SARS-CoV-2 Mutation Detection HV 69-70 Deletion
PRODUCT CODE: X-KIT-101
STORAGE: < -20 °C
SARS-CoV-2 Mutation Detection HV 69-70 Deletion Assay is an isothermal RT-LAMP laboratory re-search use assays for detection of spike gene muta-tion HV 69-70 Deletion found in the SARS-CoV-2 lineage B.1.1.7
BioThinx warrants, that at the time of the quality release or subsequent retest date this product conformed to the information contained in this publication. Purchaser must determine the suitability of the product(s) for its particular use. Additional terms and conditions may apply. Please see reverse side of the invoice or packing slip. No other warranties, express or implied, are granted, including without limitation, implied warranties of merchantability, fitness for any particular purpose, or non-infringement
SARS-CoV-2 Mutation Detection – HV 69-70 Dele-tion assay is an isothermal RT-LAMP laboratory re-search use assay for detection of spike gene muta-tion HV 69-70 Deletion found in the SARS-CoV-2 lineage B.1.1.7 Genetic variants of SARS-CoV-2 RNA can be found in the liquid from upper or lower respiratory tracts of infected individuals. Samples can be obtained from isolated viral RNA from naso- or oropharyngeal, swabs. Infection with any variant of SARS-CoV-2 can occur without showing any symptoms. Negative RT-LAMP results do not exclude present or hinder future infection with SARS-CoV-2 virus or any genetic variations and the result should always be combined with clinical observations, patient his-tory, and epidemiological information. SARS-CoV-2 Variant Detection Assays are intended for use by science and health professionals or quali-fied laboratory personnel specifically instructed and trained in molecular testing techniques as well as proficient in handling biological samples.
There is an urgent need for high throughput and rapid detection of SARS-CoV-2 infections and identi-fication of variants of concern. To address this chal-lenge, BioThinx has developed a new line of re-search use RT-LAMP assay systems which can de-tect marker mutations found in SARS-CoV-2 vari-ants of concern: cluster B.1.1.7, cluster B.1.351, cluster P.1. An additional assay is intended for SARS-CoV-2 general detection without mutation dif-ferentiation. All assays come in an easy to handle high-throughput format. Molecular testing uses one-step reverse transcrip-tion and loop-mediated isothermal amplification (RT-LAMP) method. The primers of this LAMP assay have been de-signed to target marker mutation HV 69/70 deletion within the spike protein gene found in SARS-CoV-2 lineage B.1.1.7 (British Variant). Initially in the United Kingdom (UK), a new variant of SARS-CoV-2 emerged with a large number of muta-tions. This variant has since been detected in nu-merous countries around the world and is associ-ated with increased transmissibility. Beside other mutations, the UK SARS-CoV-2 lineage B.1.1.7 has multiple mutations in the spike glycoprotein’s gene S. Five are amino acid replacements (D614G, A222V, N439K, Y453F and N501Y), and one dele-tion (HV 69-70).
This product is for LABORATORY RESEARCH USE ONLY, not for diagnostic, therapeutic, drug, house-hold, or other uses. Please consult the Material Safety Data Sheet for information regarding hazards and safe handling practices. This test kit should be handled only by qualified per-sonnel trained in laboratory procedures and familiar with their potential hazards. Wear appropriate protec-tive clothing, gloves and eye/face protection and han-dle appropriately with the requisite Good Laboratory Practices. Treat all biological specimens, including used assay tubes and transfer pipettes, as if capable of transmit-ting infections agents Follow your institution’s safety procedures for work-ing with chemicals and handling biological samples. Wear laboratory coats, laboratory gloves and eye protection when handling biological samples and re-agents. Remove gloves and wash hands thoroughly after handling samples and reagents. Do not open the tubes or unseal wells during or after amplification. Dispose of all specimens and material used to per-form the test as though they contain an infectious agent. Laboratory, chemical or biohazardous wastes must be handled and discarded in accordance with all local, state and federal regulations for hazardous ma-terial.
5 Strips – Loop amplification reaction tubes. Clear PCR single Cap 8-tube strips 200 μl; optical clear flat cap; high profile; cut able; filled with dried stabilized loop amplification and detection mix.
1 vial – LAMP reaction buffer (1000 μl)
1 vial – Negative Control (50 μl)
1 vial – Positive Control (50 μl)
45 min. at 65 °C)
– 20 °C
12 months after manufacturing or until the expiration date
6.1 INSTRUMEMTATION AND MATERIALS REQUIRED
- PCR instrument or incubator programmable at 65 °C constant temperature
- Pipettes for 1-20 μl
- Pipette tips with aerosol filters
- Collection Kits: Nasopharyngeal Swab Nasal Swab
Extraction Kit: Viral DNA/RNA Extraction Kit
6.2 – SAMPLE COLLECTION / PREPARATION
Preferentially, use the same RNA sample from which SARS-CoV-2 was detected, to genotype it for muta-tions. Ineffective or inappropriate sample collection can result in false test results. Extracted RNA should always be stored at -70°C or lower in an RNase free environment.
6.3 REAGENT PREPARATION / STORAGE
The SARS-COV-2 Mutation Detection Assay is stabi-lized and shipped at ambient temperature. Reagents must be stored at -20 °C upon arrival.
The reaction tubes are prefilled with LAMP reaction mixture and are supplied in a convenient dry stabi-lized format, containing all buffer reagents, primers, dNTPs and enzymes to perform the amplification and detection.
LAMP reaction buffer, positive and negative control are liquid and ready to use.
When stored refrigerated at -20 °C the components are stable for at least 30 days after opening or until the expiration date printed on the labels.
Remaining reaction tubes should be stored refriger-ated at -20 °C protected from moisture; store together with desiccant in the resealable bag (ZipLoc).
6.4 TECHNICAL NOTES
External control RNA should routinely be assayed as unknowns to check performance of the reagents and the assay.
Use disposable filter tips to dispense reaction buffer and samples. To avoid carryover contamination, change the tip between each sample.
6.5 ASSAY PROCEDURE
Strictly follow the procedure and Good Laboratory Practice.
Please adhere strictly to the sequence of pipetting steps provided in this protocol. Observe the guide-lines for performing quality control in medical labora-tories by assaying external controls.
All reagents should be stored refrigerated at -20 °C in their original container.
Do not exchange kit components from different lots. The expiration dates stated on the labels of the ship-ping container and all vials have to be observed. Do not use kit components after their expiration dates.
Reagents removed from refrigerator should be brought to room temperature
Prepare a sufficient number of microtubes to accom-modate samples and controls.
- Pipette 20 μl of LAMP reaction buffer into the tubes.
- Incubate for 5 minutes at room temperature (18 – 28°C).
- For each test vial add 2 μl sample (isolated RNA) or positive- or negative controls.
- Close reaction tubes
- Mix until dried reagent mixture is completely dis-solved
- Incubate for 45 min at 65 °C.
- Cool for 5 minutes at room temperature
- Evaluate samples, the positive control must be blue-green and the negative control pink. Read in-dividual samples according the negative and pos-itive control.
6.6 INTERPRETATION OF RESULTS
Possible results of the different SARS-COV-2 Detec-tion Assays:
- Incubate for 5 minutes at room temperature
- Add 2 μl RNA sample or control
- Close reaction tubes
- Mix until dried reagent mixture is completely dis-solved
- Incubate for 45 minutes at 65 °C temperature
- Cool for 5 minutes at room temperature Read color for negative reaction (slight pink or positive reaction (blue-green)
- WHO. 2020. Coronavirus disease 2019. World Health Organization, Geneva,
- Lu R, et al., Genomic characterisation and epidemi-ology of 2019 novel coronavirus: implications for vi-rus origins and receptor binding.
Lancet 395:565–574. (2020)
- Notomi T., et al: Loop-mediated isothermal amplifi-cation of DNA, Nucleic Acids Research, 28(12), e63, 2000.
- Mori Y., et al: Detection of loop-mediated isothermal amplification reaction by turbidity derived from mag-nesium pyrophosphate formation, Biochem. Bio-phys. Res. Commun., 289(1), 150-154, 2001.
- Tomita N., et al: Loop-mediated isothermal amplifi-cation (LAMP) of gene sequences and simple visual detection of products, Nat Protoc., 3(5), 877-882, 2008