Comparison of ELISA and PCR vis-à-vis cultural methods for detecting Aeromonas spp. in foods of animal origin.
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.
Arora
S
S
Division of Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar, Bareilly (UP)-243 122, India. drsarora@rediffmail.com
Agarwal
R K
RK
Bist
B
B
D000333:Aeromonas / Q000302:isolation & purification*; D000818:Animals; D002645:Chickens; D015169:Colony Count, Microbial / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005506:Food Contamination / Q000032:analysis; D008460:Meat / Q000382:microbiology*; D008892:Milk / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity