Rapid detection of α‐thalassaemia variants using droplet digital <scp>PCR</scp>

Lee, T.-Y.; Lai, Mei I; Ramachandran, Vasudevan; Tan, Ji; Teh, Lai Kuan; Othman, R; Hussein, N H; George, Elizabeth

doi:10.1111/ijlh.12520

Cited by 7 publications

(5 citation statements)

References 22 publications

(26 reference statements)

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“…The data showed that ddPCR could precisely recognize the ΔF508-MUT CFTR allele in cffDNA of all proband fetuses and exclude unaffected control fetuses with high sensitivity and cost-effectiveness. In 2016, Lee et al detected both common and rare deletions in α thalassemia using ddPCR, which showed a detection limit of ~1 ng and a rapid detection of α thalassemia variants in a Malaysian population [45].…”

Section: Prenatal Diagnosis Of Genetic Diseasementioning

confidence: 99%

Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation

Yu¹,

Shen²,

Jiang³

et al. 2017

Cell Physiol Biochem

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Gene mutation has been considered a research hotspot, and the rapid development of biomedicine has enabled significant advances in the evaluation of gene mutations. The advent of digital polymerase chain reaction (dPCR) elevates the detection of gene mutations to unprecedented levels of precision, especially in cancer-associated genes. dPCR has been utilized in the detection of tumor markers in cell-free DNA (cfDNA) samples from patients with different types of cancer in samples such as plasma, cerebrospinal fluid, urine and sputum, which confers significant value for dPCR in both clinical applications and basic research. Moreover, dPCR is extensively used in detecting pathogen mutations related to typical features of infectious diseases (e.g., drug resistance) and mutation status of heteroplasmic mitochondrial DNA, which determines the manifestation and progression of mtDNA-related diseases, as well as allows for the prenatal diagnosis of monogenic diseases and the assessment of the genome editing effects. Compared with real-time PCR (qPCR) and sequencing, the higher sensitivity and accuracy of dPCR indicates a great advantage in the detection of rare mutation. As a new technique, dPCR has some limitations, such as the necessity of highly allele-specific probes and a large sample volume. In this review, we summarize the application of dPCR in the detection of human disease-associated gene mutations.

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Section: Prenatal Diagnosis Of Genetic Diseasementioning

confidence: 99%

Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation

Yu¹,

Shen²,

Jiang³

et al. 2017

Cell Physiol Biochem

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“…At present, the conventional methods to detect large deletional mutation in α-globin cluster include Multiplex ligation-dependent probe amplification (MLPA) [ 6 ], gap-Polymerase Chain Reaction (gap-PCR) [ 7 ] and droplet digital polymerase chain reaction (ddPCR) [ 7 – 9 ]. However, these methods have several limitations.…”

Section: Introductionmentioning

confidence: 99%

Identification of four novel large deletions and complex variants in the α-globin locus in Chinese population

et al. 2023

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Background At present, the methods generally used to detect α-thalassemia mutations are confined to detecting common mutations, which may lead to misdiagnosis or missed diagnosis. The single-molecule real-time (SMRT) sequencing enables long-read single-molecule sequencing with high detection accuracy, and long-length DNA chain reads in high-fidelity read mode. This study aimed to identify novel large deletions and complex variants in the α-globin locus in Chinese population. Methods We used SMRT sequencing to detect rare and complex variants in the α-globin locus in four individuals whose hematological data indicated microcytic hypochromic anemia. However, the conventional thalassemia detection result was negative. Multiplex ligation-dependent probe amplification and droplet digital polymerase chain reaction were used to confirm SMRT sequencing results. Results Four novel large deletions were observed ranging from 23 to 81 kb in the α-globin locus. One patient also had a duplication of upstream of HBZ in the deletional region, while another, with a 27.31-kb deletion on chromosome 16 (hg 38), had abnormal hemoglobin Siriraj (Hb Siriraj). Conclusion We first identified the four novel deletions in the α-globin locus using SMRT sequencing. Considering that the conventional methods might lead to misdiagnosis or missed diagnosis, SMRT sequencing proved to be an excellent method to discover rare and complex variants in thalassemia, especially in prenatal diagnosis.

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“…At present, the conventional methods to detect large deletional mutation in α-globin cluster include MLPA [6] , gap-Polymerase Chain Reaction (gap-PCR) [7] and ddPCR [7][8][9] . However, these methods have certain limitations.…”

Section: Introductionmentioning

confidence: 99%

Four novel large deletions and complex variants were identified in α-globin locus in Chinese population

Bao

Wang

Qin

et al. 2022

Preprint

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Background: At present, the usual methods used to detect a-thalassemia mutations are confined to detect the common mutations, which may easy to result in misdiagnosis or missed diagnosis. Single molecule real time sequencing (SMRT) enables long-read single-molecule sequencing with high detection accuracy, and long-length DNA chain reads in high-fidelity reads mode. Methods: Herein, we used SMRT to detect rare and complex variants in a-globin locus in four individuals whose hematological data indicated microcytic hypochromic anemia but conventional thalassemia detecting result was negative. Multiplex ligation-dependent probe amplification (MLPA) and droplet digital polymerase chain reaction (ddPCR) were used to confirm the results of SMRT. Results: We found 4 novel large deletions ranging from 23 kb to 81 kb in a-globin locus, among which one patient also has a duplication inserted in the deletional fragment. In addition, one patient with 27.31 kb deletion on chromosome 16 (hg 38) was also detected to be abnormal hemoglobin Siriraj (Hb Siriraj). Conclusion: We first identified the four novel deletions in a-globin locus by using SMRT. Given that conventional methods may lead to misdiagnosis or missed diagnosis, SMRT served as a good method to discover rare and complex variants in thalassemia, especially in prenatal diagnosis.

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Rapid detection of α‐thalassaemia variants using droplet digital PCR

Abstract: In conclusion, ddPCR is an alternative method for rapid detection of alpha thalassaemia variants in Malaysia.

Cited by 7 publications

References 22 publications

Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation

Application of Digital PCR in Detecting Human Diseases Associated Gene Mutation

Identification of four novel large deletions and complex variants in the α-globin locus in Chinese population

Four novel large deletions and complex variants were identified in α-globin locus in Chinese population

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