LabTurboTM AIO COVID-19 RNA Testing Kit
SARS-CoV-2 nucleic acid detection
The kit should be stored at -20°C and the number of freeze-thaw is no more than three times and shipped on wet ice. The storage condition of all the components in the kit after open is at 4~8°C for 6 days. The mixtures prepared from components for target tube 1 and control tube 3 can be stored at 4°C~RT for 48 hours.
The kit can be used with LabTurboTM AIO SP-qPCR System (LabTurbo AIO) and LabTurboTM Viral DNA/RNA extraction kit for fully automated workflow from viral RNA extraction of NP swab, reaction setup, RT-qPCR, to CT result and report. It can also be used with other extraction systems and equipment with proper testing performance validation regulated by relative authorities.
TaqMan®is registered by Roche Molecular Systems Inc.
Table 1、Kit Contents and Storage
LabTurboTM AIO COVID-19 RNA Testing Kit (AIOQS3480; 480 reactions)
| Component | Description | Quantity | Storage |
| Primer/Probe mixture I (PM1) | Target tube I mixture (N1 gene) | 4 X 200 µl (10 X) | Store at -20 ⁰C for up to one year Ship at 4 to 8⁰C Store at 4 to 8⁰C for up to 6 days after open Avoid freeze-thaw cycle for more than 3 times. |
| Primer/Probe mixture III (PM3) | Control tube III mixture (EC, IC) | 4 X 200 µl (10 X) | |
| Reverse Transcription (RT) | For reverse transcription of tube I mixture and control tube III mixture | 4 X 100 µl (20 X) | |
| PCR Master Mix (MM) | For preparing target tube I mixture and control tube III mixture | 4 X 2.0 ml (2 X) | |
| Extraction Control RNA (EC) | 1000 copies/ul | 4 X 1.3 ml (50X) |
|
| Positive Control RNA (PC) | 1 copy/ul | 4 X 40 µl (4-5x LOD) |
Table 2、Required Materials Not Supplied
| Equipment | Description | P/N |
| LabTurboTM AIO SP-qPCR Automation System | For sample-to-result nucleic acid testing (nucleic acid extraction, assay setup, RT-QPCR reaction) | A2410, A2420 |
| LabTurboTM AIO SP-qPCR Dual-Panel Automation System | For sample-to-result nucleic acid testing (nucleic acid extraction, assay setup, RT-QPCR reaction) | AIO48S-144 |
| Laboratory refrigerators 4°C to 10°C |
For sample and reagent storage | N/A |
| Laboratory freezers –10°C to –30°C & ≤ –70°C | For sample and reagent storage | N/A |
| Laboratory Mixer or Vortex | For sample mixing | N/A |
| Microcentrifuge | For sample spin-down | N/A |
| Pipettes (1-1,000 µL) | For liquid handling | N/A |
| Reagent | Description | P/N |
| LabTurboTM Viral DNA/RNA Extraction AIO Kit | For viral RNA extraction (480 reactions, 500 μl sample input) | AIOLVX500 |
| 100% Ethanol | For RNA extraction | N/A |
| Human Specimen Control (HSC) | For quality control | NA |
| Consumables | Description | P/N |
| PCR 8-Strip Tubes (120) & Caps | For LabTurboTM AIO SP-qPCR Automation System, 120 strips/case | A0130 |
| LabTurboTM AIO Robotic Filter Tip 1100 μl (4800) | 96 tips/rack, 10 racks/pack, 5 pack/case | S0650 |
| LabTurboTM AIO Robotic Filter Tip 1100 μl (960) | 96 tips/rack, 10 racks/pack, 1 pack/case | S0610 |
| Nasopharyngeal swab | 480/pack | S091480 |
| Screw cap tube (2 ml) | 480/pack | S092480 |
Intended Use
LabTurboTM AIO COVID-19 RNA Testing Kit is a real-time reverse transcription polymerase chain reaction (RT-PCR) diagnostic product for SARS-CoV-2 RNA detection. The nasopharyngeal swab from individuals suspected of COVID-19 were provided by healthcare following local sampling standard. LabTurboTM AIO COVID-19 RNA Testing Kit for COVID-19 detection has been validated, but FDA’s independent review of this validation is pending.
The positive detection result represents that SARS-CoV-2 RNA has been identified. Laboratories are required to report all positive results to the appropriate public health authorities. The LabTurboTM AIO COVID-19 RNA Testing Kit does not preclude the chance of false positive result. Please contact your healthcare provider to determine how best to care for you based on the test results, medical history, and your symptoms.
The Negative results could not fully exclude the infection of SARS-CoV-2. Additional clinical observations, epidemiological information, and traveling history are recommended for comprehensive evaluation. It is possible for this test to give a negative result that is incorrect (false negative) in some people with COVID-19. This means that a patient could possibly still have COVID-19 even though the test is negative. If this is the case, the healthcare provider will consider the test result together with symptoms, possible exposures, and geographical location of places recently traveled in deciding on care.
Warnings and Precautions
In all test procedure, well laboratory training is essential to guarantee the test accuracy and safety. Independent area is needed to prevent contamination and false positive results. Inactivation step of specimens is required and must be handled under biosafety hood.
1. The LabTurboTM AIO COVID-19 RNA Testing Kit has been validated, but FDA’s review of this validation is pending.
2. Specimen sampling and processing should be performed according to the Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens for COVID-19. (https://www.cdc.gov/coronavirus/2019-ncov/lab/guidelines-clinical-specimens.html)
3. Personal protective equipment is recommended.
4. All patient specimens and wastes should be considered biohazardous, and sterilization is needed after testing.
5. RNase and DNase free pipette tips with aerosol barriers are recommended to handle infectious specimens.
6. Avoid swallowing or contacting skin and eye with Proteinase K/Lysis/Wash Buffer. If accidental swallowing or contact occur, flushing with tap water and medical care are needed.
Quality Control
1. Quality control requirements must be performed in conformance with local, state, and federal regulations or accreditation requirements and the user’s laboratory’s standard quality control procedures. For further guidance on appropriate quality control practices, refer to 42 CFR 493.1256.
2. Quality control procedures are intended to monitor reagent and assay performance.
3. Test all positive controls prior to running diagnostic samples with each new kit lot to ensure all reagents and kit components are working properly.
4. Always include a negative control (NTC or NEC) and all positive controls (PC, EC, IC) in each PCR run for clinical samples to confirm the quality of workflow including specimen collection, RNA extraction, Reaction setup, and RT-qPCR performance.
Pretreatment Protocol for Nasopharyngeal (NP) Swab Specimens
For specimen sampling, storage, and shipping and handling, please refer to the guidance issued by CDC. (https://www.cdc.gov/coronavirus/2019-ncov/lab/guidelines-clinical-specimens.html)
Procedure with virus inactivation before using LabTurboTM
Caution: Please handle specimens in biosafety hood!
Step 1: Vortex NP swab specimen with storage buffer for 30 seconds
Step 2: Transfer 0.5 ml of storage buffer of NP swab specimen into a Sample 6-tube Strip or 2 ml screw tube containing 10 μl of BE and 25 μl of proteinase K (The Sample 6-tube Strip, BE,,and proteinase K buffer are supplied in LabTurboTM Viral DNA/RNA Extraction AIO Kit).
Step 3: Incubate at room temperature for 10 minutes.
Step 4: Ready for nucleic acid extraction.
Guide for the Preparation of Reagents
Precautions
- Clear and RNase-free dedicated space is needed to prevent contamination. Reagents should avoid direct exposure from light.
- Each reagent should be mixed by vortex and centrifuge before open.
- Extraction control, positive control (SARS-CoV-2 template RNA), and no template control (NTC) are necessary for each run of RT-qPCR reaction.
Before running RT-qPCR by LabTurboTM AIO SP-qPCR System, prepare the materials (RT mixture, PCR Master Mix, Primer/probe mixture) in the 2 ml tube; PC and EC template are all ready to use.
Table 3 、Formula of RT-qPCR preparation
| Component | Target Tube I | Control Tube III |
| Reverse transcription (RT) mixture | 0.8 µl | 0.8 µl |
| 2X PCR Master Mix | 8.0 µl | 8 µl |
| Primer/probe mixture I | 1.6 µl | - |
| Primer/probe mixture III | - | 1.6 µl |
| Target Tube I Mixture* | 10.4 µl | |
| Control Tube III Mixture* | 10.4 µl | |
| Total Mixture volume for n reaction (+3 are for PC, NC, and dead volume) |
10.4 x (n +3) | 10.4 x (n +3) |
| Elution of extraction for each reaction | 5.6 ul | 5.6 ul |
| Total volume for each reaction | 16 ul | 16 ul |
Table 4、Conditions for RT-PCR
| RT reaction | Activation | Denature | Anneal & elongation |
|
| Temperature (⁰C) | 55 | 95 | 95 | 60 |
| Time (sec) | 600 | 60 | 10 | 15 |
| Cycle (s) | 1 | 1 | 40 | |
Workflow Using LabTurboTM AIO SP-qPCR System
LabTurboTM SP-qPCR All-in-one System evolves from the automated membrane-based isolation system (Taigen Bioscience)*. LabTurboTM AIO SP-qPCR System integrates the nucleic acid extraction, reaction setup, RT-qPCR, and Ct report procedure with the use of the LabTurboTM AIO COVID-19 RNA Testing Kit. The detection workflow from RNA extraction to RT-qPCR Ct report can be automatically completed in a single LabTurboTM AIO SP-qPCR System. The LabTurboTM viral DNA/RNA AIO extraction Kit is necessary for automated viral RNA extraction.
| *Screening for Babesia microti in the U.S.Blood Supply DNA was extracted with the use of an automated membrane-based isolation system (Taigen Bioscience) N Engl J Med. 2016 Dec 8;375(23):2236-2245 |
Operation of LabTurboTM AIO SP-qPCR System
LabTurboTM AIO Viral DNA/RNA Extraction Kit (Cat. No. AIOLVX500) is based on the column-based membrane extraction method. LabTurboTM AIO SP-qPCR System equips with active reagent vending function. Load the reconstituted reagents in their original bottles to the reagent bottle holders on the system safety door by following the instruction below:
- Follow the LW1 buffer reconstitute instruction provided on the LW1 reagent label. Add correct amount of absolute EtOH into the bottle and mix well. Place the LW1 bottle on the LW1 bottle holder.
- Add absolute EtOH to EtOH reagent bottle and place the EtOH bottle on the EtOH bottle holder.
- Place CCEB bottle (ready to use) on the CCEB bottle holder.
- Place VXL bottle (ready to use) on the VXL bottle holder.
- The first bottle is to be left empty.
- Turn on the machine, then click the LabTurbo AIO icon on the desktop to launch the system software.
- Select the sample numbers and choose the program “qPCR-COVID19-IC-N-1.5-LVX40-In0.5-E60-QS”, then click “Next”.
- Follow the worktable loading check instruction and make sure each consumable is placed correctly, then click “Next”
- Close the safety door. Press “Start”.
- When the program finishes, the Ct results will display on the monitor and can be exported.
| Workflow | Description | |||
| Specimen sampling, storage, and shipping and handling | Please refer to the guidance issued by CDC. (https://www.cdc.gov/coronavirus/2019-ncov/lab/guidelines-clinical-specimens.html) | |||
| Pretreatment of NP swab specimen | As described on Page 8 | |||
| AIO Startup | Turn on AIO instrument & launch system software | |||
| AIO Worktable setup | Flowing the guidance displayed on the system software to load the plastic consumables, reagents, Sample ID (by barcode or typing) and specimens, | |||
| AIO Start | Press “Start” to initiate the procedure | |||
| AIO viral RNA extraction | Input volume: 500 μl Elution volume: 60 μl |
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| AIO Reaction setup | Detection tube | Tube I | Tube III | |
| Target Tube I Mixture | 10.4 µl | - | ||
| Control Tube III Mixture* | - | 10.4 µl | ||
| Volume of elution product | 5.6 μl | 5.6 μl | ||
| AIO RT-qPCR reactive condition | RT reaction | Activation | Denature | Annealing/Elongation |
| 55⁰C | 95⁰C | 95⁰C | 60⁰C | |
| 600 sec | 60 sec | 10 sec | 15 sec | |
| 1 | 1 | 40 cycles | ||
| AIO qPCR growth curve |  |
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| AIO Ct Report |  |
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Each test result should be assessed by positive, negative, and internal control. These control reactions are necessary for each RT-qPCR reaction. In target tube 1, FAM signal shows SARS-CoV-2 has been detected. In control tube-3, FAM signal shows human cell internal control (IC) gene is detected and Cy5 signal shows RNA extraction control (EC) is detected. The result interpretation principle is as below:
Table 6、Result interpretation and actions
| N1 gene | EC | IC | Result | Interpretation and action |
| Target Tube 1 (FAM) |
Control Tube 3 (Cy5) |
Control Tube 3 (FAM) |
||
| + | + | +/- | Valid | SARS-CoV-2 RNA was Detected |
| + | - | + | Valid | |
| - | + | +/- | Valid | SARS-CoV-2 RNA was not Detected |
| - | - | + | Valid | |
| + | - | - | Invalid | Repeat extraction and RT-qPCR. If the repeated result remains invalid, consider collecting a new specimen from the patient. Consult the manufacturer for technical support and report it to regulatory authorities. |
| - | - | - | Invalid |
Cross Reactivity Testing of SARS-CoV-2 Specific Primer/Probe
In silico analysis of primers and probes in LabTurboTM AIO COVID-19 RNA Testing Kit were analyzed from NCBI database.
The following species were included for in silico analysis:
Table 7、Inclusivity (analytical sensitivity) for SARS-CoV-2
| SARS-CoV-2 strain | Country | N1 gene |
| GenBank: MT126808.1 | BRA | 100% |
| GenBank: MT123290.1 | CHN | 100% |
| GenBank: MT093571.1 | SWE | 100% |
| GenBank: MT066176.1 | TWN | 100% |
| GenBank: MT049951.1 | CHN | 100% |
| GenBank: MT163721.1 | USA | 100% |
| GenBank: LC534419.1 | JPN | 100% |
| GenBank: MT007544.1 | ANZ | 100% |
| GenBank: MT066156.1 | ITA | 100% |
| GenBank: MT233519.1 | ESP | 100% |
Table 8、in silico cross-reactivity analysis for SARS-CoV-2
| Strains | In Silico Analysis for % Identity to SARS-CoV-2 N1 gene priming site By AIO |
| Human coronavirus 229E | No alignment |
| Human coronavirus OC43 | No alignment |
| Human coronavirus HKU1 | No alignment |
| Human coronavirus NL63 | No alignment |
| SARS-coronavirus | 65% |
| MERS-coronavirus | No alignment |
| Adenovirus | No alignment |
| Human Metapneumovirus | No alignment |
| Parainfluenza virus 1 | No alignment |
| Parainfluenza virus 2 | No alignment |
| Parainfluenza virus 3 | No alignment |
| Parainfluenza virus 4 | No alignment |
| Influenza A | No alignment |
| Influenza B | No alignment |
| Influenza C | No alignment |
| Enterovirus | No alignment |
| Respiratory syncytial virus | No alignment |
| Rhinovirus | No alignment |
| Chlamydia pneumoniae | No alignment |
| Haemophilus influenzae | No alignment |
| Legionella pneumophila | No alignment |
| Mycobacterium tuberculosis | No alignment |
| Streptococcus pneumoniae | No alignment |
| Streptococcus pyogenes | No alignment |
| Bordetella pertussis | No alignment |
| Mycoplasma pneumoniae | No alignment |
| Pneumocystis jirovecii (PJP) | No alignment |
| Candida albicans | No alignment |
| Pseudomonas aeruginosa | No alignment |
| Staphylococcus epidermis | No alignment |
| Staphylococcus salivarius | No alignment |
Performance Characteristics (I)
The Limit of detection (LoD) test was performed by spiking different concentrations of the Heat Inactivated 2019 Novel Coronavirus from ATCC (VR-1986HKTM, lot number: 70035039, viral concentration: 1.6 x 105 TCID50/mL, RNA copy number by ddPCR: 3.75 x 105 genome copies/μL) into negative clinical NP swab preservation medium (VTM) of Puritan UniTranz-RT Transport System following the pretreatment protocol described on page 7 to evaluate the lowest viral copy number with 95% detection rate (19 positive detections from 20 contrived samples). The test was performed following the “Run COVID-19 RNA test on LabTurboTM AIO SP-qPCR System” on Table 5.
Information of ATCC Heat Inactivated 2019 Novel Coronavirus lot 70035039: https://www.atcc.org/~/media/Files/Certificates%20of%20Analysis/3/B/4/1/VR-1986HK_70035039.ashx
Table 9、Limit of Detection (LoD) - Analytical Sensitivity:
| N1 gene | ||||
| Concentration | ||||
| Repeat | 1 | 2.5 | 5 | 10 |
| 1 | No Ct | 31.3 | 29.97 | 27.9 |
| 2 | No Ct | 31.01 | 30.74 | 28.86 |
| 3 | No Ct | 31.62 | 29.48 | 29.07 |
| 4 | 32.79 | 30.98 | 29.17 | 28.36 |
| 5 | 35.43 | 31.4 | 30 | 29.16 |
| Average | 32.79 | 31.2275 | 29.84 | 28.5475 |
| Std | N/A | 0.2988 | 0.6844 | 0.5244 |
| CV | N/A | 0.0096 | 0.0229 | 0.0184 |
| Detection rate | 3/5 | 5/5 | 5/5 | 5/5 |
Performance Characteristics (II)
The Clinical evaluation testing was performed to evaluate the detection performance in artificial samples. In this study, negative clinical nasopharyngeal swab specimens in VTM of Puritan UniTranz-RT Transport System were collected and spiked with different concentrations of the Heat Inactivated 2019 Novel Coronavirus from ATCC (VR-1986HKTM, lot number: 70035039, viral concentration: 1.6 x 105 TCID50/mL, RNA copy number by ddPCR: 3.75 x 105 genome copies/μL) and EC/IC to obtain specimens of 1x, 2x, and 4x of LoD. The test was performed using the method listed on table 5 “Run COVID-19 RNA test on LabTurboTM AIO SP-qPCR System”. 30 negative clinical nasopharyngeal swab specimens in VTM were used as negative specimens. NTC was used as negative control; N.D.=Not detected.
Table10、Clinical evaluation study in Primer/Probe Mixture I (N1 gene) using NP swab sample.
| Replicate | Effective copy number in Nasopharyngeal specimens | Acceptance Criteria | |||
| 1X LoD 2.5 copies/ul |
2 X LoD 5.0 copies/ul |
4 X LoD 10.0 copies/ul |
Negative specimen | ||
| 1 | 31.34 | 30.98 | 30.97 | N.D. | Positive Ct < 40 Negative Ct > 40 (N.D.) |
| 2 | 30.85 | 32.78 | 32.43 | N.D. | |
| 3 | 33.28 | 32.64 | 30.64 | N.D. | |
| 4 | 33.22 | 31.09 | 31.98 | N.D. | |
| 5 | 32.93 | 30.81 | 29.09 | N.D. | |
| 6 | 33.69 | 30.96 | 30.18 | N.D. | |
| 7 | 31.69 | 30.9 | 31.6 | N.D. | |
| 8 | 32.21 | 31.7 | 31.17 | N.D. | |
| 9 | 32.04 | 31.27 | 32.25 | N.D. | |
| 10 | 32.97 | 33.23 | 30.98 | N.D. | |
| 11 | 30.36 | 30.8 | 33.36 | N.D. | |
| 12 | 31.25 | 31.4 | 30.79 | N.D. | |
| 13 | 32.3 | 31.01 | 31.28 | N.D. | |
| 14 | 32.79 | 30.9 | 32.45 | N.D. | |
| 15 | 31.57 | 31.83 | 32.26 | N.D. | |
| 16 | 31.63 | 31.38 | 31.42 | N.D. | |
| 17 | 33.68 | 33.18 | 31.66 | N.D. | |
| 18 | 31.43 | 30.7 | 31.34 | N.D. | |
| 19 | 33.01 | 32.43 | 33.86 | N.D. | |
| 20 | 31.99 | 33.31 | 31.16 | N.D. | |
| 21 | N.D. | ||||
| 22 | N.D. | ||||
| 23 | N.D. | ||||
| 24 | N.D. | ||||
| 25 | N.D. | ||||
| 26 | N.D. | ||||
| 27 | N.D. | ||||
| 28 | N.D. | ||||
| 29 | N.D. | ||||
| 30 | N.D. | ||||
| Average | 32.21 | 31.67 | 31.54 | - | |
| Standard deviation | 0.9528 | 0.9140 | 1.0715 | - | |
| CV | 0.0296 | 0.0289 | 0.0340 | - | |
| Positive samples | 20/20 (100%) | 20/20 (100%) | 20/20 (100%) | 0/30 (0%) |
|
| Concentration (Copies/ul) | Valid results | N1 gene | ||
| n | Average | Detection rate | ||
| 2.5 | 20 | 20 | 32.21 | 100% |
| 5 | 20 | 20 | 31.67 | 100% |
| 10 | 20 | |||
Clinical Confirmatory Study
For the clinical validation, 5 positive and 5 negative uncontrived NP swab samples from patients identified by this assay were sent to Bureau of Laboratories of Pennsylvania Department of Health to confirm the testing results. The comparator device uses US FDA EUA granted CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel.
Table 12 Clinical confirmatory study results
| CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel | |||
| Positive | Negative | ||
| LabTurboTM AIO COVID-19 RNA Testing Kit | Positive | 5 | 0 |
| Negative | 0 | 5 | |
| Positive percent agreement: 5/5 = 100% Negative percent agreement: 5/5 = 100% |
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Limitations
- The LabTurboTM AIO COVID-19 RNA Testing Kit has been validated, but FDA’s independent review of this validation is pending.
- The test was limited for use with nasopharyngeal (NP) swab specimen.
- False negative results might be caused by incorrect specimen sampling, handling, and transportation.
- False positive results might be caused by specimen cross-contamination during sampling or handling and/or RT-qPCR reagents contamination.
- Do not use any expired reagent for testing.
| File Name | Explanation | Tpye | Upload Date | Download |
|---|---|---|---|---|
| LabTurbo AIO COVID-19 RNA Testing Kit User Manual | Automated workflow for SARS-CoV-2 nucleic acid detection | 2020-06-08 |  |