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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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The analysis of Shiga toxin-producing Escherichia coli (STEC) strains: 286/00 (stx1−stx2+) (Lanes 1–2), 201/01 (stx1−stx2+) (Lanes 3–4), 319/01 (stx1+stx2−) (Lanes 5–6), 44/02 (stx1+stx2+) (Lanes 7–8) and 174/03 (stx1−stx2+) (Lanes 9–10) for the presence of the gene encoding (A) Shiga toxin 1 and (B) Shiga toxin 2, using PCR with a FAM- and BHQ-1-labelled probe (+) or without such a probe (−). Detection of the signal from the tox1probe or tox2probe, complementary to the gene encoding Shiga toxin 1 or Shiga toxin 2, respectively, was performed using: (I) the gel documentation system Gel Doc XR, Bio-Rad; (II) analysis of PCR products by agarose gel electrophoresis; (III) a UV transilluminator; or (IV) measurement of the fluorescence signal at 485/535 nm (means of three experiments ± SD) in a plate reader. The control reaction performed without the DNA template (Lane 11) and the reaction performed with genomic DNA of the E. coli MG1655 strain, which does not contain genes coding for Shiga toxins 1 and 2 (Lane 12), are also shown.
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toxins-07-04745-f002: The analysis of Shiga toxin-producing Escherichia coli (STEC) strains: 286/00 (stx1−stx2+) (Lanes 1–2), 201/01 (stx1−stx2+) (Lanes 3–4), 319/01 (stx1+stx2−) (Lanes 5–6), 44/02 (stx1+stx2+) (Lanes 7–8) and 174/03 (stx1−stx2+) (Lanes 9–10) for the presence of the gene encoding (A) Shiga toxin 1 and (B) Shiga toxin 2, using PCR with a FAM- and BHQ-1-labelled probe (+) or without such a probe (−). Detection of the signal from the tox1probe or tox2probe, complementary to the gene encoding Shiga toxin 1 or Shiga toxin 2, respectively, was performed using: (I) the gel documentation system Gel Doc XR, Bio-Rad; (II) analysis of PCR products by agarose gel electrophoresis; (III) a UV transilluminator; or (IV) measurement of the fluorescence signal at 485/535 nm (means of three experiments ± SD) in a plate reader. The control reaction performed without the DNA template (Lane 11) and the reaction performed with genomic DNA of the E. coli MG1655 strain, which does not contain genes coding for Shiga toxins 1 and 2 (Lane 12), are also shown.

Mentions: The specific PCR amplification of fragments of genes encoding Shiga toxins 1 and 2, in the presence of probes labeled with the fluorescent agent FAM and the fluorescence quencher BHQ-1, was performed according to a previously published method [16,17]. In the procedure described in this report, we propose to detect the specific fluorescent signal by a simple observation of the reaction tube over a UV transilluminator (Figure 1B). We found that detection of such a signal is unequivocal, and the signal is specific. When 20 different E. coli strains (Table 1) were investigated, the fluorescence occurred only when genomes of the tested bacteria contained the target gene(s) (either stx1, or stx2, or both). We observed 100% compatibility between results obtained by the proposed method and results presented in Table 1, obtained by methods determined as “gold standards”. Figure 2 shows examples of these experiments, with controls, including analyses of PCR products by agarose gel electrophoresis and the results of measurement of the fluorescence signal generated at 485/535 nm, during each PCR reaction. The fluorescence light, emitted in response to UV exposure, was compatible with the agarose gel band patterns and measurements of the fluorescence signal in a plate reader. As described previously [31], the FAM-BHQ-labelled probe, which is not degraded during the PCR reaction (because of the lack of the target DNA), exhibits some level of background fluorescence. In negative controls, the background fluorescence comes from the unhybridized probe itself and is typical for such linear probes because of the relatively long distance between the fluorescence reporter and quencher, which results in inefficient quenching. The level of the background fluorescence can be detected by sensitive fluorescence detectors, like real-time PCR cyclers or plate readers, as in this particular case (Figure 2A, Row IV, Columns 1, 3, 9, 11, 12 and Figure 2B, Row IV, Columns 5, 11, 12). Interestingly, as indicated in Figure 2 (A and B, Row I), in the proposed method, the level of the background fluorescence is low enough and not visible during the observation of the tubes under UV light.


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 Shiga toxin-producing Escherichia coli (STEC) strains: 286/00 (stx1−stx2+) (Lanes 1–2), 201/01 (stx1−stx2+) (Lanes 3–4), 319/01 (stx1+stx2−) (Lanes 5–6), 44/02 (stx1+stx2+) (Lanes 7–8) and 174/03 (stx1−stx2+) (Lanes 9–10) for the presence of the gene encoding (A) Shiga toxin 1 and (B) Shiga toxin 2, using PCR with a FAM- and BHQ-1-labelled probe (+) or without such a probe (−). Detection of the signal from the tox1probe or tox2probe, complementary to the gene encoding Shiga toxin 1 or Shiga toxin 2, respectively, was performed using: (I) the gel documentation system Gel Doc XR, Bio-Rad; (II) analysis of PCR products by agarose gel electrophoresis; (III) a UV transilluminator; or (IV) measurement of the fluorescence signal at 485/535 nm (means of three experiments ± SD) in a plate reader. The control reaction performed without the DNA template (Lane 11) and the reaction performed with genomic DNA of the E. coli MG1655 strain, which does not contain genes coding for Shiga toxins 1 and 2 (Lane 12), are also shown.
© Copyright Policy
Related In: Results  -  Collection

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

toxins-07-04745-f002: The analysis of Shiga toxin-producing Escherichia coli (STEC) strains: 286/00 (stx1−stx2+) (Lanes 1–2), 201/01 (stx1−stx2+) (Lanes 3–4), 319/01 (stx1+stx2−) (Lanes 5–6), 44/02 (stx1+stx2+) (Lanes 7–8) and 174/03 (stx1−stx2+) (Lanes 9–10) for the presence of the gene encoding (A) Shiga toxin 1 and (B) Shiga toxin 2, using PCR with a FAM- and BHQ-1-labelled probe (+) or without such a probe (−). Detection of the signal from the tox1probe or tox2probe, complementary to the gene encoding Shiga toxin 1 or Shiga toxin 2, respectively, was performed using: (I) the gel documentation system Gel Doc XR, Bio-Rad; (II) analysis of PCR products by agarose gel electrophoresis; (III) a UV transilluminator; or (IV) measurement of the fluorescence signal at 485/535 nm (means of three experiments ± SD) in a plate reader. The control reaction performed without the DNA template (Lane 11) and the reaction performed with genomic DNA of the E. coli MG1655 strain, which does not contain genes coding for Shiga toxins 1 and 2 (Lane 12), are also shown.
Mentions: The specific PCR amplification of fragments of genes encoding Shiga toxins 1 and 2, in the presence of probes labeled with the fluorescent agent FAM and the fluorescence quencher BHQ-1, was performed according to a previously published method [16,17]. In the procedure described in this report, we propose to detect the specific fluorescent signal by a simple observation of the reaction tube over a UV transilluminator (Figure 1B). We found that detection of such a signal is unequivocal, and the signal is specific. When 20 different E. coli strains (Table 1) were investigated, the fluorescence occurred only when genomes of the tested bacteria contained the target gene(s) (either stx1, or stx2, or both). We observed 100% compatibility between results obtained by the proposed method and results presented in Table 1, obtained by methods determined as “gold standards”. Figure 2 shows examples of these experiments, with controls, including analyses of PCR products by agarose gel electrophoresis and the results of measurement of the fluorescence signal generated at 485/535 nm, during each PCR reaction. The fluorescence light, emitted in response to UV exposure, was compatible with the agarose gel band patterns and measurements of the fluorescence signal in a plate reader. As described previously [31], the FAM-BHQ-labelled probe, which is not degraded during the PCR reaction (because of the lack of the target DNA), exhibits some level of background fluorescence. In negative controls, the background fluorescence comes from the unhybridized probe itself and is typical for such linear probes because of the relatively long distance between the fluorescence reporter and quencher, which results in inefficient quenching. The level of the background fluorescence can be detected by sensitive fluorescence detectors, like real-time PCR cyclers or plate readers, as in this particular case (Figure 2A, Row IV, Columns 1, 3, 9, 11, 12 and Figure 2B, Row IV, Columns 5, 11, 12). Interestingly, as indicated in Figure 2 (A and B, Row I), in the proposed method, the level of the background fluorescence is low enough and not visible during the observation of the tubes under UV light.

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