Ustar Easynat Monkeypox

EasyNAT Monkeypox Virus Assay

Instructions for Use 

 EasyNAT® Monkeypox Virus Assay

U202028-20 

 Molecular Testing Anywhere 20 

EasyNAT® Monkeypox Virus Assay is intended for rapid and qualitative detection of Monkeypox Virus DNA in blood and skin lesion exudate sample. This assay is for professional use only. Not for self-testing. 

 Do not mix materials from different lots. IVD REF Name MPXV-Cartridge MPXV-DNA Extraction Solution MPXV-Positive Control MPXV-Negative Control Specification 1 test/cartridge 1.2 mL/tube 1 mL/tube 1 mL/tube Quantity 20 cartridges 20 tubes 1 tube 1 tube Main compositions Primers and probes specific to Monkeypox Virus, primers and probes specific to internal control, deoxyribonucleoside triphosphate (dNTP), DNA polymerase Guanidine salt, magnetic beads, internal control Nucleic acid containing Monkeypox Virus target gene Normal saline

Cell lysis, nucleic acid binding magnetic bead region Magetic beads-nucleic acid cleaning area Nucleic acid elution area, CPA amplification area In the MPXV-Cartridge in this assay is equipped with multiple hydrophobic separation layers to separate the lysis buffer, washing buffer and reaction solution. Heated by an external applicable instrument, the pathogen in the extraction solution will be chemically lysed and releases nucleic acids. Then, controlled by the magnets in external instrument, the released nucleic acids will pass through different layers respectively and finally be eluted in the cartridge legs for subsequent amplification reaction. Thus, the lysis, washing, elution and amplification reactions are completed in an enclosed cartridge. This assay uses Cross Priming Amplification (CPA)¹ technology to detect the conserved F3L gene sequence of Monkeypox Virus. Through specific amplification primers, fluorescent probes and DNA polymerases of high strand displacement activity, the reaction scheme can complete the specific amplification process of Monkeypox Virus at a constant temperature at a single time. During the process, the external applicable instrument will detect fluorescence signal and automatically generate real-time fluorescence curve in real time. The assay also contains an internal control (IC) that consists of a CPA system to monitor the effectiveness of sample extraction, purification and amplification. To run a test, the user only needs to add into cartridge the MPXV-DNA Extraction Solution and the sample. Under the control of applicable instrument, the nucleic acid will be extracted and purified automatically. The purified nucleic acid, together with CPA reaction reagent in the cartridge, will be mixed and heated by the instrument for isothermal amplification reaction. Meanwhile, fluorescent probe will specifically bind to target sequence and produce fluorescence signal, changes of which will be detected and analyzed by the instrument to determine and report the test result automatically. 

Comparison Analysis


SC Ustar
1. Mechanism of COVID-19 Point-of-care testing product Polymerase Chain Reaction(PCR)—for in vitro nucleic acid extraction and amplification Polymerase Chain Reaction (PCR) used for template amplification, similar to in situ PCR Isothermal cross priming amplification
2. All-in-one system? Not an all-in-one system

lUsers need to prepare and split reaction reagent before test.

lAfter sample processing, users have to take out PCR tube from the reagent carrier tank and centrifuge it before placing it into the amplification module in the workstation.

Users need to dilute sample, equilibrate reaction reagent from -20°C to room temperaNot an all-in-one system

ture, and ensure less than four freeze-thaw cycles.

 All-in-one system: result in, result out

lIntegrate nucleic acid extraction, amplification and detection in a self-contained instrument.

lNo manual operations needed after sample loading.

lAutomatic and intuitive report of result.
3. Are there any manual operations required for testing? 1.Prepare and load reaction reagent;

2.Load sample into PCR 4-tube strip;

3.Load into reagent carrier tank;

4.Take out PCR tube;

5.Cover the tube；

6.Centrifuge；

7.Insert the PCR tube into the PCR amplification module;

8.Amplification and detection.

 (PCR can be done as follows without nucleic acid extraction)

1．Equilibrate frozen reaction reagent at room temperature;

2．Prepare amplification reagent in bio-safety cabinet;

3．Divide reagent into small volume, leading to repeated freeze-thaw cycle;

4．Dilute or equilibrate sample;

5．Centrifuge;

6．Perform template and reagent loading process in bio-safety cabinet;

7．Load sample into the instrument.

 Three steps only:

1．Add extraction solution to cartridge;

2．Add sample to cartridge；

3．Close cartridge, scan QR code on cartridge cap, and the system will run test program and report result automatically.
4. Is single sample test available? Any reagent wasted? lOne sample, one single test.

lPotential reagent waste during reagent preparation.

 lPotential reagent waste during manual process of reagent preparation and addition.

lRepeated freeze-thaw cycle may cause a change in reagent volume and concentration, resulting in a change of reaction scheme concentration.

 For each sample, one single test provides one enclosed cartridge and independent extraction solution
5. Hands-on time ~20 min ~20 min 1 min
6. Time to get result 60~80 min 60~80 min 55 min
7. Is inactivated VTM applicable for the product? Not applicable Not applicable Yes
8. How to process collected samples? 1．Applicable to non-inactivated VTM.

2．Inactivate sample for 30min at 56℃ before use.

 1．Only non-inactivated VTM is allowed.

2．Inactivate sample for 30min at 56℃ before being loaded into the workstation.

 1．Inactivated VTM: directly collect sample with VTM and load it into the instrument for detection.

2．Non-inactivated VTM: inactivate sample for 30min at 56℃ before detection.
9. Is there any measures to ensure bio-safety? High risk of aerosol leakage while transporting and centrifuging samples. High risk of exposure to bio-hazard substances due to relatively long time of sample loading and reagent preparation Low risk.

Extraction, amplification completed in one enclosed cartridge.
10. Is any special space required in addition to bio-safety cabinet? Yes, because it is not an integrated instrument. lNot point-of-care testing.

lSimilar to in situ PCR Assay, this product does not extract nucleic acid, which is viable for many other ordinary PCR products.

lNo extraction and purification. False positive and false negative result may occur due to inhibitors.

 Integrated instrument without the need of extra testing space.
11. Can the product test Orf1ab, N genes and internal control simultaneously? Yes Yes Yes
12. Are there any preliminary performance data (sensitivity, specificity, etc.)? Yes Yes Yes
13. How many samples can be tested at a time? Can samples be tested separately or at any time? Four samples each time Four samples each time Available for 4/8/16 modules.

Each module runs independently, providing instant test for clinical patients as soon as they arrive.
14. Applicable test kit (s) Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing)

DirectDetect™ SARS-CoV-2 Detection Kit

 lDiagnostic Kit for M. tuberculosis Complex DNA (CPA Assay)

lDiagnostic Kit for Novel-Coronavirus (2019-nCoV) RNA (Isothermal Amplification-Real Time Fluorescence Assay)
15. Sample loading/pretreatment Users need to place tips in Hole H of reagent strip in advance, and pipetting is an open process. In situ PCR.

No need to extract sample, but it takes a long time to liquefy sputum sample.

 Users do not need to open the cartridge after sample loading. After test, used cartridge is directly disposed of as medical waste at high temperature and pressure.

1. Storage conditions: 2. Validity period: 6 months. For production date and expiry date, please refer to the label. 3.The product performance will not be affected by ambient temperature ( ) transportation within 15 days. [SAMPLE COLLECTION AND HANDLING] 1. Applicable sample types: Blood, skin lesion exudate 2. Sample collection (1) Skin lesion exudate: wipe the exudate with a sterile swab. If there is no exudate around skin lesion tissue, prick the lesion with a sterile blood collection needle and collect the exudates with a swab. Put the swab sample in a sample storage tube for subsequent testing. (2) Blood: Collect venous blood in an EDTA anticoagulant blood collection tube (*DO NOT use heparin anticoagulant tube). Gently turn the tube upside down 5-10 times to mix the anticoagulant and blood well. 3. Sample transportation Transport samples by ice pot or bubble chamber with ice, and keep the package sealed. 4. Sample storage Collected samples should be sent for testing as soon as possible. Samples which can be tested within 24 hours should be stored at ; If they cannot be tested within 24 hours, they should be stored at -70℃ or below (if -70℃ condition is not available, refrigerate at -20℃ for temporary storage). Avoid repeated freezing and thawing. 8℃ 2℃ 8℃ 2℃ 30℃ -25℃ [APPLICABLE INSTRUMENT] Nucleic Acid Amplification and Detection Analyzer (UC0102/UC0104/UC0108/UC0116) produced by Ustar Biotechnologies (Hangzhou) Ltd. 

 Please test in accordance with this IFU. 1. Sample Testing 1.1 Sample loading 1.1.1 Resuspend the MPXV-DNA Extraction Solution, and then transfer all the solution into the MPXV-Cartridge. 1.1.2 Add 200μL sample to MPXV-Cartridge, cover the cartridge cap, mix it well (paraffin may float but will not influence subsequent steps) and the cartridge is ready for testing. 1.2 Testing Note: This section lists the basic steps for running a test. Refer to the Operator Manual of applicable instrument for details. 1.2.1 Input cartridge information Put the QR code of the cartridge (located on the cartridge cap) prepared according to [TEST PROCEDURE] 1.1 in the scanning area of the instrument, and the system will automatically input the cartridge information and run the program MPXV0 used for amplification testing. If the scanning fails, click the [Scan QR code on the cartridge] button on the touch screen to manually input it. 1.2.2 Input sample information Put the sample barcode in the scanning area of the same instrument, and the instrument will automatically input the sample information. If the scanning fails, click the [Scan the sample barcode] button on the touch screen to manually input it. 1.2.3 Start the testing program Put the cartridge into the module of applicable instrument, close the module cover, and click the [Start] button on the touch screen to start the testing program, and time countdown will be displayed on the screen. 1.3 Viewing the result Test results will be displayed and saved automatically by instrument at the end of testing. Please refer to the [INTERPRETATION OF RESULTS] for details. 2. Quality control testing 2.1 Resuspend the MPXV-DNA Extraction Solution and transfer all the solution into the MPXV-Cartridge. 2.2 Add 200μL MPXV-Positive Control or MPXV-Negative Control to MPXV-Cartridge, cover the cap, shake the cartridge gently to mix it well (paraffin may float but will not influence subsequent steps), and the cartridge is ready for testing. 2.3 Follow the steps described in [TEST PROCEDURE] 1.2-1.3. [CUT-OFF VALUE] Upon completion of a test, results of monkeypox virus and IC will be displayed respectively, presenting typical S-type amplification curves (including the S-curve before the plateau phase). ●If the Tt value of MPXV≤ 30, test result will be MPXV Positive; Otherwise, it will be Negative. ●If the Tt value of an IC≤ 30, test result for corresponding IC will be Positive; Otherwise, it will be Negative. 

Result "Positive" "Negative" "Invalid" "No result" Interpretation Monkeypox Virus DNA was detected in the sample. No Monkeypox Virus DNA was detected in the sample, and the IC testing met the acceptance criteria. The IC testing did not meet the acceptance criteria, and it was uncertain whether Monkeypox Virus DNA was in the sample. The detection system did not obtain enough data. 1. Conditions Require Retesting If any of the following conditions occur, use a new MPXV-Cartridge to retest the rest samples. ●Invalid: inappropriate handling or collecting of samples, test reagent is inhibited, or product is expired. ●No result: the test is terminated before the due time. ●Abnormal results of external quality control (MPXV-Positive Control and MPXV-Negative Control): for example, if the negative control reports a positive result, there may exist contaminants. 2. Quality Control The assay includes internal quality control and external quality control. 2.1 Internal quality control The internal quality control, or the internal standard system, is used to monitor the occurrence of sample processing failure, amplification reagent failure and instrument failure. If the test result is positive, no requirement is made for the IC test result; if the test result is negative, the IC test result shall meet the acceptance criteria, otherwise the test result is invalid. 2.2 External quality control The MPXV-Positive Control is the nucleic acid containing the specific sequences of Monkeypox Virus at a concentration used to rule out amplification reagent failure or applicable instrument failure; the MPXV-Negative Control is normal saline used to monitor for contamination of the reagent or environment. Test result of MPXV-Positive Control shall be "Positive", and test result of MPXV-Negative Control shall be "Negative". 

1.The test results of this assay are for clinical reference only. The clinical diagnosis and treatment of patients should be considered in combination with their symptoms, signs, medical history, other laboratory tests and treatment effect. 2.Inappropriate collection, transport and treatment of samples or low virus levels may produce false negative result. 3.Mutation of target nucleic acid to be tested or the sequence alteration caused by other reasons may yield false negative result. 4.Other unverified interference or amplification inhibitors may yield false negative results. 

1.Limit of detection (LOD) 1,000 copies/mL 2. Agreement with commercial reference panels 2.1 Positive reference panels: P1-P2 were positive. 2.2 Negative reference panels (N1-N2): all negative. 2.3 Repeatability reference panel (J1-J10): all positive. 2.4 LOD reference panel: S1-S2 were positive. [PRECAUTIONS]

1. Please read this IFU carefully before use. For professional use only. Ustar shall not assume any responsibility for the false results and corresponding consequences due to improper handling of the assay or any problems not derived from the performance defects of the assay. 2.This assay does not contain human-derived materials. 3. For in vitro diagnostic use of Monkeypox Virus infection only. The diagnosis and treatment of should integrate patient symptoms, signs and other diagnostic methods for comprehensive consideration. 4. This assay is a disposable product, please do not reuse. 5. All samples and other materials after use should be disposed of in compliance with the [Medical Waste Management Regulations]². 6.Disinfect work table and required items regularly with 1% sodium hypochlorite solution, 75% alcohol solution or ultraviolet lamp. 7.The MPXV-DNA Extraction Solution contains insoluble particles. Please mix well before pipetting. 8.Make sure the QR code of the MPXV-Cartridge is clean and clear. Do not scribble or cover it to ensure the code can be read. 9.The MPXV-Cartridge should be used immediately once opened. 10.Do not open the module when the test is running. 11.Test results will be saved automatically in the instrument, please check on the [View] interface. 12.Do not squeeze the middle and lower part of the MPXV-Cartridge during operation. 13.Please operate in strict accordance with the instructions. Do not load the MPXV-Cartridge first. 14.Performance alteration during the storage, transportation, and use of reagents may occur due to improper storage, transportation, collection, processing of samples and non-standard test procedures. Please strictly follow the instructions. [REFERENCE] 1.Xu G, Hu L, Zhong H, et al. Cross priming amplification: mechanism and optimization for isothermal DNA amplification. Sci. Rep. 2012; 2:246. 2. Medical waste management regulations: The State Council of the People's Republic of China promulgated on June 16, 2003. [EXPLANATION OF SYMBOLS] IVD 

LOT Use-by date Caution Temperature limit Authorized representative in the European Community Keep away from sunlight Date of manufacture Do not re-use Consult instructions for use Manufacturer Batch code Keep dry Do not use if package is damaged Biological risks The product meets the basic requirements of European in vitro diagnostic medical devices directive 98/79/EC. IVD 

1st Dec 2022 Lieven Gevaert

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