A singleplex real-time fluorescence resonance energy transfer PCR with melting curve analysis for the differential detection ofParagonimus heterotremus, Echinostoma malayanumandFasciola giganticaeggs in faeces

2016 ◽  
Vol 110 (1) ◽  
pp. 74-83 ◽  
Author(s):  
Chairat Tantrawatpan ◽  
Weerachai Saijuntha ◽  
Sirikul Manochantr ◽  
Pakpoom Kheolamai ◽  
Tongjit Thanchomnang ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Real Time ◽  
Fluorescence Resonance Energy Transfer ◽  
Melting Curve ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Differential Detection ◽  
Fluorescence Resonance

scholarly journals Hepatitis C Genotype Determination by Melting Curve Analysis with a Single Set of Fluorescence Resonance Energy Transfer Probes

2002 ◽  
Vol 48 (12) ◽  
pp. 2147-2154 ◽  
Author(s):  
Grant C Bullock ◽  
David E Bruns ◽  
Doris M Haverstick
Keyword(s):  
Energy Transfer ◽  
Hepatitis C ◽  
Real Time ◽  
Melting Curve ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Comparison Method ◽  
Curve Analysis ◽  
Melting Curve Analysis

Abstract Background: The genotype of hepatitis C virus (HCV) is a predictor of antiviral therapeutic response. We describe an approach for HCV genotype determination by real-time PCR and melting curve analysis. Methods: After automated nucleic acid extraction, we used reverse transcription-PCR in a block cycler to amplify nucleotides 6–329 of the 5′-untranslated region of HCV. The product was further amplified by single-tube real-time seminested PCR in a LightCyclerTM instrument (Roche). The final product was analyzed by melting curves with the use of fluorescence resonance energy transfer (FRET) probes. The FRET sensor probe was directed at nucleotides 151–170 of type 1 HCV and was designed to distinguish types 1a/b, 2a/c, 2b, 3a, and 4, with melting temperatures (Tms) predicted to differ by 1 °C. Genotypes were compared in a blinded fashion with those of the INNO-LiPATM test (Bayer Diagnostics) on 111 serum samples. Results: In preliminary experiments, the Mg2+ concentration was found to be critical in allowing clear separation of melting points, with the best separation at a Mg2+ concentration of 2 mmol/L. The results for 111 samples clustered at expected Tms for genotypes 1a/b (n = 78), 2a/c (n = 2), 2b (n = 11), 3a (n = 14), and 4 (n = 2). Of the 111 samples, results for 110 were concordant with the comparison method at the level of type 1, 2, 3, or 4. Subtyping results were discordant for two samples, both of type 2. For 108 samples concordant with INNO-LiPA at the genotype and subtype levels, the mean Tms were 64.1, 59.5, 54.2, 52.6, and 50.1 °C for types 1a/b, 2a/c, 4, 2b, and 3a, respectively, with SDs of 0.2, 0.3, 0.3, 0.2, and 0.3 °C. All 78 samples identified as type 1 were concordant with results of the comparison method. Conclusions: Melting analysis with a single pair of FRET probes can rapidly provide information about HCV genotypes and identifies type 1 samples with high specificity.

scholarly journals Rapid detection of the factor XIII Val34Leu (163 G→T) polymorphism by real-time PCR using fluorescence resonance energy transfer detection and melting curve analysis

2004 ◽  
Vol 42 (8) ◽  
Author(s):  
Amir H. Shemirani ◽  
László Muszbek
Keyword(s):  
Energy Transfer ◽  
Real Time ◽  
Allele Frequency ◽  
Real Time Pcr ◽  
Factor Xiii ◽  
Melting Curve ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Curve Analysis ◽  
Melting Curve Analysis

AbstractThe Val34Leu polymorphism in the A subunit of blood coagulation factor XIII (FXIII-A) is located in the activation peptide, just three amino acids upstream of the thrombin cleavage site. The Val→Leu replacement accelerates the rate of the proteolytic activation of FXIII and it seems to provide protection against myocardial infarction. Methods available for the assessment of the FXIII-A Val34Leu polymorphism are rather time-consuming, laborious and not easily applicable for large-scale studies. In this study a new method based on real-time PCR with fluorescence resonance energy transfer (FRET) detection and melting curve analysis was developed. The rapid, simple method was adapted to the widely used real-time PCR instrument, LightCycler (Roche Diagnostics). The results showed 100% coincidence with those obtained by the traditional PCR-restriction fragment length polymorphism (RFLP) assay and fluorescent DNA sequencing. Using this method, an allele frequency of 24.2% was obtained (n = 113), which well agrees with the allele frequency obtained by PCR-RFLP on a different group of the same ethnic Hungarian population (25.9%).

Rapid detection of Dirofilaria immitis in mosquito vectors and dogs using a real-time fluorescence resonance energy transfer PCR and melting curve analysis

2010 ◽  
Vol 168 (3-4) ◽  
pp. 255-260 ◽  
Author(s):  
Tongjit Thanchomnang ◽  
Pewpan M. Intapan ◽  
Viraphong Lulitanond ◽  
Somboon Sangmaneedet ◽  
Sudchit Chungpivat ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Real Time ◽  
Rapid Detection ◽  
Dirofilaria Immitis ◽  
Melting Curve ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
Mosquito Vectors
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