Rapid diagnosis of alpha(o)-thalassemia using the relative quantitative PCR and the dissociation curve analysis.
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.
Sangkitporn
S K
SK
Department of Medical Sciences, National Institute of Health, Nonthaburi, Thailand.
Wangkahat
K
K
Sangnoi
A
A
Songkharm
B
B
Charoenporn
P
P
Sangkitporn
S
S
D052160:Benzothiazoles; D004396:Coloring Agents; D003959:Diamines; D017353:Gene Deletion; D005838:Genotype; D005914:Globins / Q000235:genetics*; D006455:Hemoglobins, Abnormal / Q000235:genetics; D006801:Humans; D009691:Nucleic Acid Denaturation; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D013696:Temperature; D013785:Thailand / Q000453:epidemiology; D013997:Time Factors; D017085:alpha-Thalassemia / Q000145:classification / Q000175:diagnosis* / Q000453:epidemiology / Q000235:genetics
D052160:Benzothiazoles; D004396:Coloring Agents; D003959:Diamines; D006455:Hemoglobins, Abnormal; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I; D005914:Globins; C030448:hemoglobin Bart's