Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit
(PCR-Fluorescence Probing)
Intended Use
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.
Summary
The definitions of "suspected cases" and "suspected clusters of patients" shall be defined by referring to the "Pneumonia Diagnosis and Treatment Program for Novel Coronavirus Infection" and "Pneumonia Case Monitoring Program for Novel Coronavirus infection" issued by China CDC (the currentversion).
Novel Coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit(PCR-Fluorescence Probing) is only used for the auxiliary diagnosis of related cases and the emergency reserve for in vitro diagnosis during the pneumonia outbreak of novel Coronavirus (2019-nCoV) infection since December2019, this kit shouldn’t be used as routine in vitro diagnostic in clinical practice.
Please follow the relevant requirements of the "Pneumonia Diagnosis and Treatment Program for Novel Coronavirus Infection", "Pneumonia Prevention and Control Program for Novel Coronavirus infection" and other documents in use.
The novel Coronavirus nucleic acid tests should comply with “Technical Guidelines for Laboratory Testing of Novel Coronavirus in China CDC” and keep good biosafety.
Test Principle
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 |
Specimen Requirements
1. Applicable specimen type: nasopharyngeal swab, oropharyngeal swab, alveolar lavage fluid, sputum, serum, whole blood and feces.
2. Collection of specimen
Nasopharyngeal swab/oropharyngeal swab: Collect sample in accordance with the relevant provisions of"Specimen Collection Method" in the "Pneumonia Laboratory Technical Guide for Novel Coronavirus Infection" from "Pneumonia Prevention and Control Plan for Novel Coronavirus Infection". It is proved that the swab made of nylon sampling head and ABS sampling rod can be selected for sample collection.
Nasopharyngeal swab: The specimen collection tube should be pasted with the barcode first, the nasopharyngeal swab should be collected within3 days after the onset of the disease as far as possible. Use swab to measure the length between apex nasi and earlobe, then mark with finger. Insert the swab into the nasal cavity in direction of perpendicular to the nose(face). The swab should be inserted at least half of the length from the earlobe to the apex nasi. Make the swab stops in the nasal for15 ~ 30 s, gently rotate3 ~ 5 times, quickly put swab into specimen collection tube cotaining2 ml Lysis Buffer(same as Lysis Buffer in the Sample Release Reagent) or Sample Storage Reagent containing RNA enzyme inhibitor. Insert the swab, then break the sterile swab rod near the top, tighten tube cap and seal with sealing film.
Oropharyngeal swab:The specimen collection tube should be pasted with the barcode first, the oropharyngeal swab should be collected within 3 days after the onset of the disease as far as possible. A sterile flocking swab should be used for sampling,moderately wipe the posterior pharyngeal wall, avoid touching the tongue. Quickly place a sterile swab into the collection tube used for collection of oropharyngeal swab. Break the sterile swab rod near the top, tighten tube cap and seal with sealing film.
Alveolar lavage fluid:Severe patients or patients with pneumonia who progress rapidly. Clinician extract≥5ml BALF into a 50ml aseptic container labeled with sample barcode and screw cap by aseptic operation. Collect specimen, then tighten tube cap and seal with sealing film.
Sputum:
The specimen collection tube should be pasted with the barcode first. Do not open the airway to collect specimens when collecting sputum. Collect.deep cough sputum into a disposable aseptic sampling cup with screw cap, load 2 mL protease K (1g/L) into sampling cup. Collect sputum, then tighten tube cap and seal with sealing film. Send to detection within 30 min as far as possible. Protease K should not be added first if specimens need to be transported over long distances.
Whole blood:
Blood samples can be collected within 7 days after the onset or critical patients, or patients considered with viremia. The specimen collection tube should be pasted with the barcode first. Collect 2~4ml of blood samples into vacuum blood collection tube containing EDTA anticoagulant.
Feces:
For patients with gastrointestinal symptoms such as diarrhea at the early stage of the disease, preserve 3~5g (soybean size) feces. The specimen collection tube should be pasted with the barcode first. Collect sample into specimen collection tube with screw cap containing 2ml normal saline (RNA enzyme inhibitor can be added when conditions permit) then seal with sealing film.
After sample collection, it is recommended to place into Sample Storage Reagent for preservation.
It has been proved that preservation solution, such as normal saline, TE buffer, 2-4M containing guanidine (such as guanidine hydrochloride) can also be used as Sample Storage Reagent for sample preservation. The Sample Storage Reagent containing guanidine cannot be directly adapted to Sample Release Reagent manufactured by Sansure Biotech Inc. for nucleic acid extraction. If necessary, it is recommended to use Nucleic Acid (DNA/RNA) Extraction or Purification Kit (Magnetic beads method) ( Reference Number : S1002E ) manufactured by Sansure Biotech Inc. or the QIAamp Viral RNA Mini Kit (50) manufactured by QIAGEN for nucleic acid extraction.
3. Storage and delivery of specimens:
Specimens to be tested can be immediately processed, specimens to be tested within 24 hours can be stored at 4℃. Specimens that cannot be detected within 24 hours should be stored at -70℃ or below (in the absence of -70℃ storage conditions, specimens to be tested 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. The inactivation of samples at 56℃ for 30min will not affect the detection of this kit.
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 “Quality Control”. Go to “Plate” window to record qualitative results.
Quality Control
| Item |
2019-nCoV-PCR-Negative Control |
2019-nCoV-PCR-Positive Control |
| Ct value |
No Ct or Ct > 40 at channel FAM, ROX and HEX
(Internal Control)
|
≤ 35 at channel FAM, ROX and HEX
(Internal Control)
|
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.
Explanation of Detection Result
| Conclusion |
Amplification results |
| 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.
Storage and Stability
1. 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.
2. Please refer to the date of manufacture and expiry date on the outer package.
3. 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.
Compatible Instrument
The diagnostic kit is applicable to SLAN-96P, ABI7500, Life Technologies QuantStudio TM5 , Roche Cobas480, MA-6000 PCR instrument.
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