ABSTRACT
BACKGROUND: Since the initial outbreak in March 2009, the novel pandemic (H1N1) 2009 virus has affected individuals worldwide and caused over 18,138 deaths. There is an urgent need for the development of an easy, accurate and simple method for the diagnosis of this novel pandemic virus. DESIGN: Reverse transcription loop-mediated isothermal amplification assay (RT-LAMP) with primers targeting the M segment was established for the rapid differential diagnosis of pandemic (H1N1) 2009 virus. The performance of this assay was characterized using 111 clinic nasopharyngeal swabs, and the diagnosis accuracy was compared with real-time reverse transcription PCR (RRT-PCR) and virus isolation, the latter being the reference standard. RESULTS: This method successfully detected pandemic (H1N1) 2009 virus with a detection limit of approximately 20 copies of the target RNA per reaction, which is a comparably sensitivity to the RRT-PCR assay. Furthermore, this assay was able to discriminate pandemic (H1N1) 2009 virus from seasonal influenza viruses, such as H1N1 and H3N2, and other respiratory viruses (parainfluenza type 2 and 3, adenovirus, echovirus 7, and coxsackievirus A10). Based on validation by virus isolation, the specificity and sensitivity of this M-specific RT-LAMP assay were 100% and 98·25%, respectively. Moreover, the RT-LAMP amplification of most positive samples (46 out of 56) was achieved in < 20 min. CONCLUSIONS: This is an accurate and fast analysis system suitable for general diagnostic laboratories with only limited equipment, e.g. first-line health care centre. This assay will help clinicians and public health officials to react effectively during an outbreak.
Subject(s)
Influenza A Virus, H1N1 Subtype/isolation & purification , Influenza, Human/diagnosis , Pandemics/prevention & control , Reverse Transcriptase Polymerase Chain Reaction/methods , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza, Human/genetics , Influenza, Human/virology , Molecular Sequence Data , Nasopharynx , RNA, Viral/genetics , Reproducibility of ResultsABSTRACT
BACKGROUND: Membranous glomerulonephritis (MGN) is one of the most common causes of nephrotic syndrome in adults. NPHS1 encoding nephrin is a transmembrane protein of the immunoglobulin family. We clarified the relationship between NPHS1 gene polymorphisms and the susceptibility or progression of MGN. METHODS: We recruited a cohort of 132 biopsy-diagnosed MGN patients and 257 healthy subjects. Genotyping of three SNPs (rs401824, rs437168 and rs3814995) at chromosome positions 41034749 (5'UTR), 41026259(exon17) and 41034052 (exon 3) was performed using a Taqman SNP genotyping assay. RESULTS: There was a significant difference in genotype frequency distribution of rs437168 polymorphism between MGN patients and controls. The results also showed that the frequency of the G allele was significantly higher in the patient group. Among the polymorphisms rs437168, rs401824 and rs3814995, no significant haplotype was shown in MGN patients. A stratified analysis revealed that a high disease progression in the AA genotype of rs401824 and GG genotype of rs437168 patients were associated with a low rate of remission. CONCLUSIONS: The presence of the different genotypes of NPHS1 was associated with susceptibility of MGN and the remission of proteinuria during disease progression after the therapy.
Subject(s)
Glomerulonephritis, Membranous/genetics , Membrane Proteins/genetics , Polymorphism, Single Nucleotide/genetics , 5' Untranslated Regions/genetics , Adult , Aged , Aged, 80 and over , Creatinine/blood , Creatinine/urine , Female , Gene Frequency/genetics , Genotype , Glomerulonephritis, Membranous/complications , Glomerulonephritis, Membranous/diagnosis , Glomerulonephritis, Membranous/drug therapy , Glomerulonephritis, Membranous/metabolism , Haplotypes/genetics , Hematuria/etiology , Hematuria/genetics , Heterozygote , Homozygote , Humans , Immunosuppressive Agents/therapeutic use , Male , Middle Aged , Open Reading Frames/genetics , Proteinuria/etiology , Proteinuria/genetics , Taiwan , Treatment OutcomeABSTRACT
OBJECTIVES: The present study was aimed at identifying hemoglobin (Hb) alpha variants. DESIGN AND METHODS: To identify Hb variants, a high-resolution melting (HRM) method was performed. RESULTS: The diagnostic strategy was found to be successful in identifying Hb alpha variants including HBA1:c.27G>T, (Hb Hekinan) HBA1:c.46G>C (Hb Ottawa), HBA2:c.31_33AG (Hb alpha2-globin gene codon del AG), HBA1:c.223G>C (Hb G-Taichung), HBA1:p.Phe118_Thr119insIle (Hb Phnom Penh), HBA2:c.369C>G (Hb Westmead), HBA2:c.364G>A (or HBA1) (Hb Owari), HBA2:c.377T>C (Hb Quong Sze), and HBA2:c.427T>C (Hb Constant Spring). Each Hb variant could be readily and easily identified through the difference in plotted curves. In addition, the Hb variants could be distinguished to be located at either HBA1 or HBA2 gene. CONCLUSIONS: The HRM analysis is found to be a good tool for identifying Hb variants in alpha globin genes.
Subject(s)
Hemoglobins, Abnormal/genetics , Sequence Analysis, DNA/methods , Exons/genetics , Humans , Models, BiologicalABSTRACT
OBJECTIVE: This study was undertaken to identify HBB gene mutation. DESIGN AND METHODS: Herein we evaluated high-resolution melting analysis in the identification of HBB mutations. RESULTS: We have successfully established a diagnostic strategy for identifying HBB gene mutations including c.-78A>G, c.-79A>G, c.2T>G, c.79_80insT, c.84_85insC, c.123_124insT, c.125_128delTCTT, c.130 G>T, c.170G>A, c.216_217ins A and c.316-197 C>T from wild-type DNA using HRM analysis. The results of HRM analysis were confirmed by direct DNA sequencing. CONCLUSIONS: In summary, we report that HRM analysis is an appealing technique for the identification of HBB mutations. We also believe that HRM can be used as a method for prenatal diagnosis of beta-thalassemia.
Subject(s)
Hemoglobins/genetics , Mutation , Base Sequence , DNA Primers , Exons , Humans , Polymerase Chain Reaction , Promoter Regions, Genetic , Sequence Analysis, DNA , beta-Thalassemia/geneticsABSTRACT
We recently observed a heterozygote for Hb Hekinan in a Taiwanese subject. The molecular lesion of Hb Hekinan is a substitution of G-->T at codon 27 of the alpha1-globin gene, which abolishes an HaeIII restriction enzyme site. Hb Hekinan [alpha27(B8)Glu-->Asp, GAG-->GAC (alpha2)] has not been found in Taiwan. This variant can be detected by high performance liquid chromatography (HPLC) but not by capillary or cellulose electrophoresis.
Subject(s)
Amino Acid Substitution/genetics , Hemoglobins, Abnormal/genetics , Point Mutation , alpha-Thalassemia/genetics , Child , Deoxyribonucleases, Type II Site-Specific/metabolism , Hemoglobins, Abnormal/chemistry , Humans , Male , TaiwanABSTRACT
An outbreak of contagious ecthyma in goats in central Taiwan was investigated. The disease was diagnosed by physical and histopathologic examinations, and the etiology of the disease was identified as orf virus by electron microscopy and polymerase chain reaction (PCR) and sequence of major envelope protein (B2L) gene. The entire protein-coding region of B2L gene were cloned and sequenced. Phylogenetic analysis of B2L amino acid sequences showed that the orf virus identified in this outbreak was closer to the Indian ORFV-Mukteswar 59/05 isolate. This is the first report on the molecular characterization of orf virus in Taiwan.
Subject(s)
Disease Outbreaks , Ecthyma, Contagious/epidemiology , Ecthyma, Contagious/virology , Orf virus/isolation & purification , Animals , DNA, Viral/chemistry , DNA, Viral/genetics , DNA, Viral/isolation & purification , Ecthyma, Contagious/pathology , Ecthyma, Contagious/physiopathology , Goats , Microscopy, Electron, Transmission , Molecular Sequence Data , Orf virus/genetics , Orf virus/ultrastructure , Phylogeny , Polymerase Chain Reaction , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Taiwan/epidemiology , Viral Envelope Proteins/genetics , Virion/ultrastructureABSTRACT
BACKGROUND: Several lipid ratios may be predictors of coronary artery disease risk. We assessed the efficacy of Monascus purpureus Went rice (red yeast rice) on lowering lipid ratios. METHOD AND RESULTS: We evaluated 79 hypercholesterolemic patients (aged 23-65 years) who received a twice-daily dose of either red yeast rice or a placebo at 600 mg for 8 weeks. The 8-week treatment with red yeast rice showed significantly greater reduction than the placebo treatment in low-density lipoprotein cholesterol levels, total cholesterol/high-density lipoprotein cholesterol, low-density lipoprotein cholesterol/high-density lipoprotein cholesterol and apolipoprotein B/apolipoprotein A-I ratios. CONCLUSIONS: Red yeast rice can reduce lipid ratios in hypercholesterolemic patients.
Subject(s)
Anticholesteremic Agents/therapeutic use , Biological Products/therapeutic use , Hypercholesterolemia/drug therapy , Lipids/blood , Monascus , Phytotherapy , Administration, Oral , Anticholesteremic Agents/administration & dosage , Apolipoprotein A-I/blood , Apolipoproteins B/blood , Biological Products/administration & dosage , Capsules , Cholesterol/blood , Double-Blind Method , Drug Administration Schedule , Female , Humans , Hypercholesterolemia/blood , Male , Middle Aged , Treatment OutcomeABSTRACT
We examined the P16 expression by immunohistochemical stain and detected the methylation by methylation specific polymerase chain reaction (MSP) in 48 primary oral squamous cell carcinoma (SCC) tissues. The results showed that 20/48 (41.7%) of cancerous tissues had CpG methylation around the promoter region, but 8/48 (17%) of the nearby non-cancerous tissues also had CpG methylation, around the promoter region. The results from immunohistochemical studies showed that reduced and heterogeneous expression of P16 were found in the tissues, which had CpG methylation around the promoter region. In conclusion, the methylation of P16 in oral SCC occurs in pre-cancerous and cancerous stage, which results in decreasing or abolishing the P16 expression, which is heterogeneous in the cancer cells.
