RESUMEN
AIM: Create a method for highly sensitive, selective, rapid and easy-to-use detection and identification of economically significant potato pathogens, including viruses, bacteria and oomycetes, be it single pathogen, or a range of various pathogens occurring simultaneously. METHODS AND RESULTS: Test-systems for real-time PCR, operating in the unified amplification regime, have been developed for Phytophthora infestans, Pectobacterium atrosepticum, Dickeya dianthicola, Dickeya solani, Ralstonia solanacearum, Pectobacterium carotovorum, Clavibacter michiganensis subsp. sepedonicus, potato viruses Y (ordinary and necrotic forms as well as indiscriminative test system, detecting all forms), A, X, S, M, potato leaf roll virus, potato mop top virus and potato spindle tuber viroid. The test-systems (including polymerase and revertase) were immobilized and lyophilized in miniature microreactors (1·2 µl) on silicon DNA/RNA microarrays (micromatrices) to be used with a mobile AriaDNA® amplifier. CONCLUSIONS: Preloaded 30-reaction micromatrices having shelf life of 3 and 6 months (for RNA- and DNA-based pathogens, respectively) at room temperature with no special conditions were successfully tested on both reference and field samples in comparison with traditional ELISA and microbiological methods, showing perfect performance and sensitivity (1 pg). SIGNIFICANCE AND IMPACT OF THE STUDY: The accurate, rapid and user-friendly diagnostic system in a micromatrix format may significantly contribute to pathogen screening and phytopathological studies.
Asunto(s)
Pectobacterium carotovorum/aislamiento & purificación , Phytophthora infestans/aislamiento & purificación , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/virología , Ralstonia solanacearum/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Virus/aislamiento & purificación , Cartilla de ADN/genética , Pectobacterium carotovorum/genética , Pectobacterium carotovorum/fisiología , Phytophthora infestans/clasificación , Phytophthora infestans/genética , Ralstonia solanacearum/genética , Ralstonia solanacearum/fisiología , Solanum tuberosum/microbiología , Solanum tuberosum/virología , Virus/clasificación , Virus/genéticaRESUMEN
The clinical observation of the successful treatment of newly diagnosed fibro-cavernous pulmonary tuberculosis caused by mycobacteria of multidrug-resistant tuberculosis and the results of control of the effectiveness of treatment with modern high-tech radiation methods are presented. The necessity of an individual approach to the treatment of tuberculosis in this category of patients is shown.
Asunto(s)
Antituberculosos/administración & dosificación , Broncoscopía/métodos , Pulmón/diagnóstico por imagen , Mycobacterium tuberculosis , Toracoplastia/métodos , Tuberculosis Resistente a Múltiples Medicamentos , Tuberculosis Pulmonar , Adulto , Antituberculosos/clasificación , Terapia Combinada/métodos , Humanos , Cuidados a Largo Plazo/métodos , Cuidados a Largo Plazo/organización & administración , Masculino , Mycobacterium tuberculosis/aislamiento & purificación , Mycobacterium tuberculosis/patogenicidad , Mycobacterium tuberculosis/fisiología , Tomografía Computarizada por Rayos X/métodos , Resultado del Tratamiento , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico , Tuberculosis Resistente a Múltiples Medicamentos/radioterapia , Tuberculosis Pulmonar/diagnóstico , Tuberculosis Pulmonar/tratamiento farmacológico , Tuberculosis Pulmonar/radioterapiaRESUMEN
Single nucleotide polymorphism (SNP) genotyping methods are widely used for the detection of hereditary thrombophilias caused by genetic defects in the coagulation system. The hereditary thrombophilias are frequently associated with higher incidences of point mutations in hemostasis (F2 20210G>A, F5 1691G>A) and folate metabolism (MTHFR 677C>Т, MTHFR 1298A>C) genes. Moreover, the combination of gene abnormalities in F2 or/and MTHFR with F5 Leiden mutation leads to increased risk of developing thrombosis. Thus, simultaneous detection of the multiple gene mutations in a sample has important clinical relevance. The microchip-based multiplex real time PCR for estimation of allele specific polymorphism in hemostatic and folate metabolism genes presented here has a high efficiency and may be used for laboratory diagnosis. The optimized protocol for estimation of 4 different types of genetic polymorphisms allowed PCR to be performed with minimal quantity of DNA template and PCR reagents including Taq polymerase and a short-term thermocycling.