Citation
Abdul Talib, Mohd Afendy
(2017)
Prevalence of Salmonella spp. in chicken meat and its products, and development of PCR-ELISA for detection of Salmonella enteritidis.
Masters thesis, Universiti Putra Malaysia.
Abstract
The increase in Salmonella infections necessitate the development of methods
for the pathogen detection. Conventional culture for Salmonella detection is
laborious and time-consuming. This has encourage the use of Polymerase
Chain Reaction (PCR) and PCR-enzyme-linked immunosorbent assay (ELISA)
that offer faster result and better sensitivity. To date, there was no published
data on the prevalence and quantities of Salmonella spp., S. Enteritidis and S.
Typhimurium in raw, processed and ready-to-eat (RTE) chicken samples by
most probable number (MPN)-mPCR & MPN-plating and no published data
on the effect of two different matrices of raw chicken meat and chicken
sausages towards PCR-ELISA performance. Thus, this study is aimed to
discover these gaps.
The multiplex polymerase chain reaction (mPCR) method was established.
Three primer sets were successfully designed to simultaneously identify
Salmonella spp., S. Enteritidis and S. Typhimurium. The mPCR containing S.
Typhimurium generated 650 bp and 500 bp amplicons, S. Enteritidis generated
500 bp and 103 bp amplicons and S. Typhi produced a 500 bp amplicon.
Subsequently, MPN-mPCR was used to determine the prevalence and
quantity of Salmonella spp., S. Typhimurium and S. Enteritidis in 130 samples
of chicken meat and its products. The prevalence of Salmonella spp., S.
Enteritidis and S. Typhimurium obtained is 25.38%, 10% and 6.92% respectively. The prevalence of Salmonella spp. in raw chicken meat was the
highest at 45% followed by RTE food at 30% while S. Enteritidis is 22.5% and
8% respectively. No contamination was detected in processed chicken meat
products. The estimated quantity of Salmonella spp., S. Enteritidis and S.
Typhimurium varied from 3 MPN/g to the 1100 MPN/g. Quantity of
Salmonella spp. was found highest in chicken breast and thigh (1100 MPN/g),
S. Typhimurium was highest detected in RTE chicken curry (210 MPN/g),
while S. Enteritidis was highest detected in chicken breast (93 MPN/g).
In subsequent chapter, PCR-ELISA was successfully established and
compared with PCR to detect S. Enteritidis in raw chicken meat & chicken
sausages. The limit of detection (LOD) by PCR-ELISA is 9.4 CFU/mL in raw
chicken meat and 2.46 CFU/mL in chicken sausage while PCR detected 9.4 X
101 CFU/mL and 2.46 X 101 CFU/mL respectively, indicating that the PCRELISA
is 10-fold more sensitive than the conventional PCR. In fact, the LOD
of PCR-ELISA calculated by GraphPad Prism is 15.23 CFU/mL for chicken
sausage and 3.28 CFU/mL for raw chicken meat.
The prevalence and quantity of Salmonella spp., S. Typhimurium and S.
Enteritidis in the samples discovered in this study indicated that the
contaminated chicken samples posed a risk to consumer as it contained high
level of contamination which some exceeded the infectious dose (103 CFU/mL)
of Salmonella to human. Meanwhile, PCR-ELISA offer as an alternative and
improved assay to the PCR and real-time PCR for detection of S. Enteritidis in
semi-quantitative and high-throughput manner. The PCR, MPN-mPCR and
PCR-ELISA method established in this study, could be used selectively as a
screening tools for surveillance and monitoring of foodborne pathogen
contamination so that appropriate actions could be taken by the relevant
parties.
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