Frilasita Aisyah Yudhaputri, MBiomedSc

Bandung, 29 April 1986

Pendidikan
S1 Biologi Molekuler – Universitas Indonesia
S2 Biomedical Science – Monash University Australia

Project Coordinator within Emerging Virus Research Unit

Lembaga Biologi Molekuler Eijkman, Jakarta

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Simulasi Pemeriksaan rRTPCR
SARS CoV 2

Frilasita Aisyah Yudhaputri

Emerging Virus Research Unit
Eijkman Institute for Molecular Biology Indonesia

Webinar PATELKI 8
Jakarta, 13 Juni 2020
 COVID-19 Molecular Testing
Confirmation of cases of COVID-19 is based on detection of unique sequences
of SARS-CoV2 RNA in Clinical specimen by NAAT such as real-time reverse-
transcription polymerase chain reaction

OUTLINE
• Biological sample Collection and processing
• Viral RNA Extraction
• real-time reverse-transcription polymerase chain reaction
• Result Interpretation

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Biological sample Collection and processing

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Sethurahman, et al. 2020
Biological sample Collection and processing (Cont)
Reccomended Specimen type
• Upper Respiratory
NP, OP, Nasal Swab, Naso wash

• Lower repiratory
Sputum dan BAL

https://coronavirusexplained.ukri.org/en/article/vdt0006/
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Wang et al., 2020
Human Cell has DNA as the genetic material that passes information

CSH DNA Learning Center 6

 -Viral RNA Extraction-

The Key to Succesful NAAT –

SARS-CoV-2 Nucleic Acid Extraction
• SARS-CoV-2 is RNA Virus and require
protection from RNAse

Understanding the kit reagents and protocol will help

ol
ensure quality RNA extraction and reproducible results henate
P n
cya
thio
ne
anidi Column based
gu
• Source of sample (Fluid, Cells, Tissue)
• Manual/Automated Extraction
• Open/Closed System
• Principle of extraction (Column Based, Trizol, Beads)

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Trizol
The Key to Succesful NAAT –
SARS-CoV-2 Nucleic Acid Extraction
COVID-19 Testing by Molecular must be carried out in accordance with
biosafety regulations following National, WHO / US-CDC guideline
recommendations

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SARS-CoV-2 Structure
• (+) stranded RNA virus, 27- 32 kb
• Crown Like (TEM)
• 4 active protein: S,M, E, N
• RdRP gene (RNA-dependent RNA Polymerase) is
conserved

Testing reccomendatio to use

at least two target gene

Gen RdRP
 REFERENCES
SARS-CoV-2 (COVID-19) Detection
Development and methods are Subject to change

• 17 January 2020 à Diagnostic detection of Wuhan coronavirus 2019 by real-time RT-PCR – Charité, Berlin
Germany
• 23 January 2020 à Detection of 2019 novel coronavirus (2019-nCoV) in suspected human cases by RT-PCR – Hong
Kong University
• 24 January 2020 à China CDC Primers and probes for detection 2019-nCoV
• 24 January 2020 à PCR and sequencing protocols for 2019-nCoV- National Institute of Infectious Diseases Japan
• 28 January 2020 à PCR and sequencing protocol for 2019-nCoV - Department of Medical Sciences, Ministry of
Public Health, Thailand
• 28 January 2020 à US CDC panel primer and probes– U.S. CDC, USAV – U.S. CDC, USA
• 28 January 2020 à US CDC panel primer and probes– U.S. CDC, USA

https://www.cdc.gov/coronavirus/2019-nCoV/lab/index.html
https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/laboratory-guidance
SARS-CoV-2 rRTPCR
• SAR-CoV-2 has RNA as their material
genetic à need to convert RNA to DNA Specific site
(Reverse Transcription)

Complementary DNA (cDNA)

• Real-time PCR/Quantitative PCR permits
the identification of specific, amplified
DNA fragments real time. The method
used is usually PCR with double-stranded
DNA-binding dyes as reporters.

• Common targeted Gene: N, E, S and RdRP

(The agent detected may not be the definite
cause of disease) 11
CSH DNA Learning Center
Internal Quality Control (QC)
rRT-PCR Procedure
Assay control should be run concurrently with all test samples

• Positive Control
• Negative Control/NTC (no template RNA/DNA)
• Internal amplification control (probe)
• Human Specimen Extraction Control (HSC)

Corrective Action
False Positive/False Negative Prevention
CLSI MM3-2A Approved guidelines 2003 12
Master Mix Preparation Simulation
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©Biology With Animation
 Samples Controls

Plate loading to Machine – Simulation 14

©Biology With Animation
SARS-CoV-2 Real Time - Reverse Transcript PCR
By adding fluorecent probe, target
of DNA can be detected real time
after each PCR cycle.

Because Genetic material in the

sample can be quantify, the Real
time PCR is also referred to as
Quantitative Real time PCR(QRT
PCR)

PCR Machine CSH DNA Learning Center 15

Real Time PCR

©Biology With 16
Animation
How To
Interprete
Result?

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False negative and False positive
Result
• False negatives caused by
o Specimen collection timing
o Poor handling or poor quality of specimen
o Strain variation
o Assay inhibition
(blood, ethanol, wooden stick, calcium alginate swab)

• False Positive caused by

DNA Contamination
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SARS-CoV-2 DETECTION at EIJKMAN INSTITUTE

*video deteksi SARS-CoV-2 di Laboratorium LBME

Courtesy : MetroTV
 Masa akhir wabah ini belum dapat di ramalkan
dengan pasti, maka kita harus tetap waspada

ATML sebagai garda terdepan sangat dibutuhkan

saat ini oleh Indonesia

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THANK YOU

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