[Detection of alpha thalassemia using real-time PCR and dissociation curve analysis].
OBJECTIVE
To establish an automatic, high throughput, quick detection method of alpha thalassemia.


METHODS
The genotypes of -alpha(4.2) and -alpha(3.7) were detected by two real-time fluorescence PCRs using SYBR-Green1 (SYBR-PCR) with the analysis of dissociation curve (DC) and melting temperature (Tm). The PCR products were recombined into T-vector and the correct cloning was selected as positive control. Sensitivities were gained from a serious dilution of the recombinant as SYBR-PCR template, from which detection deadlines for two kinds of alleles could be determined. Totally 110 samples were detected by this technique.


RESULTS
The length of product of -alpha(4.2) and -alpha(3.7) were 1.65 kb and 1.9 kb respectively, and the Tm were (81.5+/-0.5) degrees C and (82.5+/-0.5) degrees C respectively. The detection deadline were 9 x 10(2 ) copies and 4.3 x 10(2) copies respectively. The sensitivity of the technique was much higher than that of regular PCR plus gel electrophoresis method, and the detection results of the technique were the same as that of multiplex PCR.


CONCLUSIONS
The genotypes of -alpha(4.2), -alpha(3.7), non-deletion alpha alpha/and --(SEA) can be sensitively, exactly diagnosed by the real-time PCR with SYBR-Green1 combined with the dissociation curve analysis. The assay is automatic, low-cost, high throughput, and easy for quality control without fluorescence probe.
Yan
Mei
M
Basic Medical Research Center, Chaoyang Hospital Affiliated to Capital Medical University, Beijing, 100020 PR China.
Wang
Li-rong
LR
Wang
Zhan-yong
ZY
Zhou
Yan
Y
Liang
Yan
Y
Xiao
Bai
B
Liu
Jing-zhong
JZ
D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017085:alpha-Thalassemia / Q000175:diagnosis* / Q000235:genetics*