title: 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: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Medical Technology, Chiang-Mai University, Chiang-Mai, Thailand. sakornmi001@gmail.com
authors.forename: Sakorn
authors.initials: S
authors.lastname: Pornprasert
authors.forename: Thanatcha
authors.initials: T
authors.lastname: Wiengkum
authors.forename: Sarinee
authors.initials: S
authors.lastname: Srithep
authors.forename: Isarapong
authors.initials: I
authors.lastname: Chainoi
authors.forename: Panthong
authors.initials: P
authors.lastname: Singboottra
authors.forename: Sanchai
authors.initials: S
authors.lastname: Wongwiwatthananukit
mesh_terms: D001210:Asia, Southeastern; D044466:Asian People / Q000235:genetics*; D052160:Benzothiazoles; D003959:Diamines; D017353:Gene Deletion; D005838:Genotype; D006801:Humans; D009930:Organic Chemicals / Q000737:chemistry*; D044367:Phase Transition; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D011933:Reagent Kits, Diagnostic; D013785:Thailand; D044366:Transition Temperature; D017085:alpha-Thalassemia / Q000175:diagnosis / Q000235:genetics*; D017086:beta-Thalassemia / Q000175:diagnosis / Q000235:genetics*
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D011933:Reagent Kits, Diagnostic; C098022:SYBR Green I