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

α-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.

www.nature.com/scientificreports/ SEA). In addition, another type of α-thalassaemia 1, the Thai type deletion (--THAI , THAI), has also been reported in Southern China and Thailand 3,5,8 . Conventional screening for α-thalassaemia trait is based on an assessment of complete blood count (CBC), red cell morphology, measurement of haemoglobin levels, and haemoglobin type classification. While Hb typing can be used for α-thalassaemia disease diagnosis, it cannot diagnose α-thalassaemia trait as the result is the same as those of normal individuals. The α-thalassaemia 1 trait shows circulating red blood cells as hypochromic microcytes, with haematological parameters similar to homozygous α-thalassaemia 2, in which only one α-globin gene deleted on each chromosome. Therefore, only DNA-based methods can be used to solve this diagnosis limitation. A widely used technique for deletional α-thalassaemia diagnosis is multiplex Gap-PCR analysis which is based on primers designed to amplify DNA across the breakpoint region of the deleted gene 12 . Although the method gives cost effective and unequivocal results, this technique requires a thermocycler and a post-amplification process. The use of expensive and specialized equipment limits its application in primary care or in rural areas in developing countries. Loop mediated isothermal amplification (LAMP) amplifies target DNA by employing a strand displacement Bst DNA polymerase and four primers that recognize six distinct sequences of the target DNA for increased specificity 13 . The amplification reaction occurs at 60-65°C, allowing the use of a simple water bath or heat block. The LAMP technique has been widely used to diagnose a wide range of infectious diseases 14,15 , pharmacogenetic and disease risk alleles 16,17 and genetic diseases [18][19][20] . In addition, the amplification products can be visualization using pH-sensitive dyes 21 , a fluorescent metal indicator 22 , fluorescent and non-fluorescent DNA intercalating dye 23,24 .
In order to cover the large populations of Southern China and Southeast Asia for screening couples at risk of having Hb Bart's hydrops fetalis, accurate screening in primary care in rural areas without sophisticated equipment is needed. Herein, we combine the features of Gap-PCR and LAMP for a rapid and reliable diagnosis of α-thalassaemia 1 based on a colorimetric Gap-LAMP technique using the DNA intercalating dye malachite green, which allows for naked eye visualization. This technique has high sensitivity, specificity, accuracy, a rapid turnaround time and does not require specialized equipment. This newly developed method could be helpful for high-throughput screening in primary care facilities and aid in the prevention and control of the disease.

Results
Detection of deletional α-thalassaemia 1 gene by Gap-LAMP. The LAMP reactions were first optimized and assessed on genomic DNA from individuals carrying known α-thalassaemia 1 genes. Three specific primer sets were used for amplification of α-thalassaemia 1 (SEA and THAI) and the normal gene (ψα2-globin gene) ( Fig. 1 and Table 1). The Gap-LAMP primers had high specificity to distinguish between normal and the two deletional α-thalassaemia 1 (Fig. 2). With the normal primer set, a blue color solution indicating a positive reaction was observed after amplification of genomic DNA from a normal subject (αα/αα), SEA trait (--SEA /αα) and THAI trait (--THAI /αα), while the solution turned colorless (no amplification) when Hb Bart's hydrops fetalis DNA (--SEA /--SEA ) was used as the genomic material ( Fig. 2A). Importantly, the SEA primer set could amplify only SEA trait and Hb Bart's hydrops fetalis as shown in Fig. 2B. In addition, the THAI primer set could amplify only THAI trait while negative reaction in normal subject, SEA trait and Hb Bart's hydrops fetalis (--SEA /--SEA ) (Fig. 2C). In agreement with the colorimetric visualization, gel electrophoresis confirmed the specificity of the three primer sets ( Fig. 2A-C, lower panel, respectively). Moreover, the normal primers, SEA primers and THAI primers show a broad range of amplification temperatures from 61 to 65°C (Fig. S1), suggesting that a regular water bath could be used. The limit of detection of normal, SEA and THAI primer sets as assessed by serial dilution of known DNA samples was 1 ng, 10 ng and 1 ng, respectively (Fig. S2).
A total of 410 genomic DNA samples from individuals carrying various α-thalassaemia genes were used for validation of the Gap-LAMP. The results were compared with those obtained from routine conventional multiplex Gap-PCR coupled with gel electrophoresis ( Table 2). The normal primers amplify the ψα2-globin gene from the normal globin allele and α-thalassaemia 2 allele gave a positive blue color signal in 404 samples of normal subjects and various α-thalassaemia 2 interactions. Moreover, there was no DNA amplification from 6 samples of amniotic DNA from HbBart's hydrops fetalis. The SEA primers gave a blue color signal from 142 samples that contained the SEA deletion, SEA trait, HbBart's hydrops fetalis and HbH disease concordant with www.nature.com/scientificreports/

Discussion
α-Thalassaemia is prevalent in southern China and Southeast Asia, and several of these countries have implemented prevention and control programs for severe thalassaemias, including Hb Bart's hydrops fetalis. In order to cover the large populations for screening couples at risk of having an affected child, accurate screening in primary care in a rural area without sophisticated equipment is needed. Herein, we report a development of a colorimetric Gap-LAMP for the visual detection of the two deletional types of α-thalassaemia 1 prevalent in southern China and Southeast Asia. In this study, three sets of Gap-LAMP primers were designed to diagnose two common α-thalassaemia 1, the SEA deletion, and the less common THAI deletion. The method described here has not been designed to detect the -α 3.7 and -α 4.2 deletions of α-thalassaemia 2. Thus, a drawback of our method is that HbH disease could not be distinguished from α-thalassaemia 1 trait. However, those two thalassaemias can be simply discriminated using haematological and clinical data. The Gap-LAMP method developed here is for population screening of the most common α-thalassaemia 1 in the Asian population for prevention and control programs. The Gap-LAMP was validated in individuals carrying various α-thalassaemia genes. A limitation is that there was only a few cases of some rare genotypes due to low frequency in the population such as THAI trait (--THAI /αα) and HbBart's hydrops fetalis (--SEA /--SEA ). Comparison of this new method with the multiplex Gap-PCR and gel electrophoresis-based method showed 100% sensitivity, specificity and accuracy of the three primer sets.
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 . The drawback of the pH-sensitive dyes is the requirement of optimal pH for the best results and the difficulty of distinguishing positive readout, changing the color of phenol red from pink to orange. The limitation of fluorescent dye is the need of develop of fluorescence under illumination by ultraviolet light. This study used malachite green dye, which provides a notable advantage as malachite green is mixed prior to amplification. The results can be observed and determined by naked eye visualization in a closed system without opening the  In summary, the Gap-LAMP using malachite green dye was found to be rapid, sensitive and reliable for screening of two common α-thalassaemia 1 that are prevalent in Asian populations. The technique allows naked eye visualization and is achieved within 60 min using a general water bath or heating block, obviating the need for a thermocycler or post-amplification processing. The technique described in this study could be applied for large-scale diagnosis of α-thalassaemia 1 in primary care facilities and rural areas.