This video will demonstrate how to perform post-run analysis of the CDC Flu SC2 Multiplex
assay on an Applied Biosystems 7500 Fast DX Real-time Instrument.
Once the Flu SC2 Multiplex Assay run has completed, select the Results tab found in the upper
left corner of the software.
In the Results tab, select the Amplification Plot tab to view the raw data. If the data
is currently in linear view, update to log view by right clicking within the Amplification
plot. This will bring up a Graph Settings control box. For Y-axis, if linear is currently
selected, select log and then click Apply to switch to log view.
To analyze the data, start by highlighting all the sample wells from the run. To do this,
click on the upper left box of the sample wells. All of the amplification curves will
appear on the graph.
On the right side of the window, ensure that the Data drop down selection is set to Delta
Rn vs Cycle. Set line color to Detector color. In the analysis settings box, Manual Ct and
Manual Baseline should both be selected, and under manual Baseline, set the start cycle
to 3 and the end cycle to 15.
To analyze the Flu SC2 Multiplex Data accurately, thresholds must be set by assay. If a single
threshold is set for all four assays, data analysis may be impacted, leading to incorrect
data interpretation. For example, if we set one threshold across all four targets in this
file, and then look at individual target data, we see that the threshold line is set too
high for the RNase P signal. To set thresholds by assay, select the first
assay, influenza A, from the Detector drop down menu on the right. When you do that,
the Amplification plot will only show the amplification curves for the influenza A detector.
Click and drag the red threshold line until it lies within the exponential phase of the
fluorescence curves and above any background signal. Click the Analyze button in the lower
right corner of the window, and the red threshold line will turn green, indicating the data
has been analyzed.
To analyze the influenza B assay, select the InfB detector in the drop down menu, and just
like the influenza A assay, click and drag the red threshold line until it lies within
the exponential phase of the fluorescence curves and above any background signal. Click
the analyze button.
Continue this process for the SARS-CoV-2 assay by selecting the SC2 detector in the drop
down menu, adjusting the threshold line and clicking analyze.
Finally, select the RNase P detector in the drop down menu, adjust the threshold line
as before, and click analyze.
At this point, all four targets of the Flu SC2 Multiplex will have independent thresholds
set. Once these thresholds are set, select the Report tab above the graph to display
the Ct values.
To filter the report by sample name, click on Sample Name at the top of the table. To
filter the report by detector, click on Detector at the top of the table.
Be sure to save the data file with the newly analyzed data by clicking on File, and then
save.
Once thresholds have been set, it is time to interpret the data from the Flu SC2 Multiplex
Assay.
Begin by reviewing data from the No template control. This control reaction uses molecular-grade
nuclease free water in the real-time RT-PCR reactions instead of RNA.
The no template control wells should not exhibit fluorescence growth curves that cross the
threshold line. If any of the no template control wells exhibit a growth curve that
crosses the threshold line, sample contamination may have occurred. Invalidate the run and
repeat the assay with strict adherence to the guidelines.
If no amplification is seen with the No template control, move on to the analysis of the Combined
Flu SC2 positive control well. Combined Flu SC2 positive control consists of RNA from
inactivated influenza A virus, influenza B virus, a synthetic SARS-CoV-2 RNA, and nucleic
acid extracted from A549 human lung epithelial cells. The Combined Flu SC2 should be positive
for all targets in the Flu SC2 Multiplex Assay – influenza A, influenza B, SARS-CoV-2,
and RNase P.
If the Combined Flu SC2 positive control reaction is positive for SARS-CoV-2, but generates
negative results for Influenza A, Influenza B, and/or RNase P, this indicates a possible
problem during specimen extraction or a problem with the real time RT-PCR reaction, and the
results should be considered invalid. Repeat extraction and testing of any clinical specimens that had
been extracted alongside the failed Combined FluSC2 PC.
If the Combined Flu SC2 positive control reaction is positive for RNase P, but negative for
influenza A, influenza B, and SARS-CoV-2, this could indicate a possible problem with
the RT-PCR reaction or the control may have been contaminated with RNase, and the results
are invalid.
Make a new FluSC2 Multiplex master mix and repeat the real-time RT-PCR testing. If the
Combined Flu SC2 positive control reaction is still only positive for RNase P, repeat
extraction and testing of any clinical specimens that had been extracted alongside the failed
Combined Flu SC2 positive control.
The Human Specimen Control, or HSC for short, is used as an RNA extraction procedural control
to demonstrate successful recovery of nucleic acid, extraction reagent integrity, and as
a control for cross-contamination during the extraction process.
The HSC control consists of non-infectious cultured A549 human lung epithelial cells.
Purified nucleic acid from the HSC control should yield a positive result with the RNase
P primer and probe set but should have a negative result for the influenza A, influenza B, and
SARS-CoV-2 agent-specific markers.
If the HSC control generates a negative result for RNase P, this indicates a potential problem
with the extraction process.
If the HSC control generates positive results for influenza A, influenza B, or SARS-CoV-2,
this may be indicative of possible cross-contamination during extraction or real time RT-PCR reaction
set-up.
If the no template control reaction is also positive for any of these targets, this strongly
suggests cross-contamination occurred in the reaction set-up. If the no template control
is negative, then cross-contamination likely occurred during extraction.
If the HSC control fails to give the expected results, invalidate the run and repeat extraction
and RT-PCR testing for all specimens that had been extracted alongside the failed HSC
control.
The no template control, Combined FluSC2 positive control, and HSC control should be evaluated
before any patient data is interpreted, or recorded. If the controls do not give
the expected results, the run should be considered invalid and a retest is necessary.
If the no template control, Combined FluSC2 positive control, and HSC control have been
examined and determined to be valid and acceptable, assessment of clinical specimen test results
can proceed.
The RNase P target in the Flu SC2 Multiplex Assay serves as an internal control for the
assay and is used in conjunction with the data from other targets for interpretation
of an individual specimen.
The RNase P target should be positive, meaning have a Ct value of less than 35, for all clinical
specimens in the absence of a signal for one of the viral targets. If RNase P is negative
in the presence of a positive result for one of the viral targets, the viral target result
should be considered valid. However, if all viral targets generate negative results and
RNase P is also negative, the test is considered invalid.
Failure to detect RNase P in clinical specimens could indicate insufficient nucleic acid extraction
from clinical samples, poor specimen quality or loss of specimen integrity, improper assay
execution, and/or reagent or equipment malfunction.
If all viral targets are negative, meaning they have a Ct of greater than or equal to
40, and RNase P generates a Ct of greater than or equal to 35, the result should be
considered invalid for the specimen.
Repeat testing of specimen nucleic acid and/or re-extract and repeat the real-time RT-PCR
assay. If all targets are negative after retest, report the specimen as invalid. Collection
of a new specimen and subsequent testing should be considered.
When all controls exhibit the expected results and one or more of the viral targets, influenza
A, influenza B, and/or SARS-CoV-2 DOES cross the threshold line BEFORE a Ct of 40.00 then
the specimen is considered positive for that virus or viruses. Multiple viruses may be
detected in a single specimen.
When all controls exhibit the expected results, a specimen is considered negative if the amplification
curves for all of the viral targets, including influenza A, influenza B, and SARS-CoV-2,
DO NOT cross the threshold line BEFORE a Ct of 40.00 AND the RP internal control DOES
cross the threshold line at a Ct value less than 35.
Laboratories should report their diagnostic results as appropriate and in compliance with
their specific reporting system.
Optimum specimen types and timing for peak viral levels during infections caused by SARS-CoV-2
have not been determined. Collection of multiple specimens from the same patient may be necessary
to detect the virus. The possibility of a false negative result should especially be
considered if the patient’s recent exposures or clinical presentation suggest that SARS-CoV-2
infection is possible, and diagnostic tests for other causes of illness, for example,
other respiratory illness, are negative. If SARS-CoV-2 infection is still suspected, retesting
should be considered in consultation with public health authorities.
Please refer to the Package Insert for the CDC Flu SC2 Multiplex Assay for detailed guidance
on assay set up, data analysis, and data interpretation. Thank you for your time and attention.