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SYBR Green I (sybr + green_i)
Selected AbstractsState transitions of Vibrio parahaemolyticus VBNC cells evaluated by flow cytometry,CYTOMETRY, Issue 5 2008Tania Falcioni Abstract Background Vibrio parahaemolyticus, in response to environmental conditions, may be present in a viable but nonculturable state (VBNC), which can still be responsible for cases of infectious diseases in humans. Methods The characterization of the cellular states of V. parahaemolyticus during entry into, persistence in, and resuscitation from the VBNC state, was assessed through plate culture method and epifluorescence microscope evaluation of actively respiring cells. Flow cytometry (FCM) in combination with SYBR Green I (SG) and propidium iodide allowed us to distinguish between viable, dead, and damaged-cells. Immunofluorescence labeling detected by FCM was used to study changes in antibody affinity. Results Two groups of bacteria, one with High Nucleic Acid (HNA) and one having Low Nucleic Acid (LNA) content, were differentiated using SG and FCM and each was correlated with cell viability. With the aging of the microcosm, the LNA bacteria population increased while the HNA population gradually disappeared. Cytofluorimetric immunofluorescence analyses showed that the bacterial cell levels dropped from 95% at day 0 to 40% at day 26 and by day 29, antibody affinity was virtually lost. FCM analyses of light scatter signals expressed by cell population highlighted morphological changes indicating a reduction in cell size, as also shown by scanning electron microscopy images and variations in cell structure. Conclusions The methodology used has provided useful data in relation to the state transitions of V. parahaemolyticus regarding cell viability, antigenic surface components, and the quantification of morphological variations during its entry into the VBNC state. © 2008 Clinical Cytometry Society [source] Loop-mediated isothermal amplification targeting the apxIVA gene for detection of Actinobacillus pleuropneumoniaeFEMS MICROBIOLOGY LETTERS, Issue 1 2009Wang Yang Abstract Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions with high specificity and efficiency. We developed a diagnostic method based on LAMP for detection of Actinobacillus pleuropneumoniae. Using six specific primers targeting the apxIVA gene, the LAMP assay rapidly amplified the target gene within 30 min, requiring only a laboratory water bath for the reaction to occur. The resulting amplificon was visualized by adding SYBR Green I to the mixture. The results obtained from testing 15 A. pleuropneumoniae reference strains and other seven bacterial species strains showed that the LAMP was as specific as and 10 times more sensitive than nested PCR. Sixty-five tonsil samples were collected from 65 healthy pigs. All the samples were negative for A. pleuropneumoniae by immunomagnetic separation-based (IMS) bacterial isolation, nested PCR and LAMP, respectively. Meanwhile, 115 tonsil samples were also collected from 115 pigs with apparent respiratory problems. Twenty-two were positive by IMS bacterial isolation. All the samples that were positive by IMS bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated exceptionally higher sensitivity than nested PCR by picking up 14 additional positive cases (,2 test, P<0.0001); we concluded that LAMP was a highly sensitive and reliable method for detection of A. pleuropneumoniae infection. [source] A flow cytometry-based assay for measuring invasion of red blood cells by Plasmodium falciparum,AMERICAN JOURNAL OF HEMATOLOGY, Issue 4 2010Amy K. Bei Variability in the ability of the malaria parasite Plasmodium falciparum to invade human erythrocytes is postulated to be an important determinant of disease severity. Both the parasite multiplication rate and erythrocyte selectivity are important parameters that underlie such variable invasion. We have established a flow cytometry-based method for simultaneously calculating both the parasitemia and the number of multiply-infected erythrocytes. Staining with the DNA-specific dye SYBR Green I allows quantitation of parasite invasion at the ring stage of parasite development. We discuss in vitro and in vivo applications and limitations of this method in relation to the study of parasite invasion. Am. J. Hematol., 2010. © 2010 Wiley-Liss, Inc. [source] Rapid detection of bordetella pertussis by real-time PCR using SYBR green I and a LightCycler instrumentJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 5 2004S. K. Poddar Abstract A polymerase chain reaction (PCR) assay in real-time for detection of B. pertussis using SYBR green I as the reporter fluorophore and LightCycler instrument (a thermocycler coupled to a fluorescence detection device) was established and evaluated. The amplified amplicon using series diluted control prototype strain (ATCC strain #9797) of B. pertussis was analyzed for the fluorescent melting profile, and melting temperature (Tm) was determined. When examined, amplicons using a representative set of clinical isolates of B. pertussis were found to have the same Tm value (86 ± 0.5°C, the specificity parameter of detection) as the control prototype strain as expected. Amplified product was also analyzed and detected by agarose gel electrophoresis. The detection limit by fluorescent profile and Tm analysis was 10-fold better than that detected by agarose gel analysis. J. Clin. Lab. Anal. 18:265,270, 2004. © 2004 Wiley-Liss, Inc. [source] | |||||||||||||