CoviDx® DirectPlex SARS-CoV-2 RT-PCR Detection Kit MATERIALS PROVIDED TEST PREPARATION/REACTION SET-UP DATA INTERPRETATION

RNA Extraction Free Screening and Confirmatory RT-PCR Test Kit for KIT COMPONENTS VOLUME 1. Thaw all components of the kit on ice, mix gently using vortex RNaseP
COVID-19 E-Gene N-Gene RdRP RESULTS
and spindown the contents for 5 secs and use it immediately. FAM HEX/VIC CY5
TexasRed/
INTERPRETATION
CM4D01 | 100 Reactions | Store at -200C 2X Master Mix 2 x 625 μL ROX
2. Calculate the number of reactions for each experiment
Expiry Date - See box label for information 20X Primer and Probe mix 125 μL including all controls with one excess reaction volume in the + + + +/- 2019-nCoV Detected
Nuclease Free dH2O 1000 μL reaction cocktail to accommodate pipetting errors. (eg:
Any one gene is 2019-nCoV Detected
Please read this manual carefully before number of reaction (n) including controls are 10 add 1 extra + Positive
+/-
Positive Test Control 200 μL
starting your experiment reaction during the preparation n+1)
Proteinase K 1000 μL 3. Prepare the reaction mix in a 1.5/2 mL tube for the calculated - + + +/- 2019-nCoV Detected
INTENDED USE TARGET REPORTER QUENCHER
number of samples in Master Mix Preparation room. Any one gene is
4. The assay should be run along with positive controls and - + 2019-nCoV Detected
Positive
CoviDx® DirectPlex SARS-CoV-2 RT-PCR Detection Kit is an E-Gene FAM BHQ1 negative controls. Presumptive positive and
in-vitro RNA extraction free, RT-PCR based qualitative assay for
N-Gene HEX/VIC BHQ2 THERMAL CYCLIC CONDITIONS | Standard 90 min + - - + sample may be positive for
the specific detection of SARS-CoV-2 in human respiratory other sarbecovirus
specimens. This kit uses one-step RT-qPCR with a hydrolysis RdRP Gene CY5 BHQ2
STEP TEMP 0C TIME DETECTION CYCLE
probe chemistry that uses 5’ nuclease activity of Taq DNA Human RNase P TEXAS RED/ROX BHQ2 - - - + 2019-nCoV NOT Detected
polymerase and enables detection of PCR product which gets 50 15 Min Off 1
Reverse Invalid results repeat the
amplified at every step during PCR cycles by producing STORAGE AND HANDLING Transcription - - - - RNA extraction and re-run
95 2 Min Off 1
fluorescence. ● Store all the kit reagents at -20°C.
● Do not repeatedly freeze-thaw reagents as it leads to reduced assay TROUBLESHOOTING
BACKGROUND sensitivity. Thaw the reagents only on ice or at 4°C. 95 15 Sec Off
Kit components are stable through the end of the expiration date indicated on PCR and
●
40 Positive control showed no amplification
Coronavirus is a kind of common virus that causes an infection in the box when stored at -20°C. Detection 58 30 Sec On Inappropriate storage of reagents
the nose, sinuses, or upper respiratory tract. Most coronaviruses
aren't dangerous. In early 2020, after a December 2019 outbreak PRECAUTIONS ● Store the reagents at recommended temperature for their optimal
● It is recommended that this product is used by personnel specially instructed THERMAL CYCLIC CONDITIONS | Fast 30 min performance.
in China, the World Health Organization identified SARS-CoV-2 as and trained in real-time PCR and in-vitro diagnostics procedures. ● Avoid repetitive freezing and thawing.
a new type of coronavirus. The outbreak quickly spread around ● Treat all the specimens as potentially infectious. STEP TEMP 0C TIME DETECTION CYCLE ● Check the expiry of reagents.
the world. ● Wear protective disposable powder-free gloves, a laboratory coat & eye Negative controls are positive
protection when handling specimens.
● Store positive and/or potentially positive material separated from all other Reverse 52 4 Min Off 1 Causes - Cross-contamination
COVID-19 is a disease caused by SARS-CoV-2 that can trigger a components of the kit. Transcription 1 ● Follow good laboratory practices to avoid contamination issues.
90 30 sec Off
respiratory tract infection. It can affect the upper respiratory tract ● Keep separate areas for master mix and template preparation and work under ● Use a new batch of reagents and repeat the experiment.
biosafety cabinets. Abnormal plot and/or low ΔRn values in amplification curve
(sinuses, nose, and throat) or lower respiratory tract (windpipe Use aerosol barrier pipette tips & frequently change the gloves. 86 1 Sec Off
●
PCR and 37 The baseline was set improperly (some samples have CT values lower
and lungs). It spreads the same way like other coronaviruses, ● Do not open the reaction tubes/plates post-amplification, to avoid
Detection than the baseline value)
contamination with amplicons. 60 1 Sec On
mainly through person-to-person contact. Infections range from ● Switch from manual to automatic baseline, or move the baseline stop
mild to deadly. SARS-CoV-2 is one of the seven types of
WASTE DISPOSAL READING TEST RESULTS / DATA ANALYSIS value to a lower CT (2 cycles before the amplification curve for the
coronavirus, including the ones that cause severe diseases like Note: Since its a direct method which uses VTM, the sample crosses the threshold)
● Dispose all the waste/remains of the reagents used in reaction mixture
Middle East respiratory syndrome (MERS) and sudden acute fluorescence obtained will be lower than the purified RNA. An amplification signal is detected in the early cycles
preparation and expired kit components along with bio-waste as per the lab
respiratory syndrome (SARS). manual/general bio-waste management instruction ● Adjust the threshold for each gene individually, for Dilute the sample to increase the CT value
The other coronaviruses cause most of the colds that affect us ● Dispose the PCR plates with patient samples “sealed” post run to avoid E-gene-FAM,
potential infection to the operators and contamination of the lab. SYMBOL MEANING SYMBOL MEANING
during the year but aren’t a serious threat for otherwise healthy ● NTCs should be negative and should not exhibit fluorescence
people. No cure for COVID-19 is present as of yet. Standard amplified curves that cross the threshold line.
SAMPLE PREPARATION In Vitro Diagnostic medical
recommendations are followed to prevent the spread of infection. ● If a false positive occurs with one or more of the primer and device
Manufacturer
The standard method for diagnosis is by Real time reverse ● Take 100 μL of patient VTM sample in a fresh 1.5 mL tube. Add 10 μL of probe in NTC reactions , it indicates sample contamination.
transcription PCR (rRT-PCR). proteinase K, vortex for 10 seconds. ● In that case, Invalidate the run and repeat the assay with Product Code/Catalogue Date of Manufacture
● Incubated the tube at 95 ℃ for 5 min. Number (MMM, YYYY)
stricter adherence to the procedure guidelines.
PRODUCT DESCRIPTION ● From this heat inactivated/ lysed sample take directly 8 μL and use it for
● Positive control should produce a positive result with an
RT-PCR, using the below provided reaction mixture preparation. Lot Number Expiry Date (MMM, YYYY)
CoviDx® DirectPlex SARS-CoV-2 RT-PCR Detection Kit is an expected Ct value for each target included in the test.
in-vitro RNA extraction free, RT-PCR based qualitative assay for ● If the samples are collected in NeoDx MTM (Molecular transport
medium) no additional sample preparation is required. Just vortex the ● If expected positive reactivity is not achieved, invalidate the run Store at -20oC Keep away from rain
the specific detection of SARS-CoV-2 in human respiratory MTM for 30 seconds and directly use 8 μL of the MTM sample as a and repeat the assay with stricter adherence to procedure
specimens which removes the burden of RNA extraction. template for RT PCR. guidelines. Contains sufficient for <n>
CoviDx® DirectPlex SARS-CoV-2 RT-PCR is a multiplex assay kit Caution
● The shelf life is one year from the date of manufacture and should be● When all controls meet stated requirements, a specimen is reaction
where detection of four genes (three SARS-CoV-2 and one used before the expiry date as given on the product package. considered positive for COVID-19 in case of reaction growth
human internal control) can be detected in a single tube Refer to Instruction for use CE Self Certified
curves that cross the threshold line within < 37 cycles for E
reaction.This CoviDx® DirectPlex Direct SARS-CoV-2 primer and gene, N gene and RdRP target. European Authorized
probe sets are designed for the detection of E-gene, N-gene Add 10 μL
proteinase K
vortex 10 sec ● When all controls display the expected performance, a Representative
and RdRP-gene of SARS-CoV-2 along with human internal specimen is considered negative if E-gene, N-gene and RdRP
control RNase P in a single tube assay, based on Berlin amplification curves are absent and the RNase P amplification MANUFACTURED BY
protocol. curve DOES cross the threshold line.
ANALYTICAL SPECIFICATIONS NeoDx Biotech Labs Pvt Ltd
RESULTS INTERPRETATION
The Specificity of the kit is 100% with 98.66% sensitivity. The SR4, 4th floor, Pragathi Plaza,
limit of detection of the kit is 10 copies/μL . The Positive Test REACTION MIXTURE - 25 μL For all 2019-nCoV genes For RNase P
2019-nCoV assay result Opp. Biocon, Hebbagodi,
E, N and RdRP gene
control is traceable to Twist Synthetic SARS-CoV-2 RNA Reagents
Bengaluru, Karnataka India - 560100
1 Rxn 25 Rxn 50 Rxn 100 Rxn
Control 2 (MN908947.3) Ct < 37 Ct ≤ 37 Positive
2X Master Mix 12.5 μL 312.5 μL 625 μL 1250 μL Obelis S.A, Bd Général Wahis, 53,
Ct = Undetermined or Ct
20X Primer and Probe mix 1.25 μL 31.25 μL 62.5 μL 125 μL ≥ 37 Ct ≤ 37 Negative B-1030 Brussels, Belgium.
Follow the WHO/US CDC Universal Precautions Nuclease Free dH2O 3.25 μL 81.25 μL 162.5 μL 325 μL Invalid. Re-purify the nucleic Issued Date : 02 Nov 2022 IFU Version : DOC_CM4D01_ 001_03
Ct = Undetermined or Ct
for the safety against Novel Coronavirus Ct ≥ 37 Undetermined acid from the sample, then
Total 17 μL 425 μL 850 μL 1700 μL ≥ 37
(SARS-COV-2) repeat the test. An ISO 13485:2016 CERTIFIED COMPANY
1 Add 8 μL of the heat inactivated VTM per reaction 2 4