Detection of α-thalassemia-1 Southeast Asian and Thai Type Deletions and β-thalassemia 3.5-kb Deletion by Single-tube Multiplex Real-time PCR with SYBR Green1 and High-resolution Melting Analysis

Abstract

BackgroundPrevention and control of thalassemia requires simple, rapid, and accurate screening tests for carrier couples who are at risk of conceiving fetuses with severe thalassemia.MethodsSingle-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.ResultsHRM 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 (Tm) 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.ConclusionsSingle-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.

Red Cell Indices and Formulas Used in Differentiation ofβ-Thalassemia Trait from Iron Deficiency in Thai Adults

“…The DNA was stored at À20 C until used. The diagnosis for a-thal-1 --SEA and --THAI type deletions were detected using quantitative real-time polymerase chain reaction (q-PCR) with SYBR Green1 and high resolution melting (HRM) analysis as previously described (14,15).…”

Quantitative detection of target cells using unghosted cells (UGCs) of DxH 800 (Beckman Coulter)

“…There is no objective guideline for classification of RBCs as target cells. [13,14]. For these reasons, various discrimination indices derived from several CBC parameters have been proposed [15][16][17][18][19][20][21].…”

The use of Taqman genotyping assays for rapid confirmation of β‐thalassaemia mutations in the Malays: accurate diagnosis with low DNA concentrations

“…In this study, Taqman probes were used instead of SYBR green, which was commonly used in earlier studies for β‐thalassaemia real‐time detection . With SYBR green detection, the probe intercalates in the minor groove of double‐stranded DNA (dsDNA) and emits a fluorescence signal.…”

“…There are no post-PCR steps as required in ARMS and RDBH, thus avoiding the possibility of cross-contamination with PCR products and also allow faster turnaround times [19]. In this study, Taqman probes were used instead of SYBR green, which was commonly used in earlier studies for b-thalassaemia real-time detection [20,21]. With SYBR green detection, the probe intercalates in the minor groove of double-stranded DNA (dsDNA) and emits a fluorescence signal.…”