle, WA). The detection limits were repeated three times and presented as the lowest DNA concentrations or lowest number of specic L. monocytogenes (DMST 17303) cells that could be detected by the assay. The statistic sensitivity, specicity, and accuracy of each assay as well as the prevalence of L. monocytogenes in raw chicken meat were calculated against the gold standard method with regard to the MEDCALC® easy-to-use statistical soware (https://www.medcalc.org/calc/diagnostic_test.php) for 2 Â 2 cross tabulated diagnostic tests 15 (Table 2).

LAMP optimization
Optimization of LAMP-turbidity was conducted at 60 AE 2 C for 60 min as a comparative analysis with 2% agarose gel electrophoresis (AGE). A positive LAMP-turbidity reaction was determined from an increase in a white magnesium precipitate generating a turbidity signal, while negative reactions remained clear ( Fig. 1A and B). The data also revealed that a 60 min incubation time was sufficient to amplify a small amount of template DNA. This could be observed by the naked eye and via a turbidimeter (Fig. 1C).
A white magnesium precipitate was formed in the reaction tube (Fig. 1c) and revealed the typical ladder-like pattern on agarose gel electrophoresis (Fig. 1d), which is a LAMP characteristic.

LAMP analytical sensitivity
The analytical sensitivity or detection limit of the hly-based LAMP-turbidity assay was determined through using 10-fold serial dilutions of L. monocytogenes (DMST 17303) template DNA and pure culture. The test revealed that the maximum T t values for detection of minimum puried DNA and culture were 46.0 and 37.3 min, corresponding to 800 pg and 2.82 Â 10 3 CFU mL À1 , respectively ( Fig. 3 and 4). No turbidity signal was detected for the negative control.

Detection of L. monocytogenes in raw food samples
Direct examination of the 200 raw chicken meat samples (without enrichment) was performed by comparing LAMPturbidity assay results to conventional PCR and LAMP results. According to this culture technique, 51 samples were positive, while 149 samples were negative. Among the 51 positive L. monocytogenes samples, 32 tested positive utilizing LAMP and LAMP-turbidity, while 36 samples tested positive utilizing PCR ( Table 3).
The diagnostic specicity, sensitivity, accuracy, and detection time of PCR, LAMP and LAMP-turbidity for detection of L. monocytogenes (based on the hly gene) were calculated in comparison to standard culture. The data clearly demonstrated that all tests showed 100% diagnostic specicity. The diagnostic sensitivity of the PCR, LAMP and LAMP-turbidity assays was 70.59%, 62.75% and 62.75%, respectively ( Table 3). The accuracy of the PCR, LAMP and LAMP-turbidity assays was 92.50%, 90.50%, and 90.50%, respectively ( Table 3). The prevalence of L. monocytogenes in raw chicken meat collected from local markets in Bangkok during the indicated time period was 25.50%. LAMP-turbidity of thirty representative raw food samples were achieved and displayed in Fig. 5.

Discussion
In this study, a one-step, qualitative, real-time, LAMP-turbidity assay was developed for the detection of L. monocytogenes from puried genomic DNA, in pure culture, and in the raw chicken meat samples. Many studies have evaluated the LAMP assay for large-scale screening of food-borne pathogens such as E. coli O157:H7, Salmonella, Shigella, and Vibrio parahaemolyticus. 10,[16][17][18] Generally, amplied products have been evaluated through agarose gel electrophoresis and subsequent staining with ethidium bromide, revealing a ladder pattern of DNA bands under ultraviolet light. However, this assay has several drawbacks, such as the use of highly toxic ethidium bromide, which is hazardous to health and the environment. 9 Turbidity analysis of the magnesium pyrophosphate precipitate allowed for persistent monitoring of accumulative DNA synthesis in the LAMP reaction with a single tube. 12 Turbidity and dye color changes are normally used to check the progress of the LAMP reaction. Changes in turbidity are mainly due to the white, focal phosphatase precipitate generated during the reaction, but this can be difficult to detect at low levels. A color change is observed aer adding calcein and SYBR Green I to the reaction mixture. 19,20 Hence, these two methods are employed in analysis to avoid the use of carcinogenic ethidium bromide. However, these dyes require equipment for observing illumination which may be an inconvenience for the user. Hence, LAMP combined with a turbidimeter can be considered as a one-step, qualitative, real-time method that can be manipulated within approximately one hour. Referring to our previous publication, the optimum LAMP was achieved at 60 min. 21 As the LAMP reaction progresses, pyrophosphate ions produced from deoxyribonucleotides (dNTPs) bind to magnesium ions and subsequently form a white precipitate of magnesium pyrophosphate. 22 The amount of the precipitate can be measured using a real-time turbidimeter, which is more appropriate for monitoring LAMP reactions because it can detect turbidity at low levels and presents no issues with aerosol contamination. In this study, it is noted that the signal and duration of turbidity are the keys of detection. Nevertheless, in certain samples of high DNA concentration or CFU, the turbidity starts to decline aer a while. This may affect the excess formation of the magnesium pyrophosphate product which precipitates and settles down to the bottom of the tube aer a while.
Therefore, this method is more reliable than other similar methods and is recommended for health management applications involving the prevention of food-borne outbreaks. This method is important and necessary to prevent the spread of L. monocytogenes in the food market, decrease mortality rates from human illness, and reduce the economic loss of farms. However, LAMP is less stable than PCR and carries a higher false-positive rate than PCR under a variety of experimental conditions. The cross-amplication of LAMP also produces magnesium pyrophosphate, leading to a false turbidity interpretation.
The hly gene, which encodes listeriolysin O of L. monocytogenes, was employed in the primer design due to its uniqueness, which was key for achieving specicity in the realtime, qualitative LAMP assay. Among 5 isolates of L. monocytogenes, 3 isolates of other Listeria species, and 27 isolates of non-Listeria spp., the one-step, real-time, hly-based LAMPturbidity assay generated 100% inclusivity and 100% exclusivity. These results suggested that the hly-based LAMPturbidity assay has both high diagnostic specicity and ampli-cation efficiency. The analytical sensitivity of hly-based PCR was ten times more sensitive than that of hly-based LAMP and real-time LAMP-turbidity. However, LAMP and real-time LAMP-turbidity were rapid when compared to the PCR assay. 21 In addition, this closed tube method can minimize the problem of carry-over contamination in less controlled environments and is effective for concurrent testing of several samples.
Considering the direct detection of L. monocytogenes using LAMP-turbidity based on the hly gene without loop primers, the limit of detection was 2.82 Â 10 3 CFU mL À1 which agrees with that of Shan X and coworkers (2.82 Â 10 3 CFU mL À1 or 6 CFU per reaction). 23 However, the authors noted that the false positive was observed due to the use of loop primers. 23 In our experiment, the intensity and length of turbidity are not related to the level of pathogen present. However, the minimum level of L. monocytogenes means that the limit of detection is the lowest quantity of a substance that can be distinguished from its absence. Regarding the limit of detection, hly-LAMP turbidity was almost 3 and 10 times less sensitive than those of Tang 25 According to Tang M. J. and coworkers, the assay was accomplished by LAMP in the presence of calcein and observation of the calcein-magnesium complex under UV/VIS light encompassing 2 steps of detection. 24 Similarly, Wang D. and coworkers also demonstrated the 2-step assay of L. monocytogenes using LAMP of the iap gene and analyzed the LAMP products on agarose gel electrophoresis which involved carcinogenic ethidium bromide in the staining step. 25 Referring to BAM of FDA's standard culture method, the 51 out of 200 raw food samples were L. monocytogenes positive. Among them, 32 tested positive utilizing LAMP and LAMP-turbidity, while 36 samples tested positive utilizing PCR. Regarding the hly-LAMPturbidity of L. monocytogenes in the raw food samples, the diagnostic sensitivity was less than that of hly-PCR. According to our previous publication, the hly-PCR assay was 10 times more sensitive than the hly-LAMP assay. 21 It is possible that the 32 samples of both LAMP-turbidity and PCR positive contain bacteria in the range of detection whereas 4 real samples of discrepancy between the two techniques (PCR positive, LAMP negative) may contain the amount of DNA less than the limit of detection of hly-LAMP. However, it was suggested that the pre-enrichment sample prior to the LAMP-turbidity assay can improve the sensitivity of the test. Nevertheless, this may not be suitable for use as a rapid screening test since it is time-consuming. As such, the direct detection of L. monocytogenes using LAMP-turbidity is convenient and easy to manipulate when compared to other assays. Furthermore, this method can be utilized as a point-of-care test since post-amplication analysis is not required. Therefore, this method is suitable for rapid screening of L. monocytogenes in food products and diminishes the risks associated with the consumption of these high-risk foods. Fig. 3 Sensitivity of the LAMP-turbidity assay using purified genomic DNA. The turbidity graphs were displayed based on three repeats corresponding to 10-fold serial dilutions of L. monocytogenes DMST 17303 purified genomic DNA ranging from 800 ng to 8 pg. R 1 , repeat 1; R 2 , repeat 2; R 3 , repeat 3; N, negative control (without the DNA template). Fig. 4 Sensitivity of the LAMP-turbidity assay using cells from a pure culture. The turbidity graphs were displayed based on three repeats corresponding to 10-fold serial dilutions of L. monocytogenes DMST 17303 cells from a pure culture ranging from 2.82 Â 10 8 to 2.82 Â 10 3 CFU mL À1 . R 1 , repeat 1; R 2 , repeat 2; R 3 , repeat 3; N, negative control (without the DNA template).