Specific and straightforward molecular investigation of β-thalassemia mutations in the Malaysian Malays and Chinese using direct TaqMan genotyping assays.
Genet Mol Res. 2013 Feb 28;12(AOP)
Authors: Kho SL, Chua KH, George E, Tan JA
Beta-thalassemia is a life-threatening inherited blood disorder. Rapid characterization of β-globin gene mutations is necessary because of the high frequency of Malaysian β-thalassemia carriers. A combination real-time polymerase chain reaction genotyping assay using TaqMan probes was developed to confirm β-globin gene mutations. In this study, primers and probes were designed to specifically identify 8 common β-thalassemia mutations in the Malaysian Malay and Chinese ethnic groups using the Primer Express software. "Blind tests" using DNA samples from healthy individuals and β-thalassemia patients with different genotypes were performed to determine the specificity and sensitivity of this newly designed assay. Our results showed 100% sensitivity and specificity for this novel assay. In conclusion, the TaqMan genotyping assay is a straightforward assay that allows detection of β-globin gene mutations in less than 40 min. The simplicity and reproducibility of the TaqMan genotyping assay permits its use in laboratories as a rapid and cost-effective diagnostic tool for confirmation of common β-thalassemia mutations in Malaysia.
PMID: 23479149 [PubMed - as supplied by publisher]
Molecular Analysis of Spinal Muscular Atrophy: A genotyping protocol based on TaqMan(®) real-time PCR.
Genet Mol Biol. 2012 Dec;35(4 (suppl)):955-9
Authors: de Souza Godinho FM, Bock H, Gheno TC, Saraiva-Pereira ML
Spinal muscular atrophy (SMA) is an autosomal recessive inherited disorder caused by alterations in the survival motor neuron I (SMN1) gene. SMA patients are classified as type I-IV based on severity of symptoms and age of onset. About 95% of SMA cases are caused by the homozygous absence of SMN1 due to gene deletion or conversion into SMN2. PCR-based methods have been widely used in genetic testing for SMA. In this work, we introduce a new approach based on TaqMan(®)real-time PCR for research and diagnostic settings. DNA samples from 100 individuals with clinical signs and symptoms suggestive of SMA were analyzed. Mutant DNA samples as well as controls were confirmed by DNA sequencing. We detected 58 SMA cases (58.0%) by showing deletion of SMN1 exon 7. Considering clinical information available from 56 of them, the patient distribution was 26 (46.4%) SMA type I, 16 (28.6%) SMA type II and 14 (25.0%) SMA type III. Results generated by the new method was confirmed by PCR-RFLP and by DNA sequencing when required. In conclusion, a protocol based on real-time PCR was shown to be effective and specific for molecular analysis of SMA patients.
PMID: 23412967 [PubMed - in process]
Novel quantitative TaqMan real-time PCR assays for detection of Cryptosporidium at the genus level and genotyping of major human and cattle-infecting species.
J Appl Microbiol. 2012 Dec 12;
Authors: Burnet JB, Ogorzaly L, Tissier A, Penny C, Cauchie HM
AIMS: Development of TaqMan MGB real-time PCR assays for quantitative typing of major cattle and human-pathogenic Cryptosporidium species. METHODS AND RESULTS: Three specific TaqMan MGB real-time PCRs, based on the SSU rRNA gene, were directed towards livestock-restricted C. andersoni and C. bovis as well as both human-pathogenic C. parvum and C. hominis. A generic TaqMan assay further identified all known Cryptosporidium species and simultaneously monitored PCR inhibition through an external amplification control. The generic and specific assays were highly reproducible and all displayed a detection limit of one oocyst per reaction. The specific TaqMan protocols also proved valuable for specifically detecting and quantifying target DNA in presence of non-target DNA in environmental samples. CONCLUSIONS: All TaqMan MGB real-time PCR assays fulfilled the required specificity and sensitivity criteria, both on laboratory strains and on a surface water matrix. © 2012The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.
PMID: 23230846 [PubMed - as supplied by publisher]
Potential clinical significance of plasma-based KRAS mutation analysis using the COLD-PCR/TaqMan(®) -MGB probe genotyping method.
Exp Ther Med. 2012 Jul;4(1):109-112
Authors: Liu P, Liang H, Xue L, Yang C, Liu Y, Zhou K, Jiang X
Despite the improved ability to detect mutations in recent years, tissue specimens cannot always be procured in a clinical setting, particularly from patients with recurrence of tumors or metastasis. Therefore, the aim of this study was to investigate whether plasma is able to be used for mutation analysis instead of tissue specimens. We collected plasma from 62 patients with colorectal cancer (CRC) prior to treatment. DNA extracted from plasma and matched tumor tissues were obtained. Mutations in KRAS were amplified from the tissue specimens and sequenced by regular polymerase chain reaction (PCR) and co-amplification at lower denaturation temperature (COLD)-PCR. Plasma KRAS gene mutation on codon 12 (GGT>GAT) was detected using a nested COLD-PCR/TaqMan(®) -MGB probe. Mutations in plasma and matched tumors were compared. KRAS mutation on codon 12 (GGT>GAT) was found in 13 (21.0%) plasma specimens and 12 (19.4%) matched tumor tissues. The consistency of KRAS mutations between plasma and tumors was 75% (9/12), which indicated a high correlation between the mutations detected in plasma DNA and the mutations detected in the corresponding tumor DNA (P<0.001; correlation index, k=0.649). Notably, four (6.5%) patients with plasma DNA mutations had no detectable KRAS mutations in the corresponding primary tumors, and three (4.8%) patients with tumor DNA mutations had no detectable KRAS mutations in the corresponding plasma DNA samples. Thus, KRAS mutations in plasma DNA correlate with the mutation status in matched tumor tissues of patients with CRC. Our study provides evidence to suggest that plasma DNA may be used as a potential medium for KRAS mutation analysis in CRC using the COLD-PCR/TaqMan-MGB probe method.
PMID: 23060932 [PubMed - as supplied by publisher]
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