| Part one Rapid detection of BCR-ABL fusion gene using a novel combined LUX primer,in-cell RT- PCR and flow cytometryObjective:To developed a new approach of dectection BCR-ABL fusion gene transcript,based on in-cell RT-PCR and LUX primer, followed by rapid analysis with a flow cytometer and the sensitivity were compared with those of BCR-ABL fusion mRNA by RT-PCR.Materials and Methods:Cell lines K562 and NB4 were wished, After cellular permeabilization and fixation of single cells in suspension,the neoplastic mRNA was reverse transcribed into cDNA, and the cDNA was amplified by PCR with LUX primer,specific for BCR/ABL.Flow cytometry was used to detect cells positive for the amplified DNA within the cell cytoplasm.After transferring the amplified cells onto slides,the positive cells for BCR-ABL cDNA were observed by fluorescent microscopy. Results:1.a strong positive yellow-green signal was observed in 99～100%cells of K562 cell lines,only red nucleus was detected in NB4 cell line and normal controls.2.A fluorescence signal was detected at 1/10~4 and was not present below this level with LUX primer combining in-cell RT- PCR and flow cytometry.3.the result of RT-PCR was negative when the ratio of K562 to normal cell was below 1/10~4Conclusion:1.LUX primers can be used to efficiently detect BCR-ABL fusion mRNA by in-cell RT-PCR.2.LUX primer combining in-cell RT- PCR and flow cytometry can be used to quantitative assays BCR/ABL fusion gene,and is equally as sensitive as RT-PCR.Part two To detect BCR/ABL fusion gene in CML patients with LUX primer combined in-cell RT- PCR and flow cytometryObjective:To detect the specificity and sensitivity of the new combined LUX primers,in-cell RT- PCR and flow cytometric methodMaterials and Methods:Twelve CML patients,including 5 newly diagnosed patients and 7 post-treated patients were admitted to the studied.Three patients had been treated with interferon-or and hydroxyurea for 6-16 months.Four patients had received imatinib mesylate for 2-12 months(No.9-12).Bone marrow mononuclear cells (BMMNCs)were isolate from 12 chronic myeloid leukemia patients, then fixed in 4%paraformaldehyde and treated with proteinase K for 15 rain,the mRNA was amplified within the cells by reverse transcription-polymerase reaction with LUX primer.Flow cytometry and fluorescent microscopy was used to detect cells positive for the amplified DNA within the cell.At last,the results were compared with the result of RT-PCR and FISH analysis.Results:1.RT-PCR results:The nine patients with CML (including 5 newly diagnosed patients,3 patients treated with interferon-αand hydroxyurea and 1 patient treated with imatinib mesylate for 2 months)had positive RT-PCR results.The other three patients,normal control and NB4 cell line had RT-PCR negative results.2.I-FISH results:The five newly diagnosed patients had 86-96.5% positive cells.Four patients,including 3 patients treated with interferon-αand hydroxyurea and 1 patient treated with imatinib mesylate,had 23～80%positive cells.Negative results were found in 3 patients treated with imatinib mesylate and NB4 cell line(positive cells＜4.11%).3.In situ RT-PCR results:In the five newly diagnosed CML patients,90-98%cells were strongly positive by in cell RT-PCR.In the three patients treated with interferon-αand hydroxyurea,the positive cells ranged from 33%to 82.5%.In the four patients treated with imatinib mesylate(No.9-12),patient 9 treated with imatinib mesylate for 2 months had 26%positive cells.While in the other three patients treated with imatinib mesylate,no positive cells were detected.In all the normal controls and NB4 cell line,no positive cells were found. The in situ RT-PCR results demonstrate complete concordance with the results of I-FISH and RT-PCR.Conclusion:Our method is a specific and sensitive way of quantitative assays BCR/ABL fusion gene.
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