Rapid detection of α-thalassaemia alleles of -- SEA /, -α 3.7 / and -α 4.2 / using a dual labelling, self-quenching hybridization probe/melting curve analysis

Gao, Lan; Liu, Qing; Sun, Manna; Zhao, Ying; Xie, Run-Gui; He, Yanan; Xu, Wanfang; Liu, Jianxin; Lin, Yangyang; Lou, Jiwu

doi:10.1016/j.mcp.2015.07.004

Cited by 4 publications

(3 citation statements)

References 19 publications

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“…Our study has found that the use of ddPCR is convenient as it allows direct quantification without the requirement of a calibration curve unlike qPCR . Secondly, our study showed that ddPCR is accurate and precise in the detection of alpha thalassaemia deletions and triplications based on the gene dosages using absolute quantification.…”

Section: Discussionmentioning

confidence: 67%

“…However, the drawbacks of this method are the requirements of post‐PCR steps such as gel electrophoresis and documentation such as the labelling the gel pictures and also gel interpretation, the possibility of PCR carryover contamination risk and the inability to detect unknown deletions . Melting curve analysis has also been used to detect common alpha deletional alleles such as ‐‐ SEA , ‐α 3.7 and ‐α 4.2, but the inability to distinguish some different genotypes accurately when it is inherited in allelic pairs due to the homologous α1 and α2 genes limits its potential to be used widely . In Thailand, a study has been carried out only on the Southeast Asian (‐‐ SEA ) deletion using ddPCR for prenatal diagnosis of Bart's hydrops foetalis .…”

Section: Discussionmentioning

confidence: 99%

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Rapid detection of α‐thalassaemia variants using droplet digital PCR

Lee

Lai

Ramachandran

et al. 2016

Int J Lab Hematology

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Section: Discussionmentioning

confidence: 67%

Section: Discussionmentioning

confidence: 99%

Rapid detection of α‐thalassaemia variants using droplet digital PCR

Lee

Lai

Ramachandran

et al. 2016

Int J Lab Hematology

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“…Recent studies have shown that the TaqMan probes can be used not only for monitoring PCR in real time but also for melting curve analysis under asymmetric PCR conditions (Huang et al, 2011). Multicolor melting curve analysis using the TaqMan probe has been successfully applied in mutant gene detection and genotyping (Botezatu et al, 2017;Mou et al, 2016;Wang et al, 2017;Xia et al, 2016;Huang et al, 2016;Gao et al, 2015;Huang et al, 2017). Nevertheless, the application to multiple virus detection has not yet been reported.…”

Section: Introductionmentioning

confidence: 99%

A novel multiplex PCR for virus detection by melting curve analysis

Liu

et al. 2018

Journal of Virological Methods

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A B S T R A C TBackground: Rapid and accurate laboratory diagnoses of viral infections are crucial for the management and treatment of patients with viral infections. Conventional methods for virus detection are labourious, time consuming, and only a single virus can be analysed in one assay. Objectives: The objective of this study was to develop a novel real-time PCR method for multiple virus detection by melting curve analysis using Taqman probes in a single reaction. Study design: As a model, six respiratory viruses were detected in one tube using three fluorophores. The specificity was assessed by cross-reaction tests with other common respiratory pathogens. The analytical sensitivity was assessed by testing the limit of detection of the assay using artificial plasmids as the positive template. The clinical evaluation of the established assay was evaluated for the detection of respiratory viruses in clinical samples, and the results were compared with direct fluorescent antibody testing (DFA). Results: The six respiratory viruses were clearly distinguished by their respective melting temperature values in the corresponding fluorescence detection channels. No cross reactions were observed by cross reaction tests. The detection limits of this assay were 2 to 2 × 10 3 copies per reaction for each virus. The clinical evaluation of this assay was demonstrated by analysing 352 clinical samples, and 67(19.0%) samples were positive for at least one virus. The accordance rate between the established PCR and DFA testing was high, and ranged from 94.57% to 100%. Conclusions: Taqman probe-based melting curve analysis is well suited for detection of multiple viruses in clinical and research laboratories because of its high throughput, reliability, and cost savings.

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Simultaneous Genotyping of α-Thalassemia Deletional and Nondeletional Mutations by Real-Time PCR–Based Multicolor Melting Curve Analysis

Huang

Wang

Tang

et al. 2017

The Journal of Molecular Diagnostics

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α-Thalassemia, which is caused by defective synthesis of the hemoglobin α-globin chains, is the most commonly inherited recessive hemoglobin abnormality. Genetic detection of a defective α-globin gene is challenging because of a variety of large deletions of the α-globin gene cluster and nondeletional mutations. Separate detections of them are often required using complex and error-prone open-tube methods. We report a novel real-time PCR-based assay that can simultaneously genotype four major deletional and three common nondeletional mutations in two parallel reactions by using multicolor melting curve analysis. The turnaround time of this closed-tube assay was within 3.5 hours, the limit of detection was 5 ng of human genomic DNA per reaction, and as low as 5% mutant DNA could be detected in the mosaic samples. The assay was evaluated using 1213 precharacterized genomic DNA samples in a double-blind manner. All seven α-thalassemia mutations were accurately genotyped, yielding a 99.3% concordance with the comparison assays. The 14 discordant samples contained the HKαα allele that was undetected by the traditional methods. Considering its rapidity, ease of use, and accuracy, we concluded that our real-time PCR assay may be recommended as an alternative screening and diagnostic tool for α-thalassemia.

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Rapid detection of α-thalassaemia alleles of -- SEA /, -α 3.7 / and -α 4.2 / using a dual labelling, self-quenching hybridization probe/melting curve analysis

Cited by 4 publications

References 19 publications

Rapid detection of α‐thalassaemia variants using droplet digital PCR

Rapid detection of α‐thalassaemia variants using droplet digital PCR

A novel multiplex PCR for virus detection by melting curve analysis

Simultaneous Genotyping of α-Thalassemia Deletional and Nondeletional Mutations by Real-Time PCR–Based Multicolor Melting Curve Analysis

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