Comparison of ELISA and PCR vis-a`-vis cultural methods for ...

More documents

Recommendations

Info

178 a duplex-PCR and to evaluate the suitability of the developed assays for detection of Aeromonas from foods of animal origin. The results of the developed assays were compared among themselves and with conventional cultural method. 2. Materials and methods 2.1. Cultural method For isolation and identification of Aeromonas from food samples 12 h enrichment in Alkaline Peptone Water supplemented with 10 mg/ml cephalothin (APW-C) was done (Sachan and Agarwal, 2000) and growth plated on Ampicillin Dextrin Agar (ADA). After 24 h incubation at 37 -C the characteristic colonies were confirmed biochemically (Agarwal et al., 2003b). 2.2. Indirect plate ELISA 2.2.1. Preparation of crude OMP The crude outer membrane protein (OMP) was extracted from the standard strain of Aeromonas hydrophila subsp. hydrophila (Microbial Type Culture Collection Strain No. 646, MTCC 646) as per the method of Crosa and Hodges (1981) as modified by Santos et al. (1996) and Sachan and Agarwal (2002). Briefly, A. hydrophila subsp. hydrophila was grown overnight in 100 ml of tryptone soya broth (TSB). The cells were recovered by centrifugation at 5000 g for 30 min at 4 -C. Cells were resuspended in 3 ml of 10 mM tris (hydroxyl methyl) amino methane buffer containing 0.3% (w/v) NaCl (pH 8.0) and sonicated with a MSE Sanyo Sonicator for 3 times at 10 Am amplitude for 45 s. After centrifugation at 10,000 g for 2 min, the supernatant was transferred to new tubes and centrifuged for 1 h at 17,000 g at 4 -C. Resulting pellets of cell envelop suspensions were incubated overnight at 4 -C with 3% (w/v) sodium lauroyl sarcosinate (sarkosyl) in 10 mM tris buffer. Outer membrane protein was obtained by centrifugation at 17,000 g for 1 h and washed twice with distilled water. The OMP was stored at 20 -C. The protein content of OMP was determined as outlined by Schacterle and Pollack (1973) with little modifications. Concisely, 5 Al of OMP was diluted with 295 Al ofDWina test tube. Different concentrations (10 Ag, 60 Ag, 120 Ag, 180 Ag, 240 Ag and 300 Ag) of bovine serum albumin (BSA) were prepared in 1% (w/v) sodium dodecyl sulfate (SDS) to a total volume of 300 Al. To all the tubes 1.2 ml of 2 Lowry concentrate was added and incubated at room temperature for 10 min. Thereafter 0.6 ml of Folin reagent was added and incubated at 55 -C for 5 min. The absorbance was measured at 750 nm. Protein concentration of OMP was determined from the standard graph drawn for optical density of BSA against different concentrations. 2.2.2. Production of antiserum against crude OMP Antiserum against crude OMP of A. hydrophila subsp. hydrophila (MTCC 646) was raised in an adult rabbit as per the S. Arora et al. / International Journal of Food Microbiology 106 (2006) 177–183 method described by Santos et al. (1996) and Sachan and Agarwal (2002). OMP (500 Ag protein) was emulsified with an equal volume of Freund’s complete adjuvant (FCA, Difco). A rabbit was injected subcutaneously in divided doses at various dorsal sites. Booster dose was given after 15 days with OMP (250 Ag protein) emulsified with equal volume of Freund’s incomplete adjuvant (IFCA, Difco) followed by a similar booster dose after another 15 days. Rabbit were bled 2 weeks after the last immunization. Preimmune serum was used as control serum. 2.2.3. Standardization of indirect plate ELISA The indirect plate ELISA was performed as per the methods described by Engvall and Perlman (1971), Sachan and Agarwal (2002) and Ghatak et al. (2003) with suitable modifications. The test was standardized by checker board analysis. The optimum titre of anti-OMP serum was found 1:1600 and thus 1:800 dilution was used in further studies. 2.3. Duplex-polymerase chain reaction 2.3.1. Preparation of DNA template DNA template was prepared by boiling followed by snap chilling into ice. One hundred microliters of the broth culture was taken and centrifuged at 5000 g for 5 min. The pellet was resuspended in 100 Al PBS. This step was repeated once and the resulting pellet dissolved in PBS was subjected to vigorous heating in a boiling water bath for about 8 min and then immediately snap chilled into ice. From this snap chilled lysate, 3 Al was used as template source in PCR. 2.3.2. Oligonucleotide primers The primers for 16S rRNA and aerolysin genes of Aeromonas spp. used in this study were got synthesized from Bangalore Genei. The details of primers used and their corresponding amplicons are given in Table 1. 2.3.3. Standardization of duplex-PCR Based on various trials, the components of the reaction mixture were optimized as follows; 3.0 Al of the bacterial cell Table 1 Details of primers used in the study Sl. No. Primer sequence Target gene Amplicon size Reference 1. Forward Primer 5-TCA TGG CTC AGA TTG AAC GCT-3 Reverse Primer 5-CGG GGC TTT CAC ATC TAA CTT ATC-3 16S rRNA 599 bp Graf (1999) 2. Forward Primer Aerolysin 252 bp Santos et al. 5-GCA GAA CCC ATC TAT CCA G-3 Reverse Primer 5-TTT CTC CGG TAA CAG GAT TG-3 (1999)
lysate, 3.0 Al of10 Taq DNA polymerase buffer, 1.5 mM MgCl 2, 0.32 mM each of dATP, dDTP, dGTP, and dCTP, 10 pM each of the two forward and two reverse primers, 1.5 U of Taq DNA polymerase and sterile milli-Q water to make up the desired volume of 25 Al. The standardized amplification reactions for the duplex- PCR started with an initial denaturation temperature at 94 -C for 2 min. These were followed by 35 cycles each of 1 min denaturations at 94 -C, 1 min annealing at 56 -C and 1 min extension at 72 -C. This was followed by a final extension for 5 min at 72 -C. 2.4. Specificity of standardized I-ELISA and duplex-PCR assays The specificity of the standardized I-ELISA and PCR assays was validated by subjected various Gram-negative and Grampositive cultures (Table 2) to I-ELISA and duplex-PCR. For assessing specificity of I-ELISA the plates were coated with 10 9 cells/ml of different organisms to be tested and treating them with 1:800 dilution of anti-OMP serum. For duplex-PCR, the bacterial cultures were inoculated in BHI broth and after incubating overnight templates were prepared and 3 Al of each bacterial lysate was subjected to duplex-PCR assay. 2.5. Experimental inoculation studies in foods Experimental inoculation studies in chicken and milk were carried out to assess the suitability of the standardized I-ELISA and duplex-PCR methods for detection of A. hydrophila subsp. hydrophila (MTCC 646). One hundred and twenty-five grams of chicken samples were collected from a retail poultry shops and were immediately transferred to laboratory under refrigerated conditions and analyzed for presence of Aeromonas within 2 h of collection. Similarly, 125 ml of milk samples were collected from unorganized dairies and were immediately transferred to laboratory under refrigerated conditions and analyzed for presence of Aeromonas within 2 h of collection. Serial 10 fold dilutions (10 0 to 10 8 ) of the standard strain of A. hydrophila subsp. hydrophila (MTCC 646) were made in sterile PBS. One milliliter of these different dilutions were inoculated in 9 g of homogenized chicken samples/9 ml milk samples along with negative controls followed by incubation in 90 ml APW-C. The experimentally inoculated chicken and milk samples were then subjected to I-ELISA, duplex-PCR and cultural methods after 12 h enrichment in APW-C broth. In total three trails were conducted. 2.6. Detection of aeromonads from naturally contaminated food samples S. Arora et al. / International Journal of Food Microbiology 106 (2006) 177–183 179 Fifty samples each of chicken (25 g) and milk (25 ml) were collected as described earlier and examined for the presence of Aeromonas spp. using I-ELISA, duplex-PCR and cultural Table 2 Bacterial strains used in the study Sl. No. Name of organism Strain number Aeromonas strains 1. Aeromonas hydrophila subsp. hydrophila MTCC 646 2. A. hydrophila Env. 1 3. A. hydrophila Env. 2 4. A. hydrophila Env. 3 5. A. hydrophila Clin. 1 6. A. hydrophila 4030 7. A. hydrophila 4048 8. A. hydrophila A308 9. A. veronii NBIMCC 150 10. A. veronii NBIMCC 1048 11. A. veronii 4074 12. A. veronii 4066 13. A. veronii 4042 14. A. caviae Ac. No. 5 15. A. caviae Ac. No. 7 16. A. caviae Ac. No. 30 17. A. caviae Sch. 3 18. A. jandaei A314 19. A. culicicula MTCC 3249 Other Gram-negative strains 20. Brucella melitensis S19 21. Citrobacter spp. A647 22. Enterobacter spp. A514 23. Escherichia coli BM-14 24. Hafnia alvei A267 25. Klebsiella spp. A340 26. Pasteurella multocida A538 27. Proteus vulgaris A088 28. Pseudomonas aeruginosa LB 01 29. Pseudomonas spp. A692 30. Salmonella spp. A598 31. Serratia spp. A550 32. Shigella flexineri A425 33. Vibrio parahaemolyticus A753 34. Yersinia enterocolitica A746 Gram positive strains 35. Bacillus cereus A431 36. Corynebacterium diphtheriae A003 37. Listeria monocytogenes MTCC 1143 38. Rhodococcus equi MTCC 1153 39. Staphylococcus aureus MTCC 1144 40. Streptococcus faecalis MTCC 439 methods simultaneously after single step selective enrichment in APW-C broth for 12 h. 3. Results and discussion Aeromonads have high ability of adaptation to different environments (Mateos et al., 1993) and possess amazing properties which enable them to survive and thrive in diverse conditions (Ascencio et al., 1995; Agarwal, 1997). This makes them widely distributed in nature having cosmopolitan occurrence both in aquatic and terrestrial environments. Recent years have witnessed motile aeromonads emerging as an important foodborne pathogen worldwide (Kirov, 1993; Merino et al., 1993). Numerous detection methods, conventional as well as rapid, have been tried for detecting Aeromonas spp. in foods
Page 1: Comparison of ELISA and PCR vis-à-
Page 5 and 6: Table 3 Results of detection of Aer
Page 7: S. Arora et al. / International Jou

Comparison of ELISA and PCR vis-a`-vis cultural methods for ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?