OBJECTIVETo establish a molecular diagnostic method for rabies virus(RV) based on TaqMan PCR.

METHODSBaseD on the rabies virus nucleoprotein gene sequences published in GenBank, RV specific primers and probe were designed by Primer Premier 5.0. The primers and probe were optimized and the sensitivity, specificity,and reproducibility of the system were tested. Quantitative standard curve of RV TaqMan PCR was established. Some RV samples were detected using this system.

RESULTSThe optimized primers and probe were 0.6 micromol/L and 0.2 micromol/L. Reproducibility test showed that coefficient variables were all less than 5% in 4 different system. Quantification standard curve based on the genomic copy was drawn. RV detection using the established method proved that TaqMan PCR was more sensitive and easier performed than traditional RT-PCR.

CONCLUSIONTaqMan PCR for RV detection had been established, which was more sensitive and specific than the general RT-PCR.

Polymerase Chain Reaction ; methods ; Rabies ; diagnosis ; Rabies virus ; genetics ; Reproducibility of Results ; Taq Polymerase

BACKGROUND: Sporotrichosis is a common deep cutaneous fungal disease caused by Sporothtix (S.) schenckii. The recent development of polymerase chain reaction (PCR) technology, in particular, arbitrarily primed PCR (AP-PCR) or random amplified polymorphic DNA (RAPD), has greatly enhanced the molecular detection and identification of various pathogenic agents, including fungi. OBJECTIVE: This study was aimed to differentiate Sporothrix schenckii, and related fungi such as S. schenckii var. luriei, S. flocculosa, S. nivea, Ophiostoma stenoceras, and clinical isolates on the basis of distinct DNA band patterns in the RAPD. METHODS: S. schendcii, S. schenckii var. luriei, S. flocculosa, S. nivea, Ophiostoma stenoceras from ATCC and KCCM, and clinical 10 isolates from Chonnam University Hospital were used for RAPD analysis. For RAPD, 3 random primers were used. Genomic DNA was extracted by Liu method. Amplification reactions were performed in volumes of 50 microL containing 10 mM Tris-HCI (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton X-100, 200microM dNTP mixture, 40 pM primer, 1 U of Taq polymerase, DNA 20 ng. RESULTS: 3 decamers (5'-TGCCGAGCTG-3', 5'-AGTCAGCCAC-3', 5'-AATCGGGCTG-3') are generated in the RAPD, distinct DNA products from S. schenckii forming characteristic band patterns upon gel electrophoresis. Each random primer amplified characteristic same band patterns in DNA from clinical 8 isolates among 10 isolates, 2 isolates have different DNA band patterns. These results suggest of being a Sporothrix anamorph different from S. schenckii in Korea. CONCLUSION: With 2 random primers (5'-TGCCGAGCTG-3', 5'-AGTCAGCCAC-3') S. schenckii and related fungi investigated produced distinct DNA band patterns on gel electrophoresis. The RAPD was a very valuable laboratory method for identification of S. schenckii isolates.
DNA* ; Electrophoresis ; Fungi* ; Jeollanam-do ; Korea ; Magnesium Chloride ; Octoxynol ; Ophiostoma ; Polymerase Chain Reaction ; Sporothrix* ; Sporotrichosis ; Taq Polymerase

One step TaqMan real-time reverse transcription polymerase chain reaction (R/T RT-PCR)using a set of primers/probes was developed for the detection of foot-and-mouth disease (FMD)virus. The gene-specific probes labeled fluorogen for the internal ribosomal entry site, Leader sequence and 2B regions were used to detect FMD virus (FMDV). This assay specifically detected FMDV both in cell culture preparations and clinical samples, and was capable of distinguishing FMD from other viral diseases similar to clinical signs (swine vesicular disease, vesicular stomatitis and bovine viral diarrhea). This assay was shown to be 1000-fold more sensitive than the conventional RT-PCR method. The detection limits of this assay was 1 TCID 50 /ml of the FMDV RNA concentration. Quantification was obtained by a standard curves plotting threshold cycle values versus known infectivity titer. The assay was sensitive, specific and rapid enough to detect FMDV RNA genome in probang samples. As such, the described method is reliable and provides faster disease diagnostics than the conventional RT-PCR procedure to detect FMDV.
Animals ; Foot-and-Mouth Disease/*diagnosis/virology ; Foot-and-Mouth Disease Virus/*isolation&purification ; Reverse Transcriptase Polymerase Chain Reaction/*methods ; Sensitivity and Specificity ; Taq Polymerase

BACKGROUND: Dermatophytoses are infections of keratinized tissues, that is, the epidermis, hair and nails, caused by a group of specialized fungi, the dermatophytes. Laboratory diagnoses of dermatophytes such as Tricophyton, Microsporum and Epidermophyton are made by microscopic examination and in vitro culture but they are either time consuming of lacking specificity. OBJECTIVE: In order to develop and apply more rapid and precise diagnostic tests for fungal pathogens to facilitate the improved identification of dermatophytes, we investigated random amplified polymorphism DNA for classification and identification of dermatophytes. METHODS: Amplification reactions were performed in volumes of 5011 containing 10mM Tris-HCl(pH 8.3), 50mM KCl, 1.5mM MgCl2, 0.01% (w/v), gelatin, 200mM dNTP mixture, 50pM primer, Taq polymerase (0.025 units/ microliter), DNA 0.001 microgram/microliter. The optimal condition to. PCR was 2 cycles (denaturing 94 degrees C 2min, annealing 33 degrees C 2min, extension 72 degrees C 4min), 40 cycles, and extension (72 degrees C 10min). RESULTS: RAPD showed interspecies polymorphism in but it had identical patterns in intraspecies. CONCLUSION: It was confirmed that RAPD PCR analysis with optimal conditions is a fast, economical and reproducible method for identification and classification of dermatophytes isolates.
Arthrodermataceae* ; Classification* ; Clinical Laboratory Techniques ; Diagnostic Tests, Routine ; DNA* ; Epidermis ; Epidermophyton ; Fungi ; Gelatin ; Hair ; Magnesium Chloride ; Microsporum ; Polymerase Chain Reaction ; Sensitivity and Specificity ; Taq Polymerase ; Tinea

BACKGROUND: Methods to detect and quanitify Mycobacterium leprae(M. leprae)are needed for studies involving the epidemiology, pathogenesis, and chemotherapy of leprosy. Serological assays and skin tests lack the sensitivity and specificity to serve as diagnostic tool for M. leprae infection. The polymerase chain reaction(PCR) based on the selective amplification of an 530-bp frangment of the gene encoding the proline-rich antigen of M. leprae was performed with sections of fixed or frozen biopsy samples from leprosy patients. OBJECTIVE: This study was done to investigate the applicability of PCR for the detection of low numbers of M. leprae in tissues and peripheral blood. METHODS: The PCR was used to amplify a 530-base-pair M. leprae DNA with the thermoxtable Taq DNA polymerase. RESULTS: The In frozen skin tissues and peripheral blood of leprosy patients. relatively high detection rates of PCR products was achieved by using direct gel analysis as well as Southern blot hybridization. CONCLUSION: These results suggest that PCR amplification for the detection of M. leprae may be useful for the epidemiologic study of large papulations as well as coinical astudies on the individual patients.
Biopsy ; Blotting, Southern ; DNA ; Drug Therapy ; Epidemiologic Studies ; Epidemiology ; Humans ; Leprosy ; Mycobacterium leprae* ; Mycobacterium* ; Polymerase Chain Reaction* ; Sensitivity and Specificity ; Skin ; Skin Tests ; Taq Polymerase

BACKGROUND: Accurate typing of Duffy blood group is important because anti-Duffy antibodies cause hemolytic transfusion reaction and hemolytic disease of the newborn. The aim of this study was to evaluate a new genotyping method using high resolution melting (HRM) analysis, a rapid and inexpensive approach for high-throughput Duffy genotyping. METHODS: A total of 20 unrelated Korean blood samples were obtained and an African-black sample was used for GATA control. Phenotyping was performed by hemagglutination (DiaMed AG, Switzerland). GATA and FYA/B PCR products were obtained by PCR-restriction fragment length polymorphism (RFLP) using Taq DNA polymerase (Promega, WI) and enzymes BanI and StyI (New England Biolab, UK). For HRM, PCR amplification was performed using LightCycler 480 ResoLight Dye (Roche, USA) and Lightcycer 480 (Roche, USA). RESULTS: Phenotyping and genotyping data using PCR-RFLP and HRM analysis were compared. Different types of HRM curves were obtained according to genotypes, FYA/FYA, FYB/FYB, and FYA/FYB, and to GATA mutations, homozygote FYB-33T (T/T), heterozygote FYB-33T/33C (T/C), and homozygote FYB-33C (C/C). Phenotypes 18 Fy(a+b-), 1 Fy(a+b+), 1 Fy(a-b+), and 1 Fy(a-b-) showed complete concordance with genotyping methods. Fy(a-b-) sample was found to be a FYB-33C homozygote by both genotyping methods. CONCLUSION: Phenotyping and genotyping showed concordant results and both genotyping methods using PCR-RFLP and HRM analysis showed good agreement in finding mutation in GATA and FY gene coding regions. HRM analysis is suitable and reliable for high-throughput screening for Duffy genotyping.
Antibodies ; Blood Group Antigens ; Blood Group Incompatibility ; Clinical Coding ; England ; Freezing ; Genotype ; Hemagglutination ; Heterozygote ; Homozygote ; Humans ; Infant, Newborn ; Mass Screening ; Phenotype ; Polymerase Chain Reaction ; Taq Polymerase

OBJECTIVETo develop a real-time polymerase chain reaction(PCR) based on TaqMan technology by using a new MGB probe for detecting enterotoxigenic Escherichia coli (ETEC) in paper.

METHODSPrimers and MGB probe were designed in the ecoding region of heat-stable toxin of ETEC. Real-time PCR detected ETEC by using the exterior standard method with protracting standard curves. The specificity, sensitivity, accuracy, stability of real-time PCR system was evaluated. An internal negative antithesis was added to the real-time PCR system in order to get rid of the false positive of system. Using UNG enzyme expelled the contamination of PCR reaction.

RESULTSPrimers and MGB probe were suited to the Real-time PCR. The assay showed that the method was quick, special, sensitive and stable. The real-time PCR system could detect ETEC in a large scale. The assay might be finished in two hour.

CONCLUSIONThese observations suggested that real-time PCR based on MGB probe should be an excellent candidate for a standard ETEC detection method.

Bacterial Toxins ; isolation & purification ; DNA Primers ; DNA Probes ; DNA, Bacterial ; Enterotoxigenic Escherichia coli ; isolation & purification ; Molecular Probe Techniques ; Polymerase Chain Reaction ; methods ; Taq Polymerase

Primer Express 2.0 software was used to design the primers and the MGB probe. With the plasmid pHaMDR1/A containing mdr1 cDNA as the template, we established a real-time fluorescent quantitative polymerase chain reaction system, which, at the template concentration of 3.061 x 10(3) to 3.061 x 10(9) cps/ml, had a correlation coefficient of 0.988243 between template concentration and threshold cycle value. This PCR method allows sensitive, specific and quantitative detection of human mdr1 gene.
ATP-Binding Cassette, Sub-Family B, Member 1 ; analysis ; genetics ; DNA Primers ; Female ; Fluorescent Dyes ; Fluorometry ; methods ; Genes, MDR ; genetics ; Humans ; Male ; Polymerase Chain Reaction ; methods ; Taq Polymerase

OBJECTIVEIn this study, orthogonal design was used to optinize DDRT-PCR amplification system on Pinellia ternata microtubers in vitro in five factors four levels respectively.

METHODP. ternata stems and microtubers in vitro were selected as explants. The effects of five kinds of factors were studied by orthogonal design method including emplate cDNA, Mg2+, dNTPs, primers and Taq DNA polymerase, and in order to establish the optimum DDRT-PCR system of P. ternata microtubers in vitro.

RESULT AND CONCLUSIONA satisfactory DDRT-PCR technique system for P. ternata microtubers in vitro with desirable repeatability and polymorphic bands was established. In a total volume of 20 microL DDRT-PCR system, it contained 10 x buffer, 150 micromol L(-1) dNTPs, 2 micromol L(-1) anchor primer, 1 micromol L(-1) arbitrary primer, 2.5 mmol L(-1) Mg2+, 0.6 U Taq DNA polymerase and 2.5 microg template cDNA. The effect of the five factors was in sequence of Taq DNA polymerase > template cDNA > dNTPs > Mg2+ > Primers. The optimum DDRT-PCR system will provide scientific reference basis for studying effecting character of P. ternata microtubers associated with genes expression.

DNA, Complementary ; genetics ; DNA, Plant ; genetics ; Electrophoresis, Polyacrylamide Gel ; Pinellia ; genetics ; Plant Tubers ; genetics ; Polymerase Chain Reaction ; methods ; Silver Staining ; Taq Polymerase ; genetics

OBJECTIVETo establish a specific TaqMan-based Real-time PCR assay for the detection of hepatitis E virus (HEV).

METHODSAccording to the references, primers-probe sets which were located in ORF2, the conservative part of HEV genome were designed and therefore we established a HEV TaqMan real-time RT-PCR assay with great performance of specificity, sensitivity and reproducibility. And then it was used in the detection of HEV RNA in clinical samples.

RESULTSThe HEV Real-time RT-PCR assay established in this study were able to detect HEV RNA with a detection limit of 10 copies/reaction. When the detection of a same sample was repeated for several times, coefficients of variation (CV) was all less than 1.53%. Our data also suggested that there were 1.87 x 10(6)-8.12 x 10(9) RNA copies in 1 ml of the clinical samples.

CONCLUSIONThe TaqMan-based Real-time PCR assay established in this study was specific and precise for the rapid detection of HEV RNA. It was applied successfully in the pathogen detection of clinical samples.

DNA Primers ; genetics ; Hepatitis E ; virology ; Hepatitis E virus ; genetics ; isolation & purification ; Humans ; RNA, Viral ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; methods ; Taq Polymerase ; metabolism