COVID-19 RT-qPCR Detection Kit (RUO)

Application

• Gene Expression (mRNA) Analysis》
• Copy Number Analysis
• SNP Genotype Analysis

Important notes

1. Shake gently before use to avoid foaming and low-speed centrifugation.
2. Reduce the exposure time.
3. During operation, always wear a lab coat, disposable gloves, and protective equipment.

FAQ

Q: Which genes does the Kit target?
A: It covers 3 genes: N, RdRP, and RP genes

Q: What are the shipment measurements?
A: We want to make sure our kits get to the destination at a cooling temperature, therefore we use Dry Ice and Ice packs in combination.

Q: Is the kit CE or FDA accredited?
A: This kit is for Research Use only.

Q: Can we run the kit in any equipment?
A: Our kit is compatible with RT-qPCR machine standards and our selected one is Bio-Rad CFX96. Please let us know which machine you are using. You are welcome to try the machine of your choice.

1. PCR Reaction: Thaw and assemble the following components in a 0.2 ml PCR tube on ice just prior to use: COVID-19 Primers, COVID-19 Probes, 2X RT-qPCR MasterMix, and RT- qPCR Enzyme Mix. Caution: Do not add more than one RNA sample into a single qPCR tube. Mix gently. If necessary, centrifuge briefly. 
   

   Component	20 μl Patient Sample	20 μl Positive Control	20 μl Positive Control	Negative Control
   RNA Sample	5 μl	0 μl	0 μl	0 μl
   COVID-19 Primers/Probes	2 μl	2 μl	2 μl	2 μl
   2X RT-qPCR MasterMix	10 μl	10 μl	10 μl	10 μl
   RT-qPCR Enzyme Mix	0.4 μl	0.4 μl	0.4 μl	0.4 μl
   RT-qPCR Enzyme Mix	0 μl	5 μl	0 μl	0 μl
   Negative Extraction Control	0 μl	0 μl	5 μl	0 μl
   Nuclease – Free H2O	2.6 μl	2.6 μl	2.6 μl	7.6 μl

   1. Use the Nuclease-free H₂O for the Negative Control while using Positive Control for the Positive Control setup. Cap tubes and place in the thermal cycler.
   2. Process in the thermal cycler for 42 cycles as follows:

   Steps	Temperature/Time	Cycle
   cDNA Synthesis	15 minutes at 42°C	1
   Pre-Denaturation	10 minutes at 95°C	1
   Denaturation	15 seconds at 95°C	40
   Annealing	60 seconds at 60°C
   Melting curve	Refer to specific guidelines for instrument used

   Note: Optimal conditions for amplification will vary depending on the primers and thermal cycler used. It may be necessary to optimize the system for individual primers, template, and thermal cycler.

   1. Detection: As three channels (FAM, ROX, HEX) are used in this one tube qPCR assay, we recommend to perform the channel calibration as requested by its manufacturer. Please refer to the instrument’s user manual to perform this calibration. Choose the FAM, ROX, and HEX channels for each sample to be tested with the LifeDireX COVID-19 RT-qPCR Detection Kit. Select “None” for ROX passive reference on any qPCR machine requiring ROX as the reference dye.

    

   Expected Performance of Controls

   Control Type	Used to Monitor	Expected Results and Ct Values
   N (FAM)	RP (HEX)	RdRP (ROX)
   Positive	Flawed assay setup and reagent failure, including degraded primer and probe	Positive Ct < 40.0	Negative Ct ND	Positive Ct < 40.0
   Negative (“NTC”)	Assay or extraction reagent contamination	Negative Ct ND	Negative Ct ND	Negative Ct ND
   Negative Extraction Control	Cross-contamination	Negative Ct ND	Positive Ct < 40.0	Negative Ct ND

   ND = Not Dtected.

   Results are considered invalid if any control does not perform as specified above.

    

   Interpretation of Results

   SARS-CoV-2	Interpretation
   N	RdRP	RP
   +	+	+/-	Positive
   If only one of the two targets is positive	+/-	Inconclusive Result
   	–	+	Negative
   –	–	–	Negative

   Note: The results from this kit should be interpreted in combination with all relevant laboratory findings.