Dataset Information

Performance and clinical evaluation of a sensitive multiplex assay for the rapid detection of common NPM1 mutations.

ABSTRACT: Determination of NPM1 mutation status has become essential for the molecular classification of acute myeloid leukemias (AML). Methods with high clinical sensitivity and specificity adapted to the molecular laboratory workflow are required for the diagnosis, prognosis, and monitoring of AML with normal karyotype. We report here the development and evaluation of a novel, streamlined, RNA-based assay for the rapid multiplex detection of common NPM1 mutations in a 96-well assay format. Using synthetic transcripts and total RNA from leukemic cell lines, we show that the assay can specifically detect NPM1 wild-type and mutants A, B, D, or J transcripts in the same reaction. Dilution experiments indicate an assay dynamic range >4 log units with an analytical sensitivity of approximately 0.01%. Evaluation of 69 clinical specimens at initial diagnosis resulted in 100% agreement with reference methods. Of patients with AML with normal karyotype, 53% carried one of four different mutations. The assay was also combined with other laboratory-developed tests to simultaneously detect NPM1 mutant transcripts and fusion transcripts resulting from t(8;21) or inv(16) in a single reaction well. Overall, these results show that the assay is a versatile and specific tool for the screening of NPM1 mutations in patients with AML. Its high analytical sensitivity further suggests potential utility for the monitoring of residual disease in AML with normal karyotype.

SUBMITTER: Hafez M

PROVIDER: S-EPMC2928427 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Performance and clinical evaluation of a sensitive multiplex assay for the rapid detection of common NPM1 mutations.

Hafez Michael M Ye Fei F Jackson Keith K Yang Zhe Z Karp Judith E JE Labourier Emmanuel E Gocke Christopher D CD

The Journal of molecular diagnostics : JMD 20100708 5

Determination of NPM1 mutation status has become essential for the molecular classification of acute myeloid leukemias (AML). Methods with high clinical sensitivity and specificity adapted to the molecular laboratory workflow are required for the diagnosis, prognosis, and monitoring of AML with normal karyotype. We report here the development and evaluation of a novel, streamlined, RNA-based assay for the rapid multiplex detection of common NPM1 mutations in a 96-well assay format. Using synthet ...[more]

PMID: 20616361

Similar Datasets

Project description:Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a major threat to public health. Rapid molecular testing for convenient and timely diagnosis of SARS-CoV-2 infections represents a challenge that could help to control the current pandemic and prevent future outbreaks. We aimed to develop and validate a multiplex and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using lyophilized LAMP reagents for sensitive and rapid detection of SARS-CoV-2. LAMP primers were designed for a set of gene targets identified by a genome-wide comparison of viruses. Primer sets that showed optimal features were combined into a multiplex RT-LAMP assay. Analytical validation included assessment of the limit of detection (LoD), intra- and inter-assay precision, and cross-reaction with other respiratory pathogens. Clinical performance compared to that of real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) was assessed using 278 clinical RNA samples isolated from swabs collected from individuals tested for COVID-19. The RT-LAMP assay targeting the RNA-dependent RNA polymerase (RdRp), membrane (M), and ORF1ab genes achieved a comparable LoD (0.65 PFU/mL, CT=34.12) to RT-qPCR and was 10-fold more sensitive than RT-qPCR at detecting viral RNA in clinical samples. Cross-reactivity to other respiratory pathogens was not observed. The multiplex RT-LAMP assay demonstrated a strong robustness and acceptable intra- and inter-assay precision (mean coefficient of variation, 4.75% and 8.30%). Diagnostic sensitivity and specificity values were 100.0% (95% CI: 97.4-100.0%) and 98.6% (95% CI: 94.9-99.8%), respectively, showing high consistency (Cohen's kappa, 0.986; 95% CI: 0.966-1.000; p<0.0001) compared to RT-qPCR. The novel one-step multiplex RT-LAMP assay is storable at room temperature and showed similar diagnostic accuracy to conventional RT-qPCR, while being faster (<45 min), simpler, and cheaper. The new assay could allow reliable and early diagnosis of SARS-CoV-2 infections in primary health care. It may aid large-scale testing in resource-limited settings, especially if it is integrated into a point-of-care diagnostic device.

Project description:BackgroundSexually transmitted infections (STIs) are some of the most common communicable conditions and exert impact on the health and lives of many hundreds of millions of people across the world every year. Screening high-risk populations and conducting comprehensive detection tests would lead to a significant improvement in preventing the transmission of STIs and help us to provide rapid treatment to those affected. Here, we successfully established and validated a novel high-throughput multiplex gene detection system (HMGS) for the simultaneous and semiquantitative detection of six important curable sexually transmitted pathogens in a single reaction from secretions samples.MethodFluorescently labeled primers were designed to target specific conserved and single-copy gene fragments of Ureaplasma urealyticum (U. urealyticum), Mycoplasma hominis (M. hominis), Chlamydia trachomatis (C. trachomatis), Neisseria gonorrhoeae (N. gonorrhoeae), Trichomonas vaginalis (T. vaginalis), and Treponema pallidum (T. pallidum). The specificity and sensitivity of the STI-HMGS was validated and optimized using plasmids and quantitative genomic DNA. Next, we validated the performances of the STI-HMGS for clinical application by testing samples of clinical secretions collected from patients who visited the gynecology and urology outpatient clinics of our reproductive medicine center. Results derived from the STI-HMGS were then compared with three approved commercialized kits that used to detect U. urealyticum, C. trachomatis and N. gonorrhoeae, respectively, followed by further validation with Sanger sequencing for all pathogens. Finally, a comprehensive analysis of epidemiology was performed among different subgroups to investigate the association between infection rates and clinically-relevant information.ResultsThe sensitivity of STI-HMGS for six target genes was 10 copies/µL. Data derived from the detection of 381 clinical secretions demonstrated that the STI-HMGS exhibited high concordance rate compared with approved commercialized kits and almost 100% sensitivity and specificity for the detection of six sexually transmitted pathogens when validated by Sanger sequencing. Semi-quantitative analysis found that STIs caused by N. gonorrhoeae had a significantly higher (P<0.05) pathogen load than the other pathogens. Infections caused by C. trachomatis were significantly more common in younger individuals (P<0.05). We also found that U. urealyticum infections were more likely to happen in females; while the males were more affected by N. gonorrhoeae (P<0.05).ConclusionsSTI-HMGS proved to be an efficient method for the semi-quantitative detection of six important curable sexually transmitted pathogens and therefore represents an alternative method for the clinical detection and monitoring of STIs.

Dataset Information

Performance and clinical evaluation of a sensitive multiplex assay for the rapid detection of common NPM1 mutations.

Publications

Performance and clinical evaluation of a sensitive multiplex assay for the rapid detection of common NPM1 mutations.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets