JP2023038883A - Method for detecting SARS-CoV-2 - Google Patents

Abstract

To provide a method for detecting SARS-CoV-2 using on-skin lipids.SOLUTION: A method for detecting SARS-CoV-2 in a subject includes the step of detecting SARS-CoV-2 for on-skin lipids taken from the subject.SELECTED DRAWING: None

Description

The present invention relates to a method for detecting SARS-CoV-2 using skin surface lipids.

SARS coronavirus-2 (Severe acute respiratory syndrome coronavirus 2; SARS-CoV-2) belongs to the betacoronavirus genus like SARS coronavirus (SARS-CoV) and MERS coronavirus (MERS-CoV), and causes acute respiratory illness. It is the SARS-related coronavirus that causes the disease (COVID-19). The outbreak was first confirmed in 2019 near Wuhan City, Hubei Province, China, and has since caused the COVID-19 pandemic.

SARS-CoV-2 is a single-stranded positive-strand RNA virus that is incapable of autonomous replication, and is known to adhere to human mucosal cells and the like, enter them, and proliferate. The multiplied virus is released with the droplets of the infected person, and other people are infected by inhaling the virus through their mouths and noses (droplet infection).
In addition, it is thought that the infection spreads through contact infection in which the virus enters through the mucous membranes when another person touches an object with the virus attached to it through the infected person's hand and touches the mouth or nose with that hand. there is

Infected individuals have been found to exhibit symptoms such as abnormal taste and smell, headache, fever, cough and difficulty breathing. Some infected people are asymptomatic, and this is one of the factors that make it difficult to prevent the spread of SARS-CoV-2. In particular, in order to prevent infection among people with underlying diseases and the elderly, there is a need to increase the number of tests, including PCR tests, and to expand the testing system.

Currently, methods for examining SARS-CoV-2 infection include genetic testing including PCR testing, antigen testing, and antibody testing. The PCR test, which is the most widely used genetic test, uses the polymerase chain reaction to amplify the virus-derived gene sequence by 100 to 10 million times or more. Extremely sensitive analysis. method. Positive or negative can be determined by the presence or absence of an amplification curve obtained by PCR reaction.

At present, it has been confirmed that nasopharyngeal swabs and saliva can be used as samples for genetic tests such as PCR tests for SARS-CoV-2. When collecting specimens from the patient's nasopharynx with a swab, there is a risk of droplet infection to healthcare workers due to sneezing, etc., so saliva, which has a low risk of droplet infection, is limited to within 9 days after the onset of symptoms. is covered by insurance.

On the other hand, techniques have been developed for investigating the physiological state of humans in vivo by analyzing nucleic acids such as DNA and RNA in biological samples. Nucleic acids derived from living organisms can be extracted from body fluids such as blood, secretions, tissues, etc. Recently, RNA contained in skin surface lipids (SSL) has been used as a sample for biological analysis. It has been reported that marker genes of epidermis, sweat glands, hair follicles and sebaceous glands can be detected from SSL (Patent Document 1).
To date, SARS-CoV-2 has been reported to be detected in the alveoli, bronchi, sputum, nasal cavity, pharynx, and feces of infected individuals, and is rarely detected in blood or urine, but detected from the skin surface. There are no reports of anything that can be done.

International Publication No. 2018/008319

The present invention relates to providing a method of detecting SARS-CoV-2 using epidermal lipids.

The present inventors have found that SARS-CoV-2 can be detected from SSL collected from COVID-19 patients with confirmed SARS-CoV-2 infection, and that SSL can be a specimen for detecting SARS-CoV-2. Found it.

That is, the present invention relates to the following 1) to 8).
1) A method for detecting SARS-CoV-2 in a subject, comprising the step of detecting SARS-CoV-2 in surface lipids collected from the subject.
2) A method for detecting SARS-CoV-2 infection in a subject, comprising the step of detecting SARS-CoV-2 in skin surface lipids collected from the subject.
3) IFI6, ISG15, IFITM1, IFITM3, IRF7, MX1, IFIT1, HLA for superficial skin lipids collected from subjects infected with SARS-CoV-2 or suspected of having SARS-CoV-2 infection - confirmation of SARS-CoV-2 infection in the subject, comprising measuring the expression level of one or more molecules selected from IFN response-related genes or expression products thereof selected from B and EGR1, B2M protein and CRP protein detection method.
4) IFN response-related genes selected from ISG15, IFITM1 and IFITM3, or their expression for surface lipids on the skin collected from subjects infected with SARS-CoV-2 or subjects suspected of being infected with SARS-CoV-2 A method of detecting severity of SARS-CoV-2 infection in a subject comprising measuring expression levels of the product.
5) A SARS-CoV-2 detection kit used in the method of 1), containing tools and reagents necessary for collecting and preserving SSL, and reagents for detecting SARS-CoV-2 from SSL.
6) containing tools and reagents necessary for collection and storage of SSL, reagents for detecting SARS-CoV-2 from SSL, and optionally further reagents for measuring expression levels of said molecules from SSL; ) SARS-CoV-2 infection detection kit used in the method.
7) A SARS-CoV-2 infectious disease confirmation and detection kit used in the method of 3), containing tools and reagents necessary for collection and storage of SSL, and reagents for measuring the expression level of said molecule from SSL.
8) Detection of the severity of SARS-CoV-2 infection used in the method of 4), which contains tools and reagents necessary for collection and storage of SSL, and reagents for measuring the expression level of said molecule from SSL. kit.

According to the present invention, SARS-CoV-2 in a subject can be detected by collecting lipids on the skin surface, and biomolecules that change due to SARS-CoV-2 infection can be detected. , allowing for the detection of SARS-CoV-2 infection with high accuracy.

Protein expression levels of HLA-B, B2M and CRP in face-derived SSL of healthy subjects (HL) and COVID-19 patients (COVID-19). Correlation between CRP amount in SSL and blood CRP concentration. Expression levels of IFN response-related genes by severity of COVID-19 patients.

All patents, non-patents, and other publications cited herein are hereby incorporated by reference in their entirety.

As used herein, the term "nucleic acid" or "polynucleotide" means DNA or RNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and "RNA" includes total RNA, mRNA, rRNA, tRNA, non-coding RNA, and synthetic RNA.

In the present invention, the term "gene" includes double-stranded DNA containing human genomic DNA, single-stranded DNA (positive strand) containing cDNA, and single-stranded DNA (complementary strand) having a sequence complementary to the positive strand. strands), and fragments thereof, which contain some biological information in the sequence information of the bases that make up the DNA.
In addition, the "gene" includes not only "gene" represented by a specific nucleotide sequence, but also nucleic acids encoding their homologues (i.e., homologues or orthologs), variants such as polymorphisms, and derivatives. be done.

In the present invention, "SARS-CoV-2" refers to SARS coronavirus 2 (Severe acute respiratory syndrome coronavirus 2), which causes COVID-19. SARS-CoV-2 is a single-stranded positive-strand RNA virus with a total length of 29.9 kb, and its genome sequence has been registered in GISAID (Global Initiative on Sharing All Influenza Data).

In the present invention, "detection of SARS-CoV-2" includes detection of SARS-CoV-2 as well as detection of the gene of SARS-CoV-2, and detection of the gene includes genomic information, preferably Detection of whole genome information is included. Therefore, detection of SARS-CoV-2 of the present invention includes detection of SARS-CoV-2 variants.

In the present invention, "SARS-CoV-2 infection" refers to respiratory tract infection, namely COVID-19, caused by human infection with SARS-CoV-2. Common symptoms include headache, loss of smell and taste, nasal congestion (congestion) and rhinorrhea, cough, muscle pain, sore throat, fever, diarrhea, and difficulty breathing. In most cases, the disease is asymptomatic or mild with cold-like symptoms and resolves spontaneously, but severe cases are accompanied by acute respiratory distress syndrome, sepsis, and multiple organ failure.

In the present invention, "detection of SARS-CoV-2 infection" means clarifying the presence or absence of SARS-CoV-2 infection. Note that the term "detection" can also be replaced by the terms inspection, measurement, determination, evaluation, or evaluation support. Note that the term "detection" in the present invention does not include judgment or evaluation by a doctor.

The method for detecting SARS-CoV-2 and the method for detecting SARS-CoV-2 infection of the present invention include the step of detecting SARS-CoV-2 in SSL collected from a subject.
Here, the subject from whom SSL is collected is not limited as long as it is a human who needs to detect SARS-CoV-2. Infected humans are included.

"SSL" refers to the fat-soluble fraction present on the surface of the skin, sometimes referred to as sebum. In general, SSL mainly contains secretions secreted from exocrine glands such as sebaceous glands in the skin, and exists on the skin surface in the form of a thin layer covering the skin surface. SSL contains subject-derived RNA expressed in skin cells (see Patent Document 1 above). In the present invention, unless otherwise specified, the term "skin" is a general term for regions including epidermis, dermis, hair follicles, sweat glands, sebaceous glands and other glands on the body surface.

Any means used to collect or remove SSL from the skin can be used to collect the SSL from the subject's skin. Preferably, an SSL absorbent material, an SSL adhesive material, or an instrument that scrapes the SSL off the skin, as described below, can be used. The SSL absorbent material or SSL adhesive material is not particularly limited as long as it has affinity for SSL, and examples thereof include polypropylene and pulp. More detailed examples of procedures for collecting SSL from the skin include a method of absorbing SSL into sheet-like materials such as blotting paper and blotting film, a method of adhering SSL to a glass plate, tape, etc., a spatula, a scraper, etc. and a method of scraping off and recovering the SSL. In order to improve the adsorptivity of SSL, an SSL absorbent material previously impregnated with a solvent having high fat solubility may be used. On the other hand, if the SSL absorptive material contains a highly water-soluble solvent or moisture, the adsorption of SSL is inhibited, so it is preferable that the content of the highly water-soluble solvent and moisture is small. The SSL absorbent material is preferably used dry. The site of the skin from which the SSL is collected is not particularly limited, and includes the skin of any site of the body such as the head, face, neck, trunk, limbs, and the like. A site with a high sebum secretion, such as the skin of the face, is preferable, but the skin of the trunk (eg, the front of the trunk, the armpit, and the back) may also be used.

The SSL collected from the subject may be stored for a period of time. The collected SSL is preferably stored under low temperature conditions as soon as possible after collection in order to minimize degradation of the contained RNA. The temperature condition for storing the RNA-containing SSL in the present invention may be 0°C or lower, preferably -20±20°C to -80±20°C, more preferably -20±10°C to -80±10°C. , More preferably -20 ± 20°C to -40 ± 20°C, more preferably -20 ± 10°C to -40 ± 10°C, more preferably -20 ± 10°C, still more preferably -20 ± 5°C . The storage period of the RNA-containing SSL under the low-temperature conditions is not particularly limited, but is preferably 12 months or less, for example, 6 hours or more and 12 months or less, more preferably 6 months or less, for example, 1 day or more and 6 months or less, More preferably, it is 3 months or less, for example, 3 days or more and 3 months or less.

Detection of SARS-CoV-2 contained in SSL is specifically performed by converting viral RNA into cDNA by reverse transcription, and then measuring the cDNA or its amplified product.
For extraction of RNA from SSL, methods commonly used to extract or purify RNA from biological samples, such as the phenol/chloroform method, the AGPC (acid guanidinium thiocyanate-phenol-chloroform extraction) method, or TRIzol® ), a method using a column such as RNeasy (registered trademark), QIAzol (registered trademark), a method using special magnetic particles coated with silica, a method using Solid Phase Reversible Immobilization magnetic particles, a commercially available method such as ISOGEN Extraction with an RNA extraction reagent or the like can be used.

The reverse transcription uses primers targeted to RNA sequences specific for SARS-CoV-2. A common reverse transcriptase or reverse transcription reagent kit can be used for the reverse transcription. Preferably, a highly accurate and efficient reverse transcriptase or reverse transcription reagent kit is used, examples of which include M-MLV Reverse Transcriptase and variants thereof, or commercially available reverse transcriptase or reverse transcription reagent kit, Examples include PrimeScript (registered trademark) Reverse Transcriptase series (Takara Bio Inc.) and SuperScript (registered trademark) Reverse Transcriptase series (Thermo Scientific). SuperScript (registered trademark) III Reverse Transcriptase, SuperScript (registered trademark) VILO cDNA Synthesis kit (both from Thermo Scientific) and the like are preferably used.
In the elongation reaction in the reverse transcription, the temperature is preferably adjusted to 42°C ± 1°C, more preferably 42°C ± 0.5°C, even more preferably 42°C ± 0.25°C, while the reaction time is preferably It is preferable to adjust the time to 60 minutes or more, more preferably 80 to 120 minutes.

Methods for detecting and measuring cDNA include the PCR method, nucleic acid amplification methods such as the SmartAmp method and the LAMP method, and hybridization methods (DNA chip, DNA microarray, dot blot hybridization, slot blot hybridization, Northern blot hybridization, etc.). hybridization, etc.), a method for determining a base sequence (sequencing), or a method combining these, it is preferable to use a nucleic acid amplification method because of the simplicity and convenience of the operation. Among them, the highly versatile PCR method is preferred, and the RT-PCR method, in which cDNA is synthesized from RNA by reverse transcription (RT) and subjected to PCR, is more preferred.

The RT-PCR method includes a 2-step method in which RT and PCR are performed separately, and a One-step RT-PCR method in which RT and PCR are performed in the same reaction solution by mixing reverse transcriptase in the reaction system in advance. However, any method may be used in the present invention. In addition, a real-time PCR method that simultaneously performs DNA amplification and quantification by PCR, a real-time RT-PCR method that combines RT, and a real-time one-step RT-PCR method can also be employed. As an example of real-time one-step RT-PCR, QuantiTect Probe RT-PCR Kit manufactured by QIAGEN is preferably used. Further, in the real-time PCR method, a multiplex real-time PCR method in which a plurality of sets of oligo-DNA type probes and primers labeled with different fluorescent dyes are mixed so as to simultaneously amplify a plurality of target sequences can be adopted.

In the real-time PCR method, PCR amplification products are usually detected by fluorescence. Fluorescence detection methods include a method using an intercalating fluorescent dye and a method using a fluorescently labeled probe. As an intercalating fluorescent dye, SYBR® Green I is used, but is not limited thereto. The intercalating fluorescent dye binds to double-stranded DNA synthesized by PCR and emits fluorescence upon irradiation with excitation light. By measuring this fluorescence intensity, the production amount of the PCR amplification product can be measured.
Fluorescently labeled probes include, but are not limited to, TaqMan probes, Molecular Beacons, cycling probes, and the like. TaqMan probes are oligonucleotides modified with a fluorescent dye at the 5′ end and a quencher substance at the 3′ end. TaqMan probes specifically hybridize to template DNA in the annealing step of PCR, but the presence of a quencher on the probe suppresses the generation of fluorescence even when irradiated with excitation light. In the subsequent elongation reaction step, when the TaqMan probe hybridized to the template DNA is decomposed by the 5'→3' exonuclease activity of Taq DNA polymerase, the fluorescent dye is released from the probe and fluorescence is generated by the quencher. is released and emits fluorescence. By measuring this fluorescence intensity, the production amount of the amplification product can be measured. Said fluorescent dyes include but are not limited to FAM, ROX, Cy5. Said quenchers include, but are not limited to, TAMRA® and MGB. In order to discriminately detect two or more types of DNA target sequences, PCR is performed using two or more types of oligonucleotide probes (eg, TaqMan probes) each bound with a different fluorescent dye.

The reaction temperature conditions for the reverse transcription reaction in the RT-PCR method and the PCR conditions (temperature, time and number of cycles) can be appropriately set according to the primers used. For example, in One-step RT-PCR (TaqMan probe method) using QIAGEN's QuantiTect Probe RT-PCR Kit, after reacting at 50°C for 30 minutes and at 95°C for 15 minutes, the reaction was carried out at 95°C for 15 seconds and at 60°C. for 45 cycles of 60 seconds.

The SARS-CoV-2 target gene (target site to be amplified) when performing the PCR method is appropriately selected in consideration of the sensitivity and specificity of detection, but mainly nucleocapsid (N), envelope protein ( E), spike protein (S), genes for each region of ORF1ab encoding a nonstructural protein, and a part thereof of RNA-dependent RNA polymerase (RdRP). For example, targeting genes to two regions of the N protein has been attempted.

In addition, primers for specifically recognizing and amplifying virus-derived nucleic acids and probes that specifically bind to virus-derived nucleic acids are designed based on the nucleotide sequence that constitutes the target gene. can be used.
When such oligonucleotides are used as primers, they only need to be capable of specific annealing and chain extension. Those having a chain length of preferably 50 bases or less, more preferably 35 bases or less are included. In addition, when used as a probe, it only needs to be capable of specific hybridization, and has a sequence of at least a part or the whole of DNA (or its complementary strand) consisting of a base sequence that constitutes a virus-derived nucleic acid, for example, 10 Those having a chain length of 15 bases or more, preferably 15 bases or more and, for example, 100 bases or less, preferably 50 bases or less, more preferably 25 bases or less are used.

PCR products can be detected and identified by electrophoresis, melting curve method, and various probe methods (Q probe, scorpion probe, hybrid probe, etc.).
Real-time measurement can be confirmed by monitoring the amplification curve of the RT-PCR product using a fluorescence filter corresponding to the fluorescent dye used. If the fluorescence intensity increases according to the number of PCR cycles, the presence of SARS-CoV-2 to be analyzed in the sample is determined to be positive, while if the fluorescence intensity does not increase in PCR, it is negative. be judged.

In addition, when detecting the genome information of SARS-CoV-2, genome analysis is performed by obtaining a sequence derived from the full-length viral genome from the amplified PCR product using a next-generation sequencer. For example, the SARS-CoV-2 genomic region is specifically amplified by multiplex PCR, and the full-length genome sequence is obtained using a next-generation sequencer.

Thus, SSL taken from a subject can be used to detect SARS-CoV-2, thereby detecting SARS-CoV-2 infection in the subject. As shown in the examples below, the detection of SARS-CoV-2 using SSL does not necessarily match the results when saliva is used as a sample, but it is not detected in saliva samples, but is detected only in SSL. Therefore, it can be said that the method of the present invention can complement the method of detecting SARS-CoV-2 using a saliva sample.

In addition, as shown in the examples described later, RNA expression analysis in SSL was performed on COVID-19 patients who were confirmed to be infected with SARS-CoV-2. , IFITM1, IFITM3, IRF7, MX1, IFIT1, HLA-B and EGR1 were upregulated. It has already been reported that the expression of IFN response-related genes is elevated in the blood of COVID-19 patients (Science. 2020 Aug 7;369(6504):718-724.), and similar changes have been observed during SSL. be done. Furthermore, in protein expression analysis, in addition to HLA-B, the expression of B2M (Beta-2-microglobulin) is significantly increased compared to healthy subjects, and it is known that the expression is increased in COVID-19 patients. An increase in CRP (C-reactive protein) (Science. 2020 Aug 7;369(6504):718-724) is also observed.

Therefore, a molecule selected from the 9 genes related to IFN response in SSL or their expression products, B2M protein and CRP protein is a marker molecule for confirming SARS-CoV-2 infection in SSL (SARS-CoV-2 It is useful as an infection confirmation marker). Therefore, SARS-CoV-2 infection can be detected with higher accuracy by measuring the expression level of the marker molecule in SSL collected from a subject in conjunction with detection of SARS-CoV-2. In addition, by using the marker molecule, humans infected with SARS-CoV-2, or humans suspected of being infected with SARS-CoV-2, such as false negatives and false positives in PCR tests , can provide more detailed information about the extent and likelihood of SARS-CoV-2 infection.

Furthermore, gene expression analysis of SSL by severity of COVID-19 patients revealed that the expression of ISG15, IFITM1, and IFITM3 was higher in patients with mild disease than in healthy subjects, and the level was higher than in patients with moderate disease. became.
Therefore, the three genes of ISG15, IFITM1, and IFITM3 in SSL or their expression products are also useful as marker molecules for determining the severity of SARS-CoV-2 infection in SSL. Therefore, by measuring the expression level of the marker molecule in SSL collected from a subject, it becomes possible to detect mild and moderate SARS-CoV-2 infected patients.

The IFN response-related genes of the present invention are IFI6 (NCBI Gene ID: 2537), ISG15 (NCBI Gene ID: 9636), IFITM1 (NCBI Gene ID: 8519), IFITM3 (NCBI Gene ID: 10410), IRF7 (NCBI Gene ID : 3665), MX1 (NCBI Gene ID: 4599), IFIT1 (NCBI Gene ID: 3434), HLA-B (NCBI Gene ID: 3106), and EGR1 (NCBI Gene ID: 1958).
In addition to the IFN-response-related genes described above, genes having substantially the same base sequences as those of the DNA constituting each gene and having the same functions are included in the marker molecules of the present invention. Here, the substantially identical base sequence, for example, using the homology calculation algorithm NCBI BLAST, expected value = 10; gaps allowed; filtering = ON; match score = 1; mismatch score = -3 It means that it has 90% or more, preferably 95% or more, more preferably 98% or more, and still more preferably 99% or more identity with the base sequence of the DNA constituting the gene when searched with .

The “expression product” of an IFN-response-related gene is a concept that includes transcription products and translation products of the gene. A "transcription product" is RNA produced by transcription from a gene (DNA), and a "translation product" means a protein encoded by a gene that is translated and synthesized based on RNA.

In the present invention, B2M protein refers to Beta-2-microglobulin (UniProtKB: P61769), and CRP protein refers to C-reactive protein (UniProtKB: P02741).

Targets for measuring the expression level of such marker molecules include cDNA artificially synthesized from RNA, DNA encoding RNA, proteins encoded by RNA, and molecules interacting with these proteins. , a molecule that interacts with the RNA, a molecule that interacts with the DNA, or the like. Here, molecules that interact with RNA, DNA or protein include DNA, RNA, protein, polysaccharides, oligosaccharides, monosaccharides, lipids, fatty acids, phosphorylated products thereof, alkylated products, sugar adducts, etc., and Any one of the above complexes may be mentioned. In addition, the expression level comprehensively means the expression level and activity of a gene or its expression product, a protein.

As the measurement of the gene expression level, in a preferred embodiment, the expression level of RNA contained in SSL is measured. Specifically, RNA is converted to cDNA by reverse transcription, and then the cDNA or its amplified product is measured. .
Extraction of RNA from SSL, conversion to cDNA, and measurement of cDNA or amplification products thereof can be performed in the same manner as described above. In addition to the PCR method, cDNA or its amplified product can be measured by hybridization methods (DNA chip, DNA microarray, dot blot hybridization, slot blot hybridization, Northern blot hybridization, etc.) using nucleic acids that hybridize to these as probes. ), a method of determining the nucleotide sequence (sequencing), or a combination thereof.

When measuring the expression level of a target gene or a nucleic acid derived therefrom using the Northern blot hybridization method, for example, the probe DNA is first labeled with a radioactive isotope, a fluorescent substance, or the like, and then the resulting labeled DNA is hybridized with biological sample-derived RNA transferred to a nylon membrane or the like according to a conventional method. After that, there is a method of measuring the formed double strand of labeled DNA and RNA by detecting a signal derived from the label.

When measuring the expression level of a target gene or a nucleic acid derived therefrom using a DNA microarray, for example, an array in which at least one nucleic acid (cDNA or DNA) derived from the target gene of the present invention is immobilized on a support. is used to bind labeled cDNA or cRNA prepared from mRNA onto a microarray, and the expression level of mRNA can be measured by detecting the label on the microarray.
The nucleic acids immobilized on the array may be nucleic acids that hybridize specifically (that is, substantially only to the target nucleic acid) under stringent conditions. It may be a nucleic acid having a sequence or a nucleic acid consisting of a partial sequence. Here, the “partial sequence” includes nucleic acids consisting of at least 15 to 25 bases. Here, stringent conditions usually include washing conditions of about "1×SSC, 0.1% SDS, 37° C.", and more stringent hybridization conditions are "0.5×SSC, 0.1% SDS. % SDS, about 42° C.”, and a more stringent hybridization condition is about “0.1×SSC, 0.1% SDS, 65° C.”. Hybridization conditions are described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Thrd Edition, Cold Spring Harbor Laboratory Press (2001) and others.

When measuring the expression level of a target gene or nucleic acid derived therefrom by sequencing, for example, analysis using a next-generation sequencer (eg, Ion S5/XL system, Life Technologies Japan Co., Ltd.) can be mentioned. RNA expression can be quantified based on the number of reads generated by sequencing (read count).

In addition, when measuring B2M protein, CRP protein, translation products (proteins) of IFN response-related genes, protein chip analysis, immunoassay (eg, ELISA, etc.), mass spectrometry (eg, LC-MS/MS, MALDI) -TOF/MS) can be used, and can be appropriately selected depending on the object. In addition, when measuring a molecule that interacts with protein, RNA or DNA, the 1-hybrid method (PNAS 100, 12271-12276 (2003)) or the 2-hybrid method (Biol. Reprod. 58, 302-311 (1998 )) can be used.
For example, when the object to be measured is a protein, it is carried out by contacting an antibody against the protein with a biological sample, detecting the protein in the sample bound to the antibody, and measuring the level. For example, according to Western blotting, after using the above antibody as a primary antibody, an antibody that binds to the primary antibody labeled with a radioisotope, fluorescent substance, enzyme, etc. is used as a secondary antibody, and the primary antibody is Labeling is performed, and signals derived from these labeling substances are measured with a radiometer, a fluorescence detector, or the like.
The antibody against the above protein may be either a polyclonal antibody or a monoclonal antibody. These antibodies can be produced according to known methods. Specifically, a polyclonal antibody is obtained by immunizing a non-human animal such as a rabbit using a protein expressed in Escherichia coli or the like and purified according to a conventional method, or by synthesizing a partial polypeptide of the protein according to a conventional method, It can be obtained from the serum of the immunized animal according to a conventional method.
On the other hand, monoclonal antibodies are obtained by immunizing a non-human animal such as a mouse with a protein expressed in Escherichia coli or the like and purified according to a conventional method or a partial polypeptide of the protein, and fusing the obtained spleen cells with myeloma cells. It can be obtained from prepared hybridoma cells. Monoclonal antibodies may also be generated using phage display (Griffiths, AD; Duncan, AR, Current Opinion in Biotechnology, Volume 9, Number 1, February 1998, pp. 102-108(7)).

Thus, the expression level of the marker molecule of the present invention in SSL collected from a subject is measured, and the confirmation and severity of SARS-CoV-2 infection are detected based on the expression level. Infection confirmation and severity detection are specifically performed by comparing the measured expression level of the marker molecule with the control level.
When analyzing the expression level of a plurality of genes by sequencing, as described above, the read count value, which is the expression level data, the RPM value obtained by correcting the read count value for the difference in the total number of reads between samples, A value obtained by converting the RPM value to a base 2 logarithmic value (Log ₂ RPM value) or a base 2 logarithmic value obtained by adding an integer 1 (Log ₂ (RPM+1) value), or a count value corrected using DESeq2 ( It is preferable to use the base 2 logarithm (Log ₂ (count+1) value) obtained by adding an integer 1 to the normalized count value) as an index. In addition, it is calculated by fragments per kilobase of exon per million reads mapped (FPKM), reads per kilobase of exon per million reads mapped (RPKM), transcripts per million (TPM), etc., which are common as quantitative values for RNA-seq. can be a value. It may be a signal value obtained by a microarray method and its correction value. In addition, when analyzing the expression level of only a specific target gene by RT-PCR, etc., the expression level of the target gene is converted into a relative expression level based on the expression level of the housekeeping gene (relative quantification ), or a method of quantifying the absolute copy number using a plasmid containing the region of the target gene (absolute quantification). It may be a copy number obtained by a digital PCR method.
Here, the "control level" includes, for example, the expression level of the marker molecule in healthy subjects. The expression level of healthy individuals may be a statistical value (eg, mean value, etc.) of the expression levels of marker molecules measured from a population of healthy individuals. For purposes of infection confirmation or severity detection, the expression level of marker molecules in mild COVID-19 patients may serve as a "control level."

In addition, confirmation of SARS-CoV-2 infection or detection of the severity of SARS-CoV-2 infection using the marker molecule of the present invention can also be performed by increasing/decreasing the expression level of the marker molecule. In this case, the expression level of the marker molecule in the subject-derived SSL is compared with the cutoff value (reference value) of the marker molecule.
The cutoff value can be determined by various statistical analysis methods. Examples include values based on ROC curve (Receiver Operating Characteristic curve) analysis (eg, Youden's index, a distance value from the upper left corner coordinates (0, 1) on the ROC curve, etc.).
In the ROC curve, the vertical axis is the probability of a positive result in positive patients (true positive rate (TPF: True Position Fraction), sensitivity), and the horizontal axis is the probability of a negative result in negative patients (specificity) from 1 The subtracted value (false positive rate (FPF: False Position Fraction)) is plotted by changing the threshold for determining which value of the measurement result is found, that is, the cutoff point as a parameter. Created by going.
Which cutoff point should be adopted as the cutoff value from the created ROC curve may be determined according to the position of infection confirmation and severity detection, and other various conditions. Usually, if the cut-off point is set at a low false-positive rate, the number of negative patients who become positive will be reduced, but on the contrary, many positive patients will be excluded, resulting in lower sensitivity. Conversely, increasing sensitivity increases the false positive rate in negative patients.
In order to generally increase both sensitivity and specificity (to approach 1), the cutoff value is the value that gives the closest point to the point (0, 1) on the ROC curve, or the "true positive (sensitivity)" - Set to the value (Youden index) that maximizes the "false positive (1-specificity)".

The detection kit for detecting SARS-CoV-2 of the present invention contains tools and reagents necessary for collecting and preserving SSL, and reagents for detecting SARS-CoV-2 from SSL.
In addition, the detection kit for detecting SARS-CoV-2 infection of the present invention includes tools and reagents necessary for collecting and storing SSL, reagents for detecting SARS-CoV-2 from SSL, and It optionally contains a reagent for measuring the expression level of the marker molecule.
In addition, the SARS-CoV-2 infectious disease confirmation or severity detection kit of the present invention contains tools and reagents necessary for collecting and storing SSL, and reagents for measuring the expression level of marker molecules from SSL. It is something to do.

Here, tools and reagents necessary for collecting and preserving SSL include, for example, an oil-removing film for collecting SSL, a reagent for preserving the collected SSL, a container for preservation, and the like.
Reagents for detecting SARS-CoV-2 and reagents for measuring the expression level of marker molecules include, for example, reagents for extracting and purifying RNA from collected SSL, SARS-CoV-2 and marker molecules Immunological reagents for nucleic acid amplification or hybridization, including oligonucleotides (e.g., primers for PCR) that specifically bind (hybridize) with the nucleic acid from which they are derived, antibodies that recognize marker molecules (proteins) In addition to reagents for measurement, labeling reagents, buffer solutions, chromogenic substrates, secondary antibodies, blocking agents, control reagents used as positive and negative controls, instruments necessary for testing, and the like are included.

Aspects and preferred embodiments of the present invention are presented below.
<1> A method for detecting SARS-CoV-2 in a subject, comprising the step of detecting SARS-CoV-2 in skin surface lipids collected from the subject.
<2> The method of <1>, wherein SARS-CoV-2 genome information, preferably whole genome information is detected.
<3> The method of <1> or <2> for detecting SARS-CoV-2 mutants.
<4> A method for detecting SARS-CoV-2 infection in a subject, comprising the step of detecting SARS-CoV-2 in skin surface lipids collected from the subject.
<5> The method according to any one of <1> to <4>, wherein the detection of SARS-CoV-2 converts viral RNA into cDNA by reverse transcription, and then the cDNA or its amplified product is measured.
<6> Furthermore, for surface lipids collected from a subject, IFN response-related genes selected from IFI6, ISG15, IFITM1, IFITM3, IRF7, MX1, IFIT1, HLA-B and EGR1 or their expression products, B2M proteins and The detection method of <4>, wherein the expression level of one or more molecules selected from CRP proteins is measured.
<7> IFI6, ISG15, IFITM1, IFITM3, IRF7, MX1, IFIT1, SARS-CoV-2 infection in the subject, comprising measuring the expression level of one or more molecules selected from IFN response-related genes or expression products thereof selected from HLA-B and EGR1, B2M protein and CRP protein Confirmation detection method.
<8> IFN response-related genes selected from ISG15, IFITM1 and IFITM3, or their A method of detecting severity of SARS-CoV-2 infection in a subject, comprising measuring the expression level of an expression product.
<9> The method according to any one of <6> to <8>, wherein the expression level of the IFN response-related gene or its expression product is the measurement of the mRNA expression level.
<10> SARS used in any of the methods <1> to <3>, containing tools and reagents necessary for collection and storage of SSL, and reagents for detecting SARS-CoV-2 from SSL- CoV-2 detection kit.
<11> Tools and reagents necessary for collection and storage of SSL, reagents for detecting SARS-CoV-2 from SSL, and optionally further reagents for measuring the expression level of the molecule from SSL, A SARS-CoV-2 infectious disease detection kit used in any of the methods <4> to <6>.
<12> SARS-CoV-2 infectious disease confirmation detection used in the method of <7>, containing tools and reagents necessary for collection and storage of SSL, and reagents for measuring the expression level of the molecule from SSL kit.
<13> Severity of SARS-CoV-2 infection used in the method of <8>, containing tools and reagents necessary for collection and storage of SSL, and reagents for measuring the expression level of said molecule from SSL degree detection kit.

EXAMPLES The present invention will be described in more detail below based on examples, but the present invention is not limited to these.
Example 1 Detection of SARS-CoV-2 from the surface of the skin 1) Subject Saliva and saliva in men and women aged 20 or older who were confirmed to be infected with SARS-CoV-2 between April 2020 and December 2020 in Japan Nine COVID-19 patients from whom both sebum specimens were available were included.

2) SSL and saliva collection After collecting sebum from the entire face of each subject using an oil blotting film (5 x 8 cm, made of polypropylene, 3M company), the oil blotting film was transferred to a vial and used for RNA extraction- Stored at 80°C. Saliva was collected on the same day as sebum or the day before.

3) Preparation of RNA The blotting film obtained in 2) above was cut into an appropriate size, and RNA was extracted using QIAzol Lysis Reagent (Qiagen) according to the attached protocol. After adding 10 μL of 3M sodium acetate and 1 μL of Ethachinmate (Nippon Gene) to 100 μL of the purified RNA solution, 250 μL of 100% EtOH was further added and vortexed. After allowing to stand at -20°C for 30 minutes (or -80°C for 15 minutes) and cooling, it was centrifuged at 15,000 rpm at 4°C for 15 minutes. After removing the solution, 700 μL of 70% EtOH was added and centrifuged again at 15,000 rpm and 4° C. for 15 minutes. The solution was completely removed, the lid of the tube was opened, and after drying for 1 minute, it was redissolved in 10 μL of RNase-free water. The resulting RNA solution was stored at -80°C.

4) Viral RNA quantification Viral RNA detection using the QuantiTect Probe RT-PCR kit (QIAGEN) and the primers in Table 1 according to the method described in the National Institute of Infectious Diseases "Pathogen Detection Manual 2019-nCoV Ver.2.9.1" carried out.
Positive Control RNA Mix (2019-nCoV) (XA0142, TAKARA) was used to prepare standard solutions of 9 steps of 5-fold dilution with the highest concentration being 10 ⁵ copies/μL. 1 μL of 10 μL RNA obtained from one oil blotting film was diluted 5-fold and used for viral RNA detection (total of 2 μL of the original solution was used for duplication). 7500 fast real-time PCR system (Applied Biosystems), reaction system 20 μL (Table 2), the following conditions: [50 ° C. for 30 min] → [95 ° C. for 15 min] → 45 cycles of [95 ° C. for 15 sec, 60 ° C. for 60 sec ] RT-PCR was performed. Viral RNA detection was performed in the same manner on RNA obtained from saliva specimens.

5) Results A positive reaction was observed in 8 out of 13 saliva specimens and 8 out of 14 sebum specimens. There were 4 cases where it was not detected in sebum but was detected only in saliva, and 3 cases where it was detected only in sebum without being detected in saliva (Table 3). This suggests the possibility that the viral RNA detection method using a sebum sample and the viral RNA detection method using a saliva sample can complement each other.

Example 2 SARS-CoV-2 Genome Analysis from Skin Surface Specimens 1) Subjects Sebum in men and women aged 20 or older who were confirmed to be infected with SARS-CoV-2 between April 2020 and December 2020 in Japan Eight specimens from 6 COVID-19 patients (2 at 2 time points) in which viral RNA was detected from

2) SSL and saliva collection After collecting sebum from the entire face of each subject using an oil blotting film (5 x 8 cm, made of polypropylene, 3M company), the oil blotting film was transferred to a vial and used for RNA extraction- Stored at 80°C. Saliva was collected on the same day as sebum or the day before.

3) Preparation of RNA The blotting film obtained in 2) above was cut into an appropriate size, and RNA was extracted using QIAzol Lysis Reagent (Qiagen) according to the attached protocol. After adding 10 μL of 3M sodium acetate and 1 μL of Ethachinmate (Nippon Gene) to 100 μL of the purified RNA solution, 250 μL of 100% EtOH was further added and vortexed. After allowing to stand at -20°C for 30 minutes (or -80°C for 15 minutes) and cooling, it was centrifuged at 15,000 rpm at 4°C for 15 minutes. After removing the solution, 700 μL of 70% EtOH was added and centrifuged again at 15,000 rpm and 4° C. for 15 minutes. The solution was completely removed, the lid of the tube was opened, and after drying for 1 minute, it was redissolved in 10 μL of RNase-free water. The resulting RNA solution was stored at -80°C.

4) Viral genome analysis Multiplex PCR method (A proposal of an alternative primer for the ARTIC Network's multiplex PCR to improve coverage of SARS-CoV-2 genome sequencing. Kentaro Ito in accordance with the protocol implemented by the National Institute of Infectious Diseases) Tsuyoshi Sekizuka, Masanori Hashino, Rina Tanaka, Makoto Kuroda. bioRxiv 2020 March 10.), the viral genome region was specifically amplified, and a sequence derived from the full-length viral genome was obtained using the next-generation sequencer MiSeq. Specifically, the sample RNA solution was reverse transcribed using SuperScript IV First-Strand Synthesis System (Thermo Fisher Scientific), and a DNA amplicon was obtained by amplifying the SARS-CoV-2 genomic region by multiplex PCR. Next, using QIAseq FX DNA Library Kit (QIAGEN), DNA amplicons were fragmented, blunt-ended, A-protruding ends were added, and adaptors were added to prepare a sequence library. Libraries were quality checked using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific). The prepared library was sequenced using MiSeq Reagent Kit v3. The resulting sequence data were mapped to the SARS-CoV-2 reference sequence (MN908947) using the analysis tool ONE CODEX.

5) Results Sequencing data with a Mean depth of 2000 x or higher, covering almost the entire SARS-CoV-2 reference sequence (99% or more), was obtained for 6 samples out of 8 samples. It was shown that genomic analysis of SARS-CoV-2 is sufficiently possible from SSL samples (Table 4).

Example 3 Gene expression analysis in SSL of COVID-19 patients 1) Subject COVID-19 patients aged 20 or older who were confirmed to have SARS-CoV-2 infection in Japan between April 2020 and December 2020 The subjects were 14 people. In addition, SSL-RNA of 18 healthy males aged 20 years or older, which was conducted in 2019, was used as a control.

2) SSL collection Sebum was collected from the entire face of each subject using an oil blotting film (5 x 8 cm, made of polypropylene, 3M company), then the oil blotting film was transferred to a vial, and -80°C until used for RNA extraction. Saved in

3) RNA Preparation and Sequencing The blotting film of 2) above was cut into an appropriate size, and RNA was extracted using QIAzol Lysis Reagent (Qiagen) according to the attached protocol. After adding 10 μL of 3M sodium acetate and 1 μL of Ethachinmate (Nippon Gene) to 100 μL of the purified RNA solution, 250 μL of 100% EtOH was further added and vortexed. After allowing to stand at -20°C for 30 minutes (or -80°C for 15 minutes) and cooling, it was centrifuged at 15,000 rpm at 4°C for 15 minutes. After removing the solution, 700 μL of 70% EtOH was added and centrifuged again at 15,000 rpm and 4° C. for 15 minutes. The solution was completely removed, the lid of the tube was opened, and after drying for 1 minute, it was redissolved in 10 μL of RNase-free water. The resulting RNA solution was stored at -80°C.
Based on the extracted RNA, reverse transcription was performed at 42° C. for 90 minutes using SuperScript VILO cDNA Synthesis kit (Life Technologies Japan Co., Ltd.) to synthesize cDNA. Random primers attached to the kit were used as primers for the reverse transcription reaction. A library containing DNA derived from the 20802 gene was prepared from the resulting cDNA by multiplex PCR. Multiplex PCR was performed using Ion AmpliSeq Transcriptome Human Gene Expression Kit (Life Technologies Japan Co., Ltd.) [99 ° C., 2 minutes → (99 ° C., 15 seconds → 62 ° C., 16 minutes) × 20 cycles → 4 ° C., Hold ]. The resulting PCR product was purified with Ampure XP (Beckman Coulter, Inc.) and then subjected to buffer reconstitution, primer sequence digestion, adapter ligation and purification, and amplification to prepare a library. The prepared library was loaded into the Ion 540 Chip and sequenced using the Ion S5/XL system (Life Technologies Japan).

4) Data Analysis The obtained read count data was analyzed using R. Among the 20802 genes targeted by the Ion AmpliSeq Transcriptome Human Gene Expression Kit, samples in which the percentage of detected genes was less than 20% were excluded from the analysis as low-quality samples. Genes detected in 90% or more samples from the sequence data obtained from the samples that cleared the criteria were normalized by the size factor in DESeq2, and expression-varied genes were extracted (False discovery rate (FDR) < 0.05, |Log2FC|>2) was performed.

5) Results Sixty-seven genes with increased expression and 20 genes with decreased expression were identified in COVID-19 patients compared to healthy subjects. Genes elevated in COVID-19 include nine genes associated with IFN response: IFI6, ISG15, IFITM1, IFITM3, IRF7, MX1, IFIT1, HLA-B, and EGR1, demonstrating that virus response can be detected. became.

Example 4 Protein expression analysis in SSL of COVID-19 patients 1) Subject COVID-19 patients aged 20 or older who were confirmed to have SARS-CoV-2 infection in Japan between April 2020 and December 2020 The subjects were 18 people. In addition, SSL-RNA of 18 healthy males aged 20 years or older, which was conducted in 2019, was used as a control.

2) SSL collection Sebum was collected from the entire face of each subject using an oil blotting film (5 x 8 cm, made of polypropylene, 3M company), then the oil blotting film was transferred to a vial, and -80°C until used for RNA extraction. Saved in

3) Protein preparation The blotting film of 1) above was cut into an appropriate size, and a protein precipitate was obtained using QIAzol Lysis Reagent (Qiagen) according to the attached protocol. From the resulting protein precipitate, using EasyPep Mini MS Sample Prep Kit (Thermo Scientific), the protein was dissolved in a lysate according to the attached protocol, and then digested with trypsin. The obtained digestive fluid was dried under reduced pressure (35° C.) and then dissolved in an aqueous solution containing 0.1% formic acid and 2% acetonitrile to prepare a peptide solution. Peptide concentration in solution was measured using a microplate reader according to the protocol of Pierce ^™ Quantitative Fluorometric Peptide Assay (ThermoFisher Scientific). The concentration of the peptide solution was adjusted to a constant value, and the protein quantification value was calculated by LC-MS/MS analysis.

4) LC-MS/MS Analysis and Data Analysis The sample peptide solution obtained in 2) above was subjected to LC-MS/MS analysis under the conditions shown in Table 6 below.

For analysis of spectral data obtained by LC-MS/MS analysis, Proteome Discoverer ver. 2.2 (ThermoFisher Scientific) was used. For protein identification, the reference database was set to Swiss Prot and Taxonomy was set to Homo sapiens, and searched using Mascot database search (Matrix Science). In the search, Enzyme was set to Trypsin, Missed cleavage to 2, Dynamic modifications to Oxidation (M), Acetyl (N-term), Acetyl (Protein N-term), and Static Modifications to Carbamidomethyl (C). Peptides satisfying a false discovery rate (FDR) of p<0.01 were searched. The identified proteins were subjected to precursor ion-based Label Free Quantification (LFQ). The protein abundance was calculated from the peak intensity of the precursor ion derived from the peptide, and if the peak intensity was below the detection limit, it was taken as a missing value. To correct experimental bias, the protein abundance was normalized by the Total peptide amount method, and the protein abundance ratio was calculated by the Summed Abundance Based method. ANOVA (Individual Based, t-test) was used to calculate the p-value indicating the significance of the existence difference between groups. Among the identified proteins, proteins with a false discovery rate (FDR) of 0.1 or higher were excluded from the analysis.

5) Results Compared to healthy subjects, 134 proteins (FDR < 0.05) whose expression fluctuates in COVID-19 patients can be identified. , and B2M (Beta-2-microglobulin, P61769) increased 21.5 times in COVID-19 patients. In addition, CRP (C-reactive protein, P02741) was identified as a protein that was not detected in healthy subjects and was detected only in COVID-19 patients (0/18 healthy subjects, 8/18 in COVID-19 patients name detection). Furthermore, it was revealed that the amount of CRP in SSL is positively correlated with the blood CRP concentration (Pearson's r=0.92). Plots of HLA-B, B2M, and CRP protein abundance (normalized abundance) in face-derived SSL of healthy subjects and COVID-19 patients are shown in Figure 1, and the correlation between CRP amount in SSL and blood CRP concentration is shown in Figure 2. Indicated.

Example 5 Detection of SARS-CoV-2 from the skin surface (comparison between face and trunk)
1) Subjects 15 COVID-19 patients aged 20 or older who were confirmed to be infected with SARS-CoV-2 in Japan between December 2020 and April 2021 and were able to collect both saliva and sebum samples. Targeted.

2) SSL and saliva collection After collecting sebum from each subject's entire face and trunk (front trunk + armpit) using one sheet of oil blotting film (5 × 8 cm, polypropylene, 3M company), the oil is blotted. Films were transferred to vials and stored at −80° C. until used for RNA extraction.

3) RNA preparation/viral RNA quantification RNA was prepared in the same manner as in Example 1, and SARS-CoV-2 was quantified.

4) Results A positive reaction was observed in 9 out of 15 facial sebum samples and 4 out of 15 trunk sebum samples (Table 7). It became clear that viral RNA can be detected even on faces other than those with a lot of sebum.

Example 6 Gene expression analysis in SSL by COVID-19 patient severity 1) Subjects SARS-CoV-2 infection was confirmed in Japan between April 2020 and April 2021 COVID -19 Subjects were 15 mild patients and 14 moderate patients, and sebum collected for the first time after hospitalization was used as a sample. In addition, 18 healthy males aged 20 or older, which was conducted in 2019, were used as controls.

2) SSL collection Sebum was collected from the entire face of each subject using an oil blotting film (5 x 8 cm, made of polypropylene, 3M company), then the oil blotting film was transferred to a vial, and -80°C until used for RNA extraction. Saved in

3) RNA Preparation and Sequencing The blotting film in 2) above was cut into pieces of appropriate size, RNA was prepared in the same manner as in Example 3, and sequencing was performed.

4) Data Analysis The obtained read count data was analyzed using R. Among the 20802 genes targeted by the Ion AmpliSeq Transcriptome Human Gene Expression Kit, samples in which the percentage of detected genes was less than 20% were excluded from the analysis as low-quality samples. Genes detected in 90% or more samples from the sequence data obtained from the samples that cleared the criteria were normalized by the size factor in DESeq2. Thereafter, Tukey-Kramer's multiple comparison test was performed on the IFN-stimulated gene expression levels in the healthy group, the mild group, and the moderate group.

5) Results It was revealed that ISG15, IFITM1, and IFITM3, which are related to IFN response, are expressed higher in mild cases than in healthy cases, and lower in moderate cases than in mild cases. It was shown that the responsiveness of IFN-related genes involved in these antivirals can predict the severity (Fig. 3).

Claims (11)

A method for detecting SARS-CoV-2 in a subject, comprising the step of detecting SARS-CoV-2 in skin surface lipids collected from the subject. 2. The method of claim 1, wherein the genome information of SARS-CoV-2 is detected. A method for detecting SARS-CoV-2 infection in a subject, comprising the step of detecting SARS-CoV-2 in skin surface lipids collected from the subject. Furthermore, the expression level of one or more molecules selected from IFN response-related genes or their expression products selected from IFI6, ISG15, IFITM1, IFITM3, IRF7, MX1, IFIT1, HLA-B and EGR1, B2M protein and CRP protein 4. The detection method according to claim 3, comprising measuring. IFI6, ISG15, IFITM1, IFITM3, IRF7, MX1, IFIT1, HLA-B for superficial skin lipids taken from subjects infected with SARS-CoV-2 or suspected of having SARS-CoV-2 infection and IFN response-related genes or expression products thereof selected from EGR1, B2M protein and CRP protein, comprising the step of measuring the expression level of one or more molecules selected from the subject, detection of confirmed SARS-CoV-2 infection in the subject Method. IFN response-related genes or their expression products selected from ISG15, IFITM1 and IFITM3 for skin surface lipids collected from subjects infected with SARS-CoV-2 or subjects suspected of having SARS-CoV-2 infection A method of detecting severity of SARS-CoV-2 infection in a subject, comprising measuring expression levels. 7. The method according to any one of claims 4 to 6, wherein the expression level of the IFN response-related gene or its expression product is the measurement of the mRNA expression level. A SARS-CoV-2 detection kit for use in the method according to claim 1 or 2, containing tools and reagents necessary for collecting and storing SSL, and reagents for detecting SARS-CoV-2 from SSL. Claim 3, containing tools and reagents necessary for collection and storage of SSL, reagents for detecting SARS-CoV-2 from SSL, and optionally reagents for measuring the expression level of said molecule from SSL. Or SARS-CoV-2 infectious disease detection kit used in the method according to 4. A kit for confirming and detecting SARS-CoV-2 infection used in the method according to claim 5, comprising tools and reagents necessary for collecting and preserving SSL, and reagents for measuring the expression level of said molecule from SSL. Detection of severity of SARS-CoV-2 infection used in the method of claim 6, containing tools and reagents necessary for collection and storage of SSL, and reagents for measuring the expression level of said molecule from SSL. kit.

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