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1.
J Clin Virol ; 61(4): 540-7, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25453331

RESUMO

BACKGROUND: High-risk (hr) human papillomavirus (HPV) infections play a causal role in the development of cervical cancer. The detection of hrHPV is, therefore, advocated in cervical cancer screening programs. OBJECTIVES: The aim of this study was to determine the performance of a novel HPV typing assay, PapillomaFinder® SMART 20. This is a one-tube-per-sample method, to be performed on standard real-time PCR platforms, using melting curve analysis to distinguish targets. The assay detects all 14 hrHPV types, of which 16, 18, 31, 33, 35, 39, 45, 52, 56 and 58 individually. HrHPV types 51, 59, 66 and 68 are detected in an hrHPV pool, and low-risk (lr) HPV types 6, 11, 40, 42, 43 and 44 in an lrHPV pool. STUDY DESIGN: The method was tested on HPV plasmid models, WHO and QCMD proficiency panels and a series of clinical cytological samples (n=45), the latter in comparison with a clinically validated real-time quantitative PCR. RESULTS: Type-specificity of the test was 100% using plasmids, the WHO and QCMD panels. Sensitivity for hrHPV in single infections was 100% using the WHO and QCMD panels and cytological samples, with an analytical sensitivity of 10-25 copies per reaction for all HPV types tested. Of the 34 HPV types present in the 8 multiple infections in the WHO panel, 30 were detected. In all cytological samples at least one hrHPV type was found, in concordance with the clinically validated method. Only when the viral load of the dominant HPV types in multiple infections greatly exceeded that of the other types in the infection, those other types were not always detected. CONCLUSIONS: PapillomaFinder® SMART 20 is a rapid, easy to perform, single tube HPV typing assay. The assay detects the 14 hrHPV types, and the 6 most important lrHPV types with a high sensitivity and type-specificity.


Assuntos
Técnicas de Genotipagem/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Papillomaviridae/classificação , Papillomaviridae/genética , Infecções por Papillomavirus/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Feminino , Humanos , Infecções por Papillomavirus/virologia , Sensibilidade e Especificidade , Fatores de Tempo
2.
J Clin Microbiol ; 47(8): 2620-2, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19553572

RESUMO

A multiplex ligation-dependent probe amplification assay for simultaneous detection of six virus species was developed and tested on clinical cerebrospinal fluid (CSF) samples. The assay, termed MeningoFinder, showed an accordance of 97%, concordance of 96%, interlaboratory sensitivity of 90%, and interlaboratory specificity of 94% compared to PCRs.


Assuntos
Técnicas Bacteriológicas/métodos , Infecções do Sistema Nervoso Central/virologia , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Viroses/diagnóstico , Vírus/isolamento & purificação , Líquido Cefalorraquidiano/virologia , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Vírus/genética
3.
Nat Biotechnol ; 16(13): 1365-9, 1998 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9853621

RESUMO

Mi-1, a Lycopersicon peruvianum gene conferring resistance to the agricultural pests, root-knot nematodes, and introgressed into tomato, has been cloned using a selective restriction fragment amplification based strategy. Complementation analysis of a susceptible tomato line with a 100 kb cosmid array yielded a single cosmid clone capable of conferring resistance both to the root-knot nematode Meloidogyne incognita and to an unrelated pathogen, the potato aphid Macrosiphum euphorbiae. This resistance was stable. The Mi-1 gene encodes a protein sharing structural features with the nucleotide-binding site leucine-rich repeat-containing type of plant resistance genes.


Assuntos
Afídeos , Genes de Plantas , Nematoides , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Solanum tuberosum/parasitologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cosmídeos , Teste de Complementação Genética , Solanum lycopersicum/parasitologia , Dados de Sequência Molecular , Proteínas de Plantas/química
4.
Plant Cell ; 10(6): 1055-68, 1998 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9634592

RESUMO

The I2 locus in tomato confers resistance to race 2 of the soil-borne fungus Fusarium oxysporum f sp lycopersici. The selective restriction fragment amplification (AFLP) positional cloning strategy was used to identify I2 in the tomato genome. A yeast artificial chromosome (YAC) clone covering approximately 750 kb encompassing the I2 locus was isolated, and the AFLP technique was used to derive tightly linked AFLP markers from this YAC clone. Genetic complementation analysis in transgenic R1 plants using a set of overlapping cosmids covering the I2 locus revealed three cosmids giving full resistance to F. o. lycopersici race 2. These cosmids shared a 7-kb DNA fragment containing an open reading frame encoding a protein with similarity to the nucleotide binding site leucine-rich repeat family of resistance genes. At the I2 locus, we identified six additional homologs that included the recently identified I2C-1 and I2C-2 genes. However, cosmids containing the I2C-1 or I2C-2 gene could not confer resistance to plants, indicating that these members are not the functional resistance genes. Alignments between the various members of the I2 gene family revealed two significant variable regions within the leucine-rich repeat region. They consisted of deletions or duplications of one or more leucine-rich repeats. We propose that one or both of these leucine-rich repeats are involved in Fusarium wilt resistance with I2 specificity.


Assuntos
Mapeamento Cromossômico , Proteínas de Ligação a DNA/genética , Fusarium/patogenicidade , Genes de Plantas , Família Multigênica , Solanum lycopersicum/genética , Solanum lycopersicum/microbiologia , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Artificiais de Levedura , Cosmídeos , Primers do DNA , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/química , Amplificação de Genes , Genoma de Planta , Imunidade Inata/genética , Dados de Sequência Molecular , Proteínas de Plantas/genética , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
5.
Nucleic Acids Res ; 23(21): 4407-14, 1995 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-7501463

RESUMO

A novel DNA fingerprinting technique called AFLP is described. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves three steps: (i) restriction of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification of sets of restriction fragments, and (iii) gel analysis of the amplified fragments. PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence. The method allows the specific co-amplification of high numbers of restriction fragments. The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution of the detection system. Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gels. The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity.


Assuntos
Impressões Digitais de DNA/métodos , Genoma , Sequência de Bases , DNA Ligases , Primers do DNA , DNA Bacteriano/genética , DNA Fúngico/genética , DNA de Plantas/genética , DNA Viral/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase
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