SIMULTANEOUS DETECTION OF FOUR FOOD BORNE BACTERIAL PATHOGENS BY METAL HYDROXIDE IMMOBILIZED MULTIPLEX PCR METHOD

Salmonella enterica, Vibrio parahaemolyticus, Escherichia coli and Listeria monocytogenes are human pathogenic bacteria which could be contaminated with many types of food, especially meat products. Testing of food for those pathogens has become routine practice all over the world. However, since the available traditional detection methods are time consuming and labor intensive, there is a need for rapid, sensitive, specific and cost effective detection technique. In this study, a rapid and sensitive single tube Multiplex PCR method for simultaneous detection of those four bacterial pathogens by amplification of target genes gyrB of V. parahaemolyticus, invA of S. enterica, eaeA of E.coli and hly of L. monocytogene was established. Concentration of bacterial cells from food samples for DNA extraction is another problem in testing of food pathogens by PCR techniques. Metal hydroxide immobilization was applied prior to DNA extraction and found to be effective step to overcome this problem.


INTRODUCTION
As consequence of many pre-harvest, harvest and post harvest factors, food and water can be contaminated by human pathogenic microorganisms.Salmonella enteritica, Vibrio parahaemolyticus, Escherichia coli and Listeria monocytogenes are four food and water borne pathogenic bacteria causing gastroenteritis of human.Salmonella is one of the major food and water borne pathogenic bacteria which cause the disease called salmonellosis.Contaminated egg, meat and poultry products are the main sources of infection by Salmonella (Wang et al. 1996;Nowak et al. 2007).Vibrio parahaemolyticus is a gram negative bacterium considered as a major cause of gastroenteritis specially associated with the consumption of raw seafood (Pham et al. 2007).E. coli is a bacterium present in the intestinal tract of warm blooded animals as its normal micro flora.Most of the E. coli strains are nonpathogenic but some serogroups, such as enterohemorrhagic O157:H7, are pathogenic and cause severe diarrhea and fever (Stender et al. 2001).E. coli can colonize the intestinal tract of cattle and other animals.During slaughtering, they may contaminate the carcass and subsequently be distributed via cut meat or raw meat materials intended for further processing.L. monocytogenes is an opportunistic intracellular pathogen causes the disease called listeriosis.It is considered as an important cause of human food and water borne infections worldwide (Liu 2006).
Conventional methods of the detection of bacterial pathogens in foods generally based on the identification of bacteria using selective culture media by their morphological, biochemical and immunological characteristics (Wang et al. 2007).These methods are tedious, time consuming, and possible to have errors in sampling and enumeration when low number of microorganisms present in the sample.Moreover, it is not possible to use these methods in detection of viable but nonculturable microorganisms (Kong et al. 2002).As a replacement for these conventional methods there are number of rapid methods developed, including automated detection methods (Peng et al. 2001), immunological methods (Wang et al. 1996;Jouy et al. 2005) and nucleic acid based analyses (Whyte et al. 2002;Nam et al. 2005;Malorny et al. 2007).However, nucleic acid based assays are more popular at present.
Out of all nucleic acid based detection methods, polymerase chain reaction (PCR) based methods have been identified as a powerful diagnostic tool for the detection of pathogenic microorganisms (Malorny et al. 2003).Compared to other detection methods, these methods are rapid, highly specific and sensitive in the identification of target organisms (Wang et al. 2007).Recently, multiplex PCR which is a modified PCR method was introduced and widely applied in microbial detection which can be used to detect more than one organism by one PCR reaction simultaneously.The establishment of Multiplex PCR protocol is a tedious and time consuming since it needs lengthy optimization *Corresponding author procedure, but after being established it is rapid and low cost method for microbial pathogen detection.The multiplex PCR method has been used by many scientists to detect different types of pathogenic microorganisms in food and water (Pham et al. 2007;Petra et al. 2007).
PCR based detection mainly depends on the purity and amount of the template DNA used (Estrada et al. 2007).The presence of PCR inhibitors in food samples and incomplete bacterial cell isolation lead to the production of false negative results in PCR based detection and the removal of PCR inhibitors, efficient bacterial cell isolation and efficient DNA extraction is important (Jenicova et al. 2000).Therefore, the application of PCR-based methods is closely linked to the selection of suitable methods for DNA extraction (Amagliani et al. 2007) and efficient isolation of bacterial cells from food samples by immobilization.
In our experiment, a multiplex PCR method was developed for the detection of four bacterial pathogens, S. enterica, V. parahaemolyticus, .E. coli and L. monocytogenes.The detection sensitivity of those pathogens from inoculated raw meat samples was also studied.Metal hydroxide immobilization prior to DNA extraction was used to bacterial cell concentration from food samples.

Bacterial cultures
V. parahaemolyticus, S. enterica, E. coli, and L. monocytogenes were cultured for 6h at 36 0 C using universal culture medium, Tryptic Soy Broth Yeast Extract medium (TSBYE).The cultures were then serially diluted (10 -1 -10 -10 ) in sterile distilled water and enumerated in Tryptic Soy Agar (TSA) plates.Bacterial concentration was estimated by calculating the average number of colonies on plates containing 30 to 300 colonies.

DNA extraction to establish Multiplex PCR method
Dilutions of each of the four bacteria which contained 10 7 cfu/mL were prepared and 1mL of each dilution was used to inoculate 25g of raw pork meat sample.Another 25g of raw pork meat sample was inoculated with 1mL of each dilution.Then each inoculated sample was placed in 225mL of TSBYE medium and homogenized using a stomacher in 90s.Those cultures were incubated for 6h at 36 0 C.After incubation, bacterial DNA was extracted from each culture following the method developed by Fontana et al. (2005) and modified by Estrada et al. 2007.Bacterial cell pellets were obtained by centrifugation of 1mL of each culture at 12,000rpm for 10min at 4 0 C. The resulting pellets were first dissolved in 200µL of Ammonium hydroxide followed by 200µL of absolute ethanol, 400µL of petrolium ether and 20µL of 10% SDS.Then they were centrifuged at 12,000rpm for 10min at 4 0 C again, and the resulted pellets were re-suspended in a solution containing 200µL of 6M urea, 200µL of absolute ethanol, 400µL of petrolium ether, 80µL of 10% SDS and 13µL of 3M Sodium acetate.Another centrifugation for 10min at 12,000rpm at 4 0 C was also performed, and the pellets were resuspended with 600 µL of pH 8.0 TE buffer (Tris -EDTA), 35µL of 10% SDS and 10 µL of 10 mg/ mL DNase-free RNase.The tubes were incubated at 37 0 C for 30min before adding 10µl of Proteinase K and then again incubated at 37 0 C for 30min.Finally, 130µL of 6M Sodium perchlorate and 500µL of phenol chloroform isoamyl alcohol (25:24:1; pH 6.7) were added for DNA extraction.The tubes were then centrifuged at 12,000rpm for 5min, the aqueous phase was collected and the nucleic acids were precipitated with absolute alcohol.The extracted DNA was dissolved in 20µL of TE buffer.

DNA extraction for the study of the sensitivity of Multiplex PCR method and the effect of metal hydroxide immobilization
Ten mixed bacterial dilution of four bacteria which contained 10 0 to 10 10 cfu/mL of each bacterium were prepared and used to inoculate ten 25g rawpork-meat samples.Then were placed in 225mL of TSBYE medium and homogenized using a stomacher in 90s and were incubated for 6h at 36 0 C. The incubated samples were used to extract DNA by the method developed by Fontana et al. (2005) and modified by Estrada et al. (2007) as mentioned above with and without metal hydroxide immobilization prior to DNA extraction.In this study, the immobilization method proposed by Lucore et al. (2000) was used with some modifications.Hundred micro liters of bacteria was added to 200µL of Zirconium hydroxide and agitated at room temperature.Then it was vortex and centrifuged at 2000 rpm for 5min at 7 0 C.After centrifugation the pellet was obtained and used for DNA extraction.

Integrity and purity of extracted DNA
Five micro liter of each extracted DNA was visualized by gel electrophoresis in a 0.9% agarose gel stained with ethidium bromide (0.5µg/mL).After electrophoresis at 80V for 40min DNA bands were visualized by using ultraviolet light and documented by a gel documentation system.The quality of the extracted nucleic acids was also investigated, and the absorbencies at 260 and 280nm were measured using spectrophotometer and the A 260 /A 280 ratios were calculated for all DNA samples.

PCR amplification
Extracted DNA were amplified by PCR using four specific forward and reverse primers, CGGCGTG GGTGTTTCGGTAGT and TCCGCTTCGCGCT CATCAATA for gyrB gene of V. parahaemolyticus (Venkateswaran et al. 1998), TCTCTACTTAAC AGTGCTGC and TGGTATAAGTAGACAGGG GC for invA gene of S. enterica (Hoa et al. 2004), GACCCGGCACAAGCATA AGC and CCACC TGCAGCAACAAGAGG for the eaeA gene of E. coli (Paton et al. 1998), and TATACCACGGA-GATGCAGTG and GCCGAAGTTTACATTCAA GC for hly gene of L. monocytogene (Hoa et al. 2004).2.5μL of each extracted DNA was used as a template for PCR amplification in a 50µL final volume of the following mixture: 25µL of Perfect-Shot TM Ex Taq (from Takara), 0.25µM of each primer and sterilized distilled water.PCR reactions were performed with the initial denaturation at 95 0 C for 3min followed by 35 cycles of denaturation at 95 0 C for 30s, annealing at 55 0 C for 60s, and extension at 72 0 C for 1.5min, and final extension for 7min at 72 0 C.

Gel electrophoresis and gel documentation
All PCR products were visualized by gel electrophoresis.Ethidium bromide containing 2.5% agarose gel in TAE buffer (20mM Tris acetate, 0.5mM EDTA, pH 7.8) was used.The gel electrophoresis processes were carried out for 2.5h at the constant voltage condition of 35mV.After gel electrophoresis, the PCR products were visualized by UV illumination using gel documentation system and images were recorded.

RESULTS AND DISCUSSION
The extraction of the adequate amount of the pure total DNA is a basic requirement in PCR based detection.In this study, the gel electrophoresis of the extracted total DNA showed clear single bands which indicate the good integrity of the extracted DNA.The purity of the extracted DNA was also in the acceptable range.The ratios of the absorbance at 260nm to the absorbance at 280nm were in the range of 1.6 to 1.8 for all extracted DNA samples.The specificity of primers used to detect target gene is very important in the detection of pathogenic organisms from a food sample which could contain a mixture of microorganisms, and this is a major concern in establishment of Multiplex PCR protocols.The primers used in this study were tested for their specificities.The sensitivity of the detection depends on the condition of the PCR reaction such as, primer annealing temperature, primer concentration, Mg 2+ concentration, extension time and characteristics of DNA polymerase used.In this study, PerfectShot TM Ex Taq (from Takara) master-mix was used with the optimum conditions based on the reported applications of the same primers.The used primers were able to amplify successfully the target genes of the extracted DNA from meat samples which were inoculated with 10 7 cfu/mL bacteria cultures and enriched for 6 h in TSBYE medium, so that no non-specific band was observed in gel electrophoresis.The bacterial DNA extracted from meat samples inoculated with the four bacteria separately were able to produce bands of 285bp, 685bp, 384bp and 482bp for the gene gyrB of Vibrio parahaemolyticus, invA of S. enterica, eaeA of E.coli and hly of L. monocytogene respectively, while the bacterial DNA extracted from meat samples inoculated with a mixture of all of the four bacteria produced the all above four bands (Fig. 1).This results indicated the suitability of the above four primers in a Multiplex PCR for the simultanious detection of four pathogens, V. parahaemolyticus, S. enterica, E. coli and L. monocytogene.
Detection sensitivity is another important factor concerned in the evaluation of microbial pathogen detection methods.This experiment assessed the ability of metal hydroxide immobilization in increasing the detection sensitivity by increasing the  M: 100bp molecular size marker, from 1 to 10: bacterial concentrations from 10 10 to 10 0 cfu/mL respectively concentration of cells from food sample.The developed Multiplex PCR method was able to detect all of the four microorganisms up to 10 4 cfu/mL for the DNA extracted without metal hydroxide immobilization (Fig. 2).But when metal hydroxide immobilization was applied, the detection sensitivity was increased by 10 fold, 10 3 cfu/mL (Fig. 3).
Although the infectious dose varies with different pathogen types, it is generally believed that most bacterial pathogens are able to cause infection when more than 10 3 infectious cells are ingested.So, the detection sensitivity of the developed Multiplex PCR method with metal hydroxide immobilization of cells prior to DNA extraction is within the infectious dose of most enteric pathogens (Kong et al. 2002).However, this developed Multiplex PCR method should be improved further to achieve higher sensitivity when concerning high risk pathogens.

CONCLUSIONS
The Multiplex PCR method developed in this study could provide a powerful supplement to conventional methods for more accurate, rapid and sensitive risk assessment and the monitoring of selected four pathogenic bacteria associated with meat and meat products.The metal hydroxide immobilization could be used as an effective application in increasing the concentration of bacterial cells from food samples in order to increase the detection sensitivity.This Multiplex PCR method should be further improved for the detection of other important food and water borne pathogenic bacteria, and the effectiveness of metal hydroxide immobilization should be tested for different food matrix.

Figure 2 :
Figure 2: PCR amplification of four pathogenic bacterial DNA extracted without metal hydroxide immobilization.M: 100bp molecular size marker, from 1 to 10: bacterial concentrations from 10 10 to 10 0 cfu/mL respectively

Figure 3 :
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