ZIKA VIRUS PCR AT

LABLINK MEDICAL
LABORATORY
 Sindrom Zika

Bilangan Hari Dari Permulaan Sindrom Zika

5–7/
Tidak Past
 Individu bersimptom

Sampel bagi Ujian Pengesanan Zika

(Serum atau Air Kencing)

Sampel dihantar ke Lablink Medical Laboratory Network

(Test performed in Molecular Diagnistcs Laboratory, Lablink
Acronym
Central)
IMR – Insttute of Medical Research
CPRC – Crisis Preparedness and
Real Time RT-PCR Response Centre

Specimens and Container

Zika Virus RNA detected Zika Virus Not Detected • Serum (Blood in Plain Tube)
- Adult (2 to 4ml)
Lablink will send sample to
- Paediatric (min 1ml)
IMR for confirmatory
• Urine (In Sterile Container)
- Adult (Min 10ml)
Report Results to CPRC and hospital - Paediatric (5 to 10ml)
Real Time RT-PCR
• Primerdesign Zika Virus Polyprotein Gene genesiq Advanced Kit.
• This kit is designed for in vitro detecton of Zika viral specific RNA genomes
based on real-tme PCR technology and to have the broadest detecton profile
possible whilst remaining specific to the Zika viral genome.
• The primers and probe sequences in this kit have 100% homology with a broad
range of Zika viral sequences based on a comprehensive bioinformatcs analysis.
• Zika virus RNA can be detected in serum typically up to 3-5 days after the
clinical onset; the viral load seems to peak when clinical signs appear (up to 10
days).
• Under optmal PCR conditons, this genesig Zika virus detecton kits have high
priming efficiencies of >95% and can detect less than 100 copies of target
template.
Kit Contents
• ZIKV specific primer/probe mix
• ZIKV positve control template
• Internal extracton control primer/probe mix
• Internal extracton control RNA
• Endogenous control primer/probe mix
• RNase/DNase free water for resuspension of primer/probe mixes
(also for negatve control)
• Template preparaton buffer for resuspension of internal control
template, positve control template and standard curve preparaton
 Real-time PCR A ZIKV
• Specific primer and probe mix is provided and this can be
detected through the FAM channel.
• The primer and probe mix provided exploits the so-called
TaqMan® principle.
• During PCR amplificaton, forward and reverse primers hybridize
to the ZIKV cDNA.
• A fluorogenic probe is included in the same reacton mixture
which consists of a DNA probe labeled with a 5`-dye and a 3`-
quencher.
• During PCR amplificaton, the probe is cleaved and the reporter
dye and quencher are separated. The resultng increase in
fluorescence can be detected on a range of qPCR platforms.
Interpreting Results
Interpreting Results
• A positve PCR result is a definite proof of current infecton and it
usually confirms the infectng pathogen as well.
• Negatve results do not preclude infecton and could potentally occur
when the concentraton of organisms in the specimen is below the
limit of detecton.
• The use of additonal laboratory testng and clinical presentaton must
be taken into consideraton in order to obtain the final diagnosis of
infectous agents.
• If PCR results are negatve, serological testng should be considered,
especially 5 days or more after the onset of symptoms.
Serology Test
• Works by detectng antbodies in body.
• Antbodies in your body are similar to antbodies you develop when
exposed to viruses in same family.
• IgM antbodies typically persist for approximately 2-12 weeks.
• The Zika Virus Antbody (IgM) assay is intended for the qualitatve
detecton of Zika virus IgM antbodies in human sample.
• Plaque-reducton Neutralizaton Tests (PRNT) can be performed to
discriminate between cross-reactng antbodies in primary flavivirus
infectons.
Igm Antibody Capture Enzyme-
linked Immunosorbent Assay (ELISA)
Ant-IgM (the capture antbody) is coated on 96-well plates.

Patent's serum added

The presence of antgen is detected by using enzyme-conjugated ant-

viral antbody.

A colorimetric result is generated by the interacton of the enzyme and

a chromogenic substrate.

Colorimetric change is detected by a spectrophotometer (ELISA reader).

 Plaque-reduction Neutralization
Tests (PRNT)
• A serological test which utlizes the
ability of a specific antbody to
neutralize a virus, in turn,
preventng the virus from causing
the formaton of plaques in a cell
monolayer.
References
• https://www.cdc.gov/zika/pdfs/non-eua-zika-mac-elisa-protocol.pdf
• https://www.rcpa.edu.au/getattachment/28a83459-1b49-489d-8629-b
e461c427d94/Linda-Hueston_Zika-Virus-Laboratory-Diagnosis.aspx
• https://www.cdc.gov/zika/pdfs/zika-mac-elisa-instructons-for-use.pdf
• https://www.cdc.gov/zika/pdfs/trioplex-real-tme-rt-pcr-assay-instruct
ions-for-use.pdf
• https://www.cdc.gov/zika/hc-providers/types-of-tests.html