BACKGROUND: In Vietnam, we observed a high incidence of carbamazepine (CBZ)-induced severe cutaneous adverse drug reactions (SCARs)-Stevens-Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN), and drug-induced hypersensitivity rash with eosinophilia and systemic symptoms (DRESS). In other Asian countries, HLA-B*1502 is an established risk factor for SCARs. OBJECTIVE: The aim of our study was to determine the frequency of HLA-B*1502 in SCARs patients at a large University Medical Center in Hanoi, Vietnam. METHODS: Thirty-eight cases of SCARs caused by CBZ and 25 patients with epilepsy tolerating CBZ were enrolled in a case-controlled study. Clinical manifestations and laboratory findings were recorded for each subject. Genomic DNA was isolated using the QIAamp DNA purification system. The combination of polymerase chain reaction and sequence specific oligonucleotide probes with the Luminex 100×MAP flow cytometry dual laser system was then used to quantitate fluorescently labelled oligonucleotides attached to colour-coded microbeads. RESULTS: Cases comprised 20 SJS (52.6%), 7 TEN (18.4%), 8 overlap syndrome (21.1%), and 3 DRESS patients (7.9%). A strong association between HLA B*1502 and bullous skin reactions such as SJS/TEN and overlap was confirmed with an odds ratio (OR) of 33.78 (95% confidence interval [CI], 7.55-151.03), p < 0.0001, Sensitivity 91.4%, Specificity 76.0%, positive predictive value 84.2%, and negative predictive value 86.4%. We did not, however, observe any correlation between the presence of this allele and CBZ-induced nonbullous skin reactions (DRESS) (OR, 6.33; 95% CI, 0.48-82.74; p = 0.1592). CONCLUSION: Our results indicate the presence of HLA-B*1502 in Vietnamese is a pharmacogenetic risk factor for developing CBZ-induced SJS/TEN.
Academic Medical Centers ; Alleles ; Asian Continental Ancestry Group ; Carbamazepine ; Case-Control Studies ; Cicatrix ; DNA ; Drug-Related Side Effects and Adverse Reactions ; Eosinophilia ; Epilepsy ; Exanthema ; Flow Cytometry ; HLA-B Antigens ; Humans ; Hypersensitivity ; Incidence ; Microspheres ; Odds Ratio ; Oligonucleotide Probes ; Oligonucleotides ; Pharmacogenetics ; Polymerase Chain Reaction ; Risk Factors ; Sensitivity and Specificity ; Skin ; Vietnam

OBJECTIVETo compare the application values of real-time quantitative PCR-Sanger sequencing and TaqMan probe method in the detection of KRAS and BRAF mutations, and to correlate KRAS/BRAF mutations with the clinicopathological characteristics in colorectal carcinomas.

METHODSGenomic DNA of the tumor cells was extracted from formalin fixed paraffin embedded (FFPE) tissue samples of 344 colorectal carcinomas by microdissection. Real-time quantitative PCR-Sanger sequencing and TaqMan probe method were performed to detect the KRAS/BRAF mutations. The frequency and types of KRAS/BRAF mutations, clinicopathological characteristics and survival time were analyzed.

RESULTSKRAS mutations were detected in 39.8% (137/344) and 38.7% (133/344) of 344 colorectal carcinomas by using real-time quantitative PCR-Sanger sequencing and TaqMan probe method, respectively. BRAF mutation was detected in 4.7% (16/344) and 4.1% (14/344), respectively. There was no significant correlation between the two methods. The frequency of the KRAS mutation in female was higher than that in male (P < 0.05). The frequency of the BRAF mutation in colon was higher than that in rectum. The frequency of the BRAF mutation in stage III-IV cases was higher than that in stageI-II cases. The frequency of the BRAF mutation in signet ring cell carcinoma was higher than that in mucinous carcinoma and nonspecific adenocarcinoma had the lowest mutation rate. The frequency of the BRAF mutation in grade III cases was higher than that in grade II cases (P < 0.05). The overall concordance for the two methods of KRAS/BRAF mutation detection was 98.8% (kappa = 0.976). There was statistic significance between BRAF and KRAS mutations for the survival time of colorectal carcinomas (P = 0.039). There were no statistic significance between BRAF mutation type and BRAF/KRAS wild type (P = 0.058).

CONCLUSIONS(1) Compared with real-time quantitative PCR-Sanger sequencing, TaqMan probe method is better with regard to handling time, efficiency, repeatability, cost and equipment. (2) The frequency of the KRAS mutation is correlated with gender. BRAF mutation is correlated with primary tumor site, TNM stage, histological types and histological grades.(3) BRAF gene mutation is an independent prognostic marker for colorectal carcinomas.

Adenocarcinoma ; genetics ; pathology ; Adenocarcinoma, Mucinous ; genetics ; pathology ; Adult ; Aged ; Aged, 80 and over ; Carcinoma, Signet Ring Cell ; genetics ; pathology ; Colonic Neoplasms ; genetics ; pathology ; Colorectal Neoplasms ; genetics ; pathology ; DNA Mutational Analysis ; Female ; Humans ; Male ; Middle Aged ; Mutation ; Neoplasm Grading ; Neoplasm Staging ; Oligonucleotide Probes ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins B-raf ; genetics ; Proto-Oncogene Proteins p21(ras) ; Real-Time Polymerase Chain Reaction ; Rectal Neoplasms ; genetics ; pathology ; Sequence Analysis, DNA ; Sex Factors ; Young Adult ; ras Proteins ; genetics

DNA microarray has become an essential medical genetic diagnostic tool for its high-throughput, miniaturization and automation. The design and selection of oligonucleotide probes are critical for preparing gene chips with high quality. Several sets of probe design software have been developed and are available to perform this work now. Every set of the software aims to different target sequences and shows different advantages and limitations. In this article, the research and development of these sets of software are reviewed in line with three main criteria, including specificity, sensitivity and melting temperature (Tm). In addition, based on the experimental results from literatures, these sets of software are classified according to their applications. This review will be helpful for users to choose an appropriate probe-design software. It will also reduce the costs of microarrays, improve the application efficiency of microarrays, and promote both the research and development (R&D) and commercialization of high-performance probe design software.
Base Sequence ; Oligonucleotide Array Sequence Analysis ; Oligonucleotide Probes ; Sensitivity and Specificity ; Software ; Software Design

OBJECTIVETo develop a novel real-time PCR for sensitively quantitative detection of JAK2 V617F allele burden in peripheral blood.

METHODSBased on the real-time allele-specific PCR (AS-qPCR), the locked nucleic acid (LNA)-modified oligonucleotide probe was used for selectively blocking amplification of wild-type alleles in AS-qPCR, and then a novel AS-LNA-qPCR method was established. The percentages of sample JAK2 V617F alleles were directly calculated by its threshold cycle (Ct) values according to the standard curve which generated by JAK2 V617F alleles with its Ct values. We validated intra- and inter-assay variability for quantifying JAK2 V617F. We also assayed 623 apparent healthy donors by our method to validate its clinical application value.

RESULTSThe quantitative lower limit of this method for JAK2 V617F was 0.01%, and the intra- and inter-assay average variability for quantifying percentage of JAK2 V617F in total DNA was 6.3% and 8.6%, respectively. Nineteen JAK2 V617F-positive individuals were identified using AS-LNA-qPCR in blood of 623 apparently healthy donors, and the range of percentages of JAK2 V617F alleles were 0.01%-5.49%.

CONCLUSIONThe AS-LNA-qPCR with highly sensitive and reproducible quantification of JAK2 V617F mutant burden can be used clinically for diagnosis as well as evaluation of disease prognosis and efficacy of therapy in patients with myeloproliferative neoplasms.

Alleles ; Humans ; Janus Kinase 2 ; genetics ; Mutation ; Oligonucleotide Probes ; genetics ; Oligonucleotides ; genetics ; Real-Time Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

OBJECTIVETo establish a highly sensitive fluorometric nanobiosensor for determination of aqueous mercury ions (Hg(2+)) using optimized mercury-specific oligonucleotide (MSO) probes and graphene oxide (GO).

METHODSThe nanobiosensor was assembled by attaching the self-designed MSO(1) (5' end labeled with fluorophore carboxyfluorescein (FAM), denoted as FAM-MSO(1)) and MSO(2) to the surface of GO through strong non-covalent bonding forces. Upon the addition of Hg(2+), the formation of the T-Hg(2+)-T configuration desorbed the FAM-MSO(1) and MSO(2) from the surface of GO, resulting in a restoration of the fluorescence of FAM-MSO(1). Using the specific mispairing of T-Hg(2+)-T and the changes in fluorescent signals in solutions, quantitative analysis of Hg(2+) could be performed.

RESULTSThe average thickness of the prepared GO sheets was only 1.4 nm. For the Hg(2+) nanobiosensor, the optimum concentrations of FAM-MSO(1) and MSO(2) were both 1 µmol/L, the optimum volume of 0.5 g/L GO was 5 µL, and the limit of detection was 10 pmol/L; it had low cross-reactivity with 10 other kinds of non-specific metal ions; the fluorescence recovery efficiency was up to 65% in the re-determination of Hg(2+) after addition of Na(2)S(2)O(3).

CONCLUSIONThe MSO/GO-based nanobiosensor is convenient to operate, highly sensitive, highly specific, highly accurate, and reusable. It can be applied to determine trace amount of Hg(2+) in aqueous solutions.

Biosensing Techniques ; Fluorometry ; Graphite ; Mercury ; analysis ; Nanotechnology ; Oligonucleotide Probes ; Water

The aim of this study was to build a gene chip system with surface plasmon resonance (SPR) technique, for which Gamma-peptide nucleic acid (Gamma-PNA) functioned as a probe, in order to improve sensitivity and its specificity. With the use of self-assembled monolayer (SAM) technology, surface chemistry of two-dimensional structure was used. Gamma-PNA was designed according to the bioinformatics, and was plated on the SPR chip modified by SAM. Subsequently, relevant parameters of the experiment were ensured and optimized. The results showed that the performances of Gamma-PNA probe was little affected by the ion concentration of buffer, and it had a strong light signal in a stable state. As the ion concentration was 0, there were still good hybrid reactions; pH value had less influence upon Gamma-PNA probe, and acid environment of buffer could be better. Gamma-PNA probe combined with sensor technologies achieved made the probe with dispensable labels and real-time detection. It also improved the efficiency of the hybridization and the stability, providing the foundation for clinical application.
Nucleic Acid Hybridization ; Nucleic Acid Probes ; Oligonucleotide Array Sequence Analysis ; methods ; Peptide Nucleic Acids ; genetics ; Sensitivity and Specificity ; Surface Plasmon Resonance

OBJECTIVETo investigate the feasibility of real-time PCR-optimized oligonucleotide probe method for detection of KRAS mutations in lung and colorectal carcinomas, as compared with Sanger sequencing method.

METHODSGenomic DNA was extracted from formalin fixed paraffin embedded samples of 221 lung carcinomas and 131 colorectal carcinomas after tumor cell content assessment and macrodissection. Real-time PCR-optimized oligonucleotide probe method and Sanger sequencing were performed to detect KRAS gene mutations. The frequency of KRAS mutation, mutation types, and their concordance were analyzed.

RESULTSKRAS mutation was detected in 6.3% (14/221) and 4.5% (10/221) of 221 lung cancer samples by using real-time PCR-optimized oligonucleotide probe method and Sanger sequencing, respectively, while in 41.2% (54/131) and 40.5% (53/131) of 131 colorectal cancer samples, respectively. There was no significant correlation between KRAS gene mutations and patients' gender and age (P > 0.05). The positive rate of KRAS codon 12 mutation was significantly higher than that of KRAS codon 13 mutation (P < 0.05). The overall concordance between real-time PCR-optimized oligonucleotide probe method and Sanger sequencing for KRAS mutation detection was 97.4%.

CONCLUSIONReal-time PCR-optimized oligonucleotide probe method provides an alternative method with high consistency and sensitivity as compared to Sanger sequencing in gene mutation detection.

Codon ; Colorectal Neoplasms ; genetics ; DNA Mutational Analysis ; methods ; Female ; Humans ; Lung Neoplasms ; genetics ; Male ; Middle Aged ; Mutation ; Oligonucleotide Probes ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins p21(ras) ; Real-Time Polymerase Chain Reaction ; methods ; Sensitivity and Specificity ; ras Proteins ; genetics

Adenocarcinoma ; genetics ; pathology ; Adult ; Aged ; Base Sequence ; Cell Line, Tumor ; DNA Mutational Analysis ; Exons ; Female ; Genes, erbB-1 ; Humans ; Lung Neoplasms ; genetics ; pathology ; Male ; Middle Aged ; Mutation, Missense ; Oligonucleotide Probes ; Polymerase Chain Reaction ; methods ; Real-Time Polymerase Chain Reaction ; methods ; Receptor, Epidermal Growth Factor ; genetics ; Sensitivity and Specificity

BACKGROUND: Dermatophytes (Trichophyton, Microsporum, and Epidermophyton) cause cutaneous mycoses called dermatophytosis. Forproper anti-dermatophytosis therapy, accurate and early diagnosis of dermatophytes is important. Laboratory diagnosis of dermatophytosis for dermatophytes still relies on microscopic and macroscopic examination of in vitro cultures and some physiological tests. These methods (conventional methods) are time-consuming (2~4 weeks) and yet, still have low sensitivity and specificity. Recently, in order to overcome such limitations of conventional methods, molecular-based methods have been developed to identify dermatophytes. The polymerase chain reaction-reverse blot hybridization assay (PCR-REBA) allows sensitive and specific identification of dermatophytes species. OBJECTIVE: This study was aimed to develop a new PCR-REBA with higher sensitivity using less amount of probe concentration, so the assay can be more practical in clinical settings. METHODS: For this, PCR primers and species-specific oligonucleotide probes were designed within the internal transcribed sequences 1 region between 5.8S and 18S rRNA. The species-specific probes designed in this study was to identify 6 species (T. rubrum, T. mentagrophytes, T. tonsurans, M. canis, M. gypseum, and E. floccosum) comprised 99% of dermatophytes isolatedin Korea. RESULTS: The detection efficiency of the PCR-REBA was compared with the microscopic method, and the results showed that the sensitivity of the PCR-REBA developed in this study is 100 times higher than previously developed one. Subsequently, the results of PCR-REBA were evaluated using clinical isolates. DNAs from a total of 68 clinical isolates were analyzed by PCR-REBA, and the inconsistent results between PCR-REBA and conventional microscopic identification results were confirmed by sequence analysis. CONCLUSION: In brief, the results showed that results of sequence analysis were identical with PCR-REBA implying newly developed PCR-REBA is very useful method for accurate and rapid identification of dermatophytes and would provide higher simplicity, specificity, sensitivity than conventional method.
Arthrodermataceae ; Chimera ; Clinical Laboratory Techniques ; DNA ; Early Diagnosis ; Microsporum ; Mycoses ; Oligonucleotide Probes ; Polymerase Chain Reaction ; Sensitivity and Specificity ; Sequence Analysis ; Tinea

OBJECTIVETo investigate the effect of microRNA-205 reduction by antagomirs on adhesion ability of normal human corneal epithelial keratinocytes (NHCEKs).

METHODSAntagomir-205, complementary and inhibitory to microRNA-205, was used to suppress endogenous microRNA-205 in NHCEKs. The adhesion ability of treated NHCEKs was then assessed by cell adhesion assay. Immunoblot and immunohistochemistry were conducted to determine the level of two focal adhesion-related proteins, focal adhesion kinase (FAK) and paxillin (Pax). Phalloidin staining was performed to measure the level of filamentous actin in antagomir-treated NHCEKs.

RESULTSAntagomir-205 markedly reduced the level of microRNA-205 in NHCEKs and significantly enhanced adhesion ability of NHCEKs (P<0.01). Further protein analysis validated that inhibition of microRNA-205 increased the number of phosphorylated FAK and phosphorylated Pax, and decreased filamentous actin.

CONCLUSIONOur findings suggest that microRNA-205 has down-regulating effect on cell motility in NHCEKs.

Base Sequence ; Cell Adhesion ; genetics ; Cells, Cultured ; Epithelium, Corneal ; cytology ; Humans ; Keratinocytes ; cytology ; MicroRNAs ; antagonists & inhibitors ; Oligonucleotide Probes