RT-PCR FOR SARS-CoV-2

(COVID-19):
RESULT INTERPRETATION,
REPORTING &
TROUBLESHOOTING
DISCLOSURES
 No potential or actual conflict of interest

Honoraria Speaker/lectures/training
(Roche, AZ, Pfizer, MSD, Sysmex)
Advisory Board Roche FMI
Clinical Trials None
Investments None
Others EIF (MSD)

DISCLAIMER

 No identifiable patient data is presented

OUTLINE OF DISCUSSION

1 •rt-PCR brief review

2 •rt-PCR result interpretation
3 •SARS-CoV-2 rt-PCR assay design & reporting

* •rt-PCR troubleshooting

Note: Assuming the assay has been properly optimized and validated
 REAL TIME PCR AMPLIFICATION PLOT
Real time PCR terms
 Baseline
 Threshold
 Cycle threshold (CT)
 Exponential phase
 Plateau

 Display: linear or log

HENRY’S CLINICAL DIAGNOSIS AND MANAGEMENT, 22nd ed
[edited by] Richard A. McPherson, Matthew R. Pincus.
REAL TIME PCR AMPLIFICATION PLOT

Log Display (Graph) Linear Display (Graph)

BASELINE

 Signal level during

initial phases of PCR
 “background noise”
 Cycles 3-15
THRESHOLD
CT – CYCLE THRESHOLD / CP – CROSSING POINT

 Level of signal that reflects statistically

significant increase over calculated baseline

 Set to distinguish “noise” from true amplification

signal

 Auto-threshold (initial) vs Manual (recommended)

THRESHOLD
CT – CYCLE THRESHOLD

 Level of signal that reflects

statistically significant increase
over calculated baseline

 Set at the exponential phase

 Set to distinguish “noise” from

true amplification signal
THRESHOLD
CT – CYCLE THRESHOLD

 Level of signal that reflects

statistically significant increase
over calculated baseline

 Set at the exponential phase

 Set to distinguish “noise” from

true amplification signal
WRONG THRESHOLD SETTINGS:
TOO HIGH
WRONG THRESHOLD SETTINGS:
TOO LOW

Note: A Ct value will be generated by the software

even if there is NO true amplification (false +)
THRESHOLD
CT – CYCLE THRESHOLD / CP – CROSSING POINT

 Level of signal that reflects statistically

significant increase over calculated
baseline
 Set to distinguish “noise” from true
amplification signal
 Auto-threshold vs Manual (recommended)
 Set threshold at the EXPONENTIAL PHASE
ALWAYS INSPECT THE RT-PCR AMPLIFICATION PLOTS
ALWAYS INSPECT THE RT-PCR AMPLIFICATION PLOTS

 Only exponential/sigmoidal curves are considered true amplification

KEY TAKE AWAYS

 STRICT ADHERENCE TO OPTIMIZED AND VALIDATED PROTOCOLS

 ALWAYS INSPECT EACH AMPLIFICATION PLOT

 MAKE SURE THRESHOLD IS SET AT THE EXPONENTIAL PHASE

 ONLY SIGMOIDAL AMPLIFICATION IS CONSIDERED POSITIVE

QUALITY CONTROLS

 QC per batch
 Positive control*
 Negative control*
 No template control
 Negative extraction control
 QC: POSITIVE CONTROL

 Included in commercial kits

 Expected Ct value (ideally)

 Monitors the RT-PCR process

 Monitors performance of the primers and probes

 Best practice: External positive control*

 Diluted to a value around the verified/validated limit of
detection of the assay

https://www.amp.org/clinical-practice/testing-resources-for-covid-19/
https://poeli.gitlab.io/collated_vendor_info/
QC: POSITIVE CONTROL

Case scenario
 Prescribed Ct value as per insert: <40
 Case scenario

QC: POSITIVE CONTROL  Usual % positive rate in your lab: 20% (QA program)
 Past week: sharp decrease to 5%

 Review of the controls showed an increasing trend in the

positive control Ct
 Risk of false negative results

 Prescribed Ct value as per insert: <40

 Expected Ct value of positive control: ~25

 Review record of lot no used, storage conditions, recent

changes in other consumables, inhibitors, thermal cycler
calibration, incorrect PCR protocol
 Best practice recommendations: Alternative test
kit/method on hand; Low positive control included
 OTHER CAUSES OF INVALID POSITIVE CONTROLS

Possible cause Corrective action

 Incorrect programming of the thermocycler  Check PCR protocol used in failed run

temperature profile
 Pipettes – out of calibration  Calibrate pipettes. Make sure tips are compatible

 Inadequate or no vortexing, or control was not

 Incorrect handling of the positive controls adequately thawed at room temperature.

 Check storage conditions and expiration date on

 Improper storage conditions the kit box. Discard the kit if necessary.
 QC: NEGATIVE CONTROLS
 Negative control (assay dependent, ie, extraction)
 Checks for contamination
Master
mix prep
Best practice recommendation, additional controls
 No template control RNA
 separate well/tube with nuclease-free water added during PCR set up/template addition extraction
 Negative extraction control
 separate “sample” that goes through extraction process
PCR
 nuclease free water instead of VTM/sample
setup
✓ Checks for contamination in different steps of the process
 PREVENTING CONTAMINATION

 Strict adherence to protocols

 Dedicated PCR hood/BSC for mastermix prep, extraction, RNA v DNA work
 Sequence of pipetting (positive control – last)
 Pipetting skills, frequent glove changes
 Briefly spin down samples, positive control tubes
 Proper decontamination
 [10% bleach > distilled water wipe downs (2) > 70% alcohol > water > dry paper towel]
 Consider DNAse/RNAse away
 Decontaminate areas before and after
 Document decontamination
QC: “INTERNAL” CONTROL (IC)

 “Internal” - Included in each sample/reaction

 Depends on the test kit

 May be added during RNA extraction (ie EAV) or

“endogenous” (ie RNAse P)
 IC Amplification may vary depending on gene target
amplification
 Invalid IC maybe due to inhibitors, pipetting issues,
inefficient extraction
TROUBLE SHOOTING - TAKE AWAYS

 Proper detailed documentation allows for easier

troubleshooting
 Decontamination Decontamination Decontamination

 Strict adherence to validated protocols

 Monitor performance indicators (QA)

 % positive
 % invalid
SARS-COV-2 RT-PCR ASSAY DESIGN & REPORTING
VARIES BY MANUFACTURER (REFER TO THE INSERT/INSTRUCTIONS FOR USE)
PERFORM IN-HOUSE EVALUATION > OPTIMIZATION > VALIDATION/VERIFICATION
 OVERALL STRUCTURE OF SARS-COV-2
 single positive-strand RNA genome
 ~30 kb in length
 96% similarity to the bat coronavirus BatCoV RaTH13
 80% similarity with SARS-CoV
 50% identity with MERS-CoV
 Genes:
 Orf - open reading frame
 N - nucleocapsid
 S - spike
 E - envelope

Örtiz-Prado, 2020.
  “Routine confirmation of cases of COVID-19 is based on
WHO GUIDANCE: detection of unique sequences of virus RNA by NAAT such
as real-time reverse-transcription polymerase chain
NUCLEIC ACID reaction (rRT-PCR) with confirmation by nucleic acid
AMPLIFICATION sequencing when necessary.”
TESTS (NAAT) FOR
COVID-19 VIRUS  The viral genes targeted so far include the N, E, S, Orf,
RdRP genes.
RESULT INTERPRETATION: VARIES BY EPIDEMIOLOGIC SETTING

 Areas with NO known COVID-19 circulation

 2 different gene targets
Or
 1 NAAT for betacoronavirus >> sequence
RESULT INTERPRETATION: VARIES BY EPIDEMIOLOGIC SETTING

 Areas with established

COVID-19 circulation
 At least 1 single
discriminatory gene target
SARS-COV-2 RT-PCR ASSAY DESIGN (QUALITATIVE)

 Test kits variations Rt-PCR kit Gene Target LoD (insert data)*
Tib MolBiol E, RdRp, N 3.8-5.2 copies/rxn
 Gene targets (primers, probes)
Sansure N, Orf1ab 200 copies/mL
 Ct cut-off
Seegene (Allplex) E, N, RdRp 1,250-4,167copies/mL
 “Internal control”
XABT E, N, Orf1ab 200 copies/ml
 Analytical limit of detection
GeneSig RdRp 0.58 copies/ul
 Manufacturer validation* SD Biosensor E, Orf1ab 0.5 copies/ul
(extraction kits, rtPCR platform)
FTD Siemens N, Orf1ab 11.5 copies/rxn
A*Star Fortitude NSP-3 12.5 copies/rxn
BD Max N1, N2 40genomic equivalents/ml
GeneXpert N1
SARS-COV-2 RT-PCR – DIAGNOSTIC GENE TARGETS

 Spike protein
 Among the structural proteins, the
highest sequence diversity between
SARS-CoV and SARS-CoV-2 occurs in the
S protein (24%
 Use of the spike protein as part of SARS-
CoV-2 diagnostic testing will provide high
specificity.

Örtiz-Prado, 2020.
SARS-COV-2 RT-PCR – DIAGNOSTIC GENE TARGETS

 N protein
 little sequence diversity in the N gene
between SARS-CoV and SARS-CoV-2
 N gene sits at the end of the coronavirus
genome, this nested strategy makes the
N gene the most abundant nucleotide
sequence during virus replication, and
therefore an excellent diagnostic target

Örtiz-Prado, 2020.
SARS-COV-2 RT-PCR – DIAGNOSTIC GENE TARGETS

 E protein
 little sequence diversity in the E gene
between SARS-CoV and SARS-CoV-2
 high positive predictive value for
infection with SARS-CoV-2

Örtiz-Prado, 2020.
 SARS-COV-2 RT-PCR – DIAGNOSTIC GENE TARGETS

 ORF1ab segment
 2/3 of the SARS-CoV-2 genome
 Encodes 16 nsp relating to the replication-
transcription complex responsible for all the
machinery associated with viral replication
 RdRp, ExoN

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988269/

Örtiz-Prado, 2020.
SARS-COV-2 RT-PCR – DIAGNOSTIC GENE TARGETS

Note: Most commercial kits do not disclose their primer and probe
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988269/ sequences but do provide analytical specificity and cross reactivity
  GeneXpert  SD Biosensor (Ct cutoff 36)
RESULT  N, E – Positive  Orf1ab, E – Positive
INTERPRETATION:  N only – Positive  Orf1ab only – Positive

 E only – Presumptive  E only – Presumptive

VARIES BY TEST KIT positive (assay repeated) positive (assay repeated)

RECOMMENDATION  XABT (Ct cutoff 38)

 Sansure (Ct cutoff 40)
 Orf and/or N – Positive
CT VALUE CUT-OFF  Orf and/or N – Positive
 E only – Presumptive
positive

Note: Most commercial kits do not disclose their primer and probe
sequences but do provide analytical specificity and cross reactivity
REPORTING

Result Notes
SARS-CoV2-RNA Detected Discriminatory gene target/s detected
SARS-CoV2-RNA Not Detected Gene targets not detected. May add comment:
This assay is designed to detect the XXXX and/or XXXX genes of SARS-CoV-2 using nucleic
acid amplification. A Not Detected result does not preclude the possibility of 2019-nCoV
infection since the adequacy of sample collection and/or low viral burden may result in the
presence of viral nucleic acids below the analytical sensitivity of this test method. Test
results should be used along with other clinical and laboratory data in making the
diagnosis.
Presumptive Positive Depends on the kit/assay design; Ex, E gene detected
May recommend repeat testing
Equivocal/Indeterminate Variable result category; May recommend repeat testing
Invalid Failure of internal controls or gene targets to amplify
Repeat specimen collection is recommended.
FACTORS AFFECTING CT VALUES

 Target gene quantity – viral load in the sample

 Specimen collection

 PCR efficiency

 RNA Extraction

 PCR inhibitors

HENRY’S CLINICAL DIAGNOSIS AND MANAGEMENT, 22nd ed

[edited by] Richard A. McPherson, Matthew R. Pincus.
QUANTITATIVE PCR

 Calibrators, standard curve are needed to

enable accurate quantitation
 BCR-ABL
 Viral loads

 ddPCR – absolute quantitation

 Research-based
 No local kits available
 Scatterplots for dynamics of cycle threshold values between
asymptomatic and symptomatic (including presymptomatic) patients
for the env gene (A), RdRp gene (B), and N gene (C). Beta value
represents a slope of decline.
 The cycle threshold values of reverse transcription–polymerase
chain reaction for SARS-CoV-2 in asymptomatic patients were
similar to those in symptomatic patients.
ADDITIONAL RESOURCES

Link Description
https://www.amp.org/AMP/assets/File/clinical- American Society for Microbiology
practice/COVID/ASM_EUA_verification_040220_FINAL. Guide in Method Verification of EUA commercial tests
pdf for Covid 19 RNA detection
https://www.finddx.org/covid-19/sarscov2-eval- Independent evaluation of molecular tests (Univ of
molecular/molecular-eval-results/ Geneva-based)
THANK YOU & STAY SAFE