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Isothermal Strand-Displacement Polymerase Reaction for Visual Detection of the Southeast Asian–Type Deletion of α-Thalassemia
Abstract
A rapid and reliable screening test for thalassemia carrier couples is the most effective strategy to decrease the risk of conceiving fetuses with severe thalassemia. We present an approach based on the isothermal strand-displacement polymerase reaction and the use of a lateral flow strip for the visual detection of an α-thalassemia Southeast Asian-type deletion. This assay was used to evaluate 86 clinical samples (72 cases of Southeast Asian-type deletions and 14 cases of other types of thalassemia), and the results obtained were 100% consistent with those obtained using conventional gap-PCR. The approach thus provides a simple, sensitive, rapid, and cost-effective method for the diagnosis of thalassemia genotypes.
... Based on CSDPR and gold nanoparticles (AuNPs), we have developed a lateral low biosensor (LFB) for the visual detection of thalassemia genes [11]. This sensor is highly sensitive (the detection limit is 0.01 fM of nucleic acid), highly speciic, and easy to use. ...
... Isothermal chain-displacement polymerase reaction (ISDPR) is a process that uses an isothermal amplification process to produce a large number of products to amplify the detection signal [18]. Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase that can catalyzes the sequential addition of dNTPs to the 3′-OH group of oligonucleotide primers to produce specific sequences such as poly-T ssDNA [19]. ...
A photoelectrochemical biosensor is described for sensitive detection of microRNA-162a. A multiple amplification strategy is employed that involves (a) isothermal strand displacement polymerase reaction; (b) terminal deoxynucleotidyl transferase-mediated extension, (c) amplification of streptavidin-coated gold nanoparticles, and (d) biotin functionalized alkaline phosphatase. Graphite-like C3N4 (g-C3N4) nanosheets were used as photoactive material. By using these amplification strategies, the detection limit is as low as 0.18 fM of microRNA, and the photocurrent increases linearly with the concentration of microRNA-162a in the range from 0.5 fM to 1 pM. The method was successfully applied to quantify the expression level of microRNA-162a in total RNA extracted from the leaves of maize seedlings after incubation with the chemical mutagen ethyl methanesulfonate. The results confirmed the applicability of the method to the analysis of practical biological samples.
Graphical AbstractSchematic of a photoelectrochemical microRNA assay based on a multiple amplification strategy involving (a) isothermal strand displacement polymerase reaction; (b) terminal deoxynucleotidyl transferase-mediated extension, (c) amplification of streptavidin-coated gold nanoparticles, and (d) biotin functionalized alkaline phosphatase.
In this work, we demonstrated an ultrasensitive approach with a dual-amplification strategy for DNA assay based on isothermal exponential amplification (EXPAR) and the hybridization chain reaction (HCR). In the presence of target DNA, the hairpin probe DNA (HP1) recognized and partially hybridized with the target DNA to form double-stranded structures containing the full recognition sequences for nicking endonuclease and then initiated EXPAR. Under the reaction of EXPAR, a large number of single-stranded DNA (ssDNA) was produced in the circle of nicking, polymerization, and strand displacement. The resulting ssDNA can bind to the surface-bound probe on the well of the microplate and trigger the hybridization chain reaction, resulting in the production of numerous double-stranded DNA concatamers with biotin labeling. In the presence of streptavidin-conjugated horseradish peroxidase (HRP), the amplified signal can be detected by a spectrophotometer via HRP-catalyzed substrate 3,3'5,5'-tetramethylbenzidine (TMB). This proposed dual-amplification method provides a detection limit of 74.48 aM, which also exhibits good linearity ranging from 0.1 fM to 100 pM.
In recent years, many nucleic acid-based lateral flow assays (NALFAs) have been developed for rapid and simple detection of various analytes including DNA sequences. In a NALFA, target molecules are applied within a small volume of a rehydrating buffer. The analyte flows laterally to reach the capture molecules at where it forms a colorimetric signal. Usually, in NALFAs, capture molecules are modified for maximized adsorption on the surface. In most cases, the modification is a biotin. The biotinylated capture DNA is held at capture line by interaction with streptavidin. However, there is a demand on methods that permit utilizing unmodified capture molecules and allow a cost-effective development process. Here, we report on a biotin- and streptavidin-free model NALFA. We also present a systematic investigation on the effect of various rehydrating buffers’ composition and concentration. In addition, the impacts of a protein blocker, detergents and chaotropic and kosmotropic agents on the intensity of the signal over background were analyzed. It has been demonstrated that simultaneous presence of sodium dodecyl sulfate and bovine serum albumin doubles the intensity of visible bands in the presented unmodified NALFA. Finally, this paper presents an optimized cost-effective model system that can be adapted for hybridization-based NALFAs.
Searching for a strategy to enhance the efficiency of nucleic acid amplification and achieve exquisite discrimination of nucleic acids at the single-base level for biological detection has become an exciting research direction in recent years. Here, we have developed a simple and universal primer design strategy which produces a fascinating effect on isothermal strand displacement amplification (iSDA). We refer to the resultant primer as “invading stacking primer (IS-Primer)” which is based on contiguous stacking hybridization and toehold-mediated exchange reaction and function by merely changing the hybridization location of the primer. Using the IS-Primer, the sensitivity in detecting the target miR-21 is improved approximately five fold compared with the traditional iSDA reaction. It was further demonstrated that the IS-Primer acts as an invading strand to initiate branch migration which can increase the efficiency of the untwisting of the hairpin probe. This effect is equivalent to reducing the free energy of the stem, and the technique shows superior selectivity for single-base mismatches. By demonstrating the enhanced effect of the IS-Primer in the iSDA reaction, this work may provide a potentially new avenue for developing more sensitive and selective nucleic acids assays.
Reliable detection of large deletions from cell-free fetal DNA (cffDNA) in maternal plasma is challenging, especially when both parents have the same deletion owing to a lack of specific markers for fetal genotyping. In order to evaluate the efficacy of a non-invasive prenatal diagnosis (NIPD) test to exclude α-thalassemia major that uses SNPs linked to the normal paternal α-globin allele, we established a novel protocol to reliably detect paternal SNPs within the (--(SEA)) breakpoints and performed evaluation of the diagnostic potential of the protocol in a total of 67 pregnancies, in whom plasma samples were collected prior to invasive obstetrics procedures in southern China. A group of nine SNPs identified within the deletion breakpoints were scanned to select the informative SNPs in each of the 67 couples DNA by multiplex PCR based mini-sequencing technique. The paternally inherited SNP allele from cffDNA was detected by allele specific real-time PCR. A protocol for reliable detection of paternal SNPs within the (--(SEA)) breakpoints was established and evaluation of the diagnostic potential of the protocol was performed in a total of 67 pregnancies. In 97% of the couples one or more different SNPs within the deletion breakpoint occurred between paternal and maternal alleles. Homozygosity for the (--(SEA)) deletion was accurately excluded in 33 out of 67 (49.3%, 95% CI, 25.4-78.6%) pregnancies through the implementation of the protocol. Protocol was completely concordant with the traditional reference methods, except for two cases that exhibited uncertain results due to sample hemolysis. This method could be used as a routine NIPD test to exclude gross fetal deletions in α-thalassemia major, and could further be employed to test for other diseases due to gene deletion.
Prevention and control of thalassemia requires simple, rapid, and accurate screening tests for carrier couples who are at risk of conceiving fetuses with severe thalassemia.
Single-tube multiplex real-time PCR with SYBR Green1 and high-resolution melting (HRM) analysis were used for the identification of α-thalassemia-1 Southeast Asian (SEA) and Thai type deletions and β-thalassemia 3.5-kb gene deletion. The results were compared with those obtained using conventional gap-PCR. DNA samples were derived from 28 normal individuals, 11 individuals with α-thalassemia-1 SEA type deletion, 2 with α-thalassemia-1 Thai type deletion, and 2 with heterozygous β-thalassemia 3.5-kb gene deletion.
HRM analysis indicated that the amplified fragments from α-thalassemia-1 SEA type deletion, α-thalassemia-1 Thai type deletion, β-thalassemia 3.5-kb gene deletion, and the wild-type β-globin gene had specific peak heights at mean melting temperature (T(m)) values of 86.89℃, 85.66℃, 77.24℃, and 74.92℃, respectively. The results obtained using single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis showed 100% consistency with those obtained using conventional gap-PCR.
Single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis is a potential alternative for routine clinical screening of the common types of α- and β-thalassemia large gene deletions, since it is simple, cost-effective, and highly accurate.
Alpha-thalassaemia is inherited as an autosomal recessive disorder characterised by a microcytic hypochromic anaemia, and a clinical phenotype varying from almost asymptomatic to a lethal haemolytic anaemia.
It is probably the most common monogenic gene disorder in the world and is especially frequent in Mediterranean countries, South-East Asia, Africa, the Middle East and in the Indian subcontinent. During the last few decades the incidence of alpha thalassaemia in North-European countries and Northern America has increased because of demographic changes. Compound heterozygotes and some homozygotes have a moderate to severe form of alpha thalassaemia called HbH disease. Hb Bart's hydrops foetalis is a lethal form in which no alpha-globin is synthesized. Alpha thalassaemia most frequently results from deletion of one or both alpha genes from the chromosome and can be classified according to its genotype/phenotype correlation. The normal complement of four functional alpha-globin genes may be decreased by 1, 2, 3 or all 4 copies of the genes, explaining the clinical variation and increasing severity of the disease. All affected individuals have a variable degree of anaemia (low Hb), reduced mean corpuscular haemoglobin (MCH/pg), reduced mean corpuscular volume (MCV/fl) and a normal/slightly reduced level of HbA2. Molecular analysis is usually required to confirm the haematological observations (especially in silent alpha-thalassaemia and alpha-thalassaemia trait). The predominant features in HbH disease are anaemia with variable amounts of HbH (0.8-40%). The type of mutation influences the clinical severity of HbH disease. The distinguishing features of the haemoglobin Bart's hydrops foetalis syndrome are the presence of Hb Bart's and the total absence of HbF. The mode of transmission of alpha thalassaemia is autosomal recessive. Genetic counselling is offered to couples at risk for HbH disease or haemoglobin Bart's Hydrops Foetalis Syndrome. Carriers of alpha+- or alpha0-thalassaemia alleles generally do not need treatment. HbH patients may require intermittent transfusion therapy especially during intercurrent illness. Most pregnancies in which the foetus is known to have the haemoglobin Bart's hydrops foetalis syndrome are terminated due to the increased risk of both maternal and foetal morbidity.
Here we have developed a sensitive DNA amplified detection method based on isothermal strand-displacement polymerization reaction.
This method takes advantage of both the hybridization property of DNA and the strand-displacement property of polymerase.
Importantly, we demonstrate that our method produces a circular polymerization reaction activated by the target, which essentially
allows it to self-detect. Functionally, this DNA system consists of a hairpin fluorescence probe, a short primer and polymerase.
Upon recognition and hybridization with the target ssDNA, the stem of the hairpin probe is opened, after which the opened
probe anneals with the primer and triggers the polymerization reaction. During this process of the polymerization reaction,
a complementary DNA is synthesized and the hybridized target is displaced. Finally, the displaced target recognizes and hybridizes
with another probe, triggering the next round of polymerization reaction, reaching a target detection limit of 6.4 × 10−15 M.
The large number of naturally occurring mutants of this well characterized locus provide an excellent opportunity for elucidating the relationship between its structure and function. Comparisons of what has been learned about the α-globin locus with complementary observations on the β-globin locus, provide a strategy for understanding the coordinate regulation of eukaryotic gene expression. From a practical point of view it is important to remember that millions of individuals throughout the world are carriers of α-thalassemia and every year many thousands of pregnancies are at risk of producing children with the severe α-thalassemia syndromes. The data summarized here provide the basis for accurately predicting the genotype in such cases and thus enabling appropriate prenatal testing. However, because this is a genetic disease that predominantly affects individuals from countries with limited health resources, simpler and cheaper methods of screening and diagnosis will have to be developed before this information has a significant impact on the attendant morbidity and mortality.
To demonstrate a method for using genetic epidemiological data to assess the needs for equitable and cost-effective services for the treatment and prevention of haemoglobin disorders. We obtained data on demographics and prevalence of gene variants responsible for haemoglobin disorders from online databases, reference resources, and published articles. A global epidemiological database for haemoglobin disorders by country was established, including five practical service indicators to express the needs for care (indicator 1) and prevention (indicators 2-5). Haemoglobin disorders present a significant health problem in 71% of 229 countries, and these 71% of countries include 89% of all births worldwide. Over 330,000 affected infants are born annually (83% sickle cell disorders, 17% thalassaemias). Haemoglobin disorders account for about 3.4% of deaths in children less than 5 years of age. Globally, around 7% of pregnant women carry b or a zero thalassaemia, or haemoglobin S, C, D Punjab or E, and over 1% of couples are at risk. Carriers and at-risk couples should be informed of their risk and the options for reducing it. Screening for haemoglobin disorders should form part of basic health services in most countries.
A simple, highly sensitive, and selective multiple microRNA (miRNA) detection method based on the graphene oxide (GO) fluorescence quenching and isothermal strand-displacement polymerase reaction (ISDPR) was proposed. The capability to discriminate ssDNA and double-stranded nucleic acid structure coupled with the extraordinary fluorescence quenching of GO on multiple organic dye allows the proposed strategy to simultaneously and selectively detect several miRNA labeled with different dyes in the same solution, while the ISDPR amplification endows the detection method with high sensitivity. The strong interaction between ssDNA and GO led to the fluorescent ssDNA probe exhibiting minimal background fluorescence. Upon the recognition of specific target miRNA, an ISDPR was triggered to produce numerous massive specific DNA-miRNA duplex helixes, and a strong emission was observed due to the weak interaction between the DNA-miRNA duplex helix and GO. A miRNA biosensor down to 2.1 fM with a linear range of 4 orders of magnitude was obtained. Furthermore, the large planar surface of GO allows simultaneous quenching of several DNA probes with different dyes and produces a multiple biosensing platform with high sensitivity and selectivity, which has promising application in profiling the pattern of miRNA expression and biomedical research.
A highly sensitive electrochemical DNA sensor that requires no probe immobilization has been developed based on a target recycling mechanism utilizing a DNA polymerase with a strand displacement activity. The electrochemical detection is realized by taking advantage of the difference in diffusivity between a free ferrocene-labeled peptide nucleic acid (Fc-PNA) and a Fc-PNA hybridized with a complementary DNA, while the DNA polymerase-assisted target recycling leads to signal generation and amplification. The hybridization of the target DNA opens up a stem-loop template DNA with the Fc-PNA hybridized to its extruded 5' end and allows a DNA primer to anneal and be extended by the DNA polymerase, which results in sequential displacement of the target DNA and the Fc-PNA from the template DNA. The displaced target DNA will hybridize with another template DNA, triggering another round of primer extension and strand displacement. The released Fc-PNA, due to its neutral backbone, has much higher diffusivity towards a negatively charged electrode, compared to that when it is hybridized with a negatively charged DNA. Therefore, a significantly enhanced signal of Fc can be observed. The outstanding sensitivity and simplicity make this approach a promising candidate for next-generation electrochemical DNA sensing technologies.
A large deletional α-thalassemia-2 (α-thal-2) allele was identified in a Thai woman with Hb H disease. The proband has α-thal-1 (SEA type) in conjunction with a 16.6 kb deletion affecting the α2-globin allele. The proband had severe anemia and required a blood transfusion during puerperium.
Here, we describe a simple and sensitive approach for visual detection of gene mutations based on isothermal strand-displacement polymerase reactions (ISDPR) and lateral flow strip (LFS). The concept was first demonstrated by detecting the R156H-mutant gene of keratin 10 in Epidermolytic hyperkeratosis (EHK). In the presence of biotin-modified hairpin DNA and digoxin-modified primer, the R156H-mutant DNA triggered the ISDPR to produce numerous digoxin- and biotin-attached duplex DNA products. The product was detected on the LFS through dual immunoreactions (anti-digoxin antibody on the gold nanoparticle (Au-NP) and digoxin on the duplex, anti-biotin antibody on the LFS test zone and biotin on the duplex). The accumulation of Au-NPs produced the characteristic red band, enabling visual detection of the mutant gene without instrumentation. After systematic optimization of the ISDPR experimental conditions and the parameters of the assay, the current approach was capable of detecting as low as 1-fM R156H-mutant DNA within 75 min without instrumentation. Differentiation of R156H- and R156C-mutant DNA on the R156 mutation site was realized by using fluorescein- and biotin-modified hairpin probes in the ISDPR process. The approach thus provides a simple, sensitive, and low-cost tool for the detection of gene mutations.
Preimplantation genetic diagnosis (PGD) is an alternative to prenatal diagnosis (PND) giving couples at risk a chance to start a pregnancy with a disease-free baby. This study aimed to develop a new PGD protocol for alpha-thalassemia(-SEA) mutation, the commonest Mendelian disorder.
Multiplex fluorescent PCR was employed for mutation, contamination and linkage analysis. A couple experienced termination of pregnancy following positive PND decided to join the project.
Novel primers for alpha-thalassemia(-SEA) mutation amplifying 5 DNA fragments were developed. Two PGD cycles were performed, resulting in an un-affected baby. PND confirmed the heterozygous result. From 24 embryos, 87.5% of affected genotype were of best quality compared to 0% and 18.2% of those with normal and heterozygous, respectively.
A novel PCR protocol for the common alpha-thalassemia(-SEA) mutation is reported. This test should be widely applicable. Interestingly, a potential effect of alpha-thalassemia(-SEA) mutation on preimplantation embryonic development was noticed.
Isothermal DNA amplification is an alternative to PCR-based amplification for point-of-care diagnosis. Since the early 1990s, the approach has been refined into a simple, rapid and cost-effective tool by means of several distinct strategies. Input signals have been diversified from DNA to RNA, protein or small organic molecules by translating these signals into input DNA before amplification, thus allowing assays on various classes of biomolecules. In situ detection of single biomolecules has been achieved using an isothermal method, leveraging localized signal amplification in an intact specimen. A few pioneering studies to develop a homogenous isothermal protein assay have successfully translated structure-switching of a probe upon target binding into input DNA for isothermal amplification. In addition to the detection of specific targets, isothermal methods have made whole-genome amplification of single cells possible owing to the unbiased, linear nature of the amplification process as well as the large size of amplified products given by ϕ29 DNA polymerase. These applications have been devised with the four isothermal amplification strategies covered in this review: strand-displacement amplification, rolling circle amplification, helicase-dependent amplification and recombinase polymerase amplification.
Nondeletional hemoglobin (Hb) H disease is caused by a deletion of both alpha-globin genes on one chromosome 16 and of an alpha(+)-thalassemia point mutation on the other chromosome 16. Patients with nondeletional Hb H disease are usually more anemic, more symptomatic, and are often transfused. Counseling and prenatal diagnosis ought to be offered to couples at risk of nondeletional Hb H disease. In this study, we report on the prenatal diagnoses of 2 cases of uncommon nondeletional Hb H disease using the PCR-reverse dot-blot method.
Alpha-thalassemia is the most common cause of hydrops fetalis among Southeast Asians. With the recent influx of Southeast Asian refugees and the rapidly growing Filipino population this will become an increasingly important obstetric problem in the United States. Homozygous alpha-thalassemia, or Bart hemoglobinopathy, is invariably fatal to the fetus and produces significant maternal morbidity. Eighteen cases of homozygous alpha-thalassemia in one hospital are reviewed. This is the largest series reported in the United States. Recommendations are made for antenatal screening, diagnosis, and management of alpha-thalassemia.
In order to provide a noninvasive prenatal diagnosis of alpha(0)-Thalassemia (Southeast Asian [SEA] deletion), we have developed a real-time quantitative semi-nested polymerase chain reaction (PCR) method for identifying the fetal alpha(0)-Thalassemia in maternal plasma. Analysis was performed using DNA extracted from 200 muL plasma from 13 pregnant women during 8-20 weeks of gestation who carried fetuses with normal (2), alpha(0)-Thalassemia carrier (8), Hb H disease (1), and homozygous alpha(0)-Thalassemia (Hb Bart's hydrops fetalis (2). The alpha(0)-Thalassemia was detected using a two-step PCR. Plasma DNA was amplified conventionally using alpha(0)-Thalassemia-specific primers and a portion of the first PCR product was subjected to a semi-nested real-time q-PCR using the SYBR green I chemistry for fluorescence detection. Calibration curve for alpha(0)-Thalassemia quantification was prepared by assaying serial dilution of genomic DNA of an alpha(0)-Thalassemia carrier. Differences in the C(T) (threshold cycle) values and calculated concentrations of amplified DNA among normal fetus, alpha(0)-Thalassemia carrier, Hb H disease, and homozygous alpha(0)-Thalassemia were clearly observed, which could help in prenatal prediction of the fetal genotype. This noninvasive prenatal detection of alpha(0)-Thalassemia in maternal plasma should enhance prenatal diagnostic options for this common genetic disorder in routine DNA diagnostic setting.
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