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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

Analysis of the DNA from Bartonella henselae strain Houston-1 for the presence of the sequence encoding 16S rRNA, using PCR amplification with a FAM- and BHQ-1-labelled probe. Detection of the signal from the 16SrRNABhprobe complementary to the gene encoding 16S rRNA was performed by analysis of PCR products by agarose gel electrophoresis and using a UV transilluminator. The control reactions were performed without the primers (Lane 1) or DNA template (Lane 2). The target PCR product size of 180 bp is indicated by the black arrow (Lane 3).
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toxins-07-04745-f004: Analysis of the DNA from Bartonella henselae strain Houston-1 for the presence of the sequence encoding 16S rRNA, using PCR amplification with a FAM- and BHQ-1-labelled probe. Detection of the signal from the 16SrRNABhprobe complementary to the gene encoding 16S rRNA was performed by analysis of PCR products by agarose gel electrophoresis and using a UV transilluminator. The control reactions were performed without the primers (Lane 1) or DNA template (Lane 2). The target PCR product size of 180 bp is indicated by the black arrow (Lane 3).

Mentions: The total time required for the procedure proposed in this report is as short as 1.5 h, including genomic DNA isolation and PCR amplification with simultaneous annealing and extension steps (see the Experimental Section for details). As the detection method is based on a simple PCR reaction, the only special equipment necessary to follow it consists of a thermocycler and a UV transilluminator, which are usually available in most laboratories (including those located in provincial hospitals), so it can be easily applied for a rapid, preliminary detection of not only STEC bacteria, but also other pathogens. Following this, we found that this procedure may be used in the detection of tick-transmitted bacteria. We performed a PCR amplification with primers and a probe that are complementary to the sequence encoding 16S rRNA of Bartonellahenselae under the same conditions as those used for the detection of stx genes (Figure 4, Lane 3). Again, a signal was detected only in the presence of B. henselae DNA, indicating the specificity of the assay (Figure 4).


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)

Analysis of the DNA from Bartonella henselae strain Houston-1 for the presence of the sequence encoding 16S rRNA, using PCR amplification with a FAM- and BHQ-1-labelled probe. Detection of the signal from the 16SrRNABhprobe complementary to the gene encoding 16S rRNA was performed by analysis of PCR products by agarose gel electrophoresis and using a UV transilluminator. The control reactions were performed without the primers (Lane 1) or DNA template (Lane 2). The target PCR product size of 180 bp is indicated by the black arrow (Lane 3).
© Copyright Policy
Related In: Results  -  Collection

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

toxins-07-04745-f004: Analysis of the DNA from Bartonella henselae strain Houston-1 for the presence of the sequence encoding 16S rRNA, using PCR amplification with a FAM- and BHQ-1-labelled probe. Detection of the signal from the 16SrRNABhprobe complementary to the gene encoding 16S rRNA was performed by analysis of PCR products by agarose gel electrophoresis and using a UV transilluminator. The control reactions were performed without the primers (Lane 1) or DNA template (Lane 2). The target PCR product size of 180 bp is indicated by the black arrow (Lane 3).
Mentions: The total time required for the procedure proposed in this report is as short as 1.5 h, including genomic DNA isolation and PCR amplification with simultaneous annealing and extension steps (see the Experimental Section for details). As the detection method is based on a simple PCR reaction, the only special equipment necessary to follow it consists of a thermocycler and a UV transilluminator, which are usually available in most laboratories (including those located in provincial hospitals), so it can be easily applied for a rapid, preliminary detection of not only STEC bacteria, but also other pathogens. Following this, we found that this procedure may be used in the detection of tick-transmitted bacteria. We performed a PCR amplification with primers and a probe that are complementary to the sequence encoding 16S rRNA of Bartonellahenselae under the same conditions as those used for the detection of stx genes (Figure 4, Lane 3). Again, a signal was detected only in the presence of B. henselae DNA, indicating the specificity of the assay (Figure 4).

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