CN116042800B - Molecular marker of RBBP6 gene and CCDC91 gene combination and application thereof - Google Patents

Molecular marker of RBBP6 gene and CCDC91 gene combination and application thereof Download PDF

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CN116042800B
CN116042800B CN202211221315.XA CN202211221315A CN116042800B CN 116042800 B CN116042800 B CN 116042800B CN 202211221315 A CN202211221315 A CN 202211221315A CN 116042800 B CN116042800 B CN 116042800B
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周晓华
李洋
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Peking University
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Abstract

The application relates to a molecular marker combined by RBBP6 gene and CCDC91 gene, a kit and application thereof, belonging to the field of biotechnology and solving at least one of the following problems: (1) The existing virus nucleic acid detection kit can not effectively screen SARS-CoV-2 virus infected persons in a window period; (2) The existing virus nucleic acid detection result is limited by a plurality of factors such as sampling positions, sampling methods, sampling periods and the like. The molecular marker comprises RBBP6 gene and CCDC91 gene. The RBBP6 gene and the CCDC91 gene have correlation with SARS-CoV-2 virus infection, and the molecular marker is suitable for early detection of SARS-CoV-2 virus infection, has easy operation and high detection sensitivity, can distinguish SARS-CoV-2 virus infection from other common respiratory tract virus infection, and shows better specificity.

Description

Molecular marker of RBBP6 gene and CCDC91 gene combination and application thereof
Technical Field
The application relates to the technical field of biology, in particular to a molecular marker for RBBP6 gene and CCDC91 gene combination, a kit and application thereof.
Background
As the SARS-CoV-2 virus continues to evolve into new varieties represented by omicuron (Omicron), leading to the appearance of a large number of asymptomatic infected ones among those infected with the virus. Importantly, more and more evidence suggests that asymptomatic infected persons are more contagious and generally susceptible to the population. Thus, early detection of SARS-COV-2 virus is a key element in the prevention and control of COVID-19.
At present, the existing detection mode of SARS-COV-2 virus infected person is mainly based on virus nucleic acid detection kit. However, a certain window period (1-14 days) exists between the virus infection and the virus nucleic acid positive, and the existing virus nucleic acid detection kit cannot effectively detect SARS-CoV-2 virus infection in the window period; in addition, the detection result of the viral nucleic acid is also limited by a plurality of factors such as a sampling position, a sampling method, a sampling period and the like. Early detection of SARS-CoV-2 virus has great significance in epidemic prevention and control.
Disclosure of Invention
In view of the above analysis, the present application aims to provide a molecular marker of RBBP6 gene and CCDC91 gene combination, a kit and application thereof, which are used for solving at least one of the following problems: (1) The existing virus nucleic acid detection kit can not effectively screen SARS-CoV-2 virus infected persons in a window period; (2) The existing virus nucleic acid detection result is limited by a plurality of factors such as sampling positions, sampling methods, sampling periods and the like.
In order to achieve the above purpose, the present application adopts the following technical scheme:
in one aspect, the present application provides a molecular marker comprising an RBBP6 gene and a CCDC91 gene.
In a second aspect, embodiments of the present application provide for the use of a molecular marker expression product of a combination of the RBBP6 gene and the CCDC91 gene in the manufacture of a tool for screening for SARS-CoV-2 virus infected persons.
In a third aspect, the present application also provides a PCR primer pair for amplifying the above molecular marker expression product, the PCR primer pair comprising a forward primer and a reverse primer;
the nucleotide sequence of the forward primer used for the RBBP6 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 2;
the nucleotide sequence of the forward primer used for the CCDC91 gene is shown as SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 4.
In a fourth aspect, the present application provides a probe for amplifying an expression product of the above molecular marker, comprising:
the probe sequence for amplifying the RBBP6 gene expression product is shown as SEQ ID NO.5, and the probe sequence for amplifying the CCDC91 gene expression product is shown as SEQ ID NO. 6.
In a fifth aspect, the application provides the use of a PCR primer pair as described above or/and a probe as described above for the preparation of a reagent for screening for SARS-CoV-2 virus infected persons.
In a sixth aspect, the present application provides a kit comprising the primer set for the RBBP6 gene and the probe for the RBBP6 gene, and the primer set for the CCDC91 gene and the probe for the CCDC91 gene.
Preferably, the concentration ratio of each primer to the corresponding probe is 1-2:1.
preferably, the working fluid concentration of each primer is 7-20. Mu.M, and the working fluid concentration of each probe is 7-20. Mu.M.
In a seventh aspect, the application provides the use of the above-described kit for detecting the expression levels of the RBBP6 gene and CCDC91 gene.
In an eighth aspect, the present application provides the use of a molecular marker expression product of a combination of the RBBP6 gene and the CCDC91 gene in the manufacture of a medicament for the treatment of covd-19.
Compared with the prior art, the application has at least one of the following beneficial effects:
1. the host will immediately elicit an immune response due to viral invasion into the host, i.e.: the application provides a molecular marker comprising a combination of RBBP6 gene and CCDC91 gene, wherein the RBBP6 gene and the CCDC91 gene are related to SARS-CoV-2 virus infection, the RBBP6 gene and the CCDC91 gene are highly expressed in a blood sample of a SARS-CoV-2 virus infected person, and whether a subject is infected by the SARS-CoV-2 virus or not can be screened by detecting the expression level of the RBBP6 gene and the CCDC91 gene, especially when the infected person is in an asymptomatic period or in a window period between the virus infection and the virus nucleic acid positive; that is, detecting the expression levels of the RBBP6 gene and the CCDC91 gene in a blood sample during a window period can determine whether to infect SARS-CoV-2 virus, and is suitable for early detection of SARS-CoV-2 virus infection, especially in a symptom-free period and in a blood sample of a high risk contactor when the SARS-CoV-2 virus targeted detection is negative.
2. The sampling part for detecting the existing virus nucleic acid is usually the pharyngeal part, the sampling method is to sample through the deep pharyngeal part of a cotton swab, the sampling mode is easy to cause uncomfortable feeling of a person to be sampled, and the matching degree is not high; in addition, the nucleic acid detection result is directly affected when the sampler is not in place, and the sampling period also affects the nucleic acid detection result. The molecular marker of the application adopts a conventional blood sampling mode, then detects the expression levels of RBBP6 gene and CCDC91 gene in a blood sample, has easy operation, is not influenced by the sampling period, has more accurate detection result and high sensitivity, can distinguish SARS-CoV-2 virus infection from other common respiratory tract virus infection, and shows better specificity.
3. The molecular marker expression product provided by the application can be applied to the preparation of a medicine for treating COVID-19, and provides a basis for developing a new SARS-CoV-2 virus infection targeted therapy.
In the application, the technical schemes can be mutually combined to realize more preferable combination schemes. Additional features and advantages of the application will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the application. The objectives and other advantages of the application may be realized and attained by the structure particularly pointed out in the written description and drawings.
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The drawings are only for purposes of illustrating particular embodiments and are not to be construed as limiting the application, like reference numerals being used to refer to like parts throughout the several views.
FIG. 1 shows the expression of RBBP6 and CCDC91 gene combinations in blood samples of asymptomatic SARS-CoV-2 virus infected persons;
FIG. 2 shows the expression of RBBP6 and CCDC91 gene combinations in blood samples of symptomatic and containing SARS-CoV-2 virus infected persons;
FIG. 3 shows the expression of RBBP6 gene in common respiratory viruses including: HRV: rhinovirus; IFV: influenza virus; RSV: respiratory syncytial virus; seasonal CoVs: seasonal coronavirus;
FIG. 4 shows the expression of CCDC91 gene in common respiratory viruses including: HRV: rhinovirus; IFV: influenza virus; RSV: respiratory syncytial virus; seasonal CoVs: seasonal coronavirus;
FIG. 5 shows the result of RBBP6 gene and CCDC91 gene combination detection of blood samples of medical workers in new coronary epidemic based on qRT-PCR method.
Detailed Description
The following detailed description of preferred embodiments of the application is made in connection with the accompanying drawings, which form a part hereof, and together with the description of the embodiments of the application, are used to explain the principles of the application and are not intended to limit the scope of the application.
The inventors have found that the host is instantaneously responsible for the immune response due to viral invasion into the host, i.e.: the expression of partial genes in the host body is greatly changed, so that the discovery of biological markers related to SARS-CoV-2 virus infection is imperative, which also helps to develop a new SARS-CoV-2 infection targeted therapeutic scheme.
Thus, in one aspect, the application provides a molecular marker, such as a molecular marker useful for screening for a SARS-CoV-2 viral infected person, comprising an RBBP6 gene and a CCDC91 gene, said RBBP6 gene and said CCDC91 gene being associated with a SARS-CoV-2 viral infection.
The host will immediately elicit an immune response due to viral invasion into the host, i.e.: the application provides a molecular marker comprising a combination of RBBP6 gene and CCDC91 gene, wherein the RBBP6 gene and the CCDC91 gene are related to SARS-CoV-2 virus infection, the RBBP6 gene and the CCDC91 gene are highly expressed in a blood sample of a SARS-CoV-2 virus infected person, and whether the patient is infected with SARS-CoV-2 virus or not can be screened by detecting the expression level of the RBBP6 gene and the CCDC91 gene, especially when the patient is in an asymptomatic period or in a window period between virus infection and virus nucleic acid positive; that is, detecting the expression levels of the RBBP6 gene and the CCDC91 gene in a blood sample during a window period can determine whether to infect SARS-CoV-2 virus, and is suitable for early detection of SARS-CoV-2 virus infection, especially in a symptom-free period and in a blood sample of a high risk contactor when the SARS-CoV-2 virus targeted detection is negative.
The sampling part for detecting the existing virus nucleic acid is usually the pharyngeal part, the sampling method is to sample through the deep pharyngeal part of a cotton swab, the sampling mode is easy to cause uncomfortable feeling of a person to be sampled, and the matching degree is not high; in addition, the nucleic acid detection result is directly affected when the sampler is not in place, and the nucleic acid detection result is affected at the sampling time. The molecular marker of the application adopts a conventional blood sampling mode, then the expression levels of RBBP6 gene and CCDC91 gene in a blood sample are detected, the operation is easy, the detection result is more accurate without being influenced by the sampling period, the method has high sensitivity, and SARS-CoV-2 virus infection and other common respiratory tract virus infection can be distinguished, thus showing better specificity. In addition, the molecular marker expression product provided by the application can be applied to the preparation of medicines for treating COVID-19, and provides a basis for developing new SARS-CoV-2 virus infection targeted therapies.
The RBBP6 gene is located on human chromosome 16 and located in the interval 24539566 to 24572863; the CCDC91 gene is located on human chromosome 12 and is located in the interval 28190456 to 28550166. The RBBP6 gene (NC_000016.10:24539566-24572863Homo sapiens chromosome 16,GRCh38.p14 Primary Assembly) and the CCDC91 gene (NC_000012.12:28190456-28550166Homo sapiens chromosome 12,GRCh38.p14 Primary Assembly) can both be queried for related sequences in the International public nucleic acid sequence database GeneBank.
Specifically, the expression level of the RBBP6 gene and the CCDC91 gene in a blood sample infected with SARS-CoV-2 virus is higher than that in a blood sample not infected with SARS-CoV-2 virus.
By detecting the expression level of the combination of RBBP6 gene and CCDC91 gene in a blood sample, it can be determined whether SARS-CoV-2 virus is infected in an early detection.
Specifically, when the expression levels of both the RBBP6 gene and the CCDC91 gene are higher than the normal expression level, infection with SARS-CoV-2 virus can be determined.
In a second aspect, the application provides the use of a molecular marker expression product of a combination of the RBBP6 gene and the CCDC91 gene in the manufacture of a tool for screening for SARS-CoV-2 virus infected persons.
Such tools include, but are not limited to, chips, kits, reagents, or high throughput sequencing platforms.
In a third aspect, the present application also provides a PCR primer pair for amplifying the above molecular marker expression product, the PCR primer pair comprising a forward primer and a reverse primer;
the nucleotide sequence of the forward primer for the RBBP6 gene is shown as SEQ ID NO.1 or a nucleotide sequence having at least 80% homology with SEQ ID NO.1, preferably having at least 85% homology, further preferably having at least 90% homology, more preferably having more than 95% homology; the nucleotide sequence of the reverse primer is shown as SEQ ID NO.2, or a nucleotide sequence having at least 80% homology with SEQ ID NO.2, preferably having at least 85% homology, further preferably having at least 90% homology, more preferably having more than 95% homology;
the nucleotide sequence of the forward primer for the CCDC91 gene is shown in SEQ ID No.3, or a nucleotide sequence having at least 80% homology with SEQ ID No.3, preferably having at least 85% homology, further preferably having at least 90% homology, more preferably having more than 95% homology; the nucleotide sequence of the reverse primer is shown as SEQ ID NO.4, or a nucleotide sequence having at least 80% homology with SEQ ID NO.4, preferably having at least 85% homology, further preferably having at least 90% homology, and more preferably having 95% homology or more.
In a fourth aspect, the present application also provides a probe for amplifying an expression product of the above molecular marker, comprising:
the probe sequence for amplifying the RBBP6 gene expression product is shown as SEQ ID NO.5, or a nucleotide sequence having at least 80% homology with SEQ ID NO.5, preferably having at least 85% homology, further preferably having at least 90% homology, more preferably having more than 95% homology, and most preferably having more than 98% homology;
the probe sequence for amplifying the CCDC91 gene expression product is a nucleotide sequence as shown in SEQ ID No.6 or having at least 80% homology with SEQ ID No.6, preferably having at least 85% homology, more preferably having at least 90% homology, more preferably having more than 95% homology, and most preferably having more than 98% homology.
In a fifth aspect, the application also provides the use of the above PCR primer pair and the above probe for preparing a reagent for screening SARS-CoV-2 virus infected person.
The expression products of the RBBP6 gene and the CCDC91 gene are amplified by adopting the PCR primer pair and the probe pair so as to judge the expression level.
In a sixth aspect, the present application also provides a kit comprising the primer pair of the RBBP6 gene and the probe of the RBBP6 gene, and the primer pair of the CCDC91 gene and the probe of the CCDC91 gene.
The primer and the probe can be directly in the form of mixed liquid, or can be respectively and independently arranged in different containers, and the primer and the probe are mixed for use when the primer and the probe are to be used; in order to simplify the operation steps, it is preferable to store the primer and the probe in a kit in the form of a mixed solution.
Specifically, the kit comprises RBBP6 gene primer probe mixed solution and CCDC91 gene primer probe mixed solution; the RBBP6 gene primer probe mixed solution comprises the primer pair of the RBBP6 gene and the probe of the RBBP6 gene, and the CCDC91 gene primer probe mixed solution comprises the primer pair of the CCDC91 gene and the probe of the CCDC91 gene.
Further, the concentration ratio of each primer to the corresponding probe is 1-2:1.
by "each primer" herein is meant a single primer, for example, a forward primer or a reverse primer of the RBBP6 gene, or a forward primer or a reverse primer of the CCDC91 gene. "probe" as used herein refers to a single probe, such as a probe for amplifying the RBBP6 gene or a probe for amplifying the CCDC91 gene.
Further, the concentration of the working solution of each primer is 7-20. Mu.M, and the concentration of the working solution of each probe is 7-20. Mu.M.
Illustratively, the method for constructing the kit comprises the following steps:
(1) The 5 end of the probe for amplifying the RBBP6 gene expression product is modified by VIC, the 3 end is modified by BHQ1, the 5 end of the probe for amplifying the CCDC91 gene expression product is modified by FAM, and the 3 end is modified by BHQ 1.
(2) The primers and probes were purified by PAGE or HPLC.
(3) The primer and probe are mixed.
Specific modification method steps and purification method steps may be conventional in the art, and are not described herein.
In a seventh aspect, the application provides the use of the kit for detecting the expression levels of the RBBP6 gene and the CCDC91 gene.
The method for detecting the expression level of the RBBP6 gene and the CCDC91 gene can be a real-time fluorescence quantitative PCR method or a high-throughput sequencing method, wherein the real-time fluorescence quantitative PCR method adopts the kit. Specific steps can be seen in the following examples.
In an eighth aspect, the present application provides the use of a molecular marker expression product of a combination of the RBBP6 gene and the CCDC91 gene in the manufacture of a medicament for the treatment of covd-19.
Example 1
Screening differential expression genes in blood samples of SARS-CoV-2 virus infected persons by a high throughput sequencing method.
1. Collection of transcriptome data from asymptome SARS-CoV-2 virus blood samples
The number is obtained by GSA-Human (https:// ngdc. Cncb. Ac. Cn/GSA-Human /). Data set of HRA 000786. The dataset contains blood sample transcriptome data from isolated individuals closely spaced from the prior SARS-CoV-2 virus epidemic in the Shanghai. Subsequent clinical manifestations of the close-fitting person were classified based on the new coronavirus diagnostic guideline (ninth edition): healthy controls, asymptomatic, and symptomatic.
2. Detection of differentially expressed genes
The reference genome (hg 38) was obtained from Ensembl (https:// asia. Ensembl. Org/index. Html) along with the annotation GTF file, completing the gene name annotation. The expression matrix was generated by Salmon and then analyzed using DESeq2 to detect differentially expressed genes. Only q-value <0.05 in the DESeq2 output, the test showed that a successful comparison was considered a differentially expressed gene.
3. Results
The transcriptome results showed that the expression level of the RBBP6 gene and CCDC91 gene combination was significantly higher in both asymptomatic (fig. 1) and symptomatic (fig. 2) blood infected with SARS-CoV-2 virus than in blood not infected with SARS-CoV-2 virus, as shown in fig. 1 and 2.
Comparative example 1
Expression of RBBP6 gene and CCDC91 gene in blood sample infected with common respiratory tract virus
1. Sample collection
10 blood samples screened as positive for viruses (including influenza virus IFV, respiratory syncytial virus RSV, rhinovirus HRV, and seasonal coronavirus CoVs) by the viral nucleic acid detection kit were collected.
2. RNA extraction
The procedure was followed according to the method scheme of Magmax-96 visual Kit, and finally elution was performed with 50. Mu.L of eluent.
3. Reverse transcription
(a) Pre-denaturation
Preheating a PCR instrument to 65 ℃ in advance
Denaturation system:
RNA template: 7 mu L
Oligo(dT) 18 Primer：1μL
Random Primer(N9)：1μL
dNTPs：1μL
Total volume: 10 mu L
The deformation system was placed in a PCR instrument and kept at 65℃for 5min.
Ice is prepared in advance until the ice bath is immediately removed.
(b) One strand synthesis
Preparation of the SSIII inversion system:
10X Buffer：2μL
25mM MgCl2：4μL
0.1M DTT：2μL
RNaseOUT：1μL
SSIII reverse transcriptase: 1 mu L
Total volume: 10 mu L
The PCR instrument procedure was as follows: after finishing 25 ℃ for 10min, 50min, 5min, 85 ℃ and 4 ℃ hold, adding 1 mu L of RNaseH and keeping the temperature at 37 ℃ for 20min.
(c) Two-chain synthesis
Heating at 85deg.C for 5min, and ice-bathing for 3min. mu.L of random hexamer was added to the sample on ice, and finally 1. Mu.L Klenow enzyme was added. The mixture was placed on a PCR apparatus again, kept at 37℃for 60min, and then the primers and Klenow enzyme were added directly to the PCR apparatus again. After holding at 37℃for 60min, the two-chain product was recovered.
(d) cDNA product purification
The two-chain product was purified using 1.8. 1.8X Beckman Ampurebeads. After the magnetic beads are added, a tube cover is covered, vortex mixing is carried out, and standing is carried out for 5-10 minutes at room temperature. Then put on a magnetic rack for 1 minute, after the magnetic beads are thoroughly adsorbed on the tube wall, suck and discard the supernatant, then add 500 μl of 75% ethanol, cover the tube cover, turn the EP tube one quarter turn every 15 seconds, turn two turns altogether, carefully wash the magnetic beads on the tube wall with ethanol evenly, and then carefully suck and discard the ethanol. The magnetic beads are washed once again with 500 mu L of ethanol, most of the ethanol is sucked and discarded, and then the magnetic beads remained on the tube wall and the tube bottom are carefully sucked or scraped by a fine gun head, so that the residual ethanol is removed as completely as possible. Then the tube cover is kept open, and the tube cover is placed for 5-15 minutes at room temperature, so that the magnetic beads are naturally air-dried. When the magnetic beads are subjected to microcracking, 30 mu L of an absorption Buffer is quickly added, the mixture is blown and mixed uniformly, the mixture is quickly vortex and mixed uniformly, the mixture is placed on a magnetic rack again after being placed at room temperature for 10 minutes, the mixture is placed on the magnetic rack again, the mixture is waited for 1-2 minutes, the magnetic beads are thoroughly adsorbed on the pipe wall, and the solution dissolved with the nucleic acid is sucked and carefully added into a new EP pipe, so that the purified nucleic acid is obtained.
4. Real-time fluorescent quantitative PCR
Preparing a PCR reaction system:
and (3) a template: 2 mu L
Forward primer (10 μm): 1 mu L
Reverse primer (10. Mu.M): 1 mu L
Probe (10 μm): 1 mu L
Takara Ex Taq：13μL
Deionized water: make up the system to 25. Mu.L
PCR reaction conditions: 95℃for 10min, (95℃30s,60℃40 s). Times.40 cycles. The PCR reaction was performed on a Light Cycler fluorescent quantitative PCR instrument while collecting fluorescent signals of FAM and VIC channels.
5. Statistical method
The experimental result data were transformed by z-score and analyzed using the Wilcox rank sum test, and were considered statistically significant when p < 0.05.
6. Results
As a result, as shown in fig. 3 and 4, it can be seen that the expression levels of CCDC91 gene (fig. 4) and RBBP6 gene (fig. 3) in the blood samples infected with the common respiratory virus were not significantly changed from the expression levels in the blood samples not infected with the common respiratory virus, suggesting that CCDC91 and RBBP6 exhibit non-specificity to the common respiratory virus.
Example 2
Detection of expression levels of PBBP6 and CCDC91 genes in blood samples by real-time fluorescent quantitative PCR
1. Sample collection
Blood samples and nasopharyngeal swab samples from the staff providing medical assistance to the covd-19 patient were collected on a weekly basis. SARS-CoV-2 virus nucleic acid detection was performed by nasopharyngeal swab samples, followed by classification of subsequent clinical manifestations by healthcare workers according to the novel coronavirus diagnostic guideline (ninth edition): asymptomatic and symptomatic.
2. RNA extraction
The procedure was followed according to the method scheme of Magmax-96 visual Kit, and finally elution was performed with 50. Mu.L of eluent.
3. Reverse transcription
(a) Pre-denaturation
Preheating a PCR instrument to 65 ℃ in advance
Denaturation system:
RNA template: 8 mu L
Primer: 1 mu L
dNTPs：1μL
Total volume: 10 mu L
The deformation system was placed in a PCR instrument and kept at 65℃for 5min.
Ice is prepared in advance until the ice bath is immediately removed.
(b) One strand synthesis
Preparation of the SSIII inversion system:
10X Buffer：2μL
25mM MgCl2：4μL
0.1M DTT：2μL
RNaseOUT：1μL
SSIII reverse transcriptase: 1 mu L
Total volume: 10 mu L
The PCR instrument procedure was as follows: after finishing 25 ℃ for 10min, 50min, 5min, 85 ℃ and 4 ℃ hold, adding 1 mu L of RNaseH and keeping the temperature at 37 ℃ for 20min.
(c) Two-chain synthesis
Heating at 85deg.C for 5min, and ice-bathing for 3min. mu.L of random hexamer was added to the sample on ice, and finally 1. Mu.L Klenow enzyme was added. The mixture was placed on a PCR apparatus again, kept at 37℃for 60min, and then the primers and Klenow enzyme were added directly to the PCR apparatus again. After holding at 37℃for 60min, the two-chain product was recovered.
(d) cDNA product purification
The two-chain product was purified using 1.8. 1.8X Beckman Ampurebeads. After the magnetic beads are added, a tube cover is covered, vortex mixing is carried out, and standing is carried out for 5-10 minutes at room temperature. Then put on a magnetic rack for 1 minute, after the magnetic beads are thoroughly adsorbed on the tube wall, suck and discard the supernatant, then add 500 μl of 75% ethanol, cover the tube cover, turn the EP tube one quarter turn every 15 seconds, turn two turns altogether, carefully wash the magnetic beads on the tube wall with ethanol evenly, and then carefully suck and discard the ethanol. The magnetic beads are washed once again with 500 mu L of ethanol, most of the ethanol is sucked and discarded, and then the magnetic beads remained on the tube wall and the tube bottom are carefully sucked or scraped by a fine gun head, so that the residual ethanol is removed as completely as possible. Then the tube cover is kept open, and the tube cover is placed for 5-15 minutes at room temperature, so that the magnetic beads are naturally air-dried. When the magnetic beads are subjected to microcracking, 30 mu L of an absorption Buffer is quickly added, the mixture is blown and mixed uniformly, the mixture is quickly vortex and mixed uniformly, the mixture is placed on a magnetic rack again after being placed at room temperature for 10 minutes, the mixture is placed on the magnetic rack again, the mixture is waited for 1-2 minutes, the magnetic beads are thoroughly adsorbed on the pipe wall, and the solution dissolved with the nucleic acid is sucked and carefully added into a new EP pipe, so that the purified nucleic acid is obtained.
4. Real-time fluorescent quantitative PCR
Preparing a PCR reaction system:
and (3) a template: 2 mu L
Forward primer (10 μm): 1 mu L
Reverse primer (10. Mu.M): 1 mu L
Probe (10 μm): 1 mu L
Takara Ex Taq：13μL
Deionized water: make up the system to 25. Mu.L
PCR reaction conditions: 95℃for 10min, (95℃30s,60℃40 s). Times.40 cycles. PCR reactions were performed on a Light Cycler fluorescent quantitative PCR instrument using SYBR Green as a fluorescent marker.
5. Statistical method
Ct value results were converted by z-score and analyzed using the Wilcox rank sum test, and were considered statistically significant when p < 0.05.
6. Results
As shown in FIG. 5, it can be seen that the expression levels of the combination of CCDC91 and RBBP6 detection in both asymptomatic and symptomatic blood samples infected with SARS-CoV-2 virus are significantly higher than those in blood samples not infected with SARS-CoV-2 virus.
The present application is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present application are intended to be included in the scope of the present application.

Claims (7)

1. Application of a molecular marker consisting of RBBP6 gene and CCDC91 gene in preparing a kit for screening SARS-CoV-2 virus infected person, wherein the RBBP6 gene is located on human chromosome 16 and located in the interval 24539566-24572863; the CCDC91 gene is located on human chromosome 12 and located in the interval 28190456 to 28550166; the RBBP6 gene is NC_000016.10:24539566-24572863Homo sapiens chromosome 16, GRCh38.p14 Primary Assembly, and the CCDC91 gene is NC_000012.12:28190456-28550166Homo sapiens chromosome 12, GRCh38.p14 Primary Assembly.
2. Application of a primer for detecting a molecular marker expression product of a combination of an RBBP6 gene and a CCDC91 gene in preparation of a kit for screening SARS-CoV-2 virus infected persons, wherein the RBBP6 gene is located on human chromosome 16 and located in a 24539566-24572863 interval; the CCDC91 gene is located on human chromosome 12 and located in the interval 28190456 to 28550166; the RBBP6 gene is NC_000016.10:24539566-24572863Homo sapiens chromosome 16, GRCh38.p14 Primary Assembly, and the CCDC91 gene is NC_000012.12:28190456-28550166Homo sapiens chromosome 12, GRCh38.p14 Primary Assembly.
3. The use according to claim 1 or 2, wherein the PCR primer pair for amplifying the molecular marker expression product comprises a forward primer and a reverse primer;
the nucleotide sequence of the forward primer used for the RBBP6 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 2;
the nucleotide sequence of the forward primer used for the CCDC91 gene is shown as SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 4.
4. The use according to claim 1 or 2, characterized in that a probe for amplifying the expression product of the molecular marker comprises:
the probe sequence for amplifying the RBBP6 gene expression product is shown as SEQ ID NO.5, and the probe sequence for amplifying the CCDC91 gene expression product is shown as SEQ ID NO. 6.
5. The application of PCR primer pair for amplifying molecular marker composed of RBBP6 gene and CCDC91 gene and probe for amplifying molecular marker composed of RBBP6 gene and CCDC91 gene in preparing reagent kit for screening SARS-CoV-2 virus infected person is characterized by that,
the RBBP6 gene is positioned on a human chromosome 16 and positioned in a 24539566-24572863 interval; the CCDC91 gene is located on human chromosome 12 and located in the interval 28190456 to 28550166; the RBBP6 gene is NC_000016.10:24539566-24572863Homo sapiens chromosome 16, GRCh38.p14 Primary Assembly, and the CCDC91 gene is NC_000012.12:28190456-28550166Homo sapiens chromosome 12, GRCh38.p14 Primary Assembly;
the PCR primer pair for amplifying the molecular marker expression product comprises a forward primer and a reverse primer;
the nucleotide sequence of the forward primer used for the RBBP6 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 2;
the nucleotide sequence of the forward primer for the CCDC91 gene is shown as SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO. 4;
a probe for amplifying said molecular marker expression product comprising:
the probe sequence for amplifying the RBBP6 gene expression product is shown as SEQ ID NO.5, and the probe sequence for amplifying the CCDC91 gene expression product is shown as SEQ ID NO. 6.
6. The use according to claim 5, wherein the concentration ratio of each primer to the corresponding probe is 1-2:1.
7. the use according to claim 5, wherein the working fluid concentration of each primer isThe working solution concentration of each probe is +.>。
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