Sample collection

1. Rinse mouth and don't eat anything 30 min prior to the collection.

2. Open the Sampling kits and label the 50-mL collection tube. 

3. Note: the preservative solution smells and is normal. 

4. Cough 2-3 times and spat the sputum into the collection tube. The total sputum will be around 1 - 2 mL.

5. Close the cap tightly and shake the tube vigorously.

6. Place the collection tube into the sample bag.

7. Pack the sample bag with another zip bag and place in the sample collection containers. 

8. The collected samples can be shipped in room temperature.  

Nucleic acid extraction

【Sample Requirements】

This product is suitable for the extraction of nucleic acids from serum, plasma, secretions, nasopharyngeal swabs, oropharyngeal swabs, reproductive tract swabs, sputum, and cell virus cultures. Samples should be extracted immediately after collection. If transportation is required, please use a curling or foam box with ice packs for storage.

9. Serum, plasma and cell virus culture samples can be directly extracted for DNA or RNA.

10. Nasopharyngeal swabs, oropharyngeal swabs, genital tract swabs were thoroughly washed in PBS, physiological saline or virus preservation solution, and the supernatant (or cell pellet) was collected for DNA or RNA extraction.

11. Sputum sample need to be liquefied, before DNA or RNA extraction can be performed.

Applicable instruments

Manual operation, automated instrument operation (e.g. Kingfisher Flex 96). Protocol for manual pipetting and automated instrument extraction.

1.The following instrument operations are based on Thermo Kingfisher™ Flex 96, other instruments please refer to manual provided by the manufacturer. Instrument operating supplies need to be purchased separately. Table 1 Instrument parameters overview for manual pipetting.

2.Take a deep well plate labeled 1 and aliquot 5μL Beads V and 20μL Proteinase K into each well.

Note: Before added, the magnetic beads should be vortexed for 15 s to be completely dispersed. When there are lots of samples, Beads V and Proteinase K can be mixed according to the required portion (used within 24 hours after mixing). After vortexing, aliquot 25μL into each well.

3. Aliquot 400μL of Lysis and Binding Solution V into the No. 1 deep well plate. After aliquoting the Lysis and Binding Solution V, the sample should be added within 45min. The Lysis and Binding Solution V can inhibit Proteinase K if the waiting time is too long.

4. Add 200μL sample to the No.1 deep well plate, and mix by pipetting up and down 3 - 5 times.

After all samples have been added, gently put the Magnetic stirring sleeve into the No. 1 deep well plate.

Take a deep well plate labeled 2 and dispense 500μL of Wash Solution V1 into each well.

Take a deep well plate labeled 3 and dispense 500μL of Wash Solution V2 into each well.

Take a deep well plate labeled 4 and dispense 500μL of Wash Solution V2 into each well.

Take a shallow well plate labeled 5 and dispense 70 - 100μL Nuclease-Free Water into each well.

Note: Wash Solution V1, Wash Solution V2, and Nuclease-free Water can be aliquoted and sealed with a film in advance, and used within 24 hours.

Select the nucleic acid extraction program "M-VirusExtra" and press start. 

After the run, transfer the nucleic acid solution for subsequent experiments. For long-term storage, store at -20 ℃ or -80 ℃。

Nucleic Acid Detection

Detection Principle

The kit is based on real-time fluorescent probe Quantitative RT-PCR technology. RT-PCR（Reverse Transcription-Polymerase Chain Reaction）is a method in which RNA reverse transcription (RT) is combined with polymerase chain reaction of cDNA. First, via reverse transcriptase, RNA fragment in COVID-19 will be synthesized to cDNA, then with cDNA as the template, target fragment will be synthesized via amplification by DNA Polymerase.  

In PCR-Fluorescent Probe method, the probe with specific binding to target sequence is added based on the forward primer and the reverse primer, Specific primers and probes are designed based on specific gene areas of Novel Coronavirus (COVID-19). Probes consist of a reporter fluorophore at 5’ and quenching fluorophore at 3’. The fluorescent signals emitted from reporter fluorophores are absorbed by the quenchers, so it doesn’t emit signals. During amplification, probes bonded to templates are cut off by Taq enzyme (5’-3’ exonuclease activity), separating reporter dye from the quencher, generating fluorescent signals, the PCR instrument will then automatically draw a real-time amplification curve based on the signal change, finally realizing the qualitative detection of Novel Coronavirus (COVID-19) at the nucleic acid level. 

Procedure

2. Reagent preparation (reagent preparation area) 

2.1 Determine the amounts of samples to be tested first, a no template control and a positive control should be included in each test run (i.e., 96-well plate). 

2.2 Thaw Reaction Mix, positive control, and RNA samples on the ice, and shake with inching on the shaker, followed by short spin on the centrifuge. RNase-free reaction tubes shall be provided with each sample. 

Reaction mixes contain RT-PCR primers, probes and reaction reagents (except enzymes). Primers and probes for Internal control (IC) gene have been included in the reaction mix. 

2.3 Using Table 1 below, prepare the PCR reaction system based on the number of samples to be tested with up to 5% allowance to make sure that there is sufficient volume for pipetting. Set up following ingredients in order:  

 PCR reaction system 

ReagentsAdded volume (μL)/test

Reaction Mix (ORF1ab/N gene/IC)14.6

RT Enzyme Mix0.4

Total volume15

2.4 After mixing the prepared PCR reaction system, pipette 15 μL into the corresponding reaction wells of a 96-well plate (or 8-tube strip), and then transfer to the sample processing area.

3. Sample loading (sample processing area)

3.1 The total RT-PCR reaction volume is 20 μL: 

（1）RNA sample: Add 5μL of RNA sample to the corresponding reaction well. 

（2）Positive control: Add 5μL of positive control to the reaction well as positive control well.

（3）Negative control: Add 5μL of Nuclease-free water to the reaction well as negative control well.

3.2 Seal the tube cap and shake with inching on the shaker several times, followed by short spin on the centrifuge.

4. PCR amplification (nucleic acid amplification area)

RT-PCR protocol as below:

Temp (℃)TimeCycles

505 min1

9520 sec1

955 sec45

6030 sec

Probe label: ORF1ab: FAM-BHQ1; N Gene: VIC-BHQ1; IC (RNase P) : CY5- BHQ3.

5. Result Analysis 

5.1 Quality Check for the Test Results 

The following requirements on value Ct of positive control well and negative control well on the reaction plate within the same reaction plate/batch: 

Quality control requirement

Positive Control reaction wellCt≤37

Negative Control reaction wellCt>37 or Undet

5.2 The experiment is invalidated, and repeat is required if the positive control and/or negative control does not meet the criteria set above.

5.3 The analysis of the Ct value of the ORF1ab, N, and IC wells in each swab or sputum specimen as follows:

ORF1ab N gene IC Interpretation

Ct≤37Ct≤37Ct≤40Positive

Ct>40 or UndetCt>40 or UndetCt≤40Negative

40>Ct>37 (Amplifiable)40>Ct>37 (Amplifiable)Ct≤40Weak positive; Retest to confirm

Ct>37 or UndetCt>37 or UndetCt>40 or undetResampling for test or other confirmation method required

Ct≤37Ct>37 or Undet

【Performance Limitation】

1. Test results only serve as clinical reference and comprehensive judgments based on clinical symptoms and other laboratory tests method should be considered by clinicians.

2. Negative results cannot completely rule out the existence of novel coronavirus. Improper sample collection, improper transportation, improper processing and insufficient initiation VL(viral load) may influences the experimental results.

3. Other unverified interferences or PCR inhibitors may cause false negative results.

Reagents and instruments required

Reagents 1Disposable Sampling Kit

Reagents 2Nucleic Acid Extraction Kit (Bead Binding)（Kit1+kit2)

Reagents 3Novel Coronavirus (COVID-19) Nucleic Acid Detection Kit

Instruments 1Nucleic acid extraction instrument

Instruments 2Fluorescence quantitative PCR instrument