DIRECT DETECTION OF LISTERIA MONOCYTOGENES BY RECOMBINASE POLYMERASE AMPLIFICATION

Listeria monocytogenes is one of the most common types of food poisoning bacteria which can cause serious foodborne diseases or even lethality. Generally, L. monocytogenes can be detected using traditional microbiology or molecular biology techniques, notably PCR. However, the application of these methods at the field is restricted due to the strict requirement of equipment and skilled personnel. In this study, recombinase polymerase amplification (RPA), an isothermal PCR assay was developed to rapidly detect L. monocytogenes in the crude samples. The results showed that the RPA reaction, without requiring complex thermal cycles, was well-performed in the optimal conditions of 39°C within only 25 minutes. The limit of detection was identified as 310 fg of L. monocytogenes genomic DNA, which was 1000-fold more sensitive than the conventional PCR. In addition, RPA also succeeded to directly detect L. monocytogenes cells at a concentration as low as 2.5 × 101 Colony Forming Unit (CFU)/ml in pure cultures and 2.5 × 102 CFU/ml in crude samples without sample extraction or processing. Therefore, RPA established in this study could be an alternative standard method to confirm the presence of L. monocytogenes in food. Accordingly, this rapid and sensitive method could be further applied to clinical testing for the diagnosis of L. monocytogenes infection, especially in areas with limited settings.


Bacterial cultivation
L. monocytogenes (laboratory collection) was cultured overnight at 37°C in Brain Heart Infusion broth (HiMedia Laboratories Pvt. Ltd, India). Through shaking bacterial cultures at 180 rounds per min (rpm), precipitation of cells was avoided. Other bacterial strains (laboratory collection) including Salmonella enterica, Staphylococcus aureus, Clostridium perfringens, Bacillus cereus, and Vibrio parahaemolyticus were cultured similarly.

DNA extraction
The cetrimonium bromide (CTAB) extraction buffer contains 2% (w/v) CTAB, 100 mM Tris-HCl (pH 8), 20 mM EDTA (pH 8), 1.4 M NaCl. The L. monocytogenes cells were harvested from 1 ml of culture by centrifugation, and supernatants were then discarded. Cells were resuspended in 800 µ l of the pre-warmed (65°C) CTAB lysis buffer and mixed thoroughly, then incubated at 65°C for 60 min. Samples were then centrifuged at 4°C for 15 min at 14000 g. Supernatants were transferred to fresh tubes and an approximately equal volume of Phenol: Chloroform: Isoamyl alcohol (PCI) was added and mixed thoroughly. Phase separation occurred by centrifugation at 14000 g for 15 min at 4°C. The upper aqueous phases were moved to new tubes and a 2.5 equal volume of ethanol 99% was added. DNA was precipitated overnight at -20°C. The DNA was then dissolved with 50 µ l of elution buffer. The concentration and purification of DNA were measured with a Genova Plus Spectrophotometer (Jenway, Staffordshire, UK). The extracted genomic DNA was then stored at -20°C until use.

Primer design
The RPA primers targeting the plcA gene of L. monocytogenes (Gene bank: CP023861.1) were designed according to the guidelines provided by TwistDx (Cambridge, UK) (http://hdl.handle.net/20.500.11794/26269). The primer set was chosen by assessing the specificity using NCBI BLAST and the amplification effects were also evaluated practically. In silico PCR analysis function available in FastPCR software (http://primerdigital.com/fastpcr.html) was additionally utilized for primer specificity analysis. The primer sequences were aligned to the reference genome sequences of other strains downloaded from NCBI. Two candidate primer pairs were commercially synthesized by IDT (Singapore). The primer pairs that produced the clearly visible bands representing for amplified product in agarose gel electrophoresis were selected and the sequences were listed in Table 1.

PCR reaction
The PCR was carried out in a reaction volume of 20 μ l in small tubes containing 0.4 µ M each of RPA primers, 4 µl of 5X Mytaq reaction buffer (Bioline, London, UK), 0.2 µl of MyTaq DNA polymerase (Bioline, London, UK) and 1 µl of DNA template. The PCR reaction was run as follows: initial denaturation stage at 95°C for 2 min, 35 cycles of 95°C for 30 seconds, 58°C for 30 seconds, 72°C for 30 seconds, and final extension stage at 72°C for 10 min. The PCR results were analyzed by a 1.5% agarose gel electrophoresis.

RPA reaction for detection of L. monocytogenes
The RPA assay was performed referring to the TwistDx's recommended protocols (http://hdl.handle.net/20.500.11794/26269). The reaction mixture containing 29.5 µ l of a rehydration buffer, 2.4 µl of 10 µ M forward and reverse primer, 12.2 µl of sterile water and 1 µl of the template was transferred to a lyophilized pellet tube. Then, 2.5 µl of magnesium acetate was added to start the reaction. Sterile water was used as a negative control sample. The tubes were mixed thoroughly and then centrifuged briefly. Subsequently, the tubes were incubated at 39°C for 25 min in BioSan Dry block thermostat Bio TDB-100. A mixing step after 4 min of incubation was carried out for better sensitivity of the assay. Finally, 50 µl of PCI was added to the tubes and vortexed lightly. The tubes were centrifuged at 14000 g for 10 min to remove the undesirable components affecting the read-out of the results. The RPA products were analyzed by a 1.5% agarose gel electrophoresis and visualized under the UV light using G: BOX Mini 6/9 (Syngene, Cambridge, UK).

Optimization of RPA reaction
According to the manufacturer's guidelines, the effective incubation temperature of TwistAmp Basic kit (https://www.twistdx.co.uk/en/products/product/twistamp-basic) used in this study ranges between 37°C -39°C. Therefore, to determine the optimal reaction temperature and incubation time of the RPA reaction, the RPA assays were performed at temperatures ranging from 35°C to 41°C for different times including 15, 20, 25 and 30 min. The amount of 31 pg of genomic DNA was used as the template for optimizing the L. monocytogenes RPA assay.

Evaluation of Specificity of RPA assay
The specificity of the RPA reaction was assessed under the optimal temperature and incubation time determined. Cross-reactivity analysis using the extracted genomic-DNAs of other typical foodborne pathogens including S. enterica, S. aureus, C. perfringens, B. cereus, and V. parahaemolyticus (laboratory collection) was also performed.

Evaluation of LOD of RPA assay
To evaluate the LOD of the RPA assay, a 10-fold serial dilution from 310 ng to 3.1 fg of the extracted genomic-DNA of L. monocytogenes was prepared. One µl of each DNA concentration was utilized as the template for RPA and PCR assays. The LOD of the RPA reaction was compared with the LOD of the PCR assay.
To determine the LOD of the direct RPA assay, various concentrations of the L. monocytogenes cell culture were prepared. L. monocytogenes was initially cultured in 10 ml of fresh Brain Heart Infusion broth at 37°C for 24 hours. The L. monocytogenes cell concentration was determined using the count plating method. Next, 1 ml of the L. monocytogenes culture was centrifuged at 4000 g for 20 min at 4°C to harvest the cells. The final pellet was washed two times and resuspended in 100 µl of 0.9% NaCl. A serial dilution of the L. monocytogenes cells was then prepared to attain samples with a final concentration ranging from 2.5 × 10 8 to 2.5 × 10 0 CFU/ml. Then one µl of each cell concentration was directly utilized as the template for the RPA assay.

Direct detection of L. monocytogenes in contaminated milk
To evaluate the efficiency of RPA assay for the direct detection of L. monocytogenes in contaminated milk, the cell pellets harvested from different concentrations of cell cultures were spiked into 1 ml of the pasteurized milk purchased from the local supermarket. The tubes were vortexed and centrifuged at 6000 g for 3 minutes. Next, 900 µ l of the upper liquid was removed gently. Then, each sample was used as the template for direct RPA assays.

RESULTS AND DISCUSSION
RPA assay for detection of L. monocytogenes RPA primers were designed to detect the plcA gene of L. monocytogenes. The target region is an important virulence gene that has been shown to have high specificity for diagnosing L. monocytogenes strain (Lida et al., 2014). The RPA reactions were performed using 3.1 ng of the genomic DNA of L. monocytogenes as a template. The result showed that the assay produced a clearly visible band at approximately 228 base pairs (bp) of the expected size when analyzed by gel electrophoresis (Fig. 1, lane 4). The size of the amplified product obtained by RPA is similar to the amplicon produced by PCR when using the same primer set (Fig. 1, lane 2), indicating that the RPA primers designed successfully amplified the target sequence as expected. The optimal temperature and incubation time of the RPA reaction for the detection of L. monocytogenes were determined. The results indicated that the highest amount of amplified product was observed at 39°C (Fig. 2A, lane 4). For incubation time, the RPA amplicon could be seen just after 15 min and got saturation after 25 min (Fig. 2B). Thus, the optimal condition of the RPA reaction for detection of L. monocytogenes genomic DNA was set at 39°C for 25 min.

Specificity of RPA assay for detection of L. monocytogenes
In silico PCR analysis showed that the designed primer pair would not amplify the genome sequences of 20 different bacterial strains, supporting that the selected primer set has high specificity for identifying L. monocytogenes (Table 2). To practically evaluate the specificity of RPA assay developed for detection of L. monocytogenes, genomic DNAs of several bacteria commonly causing food poison were extracted and used as the template for RPA reactions. The results indicated that no cross-reactivity was observed with the foodborne bacterial strains examined including S. enterica, S. aureus, C. perfringens, B. cereus, and V. parahaemolyticus (Fig. 3). Thus, the RPA primer pair designed is highly specific for L. monocytogenes.

Detection limit of RPA assay for detection of L. monocytogenes
Evaluation of the sensitivity of L. monocytogenes RPA assay developed was performed using a ten-fold dilution of extracted DNA of L. monocytogenes. The results showed that the lowest amount of extracted-genomic DNA that RPA could detect was 310 fg/reaction which equivalents to 99 genome copies per reaction (Fig. 4A). Meanwhile, the LOD value of PCR utilizing the same primer set was identified at 310 pg/reaction (Fig. 4B). The RPA assay is thus approximately 1000 times more sensitive than the PCR reaction in this study.

Direct RPA assay for detection of L. monocytogenes
We attempted to detect L. monocytogenes cells from unextracted samples by the RPA assay developed. To this end, the cell culture of L. monocytogenes was used directly as the template for RPA reactions. As expected, the RPA assay could detect the presence of L. monocytogenes genomic DNA without the sample extraction process (Fig. 5A). Next, the LOD of direct RPA for detection of L. monocytogenes was determined using the serial dilution of L. monocytogenes cell culture. The results showed that the amplified products could be observed at the cell concentrations ranging from 2.5 × 10 6 to 2.5 × 10 1 CFU/ml (Fig. 5B). There was no RPA amplicon produced at 2.5 CFU/ml (Fig. 5B, lane 7). Thus, the LOD of L. monocytogenes direct RPA was determined at 2.5 × 10 1 CFU/ml.

Direct detection of L. monocytogenes cells by RPA in contaminated milk
To examine the ability of direct RPA assay using food sample, the artificially contaminated milk was prepared by spiked with L. monocytogenes cells at low concentrations ranging from 2.5 × 10 2 to 2.5× 10 0 CFU/ml. Without the need for DNA extraction or sample processed or cell enrichment, the LOD of direct RPA assay using milk samples was defined at 2.5 × 10 2 CFU/ml (Fig. 6). The LOD value was ten times higher than that of direct RPA using cell culture, indicating that there are certain components in the milk sample interfering with the RPA reaction to some extent. In this study, the RPA assay was developed to specifically detect L. monocytogenes in direct crude samples. The RPA amplification efficiency depends on the target sequence, amplicon size, and quality and type of sample tested (Daher et al., 2016).
Most previous studies analyzed the RPA performance using genomic DNA extracted from enrichment solution or using the spiked-L. monocytogenes food samples that were boiled or pretreated with lysis buffer to release the DNA (Gao et al., 2017; Du et al., 2018). These steps made the RPA assays previously developed more time-consuming, limiting their application at the field. In this study, we eliminated the genomic DNA extraction and used contaminated milk directly for the RPA reaction. The approach makes the testing handling simpler and faster in the diagnosis of L. monocytogenes. The L. monocytogenes RPA assay is advantageous due to no requirement for a specialized thermocycler. The assay could efficiently amplify the target sequence within 25 min at a low temperature of 39°C. The short testing time and low incubation temperature are beneficial for the early detection of L. monocytogenes in practical application. These advantages make the RPA assay developed to detect L. monocytogenes time-saving and cost-effective at the field with restricted resources. The developed RPA assay had high specificity and sensitivity. No cross-reactivity was observed with several bacteria tested, which is in agreement with the previous study. Besides, the method has been proven that it was extremely sensitive compared to the PCR assay in this study. The LOD of the RPA assay was 310 fg of extracted DNA, indicating a 1000-fold higher sensitivity than PCR. Also, the RPA assay could directly detect as low as 25 CFU/ml of L. monocytogenes cells in medium cultures and 2.5 × 10 2 CFU/ml of L. monocytogenes cells in contaminated milk. The obtained results agree with the previous studies, showing that RPA assays succeeded to directly detect the target bacteria in simulated clinical samples without the need for genomic DNA extraction ( In summary, the direct RPA assay developed is a specific and rapid approach to alternate the traditional methods for efficiently and accurately diagnosing L. monocytogenes in food. Further evaluation of the assay with different types of crude samples by clinical testing for the diagnosis of L. monocytogenes infection, particularly in areas with restricted settings should be performed.

CONCLUSION
The direct RPA assay which is rapid and sensitive developed in this study could be an alternative method for the diagnosis of L. monocytogenes infection, especially in areas with limited resources.