TAIGEN Bioscience Corporation

LabTurboTM AIO COVID-19 RNA Testing Kit

SARS-CoV-2 nucleic acid detection

LabTurboTM AIO COVID-19 RNA Testing Kit contains oligonucleotide primers and dual-labeled hydrolysis probes (TaqMan®) and control material used in RT-qPCR for the in vitro qualitative detection of 2019-nCoV RNA in respiratory specimens. It uses one set of SARS-CoV-2 specific primer pair and probe that specifically recognize SARS-CoV-2 RNA sequence (N1 gene) and is designed to achieve high sensitivity (LOD 2.5 copies/ul) in the examination. The kit includes RNA Extraction Control (EC) for the confirmation of the RNA extraction, reverse transcription, and qPCR procedure for individual samples in batch operation. Furthermore, the Internal Control (IC) is provided in the kit to validate cells that were collected from sampling. They are provided in two individual reaction tubes including N1 gene detection in target tube I (1) and EC/IC detection in control tube III (3). Furthermore, the Positive Control (PC) is provided in the kit to validate the RT-qPCR and the buffer CCEB is used as no template control (NTC) and negative control (NC). The controls have to be included in each batch testing.

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 8C
 
Store at
4 to 8C
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

  1. Clear and RNase-free dedicated space is needed to prevent contamination. Reagents should avoid direct exposure from light.
  2. Each reagent should be mixed by vortex and centrifuge before open.
  3. 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
 
*The storage and in-use of Target Tube I Mixture and Control Tube III Mixture is at 4oC~RT for 48 hours
 
Table 4、Conditions for RT-PCR
  RT reaction Activation Denature Anneal &
elongation
Temperature (C) 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:
  1. 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.
  2. Add absolute EtOH to EtOH reagent bottle and place the EtOH bottle on the EtOH bottle holder.
  3. Place CCEB bottle (ready to use) on the CCEB bottle holder.
  4. Place VXL bottle (ready to use) on the VXL bottle holder.
  5. The first bottle is to be left empty.
  1. Turn on the machine, then click the LabTurbo AIO icon on the desktop to launch the system software.
  2. Select the sample numbers and choose the program “qPCR-COVID19-IC-N-1.5-LVX40-In0.5-E60-QS”, then click “Next”.
  3. Follow the worktable loading check instruction and make sure each consumable is placed correctly, then click “Next
  4. Close the safety door. Press “Start”.
  5. When the program finishes, the Ct results will display on the monitor and can be exported.
Table 5、Run COVID-19 RNA Testing on LabTurboTM AIO SP-qPCR System
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
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
55C 95C 95C 60C
600 sec 60 sec 10 sec 15 sec
1 1 40 cycles
 AIO qPCR growth curve
 AIO Ct Report
 
 

 
Results and Interpretation

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
 
N1 gene +: means CT <40; -: means not detected or CT >40;IC +: means CT <40; -: means not detected or CT >40;CT of PC in the range of 29-32 ; CT of EC in the range of 25-30; NTC should be CT >40 (N.D).


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
* The target sequences were blasted against NCBI Database and no alignment results were found. No potential unintended cross reactivity is expected based on this in silico analysis.


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
The result of LoD for N1 gene is 2.5 copies/ul


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%)
 
Table、11 Clinical evaluation study summary
 
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%
Testing results were confirmed correct without false positive or false negative results from detections using LabTurboTM AIO COVID-19 RNA Testing Kit.


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 pdf 2020-06-08