PROPIDIUM MONOAZIDE (PMA) QUANTITATIVE POLYMERASE CHAIN REACTION (q PCR) FOR DETECTION OF LIVE BACTERIA IN FOOD SAMPLES
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Date
2018
Authors
Shathish Sharma, BJ
Rathnapraba, S
Meignanalakshmi, S
TANUVAS
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Abstract
The present study was aimed to investigate the presence of foodborne bacterial pathogens in meat products. A total of 100 meat samples (including raw chicken, chicken nuggets, sausage, Kheema, cutlet, meat balls, pakoda, raw mutton, mutton balls, pork sausage, kheema, khabab, ham bacon, beef khabab, cubes, kheema and sausage) have been collected from different retail outlets in Chennai. The overall occurrence of B. cereus, S. aureus and E. coli in different meat samples were found to be 15%, 20% and 14%respectively. All the presumptive colonies were subjected to molecular screening by targeting the Hemolysin gene, fem A gene and mal B gene in B. cereus, S. aureus and E. coli respectively. Based on the molecular characterization, about 11, 5 and 5 isolates of B. cereus, S. aureus and E. coli were identical in screened meat samples. The isolates from the meat and meat products were subjected to antibiotic susceptibility test and the results revealed that all the isolates showed 100% resistance to Vancomycin and Penicillin. Staphylococcus aureus shows 100% resistance to Methicillin and 68.8% sensitivity to
ciprofloxacin. In B. cereus they showed 100% sensitivity to methicillin whereas E. coli showed 100% resistance to ampicillin and 100% sensitivity to ciprofloxacin. PMA-qPCR was developed to quantify the viable foodborne bacterial pathogen in meat products. In the present study 2.5 mM concentration of PMA dye was used, which will help to prevent the amplification of DNA from the dead cells. The developed PMA-qPCR had the detection limit of 1X101.25 CFU/g, 1X100.80CFU/g and 1X 100.4 CFU/g forB. cereus, S.aureus and E. coli respectively. These PMA qPCR results were compared with conventional qPCR. These results indicated that the developed PMA-qPCR was more sensitive than the conventional culture method. The sensitivity, specificity and accuracy of developed PMA-qPCR were calculated using Kappa statistics method. The sensitivity of PMA-qPCR for Staphylococcus aureus, Bacillus cereus and Escherichia coli were found to be 60%, 54.4% and 60% respectively. Specificity of the PMA qPCR was found to be 66.7%, 66.7% and 64.4% for Bacillus cereus Staphylococcus aureus and Escherichia coli respectively. The PMA q-PCR could discriminate among viable and dead bacteria it detect only live bacteria from the food samples.
Description
TNV_TH_MSC(M)16002_2018
Keywords
Veterinary Science