Diagnostic Testing for SARS-CoV-2:

Real-Time Reverse-Transcription
Polymerase Chain Reaction

Anna Sanawati
Peneliti – PT. Bio Farma
Desember 2020
SARS-CoV-2

Diagnostic Testing for SARS-CoV-2

Real-Time Reverse-Transcription PCR

Laboratory Requirements for SARS-CoV-2 Testing

SARS-CoV-2
Single-stranded RNA virus,
belonging to the genus
SARS-CoV-2
β-Coronavirus

Genome: + 30000 nucleotides

Encoding 4 structural protein

(S, E, M and N) and
16 non structural protein

Cell Entry:
RBD binding to ACE2 Receptor Source: CDC

Source: Rabi et.al (2000). SARS-CoV-2 and Coronavirus Disease 2019:

What We Know So Far. Pathogens, 9(3), 231.
DIAGNOSTIC TESTING FOR SARS-CoV-2

Testing for SARS-CoV-2

Nucleic Acid Amplification Test (NAAT) Antigen Detection Antibody Testing

 rRT-PCR (Real-Time Reverse-  LFI (Lateral Flow Immunoassays)  LFI (Lateral Flow Immunoassays)
Transcription Polymerase Chain
Reaction)

 ELISA (Enzyme-Linked Immunosorbent

Assay)
 RT-LAMP (Reverse Transcription Loop-
Mediated Isothermal Amplification)
Real-Time Reverse-Transcription PCR

rRT-PCR Testing for SARS-CoV-2

Specimen Collection RNA Extraction Sample Preparation rRT-PCR Data Analysis

Specimen Collection RNA Extraction

Nasopharyngeal swab
Oropharyngeal swab
Source: Geneaid
Real-Time Reverse-Transcription PCR

Sample Preparation

Probes

Source: Thermo Fisher Scientific

rRT-PCR

Source: Gordon, D.E. et al. 2020. A SARS-CoV-2 protein interaction map reveals
Source: CDC Source: IAIE targets for drug repurposing. Nature. 583.
Real-Time Reverse-Transcription PCR

rRT-PCR Data Analysis

Source: IAEA

Source: Geneaid

Source: Bitesize Bio

Laboratory Requirements for SARS-CoV-2 Testing

WHO Guidance for Diagnostic Testing for SARS-CoV-2

• Laboratory should adhere strictly to the appropriate biosafety practices

• Testing specimens should be performed in appropriately equipped laboratories by staff trained in the relevant technical
and safety procedures
• Specimen handling for molecular testing using standard rRT-PCR (non-propagative diagnostic laboratory work) requires
BSL-2 or equivalent facilities with the use of BSC or a primary containment device that is recommended for sample
manipulation before inactivation
• Virus isolation and propagative work (virus culture or neutralization assays) require BSL-3 facilities at a minimum

Surat Edaran Nomor HK.02.01/MENKES/234/2020

Pedoman Pemeriksaan Uji Real Time-Polymerase Chain Reaction (RT-PCR) SARS-CoV-2 bagi Laboratorium di
Lingkungan Rumah Sakit dan Laboratorium Lain yang Melakukan Pemeriksaan Coronavirus Disease 2019
(COVID-19)
Laboratorium harus memenuhi persyaratan prasarana (laboratorium BSL-2), peralatan, BSC, dan sumber daya manusia
serta persyaratan lainnya untuk uji RT-PCR SARS-CoV-2
TERIMA KASIH