Novel Coronavirus (COVID-19) Nucleic Acid Detection kit

Novel Coronavirus (COVID-19) Nucleic Acid Detection Kit is designed for qualitative detecting

the RNA of SARS-CoV-2 in the samples of nasal/throat swab.

For suspected cases, suspected cluster cases, or other cases that require the diagnostic

and differential diagnosis of SARS-CoV-2 infection, which helps the clinical diagnosis

of the infection of SARS-CoV-2.

【PACKING】

50 tests/kit & 200 tests/kit

【INTENDED USE】

This kit is used for the detection of COVID-19 (ORF1ab/N Gene) Nucleic Acid and assisted

diagnosis and epidemiological surveillance of COVID-19.

【TEST PRINCIPLE】

The kit adopts the principle of real-time fluorescent quantitative PCR technology, designs

specific primers and probes for COVID-19 (ORF1ab/N gene), and combines them with

real-time fluorescent quantitative PCR instrument to detect the nucleic acid of COVID-19

virus. So as to realize the qualitative detection of COVID-19 virus nucleic acid. In addition,

the PCR detection system uses the positive internal control, which monitors the presence

of PCR inhibitors in test specimens by detecting whether the internal control signal is

normal, to avoid a false negative result.

【COMPONMENTS OF THE DIGNOSTIC KIT】

Note:

1. Do not mix or exchange components from different kit lots.

2. The CODID-19 Negative Control is sterile nuclease-free water, and the COVID-19

Positive Control is transcribed RNA in vitro which contains target genes ORF1ab,

N gene, and Internal Control gene.

【STORAGE AND STABILITY】

1. The diagnostic kit should be stored in a sealed pouch at -20 ± 5℃. The expiry date

is 12 months.

2. Please refer to the date of manufacture and expiry date on the outer package.

3. The reagent keep valid and stable within the expiry date if not used. The kit should not be

frozen and thawed more than 5 times.

【APPLICABLE INSTRUTMENT】

The diagnostic kit is applicable to MA-6000, ABI series, Bio-Rad series, Roche Light Cycler

R480, Cepheid Smart Cycler, Rotor-Gene series and other multi-channel real-time quantitative

PCR instruments.

【SPECIMEN REQUIREMENTS】

1. Sample types: Upper respiratory tract specimens (including throat swabs, nasal swabs,

nasopharyngeal extracts, deep cough sputum), lower respiratory tract specimens (including

respiratory tract extracts, bronchial lavage fluid, alveolar lavage fluid, lung tissue biopsy

specimens), Tissue culture and other samples.

2. Storage conditions: The collected specimens should be submitted for inspection in a timely

manner, and the specimens should be stored at 4°C within 24 hours. It is best to store at -70°C

for more than 24 hours, and avoid repeated freeze-thaw cycles.

【TEST METHOD】

1. Reagent Preparation(performed at “reagent preparationregion”)

1. 1 Take out each components 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.

1.2 According to the quantity of test specimens, COVID-19 Positive Control and COVID-19

Negative Control, pipette appropriate quantity of COVID-19 PCR Mix and COVID-19

Enzyme Mix ( COVID-19 PCR Mix 15 μl/test+ COVID-19 Enzyme Mix 5 μl/test), mix them

thoroughly to make a PCR-Master mix, centrifuge it instantaneously for later use.

1.3 Transfer the above-prepared reagents to the “specimen processing region” for later use.

2. Processing and loading of specimens (performed at “specimen processing

region”)

2.1 This diagnostic kit does not include Viral RNA&DNA Extraction Kit.

It is recommended to use Viral RNA&DNA Extraction Kit to extract viral RNA.

The specific operation is in accordance with its instructions.

2.2 Add 20 μl PCR-Master mix into PCR reaction tube with 5 μl above processed sample,

COVID-19 Positive Control and COVID-19 Negative Control, and cap the tube. Carry out

fluorescence quantitative PCR detection on fluorescence PCR instrument.

3. PCR Amplification (performed at “nucleic acid amplification area”)

3.1 Place PCR reaction tubes into the specimen wells of the amplification

equipment. Setup COVID-19 Positive Control, COVID-19 Negative

Control and specimens to be tested in the corresponding sequence

and input specimen name.

3.2 Set cycle parameters according to the following table for PCR amplification.

Note:

1)The fluorescence collection is set at "Step 3: 55 ° C, 30s". Selection of detection channels:

FAM, HEX and Cy5, where FAM channel is ORF1abgeneand HEX channel is N gene. Cy5

channel is the internal control gene, and the reaction system is set to 25 μl.

ABI series fluorescent PCR instruments do not select ROX calibration and select None for

the quenching group.

【Positive Judgement Value and Reference Interval】

1. Condition Setting for Result Analysis

The adjustment principle of Baseline and Threshold is generally based on the results of the

automatic analysis of the instrument. When the overall slope of the curve appears, the Start,

End, and Threshold values of the Baseline can be adjusted according to the image. Usually,

the user can adjust it according to the actual situation. The Start value can be set to 3 - 15,

and the End value can be set to 5 - 20. Adjust the amplification curve of the negative control

to make it straight or below the threshold line.

2. The Validity of The Kit

2.1 The COVID-19 Positive Control: FAM, HEX and Cy5 channels have typical S-type

amplification curves and Ct≤32.

2.2 The COVID-19 Negative Control: FAM, HEX and Cy5 channels have no Ct or Ct >38.

2.3 Note: The above conditions must be met at the same time, otherwise this experiment is

invalid and needs to be repeated.

3. The Positive Judgment Value

Through the study of reference values, it was determined that the Ct reference value of the

target gene and the internal control gene detected by this kit was both 38.

The Sample Result Judgment

1. If the test sample detects a typical S-type amplification curve in the FAM, HEX and Cy5

channels and the Ct is ≤38, the sample can be judged to be COVID-19 positive.

2. If the test sample has no Ct or Ct > 38 in the FAM and HEX channels, and there is a typical

S-type amplification curve in the Internal Control channel (Cy5), Ct≤38, the sample can be

judged to be COVID-19 negative.

3. If the test sample only has a Ct value ≤38 in a single channel of the FAM or HEX channel,

and there is a typical S-type amplification curve in the Internal Control channel (Cy5),

Ct ≤38, the results need to retest. If the results repeated are consistent, the sample can be

judged as COVID-19 positive, the results repeated are negative except for the typical S-type

amplification curve of the Internal Control channel(Cy5), Ct≤38, which could be judged as

COVID-19 negative.

4. If no typical S-type amplification curve (No Ct value) or Ct value> 38 is detected in the FAM,

HEX, and Cy5 channels of the test sample, it means that there is a problem with the quality

of the sample or a problem with the operation. If the result is invalid, you should find and

eliminate the cause, collect the sample again, and repeat the test (if the test result is still

invalid, please contact the company).

【Limitations of Detection Method】

1. Test results of the diagnostic kit can be used only for clinic reference. The clinical diagnosis

and treatment of patients should be considered in conjunction with their symptoms, signs,

medical history and other related conditions.

2. False negative results may occur when the concentration of the detected nucleic acid in the

test sample is below the minimum detection limit of this kit.

3. Improper handling of the tested sample during collection, transportation, storage, and

processing can easily result in RNA degradation and false negative results​.

4. When samples are cross-contaminated during collection, transportation, storage, and

processing, it is easy to get false positive results.

【Product Performance Index】

1. LOD: The limit of detection is 200 copies/ml.

2. Precision: Coefficient of variation(CV%) of precision Ctvaluewithinbatch≤3%.

3. Specificity: There is no cross reaction between the kit and positive samples, such as Human

Coronavirus HCoV- NL63, Human Coronavirus HCoV-OC43, SARS Coronavirus, MERS

Coronavirus, Influenza A Virus, Influenza B Virus Yamagata, Victoria, H1N1 Influenza Virus,

H3N2 Influenza Virus, H5N1 Influenza Virus, H7N9 Influenza Virus, Respiratory Syncytial

Virus A, Adenovirus(type 2), Adenovirus (type 2), Mycoplasma Pneumoniae, Chlamydia

Pneumoniae, Pertussis, Streptococcus Pneumoniae, Rhinovirus(Type A) etc.

【PRECAUTIONS】

1. The entire detection process should be performed strictly in accordance with the requirements

of this manual in the reagent preparation area, sample processing area, and PCR amplification

area, and the experimental clothes, instruments, and consumables in each area should be

used independently and cannot be mixed.

2. To avoid RNA degradation, the sample processing process should be operated at 0-4℃, and

the test should be performed immediately after the experiment is completed. Utensil

consumables used in sample processing should be nuclease-free.

3. Negative and positive controls should be set for each experiment.

4. All reagents in the kit should be fully thawed and mixed at room temperature and centrifuged

immediately before use.

5. All negative and positive controls in the kit should be transferred to the sample preparation

area and stored separately before the first use.

6. To prevent fluorescence interference, avoid touching the PCR reaction tube directly with bare

hands, and avoid any markingonthe PCRreactiontube.

7. The instrument amplification related parameters should be set in accordance with the relevant

requirements of this manual, and different batches of reagents cannot be mixed.

8. The product waste during the experiment should be detoxified before being discarded.

【INDEX OF SYMBOL】

【EXPORTER】

Magnus Internationa Limited

F12, New City Internationa Mansion A, 234 Huapao Ave.

Liuyang, Hunan Province 410300 China

Contact: Goodwellmedical@gmail.com

【AUTHORIZED REPRESENTATIVE】

Lotus NL B.V.

Koningin Julianaplein 10, 1e Verd, 2595AA, The Hague, Netherlands.