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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

(A) A scheme for the procedure of the detection of specific DNA sequences reported in this work. The procedure is based on the already known method employing PCR with DNA polymerase (designated as P), appropriate primers and a sequence-specific DNA probe labeled with the fluorescent agent FAM (designated as F) at the 5′ end and the fluorescence quencher BHQ-1 (black hole quencher; designated as Q) at the 3′ end. (B) Visual observation of the fluorescence under UV light at 302 nm directly in the reaction tube is proposed, instead of detection of the fluorescence signal with the use of real-time PCR cyclers, fluorescence/luminescence spectrometers or fluorescence polarization readers and as an alternative to the gel electrophoresis technique. PCR reactions with (+) and without (−) targeted pathogenic DNA were analyzed.
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toxins-07-04745-f001: (A) A scheme for the procedure of the detection of specific DNA sequences reported in this work. The procedure is based on the already known method employing PCR with DNA polymerase (designated as P), appropriate primers and a sequence-specific DNA probe labeled with the fluorescent agent FAM (designated as F) at the 5′ end and the fluorescence quencher BHQ-1 (black hole quencher; designated as Q) at the 3′ end. (B) Visual observation of the fluorescence under UV light at 302 nm directly in the reaction tube is proposed, instead of detection of the fluorescence signal with the use of real-time PCR cyclers, fluorescence/luminescence spectrometers or fluorescence polarization readers and as an alternative to the gel electrophoresis technique. PCR reactions with (+) and without (−) targeted pathogenic DNA were analyzed.

Mentions: One of the previousy described methods for the detection of the presence of certain DNA sequences is based on the PCR reaction with appropriate primers and specific probes 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. FAM is a fluorescein derivative with an excitation peak at 492 nm and an emission peak at 517 nm [16,17,18,19]. According to this method, fluorescence may appear only if the probe hybridizes to the target DNA region and is subsequently degraded by the Taq DNA polymerase extending a specific primer designed to amplify a DNA fragment that includes the probe binding site. In such a case, FAM is no longer located in the proximity of BHQ-1; thus, its fluorescence is not quenched, and light might be emitted after excitation at the appropriate wavelength [16,18,19] (Figure 1A). However, in previously-reported procedures employing this phenomenon, the fluorescence emitted by FAM was detected using light excitation sources of a wavelength in the range of the visible spectrum, near the excitation maximum of FAM (~492 nm), such as lasers and diodes in real-time PCR cyclers, fluorescence/luminescence spectrometers or fluorescence polarization readers [18,20,21], which made them relatively expensive. Thus, we asked if it is possible to simplify the detection procedure, to make it inexpensive and rapid, while still keeping its sensitivity at an acceptable level. We would like to propose a different detection manner of fluorescence emitted by FAM, released from the BHQ-1 influence, based on the excitation with light outside the visible range, i.e., ultraviolet light, directly in the reaction tube (Figure 1B). The modification we aimed to introduce allows confirmation of the presence of the target DNA, amplified during PCR reaction in the test tube, without the need of running agarose gel electrophoresis. Such a method would be particularly desirable in detection of pathogenic microorganisms, especially those requiring rapid diagnostic methods.


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)

(A) A scheme for the procedure of the detection of specific DNA sequences reported in this work. The procedure is based on the already known method employing PCR with DNA polymerase (designated as P), appropriate primers and a sequence-specific DNA probe labeled with the fluorescent agent FAM (designated as F) at the 5′ end and the fluorescence quencher BHQ-1 (black hole quencher; designated as Q) at the 3′ end. (B) Visual observation of the fluorescence under UV light at 302 nm directly in the reaction tube is proposed, instead of detection of the fluorescence signal with the use of real-time PCR cyclers, fluorescence/luminescence spectrometers or fluorescence polarization readers and as an alternative to the gel electrophoresis technique. PCR reactions with (+) and without (−) targeted pathogenic DNA were analyzed.
© Copyright Policy
Related In: Results  -  Collection

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

toxins-07-04745-f001: (A) A scheme for the procedure of the detection of specific DNA sequences reported in this work. The procedure is based on the already known method employing PCR with DNA polymerase (designated as P), appropriate primers and a sequence-specific DNA probe labeled with the fluorescent agent FAM (designated as F) at the 5′ end and the fluorescence quencher BHQ-1 (black hole quencher; designated as Q) at the 3′ end. (B) Visual observation of the fluorescence under UV light at 302 nm directly in the reaction tube is proposed, instead of detection of the fluorescence signal with the use of real-time PCR cyclers, fluorescence/luminescence spectrometers or fluorescence polarization readers and as an alternative to the gel electrophoresis technique. PCR reactions with (+) and without (−) targeted pathogenic DNA were analyzed.
Mentions: One of the previousy described methods for the detection of the presence of certain DNA sequences is based on the PCR reaction with appropriate primers and specific probes 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. FAM is a fluorescein derivative with an excitation peak at 492 nm and an emission peak at 517 nm [16,17,18,19]. According to this method, fluorescence may appear only if the probe hybridizes to the target DNA region and is subsequently degraded by the Taq DNA polymerase extending a specific primer designed to amplify a DNA fragment that includes the probe binding site. In such a case, FAM is no longer located in the proximity of BHQ-1; thus, its fluorescence is not quenched, and light might be emitted after excitation at the appropriate wavelength [16,18,19] (Figure 1A). However, in previously-reported procedures employing this phenomenon, the fluorescence emitted by FAM was detected using light excitation sources of a wavelength in the range of the visible spectrum, near the excitation maximum of FAM (~492 nm), such as lasers and diodes in real-time PCR cyclers, fluorescence/luminescence spectrometers or fluorescence polarization readers [18,20,21], which made them relatively expensive. Thus, we asked if it is possible to simplify the detection procedure, to make it inexpensive and rapid, while still keeping its sensitivity at an acceptable level. We would like to propose a different detection manner of fluorescence emitted by FAM, released from the BHQ-1 influence, based on the excitation with light outside the visible range, i.e., ultraviolet light, directly in the reaction tube (Figure 1B). The modification we aimed to introduce allows confirmation of the presence of the target DNA, amplified during PCR reaction in the test tube, without the need of running agarose gel electrophoresis. Such a method would be particularly desirable in detection of pathogenic microorganisms, especially those requiring rapid diagnostic methods.

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