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A novel multiplex PCR method for the detection of virulence-associated genes of Escherichia coli O157:H7 in food

View Article: PubMed Central - PubMed

ABSTRACT

Shiga toxin-producing Escherichia coli O157:H7 (E. coli O157:H7) strains are foodborne infectious agents that cause a number of life-threatening diseases, including hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Shiga toxin 1 (stx1), shiga toxin 2 (stx2), or a combination of both are responsible for most clinical symptoms of these diseases. Hence, various diagnostic methods have been developed so far to detect shiga toxins such as cell culture, ELISA, Rapid Latex Agglutination (RPLA) and hybridization, but due to high costs and labor time in addition to low sensitivity, they have not received much attention. The aim of this study was to develop a complete, rapid and reliable multiplex PCR (mPCR) method by using two pairs of specific primers to detect either the stx1 or the stx2 gene confirms the presence of E.coli O157:H7. The study results show that stx1F/stx1R primers are specific for stx1 and primers stx2F/stx2R are specific for stx2 genes in E. coli O157:H7. The mPCR method with two pairs of primers for amplifying the stx1, stx2 target genes to detect E. coli O157:H7 in food has been set up successfully. Complete method performed well in both types of food matrices with a detection limit of 3 CFU/25 g or mL of food samples. Tests on 180 food samples have shown a specificity value of 93.75 % (95 % confidence interval [CI], 82.83–100), a sensitivity of 100 % (95 % CI, 83.79–99.85 %), and an accuracy of 96.66 % (CI 95 %, 83.41–99.91 %). Interestingly, results indicate that the mPCR performed as well as the traditional culture methods and can reduce the diagnosis time to 2 days. Finally, complete mPCR method was applied to natural samples covering a wide variety of food types proving that the mPCR method was a rapid and reliable screening method for detection of E. coli O157:H7 in food and environmental samples.

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Specificity of primer pair stx1, stx2 in the optimized PCR for amplification of E. coli O157:H7. Lanes MR, 100-bp DNA ladder; 1, E. coli ATCC 25922; 2, E. coli ATCC 11775; 3, Salmonella enterica ATCC 14028; 4, Shigella sonnei ATCC 9290; 5, Vibrio cholerae ATCC 17802; 6, E. coli O157:H7 (NLU); 7, Water; 8–15, the strains of E1 to E8, respectively
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Fig3: Specificity of primer pair stx1, stx2 in the optimized PCR for amplification of E. coli O157:H7. Lanes MR, 100-bp DNA ladder; 1, E. coli ATCC 25922; 2, E. coli ATCC 11775; 3, Salmonella enterica ATCC 14028; 4, Shigella sonnei ATCC 9290; 5, Vibrio cholerae ATCC 17802; 6, E. coli O157:H7 (NLU); 7, Water; 8–15, the strains of E1 to E8, respectively

Mentions: Shiga toxins are produced by E. coli O157:H7 strains. Shiga toxin might be produced from strains carrying stx1 or stx2 gene only or both. Therefore, we have combined two primers for simultaneous detection of stx1 and stx2 in the same PCR amplification reaction. The reaction conditions for the multiplex PCR assay were optimized to ensure that all of the target gene sequences were satisfactorily amplified. The primers were designed with care to avoid areas of homology with other organisms. The primers had almost equal annealing temperature, which reduced the possibility of nonspecific amplification. The annealing temperature of 54 °C was finally selected based on nearly equal intensity of PCR products. Figure 3 shows the presence of amplified products size 520 and 780 bp after agarose gel electrophoresis. Reliable amplification of two bands of stx1 and stx2 was obtained in standard E. coli 0157:H7 strain (NLU). As a negative control multiplex PCR as water, other microbiologisms were tested no amplicons were observed.Fig. 3


A novel multiplex PCR method for the detection of virulence-associated genes of Escherichia coli O157:H7 in food
Specificity of primer pair stx1, stx2 in the optimized PCR for amplification of E. coli O157:H7. Lanes MR, 100-bp DNA ladder; 1, E. coli ATCC 25922; 2, E. coli ATCC 11775; 3, Salmonella enterica ATCC 14028; 4, Shigella sonnei ATCC 9290; 5, Vibrio cholerae ATCC 17802; 6, E. coli O157:H7 (NLU); 7, Water; 8–15, the strains of E1 to E8, respectively
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4697910&req=5

Fig3: Specificity of primer pair stx1, stx2 in the optimized PCR for amplification of E. coli O157:H7. Lanes MR, 100-bp DNA ladder; 1, E. coli ATCC 25922; 2, E. coli ATCC 11775; 3, Salmonella enterica ATCC 14028; 4, Shigella sonnei ATCC 9290; 5, Vibrio cholerae ATCC 17802; 6, E. coli O157:H7 (NLU); 7, Water; 8–15, the strains of E1 to E8, respectively
Mentions: Shiga toxins are produced by E. coli O157:H7 strains. Shiga toxin might be produced from strains carrying stx1 or stx2 gene only or both. Therefore, we have combined two primers for simultaneous detection of stx1 and stx2 in the same PCR amplification reaction. The reaction conditions for the multiplex PCR assay were optimized to ensure that all of the target gene sequences were satisfactorily amplified. The primers were designed with care to avoid areas of homology with other organisms. The primers had almost equal annealing temperature, which reduced the possibility of nonspecific amplification. The annealing temperature of 54 °C was finally selected based on nearly equal intensity of PCR products. Figure 3 shows the presence of amplified products size 520 and 780 bp after agarose gel electrophoresis. Reliable amplification of two bands of stx1 and stx2 was obtained in standard E. coli 0157:H7 strain (NLU). As a negative control multiplex PCR as water, other microbiologisms were tested no amplicons were observed.Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

Shiga toxin-producing Escherichia coli O157:H7 (E. coli O157:H7) strains are foodborne infectious agents that cause a number of life-threatening diseases, including hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Shiga toxin 1 (stx1), shiga toxin 2 (stx2), or a combination of both are responsible for most clinical symptoms of these diseases. Hence, various diagnostic methods have been developed so far to detect shiga toxins such as cell culture, ELISA, Rapid Latex Agglutination (RPLA) and hybridization, but due to high costs and labor time in addition to low sensitivity, they have not received much attention. The aim of this study was to develop a complete, rapid and reliable multiplex PCR (mPCR) method by using two pairs of specific primers to detect either the stx1 or the stx2 gene confirms the presence of E.coli O157:H7. The study results show that stx1F/stx1R primers are specific for stx1 and primers stx2F/stx2R are specific for stx2 genes in E. coli O157:H7. The mPCR method with two pairs of primers for amplifying the stx1, stx2 target genes to detect E. coli O157:H7 in food has been set up successfully. Complete method performed well in both types of food matrices with a detection limit of 3 CFU/25 g or mL of food samples. Tests on 180 food samples have shown a specificity value of 93.75 % (95 % confidence interval [CI], 82.83–100), a sensitivity of 100 % (95 % CI, 83.79–99.85 %), and an accuracy of 96.66 % (CI 95 %, 83.41–99.91 %). Interestingly, results indicate that the mPCR performed as well as the traditional culture methods and can reduce the diagnosis time to 2 days. Finally, complete mPCR method was applied to natural samples covering a wide variety of food types proving that the mPCR method was a rapid and reliable screening method for detection of E. coli O157:H7 in food and environmental samples.

No MeSH data available.


Related in: MedlinePlus