scholarly journals Novel multiplex RT-PCR assay to detect BCR/ABL mRNA variants

2020 ◽  
pp. 29-35
Author(s):  
K Shires ◽  
A Rust
Keyword(s):  
Kinase Inhibitors ◽  
Residual Disease ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Early Therapy ◽  
Mrna Variants ◽  
Qualitative Assay ◽  
Therapy Intervention ◽  
Transcript Type ◽  
Selection Of

The detection of the t(9;22) translocation, which results in the formation of the BCR/ABL oncoprotein, in patients diagnosed with chronic and acute leukaemias, allows for the administration of highly effective tyrosine kinase inhibitors such as imatinib and nilotinib. For the effective management of these patients it is important to monitor for minimal residual disease, allowing early therapy intervention prior to haematological relapse. Currently this is achieved through very sensitive quantitative real-time PCR assays. Unfortunately, these assays are highly specific to the BCR/ABL variant expressed: p210, p190 or p230 and require identification of the transcript type prior to selection of the correct monitoring assay. We have developed a novel multiplex BCR/ABL variant PCR assay that can identify the variant in a single qualitative assay, creating both a cost and time effective way to identify the most appropriate monitoring methodology for each patient.

scholarly journals Frequency ofBCR-ABLTranscript Types in Syrian CML Patients

2016 ◽  
Vol 2016 ◽  
pp. 1-5 ◽  
Author(s):  
Sulaf Farhat-Maghribi ◽  
Wafa Habbal ◽  
Fawza Monem
Keyword(s):  
Kinase Inhibitors ◽  
Clinical Findings ◽  
Molecular Response ◽  
Imatinib Treatment ◽  
Monitoring Methods ◽  
Rt Pcr ◽  
Treatment Protocols ◽  
Three Samples ◽  
Pcr Method ◽  
Transcript Type

Background. In Syria, CML patients are started on tyrosine kinase inhibitors (TKIs) and monitored until complete molecular response is achieved.BCR-ABLmRNA transcript type is not routinely identified, contrary to the recommendations. In this study we aimed to identify the frequency of differentBCR-ABLtranscripts in Syrian CML patients and highlight their significance on monitoring and treatment protocols.Methods. CML patients positive forBCR-ABLtranscripts by quantitative RT-PCR were enrolled.BCR-ABLtranscript types were investigated using a home-made PCR method that was adapted from published protocols and optimized. The transcript types were then confirmed using a commercially available research kit.Results. Twenty-four transcripts were found in 21 patients. The most common was b2a2, followed by b3a2, b3a3, and e1a3 present solely in 12 (57.1%), 3 (14.3%), 2 (9.5%), and 1 (4.8%), respectively. Three samples (14.3%) contained dual transcripts. While b3a2 transcript was apparently associated with warning molecular response to imatinib treatment, b2a2, b3a3, and e1a3 transcripts collectively proved otherwise (P=0.047).Conclusion. It might be advisable to identify theBCR-ABLtranscript type in CML patients at diagnosis, using an empirically verified method, in order to link the detected transcript with the clinical findings, possible resistance to treatment, and appropriate monitoring methods.

Enumeration Methods and Production of Enterotoxins in Food-Derived Staphylococcus aureus

2012 ◽  
Vol 95 (1) ◽  
pp. 105-110 ◽  
Author(s):  
Chi Zhang ◽  
Deqin Zhang ◽  
Jun Yang ◽  
Jungui Zhou ◽  
Qilong Hu ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Culture Media ◽  
Food Poisoning ◽  
Food Samples ◽  
Rabbit Plasma ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Significant Difference ◽  
Statistical Relationships ◽  
Selection Of

Abstract Staphylococcal food poisoning is one of the most common foodborne diseases worldwide; it results from the ingestion of staphylococcal enterotoxins (SEs) in food, mainly Staphylococcus aureus. This study investigated the statistical relationships among morphological enumerations of food-derived S. aureus and production of SEs using different methodologies. Food samples naturally contaminated with coagulase-positive S. aureus were submitted for enumeration on Baird-Parker (BP) agar, Rabbit Plasma Fibrinogen agar (RPFA), and PetrifilmTM Staph Express count system (STX), and the morphologically typical colonies were isolated for VIDAS and real-time (RT) PCR tests. RPFA and STX displayed better performance for the enumeration of SE-positive S. aureus when compared with BP, including higher frequencies of SE-positive isolates and better correlation indices between typical and SE-positive counts. Among all the evaluated culture media, no significant difference (P > 0.05) was shown on the frequencies of typical colonies that carried 11 individual se genes. In addition, results for SE identification between VIDAS and RT-PCR assay were unparalleled. These data will be valuable for the selection of methods for inspection of food-derived S. aureus.

Detection of BCR-ABL Kinase Domain Mutations in Chronic Myeloid Leukemia Patients Treated with Tirosin-Kinase Inhibitors

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4261-4261
Author(s):  
Gonzalo Manrique ◽  
Roberta Bittencout ◽  
Verónica Pérez ◽  
Vanesa Sholl ◽  
Monica Cappetta ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Direct Sequencing ◽  
Low Cost ◽  
Mutation Screening ◽  
Point Mutations ◽  
Kinase Domain ◽  
Rapid Screening ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Allele Specific

Abstract Background. Point mutations in the kinase domain (KD) of the BCR-ABL are the most frequent mechanism of drug resistance in CML patients treated with kinase inhibitors (TKI). More than 80 mutations with different frequency and clinical significance have been reported. One of them, the T315I confers resistance to all TKIs available. The detection of mutations in KD allows early identification of high-risk patients and therefore guides clinical therapy decisions. Aim. To assess the mutation status of a group of CML pts resistant to TKI from Uruguay (n=35) and Brazil (n=30). Methods. KD mutation screening was performed by RT-PCR and direct sequencing according to Branford et al. (2002). Additionally, we developed a rapid, specific, sensitive and low cost allele specific (AS)-RT-PCR assay to identify T315I, using Branford’s KD amplification primers in combination with an allele specific primer for the T315I point mutation detection. BCR-ABL transcript levels were also measured by RQ-PCR according to international recommendations. Results and Discussion. RT-PCR and direct sequencing analyses performed in all pts showed the presence of T315l mutation in 3/65 cases. Other 11 showed the alternative mutations Y253H (n=2), E450A, G250E (n=2), E459K (n=2), E450G, F317L (n=2) and E255K; and the remaining 55 showed no mutations in the ABL KD. All 65 samples together with cDNA from 15 non-resistant CML pts and 10 cDNA from non-CML were analyzed by AS-RT-PCR assay for T315l mutation in order to validate the method. T315l was identified in the 3 samples in which the mutation was previously detected by direct sequencing and in 1 pt that had been classified as KD mutation negative. This result was then confirmed by direct sequencing of the AS-PCR product. T315 was neither detected in samples positive for other mutations nor in samples of non-resistant CML and non-CML patients, supporting the specificity of the method. Assessment of the sensitivity of the AS-RT-PCR was performed on serial dilutions experiments using RNA from T315 positive pt into RNA from CML-T315l negative pt, showing that the T315I mutation was detectable to a level of 0.01 % by AS-PCR, while through direct sequencing method the sensitivity was 10–20%. The prevalence of mutations in our study was 15/65 (23%). Conclusions. Our results showed that the AS-RT-PCR described here is a convenient and easy tool to be used in a clinical routine laboratory for rapid screening for BCR-ABL T315. This, together with direct sequencing, constitutes a suitable approach for CML resistance monitoring and therapeutic choice.

Multiplex reverse transcriptase–polymerase chain reaction screening in childhood acute myeloblastic leukemia

Blood ◽  
2001 ◽  
Vol 97 (3) ◽  
pp. 805-808 ◽  
Author(s):  
Sabine Strehl ◽  
Margit König ◽  
Georg Mann ◽  
Oskar A. Haas
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcriptase ◽  
Cytogenetic Analysis ◽  
Residual Disease ◽  
Acute Myeloblastic Leukemia ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Essential Information ◽  
Polymerase Chain

Abstract To determine the incidence of leukemia-specific rearrangements, 60 cases of childhood acute myeloblastic leukemia and transient myeloproliferative disorder were screened with a novel multiplex reverse transcriptase–polymerase chain reaction (RT-PCR) assay, and the results were correlated with the cytogenetic findings. The RT-PCR assay detects 28 different fusion genes and more than 80 different fusion transcript variants. RNA was isolated from methanol/acetic acid–fixed cells that had been routinely prepared for cytogenetic analysis. Nine different fusion transcripts were found in 40% of the cases, whereas 78.3% of the cases had abnormal karyotypes. Two cases with a t(6;11) and an MLL/AF6 gene fusion were missed cytogenetically. Conversely, cytogenetic analysis revealed 10 other well-defined chromosome rearrangements. Although cytogenetic analysis reveals a much broader range of abnormalities, multiplex RT-PCR serves as quality control and provides the essential information for minimal residual disease studies. Moreover, discrepant findings lead to the detection of new rearrangements on the molecular genetic level.

TaqMan RT-PCR assay coupled with capillary electrophoresis for quantification and identification of bcr-abl transcript type

2003 ◽  
Vol 17 (1) ◽  
pp. 96-103 ◽  
Author(s):  
Rajyalakshmi Luthra ◽  
Beatriz Sanchez-Vega ◽  
L Jeffrey Medeiros
Keyword(s):  
Capillary Electrophoresis ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Transcript Type

TaqMan RT-PCR assay coupled with capillary electrophoresis for quantification and identification of bcr-abl transcript type

2003 ◽  
Vol 17 (1) ◽  
pp. 96-103 ◽  
Author(s):  
Rajyalakshmi Luthra ◽  
Beatriz Sanchez-Vega ◽  
L Jeffrey Medeiros
Keyword(s):  
Capillary Electrophoresis ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Transcript Type
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