A Melting Curve Analysis–Based PCR Assay for One-Step Genotyping of β-Thalassemia Mutations

Abstract

The increasing number of disease-causing mutations demands a simple, direct, and cost-effective diagnostic genotyping technique capable of detecting multiple mutations. This study validated the efficacy of a novel melting curve analysis-based genotyping assay (MeltPro HBB assay) for 24 ␤-thalassemia mutations in the Chinese population. The diagnostic potential of this assay was evaluated in 1022 pretyped genomic DNA samples, including 909 clinical cases of ␤-thalassemia minor or major, using a double-blind analysis in a multicenter validation study. Reproducibility of the assay was 100%, and the limit of detection was 10 pg per reaction. All 24 ␤-thalassemia mutations were accurately genotyped, and ␤-thalassemia genotypes were correctly determined in all 1022 samples, yielding overall sensitivity and specificity of 100%. The concordance rate was 99.4% between this assay and the reference method. It was concluded that the MeltPro HBB assay is useful for reliable genotyping of multiple ␤-thalassemia mutations in clinical settings and may have potential as a versatile method for rapid genotyping of known mutations because of its high throughput, accuracy, ease of use, and low cost.

Detection of Anti-Hepatitis B Virus Drug Resistance Mutations Based on Multicolor Melting Curve Analysis

“…Furthermore, the 6 samples with inconsistent results were found to contain minor mutant subpopulations undetected by PCR Sanger sequencing, and their presence was confirmed by COLD-PCR Sanger sequencing. These results are in line with those of our previous studies that MMCA never missed a single mutation detected by PCR Sanger sequencing (18). The three groups of samples were representative of those from two types of patients, treated (group A) and untreated (groups B and C).…”

“…Up to 16 genetic mutations could be detected in a single reaction by this strategy (18). In the present study, we describe a two-reaction MMCA assay that can detect 24 anti-HBV drug resistance mutations at 10 amino acid positions.…”

Rapid Detection of Isoniazid Resistance in Mycobacterium tuberculosis Isolates by Use of Real-Time-PCR-Based Melting Curve Analysis

“…The MeltPro TB/INH assay is the first officially approved assay dedicated solely to detection of INH resistance. This assay is based on melting curve analysis that uses unique dually labeled, selfquenched probes, which have been proved to be accurate, sensitive, and flexible in the detection of multiple mutations in a single reaction (11,(27)(28)(29). The assay can detect 20 mutant sites that involve 30 mutation types in the inhA promoter region (positions Ϫ17 to Ϫ8), katG position 315 (katG 315), the ahpC promoter, and inhA position 94 (inhA 94) of M. tuberculosis, constituting the largest number of INH resistance mutations.…”

The Experiences of Prenatal Diagnosis in China

“…Fortunately, most of the thalassemic alleles were -thal-2 (-3.7 / or -4.2 /) , only 0.0058 for -thal-1 allele (--SEA /) [15]. The estimated incidence of carriers of -thalassemia is as high as 6.43% in Guangxi, and 1% to 2.54% in populations in the endemic areas of southern China [13,16].…”

“…It is a qualitative in vitro diagnostic method designed to genotype a panel of 24 single-nuclueotide mutations and small indels in the HBB gene that cause -thalassemia or abnormal hemoglobin. The test kit is based on a proprietary, multicolored, self-quenched, probe-based melting curve analysis performed with a standard real-time PCR instrument, from which genotype information for each mutation is retrieved based on the melting temperature (Tm) or difference in Tm between wild-type and mutant DNA samples [16].…”