Rapid detection of M. bovis and M. avium in cytological smears and tissue sections by PNA-FISH

Background: Bovine Tuberculosis is globally the paramount cause of death from single pathogen in cattle and other species. Rapid and explicit identification of mycobacteria is essential to hold back tuberculosis in bovines. We performed a fluorescence Peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) procedure for specific detection of Mycobacterium bovis and Mycobacterium avium in bovine was optimized on cytological smears and tissue sections of bovines suspected for bovine tuberculosis.Results: PNA-FISH was performed on lung and lymph node tissues impression smears. The probes were standardized for standard bovine mycobacterial cultures at 50% formamide concentration for M.bovis and 30% formamide concentration for M.avium. All the cytological smears were positive from M.bovis probe (MTBCcy3) which was standardized at hybridization conditions of (55oC and 40% formamide) concentrations. Results revealed four out of twenty five were positive in tissue sections with a bright red fluorescence with cy3 filter (MTBC probe). No results were seen with (MAVTAMRA) probe for M.avium which was standardized at hybridization conditions of (55oC and 30% formamide) respectively. No fluorescence was seen in control tissue sections .In addition, results were juxtaposed to other commonly used detection methods like IHC and PCR by targeting esxA gene. None of the sample was found positive for M. avium.Conclusion: PNA-FISH can be used in cytological impression smears and tissue sections. It is less time consuming in diagnosis of bTB in post mortem cases than PCR.

Rapid Detection of M. Bovis and M. Avium in Cytological Smears and Tissue Sections by PNA-FISH

Background: Bovine Tuberculosis is globally the paramount cause of death from single pathogen in cattle and other species. Rapid and explicit identification of mycobacteria is essential for the control of bovine tuberculosis. We performed a fluorescence Peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) procedure for specific detection of Mycobacterium bovis and Mycobacterium avium in bovine was optimized on cytological smears and tissue sections of bovines suspected for bovine tuberculosis.Results: PNA-FISH was performed on lung and lymph node tissues impression smears. The probes were standardized for standard bovine mycobacterial cultures at 50% formamide concentration for M.bovis and 30% formamide concentration for M.avium. All the cytological smears were positive from M.bovis probe (MTBCcy3) which was standardized at hybridization conditions of (55oC and 40% formamide) concentrations. Results revealed 4 out of 25 were positive in tissue sections with a bright red fluorescence with cy3 filter (MTBC probe). No results were seen with (MAVTAMRA) probe for M.avium which was standardized at hybridization conditions of (55oC and 30% formamide) respectively. No fluorescence was seen in control tissue sections .In addition, results were compared to other commonly used detection methods like IHC and PCR by targeting esxA gene. None of the sample was found positive for M. avium.Conclusion: PNA-FISH can be used in cytological impression smears and tissue sections. It is less time consuming in diagnosis of bTB in post mortem cases than PCR.

Optimization and Application of Fluorescence in Situ Hybridization Assay for Detecting Polyphosphate - Accumulating Microorganisms

Laboratory-scale sequencing batch reactors (SBRs) were operated on activated sludge processes were used to study enhanced biological phosphorus removal (EBPR) from wastewater. Polyphosphate-accumulating microorganisms (PAOs) play an important role during the enhanced biological phosphorus removal (EBPR) process. Fluorescence in situ hybridization (FISH) was applied to assess the proportions of microorganisms in the sludge. The aim of this study was to optimize hybridization of PAOMIX and RHC439 probes by orthogonal design. Orthogonal optimization test of the four factors were conducted under the individual three levels. The optimal hybridizition conditions were as follow: hybridization temperature 46°C, hybridization time 2.5h, washing time 15min, formamide concentration 35%(PAOMIX probe); hybridization temperature 50°C, hybridization time 2.5h, washing time 20min, formamide concentration 20% (RHC439 probe).

HIERARCHICAL Zn5(OH)6(CO3)2 STRUCTURE ASSEMBLED IN AN AQUEOUS SOLUTION

Hydrothermal technique is demonstrated for achieving Zn 5( OH )6( CO 3)2 microstructure consisting of nanosheets on zinc foils in the presence of formamide. Formamide concentration and reaction temperature have significant influences on the size and shape of hierarchical Zn 5( OH )6( CO 3)2 structures. A self-assembly mechanism is proposed to explain the growth of Zn 5( OH )6( CO 3)2 microstructure, which can be transformed into sheet-like dense ZnO architectures without loss of the shape features by calcining in air.

Emission Characteristics of Fluorescent Labels with Respect to Temperature Changes and Subsequent Effects on DNA Microchip Studies

ABSTRACT The effects of temperature, salt concentration, and formamide concentration on the emission characteristics of commonly used fluorescent labels were evaluated on DNA microchips. The emission intensities of different fluorophores without hybridization were observed to vary, each to a different extent, to mainly temperature changes. Rhodamine red, TAMRA (tetramethylrhodamine), and dyes from the carbocyanide group exhibited the largest variations, and Texas Red and Oregon Green exhibited the smallest variations. This temperature dependency was shown to affect results obtained during melting curve analysis in DNA microarray studies. To minimize the bias associated with the temperature-dependent emission of different fluorescent labels, a normalization step was proposed.

Sequence-Specific Cleavage of Small-Subunit (SSU) rRNA with Oligonucleotides and RNase H: a Rapid and Simple Approach to SSU rRNA-Based Quantitative Detection of Microorganisms

ABSTRACT A rapid and simple approach to the small-subunit (SSU) rRNA-based quantitative detection of a specific group of microorganisms in complex ecosystems has been developed. The method employs sequence-specific cleavage of rRNA molecules with oligonucleotides and RNase H. Defined mixtures of SSU rRNAs were mixed with an oligonucleotide (referred to as a “scissor probe”) that was specifically designed to hybridize with a particular site of targeted rRNA and were subsequently digested with RNase H to proceed to sequence-dependent rRNA scission at the hybridization site. Under appropriate reaction conditions, the targeted rRNAs were correctly cut into two fragments, whereas nontargeted rRNAs remained intact under the same conditions. The specificity of the cleavage could be properly adjusted by controlling the hybridization stringency between the rRNA and the oligonucleotides, i.e., by controlling either the temperature of the reaction or the formamide concentration in the hybridization-digestion buffer used for the reaction. This enabled the reliable discrimination of completely matched rRNA sequences from single-base mismatched sequences. For the detection of targeted rRNAs, the resulting RNA fragment patterns were analyzed by gel electrophoresis with nucleotide-staining fluorescent dyes in order to separate cleaved and intact rRNA molecules. The relative abundance of the targeted SSU rRNA fragments in the total SSU rRNA could easily be calculated without the use of an external standard by determining the signal intensity of individual SSU rRNA bands in the electropherogram. This approach provides a fast and easy means of identification, detection, and quantification of a particular group of microbes in clinical and environmental specimens based on rRNA.