Sansure Biotech

Novel Coronavirus (2019-nCoV) Nucleic Acid

Diagnostic Kit (PCR-Fluorescence Probing)

For Emergency Use Only

Instructions for Use

(24 Tests/kit and 48 Tests/kit)

For in vitro Diagnostic (IVD) Use

For Prescription Use only
For Emergency Use Authorization only

Doc. #: 2019-nCoV IFU

Doc. Version: V02
Revision Date: March 25, 2022
Sansure Biotech Inc.
No. 680, Lusong Road
Yuelu District
Changsha, Hunan Province, 410205
PEOPLE’S REPUBLIC OF CHINA
+86-731-88883176
http://eng.sansure.com.cn/
 Table of Contents

1. Reference Number........................................................................... 4
2. Package Specification....................................................................... 4
3. Intended Use................................................................................... 4
4. Product Overview/Test Principle........................................................ 5
5. Components Included within the Kit................................................... 5
6. Reagent Stability and Transportation .................................................. 6
7. Components Required But Not Included within the Test ....................... 6
8. Warnings and Precautions................................................................. 6
9. Controls Materials ........................................................................... 5
10. Sample Collection, Storage and Transportation .................................. 8
11. Laboratory Procedure ....................................................................10
12. Interpretation of Results.................................................................18
13. Limitations...................................................................................19
14. Troubleshooting............................................................................21
15. Conditions of Authorization ...........................................................21
16. Performance Evaluation.................................................................23
17. Symbols.......................................................................................38
18. Contact Information and Product Support.........................................38
1. Reference Number
S3104E

2. Package Specification
24 tests/kit, 48 tests/kit

3. Intended Use
The Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing) is
a real-time RT-PCR test intended for the qualitative detection of nucleic acid from SARS- CoV-2
in nasopharyngeal swabs, oropharyngeal (throat) swabs, anterior nasal swabs, mid- turbinate
swabs, nasal washes and nasal aspirates from individuals who are suspected of COVID- 19 by their
healthcare provider. Testing is limited to laboratories certified under the Clinical Laboratory
Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, that meet requirements to perform
high complexity tests.

Results are for the identification of SARS-CoV-2 RNA. The SARS-CoV-2 RNA is generally
detectable in respiratory specimens during the acute phase of infection. Positive results are
indicative of the presence of SARS-CoV-2 RNA; clinical correlation with patient history and other
diagnostic information is necessary to determine patient infection status. Positive results do not
rule out bacterial infection or co-infection with other viruses. The agent detected may not be the
definite cause of disease.

Laboratories within the United States and its territories are required to report all results to the
appropriate public health authorities.

Negative results do not preclude SARS-CoV-2 infection and should not be used as the sole basis
for patient management decisions. Negative results must be combined with clinical observations,
patient history, and epidemiological information.

The Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing) is
intended for use by qualified, trained clinical laboratory personnel specifically instructed and
trained in the techniques of real-time PCR and in vitro diagnostic procedures. The Novel
Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing) is only for
use under the Food and Drug Administration’s Emergency Use Authorization (EUA).

4
4. Product Overview/Test Principle
During the 2019-nCoV pneumonia epidemic that happened in China, Sansure Biotech developed
a fast and simple NAT kit based on its advanced RNA fast-releasing technology. The Novel
Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing) is a real- time
reverse transcription polymerase chain reaction (rRT-PCR) test. The 2019-nCoV primer and probe
sets are designed to detect RNA from SARS-CoV-2 in respiratory specimens from patients who
are suspected of COVID-19 by their healthcare provider. This kit is used for qualitative
detection of the ORF1ab and N genes of SARS-CoV-2 RNA. By one simple step of centrifugation
and lysis, the sample mixture can be directly added to the 2019-nCoV-PCR master mix (2019-
nCoV-PCR Mix + 2019-nCoV-PCR-Enzyme Mix) to carry out rRT-PCR amplification. QIAamp
Viral RNA Mini Kit (50, Catalogue No. 52904) can be used as alternative extraction method.
Internal control targeting the RNase P gene monitor the sample collection, sample handling and
rRT-PCR process to avoid false-negative results. The LoD of the kit is 200 copies/mL.

The Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing) is
a real-time reverse transcription polymerase chain reaction (rRT-PCR) test. The 2019-nCoV primer
and probe set(s) is designed to detect RNA from SARS-CoV-2 in respiratory specimens from
patients with signs and symptoms of infection who are suspected of COVID-19.

5. Components Included within the Kit

Spec. & Qty.

No. Reagent Name Main Ingredients
24 T 48 T

1 Sample Release Reagent 1200 μL/tube x 1 1200 μL/tube x 2 Lysis buffer(S03)

Primers, Probes, dNTPs,

2 2019-nCoV-PCR Mix 624 μL/ tubex 1 1248 μL/ tube x 1
MgCl2, Rnasin, PCR buffer
2019-nCoV-PCR-Enzyme
3 96 μL/ tube x 1 192 μL/ tubex 1 RT Enzyme, Taq Enzyme
Mix
In vitro transcriptional RNA
2019-nCoV-PCR-Positiv e
4 Control 500 μL/tubex 1 500 μL/tubex 1 for ORF1ab, N gene and
internal control RNase P gene
2019-nCoV-PCR-
5 500 μL/tubex 1 500 μL/tubex 1 Saline
Negative Control
2.0 mL/tube × 24 ×
6 Sample Storage Reagent 2.0 mL/tube × 24 0.9% saline, Rnasin
2

5
6. Reagent Stability and Transportation
The diagnostic kit (in small box) should be stored at -20 ±5℃ in the dark and should be transported
in a sealed foam box with ice packs. The Sample Storage Reagent (in big and white box) should
be stored and transported at room temperature or 2 - 8℃ or below. The kit should be stored at -20
±5℃. Unpacked kits should avoid repeated freeze-thaw cycles.

7. Components Required But Not Included within the Test

Alternative extraction reagents:
QIAamp Viral RNA Mini Kit (50, Catalogue No. 52904, Qiagen), which is equivalent to the
QIAamp DSP Viral RNA Mini Kit in the U.S.A.

Consumables not supplied:

 Swab specimens with a synthetic tip, such as nylon or Dacron®, and an aluminum or plastic
shaft.
 1.5 mL DNase-free and RNase-free Eppendorf tube
 0.2 mL PCR tube or strip
 Various models of pipettes and pipette tips (10μL, 200μL and 1000μL tips with filters)
 Centrifuge (can reach to 12,000 rpm)
 Microcentrifuge
 desktop vortex mixer
 0.9% saline
 -20℃ cold blocks
 10% bleach
 DNAZap TM (Ambion, cat. #AM9890)
 Disposable powder-free gloves and surgical gowns

Real-Time PCR Instrument(s):

ABI 7500 Real-Time PCR System

8. Warnings and Precautions

Federal Law restricts this device to sale by or on the order of a licensed practitioner.

6
For emergency use only.
For in vitro diagnostic use only (IVD).

Follow standard precautions. All patient specimens and positive controls should be considered
potentially infectious and handled accordingly.

This test has not been FDA cleared or approved; This test has been authorized by FDA under an
EUA for use by authorized laboratories certified under the Clinical Laboratory Improvement

Amendments of 1988 (CLIA), 42 U.S.C. §263a, to perform high complexity tests.

This test has been authorized only for the detection of nucleic acid from SARSCoV-2, not for any
other viruses or pathogens.

This test is only authorized for the duration of the declaration that circumstances exist justifying the
authorization of emergency use of in vitro diagnostics for detection and/or diagnosis of COVID-19
under Section 564(b)(1) of the Act, 21 U.S.C. § 360bbb-3(b)(1), unless the authorization is
terminated or revoked sooner.

Do not eat, drink, smoke, apply cosmetics or handle contact lenses in areas where reagents and
human specimens are handled.

Handle all specimens as if infectious using safe laboratory procedures. Refer to Interim Laboratory
Biosafety Guidelines for Handling and Processing Specimens Associated with 2019- nCoV
https://www.cdc.gov/coronavirus/2019-nCoV/lab-biosafety-guidelines.html. Dispose of
hazardous or biologically contaminated materials according to the practices of your institution.

Please read the package insert carefully prior to operation. The Novel Coronavirus (2019-nCoV)
Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing) is only for emergency use with a
prescription, as an in vitro diagnostic (IVD) test. Each step of operation, from specimen collection,
storage and transportation, and laboratory testing, should be strictly conducted in line with relevant
biosafety regulations and molecular laboratory management.

Separate laboratory areas, dedicated to performing predefined procedures of the assay, are required.
a) 1st Area: Preparation Area—Prepare testing reagent: b) 2nd Area: specimen processing—
7
Process the specimen and controls: c) 3rd: Amplification Area—PCR conducted.

Sample Release Reagent has not been validated with specimens stored in U.S.A commercialized
sample storage, preservation, or transport media (VTM/UTM) and may cause false negative results.

The clinical laboratory should be equipped with instruments and operators in strict accordance with
relevant requirements outlined in local, state and national regulations. After the assay procedures,
the workbench and lab supplies should be cleaned and disinfected immediately.

All contents in this package are prepared and validated for the intended testing purpose.
Replacement or modification of any of the package contents will affect the testing performance of
the kit and is in violation of the product Emergency Use Authorization. Components contained
within a kit are intended to be used together. Do not mix or exchange components from different
kit lots. Prior to begin each assay, each component must be thoroughly thawed and briefly
centrifuged. Avoid repeated freeze-thaw cycles.

All pipette tips and centrifuge tubes in the assay should be sterile and DNase/RNase-free. To
prevent contamination, filtered pipette tips are required and should be replaced after the addition
of each reagent or sample.

Dispose of waste in compliance with local, state, and federal regulations.

All lab workbench and supplies should be cleaned and disinfected regularly using 70% Ethanol or
10% bleach.

Avoid exposure to light of the 2019-nCoV-PCR Mix.

Laboratories within the United States and its territories are required to report all results to the
appropriate public health authorities.

9. Controls Materials
9.1 2019-nCoV-PCR-Negative Control: A “no template” (negative) control is used to monitor
whether there is contamination for the rRT-PCR process and is used in each detectionrun.

8
9.2 2019-nCoV-PCR-Positive Control: A positive template control is used to monitor whether the
rRT-PCR process works properly and is used in each detection run.

9.3 An internal control for RNase P gene is used to monitor the sample collection, handling and
rRT-PCR process and is used in each sample amplification.

10. Sample Collection, Storage and Transportation

10.1 Equipment preparation
Clean and decontaminate all work surfaces, pipettes, centrifuges, and other equipment prior to
use. Decontamination agents, such as 10% bleach, 70% ethanol, and DNAzap™, should be used
to minimize the risk of nucleic acid contamination.

10.2 Specimen collection

The Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing) is intended
for the qualitative detection of nucleic acid from SARS-CoV-2 in respiratory specimens.

Collection should avoid possible contamination in collection, storage, and transportation. The
specimen should be presumed contagious and be handled according to relevant regulations.

Collection swabs should have a synthetic tip, such as nylon or Dacron®, and an aluminum or plastic
shaft. Calcium alginate swabs are unacceptable and cotton swabs with wooden shafts are not
recommended. After sample collection, swabs should be stored in Sample Storage Reagent
immediately. When using the Sample Storage Reagent provided by the manufacturer, the user is
able to directly lyse the sample using the Sample Release Reagent RNA fast-releasing technology
provided in this kit.

10.3 Storage and delivery of specimens:

Specimens can be immediately processed. Specimens should be tested within 24 hours if stored at
4℃. Specimens that cannot be tested within 24 hours should be stored at -70℃ or below (in the
absence of -70℃ storage conditions, specimens can be stored at -20℃ for 10 days, nucleic acid
can be stored at -20±5℃ for 15 days). Multiple freeze/thaw cycles should be avoided. Specimens
should be transported in a sealed frozen pitcher with ice or in a sealed foam box with ice.

9
11. Laboratory Procedure
11.1 Sample extraction
11.1a: Fast and simplified sample extraction method:
For clinical specimens preserved in Sample Storage Reagent, sample processing can use the
Sample Release Reagent RNA fast-releasing technology provided in the kit. Pipet 200 μL of
specimen into 1.5 mL EP tube, centrifuge at 12,000 rpm for 5 min, and then discard the supernatant
fluid carefully,avoid removing the precipitation in the bottom. Add 50 μL Sample Release Reagent
into each tube, vortex for 5 second. The lysed sample can be directly added to the rRT-PCR
reaction.

Precautions: Sample Release Reagent has not been validated with specimens stored in U.S.A
commercialized sample storage, preservation, or transport media (VTM/UTM) and may cause false
negative results.

11.1b: Qiagen QIAamp Viral RNA Mini Kit extraction method:

Qiagen Viral RNA Mini kit may be used as an alternative extraction method using specimens
preserved in Sample Storage Reagent provided or other U.S. commercialized sample storage,
preservation, or transport media (e.g., VTM/UTM). The extraction procedure should be performed
according to the manufacturer’s instructions: load 140 μL of specimen to each column and eluted
with 50 μL solution. The extracted RNA can be directly added to the rRT-PCR reaction
immediately or store at -70℃.

11.2. Preparation of reagents

11.2.1 Take out each component from the diagnostic kit and place them at room temperature. Allow
the reagents to equilibrate at room temperature, then vortex each of them respectively for later use.

11.2.2 Prepare the 2019-nCoV-PCR Master Mix (26 μL 2019-nCoV-PCR Mix + 4 μL 2019- nCoV-
PCR-Enzyme Mix) based on the total number of specimens, 2019-nCoV-PCR-Positive Control
and 2019-nCoV-PCR-Negative Control and mix thoroughly. The remaining reagent must be stored
at -20°C immediately.

10
 Table 1. Master mix preparation

1 sample 10 samples 24 samples 48 samples

2019-nCoV-PCR Mix (μL) 26 260 624 1248
2019-nCoV-PCR-Enzyme Mix (μL) 4 40 96 192
Note: The above configuration is for reference only.

11.2.3 Add 30 μL of 2019-nCoV-PCR Master Mix into each well. Cover the wells and transfer to
the sample processing area. Add 20 μL of the extracted RNA to the well pre-filled with reagent
mix in the following order: 2019-nCoV-PCR-Negative Control, patient specimen(s), and 2019-
nCoV-PCR-Positive Control. Cover each well, centrifuge at 2000 rpm for 10 seconds, and place
into Applied Biosystems ABI 7500 real-time RT-PCR system and record the exact location of
controls and each specimen.

11.3. Running a PCR amplification on ABI 7500 using 7500 softwarev1.5:

11.3.1. Start ABI 7500 real time PCR system: Turn on the computer connected to the system first,
then turn on ABI 7500 real time PCR system.
11.3.2. Load the instrument: Push the tray door to open it, load the prepared plate containing
samples and controls into the plate holder in the instrument. Ensure that the plate is properly aligned
in the holder. Close the tray door.
11.3.3. Set up the experiment run:
11.3.3.1. Double-click ABI 7500 icon (7500 software v1.5) on the desktop. A new window should
appear, select Create New Document from the menu.

11
 Re w Docuaent Wizard [8J
Defiae Doc'OaeDl
Sehet the uu,y, container, u.d template for the doeu.•nt, and enter th• operator namt and
eo.aents.

Assay: !standard Curve <Absolute Quanlihti on) .:.l

Container: 196• '1tll Cltlr' .:.l
Teaiplate: )Blank Document iJ ~rowse . .

Run Mode: !s t andard 7500 .:.l

Operator : !Admini s trator

Plate Name: Pl ate!

B Next > Finish Cancel

11.3.3.2. Click Next and a new screen will appear as below.

New Document Wi zar d [g)
Selecl Detecto,;
Selecl lhe c'etectois:j,OJ wil be us:ing in 1te dca..r rien:

FiriJ. j .:::.I
Dctcc1or Nom e I Dcacriplion I R<lportcr I Quencher Detectors in Do:::urneM I

« Rern:,-,e

< ~I
New Detectc,... 1

< RN'l< f.Anr.P.I

12
11.3.3.3. Click New Detector and a new screen will appear as below.
-

New Document Wizard X

Select Detectors
Select lhe detectors you

: jROX

Detector Name ocu

Reporter D~e:

Quencher D~e: !Inane)

Color: 
Notes:

.i.. ======-1
New Detector .. I
Create Another I OK Cancel

<J!.ack !!.ext > Finish Cancel

11.3.3.4. Start by creating the ORF1ab Detector. Include the following:

a. Name: ORF1ab
b. Description: leave blank
c. Reporter Dye: FAM
d. Quencher Dye: (none)
e. Color: to change the color of the detector indicator do the following:
① Click on the color square to reveal the color chart
② Select a color by clicking on one of the squares
③ After selecting a color click OK to return to the New Detector screen
f. Click the OK button of the New Detector screen to return to the screen shown above.

11.3.3.5. Repeat step 11.3.3.3 - 11.3.3.4 for each target in the panel.

Name Reporter Dye Quencher Dye

ORF1ab FAM （none）
N ROX （none）
IC CY5 （none）

13
11.3.3.6. After each Detector is added, the Detector Name, Description, Reporter and Quencher
fields will become populated in the Select Detectors screen. Before proceeding, the newly created
detectors must be added to the document. To add the new detectors to the document, click Add.
Detector names will appear on the right hand side of the Select Detectors window. Once all
detectors have been added, select (none) for Passive Reference at the top right hand drop down
menu.

Hew Doc Uien t Wi zar d fg]

So1oct Doloclors
Sel ec t t he de t ec tors y ou wi l l be usln;: in the docu enl .

l_ass;ive Refe renc e '. !ROX

D1t 1c t or 1 in Doc1Jm1nt.

« R.. ovo

< >
:iew Detect or , .. ,

<Bad< Next>

He w DocUi ent Wizard [8J

Se1ect De tectors
Selo ct th e do toct ors you wi ll be u~in, in t he docwn ent.

i nd ; ..:J _:j
:>etector :S:a:Ee De:;;;;;criptio Reporter I Q-..iend::aar
F~ll ncn •

< >
lew Det ector .. j

< Back Nei<t > Fnsh Cancel

14
11.3.3.7. Click Next, select the well containing the samples and controls, and then click the
Detector.
Rew Docuaent Wizard ~
Se t Up Suple Flat o
Setup the s ample plat e wi th t asks, quantit i e s and de te ctors.

Detector Que:1cher task Quant it~

ner.t
'ncr.t

< >

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<B!ICk Next > Fnsh Cancel

11.3.3.8. Click Finish.

11.3.3.9. Select the Instrument tab. Set the parameters as follows:
 Stage 1: 50°C for 30 min, 1 cycle;
 Stage 2: 95°C for 1 min, 1 cycle;
 Stage 3: 95°C for 15 sec, 60°C for 31 sec, 45 cycles.
 Stage 4: 25°C for 10 sec, 1 cycle;
 Sample Volume: 50
 Data Collection at Stage 3, Step 2 (60.0 @ 0:31)

15
Instrument Control - - - - - - - - - - - - ~
Start Estimated Time Remaining Heat
Block
Stop
Cycle - - - - - - - - - - - - - ~
D1 sconnect Status Stage : Rep :
Time St ep ·
Extend State ·

Add Di ssoci ati on Stage

'60. 0@ 0 31

16
11.3.3.10. Save the document and then click Start to run the evaluation.

11.4 Data Analysis

See below for step-by-step operation of ABI 7500 using 7500 software v1.5 for Data analysis:
11.4.1 After the run is completed, click Results. Click Amplification Plot tab and view and
adjust the raw data.
 In the Data window, Delta Rn vs Cycle should be selected.
 In the Detector window, “ORF1ab” “N” and “IC” should be selected.
 The Start (cycle) window should be “3-15.” The End (cycle) window should be 5-20.
Users can adjust the values according to the actual situation.
 Adjust the threshold just above the curve from NTC (noise) .
 Lastly, be sure to click “Analyze” icon to update the analysis.

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17
11.4.2 Click Report icon above the graph to display the cycle threshold (Ct) values.
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12. Interpretation of Results

All test controls should be examined prior to interpretation of patient results. If the controls are not
valid, the patient results cannot be interpreted. The Ct cutoff value of this kit is set as 40 and the
end user is required to review fluorescent curves before final interpretation. All the positive curves
should be typical S-shape amplification curves or without plateau for weakly positive samples.

1) Positive and Negative Controls

The positive control and negative control for each run are interpreted as described in Table 2 below.

Table 2. Positive and Negative Control Interpretation.

2019-nCoV-PCR-Positive Control 2019-nCoV-PCR-Negative Control

ORF1ab N IC ORF1ab N IC Results Actions

(FAM) (ROX) (CY5) (FAM) (ROX) (CY5)

Continue to result
+ + + - - - Valid
interpretation

Any one of them shows negative Not considered rRT-PCR failed, re-run

Invalid Extraction, rRT-PCR

Not considered Any one of them shows positive
contaminated, re-run

Result of (-): Ct value >40 or Undetermined

Result of (+): Ct value ≤ 40

If there is contamination for the re-run, please perform decontamination procedures.

18
2) Examination and Interpretation of Patient Specimen Results:
Assessment of clinical specimen test results should be performed after the positive and negative
controls have been examined and determined to be valid and acceptable. If the controls are not
valid, the patient results cannot be interpreted. Table 3 below describes the results interpretation
concerning the use of the controls provided with the test. The Ct cutoff value of this kit is set as 40
and the end user is required to review fluorescent curves before final interpretation. All positive
curves should be typical S-shape amplification curves or without plateau for weakly positive
samples (38≤Ct≤40).

Table 3. Interpretation of Results based on Controls.

ORF1ab N IC
(FAM) (ROX) (CY5) Results

+ +

+ - Not considered 2019-nCoV Positive

- +

- - + 2019-nCoV Negative

- - - Invalid

Result of (-): Ct value >40 or Undetermined

Result of (+): Ct value ≤ 40
Invalid Result: There is no typical S-shape amplification curve or Ct > 40 or No Ct detected for
ORF1ab gene (FAM), N gene (ROX) and internal control (CY5), indicating that the specimen
concentration is too low, or there are interfering substances that inhibit the reaction. If upon
retest, the result is invalid again, another fresh sample should be collected and tested.

13. Limitations
The use of this assay as an in vitro diagnostic under the FDA Emergency Use Authorization (EUA)
is limited to laboratories that are certified under the Clinical Laboratory Improvement Amendments
of 1988 (CLIA), 42 U.S.C. § 263a, to perform high complexity tests.

False positive and false negative results can be caused by poor specimen quality, improper sample
collection, improper transportation, improper laboratory processing, or a limitation of the testing
technology.

Mutation in the target sequence of SARS-CoV-2 or change in the sequence due to virus evolution
may lead to false negative results. The performance of this test was established based on the
evaluation of a limited number of clinical specimens. Clinical performance has not been established

19
with all circulating variants but is anticipated to be reflective of the prevalent variants in circulation
at the time and location of the clinical evaluation. Performance at the time of testing may vary
depending on the variants circulating, including newly emerging strains of SARS-CoV-2 and their
prevalence, which change over time.

Improper reagent storage may lead to false negative results.

Use of this assay is limited to personnel who are trained in the procedure. Failure to follow these
instructions may result in erroneous results.

The performance of the Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-
Fluorescence Probing) was established using nasopharyngeal/oropharyngeal swabs. Nasal swabs,
mid-turbinate nasal swabs, and bronchoalveolar lavage fluid specimens are also considered
acceptable specimen types for use with the kit. but performance has not been established.

Test results of the diagnostic kit can only be used as an aid in clinical diagnosis. Symptoms and
physical signs, disease history, other laboratory examinations and therapeutic reactions of the
patients should be comprehensively considered for the clinical diagnosis and treatment.

Unverified interfering substances or PCR inhibitors may lead to false negative or invalid results.

The fast-releasing technology using Sample Releasing Reagent has been evaluated only for use in
combination with the Sample Storage Reagent provided in the Novel Coronavirus (2019-nCoV)
Nucleic Acid Diagnostic Kit. Sample Release Reagent used with specimens stored in other storage,
preservation, or transport media (VTM/UTM) not provided in the kit has not been fully validated and
may cause false negative results.

The Orf1ab and N gene primer/probes may detect bat coronaviruses and the N gene primer/probes
may detect pangolin coronaviruses based on in silico analysis.

20
14. Troubleshooting

Problems Possible Causes Action

Verify each component and ensure the

No fluorescent volumes of reagent dispensed during
Error in the preparation of the
signal is detected preparation of the master mixture are correct.
master mixture
in any samples, Repeat PCR mixture preparation.
including positive
Verify the rRT-PCR instrument settings are
control Instrument settings error
correct.

If the fluorescent Clean surfaces and instruments with aqueous

Contamination of the
signal is detected detergents, wash lab coats, and replace test
in a negative extraction/preparation area tubes and tips in use.
control reaction
PCR tube not properly sealed Ensure plates are sealed correctly.
Components degraded Use a new batch.

If the fluorescent
signal does not Poor quality of RNA samples Repeat the test with the neat extracted RNA
display the
carrying interferences and 1:10 dilution of the extracted RNA.
sigmoidal
characteristic Repeat the test or contact the equipment
PCR equipment failure
supplier

15. Conditions of Authorization for the Laboratory

The Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing)
Letter of Authorization, along with the authorized Fact Sheet for Healthcare Providers, the
authorized Fact Sheet for Patients, and authorized labeling are available on the FDA website:
https://www.fda.gov/medical-devices/coronavirus-disease-2019-covid-19-emergency-use-
authorizations-medical-devices/in-vitro-diagnostics-euas.

However, to assist clinical laboratories using the Novel Coronavirus (2019-nCoV) Nucleic Acid
Diagnostic Kit (PCR-Fluorescence Probing) (“your product” in the conditions below), the
relevant Conditions of Authorization are listed below:

A. Authorized laboratories1 using your product will include with test result reports, all
authorized Fact Sheets. Under exigent circumstances, other appropriate methods for

21
 disseminating these Fact Sheets may be used, which may include mass media.

B. Authorized laboratories using your product will use your product as outlined in the
Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-Fluorescence
Probing) Instructions for Use. Deviations from the authorized procedures, including the
authorized instruments, authorized extraction methods, authorized clinical specimen
types, authorized control materials, authorized other ancillary reagents and authorized
materials required to use your product are not permitted.

C. Authorized laboratories that receive your product will notify the relevant public health
authorities of their intent to run your product prior to initiatingtesting.

D. Authorized laboratories using your product will have a process in place for reporting test
results to healthcare providers and relevant public health authorities, as appropriate.

E. Authorized laboratories will collect information on the performance of your productand

report to DMD/OHT7-OIR/OPEQ/CDRH (via email: CDRH-EUA-
Reporting@fda.hhs.gov) and You (Young Wang, (443)538-5780)) any suspected
occurrence of false positive or false negative results and significant deviations from the
established performance characteristics of your product of which they becomeaware.

F. All laboratory personnel using your product must be appropriately trained in PCR
techniques and use appropriate laboratory and personal protective equipment when
handling this kit, and use your product in accordance with the authorized labeling.

G. Sansure Biotech Inc., authorized distributors, and authorized laboratories using your
product will ensure that any records associated with this EUA are maintained until
otherwise notified by FDA. Such records will be made available to FDA for inspection
upon request.

1
The letter of authorization refers to, “Laboratories certified under the Clinical Laboratory Improvement
Amendments of 1988 (CLIA), 42 U.S.C. §263a, to perform high complexity tests” as “authorized laboratories.”

22
16. Performance Evaluation

1) Limit of Detection (LoD) - Analytical Sensitivity:

LoD studies were used to determine the lowest detectable concentration of SARS-CoV-2 RNA
at which approximately 95% of all (true positive) replicates test positive. The LoD was
determined by limiting dilution studies using characterized samples.

Preparation of the manufacturer’s standards:

RNA was extracted from a clinical specimen positive for SARS-CoV-2 RNA (oropharyngeal
swab, confirmed by gene sequencing) and from a 1:10 dilution of the same specimen using the
QIAamp Viral RNA Mini Kit (50, Catalogue No. 52904). The RNA concentration of the neat
and diluted specimen was determined by the median value of six replicates using digital PCR
(TD-1 digital PCR system). The final concentration of the positive sample was set as 5.4×105
copies/mL using the median value of ORF1ab gene and N gene.

LoD with Clinical Specimen:

The LoD of the Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-
Fluorescence Probing) was estimated by testing standardized dilutions of the positive specimen
serially diluted to 2.0×10 4 copies/mL, 2.0×10 3 copies/mL, 2.0×10 2 copies/mL, and 20
copies/mL (n = 5 each) using negative specimen matrix (a negative oropharyngeal swab
specimen in Sample Storage Reagent). The lowest concentration at which all 5 replicates were
positive was treated as the tentative LoD for each test. The LoD of each test was then confirmed
by testing 20 replicates with concentrations at the tentative limit of detection. The final LoD of
each test was determined to be the lowest concentration resulting in positive detection of 19 out
of 20 replicates.

The LoD of the Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-
Fluorescence Probing) was established using both extraction methods: the Sample Release
Reagent RNA fast-releasing technology (Tables 4 and 5) and Qiagen QIAamp Viral RNA Mini
Kit (Table 6). The results demonstrated that the LoD of the two extraction methods are
equivalent, which is 200 copies/mL.

23
Table 4. Tentative LoD Detection Results of 2019-nCoV Using Sample Release Reagent

Lowest Limit of

Concentration in
Dilution Tested
Stock 2019-nCoV

[copies/mL]
Concentration Detection

Dilution Factor

Replicate 1 Ct

Replicate 2 Ct

Replicate 3 Ct

Replicate 4 Ct

Replicate 5 Ct
Serial 10-Fold
2019- with Uniform (LoD) per

Call Rate
Titer
Positivity per Virus
nCoV Analyte Strain
Strain
Tested

ORF1 2.7×10-1 20000 30.89 30.96 31.14 31.57 31.24 100%

ab
gene 2.7×10-2 2000 33.67 34.26 34.55 35.11 35.02 100%
5.4 200 200
2.7×10-3 200 36.63 39.07 36.65 36.65 37.62 100%
×10 5
copies/mL copies/mL
2.7×10-4 20 38.07 Undet Undet 40.43 Undet 40%
copies/
mL

N 2.7×10-1 20000 29.97 30.53 30.21 31.03 30.66 100%

gene
2.7×10-2 2000 34.36 34.04 33.61 33.76 33.62 100%
5.4
200 200
×105 2.7×10-3 200 35.60 37.11 38.02 35.67 36.23 100%
copies/mL copies/mL
copies/ 2.7×10-4 20 37.88 Undet Undet 37.34 Undet 40%
mL

24
Table 5. LoD Detection Results of 2019-nCoV Using Sample Release Reagent

Concentration(copies/mL)

Test No. ORF1ab gene N gene

400 200 100 400 200 100

1 36.78 36.26 39.55 35.64 36.50 38.28

2 36.32 36.52 38.65 38.61 36.93 38.19

3 36.84 37.36 37.05 36.19 37.27 37.50

4 36.18 37.59 Undet 35.81 36.72 Undet

5 36.51 36.28 37.81 36.08 36.64 37.23

6 34.75 36.73 Undet 35.56 36.13 Undet

7 36.79 38.18 39.13 35.91 37.44 38.42

8 35.59 38.27 Undet 35.59 36.99 Undet

9 35.27 36.74 Undet 35.54 36.88 Undet

10 36.86 37.85 37.09 35.87 36.89 42.13

11 36.99 36.85 Undet 36.04 36.86 36.71

12 36.76 Undet Undet 35.43 Undet Undet

13 37.38 36.90 39.07 35.90 37.33 38.47

14 35.76 37.88 Undet 36.27 37.81 Undet

15 36.51 38.03 Undet 35.23 36.54 Undet

16 36.54 36.06 38.33 35.49 39.35 Undet

17 36.32 37.63 37.20 36.13 36.00 38.65

18 36.23 37.92 Undet 35.00 38.54 Undet

19 35.46 37.95 Undet 35.30 36.04 Undet

20 36.35 37.95 39.08 36.67 37.65 38.25

Call rate 100% 95% 50% 100% 95% 45%

25
Table 6. LoD Detection Results of 2019-nCoV Using Qiagen QIAamp Viral RNA Mini
Kit (50, Catalogue No. 52904)

Concentration copies/mL

Test No. ORF1ab gene N gene

400 200 100 400 200 100

1 35.83 38.49 37.93 35.77 37.39 37.87

2 36.22 36.35 Undet 37.00 35.99 Undet

3 37.56 36.82 37.45 35.75 39.59 37.66

4 36.72 36.44 Undet 36.10 36.69 Undet

5 36.41 38.29 35.52 35.92 37.27 37.67

6 35.59 Undet Undet 36.01 Undet Undet

7 35.16 36.54 38.59 34.84 36.97 36.22

8 36.34 36.86 38.80 35.10 37.29 37.46

9 35.63 36.59 39.01 36.03 38.05 36.78

10 35.72 38.42 Undet 35.53 36.03 Undet

11 37.41 39.39 Undet 36.10 38.08 Undet

12 35.10 35.66 Undet 36.07 37.03 Undet

13 35.29 37.35 Undet 35.01 36.03 Undet

14 36.32 36.03 36.73 35.13 36.11 Undet

15 35.94 36.01 37.11 34.34 36.40 36.65

16 35.38 36.43 38.06 34.49 36.32 37.54

17 36.01 36.70 Undet 34.72 35.32 Undet

18 38.02 36.82 Undet 34.91 37.05 Undet

19 35.89 35.55 Undet 35.45 36.70 38.10

20 36.03 34.75 Undet 36.69 35.60 41.39

Call rate 100% 95% 45% 100% 95% 45%

26
2) Inclusivity (analytical sensitivity):

Inclusivity of the primer/probe set used in the Novel Coronavirus (2019-nCoV) Nucleic Acid
Diagnostic Kit (PCR-Fluorescence Probing) was analyzed in silico based on SARS-CoV-2
sequences from GISAID (6442390 sequences), NGDC 2019nCoVR (6563698 sequences), NCBI
(2957763 sequences) database accessed on December 23, 2021. The primer/probe sets for ORF1ab
gene and N gene sequencing alignment analysis demonstrate 100% inclusivity for SARS-CoV-2
sequences identified from patient samples. The representative alignment results for both genes are
shown in Table 7.

Table 7. Representative results of In Silico Analysis for 2019-nCoV primers/probes against

the reported 2019-nCoV sequences by 2021-12-23.

% Homology % Homology % Homology

Strain Target Accession
Test FP% Test RP% Test Probe%

BetaCoV/Wuhan/WH-01/2019 ORF1ab gene CNA0007332 100 100 100

BetaCoV/Wuhan/WH-01/2019 N gene CNA0007332 100 100 100

hCoV-19/Wuhan/IVDC-HB-01/2019 ORF1ab gene EPI_ISL_402119 100 100 100

hCoV-19/Wuhan/IVDC-HB-01/2019 N gene EPI_ISL_402119 100 100 100

hCoV-19/Wuhan/WIV04/2019 ORF1ab gene EPI_ISL_402124 100 100 100

hCoV-19/Wuhan/WIV04/2019 N gene EPI_ISL_402124 100 100 100

hCoV-19/Guangdong/20SF012/2020 ORF1ab gene EPI_ISL_403932 100 100 100

hCoV-19/Guangdong/20SF012/2020 N gene EPI_ISL_403932 100 100 100

hCoV-19/Nonthaburi/61/2020 ORF1ab gene EPI_ISL_403962 100 100 100

hCoV-19/Nonthaburi/61/2020 N gene EPI_ISL_403962 100 100 100

hCoV-19/USA/IL1/2020 ORF1ab gene EPI_ISL_404253 100 100 100

hCoV-19/USA/IL1/2020 N gene EPI_ISL_404253 100 100 100

hCoV-19/USA/CA1/2020 ORF1ab gene EPI_ISL_406034 100 100 100

hCoV-19/USA/CA1/2020 N gene EPI_ISL_406034 100 100 100

hCoV-19/France/IDF0372/2020 ORF1ab gene EPI_ISL_406596 100 100 100

hCoV-19/France/IDF0372/2020 N gene EPI_ISL_406596 100 100 100

hCoV-19/Australia/VIC01/2020 ORF1ab gene EPI_ISL_406844 100 100 100

hCoV-19/Australia/VIC01/2020 N gene EPI_ISL_406844 100 100 100

hCoV-19/Germany/BavPat1/2020 ORF1ab gene EPI_ISL_406862 100 100 100

hCoV-19/Germany/BavPat1/2020 N gene EPI_ISL_406862 100 100 100

hCoV-19/Singapore/1/2020 ORF1ab gene EPI_ISL_406973 100 100 100

hCoV-19/Singapore/1/2020 N gene EPI_ISL_406973 100 100 100

27
 hCoV-19/England/01/2020 ORF1ab gene EPI_ISL_407071 100 100 100

hCoV-19/England/01/2020 N gene EPI_ISL_407071 100 100 100

hCoV-19/Finland/1/2020 ORF1ab gene EPI_ISL_407079 100 100 100

hCoV-19/Finland/1/2020 N gene EPI_ISL_407079 100 100 100

hCoV-19/Japan/AI/I-004/2020 ORF1ab gene EPI_ISL_407084 100 100 100

hCoV-19/Japan/AI/I-004/2020 N gene EPI_ISL_407084 100 100 100

hCoV-19/South Korea/KCDC03/2020 ORF1ab gene EPI_ISL_407193 100 100 100

hCoV-19/South Korea/KCDC03/2020 N gene EPI_ISL_407193 100 100 100

Figure 1. The schema of mutations with frequency higher than 1% until December 2021.
Frequency

~] () 1-~ t,,~
I 6~
11 1 ,I I I
'o~ \ 0~ \ t-~ \ t,,~
.I LI .
\ O~ \ 'o~
I
1,0~
Iii.I 111
1,1-~ 1,t,,~
II
1,6~
1111111.
1,'o~

c : : : : = = =~ o a === Spike 3a Mf-• N 10

Specifically, an in silico inclusivity analysis of the primer/probe sets was performed using complete
genomes with high coverage in the GISAID database from 23 Oct 2021 to 23 Dec 2021, which
includes the following variants of concerns currently designated by WHO:

 18,195 genomes of the Alpha variant

 1,816 genomes of the Beta variant
 126,1398 genomes of the Delta variant
 18,002 genomes of the Gamma variant
 33,288 genomes of the Omicron variant

The analysis demonstrated that all genomes for each variant were predicted unlikely to impact the
detection of SARS-CoV-2.

The wet testing of inclusivity using the Sample Release Reagent RNA fast-releasing technology
was evaluated as supplemental data by testing three SARS-CoV-2 positive specimens from
different areas in China. These specimens were confirmed positive by China CDC suggested rRT-
PCR kit. Each specimen was diluted to 1×LoD in negative specimen matrix (oropharyngeal swab
specimen in Sample Storage Reagent) and tested in triplicate (Table 8).

28
 Table 8. Reactivity: Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-
Fluorescence Probing)

Ct of
2019-nCoV Ct of N
ORF1ab
Source/Sample Type* Concentration gene
Strain/Isolate
gene
35.24 37.77
Specimen 1 from oropharyngeal swab,
Wuhan inactivated 200 copies/mL 34.30 36.65
36.11 35.99
35.42 38.34
Specimen 2 from oropharyngeal swab,
Beijing inactivated 200 copies/mL 35.61 36.73
35.14 36.48
37.36 37.44
Specimen 3 from oropharyngeal swab,
Hunan inactivated 200 copies/mL 34.93 37.85
34.95 35.26
* Samples were inactivated at 50 ℃ for 30 minutes.

3) Cross-reactivity (Analytical Specificity):

Cross Reactivity: Cross-reactivity of the Novel Coronavirus (2019-nCoV) Nucleic Acid

Diagnostic Kit (PCR-Fluorescence Probing) was evaluated by both in silico analysis and by wet
testing potentially cross-reactive whole pathogens or purified nucleic acid from clinical
specimens. For wet-testing, each sample was diluted to a specific concentration in negative
specimen matrix (a negative oropharyngeal swab specimen in Sample Storage Reagent) and
tested in triplicate using Sample Release Reagent RNA fast-releasing technology (Table 9). No
cross-reactivity was detected. The in silico mapping analysis of each primer/probe against 27
pathogens is based on the NCBI nr/nt database accessed March 25, 2020 using the online
BLASTN 2.10.0+ and the representative results are shown in Table 10. The Orf1ab and N gene
primer/probes may detect bat coronaviruses and the N gene primer/probes may detect pangolin
coronaviruses based on this in silico analysis. No cross reactivity was observed for other listed
respiratory pathogens based on both in silico and wet-testing.

29
Table 9. Cross-Reactivity of Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit
(PCR-Fluorescence Probing)

Ct Value
Source/ Sample
(ORF1ab gene/N
Virus/Bacteria/Parasite Strain type Concentration gene)
Human coronavirus 229E 229E Clinica l specimen 1.0×10 6copies/mL Undet/Undet
Human coronavirus OC43 OC43 Clinica l specimen 1.0×10 6copies/mL Undet/Undet

Human coronavirus HKU1 HKU1 Clinica l specimen 1.0×10 6copies/mL Undet/Undet

Human coronavirus NL63 NL63 Clinica l specimen 1.0×10 6copies/mL Undet/Undet
SARS-coronavirus RNA 1.0×10 6copies/mL Undet/Undet

MERS-coronavirus Pseudovirus 1.0×10 6copies/mL Undet/Undet

Adenovirus 1 Clinica l specimen 1.0×10 6copies/mL Undet/Undet

Human Metapneumovirus (hMPV) Clinica l specimen 1.0×10 6copies/mL Undet/Undet
Parainfluenza virus 1-4 Clinica l specimen 1.0×10 6copies/mL Undet/Undet

Influenza A Na tional standard 1.0×10 6TCID50/mL Undet/Undet

Influenza B Clinica l specimen 1.0×10 6copies/mL Undet/Undet
Enterovirus (EV-C95) Clinica l specimen 1.0×10 6copies/mL Undet/Undet

Respiratory syncytial virus Clinica l specimen 1.0×10 6copies/mL Undet/Undet

Rhinovirus Clinica l specimen 1.0×10 6copies/mL Undet/Undet
Chlamydia pneumonia Clinica l specimen 1.0×10 6copies/mL Undet/Undet

Haemophilus influenzae Na tional standard 1.0×10 6CFU/mL Undet/Undet

Legionella pneumophila Na tional standard 1.0×10 6CFU/mL Undet/Undet
Mycobacterium tuberculosis Clinica l specimen 1.0×10 6copies/mL Undet/Undet
Streptococus pneumoniae Na tional standard 1.0×10 6CFU/mL Undet/Undet

Streptococcus pyogenes Clinica l specimen 1.0×10 6copies/mL Undet/Undet

Bordetella pertussis Clinica l specimen 1.0×10 6copies/mL Undet/Undet
Mycoplasma pneumoniae Clinica l specimen 1.0×10 6copies/mL Undet/Undet
Pneumocystis jirovecii (PJP) Clinica l specimen 1.0×10 6copies/mL Undet/Undet
Pooled human nasal wash - to
represent diverse microbial flora
Clinica l specimen 15ng/μL Undet/Undet
in the human respiratory tract

30
Table 10. The In Silico Specificity Analysis of Primer and Probe Sets for Other Respiratory
Pathogens.

Pathogen GenBank % Homology % Homology % Homology

Strain Target Acc# Test FP Test RP Test Probe
(Taxonomy ID)

Human coronavirus MF593473.

camel/Abu Dhabi/B38 N gene 55.00% 46.20% 60.90%
229E (11137)
1

Human coronavirus ORF1ab MF593473.

camel/Abu Dhabi/B38 50.00% 52.90% 36.00%
229E (11137) gene
1

Human coronavirus HCoV_OC43/Seattle/USA/SC9 MN310476.

N gene 45.00% 38.50% No Sig.
OC43 (31631) 428/2018
1

Human coronavirus HCoV_OC43/Seattle/USA/SC9 ORF1ab MN310476.

41.70% 76.50% 40.00%
OC43 (31631) 428/2018 gene
1

Human coronavirus KY983584.

HKU1 SC2628 N gene 40.00% 42.30% 43.50%
HKU1 (290028)
1

Human coronavirus ORF1ab KY983584.

HKU1 SC2628 45.80% 52.90% 36.00%
HKU1 (290028) gene
1

Human coronavirus HCoV_NL63/Seattle/USA/SC0 MN306018.

N gene 40.00% 42.30% 43.50%
NL63 (277944) 179/2018
1

Human coronavirus HCoV_NL63/Seattle/USA/SC0 ORF1ab MN306018.

41.70% 52.90% 40.00%
NL63 (277944) 179/2018 gene
1

MERS-CoV (1335626) BtVs-BetaCoV/SC2013 N gene KJ473821.1 No Sig. 42.30% No Sig.

ORF1ab
MERS-CoV (1335626) BtVs-BetaCoV/SC2013 KJ473821.1 41.70% No Sig. No Sig.
gene

AB568098.
Adenoviridae (10508) 53/FS161/Fukui/2004 N gene 55.00% 42.30% 47.80%
1

ORF1ab MK625182.
Adenoviridae (10508) ITA/2018/251170-16 58.30% 70.60% No Sig.
gene
1

Human
MN745086.
metapneumovirus bj0154 N gene 50.00% No Sig. 47.80%
1
(162145)

Human
ORF1ab KM408077.
metapneumovirus
C-85473 gene 45.80% No Sig. No Sig.
1
(162145)

31
 Pathogen GenBank % Homology % Homology % Homology
(Taxonomy ID) Strain Target Acc# Test FP Test RP Test Probe

Paramyxoviridae MK513627.
MVs/Venezia.ITA/22.17/3[D8] N gene No Sig. 73.10% No Sig.
(11158)
1

Paramyxoviridae ORF1ab KF687324.

HPIV3/MEX/2822/2006 No Sig. 76.50% No Sig.
(11158) gene
1

Orthomyxoviridae A/Homo MT106847.

N gene No Sig. No Sig. 56.50%
(11308) sapien/China/LS314/2019
1

Orthomyxoviridae A/sanderling/New ORF1ab CY117434.

No Sig. 76.50% No Sig.
(11308) Jersey/756/1986 gene
1

Influenza A virus A/Homo MT106847.

N gene No Sig. No Sig. 56.50%
(11320) sapien/China/LS314/2019
1

Influenza A virus A/sanderling/New ORF1ab CY117434.

No Sig. 76.50% No Sig.
(11320) Jersey/756/1986 gene
1

Influenza B virus MK999210.

B/New York/20/2018 N gene No Sig. 61.50% No Sig.
(11520)
1

Influenza B virus ORF1ab MT029398.

B/Alabama/12/2019 50.00% No Sig. No Sig.
(11520) gene
1

AY421766.
Enterovirus (12059) Donovan N gene No Sig. 69.20% No Sig.
1

ORF1ab DQ092794.
Enterovirus (12059) PS87/Belfast; ATCC VR-774 70.80% No Sig. No Sig.
gene
1

Respiratory syncytial
B/WI/629-Q0306/10 N gene JN032121.2 45.00% No Sig. 47.80%
virus (12814)

Respiratory syncytial ORF1ab MK947359.

99-901 No Sig. 47.10% No Sig.
virus (12814) gene
1

AY421766.
Rhinovirus (12059) Donovan N gene No Sig. 69.20% No Sig.
1

ORF1ab
Rhinovirus (12059) PS-87 X79368.1 70.80% No Sig. No Sig.
gene

Chlamydia pneumoniae LN847257.

- N gene 65.00% 46.20% 47.80%
(83558)
1

Chlamydia pneumoniae ORF1ab LN847257.

- 54.20% 64.70% 52.00%
(83558) gene
1

Haemophilus influenzae CP043770.

- N gene No Sig. 53.80% 47.80%
(727)
1

Haemophilus influenzae ORF1ab CP043770.

- No Sig. 76.50% 48.00%
(727) gene

32
 1

Legionella pneumophila CP011105.

- N gene No Sig. No Sig. 65.20%
(446)
1

Legionella pneumophila ORF1ab CP011105.

- 62.50% No Sig. No Sig.
(446) gene
1
Mycobacterium CP045962.
- N gene 60.00% 46.20% 52.20%
tuberculosis (1773)
1

Mycobacterium ORF1ab CP045962.

- No Sig. 76.50% 48.00%
tuberculosis (1773) gene
1

Streptococcus CP038808.
- N gene 60.00% 53.80% 52.20%
pneumoniae (1313)
1

Streptococcus ORF1ab CP038808.

- 54.20% 64.70% No Sig.
pneumoniae (1313) gene
1

Streptococcus pyogenes CP036530.

- N gene 65.00% 50.00% No Sig.
(1314)
1

Streptococcus pyogenes ORF1ab CP036530.

- No Sig. No Sig. 68.00%
(1314) gene
1

Bordetella pertussis CP033419.

- N gene 65.00% No Sig. 52.20%
(520)
1

Bordetella pertussis ORF1ab CP033419.

- No Sig. 70.60% No Sig.
(520) gene
1

33
 Pathogen GenBank % Homology % Homology % Homology
(Taxonomy ID) Strain Target Acc# Test FP Test RP Test Probe

Pneumocystis jirovecii XM_01837

RU7 N gene No Sig. 50.00% No Sig.
(42068) 2654.1

Pneumocystis jirovecii ORF1ab XM_01837

RU7 No Sig. No Sig. 60.00%
(42068) gene 3664.1

CP032019.
Candida albicans (5476) - N gene 60.00% 53.80% 60.90%
1

ORF1ab CP032019.
Candida albicans (5476) - 54.20% No Sig. 48.00%
gene
1

Pseudomonas CP047697.
- N gene 70.00% No Sig. No Sig.
aeruginosa (287)
1

Pseudomonas ORF1ab CP047697.

- No Sig. No Sig. 52.00%
aeruginosa (287) gene
1

Staphylococcus CP035643.
- N gene 55.00% 53.80% 56.50%
epidermidis (1282)
1

Staphylococcus ORF1ab CP035643.

- No Sig. 70.60% 48.00%
epidermidis (1282) gene
1
Streptococcus salivarius CP018186.
- N gene 65.00% 50.00% 52.20%
(1304)
1

Streptococcus salivarius ORF1ab CP018186.

- 58.30% 70.60% 48.00%
(1304) gene
1
AB257344.
SARSr-CoV (694009) SARS coronavirus Frankfurt 1 N gene 45.00% 80.80% 78.30%
1

ORF1ab AB257344.
SARSr-CoV (694009) SARS coronavirus Frankfurt 1 45.80% 64.70% 64.00%
gene
1
hCoV-
EPI_ISL_402
- 131
19/bat/Yunnan/RaTG13/20 N gene 100 100 86.956
13

hCoV-
EPI_ISL_402
19/bat/Yunnan/RaTG13/20 -
ORF1ab gene 131 100 100 100
13

hCoV-
EPI_ISL_410
19/pangolin/Guangxi/P4L/
- N gene 538 100 100 95.652
2017

hCoV-
EPI_ISL_410
19/pangolin/Guangxi/P4L/
- ORF1ab gene 538 95.83 88.24 -
2017

34
b) Interference Studies: The following potential interfering substances were investigated. Each
potential interfering substance was added to a contrived positive and a negative oropharyngeal
specimen in the Sample Storage Reagent and tested in triplicate using Sample Release Reagent
RNA fast-releasing technology. No interference was detected. Tables 11 through 13 provide
the results of these studies.

Table 11. Interfering Substances: Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic
Kit (PCR-Fluorescence Probing) Evaluation Test Using Negative Specimen
Results
Potential Interfering Substance Concentration
(Detected X/3)

Mucin: bovine submaxillary

20μg/mL 0/3
gland, type I-S

Blood (human) 5%(v/v) 0/3

Nasal sprays or drops 100μg/mL 0/3

Nasal corticosteroids 50μg/mL 0/3

Results
Potential Interfering Substance Concentration
(Detected X/3)

FluMist 100μg/mL 0/3

Homeopathic allergy relief

200μg/mL 0/3
medicine

Anti-viral drugs 300U/mL 0/3

Antibiotic, nasal ointment 100μg/mL 0/3

Antibacterial, systemic 100μg/mL 0/3

Table 12. Interfering Substances: Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic
Kit (PCR-Fluorescence Probing) Evaluation Test Using Positive Specimen
Potential Interfering Viral Strain Level Results
Concentration
Substance (In multiples of LoD) (Detected X/3)

Mucin: bovine submaxillary

20μg/mL 3xLoD 3/3
gland, type I-S

Blood (human) 5%(v/v) 3xLoD 3/3

Nasal sprays or drops 100μg/mL 3xLoD 3/3

Nasal corticosteroids 50μg/mL 3xLoD 3/3

FluMist 100μg/mL 3xLoD 3/3

35
 Homeopathic allergy relief 3xLoD
200μg/mL 3/3
medicine

Anti-viral drugs 300U/mL 3xLoD 3/3

Antibiotic, nasal ointment 100μg/mL 3xLoD 3/3

Antibacterial, systemic 100μg/mL 3xLoD 3/3

Table 13. Ct Values for Interfering Substances Evaluation Using Positive Specimen

Interfering substances Mean Ct Value of 3 replicates

ORF1ab gene N gene

Mucin: bovine submaxillary gland, type I-S 34.89 35.29

Blood (human) 36.05 35.68

Nasal sprays or drops 35.92 35.69

Nasal corticosteroids 35.86 35.54

FluMist 35.66 35.52

Homeopathic allergy relief medicine 35.09 35.25

Anti-viral drugs 35.45 35.85

Antibiotic, nasal ointment 35.73 34.93

Antibacterial, systemic 35.08 35.66

4). Matrix Equivalency

The matrix equivalency between oropharyngeal swabs and nasopharyngeal swabs stored in the
Sample Storage Reagent was carried out using an oropharyngeal swab positive specimen
(confirmed by gene sequencing). The positive specimen was diluted to 1×LoD and 5×LoD using
oropharyngeal swab and nasopharyngeal swab negative specimens collected from the same patient
(20 patients in total). An additional 10 pairs of oropharyngeal swab and nasopharyngeal swab
negative specimens were collected and tested using the Sample Release Reagent RNA fast-
releasing technology. The results in Table 14 show that the specimen matrices are equivalent.

36
 Table 14. The matrix equivalency evaluation between oropharyngeal and nasopharyngeal
swabs.
ORF1ab gene N gene
Sample No. of
Type Concentration Test Positive Average Ct Positive Average Ct
rate % Value rate % Value
NPS 100 36.07 100 36.74
1xLoD 20
OPS 100 36.05 100 36.30
NPS 100 34.33 100 34.14
5xLoD 20
OPS 100 34.15 100 33.80
NPS 0 n/a 0 n/a
Negative 10
OPS 0 n/a 0 n/a

5). Clinical Evaluation

The clinical performance of Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-
Fluorescence Probing) was established using 246 oropharyngeal swab specimens (in Sample
Storage Reagent) collected from patients who were suspected of COVID-19. The comparator
method was the Real-Time Fluorescent RT-PCR kit for Detecting SARS-2019-nCoV from BGI
Genomics, which received Emergency Use Authorization from the US Food and Drug
Administration on March 26, 2020. The Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic
Kit (PCR-Fluorescence Probing) used the Sample Release Reagent RNA fast- releasing technology
and BGI Genomics comparator assay extraction method was the Qiagen QIAamp Viral RNA Mini
Kit. Both assays were run on Applied Biosystems ABI 7500 with SDS software version 1.5. The
results are summarized in Table 15 and demonstrated a PPA of 94.34% and NPA of 98.96%.

Table 15. Clinical evaluation between Sansure Biotech Novel Coronavirus (2019-nCoV)
Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing) and BGI Genomics Comparator
Method

BGI Genomics Comparator

Test Total
Positive Negative

Sansure Positive 50 2 52
Biotech Negative 3 191 194

Total 53 193 246

Positive Agreement Rate: 50/53 ×100%＝94.34% (95% CI: 84.34% ~ 98.82%);

Negative Agreement Rate: 191/193×100%= 98.96 % (95% CI: 96.31% ~ 99.87 %);

37
17. Symbols

Symbols Meanings Symbols Meanings

In Vitro Diagnostic Medical Device Date of Manufacture

11vol d
~ Use By
cm Consult Instructions for Use

%
ILOTI
Temperature Limitation

Lot Number
•
IREFI
Manufacturer

Reference Number

w Number of Tests
&
Any warnings and/or precautions to take

Rx only Prescription only

18. Contact Information and Product Support

SANSURE BIOTECH INC.

No.680, Lusong Road, Yuelu District, Changsha, Hunan Province, 410205, P.R. China
Online manual: http://eng.sansure.com.cn/index.php?g=portal&m=article&a=index&id=81
Service call:(+86) 4008716677

Principle Distributor:
Karen DeVincent
Executive Director of Regulatory Affairs and Quality Assurance
Trividia Health, Inc.
2400 NW 55th Ct.,
Ft. Lauderdale, FL 33309
Phone:（ 800) 342-7226x3019
38
Email: kdevincent@trividiahealth.com

Distributor:
Su Xu
General Manager
BioSci Inc.
3460 Robin Lane, Suite 1
Cameron Park, CA 95682
Phone: (916) 850-5188
Fax: (916) 983-9911
Email: robert.xu@biosciinc.com

39
Revision History

Version Revision Date Remarks

V00 05-04-2020 Initial Release Version

1. Doc.Version: V00 updated to V01.

Revision date: 05-04-2020 updated to
December 16, 2021.
2. Add the limitation description of
V01 performance test in section 13 and
December 23, 2021 "RX Prescription only" in section 17.
3. Add “Revision History”.
4. Update the Inclusivity analysis

1. Update the Inclusivity analysis.

V02 March 24, 2022

2. Update Warnings and Precautions in
section 8.

3. Update the Limitations in section 13.

40