Development of Visual Detection of α-Thalassemia-1 (the – –SEA Deletion) Using pH-Sensitive Loop-Mediated Isothermal Amplification

Chomean, Sirinart; Pholyiam, Kanokporn; Thamwarokun, Areenuch; Kaset, Chollanot

doi:10.1080/03630269.2018.1488723

Cited by 13 publications

(9 citation statements)

References 27 publications

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“…10 A previous study detected the -SEA (Southeast Asian) deletion, and IVSII-I G>A mutation indicating thalassemia using LAMP assays. 11,12 Thus, new LAMP methods capable of detecting thalassemia could be alternative methods to other techniques, and could be used in primary hospitals and rural areas.…”

Section: Introductionmentioning

confidence: 99%

Establishment and Evaluation of a Novel Method Based on Loop-Mediated Isothermal Amplification for the Rapid Diagnosis of Thalassemia Genes

Lin¹,

Li²,

Huang³

et al. 2020

RMHP

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Currently, thalassemia is commonly detected using gap-polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) reverse dot blot, which have high requirements of space, instruments, and personnel. Therefore, it is necessary to develop a new method for thalassemia detection with high sensitivity, low cost, and simple and fast operation. In this study, we aimed to design and evaluate a new method for detecting three α-thalassemia genes including-Southeast Asian (SEA),-α3.7, and-α4.2 and five β-thalassemia genes including 654M, 41/42M, −28M, 17M, and 27/28M based on loop-mediated isothermal amplification (LAMP). Methods: Primer sequences were designed using Primer Explorer V4 software. Blood samples (5 mL) were collected from all participants in EDTA. DNA was extracted using Chelex 100 and was subjected to LAMP. LAMP products were detected by fluorescence development in ultraviolet light. Results: We found that LAMP assays for positive samples of thalassemia reached a plateau before 60 minutes, whereas the negative control samples entered the plateau after 70 minutes or showed no amplification. The concentration range of positive reactions was between 20-60 pg/μL and 20-60 ng/μL. Additionally, there were no cross-reactivities among 8 thalassemia subtypes. For clinical samples, the positive sample tube showed strong green fluorescence, whereas the negative tube showed light green fluorescence. According to these results, the LAMP method has high sensitivity for detecting thalassemia (252/254). However, 43 false-positive results were obtained in the LAMP test. The LAMP assay was also of low cost and with simple and fast operation. Conclusion: The novel LAMP assay can be completed within 60 min using a heating block or a water bath, and the result can be read visually based on color change to detect thalassemia. The LAMP assay fulfills the requirements of field application and resourcelimited areas, especially those with primary hospitals and rural areas.

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Section: Introductionmentioning

confidence: 99%

Establishment and Evaluation of a Novel Method Based on Loop-Mediated Isothermal Amplification for the Rapid Diagnosis of Thalassemia Genes

Lin¹,

Li²,

Huang³

et al. 2020

RMHP

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“…This association showed high specificity in detecting abnormal α-globin genes. Moreover, there is no cross-reactivity with α 4.2 and α 3.7 deletion 50 .…”

Section: Loop-mediated Isothermal Amplificationmentioning

confidence: 90%

“…Loop-mediated isothermal amplification (LAMP) is a novel method for nucleic acid amplification in molecular diagnostics as it is highly sensitive, specific, rapid, accurate, and cost-effective 50 . This technique requires four to six primers for six regions on the targeted gene, and the primer sets are specific to the target areas 51 .…”

Section: Loop-mediated Isothermal Amplificationmentioning

confidence: 99%

Molecular Detection of alpha Thalassemia: A Review of Prevalent Techniques

Vijian¹,

Rahman²,

Kannan³

et al. 2021

MMJ

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Alpha thalassemia (α-thalassemia) is an autosomal recessive disorder due to the reduction or absence of α globin chain production. Laboratory diagnosis of α-thalassemia requires molecular analysis for the confirmatory diagnosis. A screening test, comprising complete blood count, blood smear and hemoglobin quantification by high performance liquid chromatography and capillary electrophoresis, may not possibly detect all the thalassemia diseases. This review focused on the molecular techniques used to detect α-thalassemia, and the advantages and disadvantages of each technique were highlighted. Multiplex gap-polymerase chain reaction, single-tube multiplex polymerase chain reaction, multiplex ligation-dependent probe amplification, and loop-mediated isothermal amplification were used to detect common deletion of α-thalassemia. Furthermore, the reverse dot blot analysis and a single tube multiplex polymerase chain reaction could detect non-deletion mutation of the α-globin gene. Sanger sequencing is widely used to detect non-deletion mutations of α-thalassemia. Recently, next-generation sequencing was introduced in the diagnosis of both deletion and point mutations of α-thalassemia. Despite the advantages and disadvantages of different techniques, the routine method employed in the laboratory should be based on the facility, expertise, available equipment, and economic conditions.

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“…We successfully designed two sets of LAMP primers, whereas the previous study only reported one set of primer used in the detection of two α genes deletion --SEA type [11]. The similarities in the sequence of current primers (F3D and B3D) with the previously reported primer [11] may be due to its design using the same web-based free software, however, different DNA templates were used.…”

Section: Discussionmentioning

confidence: 99%

“…LAMP meets all of the criteria for an ideal diagnostic test because of its sensitivity, specificity, low cost, simplicity, speed, adaptability to all kinds of climatic changes, and the availability of instruments used, as suggested by the World Health Organization [10] in a report of their findings. The previous study reported the application of LAMP for the detection of α 0 thalassemia carrier --SEA type using different intercalating dyes such as pH-sensitive dyes [11]. Based on the advantage of using HNB as a DNA intercalating dye addition before amplification and colorimetric using naked eye detection [12], we tried to develop the simplest method, HNB-LAMP, for the detection of the α 0 thalassemia carrier --SEA type.…”

Section: Introductionmentioning

confidence: 99%

Naked Eye Detection of α0 Thalassemia --SEA Type using Hydroxynaphthol Blue Loop-mediated Isothermal Amplification

Phraephan

Watanasatitarpa

Intorasoot

2020

Open Access Maced J Med Sci

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BACKGROUND: Alpha-thalassemia is a major health problem for worldwide populations due to its recent large-scale global movements to many other parts of the world. The most effective method to prevent the spread of the worst form of α-thalassemia disorder, hemoglobin Bart’s hydrops fetalis, is to identify the genotypes of an α0 thalassemia carrier and then conduct subsequent genetic counseling. AIM: This research was aimed to develop a new method for the detection of α0 thalassemia (Southeast Asia or SEA) --SEA type using hydroxynaphthol blue loop-mediated isothermal amplification (HNB-LAMP) and by visually reading results using the naked eye. METHODS: This experimental research was conducted at Rangsit University, Pathum Thani Provinces, Thailand, from 2016 to 2018. Two sets of LAMP primers were used, one set of primer was used to detect the two existing α genes and the other set used in the detection of two α genes deletion --SEA types which were designed based on the nucleotide sequence of two α genes deletion from four cases of the α0 thalassemia carrier --SEA type. HNB-LAMP was developed, and then, 77 cases from subjects were evaluated by comparing them with a conventional polymerase chain reaction (PCR). RESULTS: The newly developed HNB-LAMP shows a high diagnostic sensitivity and diagnostic specificity, 100% in correct interpretation, from 10 cases of α0 thalassemia carriers --SEA type, 29 cases of α+ thalassemia carrier -α3.7 type, 8 cases of hemoglobin constant spring/hemoglobin Pakse′ (HbCS/HbPS), and 30 standard cases. CONCLUSION: This newly developed HNB-LAMP is suitable for in the field applications for detecting the presence of α0 thalassemia --SEA type due to its high diagnostic sensitivity, high diagnostic specificity, and, most notably, the ability to be detected with the naked eye.

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Development of Visual Detection of α-Thalassemia-1 (the – –^SEA Deletion) Using pH-Sensitive Loop-Mediated Isothermal Amplification

Cited by 13 publications

References 27 publications

<p>Establishment and Evaluation of a Novel Method Based on Loop-Mediated Isothermal Amplification for the Rapid Diagnosis of Thalassemia Genes</p>

<p>Establishment and Evaluation of a Novel Method Based on Loop-Mediated Isothermal Amplification for the Rapid Diagnosis of Thalassemia Genes</p>

Molecular Detection of alpha Thalassemia: A Review of Prevalent Techniques

Naked Eye Detection of α0 Thalassemia --SEA Type using Hydroxynaphthol Blue Loop-mediated Isothermal Amplification

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