Developing SARS-CoV-2 rRT-PCR Test

Francisca Srioetami Tanoerahardjo

Outline

•How to choose the instrument and method

•How to design PCR lab and its instruments

•How to preparing your staff

Developing Molecular Laboratory

SERVICE only SERVICE → R n D

Small scale

High throughput

DevelopmentEvaluation

Data &

efficiencyProduct

How to choose the instrument and method

•PCR Instrument

• Extraction

• Amplification

• Detection

•Basic Laboratory Equipment

• Centrifuges

•Micropipette

• etc

PCR = Polymerase Chain Reaction

PCR steps:

• Denaturing

• Annealing

• Synthesizing

“temperature”

ISO-THERMAL PCR

“enzyme”

DNA Amplification

LANGKAH-LANGKAH PEMERIKSAAN RT-PCR

• Persiapan bahan pemeriksaan / Spesimen

• Swab Nasofaring / Orofaring; Sputum; Broncho alveolar lavage (BAL); dll

• Mengandung virus (RNA)

• Ekstraksi RNA

• Proses inaktivasi

• Reverse trancriptase → cDNA

• Amplifikasi cDNA dan Deteksi denganProbe

• Denaturasi → annealing →amplifikasi →probe detection

• RT-PCR:

• one-step → contoh: TCM SARS-CoV-2

• two-step → Ekstrasi RNA→cDNA dan PCR & deteksi

PCR Instrument for SARS-CoV-2 molecular detection

THERMALCYCLER

Close System

Open System

PCR Instrument for SARS-CoV-2 molecular detection

THERMALCYCLER & Detection SystemClose System

with

Cartridge

Close System without

Cartridge (+ extraction)Open System

Gambaran Alat – Reagen RT-PCR SARS-CoV-2

GeneXpert

(Cepheid)

ALAT

REAGEN

LightCycler

LC480 (Roche)

AB7500 FAST(Thermo Fisher)

CFX96

(BioRad)

PowerAmp96

(Kogene)

Cartridge

SARS-CoV-2

(Cepheid)

Abbott

m2000

Abbott

Real

time

SARS

CoV2

Liferiver

Novel

Coronavirus

COVID-19

(2019 nCoV)

Real Time

Multiplex kit

Acu-

CORONA

Acumen

Research

Lab

Singapore

PowerCheck

2019-nCoV

Real time

PCR Kit

A*STAR

Fortitude

Kit

SD Biosensor

Standar M

nCoV Real

time

Detection

Kit

How to choose the instrument and method

• Insert kit – details

• ISO20395:2019(E) 1st ed. 2019-08• Biotechnology – Requirements for evaluating the performance of quantification methods for nucleic acid target sequences – qPCR and dPCR

• Design of measurement procedure• Quantification method

• qPCR – NA concentration → calibration curve• dPCR – copy number → molecular counting

• Sample QC• Total NA quantity• Integrity• Purity

How to choose the instrument and method 1

• Assay design and quantification of NA target seq• Assay design

• Target• Primers and probe• Specification

• Assay optimation• Fluorescence signal• Amplification efficiency• specificity

How to choose the instrument and method 2

• Presicion

• LoQ

• LoD

• Linearity

• Trueness

How to choose the instrument and method 3

Nucleic Acid quantification measurements

• Robustness

• Repeatability

• Reproducibility

•Method of validation

• Data QC & analysis• Acceptance criteria

• Threshold setting

• Data pre processing (Calibration curve)

•NA quantification measurements traceability and comparability

•Measurement uncertanty

How to choose the instrument and method 4

Limit of Detection

• Estimation of LOD shall take account of both the statistical distribution of false-positive results and true positive samples.

• The probability of false positive detection (β) and false negative detection (α) (for example p = 0,05) or confidence of classifying a true positive (equal to 1 – α) or false negative (equal to 1 – β) shall be specified

Linearity

• The degree of linearity within this range shall be characterized in terms of slope and correlation coefficient (for example, Pearson’s correlation coefficient, R) by regression analysis of expected nucleic acid quantity vs. observed nucleic acid quantity. The measurand shall be the quantity measured by the method

Limit of Blanc

Precision

• Precision is a measure of the variability in independent measurement results obtained for the same sample under stipulated conditions

Specificity

• Extraction from clinical samples• Pos cases

• Neg cases

• Ct value from pos sample compare with reference Ct value

• Ct value of known negative sample

• All results are concordant with the actual clinical diagnoses

How to design PCR lab and its instruments

•Unidirectional workflow

•Pressure• Avoid contamination

Molecular Laboratory Design

Molecular Laboratory Design

Basic Equipment

• BSC class II

• Laminair hood (PCR hood)

• Refrigerator

• Deep freezer

• Centrifuge biocontainment

• Autoclave

How to preparing your staff

•Dedicated Staff• ATML, Biologist, Biotechnologist, Chemist, Pharmacist, Clinical Pathologist, Microbiologist

•Certified Technical Training – GLP

•Fellowship

•Biosafety and Biosecurity Training

Terima Kasih

Terima Kasih