&lt;p&gt;Establishment and Evaluation of a Novel Method Based on Loop-Mediated Isothermal Amplification for the Rapid Diagnosis of Thalassemia Genes&lt;/p&gt;

Lin, Min; Li, Hailiang; Huang, Junyun; Chen, Jiangtao; Fang, Xiansong; Huang, Dongmei; Xi, Xuxiang; Zhao, Qian; Song, Fangli; Huang, S.; Zhong, Tianyu

doi:10.2147/rmhp.s241399

Cited by 13 publications

(5 citation statements)

References 20 publications

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“…We were unable to isolate and sequence DNA fragments from these samples, but hypothesize that amplification in negative controls may result from contamination or amplification of eDNA from other sources. The incidence of false positives in LAMP studies has been previously documented in disease prevalence studies including that of malaria and thalassemia (Kollenda et al, 2018;Wang et al, 2020). The addition of quenched fluorescent primers to LAMP has been suggested to reduce the number of false positives (Hardinge & Murray, 2019).…”

Section: Discussionmentioning

confidence: 99%

Surveillance of a federally protected freshwater fish using loop-mediated isothermal amplification (LAMP) and eDNA

Fast

Popp

Neil

et al. 2020

Preprint

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Environmental DNA (eDNA) has increasingly been used in the surveillance of imperiled aquatic species. Despite recent efforts in drawing genetic material from the environment, there are still pitfalls surrounding this field. We created a novel protocol which implements loop-mediated isothermal amplification (LAMP) to detect target DNA. Our methods are applied here in the surveillance of Etheostoma trisella, the Trispot Darter, a freshwater fish that recently received protection under the U.S. Endangered Species Act. Water samples (n = 256) were collected at sites in Alabama and Georgia to determine whether E. trisella still occupies historic sites and whether it inhabits previously unknown areas. We found evidence of E. trisella presence in 69 water samples while 187 were negative. Our LAMP protocol is capable of amplifying low quantities of DNA in the water, and is a robust technique for freshwater species surveillance. Verification of positive results from eDNA experiments is essential to confirm reaction reliability. Application of methods such as ours are necessary for recognizing species under threat that require conservation.Surveillance of a federally protected freshwater fish using loop-mediated isothermal amplification (LAMP) and eDNA

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Section: Discussionmentioning

confidence: 99%

Surveillance of a federally protected freshwater fish using loop-mediated isothermal amplification (LAMP) and eDNA

Fast

Popp

Neil

et al. 2020

Preprint

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“…Diagnosis of the SEA deletion allele by LAMP has been reported using pH-sensitive dyes or fluorescence dyes 18 , 20 , 25 . Recently, methods for detecting the THAI deletion by LAMP using pH-sensitive dyes was reported 20 .…”

Section: Discussionmentioning

confidence: 99%

Diagnosis of α-thalassaemia by colorimetric gap loop mediated isothermal amplification

Chumworathayee

Munkongdee

Buasuwan

et al. 2023

Sci Rep

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Abstractα-Thalassaemia is an inherited haemoglobin disorder that results from the defective synthesis of α-globin protein. Couples whom both carry the α-thalassaemia 1 gene are at risk of having a foetus with the most severe thalassaemia, Hb Bart’s hydrops fetalis, with a risk of maternal mortality. However, haematological parameters alone cannot distinguish between a α-thalassaemia 1 carrier and a homozygous α-thalassaemia 2, in which one α-globin gene has been deleted on each chromosome. A rapid and accurate molecular detection assay is essential for prevention of the disease in populations where α-thalassaemia 1 is common. Multiplex Gap-PCR analysis is widely used for diagnosis of α-thalassaemia. However, the technique requires a thermocycler and post-amplification processing, which limits its application in primary care or in rural areas in developing countries. Loop mediated isothermal amplification (LAMP) amplifies target DNA at a constant temperature and does not require a thermocycler. This study developed a colorimetric Gap-LAMP using malachite green to allow naked eye visualization of two deletional α-thalassaemia 1 commonly found in Asian populations, the Southeast Asian type (--SEA) and the Thai type (--THAI) deletions. The Gap-LAMP was performed on DNA samples from 410 individuals carrying various α-thalassaemia gene defects with 100% concordance with conventional Gap-PCR analysis. This method eliminates post-amplification processing or the use of expensive sophisticated equipment and allows screening large populations for the prevention and control of α-thalassaemia.

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“…In addition, it seems that the change of color from pink to orange is quite challenging to distinguish between negative and positive results [ 28 ]. In another study by Wang et al [ 29 ], the LAMP assay has been developed using fluorescence color development under ultraviolet (UV) illumination. The assay was performed on isothermal at 56 °C, 45 minutes and tested on 400 samples including–SEA, -α3.7, and -α4.2, and five β-thalassemia mutations including 654M, 41/42M, −28M, 17M, and 27/28M.…”

Section: Discussionmentioning

confidence: 99%

Loop-mediated isothermal amplification (LAMP) colorimetric phenol red assay for rapid identification of α0-thalassemia: Application to population screening and prenatal diagnosis

et al. 2022

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Background Identification of α0-thalassemia (SEA and THAI deletions) is essential in preventing and controlling of severe thalassemia diseases. We have developed the LAMP colorimetric assays for the detection of these two thalassemia defects and validated them in population screening and prenatal diagnosis. Methods Three LAMP colorimetric assays specific for α0-thalassemia (SEA deletion), α0-thalassemia (THAI deletion) and normal DNA sequence were developed. These assays were validated on 341 subjects who had initial thalassemia screening positive and various thalassemia genotypes. Prenatal diagnosis of α0-thalassemia (SEA deletion) was done on 33 fetuses at risk of having Hb Bart’s hydrops fetalis syndrome. Results The LAMP colorimetric assays for α0-thalassemia (SEA and THAI deletions) could be clearly interpreted by naked eyes. The assay for α0-thalassemia (SEA deletion) showed a 100% (62/62 x 100) sensitivity and 98.2% (274/279 x 100) specificity whereas, that of the α0-thalassemia (THAI deletion) showed 100% (1/1 x 100) sensitivity and 99.7% (339/340 x 100) specificity. We obtained a 100% concordant prenatal diagnosis results using LAMP assays of α0-thalassemia (SEA deletion) in 33 fetuses as compared to the conventional PCR analysis. Conclusions The LAMP colorimetric assays developed are simple, rapid, and do not require sophisticated equipment. Inclusion of the LAMP tests in the existing screening protocol significantly reduce the screening cost and the molecular analysis workload, which should prove useful in the prevention and control program of hemoglobinopathies in the region.

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

Cited by 13 publications

References 20 publications

Surveillance of a federally protected freshwater fish using loop-mediated isothermal amplification (LAMP) and eDNA

Surveillance of a federally protected freshwater fish using loop-mediated isothermal amplification (LAMP) and eDNA

Diagnosis of α-thalassaemia by colorimetric gap loop mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) colorimetric phenol red assay for rapid identification of α0-thalassemia: Application to population screening and prenatal diagnosis

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