• 제목/요약/키워드: PCR primers

검색결과 1,532건 처리시간 0.025초

Multiplex Polymerase Chain Reaction Assay for Simultaneous Detection of Candida albicans and Candida dublinensis

  • Lim, Young-Hee;Lee, Do-Hyun
    • Journal of Microbiology
    • /
    • 제40권2호
    • /
    • pp.146-150
    • /
    • 2002
  • A multiplex polymerase chain reaction (PCR) assay was developed for the identification of two Candida species-albicans and dubliniensis. Three sets of primers were selected from different genomic sequences to specifically amplify a 516 bp fragment within the tops gene, specific for several species of the genus Candida (CCL primers); a 239 bp fragment within the $\alpha$INT1 gene, specific for Candida albicans (CAL primers); and a 175 bp fragment within the ALSD1 gene, specific for Candida dubliniensis (CDL primers). Using the primers in conjunction (multiplex PCR), we were able to detect both C. albicans and C. dubliniensis and to differentiate between them. The detection limit of the PCR assay was approximately 10 cells per milliliter of saline. Thus, this multiplex PCR assay can be applied for differentiation of C. albicans and C. dubliniensis from clinical specimens.

중합효소연쇄반응(Polymerase Chain Reaction)을 이용한 Fusobacterium nucleatum 및 Fusobacterium necrophorum의 동점에 관한 연구 (IDENTIFICATION OF FUSOBACTERIUM NUCLEATUM AND FUSOBACTERIUM NECROPHORUM USING POLYMERASE CHAIN REACTION(PCR))

  • 강창우;박동성;윤수한
    • Restorative Dentistry and Endodontics
    • /
    • 제24권1호
    • /
    • pp.40-48
    • /
    • 1999
  • This study was designed to examine the specificities of the designed primers for F. nucleatum and F. necrophorum and to compare the PCR results using clinical samples with those of the anaerobic culture method. F. nucleatum and F. necrophorum spp. are frequently isolated in infected root canals, and they are related to periapical diseases. F. nucleatum(VPI 10197) and F. necrophorum(ATCC 25286) were used as references for PCR reaction, and thirty five teeth with one canal and periapical lesion were used. The samples were cultured anaerobically and identified using Rapid ID 32A(BioMerieux Vitek, Inc., France) as biochemical battery. In the GenBank database, species-specific PCR primers(nuc1/nuc2 primers for F. nucleatum and nec1/nec2 primers for F. necrophorum) were designed from the 16S ribosomal DNA(rDNA) sequences of F. nucleatum(accession number M58683) and F. necrophorum(accession number AF044948). PCR procedures of F. nucleatum(VPI 10197) and F. necrophorum (ATCC 25286) were simulated on a computer software. Amplify(v.1.2${\beta}$ for Macintosh). 820 bps and 817 bps of nucleotides were expected, respectively. Using extracted DNAs with QiaAmp tissue kit(Qiagen co., Germany), PCR was done. The results were as follows : 1. The nuc1/nuc2 primers produced an amplicon of 820 bps and the nec1/nec2 primers produced an amplicon of 817 bps. 2. The nuc1/nuc2 primers and the nec1/nec2 primers were specific and did not react with species other than the designated ones(i.e. nuc1/nuc2 primers did not produce amplicons for F. necrophorum, and vice versa.). And the PCR products of Porphyromonas endodontalis(ATCC 35406), Porphyromonas gingivalis(ATCC 33277), Prevotella intermedia(ATCC 25611), and Prevotella nigrescens(ATCC 33563), frequently isolated in infected root canals and periapical lesions, were not amplified by the primers specific for Fusobacterium nucleatum and Fusobacterium necrophorum. 3. This method utilizing PCR could detect F. nucleatum and F. necrophorum in clinical samples, while anaerobic culture method could detect neither.

  • PDF

Multiplex RT-PCR Assay for the Detection of Apple stem grooving virus and Apple chlorotic leaf spot virus in Infected Korean Apple Cultivars

  • Park, Hong-Lyeol;Yoon, Jae-Seung;Kim, Hyun-Ran;Baek, Kwang-Hee
    • The Plant Pathology Journal
    • /
    • 제22권2호
    • /
    • pp.168-173
    • /
    • 2006
  • To develop the diagnostic method for the viral infection in apple, the partial genes corresponding to the N-terminal region of RNA polymerase of Apple stem grooving virus (ASGV) and coat protein of Apple chlorotic leaf spot virus (ACLSV) were characterized from the infected apple cultivars in Korea. Based on the nucleotide sequences of the characterized partial genes, the virus gene-specific primers were designed for the detection of ASGV and ACLSV infected in species of Malus. The RT-PCR using the primers for the genes of ASGV and ACLSV successfully gave rise to 404 and 566 bp DNA fragments, respectively. Using those viral gene-specific primers, the multiplex RT-PCR assays were also established to diagnose the mixed infection by ASGV and ACLSV simultaneously. Furthermore, the control primers, which have to be included for the RT-PCR as an internal control, were designed using the nucleotide sequence of the gene encoding elongation factor $1{\alpha}(EF1{\alpha})$. This multiplex RT-PCR including the control primers provides more reliable, rapid and sensitive assay for the detection of ASGV and ACLSV infected in Korean apple cultivars.

PCR기법을 이용한 오이 모자이크 바이러스 개나리 분리주(CMV-Fk)의 동정과 구분 (Identification and Differentiation of Cucumber Mosaic Virus Isolated from Forsythia koreana (CMV-Fk) Using PCR Techniques)

  • 이상용;박선정;최장경
    • 한국식물병리학회지
    • /
    • 제14권4호
    • /
    • pp.308-313
    • /
    • 1998
  • Reverse transcription and polymerase chain reaction (RT-PCR) techiniques were used to identification and differentiation of cucumber mosaic virus isolated from Forsythia koreana (CMV-Fk). RT-PCT used by two set of 20-mer primers one was CMV-common primers and another was CMV subgroup I-specific primers designed in a conserved region of the 3' end of CMV RNA3, amplified about 490 bp and 200 bp DNA fragments from CMV-Fk, respectively. CMV could be detected by RT-PCR at a dilution as low as 10-4 in forsythia crude sap extracts. Restriction enzyme analysis of RT-PCR products using EcoRI and MspI showed that CMV-Fk belonged to CMV subgroup I. But, analysis of RNA fingerprinting by arbitrarily primed polymerase chain reaction (RAP-PCR) showed heterogeneity of RNA3 between CMV-Fk and CMV-Y as a member of subgroup I.

  • PDF

Rapid Screening of Apple mosaic virus in Cultivated Apples by RT-PCR

  • Ryu, Ki-Hyun;Park, Sun-Hee
    • The Plant Pathology Journal
    • /
    • 제19권3호
    • /
    • pp.159-161
    • /
    • 2003
  • The coat protein (CP) gene of Apple mosaic virus (ApMV), a member of the genus Ilarvirus, was selected for the design of virus-specific primers for amplification and molecular detection of the virus in cultivated apple. A combined assay of reverse transcription and polymerase chain reaction (RT-PCR) was performed with a single pair of ApMV-specific primers and crude nucleic acid extracts from virus-infected apple for rapid detection of the virus. The PCR product was verified by restriction mapping analysis and by sequence determination. The lowest concentration of template viral RNA required for detection was 100 fg. This indicates that the RT-PCR for detection of the virus is a 10$^3$times more sensitive, reproducible and time-saving method than the enzyme-linked immunosorbent assay. The specificity of the primers was verified using other unrelated viral RNAs. No PCR product was observed when Cucumber mosaic virus (Cucumovirus) or a crude extract of healthy apple was used as a template in RT-PCR with the same primers. The PCR product (669 bp) of the CP gene of the virus was cloned into the plasmid vector and result-ant recombinant (pAPCP1) was selected for molecule of apple transformation to breed virus-resistant transgenic apple plants as the next step. This method can be useful for early stage screening of in vitro plantlet and genetic resources of resistant cultivar of apple plants.

PCR법에 의한 잠실먼지 중 핵다각체병 바이러스의 검출 (Detection of Bombyx mori Nuclear Polyhedrosis Virus(BmNPV) in dust of Silkworm rearing room by PCR)

  • 남성희;한명세
    • 한국잠사곤충학회지
    • /
    • 제39권1호
    • /
    • pp.30-35
    • /
    • 1997
  • A rapid and sensitive detection of BmNPV contamination in silkworm rearing room was carried by Plymerase chain reaction(PCR). Silkworm nuclear polyhedra were dissolved for the extraction of viral DNA within 30 minutes followed by the treatment of alkaline solution. The combination of primers of NP3 and NP2 was superior in PCR to the other 7 primers applied. Each primer was designed with 20 base in size and Newly designed NP3 of sense and the already reported NP2 for antisense were better in reaction than other primers. PCR products appeared 500bp in size. And annealing was confirmed proper at 55$^{\circ}C$ condition. Amplifiable template DNA amount was confirmed at least 100 ng to 0.1 ng and regarded as applicative for the assay of silkworm rearing environmental condition of sericultural farm. In case of the detection of BmNPV from the dust, sensitivity by PCR was as high as 1,000,000 times than that of microscopic observation.

  • PDF

Fingerprinting of Rice Genomes Using PCR with Arbitrary Primers

  • Park, Kyong-Hee
    • Preventive Nutrition and Food Science
    • /
    • 제3권2호
    • /
    • pp.198-202
    • /
    • 1998
  • The arbitrary primed polymerase chain reaction (AP-PCR) has been used to detect the genetic alternations in the related species. Simple and reproducible fingerprints of complex genomes can be generated using single arbitrary chosen primers and the PCR. The technique was applied to the Oryza species and characterized the relationship among three cultivars of rice species based on theresult of genomic DNA fingerprints. The results indicated that the polymorphism revealed in rice strains and the differences in the PCR product pattern could be represented for each strainis. There was many variationsin the PCR product pattern between cv. Dongin(japonica type)and cv.Hyangdo (indica type), and our chosen AP-primers can ge as markers for strain identification and verfication.

  • PDF

PCR Based Detection of Phellinus linteus using Specific Primers Generated from Universal Rice Primer(URP) Derived PCR Polymorphic Band

  • Kang, Hee-Wan;Park, Dong-Suk;Park, Young-Jin;Lee, Byoung-Moo;Cho, Soo-Muk;Kim, Ki-Tae;Seo, Geon-Sik;Go, Seung-Joo
    • Mycobiology
    • /
    • 제30권4호
    • /
    • pp.202-207
    • /
    • 2002
  • This study was carried out to develop specific primers for PCR detection of Phellinus linteus. Diverse genomes of 15 Phellinus spp. including five Phellinus linteus isolates were fingerprinted by Primer Universal rice primer(URP)1F. The URP-PCR pattern differentiated P. linteus isolates from other phellinus spp. A polymorphic band(2.8 kb), which is unique for P. linteus isolates, was isolated and sequenced. Twenty four-oligonucleotide primer pairs were designed based on information of DNA sequence. The primer set(PLSPF2/PLSPR1) amplified single band(2.2 kb) of expected size with genomic DNA from seven Phellinus linteus, but not with that of other Phellinus species tested. The primers could be used identically in both DNA samples from mycelium and fruit bodies. This specific primers could offer a useful tool for detecting and identifying P. linteus rapidly.

Pathogenic and Molecular Characteristics of Agrobacterium vitis strains isolated from Grapevine in Korea

  • Kim, J.G.;Kim, S.H.;Choi, J.E.;Lee, Y.K.;Kang, H.W.
    • 한국식물병리학회:학술대회논문집
    • /
    • 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
    • /
    • pp.120.2-120
    • /
    • 2003
  • Agrobacterium vitis is a causal agent of crown-gall disease on grapevine. In Korea, grapevine variety (GeoBong) have severely been infected by the bacteria since stems of the variety were buried in soil for overwintering. Infection ratio over 70-80% was observed on 7 years old GeoBong grapevine in Ansung and Cheonan. PCR specific primers for A. vitis strains were designed using nucleotide sequences of vir A gene in Ti-Plasmid, pheA gene in chromosomal DNA and a URP-PCR polymorphic band. Three hundred bacterial strains were isolated from the different 80 galls formed on GeoBong grapevine in Cheonan and Ansung of Korea and were screened to identify A. vitis using the three specific PCR primers for Agrobacterium vitis. Twenty-four bacterial strains that are detected by the primers were further confirmed by pathogenicity and biochemical methods. To investigate the genomic diversity of the bacterial strains, twenty primers of 20 mer referred to universal rice primers (URP) were applied for PCR fingerprinting, Of them, URP2R and URP2F primers could effectively be used to detect polymorphism within the bacterial strains.

  • PDF

AP-PCR을 이용한 다제내성 Staphylococcus aureus의 유전형 분석 (Genotypic Analysis of Multi-drug Resistant Staphylococcus aureus by Arbitrarily Primed Polymerase Chain Reaction)

  • 신경현;홍승복;손승렬
    • 대한임상검사과학회지
    • /
    • 제36권2호
    • /
    • pp.89-97
    • /
    • 2004
  • Many strains of Staphylococcus aureus were isolated from pus samples from primary, secondary, and tertiary medical institutions and were subjected to an antibiotic sensitivity test. Ciprofloxacin, clindamycin, erythromycin, gentamicin, oxacillin penicillin, tetracycline, trimethoprim/sulfamethoxazole, vancomycin and teicoplanin were used for the antibiotic sensitivity test. The strains showed hightest resistance to penicillin(91%), but all of strains tested were susceptible to vancomycin and teicoplanin. The isolated multi-drug(penicillin-tetracycline-ciprofloxacin-clindamycin-erythromycin- oxacillin-gentamicin) resistant S. aureus were analyzed genotypically using an AP-PCR(Arbitrarily Primed polymerase chain reaction) with an arbitrary 3 primers. Based on the result for genotype analysis, the genotypes identified by S1 primer did not coincide with those of S2 or E2 primers. Genotypes identified by S2 primer did not coincide with those of S1 or E2 primers. Also genotypes identified by the E2 primer did not coincide with those of S1 or S2 primers. Therefore, an analysis of AP-PCR test with multiple primers will provide more sensitive identification. A strain from a secondary medical institution and a strain from a tertiary medical institution which showed the same genotype for S1, S2, and E2 primers are required for further epidemiological study.

  • PDF