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

Comparison of ELISA and PCR vis-à-vis cultural methods for detecting
Aeromonas spp. in foods of animal origin
Abstract
S. Arora a, *, R.K. Agarwal a , B. Bist a,b
a Division of Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar, Bareilly (UP)-243 122, India
b Department of Veterinary Public Health, College of Veterinary Science and Animal Husbandry, Mathura (UP)-281 001, India
Received 16 December 2004; received in revised form 6 April 2005; accepted 30 June 2005
The present study was conducted to assess the best method of the most commonly used methods for detection of aeromonads in foods of
animal origin. With this objective an OMP based indirect plate ELISA and a duplex-PCR using primers targeting aerolysin gene and 16S rRNA
gene and yielding amplicons of 252 bp and 599 bp, respectively, were standardized. The standardized protocols and the conventional cultural
method were then compared for their respective sensitivities and specificities for detecting aeromonads from chicken and milk samples. Both the
standardized assays were found to be highly specific for Aeromonas. The efficiency of the standardized indirect-ELISA and duplex-PCR protocols
was assessed by artificial inoculation studies with varying concentrations of Aeromonas cells inoculated in chicken and milk samples followed by
enrichment in Alkaline Peptone Water supplemented with 10 mg/ml cephalothin (APW-C) for 12 h. The results revealed that indirect-ELISA was
able to detect a minimum of 10 3 cells/ml or g of Aeromonas cells in spiked milk and chicken samples, respectively. Whereas, duplex-PCR and
cultural method were able to detect as low as 1 cell/ml or g of Aeromonas cells in spiked milk and chicken samples. The developed assays were
also tested for their efficiency to detect Aeromonas spp. in naturally contaminated milk and chicken samples. Out of a total 50 milk samples
screened for presence of Aeromonas by the three methods viz., indirect-ELISA, duplex-PCR and cultural method only 1 (2%) turned out to be
positive showing positive results by all three methods. Similarly, 50 samples of chicken were tested by all three methods. Three samples (6%)
turned out to be positive and here again by all the three methods.
D 2005 Published by Elsevier B.V.
Keywords: Aeromonas; PCR; ELISA; Chicken; Milk
1. Introduction
The culminating quarter of the previous century witnessed
the explosion of scientific interest in members of the genus
Aeromonas as human and animal pathogens. Aeromonads
have been implicated as potential food poisoning agents
(Buchanan and Palumbo, 1985). Foods of animal origin like
fishes and other seafoods, meat and meat products, eggs,
milk and milk products have been reported to be contaminated
by Aeromonas spp. (Bachhil and Bhilegaonkar, 1995;
Tsai and Chen, 1996; Agarwal et al., 2000; Kumar et al.,
2001; Neyts et al., 2000; Yucel and Citak, 2003; Yucel and
Erdem, 2004).
0168-1605/$ - see front matter D 2005 Published by Elsevier B.V.
doi:10.1016/j.ijfoodmicro.2005.06.019
International Journal of Food Microbiology 106 (2006) 177 – 183
* Corresponding author. High Security Animal Disease Laboratory
(HSADL), Kokta Road, Anand Nagar, Bhopal (MP), 462 021, India. Tel.:
+91 9826570605.
E-mail address: drsarora@rediffmail.com (S. Arora).
www.elsevier.com/locate/ijfoodmicro
Difficulty in recognizing potentially significant Aeromonas
strains in foods poses a dilemma for public health authorities
(Kirov, 1993). The conventional microbiological procedures
for isolating and identifying Aeromonas spp. from foods are
laborious and time consuming (ICMSF, 1996; Agarwal and
Bhilegaonkar, 2003). Some novel approaches like polymerase
chain reaction (PCR) (Kingombe et al., 1999; Wang et al.,
2003), outer membrane protein based immunoassays (Sachan
and Agarwal, 2002; Ghatak et al., 2003), DNA/RNA probes
(Dorsch et al., 1994; Ludwig et al., 1994) and flow cytometry
(Diaper et al., 1992) have been used for detection and/or
identification of Aeromonas spp. from foods or environmental
or clinical samples.
There is a need to develop rapid techniques, and to assess
the best technique out of them, for detection of Aeromonas spp.
from food samples and to compare their results with the
conventional cultural procedures. Thus, the present work was
carried out to standardize an OMP based indirect plate ELISA,

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

180
(Agarwal and Bhilegaonkar, 2003; Agarwal et al., 2003a). Here
three different methods viz., indirect-ELISA, duplex-PCR and
conventional cultural method were compared for their specificity
and sensitivity in detecting Aeromonas spp. in foods of
animal origin.
3.1. Indirect plate ELISA
OMPs have attracted significant attention in recent years by
numerous workers for their diagnostic potentials (Kuijper et al.,
1989; Santos et al., 1996; Sachan and Agarwal, 2002; Ghatak
et al., 2003). OMPs have also been used for development of
immunoassays for detecting Aeromonas providing promising
results (Sachan and Agarwal, 2002; Ghatak et al., 2003).
For standardization of OMP based ELISA, OMP were
extracted by ultrasonication and centrifugation. Separation of
inner and outer membranes was achieved by incubation with
sarkosyl. This method of separating OMPs is known to give full
recovery of OMPs (Kuijper et al., 1989; Ghatak et al., 2001).
Sera were raised against the OMPs in rabbit and this method
has been successfully used by previous workers for raising
antiserum against outer membrane proteins of the bacteria
(Sachan and Agarwal, 2002; Ghatak et al., 2003).
The antiserum raised against OMP was found to be of high
antibody titre as was reported earlier by Sachan and Agarwal
(2002). This high titre of antiserum is suggested to have an
advantage of avoiding cross-reactions by dilution of antiserum
(Sachan and Agarwal, 2002) as evident in this study where no
cross-reaction was observed at the low dilution of serum being
used.
3.2. Duplex-PCR
Polymerase chain reaction (PCR) has been one of the most
preferred tools in food microbiology in recent years for
detection of food borne pathogens due to its rapid nature and
high specificity and sensitivity (Olsen, 2000). However, for
Aeromonas spp., the PCR has been mostly used for the
characterization of isolates either for their phylogenetic
position or for virulence characterization (Graf, 1999; Kingombe
et al., 1999; Sechi et al., 2002; Abdullah et al., 2003;
Datta et al., 2003). There are hardly any reports on the use of
PCR for detection of Aeromonas from foods (Gonzalez-
Rodriguez et al., 2002; Porteen, 2004).
In this study duplex-PCR system was standardized using
primers targeted for 16S rRNA gene, which is genus specific
primer for detecting all species of Aeromonas, and for
aerolysin gene a major virulence factor of Aeromonas spp.
(Graf, 1999; Gonzalez-Rodriguez et al., 2002). The standardized
duplex-PCR was analyzed for detecting aeromonads in
spiked food samples and the results obtained were validated
with the natural samples.
The standardized duplex-PCR revealed the presence of two
bands after agarose gel electrophoresis. One amplicon of 252
bp corresponding to aerolysin gene (Santos et al., 1999) and
other of 599 bp corresponding to 16S rRNA gene (Graf, 1999)
were obtained.
S. Arora et al. / International Journal of Food Microbiology 106 (2006) 177–183
The boiling and snap chilling method was used for bacterial
lysis. This method has been reported to be a simple, rapid and
reliable method (Surendran, 2002). It does not require any
sophisticated equipment and time of processing is very less as
compared to other methods like sonication. Thus, it is apt for
large scale testing of samples. Also there are very less chances
of contamination of DNA (Surendran, 2002; Balamurugan,
2002). This method also provides freedom from any possible
inhibitory effect on PCR as mediated by detergents (SDS or
TritionX-100) used in certain other methods of DNA extraction
(Surendran, 2002). The bacterial lysate prepared after two
washings in PBS was used as it is reported to give bands of
higher intensities (Porteen, 2004).
Direct application of PCR to food samples often results in
absence of detectable amplification product or poor sensitivity
(Dickinson et al., 1995) as a result of the inhibitory effect
exerted on PCR by the complex composition of the food
matrices leading to false negative results (Lantz et al., 1994;
Wang et al., 1997). The inclusion of an enrichment step before
PCR helped in overcoming this problem.
In addition to reducing the negative influence of food matrix
by dilution, the enrichment step also enhances the sensitivity of
the assay by increasing the number of microorganisms and
eliminates the risk of amplifying DNA belonging to the dead
and non-culturable cells (Josephson et al., 1993). The
enrichment step increases the number of viable cells whereas
the dead and non-culturable cells do not grow in the
enrichment broth and thus unlikely to be detected by PCR
assay (Surendran, 2002). A single step enrichment of samples
(spiked or natural) in APW-C was thus followed; as had been
recommended by numerous workers (Wang et al., 1997; Olsen,
2000; Porteen, 2004). This enrichment step of only 12 h still
allows the whole procedure to be completed within a single
working day schedule.
Use of APW-C as enrichment broth has an additional
advantage in the cephalothin added to it which suppresses
significant initial levels of background micro flora that can
interfere with the PCR. Sachan and Agarwal (2000) found out
that cephalothin in concentration of 10 mg/l had no inhibitory
effect on various strains of Aeromonas and at the same time
exerted inhibitory effect on the most of the other related
foodborne pathogens tested.
3.3. Specificity of I-ELISA and duplex-PCR
Seven different strains of A. hydrophila, 5 different strains
of A. veronii, 4 different strains of A. caviae, a single strain
each of A. jandaei and A. culicicula (Table 2) were subjected
to the standardized indirect-ELISA. All the strains gave
positive reactions and thus strengthening the claim of the
standardized protocol for detecting Aeromonas irrespective of
type of stain involved as was reported by Sachan and
Agarwal (2002). Using duplex-PCR also these strains tested
positive. Graf (1999) was also able to detect different species
of Aeromonas using the primer targeted for 16S rRNA
primer set. Similarly, Gonzalez-Rodriguez et al. (2002)
reported primer set targeted against aerolysin gene to be

Table 3
Results of detection of Aeromonas from artificially inoculated food samples
Sl. No. Aeromonas
concentration/g of
meat or (ml of milk)
I-ELISA Duplex-PCR Cultural
method
1. 1 cell/g + +
2. 10 cells/g + +
3. 10 2 cells/g + +
4. 10 3 cells/g + + +
5. 10 4 cells/g + + +
6. 10 5 cells/g + + +
7. 10 6 cells/g + + +
8. 10 7 cells/g + + +
9. Negative control
species specific after analyzing it for various species of
Aeromonas.
Six Gram-positive strains and 15 other Gram-negative
bacteria (Table 2) were also tested for a possible misdetection
by these standardized protocols. None of the other bacteria
gave positive results either by indirect-ELISA or by duplex-
PCR. Sachan and Agarwal (2002) also reported none of the
strains used in this study to cross-react in the OMP based
indirect-ELISA standardized by her. Graf (1999) tested many
bacterial strains other than that of Aeromonas in his study using
16S rRNA primers; but none of them gave positive products.
3.4. Analysis of results of artificial inoculation studies
S. Arora et al. / International Journal of Food Microbiology 106 (2006) 177–183 181
The results of detection of Aeromonas from spiked samples
by cultural methods revealed that after a single step enrichment
of spiked food samples at 37 -C for 12 h in APW-C when the
plating was done on ADA it detected up to 1 cell/ml or g of
food samples after incubating the ADA plates at 37 -C for 24 h.
Porteen (2004) was also able to detect a minimum of 1 cell/ml
or g of food in 40% of the trials conducted by him.
The results of detection of Aeromonas from spiked samples
by indirect-ELISA (Table 3) revealed a minimum detection
level of 10 3 cells/ml or g of milk and chicken samples,
respectively. Sachan and Agarwal (2002) observed that OMP
based indirect-ELISA gave optimum results at cell concentrations
of 10 6 or more. But in the present study a single step
enrichment of the artificially inoculated food at 37 -C for 12
h in APW-C was involved, which was not used by her. Thus,
the final cell concentration of the Aeromonas cells in the
enrichment broth tube containing food sample inoculated with
10 3 cells/ml or g might have reached up to 10 6 cells/ml or g.
Analyzing the results of detection of Aeromonas from
spiked samples by duplex-PCR revealed its superiority in
rapidly detecting Aeromonas in foods at a concentration of
single cell/ml or g. Detection limits of 0.5 10 2 cells of
Aeromonas from spiked food samples using 16S rDNA PCR
were reported earlier by Khan and Cerniglia (1997) while
Porteen (2004) reported a minimum detection level of 1 cells/
ml or g using 16S rRNA PCR in a single trial out of five trails
conducted by him to detect Aeromonas in artificially inoculated
milk and chicken samples after 12 h enrichment at 37 -C in
APW-C. Using the aerolysin primers Gonzalez-Rodriguez et al.
(2002) reported a minimum detection level of 6.91 cfu/g of
initial inoculation, after 16–18 h enrichment, from artificially
inoculated fish meat samples. On, the other hand Porteen
(2004) reported a minimum detection level of 10 2 cells/g from
spiked meat samples and 10 3 cells/ml from spiked milk
samples, after 12 h enrichment at 37 -C in APW-C in a single
trial out of the five trails conducted. Variations in results from
spiked food samples may be attributed to the overgrowth of
other competing micro flora (Lee et al., 1990).
The statistical analysis of the results of the artificial
inoculation studies in chicken for determining the sensitivity
for I-ELISA, duplex-PCR and cultural method revealed the
sensitivity values for both duplex-PCR and cultural method to
be 100% as no false negative result was obtained. The
sensitivity for I-ELISA was found to be 66% as it gave false
negative results in dilutions in concentrations of 10 2 to 10cells/ml.
Specificity of all the three methods viz., I-ELISA, duplex-
PCR and cultural method for detecting Aeromonas spp. in
artificial inoculation studies in chicken was found to be 100%
there was no false positive reaction in the negative controls.
3.5. Detection of aeromonads from naturally contaminated
food samples
Three samples out of the 50 chicken samples screened 3
were found positive for Aeromonas spp. by duplex-PCR (Fig.
1) as well as I-ELISA. These samples also found positive by
cultural methods. Thus, the incidence of Aeromonas in chicken
samples turned out to be 6%; which were comparable to the
incidence rate (4.0%) reported by Porteen (2004). Higher
incidence rates ranging from 16.67% to 70% were recorded by
other workers (Bok et al., 1986; Khurana and Kumar, 1997;
Neyts et al., 2000). The environmental conditions prevailing at
the time of sampling and the difference in geographical
Fig. 1. Duplex-PCR results for screening of natural samples. Lane M: 100 bp
DNA ladder plus; Lane 1: Milk isolate; Lane 2: Chicken isolate-2; Lane 3:
Chicken isolate-1 Lane 4: Chicken isolate-3.

182
locations may be the possible reasons for these variations in
incidence rates of Aeromonas in poultry.
One of the 50 milk samples screened turned out to be
positive for Aeromonas. This also gave positive result with all
three methods viz., duplex-PCR (Fig. 1), I-ELISA as well as
cultural method. The incidence rate of Aeromonas in milk thus
was 2.0% which was comparable to the isolation rate of 2.85%,
5.56% and 4.0% reported by Agarwal et al. (2000), Kumar et
al. (2001) and Porteen (2004), respectively.
The study revealed that there was a good correlation
between duplex-PCR and cultural method but indirect-ELISA,
though detecting all naturally contaminated chicken and milk
samples, was not able to detect Aeromonas below the
concentrations of 10 3 cells/ml or g in the spiking studies. This
limits the use of indirect-ELISA as a fool-proof detection
method for aeromonads in foods when compared to PCR.
Thus, it can be concluded that both OMP based indirect
plate ELISA as well as duplex-PCR, following 12 h enrichment
in APW-C, are reliable methods for detection of Aeromonas
from foods of animal origin on account of their specificity; and
duplex-PCR, following 12 h enrichment in APW-C, is the
method of choice amongst the three methods compared in this
study on account of its rapidity and sensitivity as well as
specificity.
Acknowledgements
The authors are thankful to the Director, Indian Veterinary
Research Institute (IVRI), Izatnagar, Bareilly (UP), India for
permitting the first author to conduct this work at IVRI.
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