Noninvasive prenatal screening test for compound heterozygous beta thalassemia using an amplification refractory mutation system real-time polymerase chain reaction technique

Suwannakhon, Narutchala; Pangeson, Tanapat; Seeratanachot, Teerapat; Mahingsa, Khwanruedee; Pingyod, Arunee; Bumrungpakdee, Wanwipa; Sanguansermsri, Torpong

doi:10.4081/hr.2019.8124

Cited by 4 publications

(7 citation statements)

References 22 publications

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“…With a normal individual, PCR product will be seen only in the reaction employing the wild-type primer set. A heterozygote will generate a band using both wild-type and mutant primer set, and an individual with homozygous mutation will be negative with the normal and positive with the mutant primer set (Figure 4) (Suwannakhon et al, 2019).…”

Section: Allele-specific Pcrmentioning

confidence: 99%

Update in Laboratory Diagnosis of Thalassemia

Munkongdee

Chen

Winichagoon

et al. 2020

Front. Mol. Biosci.

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Section: Allele-specific Pcrmentioning

confidence: 99%

Update in Laboratory Diagnosis of Thalassemia

Munkongdee

Chen

Winichagoon

et al. 2020

Front. Mol. Biosci.

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“…Several studies described the noninvasive prenatal diagnosis in thalassemia disease [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25]. In case that the parents are carriers of thalassemia with different mutations, the presence of paternal mutation in maternal plasma indicates that the fetus carries the mutation and either is a carrier of the disease or, when also inherits the maternal mutation, has a disease.…”

Section: Introductionmentioning

confidence: 99%

“…In case that the parents are carriers of thalassemia with different mutations, the presence of paternal mutation in maternal plasma indicates that the fetus carries the mutation and either is a carrier of the disease or, when also inherits the maternal mutation, has a disease. Methods for the detection include quantitative real-time polymerase chain reaction (qPCR) [10,13,16,22], PCR and MassARRAY assays [12,15], droplet digital PCR (dPCR) [21,23], and next-generation sequencing (NGS) [18,19,24]. The mass spectrometry has a higher sensitivity when comparing to qPCR and is a preferable method.…”

Section: Introductionmentioning

confidence: 99%

“…The final PCR product will be detected by fluorescence probes, and the absolute count of the rare mutation among wild-type DNAs will be obtained [27,28]. The method has been used in various clinical conditions, such as quantification of chimerism, gene expression in cancers, and noninvasive prenatal diagnosis of thalassemia [21,22,[29][30][31].…”

Section: Introductionmentioning

confidence: 99%

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Noninvasive Prenatal Diagnosis of Beta-Thalassemia Disease by Using Digital PCR Analysis of Cell-Free Fetal DNA in Maternal Plasma

et al. 2022

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Introduction: Prenatal diagnosis of thalassemia disease usually based on invasive technique. Non-invasive diagnosis using cell-free fetal DNA (cff-DNA) were described with various laboratory technique. The aim of this study to identify the performance of dPCR for analyzing cff-DNA in maternal plasma to diagnose fetal beta-thalassemia diseases. Methods: Thirty-five couples at risk of fetal beta-thalassemia disease caused by four common mutations of HBB were enrolled at 12-18 weeks. The dPCR assay was designed to detect and quantify paternally-inherited beta-thalassemia allele (PIB-M) and maternally-inherited beta-thalassemia allele (MIB-M) from cff-DNA in maternal plasma. Results: Of 29 couples with different paternal/maternal mutations, all cases who inherited paternal mutation had detectable PIB-M. The MIB-mutant/wild-type (MIB-M/MIB-N) ratio in the mothers whose fetuses did not inherit maternal mutation was 0.87+/-0.07 which was significantly lower than that of the mothers whose fetuses inherited maternal mutation, 1.01+/-0.05. The sensitivity and specificity of MIB-M/MIB-N ratio >0.95 in predicting fetus inheriting maternal mutation was 100 and 92.3%, respectively. In four couples with same paternal/maternal mutation, IB-M/IB-N ratio of >0.95 correctly predicted the presence of an inheritance of at least one beta-thalassemia allele. In two couples with paternal Hb E/beta-thalassemia, the presence of PIB-M and the MIB-M/MIB-N ratio of >0.95 correctly predicted the presence of paternal/maternal mutations, respectively. Conclusions: The method of analyzing cff-DNA in maternal plasma by dPCR is efficient for prenatal diagnosis of beta-thalassemia.

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“…The genes of genetically modified organisms are searched in food control . Paternally inherited disease-associated sequences of DNA are investigated in the background of cell-free DNA in the plasma of pregnant females, and they are used in noninvasive prenatal diagnostics. , The correct quantification of both mixture components by qPCR is complicated once there are large differences in their representation in the mixture.…”

Section: Introductionmentioning

confidence: 99%

Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel

et al. 2021

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Real-time (quantitative) polymerase chain reaction (qPCR) has been widely applied in molecular diagnostics due to its immense sensitivity and specificity. qPCR multiplexing, based either on fluorescent probes or intercalating dyes, greatly expanded PCR capability due to the concurrent amplification of several deoxyribonucleic acid sequences. However, probe-based multiplexing requires multiple fluorescent channels, while intercalating dye-based multiplexing needs primers to be designed for amplicons having different melting temperatures. Here, we report a single fluorescent channel-based qPCR duplexing method on a model containing the sequence of chromosomes 21 (Chr21) and 18 (Chr18). We combined nonspecific intercalating dye EvaGreen with a 6-carboxyfluorescein (FAM) probe specific to either Chr21 or Chr18. The copy number ( cn ) of the target linked to the FAM probe could be determined in the entire tested range from the denaturation curve, while the cn of the other one was determined from the difference between the denaturation and elongation curves. We recorded the amplitude of fluorescence at the end of denaturation and elongation steps, thus getting statistical data set to determine the limit of the proposed method in detail in terms of detectable concentration ratios of both targets. The proposed method eliminated the fluorescence overspilling that happened in probe-based qPCR multiplexing and determined the specificity of the PCR product via melting curve analysis. Additionally, we performed and verified our method using a commercial thermal cycler instead of a self-developed system, making it more generally applicable for researchers. This quantitative single-channel duplexing method is an economical substitute for a conventional rather expensive probe-based qPCR requiring different color probes and hardware capable of processing these fluorescent signals.

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Noninvasive prenatal screening test for compound heterozygous beta thalassemia using an amplification refractory mutation system real-time polymerase chain reaction technique

Cited by 4 publications

References 22 publications

Update in Laboratory Diagnosis of Thalassemia

Update in Laboratory Diagnosis of Thalassemia

Noninvasive Prenatal Diagnosis of Beta-Thalassemia Disease by Using Digital PCR Analysis of Cell-Free Fetal DNA in Maternal Plasma

Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel

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