To detect Zika virus more rapidly and accurately, we developed a novel method that utilized a real-time fluorescence reverse transcription loop-mediated isothermal amplification (LAMP) technique. The NS5 gene was amplified by a set of six specific primers that recognized six distinct sequences. The amplification process, including 60 min of thermostatic reaction with Bst DNA polymerase following real-time fluorescence reverse transcriptase using genomic Zika virus standard strain (MR766), was conducted through fluorescent signaling. Among the six pairs of primers that we designate here, NS5 was the most efficient with a high sensitivity of up to 3.3 ng/μl and reproducible specificity on eight pathogen samples that were used as negative controls. The real-time fluorescence reverse transcription LAMP detection process can be completed within 35 min. Our study demonstrated that real-time fluorescence reverse transcription LAMP could be highly beneficial and convenient clinical application to detect Zika virus due to its high specificity and stability.
Background
Alterations of 11q23/KMT2A are the most prevalent cytogenetic abnormalities in acute myeloid leukemia (AML) and the prognostic significance of 11q23/KMT2A‐rearranged AML based on various translocation partners varies among different studies. However, few studies evaluated the molecular characteristics of 11q23/KMT2A‐rearranged pediatric AML. We aim to analyze the mutational landscape of 11q23/KMT2A‐rearranged AML and assess their prognostic value in outcomes.
Methods
The mutational landscape and clinical prognosis of 105 children with 11q23/KMT2A‐rearranged AML in comparison with 277 children with non‐11q23/KMT2A‐rearranged AML were analyzed using publicly accessible next‐generation sequencing data from Therapeutically Applicable Research to Generate Effective Treatments (TARGET) dataset.
Results
Pediatric AML patients with 11q23/KMT2A‐rearrangements harbored a low number of mutations (Median, 1 mutation/patient, range, 1‐22), 58% of which involved in RAS pathway mutations (KRAS, NRAS, and PTPN11) and 10.5% of which comprised of SETD2 mutations. Compared with non‐11q23/KMT2A‐rearranged AML, the incidence of KRAS (32.4% vs. 10.1%, P〈0.001) and SETD2 (10.5% vs. 1.4%, P=0.001) gene mutations in 11q23/KMT2A‐rearranged AML was significantly higher. Both KRAS and SETD2 mutations occurred more often in t(10;11)(p12;q23). KRAS mutations were correlated with worse 5‐year event‐free survival [EFS] (Plog‐rank = 0.001) and 5‐year overall survival [OS] (Plog‐rank = 0.009) and the presence of SETD2 mutations increases the 5‐year relapse rate (PGray = 0.004). Multivariate analyses confirmed KRAS mutations in 11q23/KMT2A‐rearranged AML as an independent predictor for poor EFS (hazard ratio [HR] = 2.10, P=0.05) and OS (HR = 2.39, P=0.054).
Conclusion
Our findings show that pediatric patients with 11q23/KMT2A rearrangements have characteristic mutation patterns and varying clinical outcomes depending on different translocation partners, which could be utilized to develop more accurate risk stratification and tailored therapies.
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