Improvement in the detection of alpha0- and deletional alpha-thalassemia by real-time PCR combined with dissociation curve analysis.
The prevailing cause of alpha-thalassemia in Southeast Asia is the presence of 3 deletion mutations in the alpha-globin genes (-SEA, -alpha(3.7) and -alpha(4.2)). Current detection methods include gap polymerase chain reaction (PCR), multiplex PCR and real-time PCR with SYBR Green 1 combined with dissociation curve analysis. To improve and simplify a previously published method that requires 4 separate reactions, a duplex PCR assay was designed to detect both the nondeletional and the -SEA alleles. This duplex PCR can successfully identify the nondeletional allele and both the -SEA carrier and homozygous genotypes. The combination of the duplex PCR and 2 gap PCRs (for detection of -alpha(3.7) and -alpha(4.2)) can diagnose all types of deletional alpha-thalassemia. Our method was validated by analysis of 195 DNA samples, the results of which were consistent with prior diagnoses. The developed assay can reliably diagnose alpha0-thalassemia and all types of deletional alpha-thalassemia. The diagnostic method is simple, rapid, accurate, automated, inexpensive and has a high throughput.
Liu
Jingzhong
J
Basic Medical Research Center, Beijing Chaoyang Hospital Affiliate of The Capital Medical University, Beijing, China. liujingzhong@hotmail.com
Tang
Ning
N
Liu
Quanzhang
Q
Wang
Lirong
L
Han
Han
H
Cai
Ren
R
Wu
Xiaoyi
X
Xiao
Bai
B
D001483:Base Sequence; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D017085:alpha-Thalassemia / Q000175:diagnosis*