Application of multiplex PCR assay based on uidR and fliCH7 genes for detection of Escherichia coli O157:H7 in milk

Kumar, Ashwani; Grover, Sunita; Batish, V. K.

doi:10.2323/jgam.59.011

Cited by 16 publications

(14 citation statements)

References 24 publications

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“…Similarly, the detection limits achieved in this study were superior to those of Martinez et al (2001) who achieved a sensibility limit of 100 CFU/mL for the same pathogen. Moreover, a detection limit of 2 CFU/mL was verified, being similar to the one of Kumar et al (2013) that detected E. coli producers of the Shiga toxin (STEC) in samples of milk without pre-enriching. Meiri-Bendek et al (2002) conducted a contamination of milk with different concentrations of S. agalactiae for later detection by PCR.…”

Section: Discussionsupporting

confidence: 67%

Molecular screening for the presence of Streptococcus agalactiae, Escherichia coli and Listeria monocytogenes in samples of milk from dairy herds

Pozzobon¹,

Eifler²,

Kich³

et al. 2015

Afr. J. Microbiol. Res.

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This study aims to verify the predominance of contamination with pathogenic microorganisms in dairy herds. In order to validate the initially used methodology, an artificial contamination was conducted in commercially acquired whole UHT milk, with strains of Listeria monocytogenes, Streptococcus agalactiae and Escherichia coli, in final concentrations from 2.10 -7 to 2.10 0 CFU/mL, which were submitted to a DNA extraction protocol and to a later amplification using the polymerase chain reaction (PCR) technique. The 702 bp fragments were identified, 884 and 524 bp corresponding, respectively, to L. monocytogenes, E. coli, S. agalactiae. In order to verify the presence of these pathogens in in natura milk, the samples were obtained directly from the teats of 125 cows from the dairy herds of four producers, and from the cooling tanks of eight producers, being submitted to DNA extraction, and posterior PCR analysis. The data were analyzed with the Chi-squared test ( 2 ) and different sensibility and specificity values were obtained for each microorganism . In cooling tanks, a prevalence of 37.5% of contamination with S. agalactiae and of 31.25% by E. coli was found. Regarding samples obtained from cow teats, we observed the presence of S. agalactiae and E. coli in 16.2 and 47.5% of the samples. No sample tested positive for L. monocytogenes. The results obtained indicate that the isolation protocol of bacterial DNA directly from the milk, and the PCR technique were efficient to detect the analyzed microorganisms, and may be incorporated as part of routine tests. Moreover, PCR may be an important mechanism to evaluate the quality of milk to be consumed.

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Section: Discussionsupporting

confidence: 67%

Molecular screening for the presence of Streptococcus agalactiae, Escherichia coli and Listeria monocytogenes in samples of milk from dairy herds

Pozzobon¹,

Eifler²,

Kich³

et al. 2015

Afr. J. Microbiol. Res.

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“…Recent advances in diagnostic techniques have made several protocols available for isolation and characterization of E. coli O157:H7 from food, fecal, and environmental samples (Deisingh & Thompson, 2004;Tamerat, Muktar, & Shiferaw, 2016). Further, to classify and elaborate the function of the main virulence genes, such as stx 1 and stx 2 , and genes encoding accessory STEC virulence factors, such as eaeA, hlyA, and flic H7 , different PCR assays have been described (Al-Ajmi et al, 2006;Gannon, King, Kim, & Thomas, 1992;Holland, Louie, Simor, & Louie, 2000;Kumar, Grover, & Kumar Batish, 2013;Pan, Chen, & Su, 2002;Sharma, 2006;Sharma & Dean-Nystrom, 2003).…”

Section: Discussionmentioning

confidence: 99%

Multiplex polymerase chain reaction detection of Shiga toxin genes and antibiotic sensitivity of Escherichia coli O157:H7 isolated from beef meat in Quetta, Pakistan

Samad

Abbas

Ahmad

et al. 2018

Journal of Food Safety

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The objective of this study was to conduct a qualitative analysis of raw beef meat sold in the city of Quetta, Pakistan for presence and drug sensitivity of the potentially pathogenic Escherichia coli strain O157:H7. The study used 200 raw beef meat samples collected from retail butcher shops. Conventional and rapid biochemical tests, latex agglutination and multiplex polymerase chain reaction (PCR) using primers designed for the rfb O157 and flic H7 genes were used to detect E. coli O157:H7. All O157:H7 isolates were also tested for Shiga toxin genes stx 1 and stx 2. The prevalence of E. coli O157:H7 in collected beef meat samples was 10%. Detection through PCR was found more sensitive than detection of O and H antigens. The quantity of E. coli O157:H7 isolates positive for Shiga toxins was 50% (20% for stx 1, 45% for stx 2 and 10% for both stx 1 and stx 2). Season wise variation showed highest E. coli O157:H7 prevalence during summer months. A further concern is that E. coli O157:H7 isolates were resistant to a range of common antibiotics. The results indicate an urgent need for applying proper food hygiene practices in the Quetta region to reduce incidence of foodborne diseases and they also emphasize the global problem of antimicrobial resistance. Practical applications E. coli O157:H7 is as a potentially threatening foodborne pathogen. A significant prevalence of E. coli O157:H7 detected in raw beef meat from retail outlets in the city of Quetta indicates an urgent need for applying proper food hygiene practices in the Quetta region to reduce the incidence of foodborne diseases. Furthermore, resistance of the E. coli O157:H7 isolates to a range of commonly used antibiotics emphasizes the global problem of antimicrobial resistance. The multiplex PCR method used here is a reliable, sensitive, and relatively rapid technique for detecting E. coli O157:H7 in food and environmental samples and important for ongoing surveillance to minimize contamination of raw meat products and associated cross contamination by E. coli O157:H7.

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“…It is noteworthy to mention the findings of Kumar et al (2013), who showed the presence of pathogenic E. coli O157:H7 in pasteurized milk samples. These authors described a higher detection signal after enrichment, suggesting the presence of viable cells after pasteurization or postpasteurization contamination.…”

Section: Preparation Of Template Dnamentioning

confidence: 93%

Molecular screening of bovine raw milk for the presence of Shiga toxin-producing Escherichia coli (STEC) on dairy farms

Vendramin¹,

Kich²,

Molina³

et al. 2014

Food Sci. Technol (Campinas)

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Application of multiplex PCR assay based on uidR and fliCH7 genes for detection of Escherichia coli O157:H7 in milk

Cited by 16 publications

References 24 publications

Molecular screening for the presence of Streptococcus agalactiae, Escherichia coli and Listeria monocytogenes in samples of milk from dairy herds

Molecular screening for the presence of Streptococcus agalactiae, Escherichia coli and Listeria monocytogenes in samples of milk from dairy herds

Multiplex polymerase chain reaction detection of Shiga toxin genes and antibiotic sensitivity of Escherichia coli O157:H7 isolated from beef meat in Quetta, Pakistan

Molecular screening of bovine raw milk for the presence of Shiga toxin-producing Escherichia coli (STEC) on dairy farms

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