sybr-green-i and alpha-Thalassemia

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.

Topics: alpha-Thalassemia; Asia, Southeastern; Asian People; Benzothiazoles; beta-Thalassemia; Diamines; Gene Deletion; Genotype; Humans; Organic Chemicals; Phase Transition; Polymerase Chain Reaction; Quinolines; Reagent Kits, Diagnostic; Thailand; Transition Temperature

Topics: alpha-Thalassemia; Base Sequence; Benzothiazoles; Diamines; DNA Primers; Hemoglobins, Abnormal; Heterozygote; Humans; Organic Chemicals; Polymerase Chain Reaction; Quinolines

Dear Sir, A single tube polymerase chain reaction (PCR) with three primers and SYBR GREEN1 combined with dissociation curve analysis was set up that can clearly differentiate between Hb Bart's hydrops fetalis, normal subjects and - -(SEA) heterozygotes. This method seems to be simpler than that using a two-tube real-time SYBR-PCR with two different primer sets followed by analyses of DeltaC(T) and C(T) ratio.

Topics: alpha-Thalassemia; Asia, Southeastern; Benzothiazoles; Diamines; Hemoglobins, Abnormal; Heterozygote; Humans; Hydrops Fetalis; Organic Chemicals; Polymerase Chain Reaction; Quinolines

Topics: alpha-Thalassemia; Asia, Eastern; Benzothiazoles; Blood; Diamines; DNA Probes; Humans; Organic Chemicals; Polymerase Chain Reaction; Quinolines; Sensitivity and Specificity; Specimen Handling

The aim of the study was to set up an automatic molecular diagnostic method for deletional alpha-thalassemia without gel electrophoresis and TaqMan probe. Four real-time polymerase chain reactions (PCRs) with SYBR Green1 and ABI7000 (SYBR-PCR) followed by dissociation curve (DC) analysis were used to detect the --(SEA), - alpha(3.7), -alpha(4.2), and non-deletion-type alleles (alpha alpha or alpha(T)alpha), respectively. Positive results of the SYBR-PCRs were defined by the special shapes of the dissociation curves and the peak height at specific Tm for each predetermined PCR at a specific Tm for each PCR amplicon > or = cutoff values. Molecular diagnosis of alpha-thalassemia was determined by combining all four SYBR-PCR results. The specific Tms for the SYBR-PCR1-4, which was used to detect the --(SEA), - alpha(3.7), -alpha(4.2), and non-deletion-type alleles were 82.5 +/- 1 degrees Celsius, 82.8 +/- 1 degrees Celsius, 81.5 +/- 1 degrees Celsius, and 83.0 +/- 1 degrees Celsius, respectively. The cutoff values of the specific peaks for the positive amplificons were 40, 20, 10, and 70. The C(T) VS log copies of a recombinant plasmid DNA showed a good linear relationship between 10(5) approximately 10(0). Sensitivity of the SYBR-PCR-based method was at least 16 times higher than the multiplex PCR (mPCR)/gel electrophoresis method. Diagnostic outcomes of the 120 alpha-thalassemia cases by using the SYBR-PCR and DC analysis techniques were shown to be the same as that by using the mPCR/gel electrophoresis methods. The SYBR-PCR combined with the DC analysis technique is an alternative assay for the routine molecular diagnosis of alpha-thalassemia.

Topics: Alleles; alpha-Thalassemia; Automation; Benzothiazoles; Diamines; Fluorescent Dyes; Gene Deletion; Genotype; Humans; Nucleic Acid Denaturation; Organic Chemicals; Polymerase Chain Reaction; Quinolines

A method for diagnosis of alpha(o)-thalassemia was developed based on detection of accumulated PCR product using SYBR Green I, a double-stranded DNA binding dye, and a fluorescence-detecting thermocycler. Primers were designed to specifically amplify - -SEA and - -Thai deletions of alpha(o)-thalassemia. Albumin was selected as the reference gene. The comparative CT method was used to quantitate the target gene relative to the albumin gene. Dissociation curve analysis was used as a qualitative tool to distinguish different types of alpha(o)-thalassemia. In this study, the melting temperatures of the - -Thai and - -SEA deletions were 83 and 86 degrees C, respectively. Application of the assay for the diagnosis of alpha(o)-thalassemia heterozygotes and homozygotes is reported. The assay is highly robust and amenable to high throughput. It is thus a useful tool for the rapid detection of alpha(o)-thalassemia in populations with a high frequency of alpha(o)-thalassemia, - -SEA and - -Thai deletions.

Topics: alpha-Thalassemia; Benzothiazoles; Coloring Agents; Diamines; Gene Deletion; Genotype; Globins; Hemoglobins, Abnormal; Humans; Nucleic Acid Denaturation; Organic Chemicals; Polymerase Chain Reaction; Quinolines; Temperature; Thailand; Time Factors