![[IP2000] IdPath™ COVID-19 Real-Time RT-PCR Kit, 100 RXN](/web/image/product.product/477/image_1024/%5BIP2000%5D%20IdPath%E2%84%A2%20COVID-19%20Real-Time%20RT-PCR%20Kit%2C%20100%20RXN?unique=380a575)
![[IP2000] IdPath™ COVID-19 Real-Time RT-PCR Kit, 100 RXN](/web/image/product.image/297/image_1024/%5BIP2000%5D%20IdPath%E2%84%A2%20COVID-19%20Real-Time%20RT-PCR%20Kit%2C%20100%20RXN?unique=ffc4c05)
![[IP2000] IdPath™ COVID-19 Real-Time RT-PCR Kit, 100 RXN](/web/image/product.product/477/image_128/%5BIP2000%5D%20IdPath%E2%84%A2%20COVID-19%20Real-Time%20RT-PCR%20Kit%2C%20100%20RXN?unique=380a575)
![[IP2000] IdPath™ COVID-19 Real-Time RT-PCR Kit, 100 RXN](/web/image/product.image/297/image_128/%5BIP2000%5D%20IdPath%E2%84%A2%20COVID-19%20Real-Time%20RT-PCR%20Kit%2C%20100%20RXN?unique=ffc4c05)
Description
The IdPath™ COVID-19 Real-Time RT-PCR Kit is a real-time RT-PCR test intended for the detection of SARS-CoV-2 virus RNA which might be extracted from the respiratory tract specimens. The Kit provides reagents for multiplex real-time RT-PCR to detect SARS-CoV-2 by one-step reaction, specifically targeting the E (Envelope), RdRP (RNA-dependent RNA polymerase) and N (Nucleocapsid protein) gene for SARS-CoV-2 virus.
The Kit contains the RT enzyme mix and qPCR master mix for reverse transcription and real-time PCR of virus RNA. The COVID-19 Control (positive control) and ddWater (negative control) are used as indicators to avoid false negative/positive results across all experimental procedures. The Primers/probes Mix contains multiplex primers and TaqMan probes specific to the N, E/RdRP genes of SARS-CoV-2 and RNase P gene of human, detected by FAM, VIC and ROX channels, respectively.
For Research Use Only
Features
High Sensitivity：5×102 copies/ml (10 copies/rxn)
High Inclusivity : >99% of currently available complete virus genomes for SARS-CoV-2 including Omicron variant
High Accuracy : Clinical validation with 100% accuracy
High Stability : 37/25°C for 2 weeks ; 4°C for 24 weeks ; 10 times of freeze-thaw cycles
High Compatibility : Suitable for most laboratory qPCR machines
Operation Control : Including internal control for quality control of total process
Convenience : Multiplex (E/RdRP and N) detection by one-step reaction
Storage
Aliquot to avoid multiple freeze-thaw cycles
Protect fluorogenic probes from light
-20°C for 12 months
High Sensitivity：5×102 copies/ml (10 copies/rxn)
The confirmatory LOD for SARS-CoV-2 is 500 copies/ml for the IdPath™ COVID-19 Real-Time RT-PCR Kit when analyzed in Bio-Rad CFX96, ABI StepOnePlus, ABI 7500 Fast, Roche LC480, Roche LC96, as well as in Qiagen Rotor Gene Q qPCR instruments.
High Inclusivity ：>99% of currently available complete virus genomes for SARS-CoV-2 including Omicron variant
Based upon BLAST analysis, the IdPath™ COVID-19 Real-Time RT-PCR Kit maps with >99.1% homology of known SARS-CoV-2 isolates in GenBank databases. Results confirmed the perfect matches to SARS-CoV-2 and inclusivity for 5 variants of concern are more than 99% in the database.
*Percentage of primer/probe matches with the sequences of SARS-CoV-2 (exhibiting ≤ 1 mismatch)
High Stability
The IdPath™ COVID-19 Real-Time RT-PCR Kit is stable at 37°C and 25°C for at least 2 weeks, and at 4°C for 24 weeks. The kit is also stable during 10 times of freeze-thaw cycles.
Clinical studies using contrived specimens
The data showed 100% agreement with the expected results in the RNA spiked specimens and 100% agreement with the expected results in the negative specimens. The results demonstrated that the IdPath™ COVID-19 Real-Time RT-PCR Kit has 100% sensitivity, 100% specificity and 100% accuracy. 
Clinical validation using patient specimens
The results of 60 positive (Ct < 30: 30 samples and Ct 30-35: 30 samples) and 30 negative samples from the patient specimens were compared to the FDA-authorized comparator real-time RT-PCR assay (CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel). The data showed that the IdPath™ COVID-19 Real-Time RT-PCR Kit has 100% sensitivity, 100% specificity and 100% accuracy. 
No Cross-Reactivity in Silico Analysis
An in silico analysis was performed to assess the potential of 47 organisms for interference with SARS-CoV-2 detection. The reference sequence (RefSeqs) in the NCBI Virus database or whole genome in the taxonomy database were used. No significant homology was observed between the SARS-CoV-2 primers/probes and other coronaviruses (except SARS-CoV) or human microflora.
No Cross-Reactivity in Wet Test
The cross-reactivity of the IdPath™ COVID-19 Real-Time RT-PCR Kit was also evaluated with the NATtrol™ Respiratory Verification Panel (Zeptometrix), which is comprised by 19 pathogens including those commonly found in respiratory tract. No cross-reactivity was observed between the SARS-CoV-2 primers/probes and 19 pathogens.
High Compatibility: Suitable for most laboratory qPCR machines
Applicable to Bio-Rad CFX96, ABI StepOnePlus, ABI 7500 Fast, Roche LC480, Roche LC96, as well as Qiagen Rotor Gene Q 5plex HRM qPCR instruments.
Product Information
Contents
Manual
Manual_IP2000_IdPath™ COVID-19 Real-Time RT-PCR Kit (2023 ver. 1.2.1)
Software Setting Guide
SDS
Flyer
IP2000 Operating Guide
Reagent Storage and Handling
Reagents, master mix, and RNA must be thawed and kept on a cold block at all times during preparation and use.
Do not push air into the assay mixtures and do not mix by violently pipetting up and down to avoid bubble generation.
Be sure not to introduce any foam or bubbles into the wells of 96-well plates when aliquoting reaction mix and nucleic acid.
As adding sample and control, immerse the tip below the surface of the liquid. Then press down the push button smoothly to the first stop.
Run on the qPCR machine immediately after mixture preparation is completed.
Recommended reaction mixture set up for qPCR
Component | 1 Reaction (Total 15 μl) | Volumes for N specimens (μl) |
Master Mix (B, Green) | 10 μl | 10 x (N+4) |
Enzyme Mix (E, Red) | 2 μl | 2 x (N+4) |
Primers/probes Mix (A, Amber) | 2 μl | 2 x (N+4) |
ddWater | 1 μl | 1 x (N+4) |
Sample | 5 μl | 5 x (N+4) |
Total volume | 20 μl | 20 x (N+4) |
Recommended qPCR Program
Fast program
Steps | Temp. | Time | Cycles |
Reverse transcription | 50°C | 10 min | 1 |
Enzyme activation | 95°C | 1 min | 1 |
Denaturation | 95°C | 3 sec | 40 |
Annealing/ Extension | 58°C | 30 sec |
Can the IdPathTM COVID-19 Real-Time RT-PCR Kit be applied to other qPCR machines not listed in the manual?
The kit could be applied to other qPCR machines not listed in the manual. However, please notice that users should use quantified SARS-CoV-2 RNAs to establish the appropriate threshold and baseline settings for their qPCR machine.
Can the threshold and baseline settings indicated in the manual for the different qPCR machines be changed? Or can I just set thresholds and baselines in an automatic mode?
The threshold and baseline settings listed in the manual for each qPCR machine are the best results obtained after numerous tests, and thus users are recommended to follow the instructions first. However, the users could use quantified SARS-CoV-2 RNAs to establish their own appropriate threshold and baseline settings. Automatic mode can also be used, as long as it meets the user's experimental needs.
Is it possible not to use the specimen as a test source?
Yes, the IdPathTM COVID-19 Real-Time RT-PCR Kit is suitable for test with viral RNA extracted from respiratory tract specimen, virus culture or other sources. However, the quality of RNA has a significant impact on the test results. Therefore, viral RNA extracted from different sources must meet the quality as pure RNA required, and not be largely fragmented or degraded.
Can the IdPathTM COVID-19 Real-Time RT-PCR Kit be stored at room temperature?
No, the kit must be stored at -20°C for long-term storage, but can be temporarily stored at 4°C for short-term storage. For multiple use, please aliquot the reagents first and store at -20° to avoid multiple freeze-thaw cycles.
What is the sensitivity and specificity of the IdPathTM COVID-19 Real-Time RT-PCR Kit?
You can find the detailed information of the analytical performance and clinical performance at the Technical/Spec section of IP2000 product in SMOBIO website (https://www.smobio.com/shop/product/ip2000-idpathtm-covid-19-real-time-rt-pcr-kit-100-rxn-477?search=IP2000#tab2).
What virus genes are targeted in the IdPathTM COVID-19 Real-Time RT-PCR Kit?
The IdPathTM COVID-19 Real-Time RT-PCR Kit targets regions of three SARS-CoV-2 genes (E, RdRP, and N gene), which exhibit low mutational entropy. This could reduce the influence of the SARS-CoV-2 susceptible mutation on the detection efficiency.
What sample collection swabs would you recommend for transport and processing?
The users can refer to the guidance on alternatives to sample collection swabs or viral transport medium (VTM) that the FDA believes to be appropriate on their website (https://www.cdc.gov/coronavirus/2019-nCoV/lab/guidelines-clinical-specimens.html).
Does each individual component have its own expiration date, or does the entire kit have the same expiration date?
The entire kit has the same expiration date. The kit has 12 months of shelf life from date of manufacture under appropriate storage.
Will the SARS-CoV-2 positive control work with assays from other manufacturers?
Our positive control is designedspecifically for the target regions in our assay only. Other manufacturers' kits may have different designs of target area in their assays.
Are there specific wells on 96-well plate that must be used for positive and negative controls?
Controls can be placed in any well of a 96-well plate, as long as they are properly assigned and defined for the interpretation software. However, please pay attention to the problem of possible cross-contamination during operation. It is recommended to place the negative control first and then place the positive control last, or refer to our manual for the recommended positions such as below.
NC: Negative control (ddWater)
PC: Positive control for the E/RdRP, N and RNase P genes
S: RNA sample
When using the IdPathTM COVID-19 Real-Time RT-PCR Kit, is it important to mix the reagent mixture well?
It is important to mix well and spin down the RT-PCR reaction plate. If it is not mixed sufficiently, this may lead to uneven reaction in the early cycles of the qPCR that can invalidate the run. However, please do not mix violently to avoid bubble generation. Before the reaction on the qPCR machine, please centrifuge the 96-well PCR plate to reduce air bubbles. The bubble may cause the amplification curve messy such as high fluctuations at the beginning, a non-standard S shaped curve or the curve downward at the end. Please refer to the link http://docs.smobio.com/document/doc/PV/IP2000 trouble shooting.pdf to get detailed information.
Can other brands of RNA extraction reagents or methods be used with IP2000?
The QIAamp Viral RNA Mini Kit (Cat. No. 52904) is one of the reagents commonly used to isolate viral RNA from respiratory samples and is currently widely used for the detection of SARS-CoV-2, the coronavirus that causes COVID-19. The viral RNA extracted with this reagent has been verified by our product IP2000 to effectively detect the coronavirus SARS-CoV-2. Therefore, the QIAamp Viral RNA Mini Kit is recommended first. The IdPathTM COVID-19 Real-Time RT-PCR Kit is suitable for test with viral RNA extracted from different extraction reagents or methods. However, the quantity and quality of RNA have a significant impact on the test results. Therefore, the RNA extraction by other brand reagents or methods must be sufficiently efficient, and the extracted viral RNA must meet the quality as pure RNA without largely fragmentation or degradation. Otherwise, the detection effect of the reagent may be affected.
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