scholarly journals A biochemical viability assay is compatible with molecular methods for species identification

2013 ◽  
Vol 66 ◽  
pp. 29-33 ◽  
Author(s):  
N.K. Richards ◽  
L.M. Winder ◽  
I.I. Iline ◽  
M.A. Novoselov ◽  
M.R. McNeill ◽  
...  
Keyword(s):  
Species Identification ◽  
Curve Analysis ◽  
Cytochrome C Oxidase I ◽  
Melt Curve Analysis ◽  
Identification Methods ◽  
Weevil Species ◽  
Viability Assay ◽  
Sitona Lepidus ◽  
Molecular Identification Methods ◽  
Listronotus Bonariensis

A biochemical viability assay was recently developed to quickly and easily assess the viability of small immobile arthropods including eggs intercepted on plant products On finding a viable specimen species identification often becomes the next hurdle This paper demonstrates that amplifiable DNA is present in a used biochemical viability assay solution and can be used for making taxonomic identifications Cryptically labelled heattreated and untreated eggs of three weevil species (Listronotus bonariensis Sitona lepidus and S discoideus) were first tested for viability then a 1135 bp fragment of the cytochrome c oxidase I gene was amplified from each viability assay solution in the presence of a fluorescent dye SYBR Green Melt curve analysis of the amplicons (n36) revealed three distinct melt profiles that correctly corresponded to each of the three weevil species This shows that the biochemical viability assay is compatible with the application of subsequent molecular identification methods which will facilitate the appropriate management response

scholarly journals Rapid Differentiation of Mycobacterium tuberculosis and M. bovis by High-Resolution Melt Curve Analysis

2010 ◽  
Vol 48 (11) ◽  
pp. 4269-4272 ◽  
Author(s):  
S. Ereqat ◽  
G. K. Bar-Gal ◽  
A. Nasereddin ◽  
K. Azmi ◽  
S. E. Qaddomi ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Resolution ◽  
Curve Analysis ◽  
High Resolution Melt ◽  
Melt Curve Analysis

High‐resolution DNA melt‐curve analysis for cost‐effective mass screening of pairwise species interactions

2013 ◽  
Vol 13 (5) ◽  
pp. 908-917 ◽  
Author(s):  
James K. McCarthy ◽  
Raphael K. Didham ◽  
Eckehard G. Brockerhoff ◽  
Katherine A. Bysterveldt ◽  
Arvind Varsani
Keyword(s):  
High Resolution ◽  
Effective Mass ◽  
Mass Screening ◽  
Species Interactions ◽  
Cost Effective ◽  
Curve Analysis ◽  
Melt Curve Analysis

scholarly journals Direct Detection of Rifampin and Isoniazid Resistance in Sputum Samples from Tuberculosis Patients by High-Resolution Melt Curve Analysis

2017 ◽  
Vol 55 (6) ◽  
pp. 1755-1766 ◽  
Author(s):  
Divya Anthwal ◽  
Rakesh Kumar Gupta ◽  
Manpreet Bhalla ◽  
Shinjini Bhatnagar ◽  
Jaya Sivaswami Tyagi ◽  
...  
Keyword(s):  
High Resolution ◽  
Direct Detection ◽  
Melting Curve ◽  
Curve Analysis ◽  
High Resolution Melt ◽  
Melt Curve Analysis ◽  
Content Type ◽  
Plasmid Library ◽  
Mdr Tb ◽  
Smear Negative

ABSTRACT Drug-resistant tuberculosis (TB) is a major threat to TB control worldwide. Globally, only 40% of the 340,000 notified TB patients estimated to have multidrug-resistant-TB (MDR-TB) were detected in 2015. This study was carried out to evaluate the utility of high-resolution melt curve analysis (HRM) for the rapid and direct detection of MDR-TB in Mycobacterium tuberculosis in sputum samples. A reference plasmid library was first generated of the most frequently observed mutations in the resistance-determining regions of rpoB , katG , and an inhA promoter and used as positive controls in HRM. The assay was first validated in 25 MDR M. tuberculosis clinical isolates. The assay was evaluated on DNA isolated from 99 M. tuberculosis culture-positive sputum samples that included 84 smear-negative sputum samples, using DNA sequencing as gold standard. Mutants were discriminated from the wild type by comparing melting-curve patterns with those of control plasmids using HRM software. Rifampin (RIF) and isoniazid (INH) monoresistance were detected in 11 and 21 specimens, respectively, by HRM. Six samples were classified as MDR-TB by sequencing, one of which was missed by HRM. The HRM-RIF, INH- katG , and INH- inhA assays had 89% (95% confidence interval [CI], 52, 100%), 85% (95% CI, 62, 97%), and 100% (95% CI, 74, 100%) sensitivity, respectively, in smear-negative samples, while all assays had 100% sensitivity in smear-positive samples. All assays had 100% specificity. Concordance of 97% to 100% (κ value, 0.9 to 1) was noted between sequencing and HRM. Heteroresistance was observed in 5 of 99 samples by sequencing. In conclusion, the HRM assay was a cost-effective (Indian rupee [INR]400/US$6), rapid, and closed-tube method for the direct detection of MDR-TB in sputum, especially for direct smear-negative cases.

A comparison of microsatellite instability analysis methods in colorectal cancer.

2019 ◽  
Vol 37 (8_suppl) ◽  
pp. 46-46
Author(s):  
Samantha Lewis ◽  
Shaun Peterson ◽  
Kathryn Oostdik ◽  
Heather Tomlinson
Keyword(s):  
Colorectal Cancer ◽  
Microsatellite Instability ◽  
Standard Method ◽  
Gold Standard ◽  
Curve Analysis ◽  
Patient Treatment ◽  
Human Colorectal Cancer ◽  
Fragment Analysis ◽  
Gold Standard Method ◽  
Melt Curve Analysis

46 Background: Use of biomarkers for patient treatment stratification is an increasingly important topic with the recent FDA approval of Pembrolizumab and Nivolumab for microsatellite instability (MSI) high/mismatch repair (MMR) deficient cancers. This ground-breaking approval allows physicians to make patient treatment decisions based on molecular biomarker status. Thus, performance of detection methods for these biomarkers is of great importance. The gold standard for MSI analysis in a research setting is PCR followed by fragment analysis to resolve DNA size. Recently, PCR followed by melt curve analysis has been presented as an alternative approach. In this set of experiments these two methods are compared using a subset of human colorectal cancer samples. Methods: A cohort of matched human colorectal cancer and adjacent normal FFPE samples were used for this comparison. Eight pairs were selected that contained subtle shifts, low tumor volume or heterozygosity at microsatellite loci. These samples were then tested for MSI by fragment or melt curve analysis. Samples were then classified as MSI-H, MSI-L or microsatellite stable (MSS) according to manufacturer specifications for melt curve analysis or by an interpretive software for fragment analysis. Results: 40 mononucleotide markers were examined for each method. Melt curve analysis was concordant with fragment analysis in 70% of markers tested (28/40). Of discordant markers, 92% (11/12) were identified as unstable with fragment analysis but were stable by melt curve analysis. Additionally, two of the samples identified as MSI-H by fragment analysis were called MSS or MSI-L using the melt curve analysis method. These preliminary results suggest there may be differences in sensitivity when using challenging samples with melt curve compared to the gold standard method, fragment analysis. Conclusions: The standard method for determining MSI status utilizes PCR and fragment analysis. Recently, melt curve analysis has been proposed as an alternative method. We present preliminary findings of decreased sensitivity with melt curve compared to fragment analysis. Additional studies with a larger, more diverse sample set will be required to define this relationship further.

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