Dengue Lab Tech Copy
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Transcript of Dengue Lab Tech Copy
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Comparison of Laboratory - BasedTechniques for the Detextion of
Dengue InfectionsPimentel,Buerano, Inoue, Matias, Natividad
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Dengue Vector disease
Aedes aegypti
Mild dengue fever (DF) or Denguehemorrhagic fever (DHF)
can lead to Dengue shock syndrome
(DSS) RNA virus
Flaviviridae
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Mechanism
Mosquito bite to salivary glands
White blood cells
Signal Proteins (interferon)
in severe infection, organs are affected fluid leak
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Tests
Serological assay
PCR
Cell culture
DIAGNOSIS
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Methodology
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Sampling 112 patients with thrombocytopeniafrom San Lazaro Hospital
Platelet count
Normal - 150,000/mm (not included)
Slight decrease - 50,000 to100,000/mm
Severe Decrease - < 50,000/mm
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Sampling 3 to 5 ml blood sample within
15 days
Centrifugated at 1,800 rpm for10 minutes at 4 degreesCelsius
Collect Plasma and Buffy Coatand disacard RBC
Wash with Phosphate BufferedSaline (PBS) at 1,300 rpm ,resusped in 50 to 100 ul PBS
Separate components
RBC does not contain virus
Osmotic Equilibrium
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u o uoresce ceAntiobody Test
(IFAT) Fix in cold acetone and airdried 20 ul of Blockace solution
Slides placed at 37 degreesCelsius and washed with PBS
Addition of 20 ul of mouse -monoclonal anti-flavivirusantibodies
20 ul of FITC - labeled anti -mouse IgG
blocking reagent to prevent non- specific binding
Optimum Temperature
Detection, binds to target
Dye, binds to antibody
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ResultA: plasma specimen (test sample)B: DEN2 virus (positive control)C: Mock-infected cells (blank)
Fluorescence (neon green color)indicates presence of dengue virus.
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Result
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Results
100% detection in first two days declined to 0%
Time dependent
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Infectivity Assay prepared BHK - 21 cells, spread
inoculum
Addition of 50 ul of human IgG
anti - dengue antibody, washingand incubation
Addition of 50 ul horse radishperoxidase conjugated anti -human IgG
Addition of 50 ul 0.05%Diaminobenzidine and Hydrogenperoxide
cell to be inoculated withinfected culture fluid
primary antiobody
secondary antibody withperoxidase, binds DAB andoxidize it, producing browncolor as stain
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Results
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IgM-capture ELISA
Each well of a 96-wellplate was coated with100 uL of goat anti-human IgM in 0.05 M
carbonate-bicarbonatebuffer, pH 9.6, with0.01% NaN3, incubate.
Wash plate with PBS-Tween with interval ofthree minutes
Preparation of wells
To remove excess
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IgM-capture ELISA
Plasma from denguepatients, (+) control, (-)control were put in
designated well,incubated for one hourat 37 degrees Celsius
Wash wells with PBSTween
Addition of testspecimens to the well
To eliminate non-specific
bindings
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IgM-capture ELISA
100 uL of tetravalentdengue viral antigenwas added to each
plate, incubate, wash
Each plate was reacted
with diluted horseradishperoxidase
To form complex
To hydrolyze thecomplex formed
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IgM-capture ELISA
Add -phenylenediaminedihydrochloride (OPD),0.03% H2O2 in 0.05Mcitrate phosphate buffer
Stop reaction, read plateat 492 nm
For color reaction
Colour development isindicative of thepresence of anti-dengueIgM antibodies in thetest sample.
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Results
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Reverse Transcription-PCR
RNA was isolated, airdried, dissolved indiethylpyrocarbonate-treated water (DEPC),incubated
Heated RNA was added
to 10 uL reversepropagation mix,incubated for 1 hr
PCR
To linearize RNA
For cDNA synthesis
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Reverse Transcription-PCR
Agarose GelElectrophoresis of PCRproducts
To detect genomesimilarities as compared
to the standards
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Results
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Results
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Antigen Sandwich ELISA
Each well was coatedwith anti-flavivirus IgG,incubated overnight,
washed Test specimens were
put
Put HRPO conjugatedanti-flavivirus IgG
View under 492 nm
Preparation of well
To complex antigen andvirus
React HRPO andantigen, so that virus is
sandwiched betweenantigens
Detect presences ofdengue virus
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Result
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