Evaluation of the BeTha Gene 1 Kit for the Qualitative Detection of the Eight Most Common Mediterranean β-Thalassemia Mutations

Autor: Renzo Galanello, Antonino Giambona, A. Loutradi, Aurelio Maggio, L. Macioni, F. Locati, Jimmie D. Lowery, Luis A. Ugozzoli, Ching-I Patsy Lin, M. Boussiou, Antonio A. Reyes, R. Bruce Wallace, A. Re
Rok vydání: 1998
Předmět:
Zdroj: American Journal of Hematology. 59:214-222
ISSN: 1096-8652
0361-8609
DOI: 10.1002/(sici)1096-8652(199811)59:3<214::aid-ajh6>3.0.co;2-z
Popis: We describe the evaluation of the Bio-Rad BeTha Gene 1 kit (Bio-Rad Laboratories, Hercules, CA), a DNA-probe assay designed for the qualitative determination of the eight most common Mediteranean β-thalassemia mutations. The kit utilizes the principle of allele-specific oligonucleotide (ASO) hybridization. Following sample preparation and in vitro DNA amplification by the polymerase chain reaction (PCR), an allele-specific detection of the amplified products by a nonradioactive enzymatic assay is performed. Genomic DNA is prepared from an individual's whole blood with a DNA purification matrix. In a second step, the β-globin gene is amplified in a multiplex PCR reaction containing four 5′ biotinylated oligonucleotide primers. In a final step, an aliquot of the PCR reaction is first chemically denatured and then captured in two eight-well strips of a 96-well enzyme-linked immunosorbent assay (ELISA) plate by hybridization to an immobilized ASO probe. Each DNA sequence at each of the eight mutation sites is represented by one normal and one mutant ASO. During this capture/hybridization step, which is performed at 37°C, only perfectly matched PCR products will be captured by an ASO. Subsequently, the allele-specific captured biotin-labeled PCR products are detected by a colorimetric enzymatic reaction. The system permits the detection of 16 β-thalassemia alleles using a high-throughput format that can be automated easily. A clinical feasibility study was performed to evaluate the functionality (method camparison study, assay validity using samples previously collected and stored at various temperatures for different periods of time, interference on kit performance, and assay validity for prenatal diagnosis) and the usability (ease of use, sample throughput) of the kit. The analysis of 110 samples previously studied with reference methods showed 100% clinical sensitivity and specificity. We demonstrate here that the procedure not only increases the throughput of β-thalassemia allele genotyping but also provides an accurate, rapid, reliable, and nonisotopic diagnostic tool. Am. J. Hematol. 59:214–222, 1998. © 1998 Wiley-Liss, Inc.
Databáze: OpenAIRE
Externí odkaz: