RU2775481C1 - SET OF SYNTHETIC OLIGONUCLEOTIDES FOR THE DETECTION OF RNA VIRUSES SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1 OF THE CORONAVIRIDAE FAMILY BY LOOP ISOTHERMAL AMPLIFICATION (LIA) - Google Patents

Abstract

FIELD: biotechnology, molecular genetics and medicine.

SUBSTANCE: invention relates to biotechnology, molecular genetics and medicine. A kit for detecting RNA of coronaviruses SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1 by loop isothermal amplification (LIA) is described.

EFFECT: invention can be used to identify these representatives of the Coronaviridae family for clinical laboratory diagnosis of coronavirus infection or for research purposes.

1 cl, 2 ex

Description

Technical field

The invention relates to biotechnology, molecular genetics and medicine, and in particular to a method for detecting RNA coronaviruses SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1 by loop isothermal amplification (LAMP). The invention can be used to identify these representatives of the Coronaviridae family for clinical laboratory diagnosis of coronavirus infection or for research purposes. The method includes a description of the structure of synthetic oligonucleotides, as well as their use for the diagnosis of RNA of the indicated pathogens of respiratory diseases.

State of the art

Coronaviruses are RNA-containing viruses ranging in size from 80 to 120 nm. The viral RNA genome is 27 to 32 kb in size, capped, polyadenylated, and enclosed in a helical nucleocapsid, which is surrounded by a protein membrane and a lipid-containing outer shell. Based on early serological and more recent genomic studies, Coronavirinae are divided into four genera: α-, β-, γ-, and δ-coronaviruses. Four types of virus (A, B, C and D) belong to the genus β-coronaviruses. Of the seven known human coronaviruses (HCoVs), HCoV-229E and HCoV-NL63 belong to the α-coronavirus genus, while HCoV-OC43 and HCoV-HKU1 belong to type A, SARS-CoV and SARS-CoV 2 are type B and MERS- CoV - to type C of the genus β-coronaviruses. The coronavirus genome is enclosed in non-segmented RNA ranging in size from 27 to 32 thousand base pairs. Genomic RNA has a 5' cap and a poly(A) tail at the 3' end; contains multiple open reading frames (ORFs). The genes are in the following order: 5'-replicase-S-E-M-N-3' with numerous small ORFs (accessory proteins). Representatives of coronaviruses are causative agents of respiratory diseases of varying severity, some cases of the disease can be fatal.

Currently, a variety of methods and approaches are used to detect coronavirus infection: detection of RNA viruses using polymerase chain reaction and other technologies based on nucleic acid amplification, detection of antigens using enzyme-linked immunosorbent assay (ELISA) and immunochromatographic analysis (ICA), as well as detection specific IgM and IgG antibodies using ELISA and ICA technologies. Among these methods, a special place is occupied by the LAMP method, a universal method characterized by high sensitivity (10–20 RNA copies), speed (the analysis time is about 30 minutes, excluding sample preparation), and relatively low cost. The LAMP method allows the use of various methods for the detection of amplification products and the use of fluorescent (intercalating fluorescent dyes, modified oligonucleotides) or colorimetric dyes, which make it possible to visually detect amplification products from the reaction mixture. In this case, the choice of the detection method may depend on the material and technical base of the laboratory conducting the analysis.

Known methods for detecting coronaviruses SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1 (for example, patent RU2734300, https://yandex.ru/patents/doc /RU2734300C1_20201014; Nascimentoetal., 2020, Trendsin MERS-CoV, SARS-CoV, and SARS-CoV-2 (COVID-19) DiagnosisStrategies: A Patent Review. Systematic Review; https://doi.org/10.3389/fpubh.2020.563095 ), however, these approaches use other technologies that are characterized by a longer study time.

The closest in essence to the invention are the methods described in the publications: Bektasetal., AccessibleLAMP-EnabledRapidTest (ALERT) forDetectingSARS-CoV-2 // Viruses. 2021 (https://doi.org/10.3390/v13050742); Anastasiou et al., Fast Detection of SARS-CoV-2 RNA Directly from Respiratory Samples Using a Loop-Mediated Isothermal Amplification (LAMP) Test // Viruses. 2021 (https://doi.org/10.3390/v13050801); Aoki et al., Colorimetric RT-LAMP SARS-CoV-2 diagnostic sensitivity relies on color interpretation and viral load // Scientific Reports (https://doi.org/10.1038/s41598-021-88506-y), etc., which are also based on the use of LAMP technology, but are only designed to detect RNA of a new coronavirus infection SARS-CoV-2.

Disclosure of invention

The objective of the present invention is to develop an oligonucleotide design for the differentiation of RNA viruses SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1 by loop isothermal amplification (LAMP), involving the use fluorescent and colorimetric detection of amplification products.

The technical result of the invention is to obtain structures of oligonucleotides: external forward (F3), external reverse (B3), internal forward (FIP), internal reverse (BIP), loop forward (LF) and loop reverse (LB) primers.

The technical problem posed is solved by choosing conservative species-specific genomic RNA fragments for reverse transcription and subsequent LAMP amplification. Targets for the detection of RNA coronaviruses:

SARS-CoV-2: N gene fragment;

SARS-CoV: N gene fragment;

MERS-CoV: E gene fragment;

HCoV-229E: E gene fragment;

HCoV-NL63: N gene fragment;

HCoV-OC43: RdRp gene fragment;

HCoV-HKU1: RdRp gene fragment.

The stated technical problem is solved by designing the structure of oligonucleotides from the following criteria:

- incubation temperature in LAMP - 65°C;

- the minimum number of hairpins, homo- and heterodimers.

To detect viruses, the design of oligonucleotides was carried out:

- to detect SARS-CoV-2: LCV2-F3 (SEQ ID NO: 1), LCV2-FIP2 (SEQ ID NO: 2), LCV2-BIP2 (SEQ ID NO: 3), LCV2-B3-2 (SEQ ID NO: 4);

- to detect SARS-CoV: LCV1-F3-1 (SEQ ID NO: 5), LCV1-FIP-1 (SEQ ID NO: 6), LCV1-BIP-2 (SEQ ID NO: 7), LCV1-B3- 1 (SEQ ID NO: 8);

- to detect MERS-CoV: LMS-F3 (SEQ ID NO: 9), LMS-FIP-1 (SEQ ID NO: 10), LMS-BIP-2 (SEQ ID NO: 11), LMS-B3-1 ( SEQ ID NO: 12);

- for the detection of HCoV-229E: CoV229-F3 (SEQ ID NO: 13), CoV229-FIP1 (SEQ ID NO: 14), CoV229-BIP1 (SEQ ID NO: 15), CoV229-B3-alt1 (SEQ ID NO: 16), CoV229-Blc (SEQ ID NO: 17)

- to detect HCoV-NL63: CoVNL63-F3 (SEQ ID NO: 18), CoVNL63-FIP2 (SEQ ID NO: 19), CoVNL63-BIP1 (SEQ ID NO: 20), CoVNL63-B3 (SEQ ID NO: 21) , CoVNL63-Flc-alt1 (SEQ ID NO: 22), CoVNL63-Blc (SEQ ID NO: 23);

- to detect HCoV-OC43: L43-F3 (SEQ ID NO: 24), L43-FIP-1 (SEQ ID NO: 25), L43-BIP-1 (SEQ ID NO: 26), L43-B3 (SEQ ID NO: 27);

- to detect HCoV-HKU1: LHKU-F3-2 (SEQ ID NO: 28), LHKU-FIP-2 (SEQ ID NO: 29), LHKU-BIP-1 (SEQ ID NO: 30), LHKU-B3- 1 (SEQ ID NO: 31).

The technical problem posed is solved by developing the composition of the reaction mixture to ensure the operability of oligonucleotide systems for LAMP reactions when using different detection systems.

Composition of the reaction for fluorescent detection: buffer (0.2 mMTris-HCl, 0.1 mM (NH ₄ ) ₂ SO ₄ , 0.15 MKCl, 0.1% Tween 20, pH 8.8), deoxynucleotide triphosphates - 1.4 mM, reversetase 200 units, Mg ₂ SO ₄ - 8 mM, LAMP FluorescentDye (NEB) - 0.2x, Bst polymerase 3.0 - 8 units, FIP and BIP oligonucleotides - 1.6 μM, F3 and B3 - 0.2 μM , LF and LB (if available) - 0.4 µM. The reaction is carried out in a volume of 25 µl, including 5 µl of the RNA template. Thermostating is carried out at a temperature of 65 ° C for 30 minutes; a positive reaction is judged by an increase in the level of fluorescence through the FAM channel.

Composition of the reaction for colorimetric detection: buffer (0.1 mM (NH ₄ ) ₂ SO ₄ , 0.15 MKCl, 0.1% Tween 20, pH 8.8), deoxynucleotide triphosphates - 1.4 mM, revertase 200 units, Mg ₂ SO ₄ - 8 mM, cresol red dye - 0.1 mM, Bst polymerase 3.0 - 8 units, FIP and BIP oligonucleotides - 1.6 μM, F3 and B3 - 0.2 μM, LF and LB (in the case of presence) - 0.4 μM (solution pH: 8.8). The reaction is carried out in a volume of 25 µl, including 5 µl of the RNA template. A positive reaction is judged by the color change of the reaction mixture from red to yellow.

The stated technical problem is solved by the development of positive control samples. Positive controls are required to monitor the progress of the reaction:

SARS-CoV-2: SEQIDNO: 32;

SARS-CoV: SEQ ID NO:33;

MERS-CoV: SEQ ID NO:34;

HCoV-229E: SEQ ID NO:35;

HCoV-NL63: SEQ ID NO:36;

HCoV-OC43: SEQ ID NO:37;

HCoV-HKU1: SEQ ID NO:38.

The invention has a number of advantages compared to analogues close in essence, since 7 types of coronaviruses are simultaneously detected in a sample within 25 minutes (not counting the sample preparation time).

Implementation of the invention

Example 1 Fluorescent Detection

The patient produced a smear from the nasopharynx, placed in 500 µl of saline. Nucleic acids were isolated from 100 µl of saline. Reaction mixtures were prepared for all 7 types of coronaviruses, with the composition: buffer (0.1 mM (NH ₄ ) ₂ SO ₄ , 0.15 MKCl, 0.1% Tween 20, pH 8.8), deoxynucleotide triphosphates - 1, 4 mM, reversease 200 units, Mg ₂ SO ₄ - 8 mM, cresol red dye - 0.1 mM, Bst polymerase 3.0 - 8 units, FIP and BIP oligonucleotides - 1.6 μM, F3 and B3 - 0.2 µM, LF and LB (if available) - 0.4 µM (solution pH: 8.8). Reactions were carried out in a volume of 25 μl. Three reactions were run with each reaction mixture: isolated RNA template, positive control (corresponding plasmid at 10,000,000 copies/ml) and negative control (deionized water).

Thermostating was carried out at a temperature of 65°C for 25 minutes in a QuantStudio cycler with detection via the FAM channel every minute. In the test tube with oligonucleotides for the detection of SARS-CoV-2, an increase in the fluorescence level was observed from the 10th minute of incubation; in all other test tubes, an increase in the fluorescence level was not observed. With all other mixtures, an increase in fluorescence is observed only in tubes with positive controls. Based on the results obtained, a conclusion was made about the presence of SARS-CoV-2 RNA in the studied patient sample.

Example 2 Colorimetric Detection

The patient produced a smear from the nasopharynx, placed in 500 µl of saline. Nucleic acids were isolated from 100 µl of saline. Reaction mixtures were prepared for all 7 types of coronaviruses, with the composition: buffer (0.2 mMTris-HCl, 0.1 mM (NH ₄ ) ₂ SO ₄ , 0.15 MKCl, 0.1% Tween 20, pH 8, 8), deoxynucleotide triphosphates - 1.4 mM, revertase 200 units, Mg ₂ SO ₄ - 8 mM, LAMP FluorescentDye (NEB) - 0.2x, Bst polymerase 3.0 - 8 units, FIP and BIP oligonucleotides - 1.6 μM , F3 and B3 - 0.2 μM, LF and LB (if available) - 0.4 μM. Reactions were carried out in a volume of 25 μl. Three reactions were run with each reaction mixture: isolated RNA template, positive control (corresponding plasmid at 10,000,000 copies/ml) and negative control (deionized water).

Thermostating was carried out at a temperature of 65°C for 25 minutes in a QuantStudio cycler. After the incubation time, the color of the test tubes of the reaction mixtures was detected. The color changed from red to yellow only in the positive control tubes and in the patient RNA tube in the SARS-CoV-2 detection mix. Based on the results obtained, a conclusion was made about the presence of SARS-CoV-2 RNA in the studied patient sample.

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Sequence listing

<110> BioTechGen Limited Liability Company

<120> A set of synthetic oligonucleotides for the detection of SARS-virus RNA

CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1

family Coronaviridae by loop isothermal amplification (LAMP)

<160> 1

<210> 1

<211> 23

<212>DNA

<223> for SARS-CoV-2 RNA detection

<400>TGAATAAGCATATTGACGCATAC

<210> 2

<211> 43

<212>DNA

<223> for SARS-CoV-2 RNA detection

<400>AGGCTTGAGTTTCATCAGCCTTCACAGAGCCTAAAAAAGGACAA

<210> 3

<211> 40

<212>DNA

<223> for SARS-CoV-2 RNA detection

<400>CTGTGACTCTTCTTCCTGCTGCCAGCACTGCTCATGGATT

<210> 4

<211> 20

<212>DNA

<223> for SARS-CoV-2 RNA detection

<400>CATGAGTTTAGGCCTGAGTT

<210> 5

<211> 24

<212>DNA

<223> for SARS-CoV RNA detection

<400>AAAGGACAAAAAGAAAAAAGACTGA

<210> 6

<211> 35

<212>DNA

<223> for SARS-CoV RNA detection

<400>ATGTCAGCCGCAGGAAGAACCTTTGCCGCAGAGAC

<210> 7

<211> 42

<212>DNA

<223> for SARS-CoV RNA detection

<400>AACTTCAAAAATTCCATGAGTGGAGCATCTGCCTTGTGTGGTC

<210> 8

<211> 20

<212>DNA

<223> for SARS-CoV RNA detection

<400>GCGAAAACGTTTACATAGCC

<210> 9

<211> 21

<212>DNA

<223> for MERS-CoV RNA detection

<400>CAAGAACGAATAGGGTTGTTC

<210> 10

<211> 47

<212>DNA

<223> for MERS-CoV RNA detection

<400>CTAGTAGCCGTAAGGAAAGCCATATTCATTTTTACCGTAGTATGTGC

<210> 11

<211> 47

<212>DNA

<223> for MERS-CoV RNA detection

<400>GTGCAATGTATGACAGGCTTCAATGGAATTTTACATAGACTGAACGT

<210> 12

<211> 18

<212>DNA

<223> for MERS-CoV RNA detection

<400>CACTCGTCAGGTGGTAGA

<210> 13

<211> 21

<212>DNA

<223> for HCoV-229E RNA detection

<400>GTTCCTTAAGCTAGTGGATGA

<210> 14

<211> 45

<212>DNA

<223> for HCoV-229E RNA detection

<400>ACATATGGCAAGTGAAACAAAGCTTGTGGTGCTTATAGTGATACT

<210> 15

<211> 49

<212>DNA

<223> for HCoV-229E RNA detection

<400>AATAGAACAGTTTATGGCCCCATTAAATCAATAACTCGTTTAGGGAAAG

<210> 16

<211> 21

<212>DNA

<223> for HCoV-229E RNA detection

<400>AATTCTTCAAATGGGTGACAA

<210> 17

<211> 29

<212>DNA

<223> for HCoV-229E RNA detection

<400>ATTTACCAATCATATATGCACATAGACCC

<210> 18

<211> 22

<212>DNA

<223> for HCoV-NL63 RNA detection

<400>CTTCCACTCCTAAGAAACCTAA

<210> 19

<211> 42

<212>DNA

<223> for HCoV-NL63 RNA detection

<400>TGATTAAAATCACGAGGACCAAAGCAACCTCGTTGGAAGCGT

<210> 20

<211> 43

<212>DNA

<223> for HCoV-NL63 RNA detection

<400>CAGAATGGTGTTGATGCCAAAGGTCAGTGCTAACCTCACTATC

<210> 21

<211> 24

<212>DNA

<223> for HCoV-NL63 RNA detection

<400>TTCTTATTATCCTTAGCTACAAGC

<210> 22

<211> 26

<212>DNA, symbol "+" - LNA nucleotide

<212>DNA

<223> for HCoV-NL63 RNA detection

<400>CT+GAATAACATTTTCCTCTC+TGGTAG

<210> 23

<211> 25

<212>DNA

<223> for HCoV-NL63 RNA detection

<400>CTGAATTGATTCCTAATCAGGCTGC

<210> 24

<211> 21

<212>DNA

<223> for HCoV-OC43 RNA detection

<400>CTGTACTTATGGGTTGGGATT

<210> 25

<211> 40

<212>DNA

<223> for HCoV-OC43 RNA detection

<400>TATCCGCTTTGCGAACAACATGTAGTGTGATCGTGCTATGC

<210> 26

<211> 45

<212>DNA

<223> for HCoV-OC43 RNA detection

<400>GGTTTTATCGACTTGCGAATGAATGACATAATAACAGCCACCACA

<210> 27

<211> 18

<212>DNA

<223> for HCoV-OC43 RNA detection

<400>ACTACTAGTGCCACCAGG

<210> 28

<211> 21

<212>DNA

<223> for HCoV-HKU1 RNA detection

<400>GGACGATATGTTACGTCATCT

<210> 29

<211> 44

<212>DNA

<223> for HCoV-HKU1 RNA detection

<400>ATGTTTGCGGGCCAAAACTAAGGATTATCCTAAATGTGATCGTG

<210> 30

<211> 46

<212>DNA

<223> for HCoV-HKU1 RNA detection

<400>GTGATAGATTTTATCGCCTTGCGAAGGCTTAACATAATAGCAACCG

<210> 31

<211> 18

<212>DNA

<223> for HCoV-HKU1 RNA detection

<400>TCACCACTGCTAGTACCCA

<210> 32

<211> 344

<212>DNA

<223> for SARS-CoV-2 RNA detection

<400>AAGTCATTTTGCTGAATAAGCATATTGACGCATACAAAAACATTCCCACCAACAGAGCCTAAAAAGGACAAAA

AGAAGAAGGCTGATGAAACTCAAGCCTTACCGCAGAGACAGAAGAAACAGCAAACTGTGACTCTTCTTCCTGCTGCA

GATTTGGATTGATTTCTCCAAACAATTGCAACAATCCATGAGCAGTGCTGACTCAACTCAGGCCTAAACTCATGCAGA

CCACACAAGGCAGATGGGCTATATAAACGTTTTCGCTTTTCCGTTTACGATATATAGTCTACTCTTGTGCAGAATGA

ATTCTCGTAACTACATAGCACAAGTAGATGTAGTTAACTTT

<210> 33

<211> 352

<212>DNA

<223> for SARS-CoV RNA detection

<400>ACGTCATACTGCTGAACAAGCACATTGACGCATACAAAAACATTCCCACCAACAGAGCCTAAAAAGGACAAAA

AGAAAAAGACTGATGAAGCTCAGCCTTTGCCGCAGAGACAAAAGAAGCAGCCCACTGTGACTCTTCTTCCTGCGGCT

GACATGGATGATTTCTCCAGACAACTTCAAAATTCCATGAGTGGAGCTTCTGCTGATTCAACTCAGGCATAAACACT

CATGATGACCACACAAGGCAGATGGGCTATGTAAACGTTTTCGCAATTCCGTTTACGATACATAGTCTACTCTTGTG

CAGAATGAATTCTCGTAACTAAACAGCACAAGTAGGTTTAGTTAACTTT

<210> 34

<211> 400

<212>DNA

<223> for MERS RNA detection

<400>TTCGTGCCTGCAACGCGCGATTCAGTTCCCTCTTCACATAATCGCCCCGAGCTCGCTTATCGTTTAAGCAGCT

CTGCGCTACTATGGGTCCCGTGTAGAGGCTAATCCATTAGTCTCTCTTTGGACATATGGAAAACGAACTATGTTACC

CTTTGTCCAAGAACGAATAGGGTTGTTCATAGTAAACTTTTTCATTTTTACCGTAGTATGTGCTATAACACTCTTGG

TGTGTATGGCTTTCCTTACGGCTACTAGATTATGTGTGCAATGTATGACAGGCTTCAATACCCTGTTAGTTCAGCCC

GCATTATACTTGTATAATACTGGACGTTCAGTCTATGTAAAATTCCAGGATAGTAAACCCCCTCTACCACCTGACGA

GTGGGTTTAACGAACTCCTT

<210> 35

<211> 302

<212>DNA

<223> for HCoV-229E RNA detection

<400>TAAGATGTTCCCTTAAGCTAGTGGATGATCATGCTTTGATTGTTAATGTACTACTCTGGTGTGTGGTGCTTAT

AGTGATACTACTAGTGTGTATTACAATAATTAAACTAATTAAGCTTTGTTTCACTTGCCATATGTTTTGTAATAGAA

CAGTTTATGGCCCCATTAAAAATGTGTACCATATTTACCAATCATATATGCACATAGACCCTTTCCCTAAACGAGTT

ATTGATTTCTAAACTAAACGACAATGTCAAACGACAATTGTACGGGTGACATTGTCACCCATTTGAAGAATTGGAA

<210> 36

<211> 400

<212>DNA

<223> for HCoV-NL63 RNA detection

<400>ACTTAGGTTTTGATAACCAGTCGAAGTCACCTAGTTCTTCTGGTACTTCCACTCCTAAGAAACCTAATAAGC

CTCTTTCTCAACCCAGGGCTGATAAGCCTTTCTCAGTTGAAGAAACCTCGTTGGAAGCGTGTTCCTACCAGAGAGGAA

AATGTTATTCAGTGCTTTGGTCCTCGTGATTTTAATCACAATATGGGGGATTCAGATCTTGTTCAGAATGGTGTTGA

TGCCAAAGGTTTTCCACAGCTTGCTGAATTGATTCCTAATCAGGCTGCGTTATTCTTTGATAGTGAGGTTAGCACTG

ATGAAGTGGGTGATAATGTTCAGATTACCTACACCTACAAAATGCTTGTAGCTAAGGATAATAAGAACCTTCCTAAG

TTCATTGAGCAGATTAGTGC

<210> 37

<211> 335

<212>DNA

<223> for HCoV-OC43 RNA detection

<400>AAATTTTATGGTGGCTGGGATGATATGTTACGCCGCCTTATTAAAGATGTTGACAATCCTGTACTTATGGGT

TGGGATTATCCTAAGTGTGATCGTGCTATGCCAAACCTACTACGTATTGTTAGTAGTTTGGTATTAGCCCGAAAACA

TGAGACATGTTGTTCGCAAAAGCGATAGGTTTTATCGACTTGCGAATGAATGCGCACAAGTTTTGAGTGAAATTGTTA

TGTGTGGTGGCTGTTATTATGTTAAGCCTGGTGGCACTAGTAGTGGTGATGCAACTACTGCTTTTGCTAATTCAGTC

TTTAACATATGTCAAGCTGTTTCAGCCAATGT

<210> 38

<211> 335

<212>DNA

<223> for HCoV-HKU1 RNA detection

<400>AAATTTTATGGTGGTTGGGACGATATGTTACGTCATCTTATAAAGGATGTTGACAACCCTGTTCTTATGGGT

TGGGATTATCCTAAATGTGATCGTGCTATGCCAAATATTTTGCGTATTGTTAGTAGTTTAGTTTTGGCCCGCAAACA

TGAATTTTGTTGTTCACATGGTGATAGATTTTATCGCCTTGCGAATGAATGTGCTCAAGTTTTGAGTGAAATAGTTA

TGTGTGGCGGTTGCTATTATGTTAAGCCTGGTGGTACTAGCAGTGGTGATGCAACTACTGCTTTTGCTAATTCTGTT

TTTAATATATGTCAGGCTGTTACTGCTAATGT

<---

Claims (77)

A set of synthetic oligonucleotides for the detection of RNA viruses SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1 of the Coronaviridae family by loop isothermal amplification (LAMP), including oligonucleotides with SEQ ID NO: 1 - SEQ ID NO: 38: SEQ ID NO: 1 Nucleotide sequence of LCV2-F3 TGAATAAGCATATTGACGCATAC SEQ ID NO: 2 Nucleotide sequence of LCV2-FIP2 AGGCTTGAGTTTCATCAGCCTTCACAGAGCCTAAAAAAGGACAA SEQ ID NO: 3 Nucleotide sequence of LCV2-BIP2 CTGTGACTCTTCTTCCTGCTGCCAGCACTGCTCATGGATT SEQ ID NO: 4 Nucleotide sequence of LCV2-B3-2 CATGAGTTTAGGCCTGAGTT SEQ ID NO: 5 Nucleotide sequence of LCV1-F3-1 AAAGGACAAAAAGAAAAAAGACTGA SEQ ID NO: 6 Nucleotide sequence of LCV1-FIP-1 ATGTCAGCCGCAGGAAGAACCTTTGCCGCAGAGAC SEQ ID NO: 7 Nucleotide sequence of LCV1-BIP-2 AACTTCAAAAATTCCATGAGTGGAGCATCTGCCTTGTGTGGTC SEQ ID NO: 8 Nucleotide sequence of LCV1-B3-1 GCGAAAACGTTTACATAGCC SEQ ID NO: 9 Nucleotide sequence of LMS-F3 CAAGAACGAATAGGGTTGTTC SEQ ID NO: 10 Nucleotide sequence of LMS-FIP-1 CTAGTAGCCGTAAGGAAAGCCATATTCATTTTTACCGTAGTATGTGC SEQ ID NO: 11 Nucleotide sequence of LMS-BIP-2 GTGCAATGTATGACAGGCTTCAATGGAATTTTACATAGACTGAACGT SEQ ID NO: 12 Nucleotide sequence of LMS-B3-1 CACTCGTCAGGTGGTAGA SEQ ID NO: 13 Nucleotide sequence of CoV229-F3 GTTCCTTAAGCTAGTGGATGA SEQ ID NO: 14 Nucleotide sequence of CoV229-FIP1 ACATATGGCAAGTGAAACAAAGCTTGTGGTGCTTATAGTGATACT SEQ ID NO: 15 Nucleotide sequence of CoV229-BIP1 AATAGAACAGTTTATGGCCCCATTAAATCAATAACTCGTTTAGGGAAAG SEQ ID NO: 16 Nucleotide sequence of CoV229-B3-alt1 AATTCTTCAAATGGGTGACAA SEQ ID NO: 17 Nucleotide sequence of CoV229-Blc ATTTACCAATCATATATGCACATAGACCC SEQ ID NO: 18 Nucleotide sequence of CoVNL63-F3 CTTCCACTCCTAAGAAACCTAA SEQ ID NO: 19 Nucleotide sequence of CoVNL63-FIP2 TGATTAAAATCACGAGGACCAAAGCAACCTCGTTGGAAGCGT SEQ ID NO: 20 Nucleotide sequence of CoVNL63-BIP1 CAGAATGGTGTTGATGCCAAAGGTCAGTGCTAACCTCACTATC SEQ ID NO: 21 Nucleotide sequence of CoVNL63-B3 TTCTTATTATCCTTAGCTACAAGC SEQ ID NO: 22 Nucleotide sequence of CoVNL63-Flc-alt1 CT+GAATAACATTTTCCTCTC+TGGTAG The symbol "+" denotes an LNA-modified (latched) nucleotide. SEQ ID NO: 23 Nucleotide sequence of CoVNL63-Blc CTGAATTGATTCCTAATCAGGCTGC SEQ ID NO: 24 Nucleotide sequence L43-F3 CTGTACTTATGGGTTGGGATT SEQ ID NO: 25 Nucleotide sequence of L43-FIP-1 TATCGCTTTGCGAACAACATGTAGTGTGATCGTGCTATGC SEQ ID NO: 26 Nucleotide sequence of L43-BIP-1 GGTTTTATCGACTTGCGAATGAATGACATATAATAACAGCCACCACA SEQ ID NO: 27 Nucleotide sequence of L43-B3 ACTACTAGTGCCACCAGG SEQ ID NO: 28 Nucleotide sequence of LHKU-F3-2 GGACGATATGTTACGTCATCT SEQ ID NO: 29 Nucleotide sequence of LHKU-FIP-2 ATGTTTGCGGGCCAAAACTAAGGATTATCCTAAATGTGATCGTG SEQ ID NO: 30 Nucleotide sequence of LHKU-BIP-1 GTGATAGATTTTATCGCCTTGCGAAGGCTTAACATAATAGCAACCG SEQ ID NO: 31 Nucleotide sequence of LHKU-B3-1 TCACCACTGCTAGTACCCA SEQ ID NO: 32 Nucleotide sequence of pNCV2-POS AAGTCATTTTGCTGAATAAGCATATTGACGCATACAAAACATTCCCACCAACAGAGCCTAAAAAGGACAAAAAGAAGAAGGCTGATGAAACTCAAGCCTTACCGCAGAGACAGAAGAAACAGCAAACTGTGACTCTTCTTCCTGCTGCAGATTTGGATGATTTCTCCAAACAATTGCAACAATCCATGAGCAGTGCTGACTCAACTCAGGCCTAAACTCATGCAGACCACACAAGGCAGATGGGCTATATAAACGTTTTCGCTTTTCCGTTTACGATATATAGTCTACTCTTGTGCAGAATGAATTCTCGTAACTACATAGCACAAGTAGATGTAGTTAACTTT SEQ ID NO: 33 Nucleotide sequence of pNCV1-POS ACGTCATACTGCTGAACAAGCACATTGACGCATACAAAACATTCCCACCAACAGAGCCTAAAAAGGACAAAAAGAAAAAGACTGATGAAGCTCAGCCTTTGCCGCAGAGACAAAAGAAGCAGCCCACTGTGACTCTTCTTCCTGCGGCTGACATGGATGATTTCTCCAGACAACTTCAAAATTCCATGAGTGGAGCTTCTGCTGATTCAACTCAGGCATAAACACTCATGATGACCACACAAGGCAGATGGGCTATGTAAACGTTTTCGCAATTCCGTTTACGATACATAGTCTACTCTTGTGCAGAATGAATTCTCGTAACTAAACAGCACAAGTAGGTTTAGTTAACTTT SEQ ID NO: 34 Nucleotide sequence of pMR-E-POS TTCGTGCCTGCAACGCGCGATTCAGTTCCTCTTCACATAATCGCCCCGAGCTCGCTTATCGTTTAAGCAGCTCTGCGCTACTATGGGTCCCGTGTAGAGGCTAATCCATTAGTCTCTCTTTGGACATATGGAAAACGAACTATGTTACCCTTTGTCCAAGAACGAATAGGGTTGTTCATAGTAAACTTTTTCATTTTTACCGTAGTATGTGCTATAACACTCTTGGTGTGTATGGCTTTCCTTACGGCTACTAGATTATGTGTGCAATGTATGACAGGCTTCAATACCCTGTTAGTTCAGCCCGCATTATACTTGTATAATACTGGACGTTCAGTCTATGTAAAATTCCAGGATAGTAAACCCCCTCTACCACCTGACGAGTGGGTTTAACGAACTCCTT SEQ ID NO: 35 Nucleotide sequence of pCoV229-POS-alt TAAGATGTTCCTTAAGCTAGTGGATGATCATGCTTTGATTGTTAATGTACTACTCTGGTGTGTGGTGCTTATAGTGATACTACTAGTGTGTATTACAATAATTAAACTAATTAAGCTTTGTTTCACTTGCCATATGTTTTGTAATAGAACAGTTTATGGCCCCATTAAAAATGTGTACCATATTTACCAATCATATATGCACATAGACCCTTTCCCTAAACGAGTTATTGATTTCTAAACTAAACGACAATGTCAAACGACAATTGTACGGGTGACATTGTCACCCATTTGAAGAATTGGAA SEQ ID NO: 36 Nucleotide sequence of pCoVNL63-POS ACTTAGGTTTTGATAACCAGTCGAAGTCACCTAGTTCTTCTGGTACTTCCACTCCTAAGAAACCTAATAAGCCTCTTTCTCAACCCAGGGCTGATAAGCCTTCTCAGTTGAAGAAACCTCGTTGGAAGCGTGTTCCTACCAGAGAGGAAAATGTTATTCAGTGCTTTGGTCCTCGTGATTTTAATCACAATATGGGGGATTCAGATCTTGTTCAGAATGGTGTTGATGCCAAAGGTTTTCCACAGCTTGCTGAATTGATTCCTAATCAGGCTGCGTTATTCTTTGATAGTGAGGTTAGCACTGATGAAGTGGGTGATAATGTTCAGATTACCTACACCTACAAAATGCTTGTAGCTAAGGATAATAAGAACCTTCCTAAGTTCATTGAGCAGATTAGTGC SEQ ID NO: 37 Nucleotide sequence of pCoV-OC-POS AAATTTTATGGTGGCTGGGATGATATGTTACGCCGCCTTATTAAAGATGTTGACAATCCTGTACTTATGGGTTGGGATTATCCTAAGTGTGATCGTGCTATGCCAAACCTACTACGTATTGTTAGTAGTTTGGTATTAGCCCGAAAACATGAGACATGTTGTTCGCAAAGCGATAGGTTTTATCGACTTGCGAATGAATGCGCACAAGTTTTGAGTGAAATTGTTATGTGTGGTGGCTGTTATTATGTTAAGCCTGGTGGCACTAGTAGTGGTGATGCAACTACTGCTTTTGCTAATTCAGTCTTTAACATATGTCAAGCTGTTTCAGCCAATGT SEQ ID NO: 38 Nucleotide sequence of pCoV-HK-POS AAATTTTATGGTGGTTGGGACGATATGTTACGTCATCTTATAAAGGATGTTGACAACCCTGTTCTTATGGGTTGGGATTATCCTAAATGTGATCGTGCTATGCCAAATATTTTGCGTATTGTTAGTAGTTTAGTTTTGGCCCGCAAACATGAATTTTGTTGTTCACATGGTGATAGATTTTATCGCCTTGCGAATGAATGTGCTCAAGTTTTGAGTGAAATAGTTATGTGTGGCGGTTGCTATTATGTTAAGCCTGGTGGTACTAGCAGTGGTGATGCAACTACTGCTTTTGCTAATTCTGTTTTTAATATATGTCAGGCTGTTACTGCTAATGT