WO2023135350A1 - SYSTEM FOR DETECTING SARS-COV-2 VARIANTS USING RT-gPCR - Google Patents

SYSTEM FOR DETECTING SARS-COV-2 VARIANTS USING RT-gPCR Download PDF

Info

Publication number
WO2023135350A1
WO2023135350A1 PCT/ES2023/070010 ES2023070010W WO2023135350A1 WO 2023135350 A1 WO2023135350 A1 WO 2023135350A1 ES 2023070010 W ES2023070010 W ES 2023070010W WO 2023135350 A1 WO2023135350 A1 WO 2023135350A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
preferably consisting
probe
mutations
cov
Prior art date
Application number
PCT/ES2023/070010
Other languages
Spanish (es)
French (fr)
Inventor
Santiago MELÓN GARCÍA
Marta Elena ALVAREZ ARGÜELLES
Susana ROJO ALBA
Marta SANDOVAL TORRIENTES
José Antonio BOGA RIVEIRO
Original Assignee
Fundación Para La Investigación E Innovación Biosanitaria En El Principado De Asturias
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fundación Para La Investigación E Innovación Biosanitaria En El Principado De Asturias filed Critical Fundación Para La Investigación E Innovación Biosanitaria En El Principado De Asturias
Publication of WO2023135350A1 publication Critical patent/WO2023135350A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention is within the field of biotechnology, particularly within the field of rapid detection of pathogenic viruses, more specifically the SARS-CoV-2 virus and its variants.
  • the present invention refers to a kit comprising a set of probes and primers for the detection of mutations at positions 417, 452, 478, 484 and/or 501 of the S protein of the virus.
  • the present invention also refers to the use of these probes and primers for the in vitro detection and/or quantification of SARS-CoV-2 variants carrying these mutations in biological samples, as well as an in vitro method for the detection of these mutations. variants.
  • SARS-CoV-2 Severe acute respiratory syndrome coronavirus type 2
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • SARS-CoV-2 can cause human respiratory tract infections and lung inflammation, known as novel coronavirus pneumonia (COVID-19).
  • COVID-19 is a new acute respiratory infectious disease, highly contagious in the population. It is mainly transmitted through respiratory droplets, secretions, and direct contact.
  • the SARS-CoV-2 genome comprises the following open reading frames, or ORFs, from its 5' end to its 3' end: ORF1a and ORFlab corresponding to the non-structural proteins that form the transcription-replication complex, and ORF-S , ORF-E, ORF-M and ORF-N corresponding to the four main proteins, the spike glycoprotein (S), the envelope protein (E), the membrane glycoprotein (M) and the nucleocapsid protein (N). ).
  • the SARS-CoV-2 RNA genome has a 5' methylated cap and a 3' polyadenylated tail, allowing the RNA to bind to the host cell's ribosome for translation.
  • ORFlab encodes a protein called RNA-dependent RNA polymerase (RdRp or nsp12), which allows the viral genome to be transcribed into new RNA copies using the host cell's machinery.
  • RdRp or nsp12 RNA-dependent RNA polymerase
  • the S protein binds to specific receptors on the surface of the target cell and enters it to replicate, thus causing infection.
  • Coronaviruses are enveloped positive single-stranded RNA viruses. RNA viruses are extremely susceptible to mutations, which may be related to their spread and toxicity. Since mid-2020, some variants of SARS-CoV-2 have emerged that present mutations in the S protein and have had a great impact on the epidemiology of the COVID-19 disease that is currently spreading throughout the world.
  • UUK Alpha or United Kingdom
  • SA Beta or South African
  • BR Gamma or Brazilian
  • the E484K mutation has been shown to enhance the ability of SARS-CoV-2 to escape both natural and vaccine-induced immune responses.
  • Serum-derived monoclonal antibodies have been shown to be 10 to 60 times less effective in neutralizing virus carrying the E484K mutation.
  • next-generation sequencing comprises the most accurate approach for the molecular characterization of SARS-CoV-2.
  • NGS next-generation sequencing
  • this technique is slow, expensive and laborious, requiring an infrastructure that does not exist in many microbiological diagnostic laboratories, which from a practical point of view restricts the characterization to a small number of samples from patients infected with SARS-CoV-2.
  • Alternative methods such as POR-based diagnostic screening assays, can also be used for early detection and estimation of the prevalence of virus variants.
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • RT-PCR Detection by RT-PCR is the most widely used method for the diagnosis of SARS-CoV-2 infection.
  • the specificity and tolerance of detection by RT-PCR depends mainly on the specificity and tolerance of the primers and probe sequences used in the reaction. Primer and probe sequences with good tolerance should be selected at a highly conserved position in the SARS-CoV-2 genome to avoid missing detection of certain SARS-CoV-2 virus strains due to genome differences at this position .
  • Multiplex PCR is also used to allow detection of multiple pathogens in one sample, which can improve detection efficiency and reduce costs.
  • SNPs single nucleotide polymorphisms
  • a melting curve based assay (Durner et al. Dent Maten 2021;37:e95-e97) or an assay based on the TaqMan probe (Sandoval Torr ⁇ entes et al. J Virol Methods. 2021 Aug; 294:114143), both targeting a single mutation that results in the substitution of amino acids at position 501.
  • a one-step real-time RT-PCR assay was designed for simultaneous, rapid, and accurate typing of SARS-CoV-2 gene mutations associated with substitutions at positions 484 and 501 of protein S ( E484K and N501Y).
  • four TaqMan probes were designed with locked nucleic acid (ANB) chemistry. Two of them were used for the differentiation between the wild-type sequence and the sequence with the ia mutation at position 484 and the other two were used for the same purpose at position 501.
  • Different fluorophores were conjugated to the 5' end of each Taqman probe to facilitate the differentiation of the respective signals of fluorescence.
  • the patent document WO2021176099A1 discloses a method for the detection of the presence and/or absence of SARS-CoV-2 in a sample based on the analysis of the RdRP (nsp12) and nsp9 genes of the SARS-CoV-2 virus and some of its variants.
  • the RNA-dependent RNA polymerase (RdRP or nsp12) and single-stranded RNA-binding protein (nsp9) genes of the virus are among the most conserved regions.
  • Two additional targets have also been identified in this paper, in the nsp6 and N genes.
  • the target in the nsp6 gene is specific for SARS-CoV-2 UK, SA and BR variants and therefore allows the detection of the presence or absence of a concerning variant of SARS-CoV-2 in a sample.
  • the target in the N gene is specific for the UK variant of SARS-CoV-2 and therefore allows detection of the presence of the UK variant SARS-CoV-2 when used alone, and the detection of the presence of the UK SARS-CoV-2 variant, SA or BR, when used in combination with the nsp6 target gene.
  • the method described in this state-of-the-art document is an RT-PCR that is based on the use of specific probes and primers for the detection of the presence or absence of the UK variant (B.1 .1 .7), that of South Africa (B.1 .351) and that of Brazil (P.1).
  • the probes and primers used in this prior art patent document are different from those described in the present invention.
  • the patent document CN111961762A discloses a fluorescent RT-PCR primer, a probe, a reagent and a method for detecting the SARS-CoV-2 coronavirus.
  • He Orflab fragment in the SARS-CoV-2 genome is used as a target to carry out the qualitative detection of SARS-CoV-2.
  • SARS-CoV-2 Fluorescence RT-PCR Detection Reagent is prepared using lyophilization technology.
  • CN112063764A discloses a composition of multiple real-time fluorescent RT-PCR primers and probes for the detection of coronavirus nucleic acids.
  • the kit includes fluorescence RT-PCR probes and primers to detect the ORFIab gene and the N gene of SARS-CoV-2.
  • Patent document CN113249522A discloses a method that makes it possible to detect variant strains of the SARS-CoV-2 virus by PCR. Thanks to the method described, the existing technical bottleneck in fluorescence PCR technology is broken. In fluorescence PCR it is difficult to perform single base identification. In this state-of-the-art document, two probes are designed so that they can detect the change in a single G-C base. Variations at position 484 of the S protein can be detected using these probes.
  • CN112575120A discloses a method that allows the detection of the D614G mutation in the S protein of the SARS-CoV-2 virus. For this, a series of specific probes and primers are designed and the RT-PCR method is used. This method has the advantages that it is simple, has a short detection period, and has high sensitivity.
  • the present invention is aimed at solving the need to provide PCR-based methods that are robust and stable over time to detect SARS-CoV-2 and its variants, which allow simultaneous, rapid and accurate typing of the different mutations in the protein. S of the SARS-CoV-2 virus.
  • the authors of the present invention have developed a set of probes and primers that allow a fast, simple and relatively cheap characterization of mutations present at positions 417, 452, 478 and/or 484 of the S protein of the SARS-CoV-2 virus.
  • the present invention also describes a kit for the detection of these mutations that comprises at least one set of these probes and primers. Based on these probes and primers, the inventors have also developed an in vitro method for the detection of variants carrying these mutations.
  • These variants are Alpha variant (B.1.1.7 lineage), Beta variant (B.1.351 lineage), Gamma variant (P.1 lineage), Kappa variant (B.1.617.1 lineage), Delta variant (B.1.617.2) and the Lambda variant (lineage C.37).
  • the method described in the present invention allows the detection of variants carrying mutations in these positions in just one hour. In addition, it allows analysis of all identified SARS-CoV-2 infected patient samples, while other methods such as sequencing only allow analysis of a small number of samples.
  • one aspect of the invention refers to a set of specific probes and primers, from now on “probes and primers of the invention", for the detection of mutations at positions 417, 452, 478 and/or 484 of the protein.
  • S of the SARS-CoV-2 virus S of the SARS-CoV-2 virus.
  • the set of probes and primers of the invention is selected from the list consisting of: a) Forward primer comprising, preferably consisting of, SEQ ID NO:1, reverse primer comprising, preferably consisting of, SEQ ID NO: 2, probe comprising, preferably consisting of, SEQ ID NO:3, probe comprising, preferably consisting of, SEQ ID NO:4, and probe comprising, preferably consisting of, SEQ ID NO:5; b) Forward primer comprising, preferably consisting of, SEQ ID NO:6, reverse primer comprising, preferably consisting of, SEQ ID NO:7, probe comprising, preferably consisting of, SEQ ID NO:8, probe comprising, preferably consisting of, SEQ ID NO:9, and probe comprising, preferably consisting of, SEQ ID NO:10; c) Forward primer comprising, preferably consisting of, SEQ ID NO:11, reverse primer comprising, preferably consisting of, SEQ ID NO:12, probe comprising, preferably consisting of, SEQ ID NO: 13, and probe compris
  • primer refers to an oligonucleotide capable of acting as a starting point for DNA synthesis when hybridized to the template nucleic acid.
  • Primers can be prepared by any suitable method, including, for example, but not limited to, cloning and restriction of appropriate sequences and direct chemical synthesis. Primers can be designed to hybridize to specific nucleotide sequences in the template nucleic acid (specific primers), as in the invention, or they can be randomly synthesized (arbitrary primers).
  • probe refers to a DNA fragment used to detect the presence of a specific DNA fragment within a sample through hybridization with double-stranded DNA.
  • the probes of the invention can be produced by methods known to those skilled in the art. For example, they can be produced from chemical synthesis.
  • Probes and primers in (a) detect the K417T and K417N mutations, (b) detect the L452R and L452Q mutations, (c) detect the T478K mutation, and (d) detect the E484K and E484Q mutations.
  • These mutations are single nucleotide polymorphisms, or SNPs.
  • SNPs are defined as a variation in DNA sequence that affects a single base (adenine (A), thymine (T), cytosine (C), or guanine (G)) of a genome sequence.
  • the S protein of the SARS-CoV-2 virus is from the Wuhan-Hu-1 isolate [NCBI reference number: YP_009724390.1]. Its amino acid sequence is defined in SEQ ID NO: 24. Its nucleotide sequence [ NCBI reference number: NCJ345512.2] is that defined in SEQ ID NO: 25.
  • the "K417T” mutation refers to a mutation at amino acid 417 of the S protein of the virus in which a change of lysine (K) to threonine (T) occurs, caused by a substitution of an (A) for an ( C). In this way, the AAG codon (coding for lysine) becomes ACG (coding for threonine).
  • the “K417N” mutation refers to a mutation at amino acid 417 of the S protein of the virus in which a change from lysine (K) to asparagine (N) occurs, caused by a substitution of a (G) for a ( T). In this way, the AAG codon (coding for lysine) becomes AAT (coding for asparagine).
  • the “L452R” mutation refers to a mutation at amino acid 452 of the S protein of the virus in which there is a change from leucine (L) to arginine (R), caused by a substitution of a (T) for a ( G). In this way, the CTG codon (which codes for leucine) becomes CGG (which codes for arginine).
  • the “L452Q” mutation is refers to a mutation in amino acid 452 of the S protein of the virus in which a change from leucine (L) to glutamine (Q) is produced, caused by a substitution of a (T) for an (A). In this way, the CTG codon (which codes for leucine) becomes CAG (which codes for glutamine).
  • T478K refers to a mutation at amino acid 478 of the S protein of the virus in which a change from threonine (T) to Usin (K) occurs, caused by a substitution of a (G) for a ( T). In this way, the TGT codon (which codes for threonine) becomes TTT (which codes for Usin).
  • the "E484K” mutation refers to a mutation at amino acid 484 of the S protein of the virus in which a change of glutamic acid (E) to Usin (K) occurs, caused by a substitution of one (G) for one. (TO). In this way, the GAA codon (coding for glutamic acid) becomes AAA (coding for Usin).
  • the "E484Q” mutation refers to a mutation at amino acid 484 of the S protein of the virus in which a change of glutamic acid (E) to glutamine (Q) occurs, caused by a substitution of a (G) for a (C). In this way, the GAA codon (coding for glutamic acid) becomes CAA (coding for glutamine).
  • the probes of the invention are labeled with at least one fluorophore, preferably VIC, NED or FAM, more preferably at either of their 5' or 3' ends, even more preferably at their 3' end.
  • fluorophore preferably VIC, NED or FAM
  • kits of the invention which comprises at least one set of probes and primers of the invention.
  • the kit of the invention comprises all sets of probes and primers of the invention.
  • the kit may comprise other useful components in the implementation of the present invention, such as buffers, material supports, positive and/or negative control components, etc.
  • the kits may also include instructions to practice the object of the invention. These instructions may be present in the aforementioned kits in a variety of forms, one or more of which may be present in the kit.
  • One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g. eg , a sheet or sheets of paper on which information is printed, on kit packaging, on a package insert, etc.
  • Another medium would be a computer-readable medium, eg, a CD, a USB, etc., on which the information has been recorded.
  • Another means that may be present is a website address that can be used over the Internet to access information at a remote site. Any convenient means may be present in the kits.
  • the kit of the invention also comprises specific probes and primers for the detection of mutations at position 501 of the S protein of the SARS-CoV-2 virus, preferably the N501Y mutation.
  • detect or “detection” has been defined or explained in previous paragraphs, and said definition is applicable to the present inventive aspect, which in this case refers to reporting or identifying the sequences of the variants of the SARS-CoV-2 virus. that present mutations at position 501 of the S protein.
  • N501Y refers to a mutation at amino acid 501 of the S protein of the virus in which there is a change from asparagine (N) to tyrosine (Y), caused by a substitution of an (A) for an ( T). In this way, the AAT codon (which codes for asparagine) becomes TAT (which codes for tyrosine).
  • the probes and primers that detect the mutation at position 501 of protein S are a forward primer comprising, preferably consisting of, the sequence SEQ ID NO:20, a reverse primer comprising, preferably consisting of , SEQ ID NO:21, a probe comprising, preferably consisting of, SEQ ID NO:22 and a probe comprising, preferably consisting of, SEQ ID NO:23.
  • the kit of the invention may comprise one or more sets of the probes and primers of the invention comprising, preferably consisting of, the sequences SEQ ID NO: 1-23.
  • the probes of the kit of the invention are marked with ai least one fluorophore, preferably VIC, NED or FAM, more preferably at either of its 5' or 3' ends, even more preferably at its 3' end.
  • ai least one fluorophore preferably VIC, NED or FAM
  • another aspect of the invention refers to the use of the set of probes and primers of the invention, or the kit of the invention, for the detection and/or quantification in vitro of SARS-CoV-2 variants carrying mutations.
  • SNP SARS-CoV-2 variants carrying mutations.
  • the mutations are selected from the group consisting of: K417T/N, L452R/Q, T478K, E484K/Q and N501Y.
  • Quantify refers to determining, in a sample, the amount of DNA or RNA template molecules (determining the viral load) of the variants of the SARS-CoV-2 virus that present mutations at positions 417 , 452, 478, 484 and/or 501 of the S protein.
  • in vitro refers to the fact that the detection and/or quantification of SARS-CoV-2 variants is performed outside the subject's body.
  • subject refers to any animal, preferably a mammal. In a more preferred embodiment, the subject is a human of any gender, age, or race.
  • the present invention also contemplates the methods directed to the detection and/or quantification of the variants of the SARS-CoV-2 carrying mutations at positions 417, 452, 478, 484 and/or 501 of the S protein.
  • another aspect of the invention refers to an in vitro method, from now on "method of the invention” for the detection and/or quantification of SARS-CoV-2 variants carrying mutations in positions 417, 452, 478, 484 and/or 501 of protein S, in an isolated sample from a subject comprising: a. extracting the viral RNA from the sample, b. subjecting the RNA extracted in (a) to a reverse transcription reaction followed by an amplification reaction using the set of probes and primers of the invention or the kit of the invention, and c. detecting the presence of said mutations in the amplified sample.
  • the detection and/or quantification of the SARS-CoV-2 variants that present mutations in positions 417, 452, 478 and/or 484 of the S protein by means of the probes and primers of the invention can be carried out in any sample that is likely to be contaminated by SARS-CoV-2.
  • a "sample” is understood as a part or small amount of something that is considered representative of the whole and that is taken or separated from it to submit it to study, analysis or experimentation.
  • said study, analysis or experimentation refers to the presence/absence of the genetic material of the SARS-CoV-2 variants.
  • sample also includes samples that have been manipulated in some way after they were obtained, for example, by treatment with reagents, solubilization, or enrichment of certain components.
  • the sample is a biological sample.
  • biological sample includes, but is not limited to, tissues and/or biological fluids from an individual, obtained by any method known to a person skilled in the art that serves such purpose.
  • the biological sample to which the invention relates comprises ribonucleic acid (RNA).
  • RNA ribonucleic acid
  • the biological sample comes from the oral or respiratory tract and has been obtained, more preferably, by means of a nasopharyngeal swab.
  • the amplification reaction of the method of the invention is an RT-PCR, more preferably a quantitative RT-PCR (qRT-PCR).
  • RT-PCR Reverse transcriptase polymerase chain reaction
  • cDNA complementary DNA
  • reverse transcriptase or reverse transcriptase an enzyme called reverse transcriptase or reverse transcriptase
  • RNA from step (a) of the method of the invention can be carried out by methods known to those skilled in the art.
  • 5 extraction methods can be used, both manual, based on the use of organic solvents or spin columns, or automated, for example, Microlab platforms (Hamilton Company, Nevada, USA) or Magnapure systems (Roche , Basel, Switzerland).
  • step (b) the reverse transcription reaction preferably consists of two steps.
  • the first step of the retrotranscription stage consists of the synthesis of copies of the viral genome in the form of cDNA.
  • the conditions employed in this first step depend on the reagent used. This first step is carried out for preferably 15 minutes at a temperature between 35 °C - 52 °C, plus
  • the conditions of the second step of the retrotranscription step are preferably 2 min at 95 °C.
  • step (b) of the method of the invention amplification is carried out to detect the variant of interest using two primers of the invention and two or three probes of the invention for each position (Taqman MGB), preferably labeled with fluorophores, more preferably VIC, NED or FAM, which recognize the wild-type sequence and the different mutations at positions 417, 452, 478, 484 and/or 501 (Tablal).
  • fluorophores more preferably VIC, NED or FAM
  • RNA previously extracted from the biological sample in step (a)
  • Reagent A is prepared by mixing the commercial reagent Taqman fast virus 1-step master mix (Roche) and the primers and probes of the invention specific for each mutation to be analyzed or the kit of the invention.
  • a forward primer that comprises, preferably consists of, SEQ ID NO:1, a reverse primer that comprises, preferably consists of, SEQ ID NO:2, a probe that comprises, 15 preferably consists of, SEQ ID NO:3, a probe comprising, preferably consisting of, SEQ ID NO:4, and a probe comprising, preferably consisting of, SEQ ID NO:5.
  • a forward primer that comprises, preferably consists of, SEQ ID NO:6, a reverse primer that comprises, preferably consists of, SEQ ID NO:7, a probe that comprises , preferably consisting of, SEQ ID NO: 8, a probe comprising, preferably consisting of, SEQ ID NO: 9, and a probe comprising, preferably consisting of, SEQ ID NO: 10.
  • a forward primer that comprises, preferably consists of, SEQ ID NO:11
  • a reverse primer that comprises, preferably consists of, SEQ ID NO:12
  • a probe that comprises, preferably consists of, SEQ ID NO:13
  • a probe comprising, preferably consists of, SEQ ID NO:14.
  • a forward primer that comprises, preferably consists of, SEQ ID NO: 15, a reverse primer that comprises, preferably consists of, SEQ ID NO: 16, a probe that comprises, preferably consists of, SEQ ID NO: 17, a probe comprising, preferably consisting of, SEQ ID NO: 18, and a probe comprising, preferably consisting of, SEQ ID NO: 19.
  • a forward primer that comprises, preferably consists of, the sequence SEQ ID NO:20, a reverse primer that comprises, preferably consists of, SEQ ID NO:21, a probe that comprises, preferably consists of, SEQ ID NO:22 and a probe comprising, preferably consists of, SEQ ID NO:23.
  • step (b) of the method of the invention and the subsequent analysis and detection of mutations is preferably carried out in a real-time PCR system type 7500 thermal cycler (ABI), Steponeplus thermal cycler (ABI) or QuantStudio 5 ( ABI).
  • ABSI real-time PCR system type 7500 thermal cycler
  • ABSI Steponeplus thermal cycler
  • ABI QuantStudio 5
  • step (c) of the method of the invention refers to detecting whether the probes and primers designed by the inventors have hybridized, bound or recognized the specific SNPs described. in the invention.
  • the amplification patterns fluorescence signals
  • the three preferred fluorophores with which the probes of the invention are labeled can be excited at a single wavelength (X) of 488 nm, but emit at clearly different wavelengths.
  • FAM has an absorption Xmax of 494 nm and an emission Amax of 518 nm
  • NED has an absorption Xmax of 546 nm and an emission Xmax of 575 nm
  • VIC has an absorption Xmax of 538 nm and an A maximum emission of 554 nm.
  • Fig. 1 shows the position of the primers of the invention for the detection of the K417T/N, L452R/Q, T478K, E484K/Q and N501Y mutations of the S protein of the virus.
  • SARS-CoV-2 SARS-CoV-2.
  • Fig. 2 shows the analysis using the developed technique of positive samples for the detection of the T478K mutation of the S protein of the SARS-CoV-2 virus (Delta variant).
  • reagent A 300 ⁇ l of Taqman fast virus 1-step master mix (Roche), 4 ⁇ l of the forward primer (100 ⁇ M) comprising, preferably consisting of, SEQ ID NO:20, 4 ⁇ l of the reagent A are added.
  • reverse primer (100 ⁇ M) comprising, preferably consisting of, SEQ ID NO:21, 1 pl of the probe (100 ⁇ M) comprising, preferably consisting of, SEQ ID NO:22 and 1 pl of the probe (100 ⁇ M) comprising, preferably consisting of, SEQ ID NO:23. Water is added to complete the milliliter of volume.
  • the amplification system used is a real-time ORP type 7500 thermal cycler (ABI), Steponeplus thermal cycler (ABI) or QuantStudio 5 (ABI).
  • the reverse transcription step has 2 steps.
  • the first step of the stage of reverse transcription (15 min at 50 °C) involves cDNA synthesis from viral RNA.
  • the conditions of the second step of the reverse transcription step are 2 min at 95 °C (Table 2).
  • the 40-cycle PCR step consists of 2 steps, a 5-second denaturation step at 95 °C and a combined 30-second annealing and extension step at 60 °C. During these cycles the PCR product is amplified (Table 2).
  • the reading of the results comes from the interpretation (by the system) of the intensity of the fluorescent signal at the moment the presence of the genetic sequence corresponding to the virus is identified.
  • An example of the detection of the T478K mutation in the S protein of the virus is shown in Fig.2. Variants carrying the wild-type allele T478 (light grey) and mutant T478K (dark grey) are observed, which allows identification of the Delta variant. This analysis was performed on a StepOnePlus system (Thermofisher).
  • the Alpha variant (B.1.1.7) is characterized because it presents the E484K, N501Y mutations
  • the Beta variant (B.1.351) is characterized because it presents the K417N, E484K and N501Y mutations
  • the Gamma variant (P.1) is characterized because it presents the K417N, E484K and N501Y mutations
  • the Kappa variant (B.1.617.1) is characterized by presenting the L452R, T478K and E484Q mutations
  • the Delta variant (B.1.617.2) is characterized by presenting the L452R and T478K
  • the Lambda variant (C.37) is characterized by having the L452Q mutation.
  • the method of the invention allows the detection of Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1.), Kappa (B.1.617.1), Delta (B.1.617.2) and Lambda (C.3/) carriers of mutations in codons 417, 452, 478, 484 and 501 in just one hour.
  • Example 5 Comparison of the results obtained after the analysis of samples using the method of the invention described in the previous examples and the whole genome sequencing method

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Radio Relay Systems (AREA)
  • Radar Systems Or Details Thereof (AREA)

Abstract

The present invention falls within the field of biotechnology, in particular within the field of rapid detection of pathogenic viruses, more specifically of the SARS-CoV-2 virus and its variants. The present invention relates to a kit comprising a set of probes and primers for detecting mutations at positions 417, 452, 478, 484 and/or 501 of the S protein of the virus. The present invention also relates to the use of these probes and primers for the in vitro detection and/or quantification of SARS-CoV-2 variants carrying these mutations in biological samples, as well as to an in vitro method for detecting these variants.

Description

DESCRIPCIÓN DESCRIPTION
SISTEMA DE DETECCIÓN DE VARIANTES DE SARS-CoV-2 MEDIANTE RT-qPCR SYSTEM FOR THE DETECTION OF SARS-CoV-2 VARIANTS BY RT-qPCR
La presente invención se encuentra dentro del campo de la biotecnología, en particular dentro del campo de detección rápida de virus patógenos, más concretamente del virus SARS-CoV-2 y sus variantes. La presente invención se refiere a un kit que comprende un conjunto de sondas y cebadores para la detección de mutaciones en las posiciones 417, 452, 478, 484 y/o 501 de la proteína S del virus. La presente invención también se refiere al uso de estas sondas y cebadores para la detección y/o cuantificación in vitro de variantes de SARS-CoV-2 portadoras de estas mutaciones en muestras biológicas, así como a un método in vitro para la detección de estas variantes. The present invention is within the field of biotechnology, particularly within the field of rapid detection of pathogenic viruses, more specifically the SARS-CoV-2 virus and its variants. The present invention refers to a kit comprising a set of probes and primers for the detection of mutations at positions 417, 452, 478, 484 and/or 501 of the S protein of the virus. The present invention also refers to the use of these probes and primers for the in vitro detection and/or quantification of SARS-CoV-2 variants carrying these mutations in biological samples, as well as an in vitro method for the detection of these mutations. variants.
ANTECEDENTES DE LA INVENCIÓN BACKGROUND OF THE INVENTION
El coronavirus de tipo 2 del síndrome respiratorio agudo severo (SARS-CoV-2), el coronavirus del síndrome respiratorio agudo severo (SARS-CoV) y el coronavirus del síndrome respiratorio de Oriente Medio (MERS-CoV) pertenecen al género de los beta coronavirus. En particular, el SARS-CoV-2 puede causar infecciones del tracto respiratorio humano e inflamación pulmonar, conocida como neumonía por nuevo coronavirus (COVID-19). COVID-19 es una nueva enfermedad infecciosa respiratoria aguda, altamente contagiosa en la población. Se transmite principalmente a través de gotitas respiratorias, secreciones y contacto directo. Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV) all belong to the beta genus. coronavirus. In particular, SARS-CoV-2 can cause human respiratory tract infections and lung inflammation, known as novel coronavirus pneumonia (COVID-19). COVID-19 is a new acute respiratory infectious disease, highly contagious in the population. It is mainly transmitted through respiratory droplets, secretions, and direct contact.
El genoma del SARS-CoV-2 comprende los siguientes marcos de lectura abiertos u ORF, desde su extremo 5 'hasta su extremo 3’: ORF1a y ORFlab correspondientes a las proteínas no estructurales que forman el complejo transcripción-replicación, y ORF- S, ORF-E, ORF-M y ORF-N correspondientes a las cuatro proteínas principales, la glícoproteína de pico (S), la proteína de envoltura (E), la glícoproteína de membrana (M) y la proteína de la nucleocápside (N). The SARS-CoV-2 genome comprises the following open reading frames, or ORFs, from its 5' end to its 3' end: ORF1a and ORFlab corresponding to the non-structural proteins that form the transcription-replication complex, and ORF-S , ORF-E, ORF-M and ORF-N corresponding to the four main proteins, the spike glycoprotein (S), the envelope protein (E), the membrane glycoprotein (M) and the nucleocapsid protein (N). ).
El genoma del ARN del SARS-CoV-2 tiene un casquete metilado en 5' y una cola poliadenilada en 3', lo que permite que el ARN se una al ribosoma de la célula huésped para su traducción. ORFlab codifica una proteína llamada ARN polimerasa dependiente de ARN (RdRp o nsp12), que permite que el genoma viral se transcriba en nuevas copias de ARN utilizando la maquinaria de la célula huésped. La proteína S se une a receptores específicos en la superficie de la célula diana y entra en ella para replicarse, causando así la infección. Algunos estudios han demostrado que el SARS-CoV-2 usa la proteína S para unirse a la enzima convertidora de angiotensina 2 (ACE2) del receptor humano para invadir las células huésped. The SARS-CoV-2 RNA genome has a 5' methylated cap and a 3' polyadenylated tail, allowing the RNA to bind to the host cell's ribosome for translation. ORFlab encodes a protein called RNA-dependent RNA polymerase (RdRp or nsp12), which allows the viral genome to be transcribed into new RNA copies using the host cell's machinery. The S protein binds to specific receptors on the surface of the target cell and enters it to replicate, thus causing infection. Some studies have shown that SARS-CoV-2 uses the S protein to bind to the human receptor angiotensin-converting enzyme 2 (ACE2) to invade host cells.
Los coronavirus son virus de ARN monocatenario positivo con envoltura. Los virus de ARN son extremadamente susceptibles a las mutaciones, que pueden estar relacionadas con su propagación y toxicidad. Desde mediados de 2020 han surgido algunas variantes de SARS-CoV-2 que presentan mutaciones en la proteína S y que han tenido un gran impacto en la epidemiología de ia enfermedad COVID-19 que actualmente se están extendiendo por todo el mundo. Algunas de estas variantes son la variante Alfa o de Reino Unido (UK) (linaje B.1.1.7; mutaciones notables N501Y, 69- 70del y P681 H); la variante Beta o de Sudáfrica (SA) (linaje B.1.351 ; mutaciones notables N501Y, E484K, K417N) y la variante Gamma o de Brasil (BR) (linaje P.1 ; mutaciones notables N501Y, E484K, K417T). Coronaviruses are enveloped positive single-stranded RNA viruses. RNA viruses are extremely susceptible to mutations, which may be related to their spread and toxicity. Since mid-2020, some variants of SARS-CoV-2 have emerged that present mutations in the S protein and have had a great impact on the epidemiology of the COVID-19 disease that is currently spreading throughout the world. Some of these variants are the Alpha or United Kingdom (UK) variant (lineage B.1.1.7; notable mutations N501Y, 69-70del and P681H); the Beta or South African (SA) variant (lineage B.1.351; notable mutations N501Y, E484K, K417N) and the Gamma or Brazilian (BR) variant (lineage P.1; notable mutations N501Y, E484K, K417T).
Todas estas nuevas variantes de SARS-CoV-2 se caracterizan por una mayor transmisibilidad de persona a persona en comparación con variantes anteriores del virus. Las variantes UK, SA y BR comparten la mutación N501Y en la región de unión al receptor (RBD). Se piensa que esta mutación aumenta la afinidad de unión de la proteína S hacia el receptor ACE2 humano (hACE). Las variantes SA y BR comparten una mutación adicional en esta región (K417T i N) que se sospecha contribuye a una mayor afinidad de unión a hACE2. All of these new variants of SARS-CoV-2 are characterized by increased person-to-person transmissibility compared to earlier variants of the virus. The UK, SA, and BR variants share the N501Y mutation in the receptor-binding region (RBD). This mutation is thought to increase the binding affinity of the S protein towards the human ACE2 (hACE) receptor. The SA and BR variants share an additional mutation in this region (K417T i N) that is suspected to contribute to higher hACE2 binding affinity.
Se ha visto que la mutación E484K mejora la capacidad del SARS-CoV-2 para escapar de la respuesta inmune, tanto la natural como la inducida por la vacuna. Se ha demostrado que los anticuerpos monoclonales derivados del suero son de 10 a 60 veces menos efectivos para neutralizar el virus que porta la mutación E484K. The E484K mutation has been shown to enhance the ability of SARS-CoV-2 to escape both natural and vaccine-induced immune responses. Serum-derived monoclonal antibodies have been shown to be 10 to 60 times less effective in neutralizing virus carrying the E484K mutation.
En consecuencia, estas variantes emergentes de SARS-CoV-2 son motivo de preocupación debido a su mayor transmisibilidad (Reino Unido, BR y SA). La sensibilidad reducida a los anticuerpos neutralizantes de las variantes que portan la mutación E484K (SA y BR) puede comprometer la eficacia de la vacuna. Además, la evolución convergente parece indicar que las cepas que albergan N501Y o E484K, o ambas, están evolucionando y extendiéndose rápidamente con un número creciente de mutaciones fuertemente asociadas y coexistentes en diferentes regiones de genes del SARS-CoV-2. Consequently, these emerging variants of SARS-CoV-2 are of concern due to their increased transmissibility (UK, BR and SA). Reduced sensitivity to neutralizing antibodies of variants carrying the E484K mutation (SA and BR) may compromise vaccine efficacy. Furthermore, convergent evolution seems to indicate that strains harboring N501Y or E484K, or both, are rapidly evolving and spreading with increasing numbers of Strongly associated and coexistent mutations in different regions of SARS-CoV-2 genes.
Sin duda, el análisis del genoma a través de la tecnología de secuenciación de próxima generación (NGS) comprende el enfoque más preciso para la caracterización molecular del SARS-CoV-2. Sin embargo, esta técnica es lenta, cara y laboriosa, exigiendo una infraestructura no existente en muchos laboratorios de diagnóstico microbiológico, lo que desde un punto de vista práctico restringe la caracterización a un pequeño número de muestras de pacientes infectados con SARS-CoV-2. También se pueden utilizar métodos alternativos, como los ensayos de detección diagnóstica basados en POR, para la detección temprana y el cálculo de la prevalencia de las variantes del virus. Dentro de los métodos basados en PCR destaca la reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR). Undoubtedly, genome analysis through next-generation sequencing (NGS) technology comprises the most accurate approach for the molecular characterization of SARS-CoV-2. However, this technique is slow, expensive and laborious, requiring an infrastructure that does not exist in many microbiological diagnostic laboratories, which from a practical point of view restricts the characterization to a small number of samples from patients infected with SARS-CoV-2. . Alternative methods, such as POR-based diagnostic screening assays, can also be used for early detection and estimation of the prevalence of virus variants. Among the PCR-based methods, the reverse transcriptase-polymerase chain reaction (RT-PCR) stands out.
La detección por RT-PCR es el método más utilizado para el diagnóstico de la infección por SARS-CoV-2. La especificidad y tolerancia de la detección por RT-PCR dependen principalmente de la especificidad y tolerancia de los cebadores y las secuencias de sonda empleados en la reacción. Las secuencias de cebador y sonda con buena tolerancia deben seleccionarse en una posición altamente conservada en el genoma del SARS-CoV-2 para evitar perder la detección de ciertas cepas del virus del SARS-CoV- 2 debido a diferencias en el genoma en esta posición. También se emplea la PCR multiplex que permite la detección de múltiples patógenos en una muestra, lo que puede mejorar la eficiencia de la detección y reducir los costes. Detection by RT-PCR is the most widely used method for the diagnosis of SARS-CoV-2 infection. The specificity and tolerance of detection by RT-PCR depends mainly on the specificity and tolerance of the primers and probe sequences used in the reaction. Primer and probe sequences with good tolerance should be selected at a highly conserved position in the SARS-CoV-2 genome to avoid missing detection of certain SARS-CoV-2 virus strains due to genome differences at this position . Multiplex PCR is also used to allow detection of multiple pathogens in one sample, which can improve detection efficiency and reduce costs.
Por otra parte, se han realizado estudios recientes para tratar de mejorar la detección de mutaciones en la proteína S del virus SARS-CoV-2. Algunos de estos ensayos son los basados en la detección de polimorfismos de nucleótidos único (SNPs), por ejemplo, un ensayo basado en la curva de fusión (Durner et al. Dent Maten 2021; 37: e95-e97) o un ensayo basado en la sonda TaqMan (Sandoval Torríentes et al. J Virol Methods. 2021 Aug; 294:114143), ambos dirigidos a una sola mutación que resulta en la sustitución de aminoácidos en la posición 501. En el estudio de Serafeím C. etal. J Virol Methods. 2021 Oct; 296:114242 se diseñó un ensayo RT-PCR en tiempo real de un solo paso para la tipificación simultánea, rápida y precisa de mutaciones del gen SARS-CoV- 2, asociadas con las sustituciones en las posiciones 484 y 501 de la proteína S (E484K y N501Y). Con este fin, se diseñaron cuatro sondas TaqMan con química de ácido nucleico bloqueado (ANB). Dos de ellas se utilizaron para la diferenciación entre la secuencia de tipo salvaje y la secuencia con ia mutación en la posición 484 y las otras dos se utilizaron para el mismo propósito en la posición 501. Se conjugaron diferentes fluoróforos en el extremo 5'de cada sonda Taqman para facilitar la diferenciación de las respectivas señales de fluorescencia. On the other hand, recent studies have been carried out to try to improve the detection of mutations in the S protein of the SARS-CoV-2 virus. Some of these assays are those based on the detection of single nucleotide polymorphisms (SNPs), for example, a melting curve based assay (Durner et al. Dent Maten 2021;37:e95-e97) or an assay based on the TaqMan probe (Sandoval Torríentes et al. J Virol Methods. 2021 Aug; 294:114143), both targeting a single mutation that results in the substitution of amino acids at position 501. In the study by Serafeím C. et al. J Virol Methods. 2021 Oct; 296:114242, a one-step real-time RT-PCR assay was designed for simultaneous, rapid, and accurate typing of SARS-CoV-2 gene mutations associated with substitutions at positions 484 and 501 of protein S ( E484K and N501Y). To this end, four TaqMan probes were designed with locked nucleic acid (ANB) chemistry. Two of them were used for the differentiation between the wild-type sequence and the sequence with the ia mutation at position 484 and the other two were used for the same purpose at position 501. Different fluorophores were conjugated to the 5' end of each Taqman probe to facilitate the differentiation of the respective signals of fluorescence.
Las pruebas de detección actuales pueden fallar en la detección de todos los pacientes positivos para COVID-19 y, además, la detección específica de cepas que albergan mutaciones en el gen S solo es válida durante un corto período de tiempo. Por lo tanto, existe la necesidad de proporcionar pruebas basadas en PCR que sean robustas y estables en el tiempo para detectar SARS-CoV-2 y que además permitan la tipificación simultánea, rápida y precisa de las mutaciones en la proteína S del virus SARS-CoV-2. Current screening tests may fail to detect all COVID-19 positive patients, and furthermore, the specific detection of strains harboring S gene mutations is only valid for a short period of time. Therefore, there is a need to provide PCR-based tests that are robust and stable over time to detect SARS-CoV-2 and that also allow simultaneous, rapid and accurate typing of mutations in the S protein of the SARS-CoV-2 virus. CoV-2.
En el estado de la técnica existen divulgaciones relacionadas con diferentes métodos y tecnologías para la detección de distintas variantes del virus SARS-CoV-2. In the state of the art there are disclosures related to different methods and technologies for the detection of different variants of the SARS-CoV-2 virus.
El documento de patente WO2021176099A1 divulga un método para la detección de la presencia y / o ausencia de SARS-CoV-2 en una muestra basado en el análisis de los genes RdRP (nsp12) y nsp9 del virus SARS-CoV-2 y de algunas de sus variantes. Los genes de la ARN polimerasa dependiente de ARN (RdRP o nsp12) y la proteína de unión a ARN monocatenario (nsp9) del virus se encuentran entre las regiones más conservadas. En este documento también se han identificado dos dianas adicionales, en los genes nsp6 y N. La diana en el gen nsp6 es específica para las variantes del SARS-CoV-2 UK, SA y BR y, por lo tanto, permite la detección de la presencia o ausencia de una variante preocupante del SARS-CoV-2 en una muestra. La diana en el gen N es especifica para la variante del SARS-CoV-2 del Reino Unido y, por lo tanto, permite la detección de la presencia de la variante SARS-CoV-2 del Reino Unido cuando se usa solo, y la detección de la presencia de la variante SARS-CoV-2 del Reino Unido, SA o BR, cuando se usa en combinación con el gen diana nsp6. El método descrito en este documento del estado del arte es una RT-PCR que se basa en la utilización de sondas y cebadores específicos para la detección de la presencia o ausencia de la variante de Reino Unido (B.1 .1 .7), la de Sudáfrica (B.1 .351 ) y la de Brasil (P.1). Las sondas y cebadores empleados en este documento de patente del estado de la técnica son distintas a las descritas en la presente invención. The patent document WO2021176099A1 discloses a method for the detection of the presence and/or absence of SARS-CoV-2 in a sample based on the analysis of the RdRP (nsp12) and nsp9 genes of the SARS-CoV-2 virus and some of its variants. The RNA-dependent RNA polymerase (RdRP or nsp12) and single-stranded RNA-binding protein (nsp9) genes of the virus are among the most conserved regions. Two additional targets have also been identified in this paper, in the nsp6 and N genes. The target in the nsp6 gene is specific for SARS-CoV-2 UK, SA and BR variants and therefore allows the detection of the presence or absence of a concerning variant of SARS-CoV-2 in a sample. The target in the N gene is specific for the UK variant of SARS-CoV-2 and therefore allows detection of the presence of the UK variant SARS-CoV-2 when used alone, and the detection of the presence of the UK SARS-CoV-2 variant, SA or BR, when used in combination with the nsp6 target gene. The method described in this state-of-the-art document is an RT-PCR that is based on the use of specific probes and primers for the detection of the presence or absence of the UK variant (B.1 .1 .7), that of South Africa (B.1 .351) and that of Brazil (P.1). The probes and primers used in this prior art patent document are different from those described in the present invention.
El documento de patente CN111961762A divulga un cebador de RT-PCR fluorescente, una sonda, un reactivo y un método para detectar el coronavirus SARS-CoV-2. El fragmento Orflab en el genoma del SARS-CoV-2 se utiliza como diana para llevar a cabo la detección cualitativa del SARS-CoV-2. El reactivo de detección de RT-PCR de fluorescencia de SARS-CoV-2 se prepara mediante una tecnología de liofilización. Por otra parte, CN112063764A divulga una composición de sondas y cebadores de RT-PCR fluorescente en tiempo real múltiple para la detección de ácidos nucleicos de coronavirus. El kit incluye cebadores y sondas de RT-PCR de fluorescencia para detectar el gen ORFIab y el gen N del SARS-CoV-2. The patent document CN111961762A discloses a fluorescent RT-PCR primer, a probe, a reagent and a method for detecting the SARS-CoV-2 coronavirus. He Orflab fragment in the SARS-CoV-2 genome is used as a target to carry out the qualitative detection of SARS-CoV-2. SARS-CoV-2 Fluorescence RT-PCR Detection Reagent is prepared using lyophilization technology. On the other hand, CN112063764A discloses a composition of multiple real-time fluorescent RT-PCR primers and probes for the detection of coronavirus nucleic acids. The kit includes fluorescence RT-PCR probes and primers to detect the ORFIab gene and the N gene of SARS-CoV-2.
El documento de patente CN113249522A divulga un método que permite detectar cepas variantes del virus SARS-CoV-2 mediante PCR. Gracias al método descrito se rompe el cuello de botella técnico existente en la tecnología de PCR de fluorescencia. En la PCR de fluorescencia es complicado realizar una identificación de base única. En este documento del estado del arte se diseñan dos sondas para que puedan detectar el cambio en una sola base G-C. Mediante estas sondas se pueden detectar variaciones en la posición 484 de la proteína S. Patent document CN113249522A discloses a method that makes it possible to detect variant strains of the SARS-CoV-2 virus by PCR. Thanks to the method described, the existing technical bottleneck in fluorescence PCR technology is broken. In fluorescence PCR it is difficult to perform single base identification. In this state-of-the-art document, two probes are designed so that they can detect the change in a single G-C base. Variations at position 484 of the S protein can be detected using these probes.
CN112575120A divulga un método que permite la detección de la mutación D614G en la proteína S del virus SARS-CoV-2. Para ello se diseñan una serie de sondas y cebadores específicos y se emplea el método RT-PCR. Este método tiene las ventajas de que es sencillo, presenta un período de detección corto y tiene una alta sensibilidad. CN112575120A discloses a method that allows the detection of the D614G mutation in the S protein of the SARS-CoV-2 virus. For this, a series of specific probes and primers are designed and the RT-PCR method is used. This method has the advantages that it is simple, has a short detection period, and has high sensitivity.
A pesar de que hay una pluralidad de documentos que describen distintas formas de detección del virus SARS-CoV-2, existe una necesidad de nuevos métodos y reactivos para identificar con especificidad y sensibilidad mejorada la presencia y/o ausencia de variantes de SARS-CoV-2 en las muestras, preferentemente de manera simultánea (en una única reacción de amplificación). Despite the fact that there are a plurality of documents that describe different ways of detecting the SARS-CoV-2 virus, there is a need for new methods and reagents to identify with improved specificity and sensitivity the presence and/or absence of SARS-CoV variants. -2 in the samples, preferably simultaneously (in a single amplification reaction).
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
La presente invención está dirigida a resolver la necesidad de proporcionar métodos basados en PCR que sean robustos y estables en el tiempo para detectar SARS-CoV- 2 y sus variantes, que permitan la tipificación simultánea, rápida y precisa de las distintas mutaciones en la proteina S del virus SARS-CoV-2. The present invention is aimed at solving the need to provide PCR-based methods that are robust and stable over time to detect SARS-CoV-2 and its variants, which allow simultaneous, rapid and accurate typing of the different mutations in the protein. S of the SARS-CoV-2 virus.
Los autores de la presente invención han desarrollado un conjunto de sondas y cebadores que permiten una caracterización rápida, sencilla y relativamente barata de mutaciones presentes en las posiciones 417, 452, 478 y/o 484 de la proteína S del virus SARS-CoV-2. En la presente invención también se describe un kit para la detección de estas mutaciones que comprende, al menos, un conjunto de estas sondas y cebadores. En base a estas sondas y cebadores, los inventores también han desarrollado un método in vitro para la detección de las variantes portadoras de estas mutaciones. Estas variantes son la variante Alfa (linaje B.1.1.7), la variante Beta (linaje B.1.351), la variante Gamma (linaje P.1), la variante Kappa (linaje B.1.617.1), la variante Delta (B.1.617.2) y la variante Lambda (linaje C.37). The authors of the present invention have developed a set of probes and primers that allow a fast, simple and relatively cheap characterization of mutations present at positions 417, 452, 478 and/or 484 of the S protein of the SARS-CoV-2 virus. The present invention also describes a kit for the detection of these mutations that comprises at least one set of these probes and primers. Based on these probes and primers, the inventors have also developed an in vitro method for the detection of variants carrying these mutations. These variants are Alpha variant (B.1.1.7 lineage), Beta variant (B.1.351 lineage), Gamma variant (P.1 lineage), Kappa variant (B.1.617.1 lineage), Delta variant (B.1.617.2) and the Lambda variant (lineage C.37).
El método descrito en la presente invención permite la detección de variantes portadoras de mutaciones en estas posiciones en apenas una hora. Además, permite analizar todas las muestras de pacientes infectados con SARS-CoV-2 identificadas, mientras que otros métodos como la secuenciación solo permiten el análisis de un pequeño número de muestras. The method described in the present invention allows the detection of variants carrying mutations in these positions in just one hour. In addition, it allows analysis of all identified SARS-CoV-2 infected patient samples, while other methods such as sequencing only allow analysis of a small number of samples.
Así un aspecto de la invención se refiere a un conjunto de sondas y cebadores específicos, de ahora en adelante “sondas y cebadores de la invención”, para la detección de mutaciones en las posiciones 417, 452, 478 y/o 484 de la proteína S del virus SARS-CoV-2. El conjunto de sondas y cebadores de la invención es seleccionado de la lista que consiste en: a) Cebador directo que comprende, preferiblemente consiste en, la SEQ ID NO:1, cebador inverso que comprende, preferiblemente consiste en, la SEQ ID NO:2, sonda que comprende, preferiblemente consiste en, la SEQ ID NO:3, sonda que comprende, preferiblemente consiste en, la SEQ ID NO:4, y sonda que comprende, preferiblemente consiste en, la SEQ ID NO:5; b) Cebador directo que comprende, preferiblemente consiste en, la SEQ ID NO:6, cebador inverso que comprende, preferiblemente consiste en, la SEQ ID NO:7, sonda que comprende, preferiblemente consiste en, la SEQ ID NO:8, sonda que comprende, preferiblemente consiste en, la SEQ ID NO:9, y sonda que comprende, preferiblemente consiste en, la SEQ ID NO:10; c) Cebador directo que comprende, preferiblemente consiste en, la SEQ ID NO:11 , cebador inverso que comprende, preferiblemente consiste en, la SEQ ID NO:12, sonda que comprende, preferiblemente consiste en, la SEQ ID NO: 13, y sonda que comprende, preferiblemente consiste en, la SEQ ID NO: 14; y d) Cebador directo que comprende, preferiblemente consiste en, la SEQ ID NO:15, cebador inverso que comprende, preferiblemente consiste en, la SEQ ID NO:16, sonda que comprende, preferiblemente consiste en, la SEQ ID NO: 17, sonda que comprende, preferiblemente consiste en, la SEQ ID NO: 18, y sonda que comprende, preferiblemente consiste en, la SEQ ID NO: 19. Thus, one aspect of the invention refers to a set of specific probes and primers, from now on "probes and primers of the invention", for the detection of mutations at positions 417, 452, 478 and/or 484 of the protein. S of the SARS-CoV-2 virus. The set of probes and primers of the invention is selected from the list consisting of: a) Forward primer comprising, preferably consisting of, SEQ ID NO:1, reverse primer comprising, preferably consisting of, SEQ ID NO: 2, probe comprising, preferably consisting of, SEQ ID NO:3, probe comprising, preferably consisting of, SEQ ID NO:4, and probe comprising, preferably consisting of, SEQ ID NO:5; b) Forward primer comprising, preferably consisting of, SEQ ID NO:6, reverse primer comprising, preferably consisting of, SEQ ID NO:7, probe comprising, preferably consisting of, SEQ ID NO:8, probe comprising, preferably consisting of, SEQ ID NO:9, and probe comprising, preferably consisting of, SEQ ID NO:10; c) Forward primer comprising, preferably consisting of, SEQ ID NO:11, reverse primer comprising, preferably consisting of, SEQ ID NO:12, probe comprising, preferably consisting of, SEQ ID NO: 13, and probe comprising, preferably consisting of, SEQ ID NO: 14; and d) Forward primer comprising, preferably consisting of, SEQ ID NO:15, reverse primer comprising, preferably consisting of, SEQ ID NO: 16, probe comprising, preferably consisting of, SEQ ID NO: 17, probe comprising, preferably consisting of, SEQ ID NO: 18, and probe comprising , preferably consists of, SEQ ID NO: 19.
En la presente invención se entiende por “detectar” o “detección” a reportar o identificar las secuencias de las variantes del virus SARS-CoV-2 que presentan mutaciones en las posiciones 417, 452, 478 y/o 484 de la proteína S. In the present invention it is understood by "detecting" or "detection" to report or identify the sequences of the variants of the SARS-CoV-2 virus that present mutations in positions 417, 452, 478 and/or 484 of the S protein.
El término “cebador” (también denominado “oligo”), se refiere a un oligonucleótido capaz de actuar como punto de inicio de la síntesis de ADN cuando híbrida con el ácido nucleico molde. Los cebadores pueden prepararse mediante cualquier método adecuado, incluyendo, por ejemplo, pero sin limitarse a, la clonación y restricción de secuencias apropiadas y la síntesis química directa. Los cebadores pueden diseñarse para hibridar con secuencias especificas de nucleótidos en el ácido nucleico molde (cebadores específicos), como en la invención, o pueden ser sintetizados al azar (cebadores arbitrarios). The term "primer" (also called "oligo"), refers to an oligonucleotide capable of acting as a starting point for DNA synthesis when hybridized to the template nucleic acid. Primers can be prepared by any suitable method, including, for example, but not limited to, cloning and restriction of appropriate sequences and direct chemical synthesis. Primers can be designed to hybridize to specific nucleotide sequences in the template nucleic acid (specific primers), as in the invention, or they can be randomly synthesized (arbitrary primers).
El término “sonda” se refiere a un fragmento de ADN utilizado para detectar la presencia de un fragmento de ADN específico dentro de una muestra a través de la hibridación con un ADN bicatenario. Las sondas de la invención se pueden producir por los métodos conocidos por el experto en la materia. Por ejemplo, pueden producirse a partir de síntesis química. The term "probe" refers to a DNA fragment used to detect the presence of a specific DNA fragment within a sample through hybridization with double-stranded DNA. The probes of the invention can be produced by methods known to those skilled in the art. For example, they can be produced from chemical synthesis.
Las sondas y cebadores de (a) detectan las mutaciones K417T y K417N, las de (b) detectan las mutaciones L452R y L452Q, las de (c) detectan la mutación T478K y las de (d) detectan las mutaciones E484K y E484Q. Estas mutaciones son polimorfismos de un solo nucleótido o SNP. Los “SNP” se definen como una variación en la secuencia de ADN que afecta a una sola base (adenina (A), timina (T), citosina (C) o guanina (G)) de una secuencia del genoma. Probes and primers in (a) detect the K417T and K417N mutations, (b) detect the L452R and L452Q mutations, (c) detect the T478K mutation, and (d) detect the E484K and E484Q mutations. These mutations are single nucleotide polymorphisms, or SNPs. “SNPs” are defined as a variation in DNA sequence that affects a single base (adenine (A), thymine (T), cytosine (C), or guanine (G)) of a genome sequence.
La proteína S del virus SARS-CoV-2 es la procedente del aislado Wuhan-Hu-1 [número de referencia en NCBI: YP_009724390.1], Su secuencia aminoacídica es la definida en la SEQ ID NO: 24. Su secuencia nucleotídica [número de referencia en NCBI: NCJ345512.2] es la definida en la SEQ ID NO: 25.
Figure imgf000009_0001
Figure imgf000010_0001
Figure imgf000011_0001
The S protein of the SARS-CoV-2 virus is from the Wuhan-Hu-1 isolate [NCBI reference number: YP_009724390.1]. Its amino acid sequence is defined in SEQ ID NO: 24. Its nucleotide sequence [ NCBI reference number: NCJ345512.2] is that defined in SEQ ID NO: 25.
Figure imgf000009_0001
Figure imgf000010_0001
Figure imgf000011_0001
La mutación “K417T” se refiere a una mutación en el aminoácido 417 de la proteina S del virus en la que se produce un cambio de lisina (K) por treonina (T), provocado por una sustitución de una (A) por una (C). De esta forma, el codón AAG (que codifica para la lisina) pasa a ser ACG (que codifica para la treonina). La mutación “K417N” se refiere a una mutación en el aminoácido 417 de la proteína S del virus en la que se produce un cambio de lisina (K) por asparagina (N), provocado por una sustitución de una (G) por una (T). De esta forma, el codón AAG (que codifica para la lisina) pasa a ser AAT (que codifica para la asparagina). Estas dos mutaciones aparecen representadas en la invención como “K417T/N”. The "K417T" mutation refers to a mutation at amino acid 417 of the S protein of the virus in which a change of lysine (K) to threonine (T) occurs, caused by a substitution of an (A) for an ( C). In this way, the AAG codon (coding for lysine) becomes ACG (coding for threonine). The “K417N” mutation refers to a mutation at amino acid 417 of the S protein of the virus in which a change from lysine (K) to asparagine (N) occurs, caused by a substitution of a (G) for a ( T). In this way, the AAG codon (coding for lysine) becomes AAT (coding for asparagine). These two mutations are represented in the invention as "K417T/N".
La mutación “L452R” se refiere a una mutación en el aminoácido 452 de la proteína S del virus en la que se produce un cambio de leucina (L) por arginina (R), provocado por una sustitución de una (T) por una (G). De esta forma, el codón CTG (que codifica para la leucina) pasa a ser CGG (que codifica para la arginina). La mutación “L452Q” se refiere a una mutación en ei aminoácido 452 de la proteína S del virus en la que se produce un cambio de leucina (L) por glutamina (Q), provocado por una sustitución de una (T) por una (A). De esta forma, el codón CTG (que codifica para la leucina) pasa a ser CAG (que codifica para la glutamina). Estas dos mutaciones aparecen representadas en la invención como “L452R/Q”. The “L452R” mutation refers to a mutation at amino acid 452 of the S protein of the virus in which there is a change from leucine (L) to arginine (R), caused by a substitution of a (T) for a ( G). In this way, the CTG codon (which codes for leucine) becomes CGG (which codes for arginine). The “L452Q” mutation is refers to a mutation in amino acid 452 of the S protein of the virus in which a change from leucine (L) to glutamine (Q) is produced, caused by a substitution of a (T) for an (A). In this way, the CTG codon (which codes for leucine) becomes CAG (which codes for glutamine). These two mutations are represented in the invention as "L452R/Q".
La mutación “T478K” se refiere a una mutación en el aminoácido 478 de la proteína S del virus en la que se produce un cambio de treonina (T) por Usina (K), provocado por una sustitución de una (G) por una (T). De esta forma, el codón TGT (que codifica para la treonina) pasa a ser TTT (que codifica para la Usina). The "T478K" mutation refers to a mutation at amino acid 478 of the S protein of the virus in which a change from threonine (T) to Usin (K) occurs, caused by a substitution of a (G) for a ( T). In this way, the TGT codon (which codes for threonine) becomes TTT (which codes for Usin).
La mutación “E484K” se refiere a una mutación en el aminoácido 484 de la proteína S del virus en la que se produce un cambio de ácido glutámico (E) por Usina (K), provocado por una sustitución de una (G) por una (A). De esta forma, el codón GAA (que codifica para el ácido glutámico) pasa a ser AAA (que codifica para la Usina). La mutación “E484Q” se refiere a una mutación en el aminoácido 484 de la proteína S del virus en la que se produce un cambio de ácido glutámico (E) por glutamina (Q), provocado por una sustitución de una (G) por una (C). De esta forma, el codón GAA (que codifica para el ácido glutámico) pasa a ser CAA (que codifica para la glutamina). Estas dos mutaciones aparecen representadas en la invención como “E484K/Q”. The "E484K" mutation refers to a mutation at amino acid 484 of the S protein of the virus in which a change of glutamic acid (E) to Usin (K) occurs, caused by a substitution of one (G) for one. (TO). In this way, the GAA codon (coding for glutamic acid) becomes AAA (coding for Usin). The "E484Q" mutation refers to a mutation at amino acid 484 of the S protein of the virus in which a change of glutamic acid (E) to glutamine (Q) occurs, caused by a substitution of a (G) for a (C). In this way, the GAA codon (coding for glutamic acid) becomes CAA (coding for glutamine). These two mutations are represented in the invention as "E484K/Q".
En otra realización preferida, las sondas de la invención están marcadas con al menos un fluoróforo, preferiblemente VIC, NED o FAM, más preferiblemente en cualquiera de sus extremos, 5' o 3', aún más preferiblemente en su extremo 3'. In another preferred embodiment, the probes of the invention are labeled with at least one fluorophore, preferably VIC, NED or FAM, more preferably at either of their 5' or 3' ends, even more preferably at their 3' end.
Un segundo aspecto de la invención se refiere a un kit, de ahora en adelante “kit de la invención”, que comprende al menos un conjunto de sondas y cebadores de la invención. A second aspect of the invention refers to a kit, hereinafter "kit of the invention", which comprises at least one set of probes and primers of the invention.
En una realización preferida, el kit de la invención comprende todos los conjuntos de sondas y cebadores de la invención. In a preferred embodiment, the kit of the invention comprises all sets of probes and primers of the invention.
Además de las sondas y cebadores de la invención, el kit puede comprender otros componentes útiles en la puesta en práctica de la presente invención, tales como, buffers, soportes del material, componentes de controles positivos y/o negativos, etc. Además de los componentes mencionados, los kits podrán incluir también instrucciones para practicar el objeto de la invención. Estas instrucciones pueden estar presentes en los kits mencionados en una variedad de formas, una o más de las cuales pueden estar presentes en el kit. Una forma en la que estas instrucciones pueden estar presentes es como información impresa en un medio o sustrato adecuado, p. ej. , una hoja u hojas de papel en las que se imprime la información, en el embalaje del kit, en un inserto de paquete, etc. Otro medio sería un medio legible por ordenador, por ejemplo, un CD, un USB, etc., en el que se haya registrado la información. Otro medio que puede estar presente es una dirección de sitio web que puede utilizarse a través de Internet para acceder a la información en un sitio remoto. Cualquier medio conveniente puede estar presente en los kits. In addition to the probes and primers of the invention, the kit may comprise other useful components in the implementation of the present invention, such as buffers, material supports, positive and/or negative control components, etc. In addition to the components mentioned, the kits may also include instructions to practice the object of the invention. These instructions may be present in the aforementioned kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g. eg , a sheet or sheets of paper on which information is printed, on kit packaging, on a package insert, etc. Another medium would be a computer-readable medium, eg, a CD, a USB, etc., on which the information has been recorded. Another means that may be present is a website address that can be used over the Internet to access information at a remote site. Any convenient means may be present in the kits.
En otra realización preferida, el kit de la invención comprende, además, sondas y cebadores específicos para la detección de mutaciones en la posición 501 de la proteína S del virus SARS-CoV-2, preferiblemente la mutación N501Y. In another preferred embodiment, the kit of the invention also comprises specific probes and primers for the detection of mutations at position 501 of the S protein of the SARS-CoV-2 virus, preferably the N501Y mutation.
El término “detectar” o “detección” ha sido definido o explicado en párrafos anteriores, y dicha definición es aplicable al presente aspecto inventivo, que en este caso se refiere a reportar o identificar las secuencias de las variantes del virus SARS-CoV-2 que presentan mutaciones en la posición 501 de la proteína S. The term "detect" or "detection" has been defined or explained in previous paragraphs, and said definition is applicable to the present inventive aspect, which in this case refers to reporting or identifying the sequences of the variants of the SARS-CoV-2 virus. that present mutations at position 501 of the S protein.
La mutación “N501Y” se refiere a una mutación en el aminoácido 501 de la proteína S del virus en la que se produce un cambio de asparagina (N) por tirosina (Y), provocado por una sustitución de una (A) por una (T). De esta forma, el codón AAT (que codifica para la asparagina) pasa a ser TAT (que codifica para la tirosina). The "N501Y" mutation refers to a mutation at amino acid 501 of the S protein of the virus in which there is a change from asparagine (N) to tyrosine (Y), caused by a substitution of an (A) for an ( T). In this way, the AAT codon (which codes for asparagine) becomes TAT (which codes for tyrosine).
En una realización más preferida, las sondas y cebadores que detectan la mutación en la posición 501 de la proteína S son un cebador directo que comprende, preferiblemente consiste en, la secuencia SEQ ID NO:20, un cebador inverso que comprende, preferiblemente consiste en, la SEQ ID NO:21 , una sonda que comprende, preferiblemente consiste en, la SEQ ID NO:22 y una sonda que comprende, preferiblemente consiste en, la SEQ ID NO:23. In a more preferred embodiment, the probes and primers that detect the mutation at position 501 of protein S are a forward primer comprising, preferably consisting of, the sequence SEQ ID NO:20, a reverse primer comprising, preferably consisting of , SEQ ID NO:21, a probe comprising, preferably consisting of, SEQ ID NO:22 and a probe comprising, preferably consisting of, SEQ ID NO:23.
De esta forma, el kit de la invención puede comprender uno o más conjuntos de las sondas y cebadores de la invención que comprenden, preferiblemente consisten en, las secuencias SEQ ID NO: 1-23. Thus, the kit of the invention may comprise one or more sets of the probes and primers of the invention comprising, preferably consisting of, the sequences SEQ ID NO: 1-23.
En otra realización preferida, las sondas del kit de la invención están marcadas con ai menos un fluoróforo, preferiblemente VIC, NED o FAM, más preferiblemente en cualquiera de sus extremos, 5' o 3', aún más preferiblemente en su extremo 3' . In another preferred embodiment, the probes of the kit of the invention are marked with ai least one fluorophore, preferably VIC, NED or FAM, more preferably at either of its 5' or 3' ends, even more preferably at its 3' end.
A lo largo de los párrafos anteriores se ha puesto de manifiesto la utilidad de las sondas y cebadores de la invención en la detección de las mutaciones en las posiciones 417, 452, 478, 484 y/o 501 de la proteína S del virus SARS-CoV-2. Throughout the preceding paragraphs, the usefulness of the probes and primers of the invention in the detection of mutations at positions 417, 452, 478, 484 and/or 501 of the S protein of the SARS-virus has been revealed. CoV-2.
Por lo tanto, otro aspecto de la invención se refiere al uso del conjunto de sondas y cebadores de la invención, o del kit de la invención, para la detección y/o cuantificación in vitro de variantes de SARS-CoV-2 portadoras de mutaciones (SNP) en las posiciones 417, 452, 478, 484 y/o 501 de la proteína S, donde las mutaciones son seleccionadas del grupo que consiste en: K417T/N, L452R/Q, T478K, E484K/Q y N501Y. Therefore, another aspect of the invention refers to the use of the set of probes and primers of the invention, or the kit of the invention, for the detection and/or quantification in vitro of SARS-CoV-2 variants carrying mutations. (SNP) at positions 417, 452, 478, 484 and/or 501 of the S protein, where the mutations are selected from the group consisting of: K417T/N, L452R/Q, T478K, E484K/Q and N501Y.
El término “detectar” o “detección” ha sido definido o explicado en párrafos anteriores, y dicha definición es aplicable al presente aspecto inventivo. The term "detect" or "detection" has been defined or explained in previous paragraphs, and said definition is applicable to the present inventive aspect.
El término “cuantificar” o “cuantificación” se refiere a determinar, en una muestra, la cantidad de moléculas molde de DNA o RNA (determinar la carga vírica) de las variantes del virus SARS-CoV-2 que presentan mutaciones en las posiciones 417, 452, 478, 484 y/o 501 de la proteína S. The term "quantify" or "quantification" refers to determining, in a sample, the amount of DNA or RNA template molecules (determining the viral load) of the variants of the SARS-CoV-2 virus that present mutations at positions 417 , 452, 478, 484 and/or 501 of the S protein.
El término “in vitro” se refiere a que la detección y/o cuantificación de las variantes del SARS-CoV-2 se realiza fuera del cuerpo del sujeto. El término “sujeto”, tal como se utiliza en la presente invención, hace referencia a cualquier animal, preferiblemente un mamífero. En una realización más preferida, el sujeto es un ser humano de cualquier sexo, edad o raza. The term "in vitro" refers to the fact that the detection and/or quantification of SARS-CoV-2 variants is performed outside the subject's body. The term "subject" as used herein refers to any animal, preferably a mammal. In a more preferred embodiment, the subject is a human of any gender, age, or race.
De forma análoga al uso de las sondas y cebadores de la invención, así como al uso del kit de la invención, descrito en párrafos anteriores, en la presente invención también se contemplan los métodos dirigidos a la detección y/o cuantificación de las variantes del SARS-CoV-2 portadoras de mutaciones en las posiciones 417, 452, 478, 484 y/o 501 de la proteína S. Similarly to the use of the probes and primers of the invention, as well as the use of the kit of the invention, described in previous paragraphs, the present invention also contemplates the methods directed to the detection and/or quantification of the variants of the SARS-CoV-2 carrying mutations at positions 417, 452, 478, 484 and/or 501 of the S protein.
De esta forma, otro aspecto de la invención se refiere a un método in vitro, de ahora en adelante “método de la invención” para la detección y/o cuantificación de variantes de SARS-CoV-2 portadoras de mutaciones en las posiciones 417, 452, 478, 484 y/o 501 de la proteína S, en una muestra aislada de un sujeto que comprende: a. extraer el ARN viral de la muestra, b. someter al ARN extraído en (a) a una reacción de transcripción inversa seguida de una reacción de amplificación utilizando el conjunto de sondas y cebadores de la invención o el kit de la invención, y c. detectar la presencia de dichas mutaciones en la muestra amplificada. Thus, another aspect of the invention refers to an in vitro method, from now on "method of the invention" for the detection and/or quantification of SARS-CoV-2 variants carrying mutations in positions 417, 452, 478, 484 and/or 501 of protein S, in an isolated sample from a subject comprising: a. extracting the viral RNA from the sample, b. subjecting the RNA extracted in (a) to a reverse transcription reaction followed by an amplification reaction using the set of probes and primers of the invention or the kit of the invention, and c. detecting the presence of said mutations in the amplified sample.
Los términos “in vitro”, “sujeto”, “detectar” o “detección”, “cuantificar” o “cuantifícación” han sido definidos o explicados en párrafos anteriores, y dichas definiciones son aplicables al presente aspecto inventivo. The terms "in vitro", "subject", "detect" or "detection", "quantify" or "quantification" have been defined or explained in previous paragraphs, and said definitions are applicable to the present inventive aspect.
La detección y/o cuantifícación de las variantes del SARS-CoV-2 que presentan mutaciones en las posiciones 417, 452, 478 y/o 484 de la proteína S mediante las sondas y cebadores de la invención puede llevarse a cabo en cualquier muestra que sea susceptible de estar contaminada por SARS-CoV-2. Se entiende por “muestra” a una parte o cantidad pequeña de una cosa que se considera representativa del total y que se toma o se separa de ella para someterla a estudio, análisis o experimentación. En la presente invención, dicho estudio, análisis o experimentación hace referencia a la presencia/ausencia del material genético de las variantes de SARS-CoV-2. Dentro del término “muestra” también se incluyen muestras que han sido manipuladas de alguna manera después de su obtención, por ejemplo, mediante el tratamiento con reactivos, la solubilización o el enriquecimiento de ciertos componentes. En una realización preferida del uso de la invención, la muestra es una muestra biológica. The detection and/or quantification of the SARS-CoV-2 variants that present mutations in positions 417, 452, 478 and/or 484 of the S protein by means of the probes and primers of the invention can be carried out in any sample that is likely to be contaminated by SARS-CoV-2. A "sample" is understood as a part or small amount of something that is considered representative of the whole and that is taken or separated from it to submit it to study, analysis or experimentation. In the present invention, said study, analysis or experimentation refers to the presence/absence of the genetic material of the SARS-CoV-2 variants. The term "sample" also includes samples that have been manipulated in some way after they were obtained, for example, by treatment with reagents, solubilization, or enrichment of certain components. In a preferred embodiment of the use of the invention, the sample is a biological sample.
El término “muestra biológica” incluye, pero sin limitarnos, tejidos y/o fluidos biológicos de un individuo, obtenidos mediante cualquier método conocido por un experto en la materia que sirva para tal fin. La muestra biológica a la que se refiere la invención comprende ácido ribonucleico (ARN). En una realización preferida, la muestra biológica procede del tracto oral o respiratorio y ha sido obtenida, más preferiblemente, mediante un hisopo nasofaríngeo. The term "biological sample" includes, but is not limited to, tissues and/or biological fluids from an individual, obtained by any method known to a person skilled in the art that serves such purpose. The biological sample to which the invention relates comprises ribonucleic acid (RNA). In a preferred embodiment, the biological sample comes from the oral or respiratory tract and has been obtained, more preferably, by means of a nasopharyngeal swab.
En otra realización preferida, la reacción de amplificación del método de la invención es una RT-PCR, más preferiblemente una RT-PCR cuantitativa (qRT-PCR). La reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR) es una variante de la PCR en la que se retrotranscribe una cadena de ARN en ADN complementario (ADNc) usando una enzima llamada transcriptasa inversa o transcriptasa reversa, y el resultado se amplifica mediante una PCR tradicional. In another preferred embodiment, the amplification reaction of the method of the invention is an RT-PCR, more preferably a quantitative RT-PCR (qRT-PCR). Reverse transcriptase polymerase chain reaction (RT-PCR) is a variant of PCR in which a strand of RNA is reverse transcribed into complementary DNA (cDNA) using an enzyme called reverse transcriptase or reverse transcriptase, and the result it is amplified by traditional PCR.
La extracción del ARN del paso (a) del método de la invención se puede realizar por los métodos conocidos por el experto en la materia. Por ejemplo, pueden emplearse 5 métodos de extracción tanto manuales, basados en el empleo de solventes orgánicos o columnas de centrifugación, como automatizados, por ejemplo, las plataformas Microlab (Hamilton Company, Nevada, EE. UU.) o los sistemas Magnapure (Roche, Basilea, Suiza). The extraction of the RNA from step (a) of the method of the invention can be carried out by methods known to those skilled in the art. For example, 5 extraction methods can be used, both manual, based on the use of organic solvents or spin columns, or automated, for example, Microlab platforms (Hamilton Company, Nevada, USA) or Magnapure systems (Roche , Basel, Switzerland).
10 En el paso (b) la reacción de transcripción inversa consta, preferiblemente, de dos pasos. El primer paso de la etapa de retrotranscripción consiste en la síntesis de copias del genoma viral en forma de cDNA. Las condiciones empleadas en este primer paso dependen del reactivo utilizado. Este primer paso es llevado a cabo durante preferiblemente 15 minutos a una temperatura de entre 35 °C - 52 °C, másIn step (b) the reverse transcription reaction preferably consists of two steps. The first step of the retrotranscription stage consists of the synthesis of copies of the viral genome in the form of cDNA. The conditions employed in this first step depend on the reagent used. This first step is carried out for preferably 15 minutes at a temperature between 35 °C - 52 °C, plus
15 preferiblemente a 50 °C. Las condiciones del segundo paso de la etapa de retrotranscripción son, preferiblemente, 2 minutos a 95 ° C. 15 preferably at 50 °C. The conditions of the second step of the retrotranscription step are preferably 2 min at 95 °C.
Posteriormente a la retrotranscripción, en el paso (b) del método de la invención se lleva a cabo la amplificación para la detección de la variante de interés usando para cada 20 posición dos cebadores de la invención y dos o tres sondas de la invención (Taqman MGB), preferiblemente marcadas con fluoróforos, más preferiblemente VIC, NED o FAM, que reconocen la secuencia salvaje y las diferentes mutaciones en las posiciones 417, 452, 478, 484 y/o 501 (Tablal). After retrotranscription, in step (b) of the method of the invention, amplification is carried out to detect the variant of interest using two primers of the invention and two or three probes of the invention for each position (Taqman MGB), preferably labeled with fluorophores, more preferably VIC, NED or FAM, which recognize the wild-type sequence and the different mutations at positions 417, 452, 478, 484 and/or 501 (Tablal).
25 Tabla 1. Cebadores y sondas para la detección de las mutaciones K417N/T, L452R/Q,25 Table 1. Primers and probes for the detection of mutations K417N/T, L452R/Q,
T478K, E484K/Q y N501Y.
Figure imgf000016_0001
Figure imgf000017_0001
T478K, E484K/Q and N501Y.
Figure imgf000016_0001
Figure imgf000017_0001
5 Para llevar a cabo la detección de las variantes del virus SARS-CoV-2 se preparan, preferiblemente, 5 reacciones, una para cada posición. En cada una de ellas se mezcla el ARN viral, previamente extraído de la muestra biológica en el paso (a), con el reactivo A. El reactivo A se prepara mezclando el reactivo comercial Taqman fast virus 1-step master mix (Roche) y los cebadores y las sondas de la invención específicas para cada 10 mutación a analizar o el kit de la invención. 5 To carry out the detection of the variants of the SARS-CoV-2 virus, preferably 5 reactions are prepared, one for each position. In each of them, the viral RNA, previously extracted from the biological sample in step (a), is mixed with reagent A. Reagent A is prepared by mixing the commercial reagent Taqman fast virus 1-step master mix (Roche) and the primers and probes of the invention specific for each mutation to be analyzed or the kit of the invention.
Para la detección de las mutaciones K417T/N se emplea un cebador directo que comprende, preferiblemente consiste en, la SEQ ID NO:1 , un cebador inverso que comprende, preferiblemente consiste en, la SEQ ID NO:2, una sonda que comprende, 15 preferiblemente consiste en, la SEQ ID NO:3, una sonda que comprende, preferiblemente consiste en, la SEQ ID NO:4, y una sonda que comprende, preferiblemente consiste en, la SEQ ID NO:5. For the detection of K417T/N mutations, a forward primer is used that comprises, preferably consists of, SEQ ID NO:1, a reverse primer that comprises, preferably consists of, SEQ ID NO:2, a probe that comprises, 15 preferably consists of, SEQ ID NO:3, a probe comprising, preferably consisting of, SEQ ID NO:4, and a probe comprising, preferably consisting of, SEQ ID NO:5.
Para la detección de las mutaciones L452R/Q se emplea un cebador directo que 20 comprende, preferiblemente consiste en, la SEQ ID NO:6, un cebador inverso que comprende, preferiblemente consiste en, la SEQ ID NO:7, una sonda que comprende, preferiblemente consiste en, la SEQ ID NO:8, una sonda que comprende, preferiblemente consiste en, la SEQ ID NO:9, y una sonda que comprende, preferiblemente consiste en, la SEQ ID NO: 10. For the detection of L452R/Q mutations, a forward primer is used that comprises, preferably consists of, SEQ ID NO:6, a reverse primer that comprises, preferably consists of, SEQ ID NO:7, a probe that comprises , preferably consisting of, SEQ ID NO: 8, a probe comprising, preferably consisting of, SEQ ID NO: 9, and a probe comprising, preferably consisting of, SEQ ID NO: 10.
25 25
Para la detección de la mutación T478K se emplea un cebador directo que comprende, preferiblemente consiste en, la SEQ ID NO:11, un cebador inverso que comprende, preferiblemente consiste en, la SEQ ID NO:12, una sonda que comprende, preferiblemente consiste en, la SEQ ID NO:13, y una sonda que comprende, preferiblemente consiste en, la SEQ ID NO:14. For the detection of the T478K mutation, a forward primer is used that comprises, preferably consists of, SEQ ID NO:11, a reverse primer that comprises, preferably consists of, SEQ ID NO:12, a probe that comprises, preferably consists of, SEQ ID NO:13, and a probe comprising, preferably consists of, SEQ ID NO:14.
Para la detección de las mutaciones E484K/Q se emplea un cebador directo que comprende, preferiblemente consiste en, la SEQ ID NO: 15, un cebador inverso que comprende, preferiblemente consiste en, la SEQ ID NO:16, una sonda que comprende, preferiblemente consiste en, la SEQ ID NO: 17, una sonda que comprende, preferiblemente consiste en, la SEQ ID NO:18, y una sonda que comprende, preferiblemente consiste en, la SEQ ID NO: 19. For the detection of the E484K/Q mutations, a forward primer is used that comprises, preferably consists of, SEQ ID NO: 15, a reverse primer that comprises, preferably consists of, SEQ ID NO: 16, a probe that comprises, preferably consists of, SEQ ID NO: 17, a probe comprising, preferably consisting of, SEQ ID NO: 18, and a probe comprising, preferably consisting of, SEQ ID NO: 19.
Para la detección de la mutación N501 Y se emplea un cebador directo que comprende, preferiblemente consiste en, la secuencia SEQ ID NO:20, un cebador inverso que comprende, preferiblemente consiste en, la SEQ ID NO:21 , una sonda que comprende, preferiblemente consiste en, la SEQ ID NO:22 y una sonda que comprende, preferiblemente consiste en, la SEQ ID NO:23. For the detection of the N501 Y mutation, a forward primer is used that comprises, preferably consists of, the sequence SEQ ID NO:20, a reverse primer that comprises, preferably consists of, SEQ ID NO:21, a probe that comprises, preferably consists of, SEQ ID NO:22 and a probe comprising, preferably consists of, SEQ ID NO:23.
La amplificación del paso (b) del método de la invención y el posterior análisis y detección de las mutaciones se realiza preferiblemente en un sistema de PCR a tiempo real tipo 7500 thermal cycler (ABI), Steponeplus thermal cycler (ABI) o QuantStudio 5 (ABI). The amplification of step (b) of the method of the invention and the subsequent analysis and detection of mutations is preferably carried out in a real-time PCR system type 7500 thermal cycler (ABI), Steponeplus thermal cycler (ABI) or QuantStudio 5 ( ABI).
En el paso (c) del método de la invención, la expresión “detectar la presencia de dichas mutaciones en la muestra amplificada” se refiere a detectar si las sondas y cebadores diseñados por los inventores han hibridado, unido o reconocido a los SNPs específicos descritos en la invención. Los diagramas de amplificación (señales de fluorescencia) varían en función del fluoróforo de las sondas. Los tres fluoróforos preferidos con los que se marcan las sondas de la invención pueden ser excitados a una sola longitud de onda (X) de 488 nm, pero emiten a longitudes de onda claramente diferentes. FAM tiene una X máxima de absorción de 494 nm y una A máxima de emisión de 518 nm, NED tiene una X máxima de absorción de 546 nm y una X máxima de emisión de 575 nm y VIC tiene una X máxima de absorción de 538 nm y una A máxima de emisión de 554 nm. In step (c) of the method of the invention, the expression "detecting the presence of said mutations in the amplified sample" refers to detecting whether the probes and primers designed by the inventors have hybridized, bound or recognized the specific SNPs described. in the invention. The amplification patterns (fluorescence signals) vary depending on the fluorophore of the probes. The three preferred fluorophores with which the probes of the invention are labeled can be excited at a single wavelength (X) of 488 nm, but emit at clearly different wavelengths. FAM has an absorption Xmax of 494 nm and an emission Amax of 518 nm, NED has an absorption Xmax of 546 nm and an emission Xmax of 575 nm and VIC has an absorption Xmax of 538 nm and an A maximum emission of 554 nm.
DESCRIPCIÓN DE LAS FIGURAS DESCRIPTION OF THE FIGURES
Fíg. 1 muestra la posición de los cebadores de la invención para la detección de las mutaciones K417T/N, L452R/Q, T478K, E484K/Q y N501Y de la proteína S del virus SARS-CoV-2. Fig. 1 shows the position of the primers of the invention for the detection of the K417T/N, L452R/Q, T478K, E484K/Q and N501Y mutations of the S protein of the virus. SARS-CoV-2.
Fig. 2 muestra el análisis mediante la técnica desarrollada de muestras positivas para la detección de la mutación T478K de la proteína S del virus SARS-CoV-2 (variante Delta). Fig. 2 shows the analysis using the developed technique of positive samples for the detection of the T478K mutation of the S protein of the SARS-CoV-2 virus (Delta variant).
EJEMPLOS EXAMPLES
A continuación, se ilustrará la invención mediante unos ensayos realizados por los inventores, que ponen de manifiesto la efectividad de las sondas y cebadores de la invención, así como del método de la invención. Next, the invention will be illustrated by means of tests carried out by the inventors, which show the effectiveness of the probes and primers of the invention, as well as the method of the invention.
Eiemplo 1: Preparación del reactivo A Example 1: Preparation of reagent A
Como se ha comentado en la descripción de la invención, se preparan 5 reacciones, una para cada posición: As mentioned in the description of the invention, 5 reactions are prepared, one for each position:
(1) Detección de las mutaciones K417T/N. Para preparar 1 mililitro de volumen del reactivo A se añaden 300 μl de Taqman fast virus 1-step master mix (Roche), 4 μl del cebador directo (100 μM ) que comprende, preferiblemente consiste en, la SEQ ID NO:1 , 4 μl del cebador inverso (100 μM ) que comprende, preferiblemente consiste en, la SEQ ID NO:2, 1 μl de la sonda (100 μM ) que comprende, preferiblemente consiste en, la SEQ ID NO:3, 1 μl de la sonda (100 μM ) que comprende, preferiblemente consiste en, la SEQ ID NO:4 y 1 μl de la sonda (100 μM ) que comprende, preferiblemente consiste en, la SEQ ID NO:5. Se añade agua hasta completar el mililitro de volumen. (1) Detection of K417T/N mutations. To prepare 1 milliliter volume of reagent A, add 300 μl of Taqman fast virus 1-step master mix (Roche), 4 μl of the forward primer (100 μM) comprising, preferably consisting of, SEQ ID NO:1, 4 µl of the reverse primer (100 µM ) comprising, preferably consisting of, SEQ ID NO:2, 1 µl of the probe (100 µM ) comprising, preferably consisting of, SEQ ID NO:3, 1 µl of the probe (100 µM) comprising, preferably consisting of, SEQ ID NO:4 and 1 µl of the probe (100 µM) comprising, preferably consisting of, SEQ ID NO:5. Water is added to complete the milliliter of volume.
(2) Detección de las mutaciones L452R/Q. Para preparar 1 mililitro de volumen del reactivo A se añaden 300 μl de Taqman fast virus 1-step master mix (Roche), 4 μl del cebador directo (100 μM ) que comprende, preferiblemente consiste en, la SEQ ID NO:6, 4 μl del cebador inverso (100 μM) que comprende, preferiblemente consiste en, la SEQ ID NO:7, 1 μl de la sonda (100 μM ) que comprende, preferiblemente consiste en, la SEQ ID NO:8, 1 μl de la sonda (100 μM ) que comprende, preferiblemente consiste en, la SEQ ID NO:9 y 1 μl de la sonda (100 μM ) que comprende, preferiblemente consiste en, la SEQ ID NO: 10. Se añade agua hasta completar el mililitro de volumen. (2) Detection of L452R/Q mutations. To prepare 1 milliliter volume of reagent A, add 300 μl of Taqman fast virus 1-step master mix (Roche), 4 μl of the forward primer (100 μM) comprising, preferably consisting of, SEQ ID NO: 6, 4 µl of the reverse primer (100 µM) comprising, preferably consisting of, SEQ ID NO:7, 1 µl of the probe (100 µM) comprising, preferably consisting of, SEQ ID NO:8, 1 µl of the probe (100 μM ) comprising, preferably consisting of, SEQ ID NO: 9 and 1 μl of the probe (100 μM ) comprising, preferably consisting of, SEQ ID NO: 10. Add water to complete the milliliter of volume .
(3) Detección de la mutación T478K. Para preparar 1 mililitro de volumen del reactivo A se añaden 300 pl de Taqman fast virus 1-step master mix (Roche), 4 pl del cebador directo (100 μM) que comprende, preferiblemente consiste en, la SEQ ID NO:11 , 4 pl del cebador inverso (100 μM) que comprende, preferiblemente consiste en, la SEQ ID NO: 12, 1 pl de la sonda (100 μM) que comprende, preferiblemente consiste en, la SEQ ID NO: 13 y 1 pl de la sonda (100 μM) que comprende, preferiblemente consiste en, la SEQ ID NO:14. Se añade agua hasta completar el mililitro de volumen. (3) Detection of the T478K mutation. To prepare 1 milliliter volume of reagent A 300 pl of Taqman fast virus 1-step master mix (Roche) are added, 4 pl of the forward primer (100 μM) comprising, preferably consisting of, SEQ ID NO:11, 4 pl of the reverse primer (100 μM) containing comprises, preferably consists of, SEQ ID NO: 12, 1 pl of the probe (100 µM) comprising, preferably consists of, SEQ ID NO: 13 and 1 pl of the probe (100 µM) comprising, preferably consists of in, SEQ ID NO:14. Water is added to complete the milliliter of volume.
(4) Detección de las mutaciones E484K/Q. Para preparar 1 mililitro del reactivo A se añaden 300 pl de Taqman fast virus 1-step master mix (Roche), 4 pl del cebador directo (100 μM) que comprende, preferiblemente consiste en, la SEQ ID NO: 15, 4 pl del cebador inverso (100 μM) que comprende, preferiblemente consiste en, la SEQ ID NO: 16, 1 pl de la sonda (100 μM) que comprende, preferiblemente consiste en, la SEQ ID NO: 17, 1 pl de la sonda (100 μM) que comprende, preferiblemente consiste en, la SEQ ID NO: 18 y 1 pl de la sonda (100 μM) que comprende, preferiblemente consiste en, la SEQ ID NO:19. Se añade agua hasta completar el mililitro de volumen. (4) Detection of E484K/Q mutations. To prepare 1 ml of reagent A, 300 µl of Taqman fast virus 1-step master mix (Roche), 4 µl of the forward primer (100 µM) comprising, preferably consisting of, SEQ ID NO: 15, 4 µl of the reverse primer (100 µM) comprising, preferably consisting of, SEQ ID NO: 16, 1 pl of probe (100 µM) comprising, preferably consisting of, SEQ ID NO: 17, 1 pl of probe (100 µM) comprising, preferably consisting of, SEQ ID NO:18 and 1 µL of the probe (100 µM) comprising, preferably consisting of, SEQ ID NO:19. Water is added to complete the milliliter of volume.
(5) Detección de la mutación N501Y. Para preparar 1 mililitro del reactivo A se añaden 300 pl de Taqman fast virus 1-step master mix (Roche), 4 pl del cebador directo (100 μM) que comprende, preferiblemente consiste en, la SEQ ID NO:20, 4 pl del cebador inverso (100 μM) que comprende, preferiblemente consiste en, la SEQ ID NO:21 , 1 pl de la sonda (100 μM) que comprende, preferiblemente consiste en, la SEQ ID NO:22 y 1 pl de la sonda (100 μM) que comprende, preferiblemente consiste en, la SEQ ID NO:23. Se añade agua hasta completar el mililitro de volumen. (5) Detection of the N501Y mutation. To prepare 1 ml of reagent A, 300 µl of Taqman fast virus 1-step master mix (Roche), 4 µl of the forward primer (100 µM) comprising, preferably consisting of, SEQ ID NO:20, 4 µl of the reagent A are added. reverse primer (100 µM) comprising, preferably consisting of, SEQ ID NO:21, 1 pl of the probe (100 µM) comprising, preferably consisting of, SEQ ID NO:22 and 1 pl of the probe (100 µM) comprising, preferably consisting of, SEQ ID NO:23. Water is added to complete the milliliter of volume.
Ejemplo 2: Preparación de la mezcla de amplificación Example 2: Preparation of the amplification mix
Se preparan 5 reacciones en las que se mezclan 5 pl de ARN viral previamente extraído y 10 pl del reactivo A previamente preparado para cada una de las reacciones según se describe en el ejemplo 1. 5 reactions are prepared in which 5 pl of previously extracted viral RNA and 10 pl of reagent A previously prepared for each of the reactions are mixed as described in Example 1.
Ejemplo 3: Amplificación Example 3: Amplification
El sistema de amplificación empleado es una POR a tiempo real tipo 7500 thermal cycler (ABI), Steponeplus thermal cycler (ABI) o QuantStudio 5 (ABI). The amplification system used is a real-time ORP type 7500 thermal cycler (ABI), Steponeplus thermal cycler (ABI) or QuantStudio 5 (ABI).
La etapa de retrotranscripción tiene 2 pasos. El primer paso de la etapa de retrotranscripción (15 minutos a 50 ° C) consiste en la síntesis de cDNA a partir del ARN viral. Las condiciones del segundo paso de la etapa de retrotrancripción son 2 minutos a 95 ° C (Tabla 2). The reverse transcription step has 2 steps. The first step of the stage of reverse transcription (15 min at 50 °C) involves cDNA synthesis from viral RNA. The conditions of the second step of the reverse transcription step are 2 min at 95 °C (Table 2).
La etapa de PCR de 40 ciclos consta de 2 pasos, un paso de desnaturalización de 5 segundos a 95 ° C y un paso combinado de hibridación y extensión de 30 segundos a 60 ° C. Durante estos ciclos el producto de PCR es amplificado (Tabla 2). The 40-cycle PCR step consists of 2 steps, a 5-second denaturation step at 95 °C and a combined 30-second annealing and extension step at 60 °C. During these cycles the PCR product is amplified (Table 2).
Tabla 2. Programa de amplificación
Figure imgf000021_0001
Table 2. Amplification program
Figure imgf000021_0001
4: Obtención e interpretación de los resultados 4: Obtaining and interpreting the results
La lectura de los resultados llega por la interpretación (por el sistema) de la intensidad de la señal fluorescente en el momento que se identifique la presencia de la secuencia genética correspondiente al virus. Un ejemplo de la detección de la mutación T478K en la proteína S del virus se muestra en la Fig.2. Se observan variantes portadoras del alelo salvaje T478 (gris claro) y mutante T478K (gris oscuro), que permite identificar la variante Delta. Este análisis se realizó en un sistema StepOnePlus (Thermofisher). The reading of the results comes from the interpretation (by the system) of the intensity of the fluorescent signal at the moment the presence of the genetic sequence corresponding to the virus is identified. An example of the detection of the T478K mutation in the S protein of the virus is shown in Fig.2. Variants carrying the wild-type allele T478 (light grey) and mutant T478K (dark grey) are observed, which allows identification of the Delta variant. This analysis was performed on a StepOnePlus system (Thermofisher).
La variante Alfa (B.1.1.7) se caracteriza porque presenta las mutaciones E484K, N501Y, la variante Beta (B.1.351) se caracteriza porque presenta las mutaciones K417N, E484K y N501Y, la variante Gamma (P.1) se caracteriza porque presenta las mutaciones K417N, E484K y N501Y, la variante Kappa (B.1.617.1) se caracteriza porque presenta las mutaciones L452R, T478K y E484Q, la variante Delta (B.1.617.2) se caracteriza porque presenta las mutaciones L452R y T478K y la variante Lambda (C.37) se caracteriza porque presenta la mutación L452Q. The Alpha variant (B.1.1.7) is characterized because it presents the E484K, N501Y mutations, the Beta variant (B.1.351) is characterized because it presents the K417N, E484K and N501Y mutations, the Gamma variant (P.1) is characterized because it presents the K417N, E484K and N501Y mutations, the Kappa variant (B.1.617.1) is characterized by presenting the L452R, T478K and E484Q mutations, the Delta variant (B.1.617.2) is characterized by presenting the L452R and T478K and the Lambda variant (C.37) is characterized by having the L452Q mutation.
Tal y como se ve en los resultados de detección de la Tabla 3, el método de la invención permite la detección de las variantes Alfa (B.1.1.7), Beta (B.1.351), Gamma (P.1.), Kappa (B.1.617.1), Delta (B.1.617.2) y Lambda (C.3/) portadoras de mutaciones en los codones 417, 452, 478, 484 y 501 en apenas una hora. As seen in the detection results in Table 3, the method of the invention allows the detection of Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1.), Kappa (B.1.617.1), Delta (B.1.617.2) and Lambda (C.3/) carriers of mutations in codons 417, 452, 478, 484 and 501 in just one hour.
Tabla 3. Detección de las variantes del virus SAR-CoV-2 mediante
Figure imgf000022_0001
sondas y cebadores de la invención.
Figure imgf000022_0002
Table 3. Detection of variants of the SAR-CoV-2 virus using
Figure imgf000022_0001
probes and primers of the invention.
Figure imgf000022_0002
Ejemplo 5: Comparación de los resultados obtenidos tras et análisis de muestras mediante el método de la invención descrito en los ejemplos anteriores y el método de secuenciación de genoma completo Example 5: Comparison of the results obtained after the analysis of samples using the method of the invention described in the previous examples and the whole genome sequencing method
En este ensayo se llevó a cabo un método de secuenciación de genoma completo usando la técnica NGS para comparar sus resultados con los previamente obtenidos por el sistema aquí propuesto, sobre 487 muestras. Como se aprecia en la Tabla 4, se observó una alta concordancia, del 99,4% (483/487), entre ambas técnicas. In this trial, a whole genome sequencing method was carried out using the NGS technique to compare its results with those previously obtained by the system proposed here, on 487 samples. As can be seen in Table 4, a high concordance of 99.4% (483/487) was observed between both techniques.
Tabla 4. Resultados del análisis de las muestras tras la realización de la técnica NGS.
Figure imgf000023_0001
Table 4. Results of the analysis of the samples after performing the NGS technique.
Figure imgf000023_0001
En negrita se remarcan las muestras discordantes, que fueron:
Figure imgf000023_0002
The discordant samples are highlighted in bold, which were:
Figure imgf000023_0002

Claims

REIVINDICACIONES
1. Kit para la detección de mutaciones en las posiciones 417, 452, 478 y/o 484 de la proteína S del virus SARS-CoV-2 que comprende todos los conjuntos de sondas y cebadores específicos de la lista que consiste en: a) Cebador directo que comprende, preferiblemente consiste en, la SEQ ID NO:1 , cebador inverso que comprende, preferiblemente consiste en, la SEQ ID NO:2, sonda que comprende, preferiblemente consiste en, la SEQ ID NO:3, sonda que comprende, preferiblemente consiste en, la SEQ ID NO:4, y sonda que comprende, preferiblemente consiste en, la SEQ ID NO:5; b) Cebador directo que comprende, preferiblemente consiste en, la SEQ ID NO:6, cebador inverso que comprende, preferiblemente consiste en, la SEQ ID NO:7, sonda que comprende, preferiblemente consiste en, la SEQ ID NO:8, sonda que comprende, preferiblemente consiste en, la SEQ ID NO:9, y sonda que comprende, preferiblemente consiste en, la SEQ ID NO:10; c) Cebador directo que comprende, preferiblemente consiste en, la SEQ ID NO:11 , cebador inverso que comprende, preferiblemente consiste en, la SEQ ID NO:12, sonda que comprende, preferiblemente consiste en, la SEQ ID NO: 13, y sonda que comprende, preferiblemente consiste en, la SEQ ID NO: 14; y d) Cebador directo que comprende, preferiblemente consiste en, la SEQ ID NO: 15, cebador inverso que comprende, preferiblemente consiste en, la SEQ ID NO:16, sonda que comprende, preferiblemente consiste en, la SEQ ID NO: 17, sonda que comprende, preferiblemente consiste en, la SEQ ID NO: 18, y sonda que comprende, preferiblemente consiste en, la SEQ ID NO:19. 1. Kit for the detection of mutations at positions 417, 452, 478 and/or 484 of the S protein of the SARS-CoV-2 virus comprising all the sets of specific probes and primers from the list consisting of: a) Forward primer comprising, preferably consisting of, SEQ ID NO:1, reverse primer comprising, preferably consisting of, SEQ ID NO:2, probe comprising, preferably consisting of, SEQ ID NO:3, probe comprising , preferably consisting of, SEQ ID NO:4, and probe comprising, preferably consisting of, SEQ ID NO:5; b) Forward primer comprising, preferably consisting of, SEQ ID NO:6, reverse primer comprising, preferably consisting of, SEQ ID NO:7, probe comprising, preferably consisting of, SEQ ID NO:8, probe comprising, preferably consisting of, SEQ ID NO:9, and probe comprising, preferably consisting of, SEQ ID NO:10; c) Forward primer comprising, preferably consisting of, SEQ ID NO:11, reverse primer comprising, preferably consisting of, SEQ ID NO:12, probe comprising, preferably consisting of, SEQ ID NO: 13, and probe comprising, preferably consisting of, SEQ ID NO: 14; and d) Forward primer comprising, preferably consisting of, SEQ ID NO: 15, reverse primer comprising, preferably consisting of, SEQ ID NO: 16, probe comprising, preferably consisting of, SEQ ID NO: 17, probe comprising, preferably consisting of, SEQ ID NO:18, and probe comprising, preferably consisting of, SEQ ID NO:19.
2. Kit según la reivindicación 1 donde las secuencias de (a) detectan las mutaciones K417T y K417N, las secuencias de (b) detectan las mutaciones L452R y L452Q, las secuencias de (c) detectan la mutación T478K y las secuencias de (d) detectan las mutaciones E484K y E484Q. 2. Kit according to claim 1 where the sequences of (a) detect the K417T and K417N mutations, the sequences of (b) detect the L452R and L452Q mutations, the sequences of (c) detect the T478K mutation and the sequences of (d ) detect the E484K and E484Q mutations.
3. Kit según cualquiera de las reivindicaciones 1 o 2, donde las sondas están marcadas con al menos un fluoróforo. Kit according to any of claims 1 or 2, wherein the probes are labeled with at least one fluorophore.
4. Kit según la reivindicación 3, donde el fluoróforo es VIC, NED o FAM. 4. Kit according to claim 3, wherein the fluorophore is VIC, NED or FAM.
5. Kit según cualquiera de las reivindicaciones 1 a 4, que además comprende sondas y cebadores específicos para la detección de mutaciones en la posición 501 de la proteína S del virus SARS-CoV-2. 5. Kit according to any of claims 1 to 4, which also comprises probes and specific primers for the detection of mutations at position 501 of the S protein of the SARS-CoV-2 virus.
6. Kit según la reivindicación 5 donde la mutación en la posición 501 es N501Y. 6. Kit according to claim 5, wherein the mutation at position 501 is N501Y.
7. Kit según cualquiera de las reivindicaciones 5 o 6 donde las sondas y cebadores que detectan la mutación en la posición 501 de la proteína S son un cebador directo que comprende, preferiblemente consiste en, la secuencia SEQ ID NO:20, un cebador inverso que comprende, preferiblemente consiste en, la SEQ ID NO:21 , una sonda que comprende, preferiblemente consiste en, la SEQ ID NO:22 y una sonda que comprende, preferiblemente consiste en, la SEQ ID NO:23. 7. Kit according to any of claims 5 or 6 where the probes and primers that detect the mutation at position 501 of protein S are a forward primer comprising, preferably consisting of, the sequence SEQ ID NO:20, a reverse primer comprising, preferably consisting of, SEQ ID NO:21, a probe comprising, preferably consisting of, SEQ ID NO:22 and a probe comprising, preferably consisting of, SEQ ID NO:23.
8. Kit según cualquiera de las reivindicaciones 5 a 7 donde las sondas para la detección de mutaciones en la posición 501 de la proteína S del virus SARS-CoV-2 están marcadas con al menos un fluoróforo. 8. Kit according to any of claims 5 to 7, where the probes for the detection of mutations at position 501 of the S protein of the SARS-CoV-2 virus are labeled with at least one fluorophore.
9. Kit según la reivindicación 8, donde el fluoróforo es VIC, NED o FAM. 9. Kit according to claim 8, wherein the fluorophore is VIC, NED or FAM.
10. Uso del kit según cualquiera de las reivindicaciones 1 a 9 para la detección y/o cuantificación in vitro de variantes del virus SARS-CoV-2 portadoras de mutaciones (SNP) en las posiciones 417, 452, 478, 484 y/o 501 de la proteína S, donde las mutaciones son seleccionadas del grupo que consiste en: K417T, K417N, L452R, L452Q, T478K, E484K, E484Q y N501Y. 10. Use of the kit according to any of claims 1 to 9 for the detection and/or quantification in vitro of variants of the SARS-CoV-2 virus carrying mutations (SNP) at positions 417, 452, 478, 484 and/or 501 protein S, where the mutations are selected from the group consisting of: K417T, K417N, L452R, L452Q, T478K, E484K, E484Q and N501Y.
11. Método in vitro para la detección y/o cuantificación de variantes del virus SARS-CoV- 2 portadoras de mutaciones en las posiciones 417, 452, 478, 484 y/o 501 de la proteína S en una muestra aislada de un sujeto, que comprende: a. extraer el ARN viral de la muestra, b. someter al ARN extraído en (a) a una reacción de transcripción inversa seguida de una reacción de amplificación utilizando el kit según cualquiera de las reivindicaciones 1 a 9, y c. detectar la presencia de dichas mutaciones en la muestra amplificada. 11. In vitro method for the detection and/or quantification of variants of the SARS-CoV-2 virus carrying mutations at positions 417, 452, 478, 484 and/or 501 of the S protein in an isolated sample from a subject, which includes: a. extracting the viral RNA from the sample, b. subjecting the RNA extracted in (a) to a reverse transcription reaction followed by an amplification reaction using the kit according to any of claims 1 to 9, and c. detecting the presence of said mutations in the amplified sample.
12. Método según la reivindicación 11 , donde la reacción de amplificación es una RT- PCR cuantitativa (qRT-PCR). 12. Method according to claim 11, wherein the amplification reaction is a quantitative RT-PCR (qRT-PCR).
13. Método según cualquiera de las reivindicaciones 11 o 12, donde la muestra aislada de un sujeto es una muestra biológica procedente de un mamífero, preferiblemente humano. A method according to any of claims 11 or 12, wherein the sample isolated from a subject is a biological sample from a mammal, preferably a human.
14. Método según cualquiera de las reivindicaciones 11 a 13, donde la muestra biológica aislada procede del tracto oral o respiratorio. 14. Method according to any of claims 11 to 13, wherein the isolated biological sample comes from the oral or respiratory tract.
PCT/ES2023/070010 2022-01-13 2023-01-11 SYSTEM FOR DETECTING SARS-COV-2 VARIANTS USING RT-gPCR WO2023135350A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ESP202230021 2022-01-13
ES202230021A ES2946232A1 (en) 2022-01-13 2022-01-13 SYSTEM FOR THE DETECTION OF SARS-CoV-2 VARIANTS BY RT-qPCR (Machine-translation by Google Translate, not legally binding)

Publications (1)

Publication Number Publication Date
WO2023135350A1 true WO2023135350A1 (en) 2023-07-20

Family

ID=85278387

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/ES2023/070010 WO2023135350A1 (en) 2022-01-13 2023-01-11 SYSTEM FOR DETECTING SARS-COV-2 VARIANTS USING RT-gPCR

Country Status (2)

Country Link
ES (1) ES2946232A1 (en)
WO (1) WO2023135350A1 (en)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111961762A (en) 2020-09-03 2020-11-20 深圳市赛格诺生物科技有限公司 Fluorescent RT-PCR primer, probe, reagent and method for detecting coronavirus SARS-CoV-2
CN112063764A (en) 2020-10-28 2020-12-11 江苏科德生物医药科技有限公司 Multiplex real-time fluorescent RT-PCR primer probe composition and kit for novel coronavirus nucleic acid detection
CN112575120A (en) 2020-11-03 2021-03-30 厦门大学 SARS-CoV-2D 614G mutation detection kit and detection method
CN113249522A (en) 2021-06-11 2021-08-13 中国科学院微生物研究所 Method for detecting SARS-CoV-2variant strain nucleic acid and its application
WO2021176099A1 (en) 2020-03-05 2021-09-10 Institut Pasteur Methods and reagents for the specific and sensitive detection of sars-cov-2
CN113881812A (en) * 2021-12-03 2022-01-04 深圳联合医学科技有限公司 Composition, kit and method for detecting SARS-CoV-2 mutant strain and use thereof
CN113897460A (en) * 2021-11-02 2022-01-07 深圳市梓健生物科技有限公司 Nucleic acid composition, kit and method for simultaneously detecting multiple mutant strains of novel coronavirus

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021176099A1 (en) 2020-03-05 2021-09-10 Institut Pasteur Methods and reagents for the specific and sensitive detection of sars-cov-2
CN111961762A (en) 2020-09-03 2020-11-20 深圳市赛格诺生物科技有限公司 Fluorescent RT-PCR primer, probe, reagent and method for detecting coronavirus SARS-CoV-2
CN112063764A (en) 2020-10-28 2020-12-11 江苏科德生物医药科技有限公司 Multiplex real-time fluorescent RT-PCR primer probe composition and kit for novel coronavirus nucleic acid detection
CN112575120A (en) 2020-11-03 2021-03-30 厦门大学 SARS-CoV-2D 614G mutation detection kit and detection method
CN113249522A (en) 2021-06-11 2021-08-13 中国科学院微生物研究所 Method for detecting SARS-CoV-2variant strain nucleic acid and its application
CN113897460A (en) * 2021-11-02 2022-01-07 深圳市梓健生物科技有限公司 Nucleic acid composition, kit and method for simultaneously detecting multiple mutant strains of novel coronavirus
CN113881812A (en) * 2021-12-03 2022-01-04 深圳联合医学科技有限公司 Composition, kit and method for detecting SARS-CoV-2 mutant strain and use thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DURNER ET AL., DENT. MATER, vol. 37, 2021, pages e95 - e97
LIANG YUANHAO ET AL: "CRISPR-Cas12a-Based Detection for the Major SARS-CoV-2 Variants of Concern", MICROBIOLOGY SPECTRUM, vol. 9, no. 3, 22 December 2021 (2021-12-22), pages 1 - 19, XP093030343, DOI: 10.1128/Spectrum.01017-21 *
SANDOVA/ TORRIENTES ET AL., J VIRO! METHODS, vol. 294, August 2021 (2021-08-01), pages 114143
SERAFEIM C ET AL., J VIRO! METT?ODS, vol. 296, October 2021 (2021-10-01), pages 114242

Also Published As

Publication number Publication date
ES2946232A1 (en) 2023-07-13

Similar Documents

Publication Publication Date Title
Watzinger et al. Detection and monitoring of virus infections by real-time PCR
ES2970603T3 (en) Methods for the specific and sensitive detection of SARS-CoV-2
Decaro et al. Genotype-specific fluorogenic RT-PCR assays for the detection and quantitation of canine coronavirus type I and type II RNA in faecal samples of dogs
Belák Molecular diagnosis of viral diseases, present trends and future aspects: A view from the OIE Collaborating Centre for the Application of Polymerase Chain Reaction Methods for Diagnosis of Viral Diseases in Veterinary Medicine
ES2439951T3 (en) Multiplex detection and quantification of internally controlled microbial nucleic acids
ES2445495T3 (en) Nucleic acid primers and probes to detect HIV-1 and HIV-2
CN111500771B (en) Primer group and kit for detecting novel coronavirus SARS-CoV-2
JP2014511177A (en) Simultaneous diagnosis kit for diseases caused by respiratory viruses
Vernet Molecular diagnostics in virology
Teng et al. Specific detection of reverse transcription-loop-mediated isothermal amplification amplicons for Taura syndrome virus by colorimetric dot–blot hybridization
CA2539703A1 (en) Detection of human papilloma virus (hpv) utilizing invasive cleavage structure assays
CN113249522B (en) Method for detecting SARS-CoV-2variant strain nucleic acid and its application
Park et al. Loop-mediated isothermal amplification for the rapid detection of Chrysanthemum chlorotic mottle viroid (CChMVd)
EA008388B1 (en) Amplification-hybridisation method for detecting and typing human papillomavirus
Lin et al. The development and application of a duplex reverse transcription loop-mediated isothermal amplification assay combined with a lateral flow dipstick method for Macrobrachium rosenbergii nodavirus and extra small virus isolated in China
Wang et al. Development of a gold nanoparticle-based oligonucleotide microarray for simultaneous detection of seven swine viruses
ES2336807T3 (en) DETECTION OF THE TYPE AND SUBTIPE OF HIV.
ES2425620T3 (en) Method for detecting respiratory viral agents in a test sample
US20040265796A1 (en) Methods and kits for detecting SARS-associated coronavirus
EP3601608A1 (en) Quantification of ngs dna by adapter sequence
ES2622908T3 (en) Neisseria gonorrhoeae trial
WO2023135350A1 (en) SYSTEM FOR DETECTING SARS-COV-2 VARIANTS USING RT-gPCR
CN113801966B (en) Fluorescent quantitative PCR method and kit for detecting novel coronavirus subgenomic
JP6592697B2 (en) Serotype 2 dengue virus detection kit, serotype 3 dengue virus detection kit, and dengue virus detection kit
EP3885455A1 (en) Method and kit for the detection of sars-cov-2 virus in a sample based on reverse transcription loop-mediated isothermal amplification (rt-lamp)

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23705618

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
NENP Non-entry into the national phase

Ref country code: DE