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Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit
Novel Coronavirus(2019-nCoV) Nucleic Acid Diagnostic Kit(PCR-Fluorescence Probing) is used for qualitative detection of the ORF1ab and N genes of novel coronavirus(2019-nCoV) in nasopharyngeal swab, oropharyngeal swab, alveolar lavage fluid, sputum, serum, whole blood and feces from suspected pneumonia cases with novel coronavirus infection, patients with suspected clusters of novel coronavirus infection, and other patients requiring diagnosis or differential diagnosis of novel coronavirus infection.
For in vitro diagnostic use only. For professional use only.
By applying Real-time fluorescence quantitative RT-PCR technology on the fluorescence quantitative PCR instrument, this test utilizes the novel coronavirus(2019-nCoV) ORF1ab and the specific conserved sequence of coding nucleocapsid protein N gene as the target regions which are designed for the conserved sequence of the double-target genes, to achieve detection of sample RNA through fluorescent signal changes.
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.
Components of the Diagnostic Kit
This kit is an amplification reaction reagent and contains the following components:
|No.||Reagent Name||Spec. & Qty||Main Ingredients|
|24 T||48 T|
|1||2019-nCov-PCR Mix||624µL/tube x 1||1248µL/tube x 1||Premiers (4.62%), Probes (1.15%), dNTPs (3.85%), MgCl2 (0.77%), Rnasin (0.48%), PCR Buffer(89.13%)|
|2||2019-nCov-PCR-Enzyme Mix||96µL/tube x 1||192µL/tube x 1||
RT Enzyme (62.5%),
Taq Enzyme (37.5%)
|3||2019-nCov-PCR-Positive Control||500µL/tube x 1||500µL/tube x 1||In Vitro Transcriptional RNA Containing Target Genes (ORF1 ab, N gene) and internal standard gene fragments (Rnase P)|
|4||2019-nCov-PCR-Negative Control||500µL/tube x 1||500µL/tube x 1||Normal Saline|
Preparation of reagent (performed at “reagent preparation region”)
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.
According to the quantity of test specimens, 2019-nCoV-PCR-Positive Control and 2019-nCoV-PCR-Negative Control, pipette appropriate quantity of 2019-nCoV-PCR Mix and 2019-nCoV-PCR-Enzyme Mix (2019-nCoV-PCR Mix 26μL/test + 2019-nCoV- PCR-Enzyme Mix 4μL/test), mix them thoroughly to make a PCR-Mastermix, centrifuge it instantaneously for later use.
|Item||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 just for your reference and to ensure enough volume of the PCR-Mastermix, more volume of the actual pipetting may be required.|
Transfer the above-prepared reagents to the “specimen processing region” for later use.
PCR Amplification (Refer to user manual of each instrument to adjust the settings.)
Place PCR reaction tubes into the specimen wells of the amplification equipment. Set up the 2019-nCoV-PCR-Positive Control, 2019-nCoV-PCR-Negative Control and specimens to be tested in the corresponding sequence and input specimen name.
Select PCR Test Channel:
a. Select FAM (ORF-1ab region) and ROX (N gene) channels to test 2019-nCoV nucleic acid.
b. Select HEX channel to test internal control.
Set Cycle Parameters
|1||Reverse transcription||50°C||30 min.||1|
|2||cDNA predenaturation||95°C||1 min.||1|
|Annealing, extension and fluorescence collection||60°C||30 sec.*|
|4||Device cooling||25°C||10 sec.||1|
When the settings are completed, save the settings and carry out the reaction procedure.
Result Analysis (Refer to user manual of instrument to adjust the settings.)
Results will be saved automatically when reactions are completed. Analyze amplification curve of target of detection and internal control. Adjust Start, End and Threshold values of Baseline of the graph according to analysis result (Users can adjust the values according to the actual situation. Start value can be set between 3-15, and End value between 5-20. Adjust the amplification curve of negative control to be flat or below threshold). Click “Analyze” to implement the analysis, make sure each parameter satisfy the requirements given in “5. Quality Control”. Go to “Plate” window to record qualitative results.
|Item||2019-nCoV-PCR-Negative Control||2019-nCoV-PCR-Positive Control|
No Ct or Ct > 40 at channel FAM, ROX and HEX
≤ 35 at channel FAM, ROX and HEX
The test result is treated as valid if all the conditions in the above-mentioned are met for the same test. Otherwise the test result is treated as invalid and needs to be re-tested.
Through the research on reference values, the Ct reference value of target gene is determined to be 40, the CT reference value of internal control is determined to be 40.
Explanation of Detection Result
|2019-nCoV Positive||There is typical S-shape amplification curve detected at FAM and/or ROX channel, and the amplification curve which is detected at HEX channel, Ct≤40.|
|2019-nCoV Negative||There is no typical S-shape amplification curve(No Ct) or Ct ＞ 40 detected at FAM and ROX channel, and the amplification curve which is detected at HEX channel, Ct ≤ 40.|
There is no typical S-shape amplification curve detected at FAM, ROX and HEX channel (No Ct), or Ct > 40. It is indicated that the specimen’s concentration is too low, or there are interfering substances that inhibit the reaction. The test result is invalid. An investigation should be performed to find out and exclude the reasons, please collect specimen again and retest the specimens.
Note: For virus cultures, there is no requirements for internal control test results.
Product Performance Index
Test enterprise positive reference, the results are all positive.
For Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing), there are also no cross-reaction with coronavirus (NL63, HKU1, 229E,OC43), SARS coronavirus, MERS coronavirus, influenza A virus, influenza B virus Type Yamagata and Type Victoria, influenza A (H1N1) virus, influenza A (H3N2) virus, influenza A (H5N1) virus, influenza A (H7N9) virus, respiratory syncytial virus type A and Type B, nasal virus Type A, Type B and Type C, adenovirus Type 1, 2, 3, 4, 5, 7 and 55, parainfluenza virus Type 1, 2 and 3, intestinal virus type A, type B, type C (EV-C95), type D(EV-D70), partial pulmonary virus, human lung virus, cryptococcus neoformans, pyogenic streptococcus, acinetobacter baumannii, pneumocystis carinii, klebsiella pneumoniae, streptococcus pneumoniae, haemophilus influenzae, pseudomonas aeruginosa, legionella pneumophilia, bordetella pertussis, staphylococcus aureus, mycoplasma pneumoniae pneumonia, streptococcus pneumoniae, klebsiella pneumoniae, chlamydia, EB virus, human cytomegalo virus, aspergillus fumigatus, candida albicans, candida glabrata, mycobacterium tuberculosis, non-tuberculous mycobacterium, norovirus, rotavirus, varicella zoster virus, measles virus, mumps virus, human genome DNA etc. positive samples. Test the enterprise negative reference, the result are all negative.
The coefficient of variation (CV%) of CT value of the within-run precision is ≤5%.
Clinical evaluation is based on the recommend method of "Novel Coronavirus Infection Pneumonia Laboratory Testing Technology Guide”, “Novel Coronavirus Infection Pneumonia Cases Monitoring Programme (second edition)" to diagnosis/exclusion result as a comparision, in the Military Cademy of Military Medical Research Institute, Hunan Disease Control and Prevention Center, Hunan Province People's Hospital, Central South University Xiangya 2nd hospital, according to clinical data collected from the four institutions, such as statistical analysis, the preliminary evaluation, basic clinical confirmed the product performance can meet the emergency needs. The types of samples for clinical evaluation included pharyngeal swabs and alveolar lavage. Further clinical data will be collected after marketing to confirm the clinical performance of the product.
Due to the characteristics of swab and other sample collection process and viral infection process itself, false negative results may be caused by insufficient sample volume, which should be combined with other clinical diagnosis and treatment information for comprehensive judgment, retest when necessary.
Storage and Stability
The diagnostic kit should be stored in a sealed pouch at-20±5°C and protected from light. The kit is provisionally valid for 6 months.
Please refer to the date of manufacture and expiry date on the outer package.
The reagents keep valid and stable within the expiry date if not used. As long as the container of the reagent is opened, the freeze/thaw cycles should not exceed three.
The diagnostic kit is applicable to SLAN-96P, ABI7500, Life Technologies QuantStudio TM5 , Roche Cobas480, MA-6000 PCR instrument.
Contact Person: Mr. Randy.Zhang