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1.
Electron. j. biotechnol ; 47: 72-82, sept. 2020. tab, ilus, graf
Article in English | LILACS | ID: biblio-1253093

ABSTRACT

BACKGROUND: Piercing/sucking insect pests in the order Hemiptera causes substantial crop losses by removing photoassimilates and transmitting viruses to their host plants. Cloning and heterologous expression of plantderived insect resistance genes is a promising approach to control aphids and other sap-sucking insect pests. While expression from the constitutive 35S promoter provides broad protection, the phloem-specific rolC promoter provides better defense against sap sucking insects. The selection of plant-derived insect resistance genes for expression in crop species will minimize bio-safety concerns. RESULTS: Pinellia ternata leaf agglutinin gene (pta), encodes an insecticidal lectin, was isolated and cloned under the 35S and rolC promoters in the pGA482 plant transformation vector for Agrobacterium-mediated tobacco transformation. Integration and expression of the transgene was validated by Southern blotting and qRT-PCR, respectively. Insect bioassays data of transgenic tobacco plants showed that expression of pta under rolC promoter caused 100% aphid mortality and reduced aphid fecundity up to 70% in transgenic tobacco line LRP9. These results highlight the better effectivity of pta under rolC promoter to control phloem feeders, aphids. CONCLUSIONS: These findings suggested the potential of PTA against aphids and other sap sucking insect pests. Evaluation of gene in tobacco under two different promoters; 35S constitutive promoter and rolC phloemspecific promoter could be successfully use for other crop plants particularly in cotton. Development of transgenic cotton plants using plant-derived insecticidal, PTA, would be key step towards commercialization of environmentally safe insect-resistant crops.


Subject(s)
Aphids/pathogenicity , Pest Control, Biological , Pinellia/chemistry , Plant Viruses , Tobacco , Blotting, Southern , Polymerase Chain Reaction , Promoter Regions, Genetic , Plants, Genetically Modified , Plant Leaves/chemistry , Transgenes , Disease Resistance , Crop Protection
2.
Mycobiology ; : 59-65, 2019.
Article in English | WPRIM | ID: wpr-760526

ABSTRACT

Agrobacterium tumefaciens-mediated transformation (ATMT), as a simple and versatile method, achieves successful transformation in the yeast-like cells (YLCs) of Tremella fuciformis with lower efficiency. Establishment of a more efficient transformation system of YLCs is important for functional genomics research and biotechnological application. In this study, an enzymolysis-assisted ATMT method was developed. The degradation degree of YLCs depends on the concentration and digestion time of Lywallzyme. Lower concentration (≤0.1%) of Lywallzyme was capable of formation of limited wounds on the surface of YLCs and has less influence on their growth. In addition, there is no significant difference of YLCs growth among groups treated with 0.1% Lywallzyme for different time. The binary vector pGEH under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter was utilized to transform the enzymolytic wounded YLCs with different concentrations and digestion time. The results of PCR, Southern blot, quantitative real-time PCR (qRT-PCR) and fluorescence microscopy revealed that the T-DNA was integrated into the YLCs genome, suggesting an efficient enzymolysis-assisted ATMT method of YLCs was established. The highest transformation frequency reached 1200 transformants per 106 YLCs by 0.05% (w/v) Lywallzyme digestion for 15 min, and the transformants were genetically stable. Compared with the mechanical wounding methods, enzymolytic wounding is thought to be a tender, safer and more effective method.


Subject(s)
Agrobacterium , Blotting, Southern , Digestion , Genome , Genomics , Methods , Microscopy, Fluorescence , Oxidoreductases , Polymerase Chain Reaction , Real-Time Polymerase Chain Reaction , Wounds and Injuries
3.
Electron. j. biotechnol ; 32: 6-12, Mar. 2018. tab, graf, ilus
Article in English | LILACS | ID: biblio-1022493

ABSTRACT

Background: Hydrophobins are small proteins secreted by filamentous fungi, which show a highly surface activity. Because of the signally self-assembling abilities and surface activities, hydrophobins were considered as candidates in many aspects, for example, stabilizing foams and emulsions in food products. Lentinus tuber-regium, known as tiger milk mushroom, is both an edible and medicinal sclerotium-producing mushroom. Up to now, the hydrophobins of L. tuber-regium have not been identified. Results: In this paper, a Class I hydrophobin gene, Ltr.hyd, was cloned from L. tuber-regium and expressed in the yeast-like cells of Tremella fuciformis mediated by Agrobacterium tumefaciens. The expression vector pGEH-GH was under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter. The integration of Ltr.hyd into the genome of T. fuciformis was confirmed by PCR, Southern blot, fluorescence observation and quantitative real-time PCR (qRT-PCR). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that recombinant hydrophobin rLtr.HYD with an expected molecular mass of 13 kDa was extracted. The yield of rLtr.HYD was 0.66 mg/g dry weight. The emulsifying activity of rLtr.HYD was better than the typical food emulsifiers sodium caseinate and Tween 20. Conclusions: We evaluated the emulsifying property of hydrophobin Ltr.HYD, which can be potentially used as a food emulsifier.


Subject(s)
Basidiomycota/metabolism , Fungal Proteins/genetics , Lentinula/genetics , Lentinula/metabolism , Transformation, Genetic , Basidiomycota/enzymology , Yeasts , Fungal Proteins/metabolism , Blotting, Southern , Cloning, Molecular , Agrobacterium tumefaciens/metabolism , Sequence Analysis , Emulsifying Agents , Electrophoresis, Polyacrylamide Gel , Real-Time Polymerase Chain Reaction , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Microscopy, Fluorescence
4.
Yonsei Medical Journal ; : 798-800, 2018.
Article in English | WPRIM | ID: wpr-716422

ABSTRACT

Unverricht-Lundborg disease (ULD) is a form of progressive myoclonus epilepsy characterized by stimulation-induced myoclonus and seizures. This disease is an autosomal recessive disorder, and the gene CSTB, which encodes cystatin B, a cysteine protease inhibitor, is the only gene known to be associated with ULD. Although the prevalence of ULD is higher in the Baltic region of Europe and the Mediterranean, sporadic cases have occasionally been diagnosed worldwide. The patient described in the current report showed only abnormally enlarged restriction fragments of 62 dodecamer repeats, confirming ULD, that were transmitted from both her father and mother who carried the abnormally enlarged restriction fragment as heterozygotes with normal-sized fragments. We report the first case of a genetically confirmed patient with ULD in Korea.


Subject(s)
Blotting, Southern , Cystatin B , Cysteine Proteases , Diagnosis , Europe , Fathers , Heterozygote , Humans , Korea , Mothers , Myoclonic Epilepsies, Progressive , Myoclonus , Prevalence , Seizures , Unverricht-Lundborg Syndrome
5.
Electron. j. biotechnol ; 25: 33-38, ene. 2017. tab, ilus
Article in English | LILACS | ID: biblio-1008414

ABSTRACT

Background: Banana (Musa spp.) is an important staple food, economic crop, and nutritional fruit worldwide. Conventional breeding has been seriously hampered by their long generation time, polyploidy, and sterility of most cultivated varieties. Establishment of an efficient regeneration and transformation system for banana is critical to its genetic improvement and functional genomics. Results: In this study, a vigorous and repeatable transformation system for banana using direct organogenesis was developed. The greatest number of shoots per explant for all five Musa varieties was obtained using Murashige and Skoog medium supplemented with 8.9 µM benzylaminopurine and 9.1 µM thidiazuron. One immature male flower could regenerate 380­456, 310­372, 200­240, 130­156, and 100­130 well-developed shoots in only 240­270 d for Gongjiao, Red banana, Rose banana, Baxi, and Xinglongnaijiao, respectively. Longitudinal sections of buds were transformed through particle bombardment combined with Agrobacterium-mediated transformation using a promoterless ß-glucuronidase (GUS) reporter gene; the highest transformation efficiency was 9.81% in regenerated Gongjiao plantlets in an optimized selection medium. Transgenic plants were confirmed by a histochemical assay of GUS, polymerase chain reaction, and Southern blot. Conclusions: Our robust transformation platform successfully generated hundreds of transgenic plants. Such a platform will facilitate molecular breeding and functional genomics of banana.


Subject(s)
Musa/growth & development , Musa/genetics , Regeneration , Transformation, Genetic , Immunohistochemistry , Blotting, Southern , Polymerase Chain Reaction , Plants, Genetically Modified , Agrobacterium tumefaciens/physiology , Musa/microbiology , Organogenesis, Plant , Glucuronidase
6.
Article in English | WPRIM | ID: wpr-151261

ABSTRACT

BACKGROUND/AIMS:: We demonstrated the role of caveolin-1 involved in high glucose (HG)-induced glomerular mesangial cells (GMCs) senescence. METHODS:: HG was used to stimulate GMCs. The telomere lengths were analyzed by Southern blot. β-Galactosidase staining was determined. The expressions of caveolin-1 and P53 proteins were determined by Western blot. RESULTS:: Treatment with high concentrations of glucose induced GMC senescence accompanied by shortened telomere length and increase of β-galactosidase staining as well as P53 protein, which was abrogated after application of caveolin-1-siRNA. CONCLUSIONS:: This study proved that HG induced cell senescence in GMCs. The caveolin-1 is involved in HG-induced mesangial cell senescence, and blocking caveolin-1 significantly reduced cell senescence. The effect of caveolin-1 is mediated by P53 pathway.


Subject(s)
Aging , Blotting, Southern , Blotting, Western , Caveolin 1 , Cellular Senescence , Glucose , Humans , Mesangial Cells , Telomere
7.
Article in Chinese | WPRIM | ID: wpr-345399

ABSTRACT

<p><b>OBJECTIVE</b>To study the pattern of CGG repeat instability within germline cells derived from two male fetuses affected with Fragile X syndrome (FXS).</p><p><b>METHODS</b>The length and methylation status of CGG repeats within the testes of a fetus carrying a full FXS mutation and another fetus carrying mosaicism FXS mutation were analyzed with Southern blotting and AmplideX FMR1 PCR. Immunohistochemistry was also applied for the measurement of FMR1 protein (FMRP) expression within the testes.</p><p><b>RESULTS</b>For the fetus carrying the full mutation, Southern blotting analysis of the PCR product has detected an expected band representing the full mutation in its brain and a premutation band of > 160 CGG repeats in its testis. Whereas the pattern of premutation/full mutation in mosaic testis was similar to that in peripheral blood and no sign of contracted fragment was found other than a band of about 160 CGG repeats. Immunohistochemistry assay with a FMRP-specific antibody demonstrated a number of FMRP-positive germ cells, which suggested a contraction from full mutation to premutation alleles.</p><p><b>CONCLUSION</b>This study has clarified the instability pattern of CGG repeat and expression of FMRP protein within the testes of fetuses affected with FXS, confirming that the CGG repeat can contract progressively within the germline. The FMRP expression in the testis is consistent with spermatogonium proliferation, and thus the contraction from full mutation to unmethylated premutations may occur for the requirement of FMRP expression during spermatogenesis. The better understanding of FMRP function during germ cell proliferation may elucidate the mechanism underlying the contraction of full FXS mutation in male germline.</p>


Subject(s)
Abortion, Eugenic , Blotting, Southern , Brain , Embryology , Metabolism , DNA Methylation , Fatal Outcome , Fetus , Cell Biology , Metabolism , Fragile X Mental Retardation Protein , Genetics , Metabolism , Fragile X Syndrome , Diagnosis , Genetics , Humans , Immunohistochemistry , Male , Mosaicism , Mutation , Polymerase Chain Reaction , Spermatozoa , Metabolism , Testis , Cell Biology , Embryology , Metabolism , Trinucleotide Repeat Expansion , Genetics
8.
Article in English | WPRIM | ID: wpr-164787

ABSTRACT

PURPOSE: We examined the prevalence and CGG/AGG repeat structure of expanded alleles of the FMR1 gene in preconceptional and pregnant Korean women. MATERIALS AND METHODS: The CGG repeats in the FMR1 genes of 1,408 women were analyzed by polymerase chain reaction and Southern blot analysis. To estimate the prevalence of expansion alleles, the individuals were divided into low risk and high risk group. RESULTS: Within this population, 98.4% had normal alleles and 1.6% had abnormal alleles including intermediate (0.6%), premutation (0.5%), full mutation (0.1%), and hemizygous (0.4%) alleles. There were 2 premutation alleles (1:666, 95% confidence interval [CI] 1:250-1,776) in the low risk group and 5 premutation alleles (1:15, 95% 1:6-36) in the high risk group. There were 8 intermediate alleles (1:167, 95% CI 1:130-213) in the low risk group and 1 intermediate alleles (1:76, 95% CI 1:11-533) in the high group. Six of the 7 premutation alleles did not contain AGG interruptions within the repeats and 1 had a single AGG interruption. Four of the 9 intermediate alleles contained 2-3 AGG, 4 had a single AGG, and 1 had no AGG interruptions. CONCLUSION: Our study demonstrates the prevalence and CGG/AGG structure of expansion alleles in Korean women. The identified premutation prevalence is higher than that of other Asian populations and lower than that of Caucasian populations. Although our study is limited by size and population bias, our findings could prove useful for genetic counseling of preconceptional or pregnant women.


Subject(s)
Alleles , Asian Continental Ancestry Group , Bias , Blotting, Southern , Carrier State , Female , Fragile X Syndrome , Gene Frequency , Genetic Counseling , Humans , Mass Screening , Polymerase Chain Reaction , Pregnant Women , Prevalence , Trinucleotide Repeat Expansion
9.
Electron. j. biotechnol ; 18(3): 161-168, May 2015. ilus, tab
Article in English | LILACS | ID: lil-750642

ABSTRACT

Background The high capacity of chloroplast genome response to integrate and express transgenes at high levels makes this technology a good option to produce proteins of interest. This report presents the stable expression of Pectin lyase (PelA gene) and the first stable expression of manganese peroxidase (MnP-2 gene) from the chloroplast genome. Results pES4 and pES5 vectors were derived from pPV111A plasmid and contain the PelA and MnP-2 synthetic genes, respectively. Both genes are flanked by a synthetic rrn16S promoter and the 3'UTR from rbcL gene. Efficient gene integration into both inverted repeats of the intergenic region between rrn16S and 3'rps'12 was confirmed by Southern blot. Stable processing and expression of the RNA were confirmed by Northern blot analysis. Enzymatic activity was evaluated to detect expression and functionality of both enzymes. In general, mature plants showed more activity than young transplastomic plants. Compared to wild type plants, transplastomic events expressing pectin lyase exhibited enzymatic activity above 58.5% of total soluble protein at neutral pH and 60°C. In contrast, MnP-2 showed high activity at pH 6 with optimum temperature at 65°C. Neither transplastomic plant exhibited an abnormal phenotype. Conclusion This study demonstrated that hydrolytic genes PelA and MnP-2 could be integrated and expressed correctly from the chloroplast genome of tobacco plants. A whole plant, having ~ 470 g of biomass could feasibly yield 66,676.25 units of pectin or 21,715.46 units of manganese peroxidase. Also, this study provides new information about methods and strategies for the expression of enzymes with industrial value.


Subject(s)
Polygalacturonase/genetics , Polygalacturonase/metabolism , Tobacco , Chloroplasts/genetics , Peroxidase/genetics , Peroxidase/metabolism , Temperature , Bacteria/enzymology , Transformation, Genetic , Cell Wall , Blotting, Southern , Polymerase Chain Reaction , Fungi/enzymology , Hydrogen-Ion Concentration , Hydrolases
10.
Electron. j. biotechnol ; 17(6): 296-303, Nov. 2014. ilus, graf, tab
Article in English | LILACS | ID: lil-730261

ABSTRACT

Background Jatropha curcas is a rich reservoir of pharmaceutically active terpenoids. More than 25 terpenoids have been isolated from this plant, and their activities are anti-bacterial, anti-fungal, anti-cancer, insecticidal, rodenticidal, cytotoxic and molluscicidal. But not much is known about the pathway involved in the biosynthesis of terpenoids. The present investigation describes the cloning, characterization and subcellular localization of isopentenyl diphosphate isomerase (IPI) gene from J. curcas. IPI is one of the rate limiting enzymes in the biosynthesis of terpenoids, catalyzing the crucial interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Results A full-length JcIPI cDNA consisting of 1355 bp was cloned. It encoded a protein of 305 amino acids. Analysis of deduced amino acid sequence predicted the presence of conserved active sites, metal binding sites and the NUDIX motif, which were consistent with other IPIs. Phylogenetic analysis indicated a significant evolutionary relatedness with Ricinus communis. Southern blot analysis showed the presence of an IPI multigene family in J. curcas. Comparative expression analysis of tissue specific JcIPI demonstrated the highest transcript level in flowers. Abiotic factors could induce the expression of JcIPI. Subcellular distribution showed that JcIPI was localized in chloroplasts. Conclusion This is the first report of cloning and characterization of IPI from J. curcas. Our study will be of significant interest to understanding the regulatory role of IPI in the biosynthesis of terpenoids, although its function still needs further confirmation.


Subject(s)
Carbon-Carbon Double Bond Isomerases/genetics , Carbon-Carbon Double Bond Isomerases/metabolism , Jatropha/enzymology , Jatropha/chemistry , Hemiterpenes/genetics , Hemiterpenes/metabolism , Phylogeny , RNA/isolation & purification , Gene Expression , Chloroplasts , Blotting, Southern , Cloning, Molecular , DNA, Complementary/chemical synthesis , Reverse Transcriptase Polymerase Chain Reaction
11.
Mem. Inst. Oswaldo Cruz ; 109(6): 712-715, 09/09/2014. graf
Article in English | LILACS | ID: lil-723999

ABSTRACT

The vanC1 gene, which is chromosomally located, confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Enterococcus faecium TJ4031 was isolated from a blood culture and harbours the vanC1gene. Polymerase chain reaction (PCR) assays were performed to detect vanXYc and vanTc genes. Only the vanXYc gene was found in the E. faecium TJ4031 isolate. The minimum inhibitory concentrations of vancomycin and teicoplanin were 2 µg/mL and 1 µg/mL, respectively. Real-time reverse transcription-PCR results revealed that the vanC1and vanXYc genes were not expressed. Pulsed-field gel electrophoresis and southern hybridisation results showed that the vanC1 gene was encoded in the chromosome. E. faecalis isolated from animals has been reported to harbour vanC1gene. However, this study is the first to report the presence of the vanC1gene in E. faecium of human origin. Additionally, our research showed the vanC1gene cannot serve as a species-specific gene of E. gallinarum and that it is able to be transferred between bacteria. Although the resistance marker is not expressed in the strain, our results showed that E. faecium could acquire the vanC1gene from different species.


Subject(s)
Humans , Bacterial Proteins/genetics , Enterococcus faecium/genetics , Genes, Bacterial/genetics , Vancomycin-Resistant Enterococci/genetics , Anti-Bacterial Agents/pharmacology , Blotting, Southern , Bacterial Proteins/blood , Electrophoresis, Gel, Pulsed-Field , Enterococcus faecalis/genetics , Enterococcus faecium/drug effects , Enterococcus/drug effects , Enterococcus/genetics , In Situ Hybridization/methods , Microbial Sensitivity Tests , Multilocus Sequence Typing , Multigene Family/physiology , Polymerase Chain Reaction , Teicoplanin/pharmacology , Vancomycin Resistance/genetics , Vancomycin/pharmacology
12.
Article in English | WPRIM | ID: wpr-110412

ABSTRACT

BACKGROUND: Clinical specificity and sensitivity are essential factors in the adoption of a human papillomavirus (HPV) test as a primary screening tool and test of cure after treatment of cervical cancer and precancerous lesions (High-Risk-Lesion). Using histologically-confirmed High-Risk-Lesion-patient specimens with postoperative follow-ups, we performed clinical validation of the AdvanSure GenoBlot Assay (GenoBlot; LG Life Sciences, Korea). METHODS: The study population included 100 cases with High-Risk-Lesion, 96 with high-risk genotype positive and cervical intraepithelial neoplasia (CIN) 1 or better, and 39 with HR-negative and better than CIN 1. Forty-eight High-Risk-Lesion cases received follow-up HPV exams after surgery. For validation as a test of cure, 48 preoperative specimens (PreOP) and 78 postoperative specimens (PostOP) from 48 subjects were separately analyzed. The results of HPV DNA chip tests (HPVDNAChip; BioMedLab Co., Korea) and sequencing were cross-compared. RESULTS: The concordance rates for each genotype between HPVDNAChip and GenoBlot were between 96.3-100%. The accuracy of HPVDNAChip and GenoBlot was 87.9% and 96.6%, respectively. Genotype-based specificity for High-Risk-Lesion detection was higher than 87% for both assays; genotype 16 showed the highest sensitivity. In the PostOP group, the positive rates for HPVDNAChip and GenoBlot were 30.8% and 47.4%, respectively. CONCLUSIONS: GenoBlot showed a higher positive rate than HPVDNAChip for each genotype, with concordance rate and accuracy being similar to previous reports. As a test of cure, GenoBlot performed better than the HPVDNAChip.


Subject(s)
Adolescent , Adult , Aged , Blotting, Southern , DNA, Viral/analysis , Female , Genotype , Humans , Middle Aged , Papillomaviridae/genetics , Papillomavirus Infections/diagnosis , Reagent Kits, Diagnostic , Real-Time Polymerase Chain Reaction , Sequence Analysis, DNA , Young Adult
13.
Article in English | WPRIM | ID: wpr-56430

ABSTRACT

Exotoxins produced by Actinobacillus (A.) pleuropneumoniae (Apx) play major roles in the pathogenesis of pleuropneumonia in swine. This study investigated the role of ApxI in hemolysis and cellular damage using a novel apxIA mutant, ApxIA336, which was developed from the parental strain A. pleuropneumoniae serotype 10 that produces only ApxI in vitro. The genotype of ApxIA336 was confirmed by PCR, Southern blotting, and gene sequencing. Exotoxin preparation derived from ApxIA336 was analyzed for its bioactivity towards porcine erythrocytes and alveolar macrophages. Analysis results indicated that ApxIA336 contained a kanamycin-resistant cassette inserted immediately after 1005 bp of the apxIA gene. Phenotype analysis of ApxIA336 revealed no difference in the growth rate as compared to the parental strain. Meanwhile, ApxI production was abolished in the bacterial culture supernatant, i.e. exotoxin preparation. The inability of ApxIA336 to produce ApxI corresponded to the loss of hemolytic and cytotoxic bioactivity in exotoxin preparation, as demonstrated by hemolysis, lactate dehydrogenase release, mitochondrial activity, and apoptosis assays. Additionally, the virulence of ApxIA336 appeared to be attenuated by 15-fold in BALB/c mice. Collectively, ApxI, but not other components in the exotoxin preparation of A. pleuropneumoniae serotype 10, was responsible for the hemolytic and cytotoxic effects on porcine erythrocytes and alveolar macrophages.


Subject(s)
Actinobacillus pleuropneumoniae/genetics , Animals , Apoptosis , Bacterial Proteins/genetics , Blotting, Southern , Exotoxins/genetics , Hemolysin Proteins/genetics , Hemolysis , Macrophages, Alveolar/metabolism , Polymerase Chain Reaction , Sequence Analysis, DNA , Swine , Virulence
14.
Chinese Medical Journal ; (24): 1084-1088, 2014.
Article in English | WPRIM | ID: wpr-253194

ABSTRACT

<p><b>BACKGROUND</b>Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystem disease caused by abnormal expansion of cytosine-thymine-guanine (CTG) repeats in the myotonic dystrophy protein kinase gene. The clinical manifestations of DM1 are multisystemic and highly variable, and the unstable nature of CTG expansion causes wide genotypic and phenotypic presentations, which make molecular methods essential for the diagnosis. So far, very few studies about molecular diagnosis in Chinese patients with DM1 have been reported. Therefore, we carried out a study using two different methods in molecular diagnosis to verify the validity in detecting CTG expansion in Chinese patients showing DM signs.</p><p><b>METHODS</b>A total of 97 Chinese individuals were referred for molecular diagnosis of DM1 using conventional polymerase chain reaction (PCR) accompanied by Southern blotting and triplet primed PCR (TP-PCR). We evaluated the sensitivity and limitation of each method using percentage.</p><p><b>RESULTS</b>By conventional PCR 65 samples showed only one fragment corresponding to the normal allele and 62 out of them were correctly diagnosed as DM1 by TP-PCR and three homologous non-DM1 samples were ruled out; Southern blotting analysis successfully made 13 out of 16 correct diagnoses with a more sensitivity using α-(32)P-labeled probes than dig-labeled probes.</p><p><b>CONCLUSION</b>Molecular analysis is necessary for the diagnosis of DM1 and TP-PCR is a reliable, sensitive, and easily performed method in molecular diagnosis which is worthy to be popularized.</p>


Subject(s)
Adult , Aged , Blotting, Southern , Female , Humans , Male , Middle Aged , Molecular Diagnostic Techniques , Methods , Myotonic Dystrophy , Diagnosis , Genetics , Polymerase Chain Reaction , Sensitivity and Specificity , Young Adult
15.
Article in English | WPRIM | ID: wpr-83944

ABSTRACT

PURPOSE: Myotonic dystrophy 1 (DM1, OMIM 160900) is an autosomal-dominant muscular disorder caused by an expansion of CTG repeats in the 3' UTR of the DMPK gene. Variable expansions of CTG repeats preclude the accurate determination of repeat size. We tried to show the clinical and analytical validity of the application of Southern blotting after long-range PCR was demonstrated in Korean DM1 patients. MATERIALS AND METHODS: The Southern blotting of long-range PCR was applied to 1,231 cases with clinical suspicion of DM1, between 2000 and 2011. PCR was performed using genomic DNA with forward 5'-CAGTTCACAACCGCTCCGAGC-3' and reverse 5'-CGTGGAGGATGGAACACGGAC-3' primers. Subsequently, the PCR fragments were subjected to gel electrophoresis, capillary transfer to a nylon membrane, hybridization with a labeled (CAG)10 probe. The correlation between clinical manifestations and the CTG repeat expansions were analyzed. RESULTS: Among a total of 1,231 tested cases, 642 individuals were diagnosed with DM1 and the range of the detected expansion was 50 to 2,500 repeats; fourteen cases with mild DM1 (75+/-14 repeats), 602 cases with classical DM1 (314+/-143 repeats), and 26 cases with congenital DM1 (1,219+/-402 repeats). The positive and negative predictive values were 100%. The age at test requested and the CTG repeat numbers were inversely correlated (R=-0.444, P<0.01). CONCLUSION: This study indicates that Southern blotting after long-range PCR is a reliable diagnostic method DM1.


Subject(s)
3' Untranslated Regions , Blotting, Southern , Chimera , Databases, Genetic , DNA , Electrophoresis, Capillary , Genetic Testing , Humans , Membranes , Myotonic Dystrophy , Nylons , Polymerase Chain Reaction
16.
Rio de Janeiro; s.n; 2013. 50 p. ilus, tab, graf.
Thesis in Portuguese | LILACS | ID: lil-751586

ABSTRACT

A leishmaniose tegumentar americana (LTA), em cães, costuma estar associada à resposta humoral baixa ou mesmo negativa, o que inviabiliza os métodos sorológicos convencionais, como ferramenta única no diagnóstico. [...] Métodos convencionais de diagnóstico parasitológico, não têm sido capazes de detectar a presença do parasito em outros sítios anatômicos diferentes da lesão cutânea, em cães naturalmente infectados por Leishmania (Viannia) braziliensis. Diante dos questionamentos sobre o papel do cão doméstico no ciclo de transmissão da LTA e sobre o valor de métodos diagnósticos aplicados na rotina, principalmente em áreas de sobreposição da forma tegumentar e visceral, faz-se necessário a avaliação desses animais sob diversos aspectos. O presente estudo teve como objetivo empregar a PCR específica associada à hibridização molecular e a técnica da reação em cadeia da polimerase com primer único em condições de baixa estringência (LSSP-PCR) visando detectar a presença de DNA parasitário e investigar a variabilidade genética de populações parasitárias presentes em diferentes tecidos de cães naturalmente infectados por L. (V.) braziliensis. Os animais foram selecionados entre os cães sororeatores para Leishmania procedentes de cidades do estado do Rio de Janeiro e encaminhados para eutanásia ao Laboratório de Pesquisa Clínica em Dermatozoonoses em Animais Domésticos do Instituto de Pesquisa Clínica Evandro Chagas da Fundação Oswaldo Cruz. Durante a necropsia, foram obtidas amostras de lesão cutânea, pele íntegra (região escapular e abdominal), baço, fígado e linfonodos (poplíteo e cervical)...


In dogs, American tegumentary leishmaniasis (ATL) is usually associated to a low humoral responseor even to negative results which turns not unfeasible the conventional serological methods. [...] Conventional methods ofparasitological diagnosis have failed to detect the presence of the parasite anatomic sites others thanthe cutaneous lesion, in dogs naturally infected by Leishmania (Viannia) braziliensis. Regarding thequestions on the rule of domestic dogs in the transmission cycle of ATL and, on the applicability ofdiagnostic methods mainly in areas where both visceral and tegumentary disease are found, theevaluation of these animals under different aspects is needed. The objective of this project is to applyspecific PCR assays associated to molecular hybridization and the technique of Low-StringencySpecific-Single Primer – PCR (LSSP-PCR) in order to detect the presence of parasite DNA andevaluate the genetic variability of parasite populations found in different tissues of dogs naturallyinfected by Leishmania (V.) braziliensis...


Subject(s)
Dogs , Leishmania braziliensis , Leishmaniasis, Cutaneous/diagnosis , Leishmaniasis, Cutaneous/epidemiology , Leishmaniasis, Cutaneous/transmission , Blotting, Southern , Polymerase Chain Reaction
18.
Chinese Medical Journal ; (24): 894-900, 2012.
Article in English | WPRIM | ID: wpr-269330

ABSTRACT

<p><b>BACKGROUND</b>The extended spectrum β-lactamase (ESBL)-producing Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) are the major pathogens causing pneumonia and have a significant impact on the clinical course. Limited data exist on molecular characterization of ESBL-producing E. coli and K. pneumoniae that cause pneumonia. The aim of this study was to investigate the comprehensive multilevel characteristics of E. coli and K. pneumoniae causing pneumonia in China for the first time.</p><p><b>METHODS</b>E. coli (17) and K. pneumoniae (21) isolates responsible for pneumonia were isolated from 1270 specimens collected in a prospective multi-center study in eight teaching hospitals in China from June to December in 2007. The susceptibilities, ESBL confirmation, sequence typing, blaCTX-M and blaSHV genes, their genetic environment and plasmid Inc/rep types were determined.</p><p><b>RESULTS</b>Sixteen E. coli (94.1%) and eleven K. pneumoniae (52.4%) isolates were ESBL producers. About 77.8% and 66.7% of them were resistance to ciprofloxacin and levofloxacin, and 100% were susceptible to imipenem. The most prevalent ESBL gene was CTX-M-14, followed by SHV-2, CTX-M-15, CTX-M-3, CTX-M-65, SHV-12, SHV-26 and SHV-28. SHV-1 and SHV-11 were also detected and coexisted with blaCTX-Ms in five strains, and three strains contained only SHV-1. All CTX-M-14 were detected ISEcp1 upstream and nine were found IS903 downstream and the majority of them (64.3%) were carried by IncF plasmids. All blaSHV were flanked by recF and deoR, located on IncF, IncN, IncX and IncH plasmids. Two SHV-2, one SHV-1 and the only SHV-28 were further preceded by IS26. Genes lacY and lacZ were detected at further upstream of two blaSHV-1. The K. pneumoniae carrying SHV-28 was susceptible to β-lactams, and no mutations or deletions in gene or promoter sequences were identified to account for susceptibility. Multilocus sequence typing experiments showed the ESBL-producing strains were genetically diverse.</p><p><b>CONCLUSIONS</b>The rate of occurrence of blaESBL in E. coli and K. pneumoniae causing pneumonia was high, and blaCTX-M-14 was dominant and probably mobilized by ISEcp1 mainly on IncF plasmids. Importantly, unexpressed blaESBL genes may occur in susceptible isolates and hence may have clinical implications.</p>


Subject(s)
Blotting, Southern , Escherichia coli , Genetics , Klebsiella pneumoniae , Genetics , Plasmids , Genetics , Pneumonia , Microbiology , Promoter Regions, Genetic , Genetics , Prospective Studies , beta-Lactams , Metabolism
19.
Article in English | WPRIM | ID: wpr-164982

ABSTRACT

Congenital myotonic dystrophy type 1 (DM1) presents severe generalized weakness, hypotonia, and respiratory compromise after delivery with high mortality and poor prognosis. We presented a congenital DM1 of premature twins in the 30th week of gestation. These twins were conceived by in vitro fertilization (IVF). Both babies presented apnea and hypotonia and had characteristic facial appearance. They were diagnosed DM1 by genetic method. They were complicated by chylothorax and expired at 100 and 215 days of age, respectively. Mother was diagnosed DM1 during the evaluation of babies. This is the first report on congenital DM1 which accompanied the chylothorax. More investigation on the association with chylothorax and congenital DM1 is recommended. With a case of severe neonatal hypotonia, congenital DM1 should be differentiated in any gestational age. Finally, since DM1 is a cause of infertility, we should consider DM1 in infertility clinic with detailed history and physical examination.


Subject(s)
Adult , Apnea/etiology , Blotting, Southern , Chylothorax/complications , Female , Fertilization in Vitro , Humans , Infant, Newborn , Infant, Premature , Microsatellite Repeats/genetics , Muscle Hypotonia/etiology , Myotonic Dystrophy/complications , Twins
20.
Article in English | WPRIM | ID: wpr-137172

ABSTRACT

PURPOSE: Fragile X carrier detection before or at early pregnancy through a wide screening program may not only confer a risk of having offspring with Fragile X syndrome (FXS), but may also confer a risk for Fragile X-associated primary ovarian insufficiency and Fragile X-associated tremor/ataxia syndrome. However, prior to the implementation of such a program, the carrier prevalence in a population and the availability of effective screening test should be evaluated. The aim of our study was to determine the prevalence of premutation carriers and to evaluate the feasibility of screening test. MATERIALS AND METHODS: The blood samples were obtained from 8,641 pregnant women with no family history of mental retardation. We performed a three-primer CGG repeat primed (RP) PCR using the AmplideX(TM) FMR1 PCR kit (Asuragen, Inc. Austin, TX, USA). Samples showing full mutation alleles were reflexed to Southern blot analysis for methylation status and sizing. RESULTS: Among the 8,641 women, we found 8 premutation carriers (1:1,090, 0.09%) and 46 women with an intermediate allele (1:190, 0.53%). No woman was found to carry the fully mutated allele. All the detected alleles were within the CGG repeat range of 8-117. Among the 8,641 samples, 29 and 30 CGG repeats represent 66.6% of all cases. The CGG RP PCR method provides robust detection of expanded alleles and resolves allele zygosity, thus minimizing the number of samples that require Southern blot analysis. CONCLUSION: This is the first study that has focused on the prevalence of FXS premutation carriers and FMR1 allele distribution in normal pregnant women. These data have important implications for population-based fragile X carrier screening in Korea.


Subject(s)
Alleles , Blotting, Southern , Female , Fragile X Syndrome , Humans , Intellectual Disability , Korea , Mass Screening , Methylation , Polymerase Chain Reaction , Pregnancy , Pregnant Women , Prevalence , Primary Ovarian Insufficiency , Reflex , Terpenes
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