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A simple and rapid procedure for the detection of genes encoding Shiga toxins and other specific DNA sequences.

Nejman-Faleńczyk B, Bloch S, Januszkiewicz A, Węgrzyn A, Węgrzyn G - Toxins (Basel) (2015)

Bottom Line: A novel procedure for the detection of specific DNA sequences has been developed.However, instead of the detection of the fluorescence signal with the use of real-time PCR cyclers, fluorescence/luminescence spectrometers or fluorescence polarization readers, as in all previously-reported procedures, we propose visual observation of the fluorescence under UV light directly in the reaction tube.It may be suitable for use in research laboratories, as well as in diagnostic units of medical institutions, even those equipped only with a thermocycler and a UV transilluminator, particularly if rapid identification of a pathogen is required.

View Article: PubMed Central - PubMed

Affiliation: Depratment of Molecular Biology, University of Gdańsk, Wita Stwosza 59, 80-308 Gdańsk, Poland. bozena.nejman@ug.edu.pl.

ABSTRACT
A novel procedure for the detection of specific DNA sequences has been developed. This procedure is based on the already known method employing PCR with appropriate primers and a sequence-specific DNA probe labeled with the fluorescent agent 6-carboxylfluorescein (FAM) at the 5' end and the fluorescence quencher BHQ-1 (black hole quencher) at the 3' end. However, instead of the detection of the fluorescence signal with the use of real-time PCR cyclers, fluorescence/luminescence spectrometers or fluorescence polarization readers, as in all previously-reported procedures, we propose visual observation of the fluorescence under UV light directly in the reaction tube. An example for the specific detection of the Shiga toxin-producing Escherichia coli (STEC) strains, by detecting Shiga toxin genes, is demonstrated. This method appears to be specific, simple, rapid and cost effective. It may be suitable for use in research laboratories, as well as in diagnostic units of medical institutions, even those equipped only with a thermocycler and a UV transilluminator, particularly if rapid identification of a pathogen is required.

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Related in: MedlinePlus

The analysis of STEC stx-positive strain (+) EDL933W and the stx-negative (−) strain MG1655 of E. coli K12 bacteria for the presence of the genes encoding Shiga toxins 1 and 2 and the occurrence of nonspecific PCR products using PCR with a FAM- and BHQ-1-labelled probe. Detection of the signal from the tox1probe (A) or tox2probe (B), complementary to the genes encoding Shiga toxin 1 or Shiga toxin 2, respectively, was performed by analysis of PCR products by agarose gel electrophoresis and using a UV transilluminator. Black arrows indicate the size of the target PCR products: 196 bp for stx1 and 211 bp for stx2.
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toxins-07-04745-f003: The analysis of STEC stx-positive strain (+) EDL933W and the stx-negative (−) strain MG1655 of E. coli K12 bacteria for the presence of the genes encoding Shiga toxins 1 and 2 and the occurrence of nonspecific PCR products using PCR with a FAM- and BHQ-1-labelled probe. Detection of the signal from the tox1probe (A) or tox2probe (B), complementary to the genes encoding Shiga toxin 1 or Shiga toxin 2, respectively, was performed by analysis of PCR products by agarose gel electrophoresis and using a UV transilluminator. Black arrows indicate the size of the target PCR products: 196 bp for stx1 and 211 bp for stx2.

Mentions: Due to the fact that phages bearing stx1 and stx2 genes can be transmitted to previously non-pathogenic E. coli bacteria [32,33], which naturally occur in the human gut, the detection of these genes might be difficult and give false positive results, as nonspecifically-primed reactions may occur [34,35]. Keeping this in mind, we decided to check how the proposed method works in a situation with increased risk of nonspecific primer binding. For that, we used DNA isolated from an EDL933W strain that is stx-positive (+) and DNA from an stx-negative (−) E. coli K12 MG1655 strain as the representative of non-pathogenic bacteria. DNA was used in different amounts: 1, 10 and 100 pg, prepared as ten-fold dilutions of 1 ng of genomic DNA, which corresponds to 168, 1680 and 16,800 CFU, respectively. Additionally, we increased the number of PCR cycles to 40, as excessive cycling increases the opportunity for nonspecific amplification [36,37]. As expected, analysis of PCR products by agarose gel electrophoresis allowed us to identify smeared and nonspecific bands, especially occurring in the case of stx2 detection (Figure 3). Note that the similar problem with nonspecific binding during PCR-based detection of the stx2 gene was also described previously by Fagan and collaborators [34]. Importantly, even in the case of the appearance of nonspecific PCR products (which is likely to occur during testing of previously unknown, natural isolates of bacteria), the fluorescence is not detected over a UV transilluminator, contrary to the presence of DNA bands and smears on an electropherogram (Figure 3). Post-PCR visual observation of the fluorescence directly in the tube that we propose here allows avoidance of false positive results in dubious situations when a nonspecific PCR product is similar in size to that of a target amplicon (Figure 3, Lane 12). The specificity of the presented method is increased in comparison to conventional PCR because of the probe, which requires an additional complementary region within the template DNA. This may indicate that interpretation of the results of the test performed according to the proposed procedure is easier than that of the traditional PCR-based assay, and thus, our method is more specific, while, as indicated in Figure 3, its sensitivity is at a similar level, equal to 10 pg for both genes.


A simple and rapid procedure for the detection of genes encoding Shiga toxins and other specific DNA sequences.

Nejman-Faleńczyk B, Bloch S, Januszkiewicz A, Węgrzyn A, Węgrzyn G - Toxins (Basel) (2015)

The analysis of STEC stx-positive strain (+) EDL933W and the stx-negative (−) strain MG1655 of E. coli K12 bacteria for the presence of the genes encoding Shiga toxins 1 and 2 and the occurrence of nonspecific PCR products using PCR with a FAM- and BHQ-1-labelled probe. Detection of the signal from the tox1probe (A) or tox2probe (B), complementary to the genes encoding Shiga toxin 1 or Shiga toxin 2, respectively, was performed by analysis of PCR products by agarose gel electrophoresis and using a UV transilluminator. Black arrows indicate the size of the target PCR products: 196 bp for stx1 and 211 bp for stx2.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663531&req=5

toxins-07-04745-f003: The analysis of STEC stx-positive strain (+) EDL933W and the stx-negative (−) strain MG1655 of E. coli K12 bacteria for the presence of the genes encoding Shiga toxins 1 and 2 and the occurrence of nonspecific PCR products using PCR with a FAM- and BHQ-1-labelled probe. Detection of the signal from the tox1probe (A) or tox2probe (B), complementary to the genes encoding Shiga toxin 1 or Shiga toxin 2, respectively, was performed by analysis of PCR products by agarose gel electrophoresis and using a UV transilluminator. Black arrows indicate the size of the target PCR products: 196 bp for stx1 and 211 bp for stx2.
Mentions: Due to the fact that phages bearing stx1 and stx2 genes can be transmitted to previously non-pathogenic E. coli bacteria [32,33], which naturally occur in the human gut, the detection of these genes might be difficult and give false positive results, as nonspecifically-primed reactions may occur [34,35]. Keeping this in mind, we decided to check how the proposed method works in a situation with increased risk of nonspecific primer binding. For that, we used DNA isolated from an EDL933W strain that is stx-positive (+) and DNA from an stx-negative (−) E. coli K12 MG1655 strain as the representative of non-pathogenic bacteria. DNA was used in different amounts: 1, 10 and 100 pg, prepared as ten-fold dilutions of 1 ng of genomic DNA, which corresponds to 168, 1680 and 16,800 CFU, respectively. Additionally, we increased the number of PCR cycles to 40, as excessive cycling increases the opportunity for nonspecific amplification [36,37]. As expected, analysis of PCR products by agarose gel electrophoresis allowed us to identify smeared and nonspecific bands, especially occurring in the case of stx2 detection (Figure 3). Note that the similar problem with nonspecific binding during PCR-based detection of the stx2 gene was also described previously by Fagan and collaborators [34]. Importantly, even in the case of the appearance of nonspecific PCR products (which is likely to occur during testing of previously unknown, natural isolates of bacteria), the fluorescence is not detected over a UV transilluminator, contrary to the presence of DNA bands and smears on an electropherogram (Figure 3). Post-PCR visual observation of the fluorescence directly in the tube that we propose here allows avoidance of false positive results in dubious situations when a nonspecific PCR product is similar in size to that of a target amplicon (Figure 3, Lane 12). The specificity of the presented method is increased in comparison to conventional PCR because of the probe, which requires an additional complementary region within the template DNA. This may indicate that interpretation of the results of the test performed according to the proposed procedure is easier than that of the traditional PCR-based assay, and thus, our method is more specific, while, as indicated in Figure 3, its sensitivity is at a similar level, equal to 10 pg for both genes.

Bottom Line: A novel procedure for the detection of specific DNA sequences has been developed.However, instead of the detection of the fluorescence signal with the use of real-time PCR cyclers, fluorescence/luminescence spectrometers or fluorescence polarization readers, as in all previously-reported procedures, we propose visual observation of the fluorescence under UV light directly in the reaction tube.It may be suitable for use in research laboratories, as well as in diagnostic units of medical institutions, even those equipped only with a thermocycler and a UV transilluminator, particularly if rapid identification of a pathogen is required.

View Article: PubMed Central - PubMed

Affiliation: Depratment of Molecular Biology, University of Gdańsk, Wita Stwosza 59, 80-308 Gdańsk, Poland. bozena.nejman@ug.edu.pl.

ABSTRACT
A novel procedure for the detection of specific DNA sequences has been developed. This procedure is based on the already known method employing PCR with appropriate primers and a sequence-specific DNA probe labeled with the fluorescent agent 6-carboxylfluorescein (FAM) at the 5' end and the fluorescence quencher BHQ-1 (black hole quencher) at the 3' end. However, instead of the detection of the fluorescence signal with the use of real-time PCR cyclers, fluorescence/luminescence spectrometers or fluorescence polarization readers, as in all previously-reported procedures, we propose visual observation of the fluorescence under UV light directly in the reaction tube. An example for the specific detection of the Shiga toxin-producing Escherichia coli (STEC) strains, by detecting Shiga toxin genes, is demonstrated. This method appears to be specific, simple, rapid and cost effective. It may be suitable for use in research laboratories, as well as in diagnostic units of medical institutions, even those equipped only with a thermocycler and a UV transilluminator, particularly if rapid identification of a pathogen is required.

Show MeSH
Related in: MedlinePlus