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Dry-reagent disposable dipstick test for visual screening of seven leukemia-related chromosomal translocations.

Kalogianni DP, Bravou V, Christopoulos TK, Ioannou PC, Zoumbos NC - Nucleic Acids Res. (2007)

Bottom Line: Upon immersion of the strip in the appropriate buffer, the solution migrates and the hybrids are captured by immobilized streptavidin at the test zone generating a characteristic red line.We studied the: t(9;22)(q34;q11), t(15;17)(q22;q21), t(11;17)(q23;q21), t(5;17)(q32;q21), t(11;17)(q13;q21), t(8,21)(q22;q22) and inv(16)(p13;q22) that generate the BCR-ABL, PML-RARa, PLZF-RARa, NPM-RARa, NuMA-RARa, AML1-ETO and CBFbeta-MYH11 fusion genes, respectively.A single K562 cell was detectable amidst 10(6) normal leukocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Patras, Patras 26500, Greece.

ABSTRACT
We report the first dry-reagent, disposable, dipstick test for molecular screening of seven chromosomal translocations associated with acute and chronic leukemia. The dipstick assay offers about 10 times higher detectability than agarose gel electrophoresis and, contrary to electrophoresis, allows confirmation of the sequence of the polymerase chain reaction (PCR) product by hybridization within a few minutes without the need of instrumentation. Biotinylated amplified DNA is hybridized with a dA-tailed probe and applied to the strip, which contains oligo(dT)-conjugated gold nanoparticles in dry form. Upon immersion of the strip in the appropriate buffer, the solution migrates and the hybrids are captured by immobilized streptavidin at the test zone generating a characteristic red line. The excess nanoparticles are captured by oligo(dA) strands immobilized at the control zone of the strip producing a second red line. We studied the: t(9;22)(q34;q11), t(15;17)(q22;q21), t(11;17)(q23;q21), t(5;17)(q32;q21), t(11;17)(q13;q21), t(8,21)(q22;q22) and inv(16)(p13;q22) that generate the BCR-ABL, PML-RARa, PLZF-RARa, NPM-RARa, NuMA-RARa, AML1-ETO and CBFbeta-MYH11 fusion genes, respectively. A single K562 cell was detectable amidst 10(6) normal leukocytes. A dipstick test was developed for actin, as a reference gene. The dipstick assay with appropriate probes can be used for identification of the fusion transcripts involved in the translocation.

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Left panel: Typical results for actin by the dipstick assay and agarose (2%) gel electrophoresis. The numbers correspond to patient numbers of Figure 3. Right panel: Typing of the fusion transcripts (b3-a2 and b2-a2) of the Philadelphia translocation. Following a positive dipstick test with probes b3-a2 and b2-a2, the PCR product is subjected to another dipstick assay using probe b3. Samples 1, 14, 16 and 22, which contain the b3-a2 transcript, give a positive result with the b3 probe. However, no signal is obtained with samples S1-S5 that contain the b2-a2 transcript.
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Figure 4: Left panel: Typical results for actin by the dipstick assay and agarose (2%) gel electrophoresis. The numbers correspond to patient numbers of Figure 3. Right panel: Typing of the fusion transcripts (b3-a2 and b2-a2) of the Philadelphia translocation. Following a positive dipstick test with probes b3-a2 and b2-a2, the PCR product is subjected to another dipstick assay using probe b3. Samples 1, 14, 16 and 22, which contain the b3-a2 transcript, give a positive result with the b3 probe. However, no signal is obtained with samples S1-S5 that contain the b2-a2 transcript.

Mentions: The clinical evaluation of the proposed method was carried out by using 22 samples from patients with the b3-a2 transcript and 5 samples containing the b2-a2 transcript. Two patients with essential thrombocythemia were also included as negative controls. The Ph1 chromosome positive patients were undergoing treatment. All blood samples were subjected to the preparation steps described in the experimental section, for isolation of leukocytes, purification of mRNA, cDNA synthesis, nested PCR and detection by the strip. Amplification products were also analyzed by agarose gel electrophoresis. The results are presented in Figure 3B and C. It is observed that the strip test compares well with the electrophoresis results. Sample No. 7 was found negative both by electrophoresis and the strip test. Figure 4 shows typical results of the dipstick assay for the reference gene actin.Figure 4.


Dry-reagent disposable dipstick test for visual screening of seven leukemia-related chromosomal translocations.

Kalogianni DP, Bravou V, Christopoulos TK, Ioannou PC, Zoumbos NC - Nucleic Acids Res. (2007)

Left panel: Typical results for actin by the dipstick assay and agarose (2%) gel electrophoresis. The numbers correspond to patient numbers of Figure 3. Right panel: Typing of the fusion transcripts (b3-a2 and b2-a2) of the Philadelphia translocation. Following a positive dipstick test with probes b3-a2 and b2-a2, the PCR product is subjected to another dipstick assay using probe b3. Samples 1, 14, 16 and 22, which contain the b3-a2 transcript, give a positive result with the b3 probe. However, no signal is obtained with samples S1-S5 that contain the b2-a2 transcript.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851627&req=5

Figure 4: Left panel: Typical results for actin by the dipstick assay and agarose (2%) gel electrophoresis. The numbers correspond to patient numbers of Figure 3. Right panel: Typing of the fusion transcripts (b3-a2 and b2-a2) of the Philadelphia translocation. Following a positive dipstick test with probes b3-a2 and b2-a2, the PCR product is subjected to another dipstick assay using probe b3. Samples 1, 14, 16 and 22, which contain the b3-a2 transcript, give a positive result with the b3 probe. However, no signal is obtained with samples S1-S5 that contain the b2-a2 transcript.
Mentions: The clinical evaluation of the proposed method was carried out by using 22 samples from patients with the b3-a2 transcript and 5 samples containing the b2-a2 transcript. Two patients with essential thrombocythemia were also included as negative controls. The Ph1 chromosome positive patients were undergoing treatment. All blood samples were subjected to the preparation steps described in the experimental section, for isolation of leukocytes, purification of mRNA, cDNA synthesis, nested PCR and detection by the strip. Amplification products were also analyzed by agarose gel electrophoresis. The results are presented in Figure 3B and C. It is observed that the strip test compares well with the electrophoresis results. Sample No. 7 was found negative both by electrophoresis and the strip test. Figure 4 shows typical results of the dipstick assay for the reference gene actin.Figure 4.

Bottom Line: Upon immersion of the strip in the appropriate buffer, the solution migrates and the hybrids are captured by immobilized streptavidin at the test zone generating a characteristic red line.We studied the: t(9;22)(q34;q11), t(15;17)(q22;q21), t(11;17)(q23;q21), t(5;17)(q32;q21), t(11;17)(q13;q21), t(8,21)(q22;q22) and inv(16)(p13;q22) that generate the BCR-ABL, PML-RARa, PLZF-RARa, NPM-RARa, NuMA-RARa, AML1-ETO and CBFbeta-MYH11 fusion genes, respectively.A single K562 cell was detectable amidst 10(6) normal leukocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Patras, Patras 26500, Greece.

ABSTRACT
We report the first dry-reagent, disposable, dipstick test for molecular screening of seven chromosomal translocations associated with acute and chronic leukemia. The dipstick assay offers about 10 times higher detectability than agarose gel electrophoresis and, contrary to electrophoresis, allows confirmation of the sequence of the polymerase chain reaction (PCR) product by hybridization within a few minutes without the need of instrumentation. Biotinylated amplified DNA is hybridized with a dA-tailed probe and applied to the strip, which contains oligo(dT)-conjugated gold nanoparticles in dry form. Upon immersion of the strip in the appropriate buffer, the solution migrates and the hybrids are captured by immobilized streptavidin at the test zone generating a characteristic red line. The excess nanoparticles are captured by oligo(dA) strands immobilized at the control zone of the strip producing a second red line. We studied the: t(9;22)(q34;q11), t(15;17)(q22;q21), t(11;17)(q23;q21), t(5;17)(q32;q21), t(11;17)(q13;q21), t(8,21)(q22;q22) and inv(16)(p13;q22) that generate the BCR-ABL, PML-RARa, PLZF-RARa, NPM-RARa, NuMA-RARa, AML1-ETO and CBFbeta-MYH11 fusion genes, respectively. A single K562 cell was detectable amidst 10(6) normal leukocytes. A dipstick test was developed for actin, as a reference gene. The dipstick assay with appropriate probes can be used for identification of the fusion transcripts involved in the translocation.

Show MeSH
Related in: MedlinePlus