COVID-19 ORF1ab Gene Isothermal Nucleic Acid Amplification Kit (RT-SRDA Fluorescent Type)

Description：
Novel coronavirus pneumonia (NCP) that was officially named by the WHO as “Corona virus disease 2019” (COVID-19), is a respiratory infection caused by a new virus that was first identified in late 2019.
This kit applies Strengthened Recombinase Displacement Amplification (SRDA) technology to carry out COVID-19’s RNA in vitro amplification. Basing on the N gene of the COVID-19, wasdesigned a pair of heterogeneous primers and then combined with a specific EXO fluorescent probe. The test can be completed within 20 minutes at 35-42 °C.
The test can be applied for clinical diagnosis.

Kit Contents:

Note: the kit components of different lots cannot be interchanged.

Storage and Transportation：
Store at -20℃±5℃. Can be stored at 4 ° C for a short period of time. Shelf life 1 year.
Repeated freezing and thawing ≤5 times.
Protect from light.

Applicable equipment：
ABI series, Bio-Rad, Twist DXT16-ISO, and other fluorescent real-time PCR instruments.

Basic Protocol：
1. Sample preparation.
     Extract RNA Nucleic acid. The nucleic acid extraction product should be stored at -20 ° C.
2. Reaction mixture preparation
     Please prepare the reaction solution according to the list below.
    The following protocol is recommended for a 50 μl reaction volume. If the volume of reaction changes, please adjust proportionally.

3. Sample adding.
Add 2 μl each of the extracted RNA, positive and of negative control to corresponding reaction tubes. After assembling all the components, cover the tube caps and gently mix the contents of the tube, mix well, and centrifuge briefly.
Note: please prepare the reaction solution as quickly as possible. First, add B Buffer on an 8-well plate’s caps, add sample and only then cover the caps.

4. Perform quantitative PCR
Place the reaction tube inside a real-time PCR instrument.
Set the channel and sample information, reaction system volume 50 μl.
Select the following fluorescence channels: FAM for detection channel, NONE for quenching channel, and NONE for reference fluorescence.

Perform quantitative PCR using recommended cycling parameters settings:

Limitations of detection method:

• The test results depend on the quality of sample collection, processing, transportation and preservation;
• During the sample extraction process, precautionary measures should be taken to avoid cross-contamination, otherwise it may lead to false positive results;
• Leakage of positive controls and amplification products my lead to false positive results;
• Contaminated consumables and equipment used during the experimentmay cause false positive results.
• Different extraction methods have different extraction efficiency, which may lead to false negative results;
• Incorrect sample collection, transfer and processing, low pathogen content in the sample or improper reagent preparation may cause false negative results;
• Variations in the target sequence of the pathogen or sequence changes caused by other reasons may lead to false negative results;
• The test results are for reference only. If a diagnosis is required, please combine with clinical symptoms and other testing methods.

Precautions:

• All operations should be performed strictly in accordance with the protocol
• The components of the kit should be thawed at room temperature before use,thoroughly mixed and centrifuged briefly;
• ORF1ab-C buffer should be protected from light;
• Use disposable tips, g tubes loves, and work clothes for each area;
• Sample processing, reagent preparation and sample addition should be performed in different areas to avoid cross-contamination;
• The workbench and various experimental items should be disinfected with 10% sodium hypochlorite, 75% alcohol, and UV lamps after each experiment.
• The test samples involved in this kit should be considered as infectious substances, and their handling and handling must meet the relevant requirements of the General Guidelines for Biosafety of Microbial Biomedical Laboratories and the Medical Waste Management Regulations of the Ministry of Health