Four-channel asymmetric Real-Time PCR hybridization probe assay: A rapid pre-screening method for critical BCR-ABL kinase domain mutations

Abstract Imatinib mesylate is a selective Bcr-Abl kinase inhibitor, effective in the treatment of chronic myelogenous leukemia. Most patients in chronic phase maintain durable responses; however, many in blast crisis fail to respond, or relapse quickly. Kinase domain mutations are the most commonly identified mechanism associated with relapse. Many of these mutations decrease the sensitivity of the Abl kinase to imatinib, thus accounting for resistance to imatinib. The role of other mutations in the emergence of resistance has not been established. Using biochemical and cellular assays, we analyzed the sensitivity of several mutants (Met244Val, Phe311Leu, Phe317Leu, Glu355Gly, Phe359Val, Val379Ile, Leu387Met, and His396Pro/Arg) to imatinib mesylate to better understand their role in mediating resistance.While some Abl mutations lead to imatinib resistance, many others are significantly, and some fully, inhibited. This study highlights the need for biochemical and biologic characterization, before a resistant phenotype can be ascribed to a mutant.

Download Full-text

Detection of BCR-ABL Kinase Domain Mutations in Chronic Myeloid Leukemia Patients

Clinical Lymphoma Myeloma & Leukemia ◽

10.1016/j.clml.2016.07.087 ◽

2016 ◽

Vol 16 ◽

pp. S60

Author(s):

Nancy Escobar ◽

Mariana Herrera ◽

Luisa Rosales ◽

Silvana Torselli ◽

Julio Caceres ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Kinase Domain ◽

Abl Kinase ◽

Kinase Domain Mutations

Download Full-text

Highly sensitive and quantitative detection of BCR-ABL kinase domain mutations by ligation PCR

Leukemia ◽

10.1038/leu.2008.180 ◽

2008 ◽

Vol 22 (12) ◽

pp. 2288-2291 ◽

Cited By ~ 20

Author(s):

O Pelz-Ackermann ◽

M Cross ◽

H Pfeifer ◽

M Deininger ◽

S-Y Wang ◽

...

Keyword(s):

Kinase Domain ◽

Quantitative Detection ◽

Abl Kinase ◽

Highly Sensitive ◽

Kinase Domain Mutations

Download Full-text

Development of PCR, LAMP and qPCR Assays for the Detection of Aflatoxigenic Strains of Aspergillus flavus and A. parasiticus in Hazelnut

Toxins ◽

10.3390/toxins12120757 ◽

2020 ◽

Vol 12 (12) ◽

pp. 757

Author(s):

Sara Franco Ortega ◽

Ilenia Siciliano ◽

Simona Prencipe ◽

Maria Lodovica Gullino ◽

Davide Spadaro

Keyword(s):

Food Safety ◽

Real Time ◽

Aspergillus Flavus ◽

Real Time Pcr ◽

Limit Of Detection ◽

Screening Method ◽

Lamp Assay ◽

High Sensitivity ◽

Isothermal Amplification

Aspergillus flavus and A. parasiticus are two species able to produce aflatoxins in foodstuffs, and in particular in hazelnuts, at harvest and during postharvest phase. As not all the strains of these species are aflatoxin producers, it is necessary to develop techniques that can detect aflatoxigenic from not aflatoxigenic strains. Two assays, a LAMP (loop-mediated isothermal amplification) and a real time PCR with TaqMan® probe were designed and validated in terms of specificity, sensitivity, reproducibility, and repeatability. The capability of the strains to produce aflatoxins was measured in vitro and both assays showed to be specific for the aflatoxigenic strains of A. flavus and A. parasiticus. The limit of detection of the LAMP assay was 100–999 picograms of DNA, while the qPCR detected 160 femtograms of DNA in hazelnuts. Both techniques were validated using artificially inoculated hazelnuts and naturally infected hazelnuts. The qPCR was able to detect as few as eight cells of aflatoxigenic Aspergillus in naturally infected hazelnut. The combination of the LAMP assay, which can be performed in less than an hour, as screening method, with the high sensitivity of the qPCR, as confirmation assay, is able to detect aflatoxigenic strains already in field, helping to preserve the food safety of hazelnuts.

Download Full-text

Different Real-Time PCR Formats Compared for the Quantitative Detection of Human Cytomegalovirus DNA

Clinical Chemistry ◽

10.1093/clinchem/45.11.1932 ◽

1999 ◽

Vol 45 (11) ◽

pp. 1932-1937 ◽

Cited By ~ 65

Author(s):

Andreas Nitsche ◽

Nina Steuer ◽

Christian Andreas Schmidt ◽

Olfert Landt ◽

Wolfgang Siegert

Keyword(s):

Real Time ◽

Human Cytomegalovirus ◽

Real Time Pcr ◽

Detection System ◽

Quantitative Detection ◽

Fluorescence Signal ◽

Hybridization Probe ◽

Sequence Detection ◽

Hybridization Probes ◽

Assay Time

Abstract Background: The aim of this study was to compare the ABI PRISM 7700 Sequence Detection System and the LightCycler to develop a quantitative real-time PCR assay for the detection of human cytomegalovirus (HCMV) DNA suitable for routine hospital application. Methods: We used one exonuclease probe and five different hybridization probe sets as sequence-specific fluorescence detection formats. For the exonuclease assay and two hybridization probe sets, reproducibility and the detection limit were determined. To keep the total assay time to a minimum, we gradually shortened individual reaction steps on both instruments. Results: The exonuclease assay can be interchangeably performed on the 7700 and the LightCycler. No change of reaction conditions is required, except for the addition of bovine serum albumin to the LightCycler reaction. The shortest possible total assay time is 80 min for the ABI PRISM 7700 Sequence Detection System and 20 min for the LightCycler. When the LightCycler is used, the exonuclease probe can be replaced by a set of hybridization probes. All assays presented here detected HCMV DNA in a linear range from 101 to 107 HCMV genome equivalents/assay (r >0.995) with low intraassay (<5%) and interassay (<10%) variation. Conclusions: The ABI PRISM 7700 Sequence Detection System as well as the LightCycler are useful instruments for rapid and precise online PCR detection. Moreover, the two principles of fluorescence signal production allow HCMV quantification with the same accuracy.

Download Full-text

Detection and identification of Acanthamoeba and other non-viral causes of infectious keratitis in corneal scrapings by real-time PCR and next-generation sequencing-based 16S-18S gene analysis

Journal of Clinical Microbiology ◽

10.1128/jcm.02224-20 ◽

2020 ◽

Author(s):

Dennis Back Holmgaard ◽

Celine Barnadas ◽

Seyed Hossein Mirbarati ◽

Lee O’Brien Andersen ◽

Henrik Vedel Nielsen ◽

...

Keyword(s):

Next Generation Sequencing ◽

Real Time ◽

Real Time Pcr ◽

Clinical Relevance ◽

Bacterial Species ◽

Screening Method ◽

Infectious Keratitis ◽

Next Generation ◽

Specificity And Sensitivity ◽

Generation Sequencing

Acanthamoeba is a free-living amoeba of extensive genetic diversity. It may cause infectious keratitis (IK), which can also be caused by bacteria, fungi, and viruses. High diagnostic sensitivity is essential to establish an early diagnosis of Acanthamoeba-associated keratitis. Here, we investigated the applicability of next-generation sequencing (NGS)-based ribosomal gene detection and differentiation (16S-18S) compared with specific real-time PCR for detection of Acanthamoeba. Two hundred DNAs extracted from corneal scrapings and screened by Acanthamoeba-specific real-time PCR were analyzed using an in-house 16S-18S NGS assay. Of these, 24 were positive using specific real-time PCR, 21 of which were positive using the NGS assay. Compared with real-time PCR; the specificity and sensitivity of the NGS assay were 100% and 88%, respectively. Genotypes identified by the NGS assay included T4 (n = 19) and T6 (n = 2). Fungal and bacterial species of potential clinical relevance were identified in 31 of the samples negative for Acanthamoeba, exemplified by Pseudomonas aeruginosa (n = 11), Moraxella spp. (n = 6), Staphylococcus aureus (n = 2), Fusarium spp. (n = 4), and Candida albicans (n = 1). Conclusively, the 16S-18S assay was slightly less sensitive than real-time PCR in detecting Acanthamoeba-specific DNA in corneal scrapings. Robust information on genotype was provided by the NGS assay, and other pathogens of potential clinical relevance were identified in 16% of the samples negative for Acanthamoeba. NGS-based detection of ribosomal genes in corneal scrapings could be an efficient screening method for detecting non-viral causes of IK, including Acanthamoeba.

Download Full-text