scholarly journals Development of a single tube 640-plex genotyping method for detection of nucleic acid variations on microarrays

2008 ◽  
Vol 36 (12) ◽  
pp. e75-e75 ◽  
Author(s):  
K. Krjutskov ◽  
R. Andreson ◽  
R. Magi ◽  
T. Nikopensius ◽  
A. Khrunin ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Single Tube ◽  
Genotyping Method

scholarly journals Gargle-Direct: Extraction-Free Detection of SARS-CoV-2 using Real-time PCR (RT-qPCR) of Saline Gargle Rinse Samples

2020 ◽  
Author(s):  
Vijay J. Gadkar ◽  
David M. Goldfarb ◽  
Virginia Young ◽  
Nicole Watson ◽  
Linda Hoang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Standard Method ◽  
Molecular Diagnostic ◽  
Heat Inactivation ◽  
Acid Extraction ◽  
Analytical Sensitivity ◽  
Sample Type ◽  
Mouth Rinse ◽  
Nucleic Acid Extraction ◽  
Single Tube

ABSTRACTBackgroundSaline mouth rinse/gargle samples have recently been shown to be a suitable option for swab-independent self-collection for SARS-CoV-2 diagnosis. We sought to evaluate a simplified process for direct reverse transcriptase PCR (RT-qPCR) testing of this novel sample type and to compare performance with routine RT-qPCR using automated nucleic acid extraction.MethodsClinical saline mouth rinse/gargle samples were subjected to automated nucleic acid extraction (“standard method”), followed by RT-qPCR using three assays including the FDA authorized US-CDC’s N1/N2 assay, which was the reference standard for determining sensitivity/specificity. For extraction-free workflow, an aliquot of each gargle sample underwent viral heat inactivation at 65 °C for 20 minutes followed by RT-qPCR testing, without an intermediate extraction step. An in-house validated RT-qPCR lab developed test (LDT), targeting the SARS-CoV-2’s S/ORF8 genes (SORP triplex assay) and the N1/N2 US-CDC assay was used to evaluate the extraction-free protocol. To improve the analytical sensitivity, we developed a single-tube hemi-nested (STHN) version of the SORP triplex assay.ResultsA total of 38 SARS-CoV-2 positive and 75 negative saline mouth rinse/gargle samples were included in this evaluation. A 100% concordance in detection rate was obtained between the standard method and the extraction-free approach for the SORP assay. An average increase of +2.63 to +5.74 of the cycle threshold (CT) values was observed for both the SORP and N1/N2 assay when extraction-free was compared between the standard method. The average ΔCT [ΔCT=CT(Direct PCR)-CT(Extracted RNA)], for each of the gene targets were: S (ΔCT= +4.24), ORF8 (ΔCT=+2.63), N1 (ΔCT=+2.74) and N2 (ΔCT=+5.74). The ΔCT for the STHN SORP assay was +1.51 and −2.05 for the S and ORF8 targets respectively, when extracted method was compared to the standard method.ConclusionOur Gargle-Direct SARS-CoV-2 method is operationally simple, minimizes pre-analytical sample processing and is potentially implementable by most molecular diagnostic laboratories. The empirical demonstration of single-tube hemi-nested RT-qPCR, to specifically address and alleviate the widely-acknowledged problem of reduced analytical sensitivity of detection of extraction-free templates, should help diagnostic laboratories in choosing Gargle-Direct protocol for high-throughput testing.

scholarly journals Detection of low copies of drug-resistant influenza viral gene by a single-tube reaction using peptide nucleic acid as both PCR clamp and sensor probe

2014 ◽  
Vol 47 (3) ◽  
pp. 254-256 ◽  
Author(s):  
Kuo-Chien Tsao ◽  
Chiuan-Chian Chiou ◽  
Tai-Long Chen ◽  
Chung-Guei Huang ◽  
Erh-Fang Hsieh ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Peptide Nucleic Acid ◽  
Viral Gene ◽  
Drug Resistant ◽  
Single Tube ◽  
Sensor Probe ◽  
Single Tube Reaction

scholarly journals A Novel Multimodal Next Generation Sequencing Assay with Total Nucleic Acid Input to Provide Comprehensive Genomic Profiling for Hematologic Malignancies

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 29-30
Author(s):  
Yongxin Yu ◽  
Francys Alarcon ◽  
Yanglong Mou ◽  
Segun C Jung ◽  
Hyunjun Nam ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Cost Effective ◽  
Hematologic Malignancies ◽  
Genomic Profiling ◽  
Gene Fusions ◽  
Total Nucleic Acid ◽  
Single Tube ◽  
Dna And Rna ◽  
Comprehensive Genomic Profiling ◽  
Generation Sequencing

Background: A cost effective and comprehensive genomic profiling (CGP) approach for diagnosis, risk stratification and therapy would be useful for the evaluation of oncologic specimens. Available approaches involving additive testing for DNA and RNA abnormalities through traditional methods (e.g. Sanger, FISH, cytogenetics, qRT-PCR) are not comprehensive, require multiple different workflows and are sample consuming, often resulting in incomplete testing. While there are next generation sequencing (NGS) assays designed for detecting DNA and RNA abnormalities, they have separate workflows that require twice the amount of sample and effort. To address this, we developed a novel total nucleic acid (TNA) extraction method and single tube workflow utilizing TNA and a custom multimodal chemistry designed for hematologic malignancies. This consolidated workflow enables an efficient discovery based approach for both DNA/RNA abnormalities including single nucleotide variants (SNVs), InDels, copy number variants (CNVs), large structural changes from DNA and gene fusions and gene expression levels from RNA. This method maximizes data derived from valuable samples while delivering a comprehensive profile of the patient's tumor which can help guide therapeutic and clinical decisions. Methods: Total nucleic acid (TNA) was extracted from bone marrow and peripheral blood of 95 patients (CML, CMML, CLL, AML and myeloid disorders). 297 genes that have DNA mutations specific to hematological cancers were targeted, along with 213 genes that were targeted for clinically significant RNA abnormalities. Enriched genomic and transcriptomic regions of interest from 85 patients were successfully sequenced with unique dual indices on an Illumina NovaSeq 6000. DNA variant detection as well as fusion detection from RNA were compared to traditional orthogonal NGS assays that use DNA input or compared to qRT-PCR and Sanger sequencing assays that use RNA as input. Results: In this study, we developed an efficient and high-quality TNA extraction method that can purify enough total nucleic acid from bone marrow, peripheral blood, cytogenetic pellets, flow suspension, and FFPE samples for the downstream NGS assay. The average OD 260/280 value was 1.9 and the OD 260/230 was 2.18. After sequencing, 256/262 (97.7% accuracy) SNV and Indel variants that were candidate pathogenic mutations were concordant from 38 patients. Meanwhile, 100% (7/7) of all BCR/ABL1 gene fusions which had an international scale (IS) value above 6.4% were concordant. In addition, 69 fusion positive samples containing 20 unique gene fusions which had been previously reported by an independent ArcherDX assay designed specifically for gene fusions were also evaluated with this chemistry. Analysis revealed a 92.5% (64/69) concordance. More importantly, the QIAseq multimodal TNA NGS assay detected both DNA and RNA abnormalities in a single tube. For example, in one myeloid leukemia patient, we not only identified pathogenic variants of ASXL1 and JAK2 which had been previously detected by a DNA NGS assay, but also detected a concurrent BCR-FGFR1 fusion which had been previously reported by a FISH assay. Moreover, we were able to provide more comprehensive genomic profiling by investigating many DNA and RNA abnormalities simultaneously. In our study, for 5 patients that previously been tested for BCR-ABL1 fusion only, we are able to assess BCR-ABL1 fusion status from RNA as well as identify pathogenic DNA variants at the same time, including JAK2 p.V617F, U2AF1 p.S34F, ASXL1 p.E635Rfs*15, BRCA p.S1982Rfs*22, and DNMT3A p.S708Vfs*71, which provides valuable information to assist diagnosis and treatment in a cost effective and efficient way. Conclusions: We developed a single tube TNA based workflow with a custom multimodal chemistry that simultaneously detects many DNA and RNA abnormalities in a cost effective and efficient way while reducing sample requirements. This unique TNA NGS assay provides comprehensive genomic profiling for hematologic malignancies and improves the diagnostic testing options for precise patient care. Disclosures Yu: NeoGenomics: Current Employment. Alarcon:NeoGenomics: Current Employment. Mou:NeoGenomics: Current Employment. Jung:NeoGenomics: Current Employment. Nam:NeoGenomics: Current Employment. Thomas:NeoGenomics: Current Employment. Keeler:NeoGenomics: Current Employment. Shinbrot:NeoGenomics: Current Employment. Magnan:NeoGenomics: Current Employment. Bender:NeoGenomics: Current Employment. Jiang:NeoGenomics: Current Employment. Agersborg:NeoGenomics: Current Employment. Weiss:Bayer: Other: speaker; Genentech: Other: Speaker; Merck: Other: Speaker; NeoGenomics: Current Employment. Ye:NeoGenomics: Current Employment. Funari:NeoGenomics: Current Employment.

scholarly journals The using of gene-oriented SCoT markers in genotyping of the Apple genus (Malus Mill.)

2021 ◽  
Vol 34 ◽  
pp. 02005
Author(s):  
Ilya Stepanov ◽  
Ilnur Balapanov ◽  
Elena Lobodina ◽  
Ivan Suprun
Keyword(s):  
Nucleic Acid ◽  
Malus Domestica ◽  
Plant Tissue ◽  
Dna Isolation ◽  
Genotyping Method ◽  
Golden Delicious ◽  
Apple Plant ◽  
Nucleic Acid Preparation ◽  
Total Dna

This paper discusses the aspects of optimization of the SCoT genotyping method for representatives of the genus Apple (Malus Mill.), including the orchard apple (Malus domestica). Special attention is paid to the methods of total DNA isolation from apple plant tissue, which is due to the sensitivity of multilocus marker systems to the quality of the nucleic acid preparation. On a sample of total DNA isolated from the leaves of the Golden Delicious cultivar, 18 SCoT markers were tested, from which 4 Malus Mill cultivars and species most promising for genotyping were selected.

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