J.Natn.Sci.Foundation Sri Lanka 2007 35 (4): 225-233 RESEARCH A R T I C L E DNase treated DNA multiplex polymerase chain reaction assay for rapid detection of viable food borne pathogens L. Mahesha N. Sigera Nadugala1*, Sudip K. Rakshit1 Food Engineering and Bioprocess Technology Programme, Asian Institute of Technology, SERD. P. 0. Box 4 Klong Luang, Pathumthani 12120, Bangkok, Thailand. Revised: 19 April 2007 ; Accepted: 15 May 2007 Abstract : The main objective of this work was to develop methods to overcome the problems associated with rapid detection of food borne pathogens using PCR based techniques. A multiplex PCR method was developed as a solution to the problem of having to test for one organism at a time. DNasel enzyme treatment followed by PCR [DNase Treated DNA (DTD) PCR] was experimented with to find a solution to the problem of false positive results obtained by amplification of DNA from dead cells. Four sets of primers were used for detection of eaeA, hly, invA and gryB genes of frequently occurring food borne pathogens Escherichia coli 0157:H7, Listeria monocytogene. Salmonella enterica and Vibrio parahaemolyticus respectively. Experiments proved that DNasel has the ability to remove DNA from dead cells without causing any damage to the DNA present inside live cells. DNasel at a level of 10U/100 uL was found to remove DNA sourced from 5x 10 7 dead cells in food systems within one hour of incubation. In the specificity test no interferences or non-specific amplification was observed when the multiplex protocol was tested with 89 strains of bacteria. The method developed was found to be sensitive to a minimum cell count of 102 cells in both pure cultures and in artificially spiked food systems. There was no interference or inhibitory actions when the protocol was applied to shrimps. Thus, this DTD multiplex PCR assay can be practically applied for simultaneous identification of viable cells of four important pathogens namely E. coli 0157:H7, S. enterica, L. monocytogenes and V. parahaemolyticus. Keywords: detection, DNasel, DTD multiplex PCR, food borne pathogens. INTRODUCTION The major cause of food poisoning results from bacteria. Ninety percent of the cases of food poisoning each year is caused by Salmonella spp., Listeria monocytogenes, Vibrio parahaemolyticus, Diarrheagenic Escherichia coli. Staphylococcus aureus, Clostridium perfringens, Campylobacter, Bacillus cereus and Shigella dysenleriae1. The percentage of transmission of Salmonella spp., L. monocytogenes, V. parahaemolyticus and Diarrheagenic E. coli through food items is 95, 99, 95"and 85% respectively2. These four bacteria are important because they cause gastrointestinal diseases in humans, paticulary since the infections may result in life threatening sequelae such as salmonellosis, listeriosis, gastroenteritis and hemolytic uremic syndrome (HUS) respectively and these outbreaks occur very frequently around the world. Increased public awareness of health related issues and economic impacts of contaminated food and resulting illness have led to greater efforts to develop more sensitive methods of pathogen detection and identification. Traditional methods, which are highly reliable and accurate to detect food borne bacteria often rely on time consuming growth in culture media, followed by isolation, biochemical identification, and sometimes serology. The absence of rapid, cost effective methods for bacterial detection in food items with a short shelf life poses particular difficulties for the implementation of effective food quality management systems in the modem market where there is a very rapid demand for food. It is well known that PCR is one of the most promising analytical tools in food safety control and clinical analysis because of its specificity and sensitivity'4. However, conventional PCR methods do not distinguish viable cells from dead cells. The DNA extracted from cells killed by heat or other treatments will serve as a template for PCR many days after cell viability has been lost'1'5. The presence of dead cells therefore limits the use of PCR for microbiological monitoring in food samples. ' Corresponding author 226 L.M.N.S. Nadugala & Sudip K. Rakshit Several authors have attempted to detect only viable cells by detection of mRNA using Reverse Transcriptase PCR (RT-PCR)6"9. DNA-PCR, however, has been found to provide stronger amplification signals compared to RT-PCR at the corresponding time points with the same PCR primer set, indicating a lower efficiency of RNA amplification compared to that of DNA 1. The selective detection of viable but not dead bacteria is a major issue in nucleic acid-based diagnostics'". DNase treated DNA PCR (DTD PCR) methods have been attempted to eliminate such contaminating DNA from dead cells prior to the isolation of template DNA, to improve the overall fidelity of such detection methods". In these preliminary experiments the harvested bacterial cells were exposed to crude DNasel treatment to degrade the exogenous DNA released from completely lysed cells or the DNA still trapped in partially lysed cells. There is a need to prove that the DNA present inside the intact cells, which are likely viable cells, are spared. Although PCR is rapid and specific, the process could become cumbersome when applied to numerous samples with various potential targets. To minimize time and materials, primers can be combined in a single reaction tube to form a multiplex-PCR (MP-PCR) 1 2 which can simultaneously detect numerous target genes in a single sample. In this study we report the development of a cheaper, faster and more sensitive alternative method than the commonly used RT-PCR method, which is more reliable than the conventional DNA based PCR method for prediction of the presence of possible viable pathogens in food. We also report the development of a highly specific four gene targeted MP-PCR assay which would be useful for simultaneous detection of the four highly problematic food bome pathogens; S. entehca. L. monocytogenes. V. parahaemolyticus and E. coli 0157:H7 targeting the following genes invA. hly, grvB, eaeA. respectively. METHODS AND MATERIALS Bacterial strains and culturing: A total of eighty nine bacterial strains (Table 4) representing nine strains of E. coli, ten strains of Listeria, twelve strains of Salmonella and nineteen strains of Vibrio were included in this study. Additionally thirty nine strains of other bacteria which include food borne pathogens (closely related to target organisms and non related target organisms) and bacteria that are important in the food industry were also included. All strains were cultured in 10 mL of tryptic soy broth yeast extract medium (TSBYE) (Difco Laboratories, Detroit, Ml, USA) and incubated under aerobic conditions overnight at 36 ± 1°C. DNA extracts preparation: DNA extracts used as templates in PCR amplifications were prepared by Triton X-100 method. Bacterial DNA extracts were prepared according to the manufacturer's instructions (Easy DNA kit, Invitrogen Co., San Diego, USAJ.The procedure involved concentrating cells from a 7 mL aliquot of late log phase culture. The final DNA extract was dissolved in 50 uL of ultra pure nuclease free water (Seromed *). Preparation of heat killed cells: In developing the DTD multiplex PCR system, heat treated cells were taken as the model cells to represent the "non cultivable cells" (dead cells with intact cell covering). According to preliminary experiments that were done in the Bioprocess lab in the Asian Institute of Technology, Thailand, it was proven that a heat treatment at 100 °C for 10 min in a constant temperature water bath produce dead cells with intact cell covering. Growth of the cells that were subjected to heat treatment was tested on TSBYE agar and cell covering was observed under light microscope. PCR primers: Seven sets of primers were selected for this study based on the virulence gene sequences and other specific gene sequences, generating amplicons ranging from 285 to 1,087 bp in length (Table I). The sequences of three primer sets (0157, eaeA, gryB) were obtained from reference materials and sequences of the other four primer sets (ehxA, hly. invA. toxR) were developed in the Bioprocess lab of Asian Institute of Technology, Thailand. Primers were prepared by Bioservice Unit of BIOTEC, Thailand and hydrated to a stock concentration of 100 pmole/uL using sterile TE buffer (10 mM Tris-HCI, 1 mM EDTA). An aliquot of the primer stock solution was diluted to a working concentration of 50 pmole/uL using sterile TE buffer. The stock and working primer solutions were stored in 50 uL aliquots at -20 UC until required. Multiplex DNA amplification: Multiplex PCR was performed in 50 uL reactions in 0.2 mL thin walled PCR tubes. The reaction mixtures contained 25 uLof Hotstar Taq master mixture (1.25 U Taq DNA polymerase, 1.5 mM MgCl,, 10X PCR buffer, dNTPs 200 uM each), 0.25 pL of each forward and reverse primers, 1 uL of template DNA and ultra pure water to bring the final volume to 50 uL. The PCR procedure was performed in a PCR Thermocycler (Biometra ™ Personel). The protocol performed consisted of three stages: Stage 1:1 cycle of 95 °C for 10 min; Stage 2: 34 cycles of 95 °C for 45 s, 45 °C for 1 min 30 s and 72 °C for 1 min and 15 s; Stage 3: 72 °C for 5 min and 4 °C holding temperature. The PCR amplification products were visualized in 2% agarose gels (Analytical grade, Promega) stained with ethidium bromide (0.2 pg/mL) using U V trans illuminator. In this December 2007 Journal of the National Science Foundation of Sri Lanka 35 (4) DNase treated DNA multiplex PCR to detect viable pathogens 227 Table 1: Summary of the primers used in this study Species Target gene PCR primers' sequences (5 ' - 3') Product size Reference E. coli 0157:H7 ehxA gene ehxAl: TCT GAG CAG CTT AAC CAG CT ehxA2: GAT TCA AGC TGC TTA OCT CG eae gene 0157-F: CAG GTC GTC GTG TCT GCT AAA 0157- F: TCA GCG TGG TTG GAT CAA CCT 398 bp 1.087 bp 13 14 * nt 2 7 ^ 10 eae A1 :GAC CCG GCA CAA GCA TAA GC of eaeA gene eae A2: CCA CCT GCA GCA ACA AGA GG 384 bp 15 [..monocytogenes My hlyl: TATACC ACG GAG ATG CAG TG gene hly2: GCC GAA GTT TAC ATT CAA GC 482 bp 13 S. enterica invA gene invAl: TCT CTA CTT AAC AGT GCT CG inv-A2: TGG TAT AAG TAG ACA GGG CG 685 bp 13 V. parahaemolyticus toxR gene toxRl: TGT ACG ATT AGG AAG CAA CG toxR2: AAC GTA GCG TTC AAT GCA CT gryB gene VP-F : CGG CGT GGG TGT TTC GGT AGT VP-R : TCC GCT TCG CGC TCA TCA ATA 622 bp 285 bp 13 lb * selected a region that is conserved between EPEC and STEC E.coli Table 2: Different combinations of primer sets tested in different multiplex PCR systems Multiplex system 1 Multiplex system 2 Multiplex system 3 Multiplex system 4 gryB (285 bp) gryR (285 bp) ehxA (398 bp) eaeA (384 bp) My (482 bp) My (482 bp) hly (482 bp) invA (685 bp) toxR (622 bp) invA (685 bp) 0157 (1087 bp) invA (685 bp) Wv<482 bp) toxR (622 bp) ;m'A(685 bp) 0 /57(1087 bp) particular experiment four different combinations of primer sets (Table 2) were tested to obtain a well defined suitable multiplex PCR system with higher specificity towards target organisms, better separation of amplicons on gels and broader range of identification. Also two commercially available PCR master mix kits from Biorad and Qiagen were tested to detect the most suitable PCR master mix for industrial application in multiplex PCR detection systems. Preparation of liquid food extract: Shrimps were shelled and whole meat was homogenized in a blender (National). 90 mL of TSBYE broth was added to 25 g of shrimp meat. Filtrate of boiled shrimp sample was taken as the liquid food extract. Sensitivity and specificity for primers: The primers were tested for their sensitivity for simultaneous detection of S. enterica, L. monocytogenes, V. parahaemolyticus and E. coli 0 1 5 7 : H 7 in various concentrations (10 n-10 9 bacterial cells mL"1 of ultra pure water/liquid food extract). They were also tested for their specificity against 89 different strains of bacteria (Table 4) which included food borne pathogens (closely related to target organisms and non related organisms) and bacteria that are important in the food industry. DTD multiplex PCR amplification: Developed DTD multiplex PCR system was tested for the conditions necessary for simultaneous detection of live target organisms and the ability to eliminate DNA that yield from dead cells. Live cells of three organisms were separately mixed with heat killed cells of one organism in ultra pure water and liquid food extract according to the matrix given in Table 3 and treated with 10 U of DNasel enzyme for 1 h before extracting DNA by Triton X-I00 methods. Extracted DNA was dissolved in 10 p.L ultra pure water and multiplex PCR amplification was done as described in the previous section. Journal of the National Science Foundation of Sri Lanka 35 (4) December 200? 225 L.M.N.S. Nadugala & Sudip K. Rakshil Table 3: Different combinations of cells used in the experiment for testing sensitivity of DNasel treated multiplex PCR for heat killed cells (HKC) and live cells (LC) Bacterial strains Combination 1 Combination 2 Combination 3 Combination 4 S. enterica HKC LC LC LC L. monocytogenes LC HKC LC LC E. coli 0157:H7 LC LC HKC LC V. parahaemolyticus LC LC LC HKC Table 4: Results of PCR specificity experiments with multiplex system against different food related micro-organisms PCR results S.No. Bacterial strains Primers (Source) gryB eaeA 0157 ehtA hly invA (285 bp) (384 bp) (1087 bp) (398 bp) (482 bp) (685 bp) 1. E. coli (ATCC 25922) 2. E. co/i (Wild type | (AFRIMS) - - - - 3. E. coli(M 109) (BPT) - - - - 4. E. coli. (DH5a) (BPT) - • - • 5. E. c«//OI57:H7 (ATCC 43890) + + + - 6. E. coli(Roseila) (BPT) - - - - 7. E. c»/f(TlSTR73) - - • - 8. E. coli (Wild type) (Mahidol) - - - • 9. E. coli • • - - 10. L. monocytogenes (ATCC 35152) - - • + 11. L .monocytogenes (DMST 17303) • • - + 12. L. monocytogenes (DMST 4553) • • + 13. L. innocua (DMST 9011) 14. L. grayi (DMST 15840) - • - 15. L. welshimen (DMST 10828) - - - - 16. L. solanaceae - - - - 17. 18. L. murrayi (DMST 4580) L. ivonivii (DMST 9012) - - - - December 2007 Journal of the National Science Foundation of Sri Lanka 35 (4) DNase treated DNA multiplex PCR to detect viable pathogens 229 PCR results S.No. Bacterial strains Primers (Source) gryQ eaeA 0157 ehxA My invA (285 bp) (384 bp) (1087 bp) (398 bp) (482 bp) (685 bp) 19. L. seeligeri (DMST) S. enterica 20. Serovar Typhimurium (ATCC 14028) 21. Serovar Typhimurium (ATCC 13311/TISTR 292) 22. Serovar Typhimurium (DMST 15674) 23. Serovar Typhimurium (ATCC 19430) 24. Serovar Choleraesuis (ATCC 10708 / DMST 8014) 25. Serovar Paratyphi A (DMST 15673) 26. Serovar Enteritidis (DMST 15676) 27. Serovar Paratyphi B (DMST 7090) 28. Salmonella sp. (TISTR 96) 29. Salmonella sp. (Burapha) 30. Salmonella sp (Burapha) 31. Salmonella sp. (Burapha) 32. Salmonella sp. (Burapha) 33. V. parahaemolyticus (AFRIMS-BH01-0124) 34. V. parahaemolyticus (Burapha 1) 35. V. parahaemolyticus (Burapha 2) 36. V. parahaemolyticus (Burapha 3) 37. V. parahaemolyticus (DMST 15285) 38. V. aiginoiyticus (DMST 14800) 39. V. cholerae 0139 (AFR1MS) 40. V. cholerae (Ogawa) (AFRIMS) Journal of the National Science Foundation of Sri Lanka 35 (4) December 2007 230 L.M.N.S. Nadugala & Sudip K. Rakshit PCR results S.No. Bacterial strains Primers (Source) gryB eaeA 0 1 5 7 ehxA hly invA (285 bp) (384 bp) (1087 bp) (398 bp) (482 bp) (685 bp) 41. V. cholerae (Inaba) (AFRIMS-56190) 42. V. vulnificus (Biotype 1) - 43. V. vulnificus (Biotype 2) - 44. V vulnificus (DMST 5852) 45. V. harvey (KCTC 2717) 46. V .campbelli (Mahidol) 47. Vibrio sp. + . . . (Burapha) 48. Vibrio sp. + . . . (Burapha) 49. Vibrio sp. + . . . 50. Vibrio sp. + . . . (Burapha) • In addition to the bacterial strains listed in this table , we also obtained negative PCR results for all the above six PCR primer sets for the following bacterial strains: Shigella dysenieriae (AFR1MS 102/2), Sh. dysenteriae (Burapha), Sh .boydii (AFRIMS), Sh. sonnei (ATCC 25931), Sh. sonnei (Burapha), Sh. ftexineri (ATCC 12022), Sh.ftexineri (Burapha), Klebsiella oxytoca (AFRIMS), Klebsiella pneumoniae (ATCC 13883), Klebsiella pneumoniae (Burapha), Staphylococcus aureus (ATCC 13565;TISTR 29), Staphylococcus aureus (ATCC 25923). Staphylococcus aureus (ATCC 2913). Streptococcus pyogenes (ATCC 19615), Streptococcus pyogenes (Burapha), Bacillus cereus (ATCC 1 1778/T1STR 686), B. circulans (ATCC 9995/TISTR 906), B. subtilis (T1STR 25), B. stearothermophilis (UPCC116/CS1RO (FS 1518)), Enterococcus faecalis (ATCC 19433), Enterobacter amnigenus (AFRIMS), Enterobacter aerogenes (ATCC 13048), Enterobacter (ATCC 23355), Pseudomonas aeruginosa (ATCC 27853), P. siutzeri (AFRIMS), P. putida (Mohidol), P. demimita (BPT), Flavobacterium colomnarv (Burapha). Flavobacterium sp. (Burapha), Serratia marcesseus (Burapha), Edv,ardsiella tarda (Burapha), Haemophilus influenzae (AFRIMS), Connebacterium glutamicum (ATCC 21475), Lactobacillusplantarum (TISTR 541), L. casei (TISTR 1463), L. brevis (TISTR 855). L lactis (TISTR 1464), L. acidophilus (TISTR 1338), Aeromonas hydrophita (Burapha) b AFRIMS-Armed Forces Research Institute of Medical Sciences, ATCC - American Type culture collection. BPT - Bioprocess Technology culture collection, DMST - Department of Medical Sciences, Thailand, KCTC - Korean Collection for Type Cultures, Burapha - Burapha University culture collection. Mahidol - Mahidol University culture collection. ' (+) - presence of expected PCR product; {-) - absence of expected PCR product RESULTS Multiplex DNA amplification Theoretical melting temperatures of all the primers used ranged from 45 to 55 (>C. Therefore individual PCR reactions were carried out at three different temperatures (45 °C, 50 n C, and 55 °C) (gel pictures not given). At December 2007 the three tested annealing temperatures all the primers produced PCR products. However at the annealing temperature of 45 °C all the primers gave better amplification and the PCR bands showed the highest intensity. Hence the annealing temperature of 45 °C was used in the multiplex system as well (gel pictures not given). Multiplex system 1 gave the expected PCR products for all four target organisms and the amplicons Journal of (he National Science Foundation of Sri Lanka 35 (4) DNase treated DNA multiplex PCR to detect viable pathogens 231 were well separated on the 2 % agarose gel. Mult iplex system 2 also gave four ampl icons , but one of the four bands was an unexpected band (200 bp approximately) which may be a result of misamplif icat ion. Mult iplex system 2 did not produce the expected band for E. coli 0 1 5 7 : H 7 at the size of 1,087 bp. Mult iplex system 3 also gave the expected P C R products for the target organisms, but separation of bands was not very clear. Mult iplex system 4 yielded only three prominent expected bands . This system also did not yield the P C R product expected for E. coli 0 1 5 7 : H 7 at the size of 1,087 bp but there were some faint extra bands presented in the gel due to misamplification. In both mult iplex sys tems (2 and 4) that used 0157 pr imer pair for detect ion of E. coli 0 1 5 7 : H 7 the expected P C R ampl icon was not produced. This may be due to pr imer-dimer formation. Based on the results of these exper iments mult iplex sys tems 2 , 3 , 4 were not chosen for further studies. Mult iplex P C R system 1 was developed with four pr imer pairs namely gryB, eaeA, hly and invA. The efficiency of amplification was ideal in the tested extension temperatures of 72 °C and 74°C. Commerc ia l P C R master mix kits from Qiagen and Biorad gave amplification products under all tested condit ions. The intensify of the P C R products was higher with Qiagen P C R master mix kit than with Biorad kit when other parameters were kept constant . Hence the Qiagen P C R master mix kit was used for further studies. Sensitivity and specificity of primers The main objective o f the sensitivity test was to find the m i n i m u m detectable level of cells of target organism from pure cul tures and from liquid food extracts . It should be noted here that theoretical ly P C R based methods are M l 2 3 4 5 6 7 8 9 Figure 1: Sensitivity of developed multiplex PCR system on different cell counts of bacteria from pure cultures. Lane M: 100 bp molecular size marker. Lane 1-9: E.coli 0157:H7, L. monocytogenes, V. parahaemolyticus and S. enterica multiplex PCR products with known concentration (10"-1 cells). Journal of the National Science Foundation of Sri Lanka 35 (4) expected to yield good results even if a single D N A is present. However , efficiency of extraction of D N A , the efficiency with which pr imers interact with the template at low concentrat ions , the random chances of occurrence in the diluted test samples etc. m a k e this difficult. This is also pointed out by the fact that enr ichment media is required in the conventional plate count method . Accord ing to the results of gel electrophoresis (Figures 1 and 2) it is clear that this system is able to detect a m i n i m u m level of 10 2 cells of all target o rganisms from pure cultures. The cell level 10' also produced very faint bands which were not clearly documen ted by the available gel documenta t ion system. However , in industrial applicat ions this will be a better starting point which can be reconfirmed by the reamplification of P C R products or by use of convent ional methods if needed. Specificity of the developed mult iplex P C R method was checked against 89 strains of bacteria. S o m e of the strains were either closely related to target bacteria , not related to target bacteria or related to other food industry bacteria. Results of this exper iment are given in Table 4. N o n e of the specific pr imer pairs gave cross amplification with the other organisms tested to date. In the case of eaeA pr imer of E. coli it produced an ampl icon with a size of 384 bp with the E. coli 0 1 5 7 : H 7 strain. It also produced a prominent band with other E. coli strains which has a size of be tween 400 - 500 bp. These results were due to the reason that eaeA p r imer pair targets a region that is conserved between enteropathogenic and shiga toxigenic E. coli s t ra ins 1 5 . This pr imer will help the detection of a wide range of diarrheagenic E. coli which are human pathogens . If further confirmation is needed with E. coli 0 1 5 7 : H 7 strain reconfirmation can be done with ehxA and 0 1 5 7 pr imer sets which are highly specific to E. coli 0 1 5 7 : H 7 . The specific pr imer (hly pr imers) M l 2 3 4 5 6 7 8 9 10 M * 1 Figure 2: Sensitivity of developed multiplex PCR system on different cell counts of bacteria on shrimp. Lane M: 100 bp molecular size marker. Lane 1-10: E.coli 0157:H7, L. monocytogenes, V. parahaemolyticus and 5. enterica multiplex PCR products with known concentration (10'- 1 cells). Lane M: 100b bp molecular size marker. December 2007 Figure 3: Sensitivity of developed DTD multiplex PCR system on different cell combinations (see Table 3) of target bacteria from pure cultures. Lane M: 100 bp molecular size marker. Lane 1: Live cells of four bacteria with DNasel treatment, Lane 2: live cells of four bacteria with out DNasel treatment. Lane 3: Combination 1 with DNasel treatment, lane 4: Combination 2 with DNasel treatment. Lane 5: Combination 3 with DNasel treatment, Lane 6: Combination 4 with DNasel treatment, Lane 7: Heat killed cells of four bacteria with DNasel treatment, Lane 8: Heat killed cells of four bacteria without DNasel treatment. Lane 9: PCR negative control. Lane M: 100 bp molecular size marker. L.M.N.S. Nadugala & Sudip K. Rakshit M 1 2 3 4 M Figure 4: Sensitivity of developed DTD multiplex PCR system on different cell combinations (see Table 3) of target bacteria on shrimp. Lane M: 100 bp molecular size marker, Lane I: Combination 1 with DNasel treatment, Lane 2: Combination 2 with DNasel treatment. Lane3: Combination 3 with DNasel treatment, Lane4: Combination4 with DNasel treatment, Lane M: 100 bp molecular size marker. used to detect L. monocytogenes was also confirmed to be highly specific in the developed mult iplex PCR system. It did not show any cross amplification with other bacteria tested in the study. The pr imer pair produced the expected band of 482 bp size only with the tested strains o f L. monocytogenes. The pr imer pair (invA) used for detect ion of Salmonella is specific to the species enterica. Species enterica includes several serovars . Most of these serovars are human pa thogens which cause sa lmonel los is . The mult iplex system was tested against six know serovars of Salmonella enterica species and five other unidentified Salmonella species. Except serovars Enteri t idis all the other serovars gave the expected PCR product 685 bp. Theoret ical ly serovar Enteri t idis also should produce the P C R product as the pr imer pair is specific to species enterica. Negat ive result may be due to the degradat ion of extracted D N A which could not be reconfirmed. All the unidentified Salmonella species produced the expected PCR product (685 bp) which helps to confirm that they be long to species enterica. The pr imer pair invA was also tested with different species of Shigella that is closely related to Salmonella. N o n e of the Shigella species tested gave PCR products . As the original paper repor ted 1 6 the pr imer pair gryB is highly specific to V. parahaemolyticus. It did not show any cross amplification with the other micro-organisms tested in the study. The pr imer pair is specific enough to differentiate be tween V. parahaemolyticus and V. alginolyticus which are reported to be closely related. All the unidentified Vibrio species showed the expected PCR product (285 bp) which confirms that they belong to species parahaemolyticus. Specificity of the developed mult iplex system was tested with many other unrelated food borne pathogens and other food industry related micro-organisms. N o n e of them produced any kind of PCR product . This confirms the specificity of the developed mult iplex system. DNase Treated DNA (DTD) multiplex PCR amplification This exper iment was conducted to test the ability of developed D T D mult iplex P C R system to detect live cell D N A of target organisms and s imultaneously digest the D N A in dead cells. The cell count was fixed at 5* 10 7 cel ls /100 ul for DNase l t reatment . The result in the Figures 3 and 4 show the ability of developed D T D mult iplex PCR system to detect live cell D N A and the ability to remove the D N A that yield from dead cells which are represented by heat killed cells in this system. DISCUSSION The D T D mult iplex system with pure cul tures and artificially spiked food samples gave expected results and did not show any kind of inhibitory effect with the shr imp samples . The DNasel t reatment (10 U, one hour) is able to remove D N A yielding from 5 x 10 7 of dead cells, which is far beyond the expected level of cells in natural food sys tems. The results of the developed Journal of Ihe National Science Foundation of Sri Lanka 35 (4) December 2007 DNase treated DNA multiplex PCR to detect viable pathogens 233 DTD multiplex PCR system reveal its ability to get rid of exogenous DNA released from completely lysed cells or partially lysed cells. DNasel was capable of penetrating through i the cell covering of non cultiva­ ble cells and degrade the DNA inside those cells too. DNasel did not however degrade the DNA inside live cells. Its efficiency in spiked shrimp samples indicates that the DNA extraction methods, DNasel activity and PCR conditions are Inot affected by the components of the shrimp. This system proved to be an effective meth­ od to be used in food systems to detect live food borne pathogens. Sensitivity studies gave the same result with pure cultures as well as artificially spiked food samples. It showed the ability of the developed tool to detect the minimum cell level of 10 2 cells. The major contribution of this study is the development of a PrjR kit using a universal culture medium and four pairs of specific primers in the same PCR conditions for detection of four important food borne pathogens that occur very commonly in food systems. 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