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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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Related in: MedlinePlus

Left panel: Illustration of the principle of the dry-reagent disposable dipstick test for visual detection of chromosomal translocations. Right panel: Scanning electron microscopy (SEM) images of the conjugate pad and the membrane of the strip. IP = immersion pad; CP = conjugation pad; M = membrane; AP = absorbent pad; TZ = test zone; CZ = control zone; SA = streptavidin; B = biotin.
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Figure 1: Left panel: Illustration of the principle of the dry-reagent disposable dipstick test for visual detection of chromosomal translocations. Right panel: Scanning electron microscopy (SEM) images of the conjugate pad and the membrane of the strip. IP = immersion pad; CP = conjugation pad; M = membrane; AP = absorbent pad; TZ = test zone; CZ = control zone; SA = streptavidin; B = biotin.

Mentions: A schematic illustration of the dry-reagent dipstick test is presented in Figure 1. The assay entails rapid hybridization of the amplified fragments (target DNA), in solution, with a dATP-tailed oligonucleotide probe, application to the sample loading area on the conjugate pad of the strip and immersion of the strip (via the immersion pad) in the developing solution. The developing solution migrates to the opposite end of the strip, by capillary forces, and causes rehydration of the poly(dT)-functionalized gold nanoparticles (Au NP), which are then connected to the probe via hybridization of the poly(dT) strands with the poly(dA) tail of the probe. As the solution passes through the test zone of the strip, the hybrids are captured by immobilized streptavidin, thus resulting in accumulation of Au NP which is detected visually as a characteristic red line. The red color of the Au NP is due to the plasmon resonance peak at 520 nm (24,25). The excess of poly(dT)-Au NP are captured by immobilized poly(dA) strands at the control zone of the strip, giving a second red line. In the absence of the translocation, no red line is observed at the test zone. A red line, however, is always formed at the control zone to confirm the proper functioning of the strip. Consequently, a sample is positive for a certain translocation when a red line is observed both in the test and the control zone. Scanning electron microscope (SEM) images of the conjugate pad (consisting of glass fibers) and the polyethersulfone membrane of the strip containing the Au NP are also presented in Figure 1.Figure 1.


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: Illustration of the principle of the dry-reagent disposable dipstick test for visual detection of chromosomal translocations. Right panel: Scanning electron microscopy (SEM) images of the conjugate pad and the membrane of the strip. IP = immersion pad; CP = conjugation pad; M = membrane; AP = absorbent pad; TZ = test zone; CZ = control zone; SA = streptavidin; B = biotin.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 1: Left panel: Illustration of the principle of the dry-reagent disposable dipstick test for visual detection of chromosomal translocations. Right panel: Scanning electron microscopy (SEM) images of the conjugate pad and the membrane of the strip. IP = immersion pad; CP = conjugation pad; M = membrane; AP = absorbent pad; TZ = test zone; CZ = control zone; SA = streptavidin; B = biotin.
Mentions: A schematic illustration of the dry-reagent dipstick test is presented in Figure 1. The assay entails rapid hybridization of the amplified fragments (target DNA), in solution, with a dATP-tailed oligonucleotide probe, application to the sample loading area on the conjugate pad of the strip and immersion of the strip (via the immersion pad) in the developing solution. The developing solution migrates to the opposite end of the strip, by capillary forces, and causes rehydration of the poly(dT)-functionalized gold nanoparticles (Au NP), which are then connected to the probe via hybridization of the poly(dT) strands with the poly(dA) tail of the probe. As the solution passes through the test zone of the strip, the hybrids are captured by immobilized streptavidin, thus resulting in accumulation of Au NP which is detected visually as a characteristic red line. The red color of the Au NP is due to the plasmon resonance peak at 520 nm (24,25). The excess of poly(dT)-Au NP are captured by immobilized poly(dA) strands at the control zone of the strip, giving a second red line. In the absence of the translocation, no red line is observed at the test zone. A red line, however, is always formed at the control zone to confirm the proper functioning of the strip. Consequently, a sample is positive for a certain translocation when a red line is observed both in the test and the control zone. Scanning electron microscope (SEM) images of the conjugate pad (consisting of glass fibers) and the polyethersulfone membrane of the strip containing the Au NP are also presented in Figure 1.Figure 1.

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