dengue lab tech copy

8/4/2019 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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