KR102653475B1 - Composition for detecting coronavirus simultaneously and method for detecting coronavirus simultaneously comprising the same - Google Patents

Abstract

According to the composition according to one aspect and the determination method using the same, it is possible to simultaneously detect and distinguish porcine epidemic diarrhea virus, porcine delta coronavirus, and porcine acute diarrhea syndrome coronavirus with only a small amount of specimen sample, and quickly and specifically Viruses can be detected and identified.

Description

Composition for detecting coronavirus simultaneously and method for detecting coronavirus simultaneously comprising the same}

It relates to a composition for simultaneously detecting porcine epidemic diarrhea virus, porcine delta coronavirus, and porcine acute diarrhea syndrome coronavirus, and a detection method containing the same. This result is funded by the Ministry of Agriculture, Food and Rural Affairs, and is supported by the Korea Institute of Agriculture, Food and Rural Affairs. The research was conducted with support from the Response Technology Upgrade Support Project (Project Number: 122012-2).

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a new swine coronavirus first reported in southern China in 2017, and is a type of porcine epidemic diarrhea virus (PEDV) and porcine delta coronavirus (PEDV). As it shows clinical symptoms similar to those of Porcine deltacoronavirus (PDCoV), prompt diagnosis and treatment of infection is necessary.

As one of the technologies for detecting porcine epidemic diarrhea virus (PEDV), domestic patent publication 10-2019-0026403 discloses a method for determining PEDV genotypes using genetic differences between genotypes of PEDV genes. A method for simultaneously detecting PDCoV and SADS-CoV is not disclosed.

Currently, due to the similar antigenicity of the three types of viruses, no technique has been developed to distinguish and diagnose the three types of viruses with just one test. Therefore, there is a need to develop a method that can distinguish and simultaneously detect the three types of viruses with just one test. This exists.

One aspect is to provide a composition for simultaneous detection of coronaviruses, including a primer set and a probe set for simultaneously detecting porcine epidemic diarrhea virus, porcine delta coronavirus, and porcine acute diarrhea syndrome coronavirus.

Another aspect is to provide a kit for simultaneous detection of coronaviruses including the composition for detecting the virus.

Another aspect includes (a) contacting the composition with a sample isolated from an individual; (b) performing a PCR reaction on a sample isolated from the individual and inducing binding between the PCR amplification product and the probe; and (c) confirming the binding between the PCR amplification product and the probe through analysis of fluorescence labeled on the probe.

One aspect is (a) a first primer pair for amplifying the region containing the base sequence of SEQ ID NO: 1, a second primer pair for amplifying the region containing the base sequence of SEQ ID NO: 2, and the base of SEQ ID NO: 3 A primer set including a third primer pair to amplify a region containing the sequence; and (b) a probe set including a first probe consisting of the base sequence of SEQ ID NO: 1, a second probe consisting of the base sequence of SEQ ID NO: 2, and a third probe consisting of the base sequence of SEQ ID NO: 3. , provides a composition for simultaneous detection of coronaviruses.

As used herein, the term “primer” refers to a primer that acts as an initiation point for template-directed DNA synthesis under suitable conditions (i.e., four different nucleoside triphosphates and polymerization enzymes) at a suitable temperature and buffer. refers to a single-stranded oligonucleotide that can be In addition, “Forward primer” and “Reverse primer” refer to primers that bind to the 3' and 5' ends, respectively, of a certain region of the template to be amplified by polymerase chain reaction.

As used herein, the term “probe” refers to a natural or modified monomer containing a deoxyribonucleotide that can hybridize to a specific polynucleotide sequence, or a linear oligomer having a bond.

A composition for simultaneous detection of coronaviruses according to one embodiment includes a primer set targeting genes in a region including the ORF1ab gene of PEDV, the M gene (Membrane gene) of PDCoV, and the N gene (Nucleocapsid gene) of SADS-CoV; It may include a probe set, and through their combination, PEDV, PDCoV, and SADS-CoV can be specifically detected and discriminated simultaneously.

The first primer pair may be for amplifying the 681st base sequence to the 812th base sequence of SEQ ID NO: 10, and the second primer pair may be for amplifying the 160th base sequence to the 322nd base sequence of SEQ ID NO: 11. may be, and the third primer pair may be for amplifying the 457th to 587th base sequence of SEQ ID NO: 12.

The SEQ ID NO: 10 corresponds to a specific gene region (2051-3317 bp) within the ORF1ab gene (Genebank Accession No. AF353511.1) region of PEDV, and SEQ ID NO: 11 corresponds to the M gene (membrane gene) of PDCoV (Genebank Accession No. KX443143.2), and SEQ ID NO: 12 corresponds to the N gene of SADS-CoV (Genebank Accession No. MH539766.1).

The first primer pair is for amplifying the gene in the region containing the ORF1ab gene of PEDV and may consist of a forward primer consisting of the base sequence of SEQ ID NO: 4 and a reverse primer consisting of the base sequence of SEQ ID NO: 5. The second primer pair is for amplifying the gene in the region containing the M gene (membrane gene) of PDCoV, and may consist of a forward primer consisting of the base sequence of SEQ ID NO: 6 and a reverse primer consisting of the base sequence of SEQ ID NO: 7. there is. The third primer pair is for amplifying the gene in the region containing the N gene of SADS-CoV, and may consist of a forward primer consisting of the base sequence of SEQ ID NO: 8 and a reverse primer consisting of the base sequence of SEQ ID NO: 9. If the primer pair is capable of amplifying the gene as described above, it has sequence homology of at least 80%, at least 85%, at least 90%, and at least 95% to the base sequences of SEQ ID NOs: 4 to 9, respectively. It can be made up of base sequences.

The probe can detect a target sequence for virus identification from a PCR amplification product using the primer pair. The probe may be labeled with a fluorescent substance. The fluorescent substances include 6-FAM (6-Carboxyfluorescein), TET (2',7'-dichloro-6-carboxy-4,7-dichlorofluorescein), Texas Red, and 6-JOE (6-carboxy-4',5 '-dichloro-2',7'-dimethoxyfluorescein), HEX(2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), Cy3(Cyanine 3), Cy5, Rhodamine (rhodamine) and VIC may be applied, but are not limited thereto.

In addition, the probe may be labeled with a fluorescent substance at the 5' end and a quencher at the 3' end for visualization using the principle of Fluorescent Resonance Energy Transfer (FRET). SFC-Q1, SFC-Q2, 6-TAMRA (6-carboxytetramethylrhodamine), BHQ (Black Hole Quencher)-1, BHQ-2, and BHQ-3 may be applied as the quenching material, but this is also limited thereto. That is not the case.

In one embodiment, the 5' ends of the first to third probes may be independently labeled with fluorescent substances having different wavelengths for virus identification and detection. Since fluorescent substances with unique excitation and emission wavelengths are labeled differently for each probe, the binding between the probe and the target sequence can be confirmed by detecting the unique signal derived from the fluorescent substance. .

In one embodiment, FAM may be labeled at the 5' end of the first probe for identifying PEDV, and VIC may be labeled at the 5' end of the second probe for identifying PDCoV. Additionally, Texas Red may be labeled at the 5' end of the third probe for identifying SADS-CoV.

In one embodiment, the 5' end of the first probe for identifying PEDV may be labeled with FAM, the 3' end may be labeled with SFC-Q1, and the 5' end of the second probe for identifying PDCoV may be labeled. VIC may be labeled, SFC-Q1 may be labeled at the 3' end, Texas Red may be labeled at the 5' end of the third probe to identify SADS-CoV, and SFC-Q2 may be labeled at the 3' end. there is.

The composition may be used to diagnose infection with one or more viruses selected from the group consisting of PEDV, PDCoV, and SADS-CoV. Specifically, the composition may be used to simultaneously diagnose PEDV, PDCoV, and SADS-CoV infections.

The composition includes one or more primer pairs selected from the primer pairs represented by SEQ ID NOs: 4 and 5, SEQ ID NOs: 6 and 7, and SEQ ID NOs: 8 and 9, which are specific for the PEDV, PDCoV, and SADS-CoV genes, respectively. It is possible to specifically diagnose whether infection is caused by a virus.

The composition may independently include one of the first to third primer pairs depending on the purpose of detecting PEDV, PDCoV, and SADS-CoV, and may simultaneously detect two or more of PEDV, PDCoV, and SADS-CoV. It may include two or more primer pairs selected from the first to third primer pairs. In order to simultaneously detect three types of PEDV, PDCoV, and SADS-CoV, all first to third primer pairs may be included. In addition, a primer pair may be additionally included to eliminate false positive diagnosis.

In addition to the primers, the composition may include probes capable of specifically binding to the PEDV, PDCoV, and SADS-CoV genes. The composition may independently contain one of the first to third probes depending on the purpose of detecting PEDV, PDCoV, and SADS-CoV, and to simultaneously detect two or more of PEDV, PDCoV, and SADS-CoV. It may include two or more probes selected from the first to third probes. All first to third probes may be included to simultaneously detect three types of PEDV, PDCoV, and SADS-CoV.

The composition may include a solution for stabilizing or preserving the polynucleotide and reagents necessary for amplification. The composition may further include DNA polymerase for performing a polymerase chain reaction. In addition to DNA polymerase, the composition may further include a reverse transcriptase for performing reverse transcription polymerase chain reaction.

Another aspect provides a kit for simultaneous detection of coronaviruses comprising the composition. In the kit for simultaneous detection of coronavirus, among the terms or elements mentioned, those that are the same as those already mentioned are as described above.

A kit for simultaneous detection of coronaviruses according to one embodiment may further include tools or reagents necessary for PCR reaction or detection of PCR amplification products in addition to the primer set or probe described above. In the case of reagents, for example, DNA polymerase, dNTP mixture (dATP, dCTP, dGTP, dTTP), PCR buffer, and DNA polymerase cofactor may be separately included.

Another aspect includes the steps of (a) contacting a sample isolated from an individual with a composition comprising a primer set and a probe set; (b) performing a PCR reaction on a sample isolated from the individual and inducing binding between the PCR amplification product and the probe; and (c) confirming the binding between the PCR amplification product and the probe through analysis of fluorescence labeled on the probe,

The primer set includes a first primer pair for amplifying the region containing the base sequence of SEQ ID NO: 1, a second primer pair for amplifying the region containing the base sequence of SEQ ID NO: 2, and the base sequence of SEQ ID NO: 3. It includes a third primer pair for amplifying the region containing, and the probe set includes a first probe consisting of the base sequence of SEQ ID NO: 1, a second probe consisting of the base sequence of SEQ ID NO: 2, and a base sequence of SEQ ID NO: 3. It provides a method for simultaneously detecting coronaviruses, including a third probe, wherein the first to third probes are labeled with a fluorescent substance at their ends.

In the method for simultaneously detecting the coronavirus, among the terms or elements mentioned, those that are the same as those already mentioned are as described above.

Samples isolated from the subject include blood and other liquid samples of biological origin, biopsy specimens, solid tissue samples such as tissue cultures, or cells derived therefrom. More specifically, examples include, but are not limited to, tissues, extracts, cell lysates, whole blood, plasma, serum, saliva, sputum, oropharyngeal and nasopharyngeal specimens, ocular fluid, cerebrospinal fluid, sweat, urine, milk, ascites fluid, synovial fluid. , peritoneal fluid, etc. The sample may be obtained from an animal, preferably a mammal, most preferably a pig. The sample may be pretreated before use for detection. For example, it may include filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, etc. Additionally, nucleic acids and proteins can be separated from the sample and used for detection.

According to one embodiment, the method for simultaneously detecting the coronavirus is to detect the region containing the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 2 of a sample isolated from an individual through a PCR reaction using the above-described primer set. A step of amplifying at least one of the region containing the base sequence and the region containing the base sequence of SEQ ID NO: 3; And it may include the step of annealing the target sequence and the probe in the PCR amplification product. The PCR reaction and annealing conditions may be carried out under conditions known in the art, and may be changed as appropriate.

The polymerase chain reaction may be quantitative real time polymerase chain reaction (qRT-PCR), and may be performed in combination with reverse transcript-polymerase chain reaction.

In the polymerase chain reaction, the concentration of the first to third primer pairs is each 0.01 to 2.0 pmol/μl, for example 0.1 to 2.0 pmol/μl, for example 0.1 to 1.0 pmol/μl, for example 0.1 to 2.0 pmol/μl. 0.9, for example 0.1 to 0.8 pmol/μl, for example 0.2 to 1.0, for example 0.2 to 0.9 pmol/μl, for example 0.2 to 0.8 pmol/μl.

In the polymerase chain reaction, the concentration of the first to third probes is each 0.01 to 2.0 pmol/μl, for example 0.1 to 2.0 pmol/μl, for example 0.1 to 1.0 pmol/μl, for example 0.1 to 0.9. , for example 0.1 to 0.8 pmol/μl, for example 0.2 to 1.0, for example 0.2 to 0.9 pmol/μl, for example 0.2 to 0.8 pmol/μl.

Binding between the target sequence and the probe through a series of reactions can be confirmed through a specific signal in the corresponding wavelength range originating from the fluorescent substance labeled at the 5’ end of the probe. Since each probe is labeled with a different fluorescent substance, porcine epidemic diarrhea virus, porcine delta coronavirus, and porcine acute diarrhea syndrome coronavirus can be easily identified at the same time through detection of unique signals derived from the fluorescent substance.

According to the composition according to one aspect and the detection method using the same, it is possible to simultaneously detect and discriminate porcine epidemic diarrhea virus, porcine delta coronavirus, and porcine acute diarrhea syndrome coronavirus with only a small amount of specimen sample, and quickly and specifically Viruses can be detected and identified.

Figure 1 shows the gene region (2051-3317 bp) in the ORF1ab (Genebank Accession No. AF353511.1) region of PEDV that is specifically amplified by the first primer pair. The regions marked with PEDV-F and PEDV-R represent the sites where the forward primer and reverse primer according to one embodiment bind, and the regions marked with PEDV-P represent the sites where the probe binds according to one embodiment.
Figure 2 shows the gene region of PDCoV specifically amplified by the second primer pair. The regions marked PDCoV-F and PDCoV-R represent the sites where the forward primer and reverse primer according to one embodiment bind, and the regions marked PDCoV-P represent the sites where the probe binds according to one embodiment.
Figure 3 shows the gene region of SADS-CoV specifically amplified by the third primer pair. The regions marked with SADS-CoV-F and SADS-CoV-R represent the sites where the forward and reverse primers according to one embodiment bind, and the regions marked with SADS-CoV-P represent the sites where the probe according to one embodiment binds. It shows the part where it is done.
Figure 4 relates to a standard curve of multiplex qRT-PCR (quantitative real time polymerase chain reaction) using primers for amplifying the gene of PEDV according to one embodiment.
Figure 5 relates to a standard curve of multiplex qRT-PCR using primers for amplifying the gene of PDCoV according to one embodiment.
Figure 6 relates to the standard curve of multiplex qRT-PCR using primers for amplifying the gene of SADS-CoV according to one embodiment.
Figure 7 relates to BLASTn results for PCR amplicons of PEDV.
Figure 8 relates to BLASTn results for PCR amplicons of PDCoV
Figure 9 relates to BLASTn results for PCR amplicons of SADS-CoV
Figure 10 relates to the standard curve of multiplex qRT-PCR for PEDV.
Figure 11 relates to the standard curve of multiplex qRT-PCR for PDCoV.
Figure 12 relates to the standard curve of multiplex qRT-PCR for SADS-CoV.
Figure 13 shows the results of multiplex qRT-PCR for PEDV and PDCOV positive specimens as an amplification curve. (IC: internal control, RFU: relative fluorescence units)
Figure 14 is an amplification curve showing the results of multiplex qRT-PCR to determine the minimum detection concentration of SADS-CoV, PDCoV, and PEDV. Purple is the result for IC, blue is the result for PEDV, yellow is the result for PDCoV, and red is the result for SADS-CoV.
Figure 15 is an amplification curve showing the results of multiplex qRT-PCR to confirm the minimum detection concentration of SADS-CoV, PDCoV, and PEDV. Purple is the result for IC, blue is the result for PEDV, yellow is the result for PDCoV, and red is the result for SADS-CoV.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1. Preparation of primer set for virus detection

In this example, a set of primers for detecting the three types of viruses was created based on NCBI's GeneBank and PEDV, PDCoV, and SADS-CoV genetic information. Targeting each of the genes of PEDV, PDCoV, and SADS-CoV, the primers are a region containing SEQ ID NO: 1 for PEDV, a region containing SEQ ID NO: 2 for SADS-CoV, and SEQ ID NO: 3 for PDCoV, respectively. It was manufactured to amplify a specific region among the regions containing.

Specifically, candidate primers were designed considering primer length, Tm, and GC content, and the Ct (Cycle Threshold) value, RFU (relative fluorescence unit) value, standard deviation, and ΔCt value between the candidate primers were compared to produce the following primers was selected. Additionally, information on the Ct value and RFU (relative fluorescence unit) value of each selected primer is shown in Tables 1 to 3 and Figures 4 to 6. Tables 1 to 3 show the results of qRT-PCR using primer sets for detecting PEDV, PDCoV, and SADS-CoV according to an example selected according to the above criteria, and other candidate primers (Comparative Example 1 The results for to 8) are shown. The graphs shown in Figures 4 to 6 all relate to the results performed with each viral RNA at a concentration of 10 ³ copies/μL.

[Table 1]

[Table 2]

[Table 3]

Through this, it was confirmed that the primer set according to the selected example as described above can simultaneously discriminate and detect PEDV, PDCoV, and SADS-CoV with excellent sensitivity. Accordingly, the base sequence of the primer set and the size of the amplification product according to the selected example are shown in Table 4 below.

[Table 4]

Example 2. Production of probe for virus identification

In this implementation, based on NCBI's GeneBank and gene information for PEDV, PDCoV, and SADS-CoV, virus discrimination probes were produced to detect each target sequence. Specifically, for PEDV, a sequence 100% identical to the 745-774th nucleotide sequence of SEQ ID NO: 10, for PDCoV, a sequence 100% identical to the 225-252nd nucleotide sequence of SEQ ID NO: 11, and for SADS-CoV. It was created to have a sequence that is 100% identical to the 497-520th base sequence of number 12.

The base sequence of the probe according to one example for virus identification is shown in Table 5 below.

[Table 5]

In addition, in order to simultaneously determine PEDV, PDCoV, and SADS-CoV in the sample, FAM was labeled at the 5' end of the first probe to determine PEDV, and SFC-Q1 was labeled at the 3' end, and PDCoV was identified. VIC was labeled at the 5' end of the second probe, SFC-Q1 was labeled at the 3' end, and Texas Red (SFC-C610) was labeled at the 5' end of the third probe to identify SADS-CoV. And SFC-Q2 was labeled at the 3' end.

Experimental Example 1. Evaluation of primers and probes

(1.1) Inclusivity evaluation

In this example, the comprehensive detection performance of the primer and probe set according to one embodiment for each target virus-derived gene, that is, the comprehensive detection performance for various mutants or strains of each PEDV, PDCoV, and SADS-CoV virus was attempted. . For this purpose, BLASTn analysis was performed on the whole genome sequences of various mutants and strains of each virus using the NCBI GeneBank Database, and as a result, the primers and It was confirmed that the probes were 100% identical to the gene sequences of each virus (Figures 7 to 9).

Through this, a set of primers and probes according to one embodiment can simultaneously detect the target viruses PEDV, PDCoV, and SADS-CoV with high sensitivity, and can also detect multiple mutants or strains of the viruses. It can be seen that comprehensive detection is possible.

(1.2) Exclusitivy evaluation

In this example, we attempted to confirm the detection specificity of the primer and probe set according to one example for the target virus. For this purpose, BLASTn analysis was performed on the full-length genome sequences of pathogens other than PEDV, PDCoV, and SADS-CoV, which are the viruses to be detected by the present invention, using the NCBI GeneBank Database.

In addition to PEDV, PDCoV, and SADS-CoV, BLASTn analysis was also performed on the full-length genome sequences of Swine enteric coronavirus (SECoV) and Porcine transmissible gastroenteritis virus (TGEV), and the results are shown in Tables 6 to 8.

Table 6 shows the results of PEDV-F, PEDV-P, and PEDV-R, Table 7 shows the results of PDCoV-F, PDCoV-P, and PDCoV-R, and Table 8 shows the results of SADS-CoV-F, SADS-CoV- This is the result of P, and SADS-CoV-R (the % homology value was obtained by multiplying the query coverage value and the identity value. Identity of less than 40% was determined to have no homology and was marked as '/ = no alignment found'. ).

[Table 6]

[Table 7]

[Table 8]

As a result, it was confirmed that the set of primers and probes for each virus according to one example showed 100% homology only to the sequence of the target virus, and showed low homology to other virus sequences. Accordingly, the primer and probe set according to one embodiment can exhibit excellent sensitivity as it shows 100% homology to the target viral gene, and shows excellent specificity as it shows low homology to other viral genes. It was confirmed that simultaneous detection and discrimination of each virus is possible.

Experimental Example 2. Evaluation of sensitivity, specificity and precision of primer and probe sets

(2.1) Perform Real-Time PCR

Real-time PCR reaction was performed according to the protocol of the CFX96™ Real-Time PCR Detection System instrument. The reaction solution used in the real-time PCR reaction was prepared with the composition shown in Table 9 below.

[Table 9]

The specific composition of the primer and probe mixture is shown in Table 10 below.

[Table 10]

The template in Table 9 was included in 5 ㎕ each at a concentration of 10 ³ Copies/㎕, 10 ² Copies/㎕, and 10 ¹ Copies/㎕ for each of the PEDV, PDCOV, and SADS-CoV standard samples.

The RT-PCR reaction conditions are as shown in Table 11.

[Table 11]

(2.2) Check the standard curve

In order to confirm the sensitivity of the composition according to one embodiment, Real-Time PCR was performed as in Experimental Example (2.1), and then a standard curve for the results was generated to determine the slope, correlation coefficient, R2), and PCR efficiency values were confirmed.

For this purpose, 1000 copies/μL of in vitro mRNA of PEDV, PDCoV, and SADS-CoV standard samples were serially diluted in 10-fold increments and mixed with the Real-Time PCR reaction mixture, and multiplex RT-PCR reaction was performed 3 times. A standard curve was created. The results are shown in Table 12 below and Figures 10 to 12.

[Table 12]

From the above results, it was confirmed that the slope value of the standard curve was -3.1 to -3.7, the correlation coefficient value was 0.98 to 1, and the PCR efficiency was 80-120%. It was confirmed that the composition for detecting a virus according to one embodiment has an excellent effect on each target gene. It was found that it was detected with sensitivity.

Experimental Example 3. Sample amplification confirmation test

In this experimental example, in order to confirm whether the composition according to one embodiment exhibits excellent sensitivity and specificity for target virus-positive samples, multiple Real Real -Time PCR was performed. If the Ct value was detected within 40, it was judged to be positive for the virus. Arabidopsis thaliana Actin gene (Exogeneus gene) was used as IC (internal control).

As a result, it was confirmed that PDCoV and SADS-COV signals were not amplified in the PEDV-positive specimen sample, and only the PEDV signal was detected. In addition, it was confirmed that SADS-COV and PEDV signals were not amplified in PDCoV positive specimen samples, and only PDCoV was detected (Figure 13).

Through this, it was confirmed that the composition according to one embodiment detects viruses with high sensitivity and specificity even in actual target virus positive samples. Through this, the composition according to one embodiment simultaneously discriminates only the target virus with excellent sensitivity and specificity. It was found that it could be detected.

Experimental Example 4. Minimum detection limit measurement

(4.1) Minimum detection limit measurement

In this experimental example, to evaluate the minimum detection limit of the multiple real-time PCR measurement method using the composition according to one embodiment, SADS-CoV, PEDV, and PDCoV RNA were 10 copies/μL, 5 copies/μL, and 2.5 copies/μL. , multiplex RT-PCR was performed by diluting to a concentration of 1 copies/μL and 0.5 copies/μL. Arabidopsis thaliana Actin gene (Exogeneus gene) was used as IC (internal control). The results are shown in Figure 14 and Tables 13 to 15 below.

[Table 13]

[Table 14]

[Table 15]

According to the above results, 5 copies/μL, which is the RNA concentration at which more than 95% of SADS-CoV, PEDV, and PDCoV were all detected, was selected as the minimum detection limit.

(4.2) Check the minimum detection limit

To confirm that the minimum detection limit of 5 copies/μL confirmed above can detect the target viral gene with high sensitivity, multiplex qRT-PCR was performed. Arabidopsis thaliana Actin gene (Exogeneus gene) was used as IC (internal control). The results are shown in Figure 15, and the Ct, average value of Ct, standard deviation, and coefficient of variation (CV%) are shown in Table 16 below.

[Table 16]

Through these results, it was confirmed that the composition for detecting viruses according to one embodiment can simultaneously detect SADS-CoV, PEDV, and PDCoV at 100% at a minimum detection limit of 5 copies/μL. This indicates that the composition for simultaneous detection of coronaviruses according to one embodiment can simultaneously discriminate and detect PEDV, PDCoV, and SADS-CoV with excellent sensitivity.

In addition, it was confirmed that the composition for virus detection according to one example showed significantly low CV% values (1.63, 2.15, and 2.42, respectively), and these results showed that the composition discriminated SADS-CoV, PEDV, and PDCoV with excellent precision and This indicates that it can be detected.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive.

Claims (12)

(a) a first primer pair for amplifying the region containing the base sequence of SEQ ID NO: 1, a second primer pair for amplifying the region containing the base sequence of SEQ ID NO: 2, and the base sequence of SEQ ID NO: 3 A primer set including a third primer pair for amplifying the region; and
(b) comprising a probe set including a first probe consisting of the base sequence of SEQ ID NO: 1, a second probe consisting of the base sequence of SEQ ID NO: 2, and a third probe consisting of the base sequence of SEQ ID NO: 3, A composition for simultaneous detection of coronaviruses,
The coronaviruses are Porcine epidemic diarrhea virus (PEDV), Porcine deltacoronavirus (PDCoV), and Swine acute diarrhea syndrome coronavirus (SADS-CoV),
The first primer pair is a forward primer consisting of the nucleotide sequence of SEQ ID NO: 4 and the reverse primer consisting of the nucleotide sequence of SEQ ID NO: 5, and the second primer pair is a forward primer consisting of the nucleotide sequence of SEQ ID NO: 6 and the reverse primer of SEQ ID NO: 7 A composition for simultaneous detection of coronaviruses, wherein the third primer pair is a forward primer consisting of a base sequence of SEQ ID NO: 8 and a reverse primer consisting of a base sequence of SEQ ID NO: 9.
delete delete The method of claim 1, wherein the first primer pair is for amplifying the gene of porcine epidemic diarrhea virus (PEDV), and the second primer pair is for amplifying the gene of porcine deltacoronavirus (PDCoV). A composition for simultaneous detection of coronaviruses, wherein the third primer pair is for amplifying the gene of swine acute diarrhea syndrome coronavirus (SADS-CoV).
The composition of claim 1, wherein the first probe is for detecting PEDV, the second probe is for detecting PDCoV, and the third probe is for detecting SADS-CoV. .
The composition for simultaneous detection of coronaviruses according to claim 1, wherein the first to third probes are labeled with a fluorescent substance.
The composition for simultaneous detection of coronaviruses according to claim 6, wherein the fluorescent substance labeled with the first to third probes is different for each probe.
delete A kit for simultaneous detection of coronaviruses, comprising the composition of any one of claims 1 and 4 to 7.
(a) contacting a sample isolated from an individual with a composition containing a primer set and a probe set;
(b) performing a PCR reaction on a sample isolated from the individual and inducing binding between the PCR amplification product and the probe; and
(c) Confirming the binding between the PCR amplification product and the probe through analysis of fluorescence labeled on the probe,
The primer set includes a first primer pair for amplifying the region containing the base sequence of SEQ ID NO: 1, a second primer pair for amplifying the region containing the base sequence of SEQ ID NO: 2, and the base sequence of SEQ ID NO: 3. Comprising a third primer pair for amplifying the region comprising,
The probe set includes a first probe consisting of the base sequence of SEQ ID NO: 1, a second probe consisting of the base sequence of SEQ ID NO: 2, and a third probe consisting of the base sequence of SEQ ID NO: 3,
The first to third probes are labeled with a fluorescent substance at their ends,
The coronaviruses are PEDV, PDCoV and SADS-CoV,
The first primer pair is a forward primer composed of the nucleotide sequence of SEQ ID NO: 4 and the reverse primer composed of the nucleotide sequence of SEQ ID NO: 5, and the second primer pair is a forward primer composed of the nucleotide sequence of SEQ ID NO: 6 and a reverse primer composed of the nucleotide sequence of SEQ ID NO: 7 A method for simultaneously detecting a coronavirus, wherein the reverse primer consists of a base sequence, and the third primer pair is a forward primer consisting of the base sequence of SEQ ID NO: 8 and a reverse primer consisting of the base sequence of SEQ ID NO: 9.
The method of claim 10, wherein the fluorescent substance labeled at the ends of the first to third probes is different for each probe.
delete