KR102072109B1 - Composition for Detecting Virus Relating Respiratory Disease and Kit for Detecting Virus Relating Respiratory Disease Comprising the Same - Google Patents

Abstract

The present invention is a respiratory virus diagnostic primer and / or probe set comprising a primer and / or a probe to confirm whether the respiratory virus infection, respiratory virus diagnostic composition comprising the primer and / or probe set, the primer and / Or it relates to a kit for diagnosing a respiratory virus comprising a probe set or composition and a method for providing information for diagnosing a respiratory virus comprising using the composition. When performing a real-time multiple reverse transcriptase chain reaction using the composition according to the present invention it is possible to quickly detect 14 kinds of respiratory viruses at the same time through the reaction of five tubes (once) to make a clinical diagnosis.

Description

Composition for detecting respiratory viruses, and kit for detecting respiratory viruses containing the same {Composition for Detecting Virus Relating Respiratory Disease and Kit for Detecting Virus Relating Respiratory Disease Comprising the Same}

The present invention relates to a composition for detecting a respiratory virus and a kit for detecting a respiratory virus comprising the same, and more particularly, the present invention is a primer for diagnosing a respiratory virus comprising a primer and/or a probe for confirming the infection of a respiratory virus. And/or a probe set, a respiratory virus diagnostic composition comprising the primer and/or probe set, a respiratory virus diagnostic kit comprising the primer and/or probe set or composition, and a respiratory virus diagnosis comprising the step of using the composition. It relates to the method of providing information for the purpose.

Respiratory viruses are the leading cause of acute respiratory infections and are the most common disease regardless of age or gender. In particular, it is known that infants, the elderly, and those with reduced cardiopulmonary function and immune function can cause sequelae and lead to death. Diseases associated with respiratory viruses most commonly start with cold symptoms, and range from pharyngitis, exacerbation of chronic asthma, and pneumonia. Early diagnosis of such a respiratory virus has the advantage of preventing unnecessary use of antibiotics, detecting the type of virus early, and performing appropriate patient treatment.

Methods for diagnosing such respiratory viruses include traditional cell culture, rapid cell culture, rapid antigen non-immunofluorescence, rapid antigen immunofluorescence, and molecular diagnosis using electrophoresis. In the case of the current standard test method, the cell culture method takes 5 to 9 days or more to check the test results, making it difficult to diagnose and treat a respiratory viral infection early, and to secure these shortcomings, while checking the results within 24 to 72 hours. As for the sensitivity, the R-mix virus culture method, which is similar to the traditional culture method, is being used, but it also shows limitations in early diagnosis and treatment. The rapid antigen non-immunofluorescence test method can confirm the result in a short time, but the sensitivity is lower than that of the traditional culture method, so there are many disadvantages of false negative. The molecular diagnosis method using the electrophoresis method solved the sensitivity problem, but it proceeds in 2 steps to be contaminated. There is a disadvantage that the problem may occur and it takes a long time.

Under this background, the present inventors made diligent efforts to find a method that can overcome the problems of sensitivity, contamination, and respiratory virus diagnostics that take a long time. As a result, each primer set capable of amplifying genes specific to 14 kinds of viruses, and By developing a probe, it was confirmed that 14 types of respiratory viruses can be diagnosed with high sensitivity and specificity by the One-Step real-time RT-PCR method, and the present invention was completed.

As a result, one object of the present invention is to provide a primer set for diagnosing respiratory viruses, including primers that enable confirmation of infection of respiratory viruses.

Another object of the present invention is to provide a composition for diagnosing respiratory viruses comprising a primer set and a probe capable of detecting a specific gene of a respiratory virus, a DNA polymerase, a reverse transcriptase, and the like.

Another object of the present invention is to provide a kit for diagnosing respiratory viruses comprising the primer set or composition.

Another object of the present invention is to provide a method of providing information for diagnosing respiratory viruses comprising the step of using the composition or kit.

As an aspect for achieving the above object, the present invention is SEQ ID NO: 1-2, 3-4, 5-6, 7-8, 9-10, 11-12, so that the infection of the respiratory virus can be confirmed. 13-14, 17-18, 19-20, 21-22, 23-24, 25-26, 32-33 and comprising a combination of the forward sequence of SEQ ID NO: 27-29 and reverse sequence of SEQ ID NO: 30-31 It provides a primer set for diagnosing respiratory viruses selected from a group consisting of a primer set.

In the present invention, the term "diagnosis" means to confirm a pathological condition. For the purpose of the present invention, the diagnosis is to confirm the onset of diseases caused by respiratory viruses and the prognosis in the treatment of antiviral drugs after the onset by checking the expression of genes specific to various existing respiratory viruses.

The term "primer" in the present invention is a nucleic acid sequence having a short free 3'hydroxyl group, which can form a complementary template and a base pair, and is a starting point for template strand copying. It refers to a short nucleic acid sequence that functions as. The primer can initiate DNA synthesis in the presence of a reagent for polymerization (DNA polymerase or reverse transcriptase) and four different deoxynucleoside triphospates (dNTPs) at an appropriate buffer and temperature. According to the present invention, the primer is preferably composed of each of the forward and reverse nucleic acids having 10 to 30 nucleotide sequences.

The primer can amplify a gene specific for a respiratory virus through a polymerase chain reaction to confirm whether the respiratory virus is infected, but the respiratory virus is not particularly limited thereto, but Corona Virus 229E, Corona Virus 229E, Corona Virus 229E Corona Virus OC43, Corona Virus NL63, Influenza A Virus, Influenza B Virus, Parainfluenza Virus 1, Parainfluenza Virus 2 ( Parainfluenza Virus 2), Parainfluenza Virus 3, Rs Virus A (Respiratory Syncytial Virus A), Rs Virus B (Respiratory Syncytial Virus B), Adeno Virus, Rhino Virus A, B, C ( Rhino Virus A, B, C), Metapneumovirus, Boca Virus, and the like, and genes that can distinguish each respiratory virus from other viruses are not particularly limited thereto, but coronavirus Nucleoprotein (N) gene of 229E, nucleoprotein (N) gene of coronavirus OC43, polyprotein (1a) gene of coronavirus NL63 ), matrix protein (M) gene of influenza A virus, hemagglutinin (HA) gene of influenza B virus, hemagglutinin of parainfluenza virus 1 -Neuraminidase (HN) gene (hemagglutinin-neuraminidase (HN) gene), Hemagglutinin-neuraminidase (HN) gene of parainfluenza virus 2, hemagglutinin-neuraminidase (HN) gene of parainfluenza virus 3 (hemagglutinin-neuraminidase) (HN) gene), Rs virus A fusion protein gene, Rs virus B fusion protein gene, adenovirus hexon protein gene, or rhinovirus A, B , C 5'UTR, metapneumovirus nucleoprotein (N) gene, Boca virus nucleocapsid protein (NP) gene, Viral protein (VP) gene, and the like.

Looking at the present invention from another point of view, the primers for confirming the infection of the respiratory virus of the present invention are not particularly limited thereto, but SEQ ID NOs: 1 and 2 capable of amplifying the nuclear protein (N) gene of corona virus 229E. A primer having a nucleotide sequence of, a primer having a nucleotide sequence of SEQ ID NO: 3 and 4 capable of amplifying the nuclear protein (N) gene of corona virus OC43, a sequence capable of amplifying the polyprotein (1a) gene of corona virus NL63 Primers having nucleotide sequences of Nos. 5 and 6, primers having nucleotide sequences of SEQ ID Nos. 7 and 8 capable of amplifying the hemeglutinin-neuramidase (HN) gene of parainfluenza virus 1, parainfluenza virus 2 The primers having the nucleotide sequences of SEQ ID NOs: 9 and 10 capable of amplifying the hemeglutinin-neuraminidase (HN) gene of, and the hemeglutinin-neuramidase (HN) gene of the parainfluenza virus 3 Primers having base sequences of SEQ ID NOs: 11 and 12 that can be amplified, primers having base sequences of SEQ ID NOs: 13 and 14 that can amplify the matrix protein (M) gene of influenza A virus, hemegluti of influenza B virus Primers having the nucleotide sequences of SEQ ID NOs: 17 and 18 capable of amplifying the nin (HA) gene, primers having the nucleotide sequences of SEQ ID NOs: 19 and 20 capable of amplifying the fusion protein gene of Rs virus A, Rs virus B Primers having the nucleotide sequences of SEQ ID NOs: 21 and 22 capable of amplifying the fusion protein gene, primers having the nucleotide sequences of SEQ ID NOs: 25 and 26 capable of amplifying the hexon protein gene of adenovirus, rhinovirus A, B, C Primers having the nucleotide sequences of SEQ ID NOs: 27 and 31 capable of amplifying the 5'UTR of, primers having the nucleotide sequences of SEQ ID NOs: 23 and 24 capable of amplifying the nucleoprotein (N) gene of metapneumovirus, Boca virus Nucleocapsid protein of It is preferable to use a primer having nucleotide sequences of SEQ ID NOs: 32 and 33 that can amplify the (NP) gene and the Viral protein (VP) gene.

Primers can incorporate additional features that do not change the basic properties of the primers serving as the starting point for DNA synthesis. In addition, the primer nucleic acid sequence of the present invention may include a label detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means, if necessary. Examples of labels include enzymes (e.g. horseradish peroxidase, alkaline phosphatase), radioactive isotopes (e.g., ³² P), fluorescent molecules, chemical groups (e.g., biotin), etc. There is this.

According to another aspect of the present invention, the present invention relates to a composition for diagnosing respiratory viruses comprising the primer set. In this case, the composition may further include a probe having nucleotide sequences of SEQ ID NOs: 34 to 40 and 42 to 52 to detect a specific gene of a respiratory virus.

In the present invention, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a few bases or hundreds of bases for a specific binding with a complementary base sequence, and is labeled so that the presence or absence of a specific base sequence You can check. The probe may be manufactured in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like. In addition, in order to perform real-time PCR, a fluorescently labeled probe may be used. More specifically, when using the TaqMan probe, an oligonucleotide modified with a fluorescent material at the 5'end and a quencher material at the 3'end is added to the PCR reaction solution. In addition, real-time PCR using a cycling probe is a highly sensitive detection method composed of a chimeric probe composed of RNA and DNA and RNAse H. Again, the 5'end of the probe is labeled with a fluorescent material and the 3'end with a quencher material.

Looking at the present invention from another point of view, the present inventors perform real-time PCR to detect the amplification product, so that the increase in the amplification product can be monitored in real time, it is possible to accurately quantify DNA and RNA, and electrophoresis is possible. Since it is not necessary, it can be interpreted quickly and easily, and the risk of contamination was small, and a fluorescently labeled probe was manufactured to perform real-time PCR. The probe is not particularly limited thereto, but a probe having a nucleotide sequence of SEQ ID NO: 34 capable of detecting the nuclear protein (N) gene of corona virus 229E, a sequence capable of detecting the nuclear protein (N) gene of corona virus OC43 A probe having a nucleotide sequence of No. 35, a probe having a nucleotide sequence of SEQ ID No. 36 capable of detecting the polyprotein (1a) gene of coronavirus NL63, hemeglutinin-neuramidase of parainfluenza virus 1 (HN ) A probe having a nucleotide sequence of SEQ ID NO: 37 capable of detecting a gene, a probe having a nucleotide sequence of SEQ ID NO: 38 capable of detecting a hemeglutinin-neuraminidase (HN) gene of parainfluenza virus 2, A probe having a nucleotide sequence of SEQ ID NO: 39 capable of detecting the hemeglutinin-neuramidase (HN) gene of parainfluenza virus 3, and a sequence number capable of detecting the matrix protein (M) gene of influenza A virus. A probe having a nucleotide sequence of 40, a probe having a nucleotide sequence of SEQ ID NO: 42 capable of detecting the hemeglutinin (HA) gene of influenza B virus, and SEQ ID NO: 43 capable of detecting a fusion protein gene of Rs virus A A probe having a nucleotide sequence of, a probe having a nucleotide sequence of SEQ ID NO: 44 capable of detecting a fusion protein gene of Rs virus B, a nucleotide sequence of SEQ ID NO: 45 capable of detecting a nuclear protein (N) gene of metapneumovirus A probe having, a probe having a nucleotide sequence of SEQ ID NO: 46-49 capable of detecting the hexon protein gene of adenovirus, a base of SEQ ID NO: 50-51 capable of detecting the 5'UTR of rhinovirus A, B, C It is preferable to use a probe having a sequence, a probe having a nucleotide sequence of SEQ ID NO: 52 capable of detecting the nucleocapsid protein (NP) gene of the Boca virus, and the Viral protein (VP) gene.

The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well known methods. Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, “encapsulation”, substitution of one or more homologs of natural nucleotides, and modifications between nucleotides, such as uncharged linkers (eg, methyl phosphonate, phosphotriester, Phosphoroamidate, carbamate, etc.) or to a charged linker (eg phosphorothioate, phosphorodithioate, etc.).

Looking at the present invention from another perspective, the composition for diagnosing respiratory viruses of the present invention may further include a DNA polymerase for performing a polymerase chain reaction as well as the probe, but the DNA polymerase is not particularly limited thereto. However, it is preferable to use Hot start Taq DNA polymerase.

Looking at the present invention from another perspective, the composition for diagnosing respiratory viruses of the present invention targets a specific gene of a respiratory virus obtained by applying RNA extracted from a sample to a reverse transcription polymerase chain reaction. The composition for diagnosing respiratory viruses may further include a reverse transcriptase for performing a reverse transcriptase polymerase chain reaction in addition to the probe and the DNA polymerase.

According to another aspect of the present invention, the present invention relates to a kit for diagnosing respiratory viruses comprising the composition for diagnosing respiratory viruses. Using the kit, it is possible to check whether a patient has developed a disease caused by a respiratory virus.

The diagnostic kit of the present invention may further include a composition, solution, or device of one or more other components suitable for an analysis method. Preferably, the diagnostic kit and diagnostic composition relate to a kit for simultaneous diagnosis of respiratory viruses comprising essential elements necessary for performing reverse transcription reaction and real-time multiplex PCR (real-time multiplex PCR). The diagnostic kit of the present invention includes a reverse transcriptase for synthesizing complementary cDNA from RNA, which is a respiratory virus gene, in addition to each primer pair specific for the detection gene, a DNA polymerase for amplifying complementary DNA, a tube or other suitable Container, reaction buffer (varies in pH and magnesium concentration), deoxynucleotides (dNTPs), enzymes such as Hot start Taq-polymerase and reverse transcription targets, DNase, RNase inhibitor, DEPC-water, sterile water And the like.

In addition, when the kit is used, information for diagnosis of respiratory viruses may be provided.

Looking at the present invention from another aspect, the method of providing information for diagnosing respiratory viruses of the present invention includes: a) mixing an enzyme composition comprising Hot start Taq DNA polymerase and reverse transcriptase in the composition for diagnosing respiratory viruses; b) adding the sample DNA, RNA or DNA and RNA to the mixture prepared in step a); And, c) performing a reverse transcription reaction and a real-time PCR reaction on the sample DNA, RNA, or a reaction solution including DNA and RNA prepared in step b). At this time, the respiratory virus is not particularly limited thereto, but corona virus 229E, corona virus OC43, corona virus NL63, influenza A virus, influenza B virus, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, Rs virus A , Rs virus B, adenovirus, rhinovirus A, B, C, metapneumovirus, Voca virus, etc. are preferably used, and the sample is not particularly limited thereto, but is not particularly limited thereto, but saliva or sputum (Nasopharyngeal swabs (NPS), Nasla It is preferable to use swabs (NS), Throat swabs (TS), Nasal aspirates (NA)) or the like. At this time, the analysis methods for measuring DNA or DNA and RNA levels in step c) may include reverse transcription polymerase chain reaction, competitive PCR, real-time PCR, RNase protection assay, Northern blotting, DNA chip, etc., and are limited thereto. It does not become. Through the above detection methods, it is possible to predict whether the respiratory virus is infected by detecting the virus genome in a patient suspected of having a respiratory virus infection.

In the present invention, the "sample" includes, but is not limited to, a sample such as saliva or sputum in which the expression level of a gene specific to each virus is different due to various respiratory virus infections.

Looking at the present invention from another perspective, the method of providing information for diagnosing respiratory viruses of the present invention targets Human RNase P to provide a criterion for determining the effectiveness of the RNA extraction process in step a). As an internal control primer, a primer having a nucleotide sequence of SEQ ID NO: 15 and 16 may be additionally included, or a probe having a nucleotide sequence of SEQ ID NO: 41 may be further included.

By using the primer set for diagnosing respiratory viruses of the present invention, it is possible to quickly detect 14 respiratory viruses at the same time through a reaction of 5 tubes (once) through a real-time multiple reverse transcription polymerase chain reaction, so that clinical diagnosis can be performed. It will be widely used for rapid diagnosis and treatment of respiratory diseases.

Hereinafter, the present invention will be further described with reference to examples, but the scope of the present invention is not limited thereto.

The following experiment was conducted on aspirates or swab samples of the nasopharynx (Nasopharyngeal swabs (NPS), Nasal swabs (NS), Throat swabs (TS), Nasal aspirates (NA))). Was performed.

Example 1 : Respiratory virus-specific primers and probes

Example 1-1 : Respiratory virus-specific primer construction

The present inventors have primers having nucleotide sequences of SEQ ID NOs: 1 and 2 capable of amplifying the nuclear protein (N) gene of corona virus 229E, and SEQ ID NOs: 3 and 4 capable of amplifying the nuclear protein (N) gene of corona virus OC43. A primer having a nucleotide sequence of, a primer having a nucleotide sequence of SEQ ID NO: 5 and 6 capable of amplifying the polyprotein (1a) gene of coronavirus NL63, hemeglutinin-neuraminidase of parainfluenza virus 1 (HN ) Primers having the nucleotide sequences of SEQ ID NOs: 7 and 8 capable of amplifying the gene, the nucleotide sequences of SEQ ID NOs: 9 and 10 capable of amplifying the hemeglutinin-neuraminidase (HN) gene of parainfluenza virus 2 A primer having, a primer having a base sequence of SEQ ID NO: 11 and 12 capable of amplifying the hemeglutinin-neuramidase (HN) gene of parainfluenza virus 3, and the substrate protein (M) gene of influenza A virus Primers having base sequences of SEQ ID NOs: 13 and 14 that can be amplified, primers having base sequences of SEQ ID NOs: 15 and 16 that provide criteria for determining the effectiveness of the RNA extraction process as an internal control targeting RNase P, influenza Primers having the nucleotide sequences of SEQ ID NOs: 17 and 18 capable of amplifying the hemeglutinin (HA) gene of the B virus, and having the nucleotide sequences of SEQ ID NOs: 19 and 20 capable of amplifying the fusion protein gene of Rs virus A. Primers, primers having base sequences of SEQ ID NOs: 21 and 22 capable of amplifying the fusion protein gene of Rs virus B, primers having base sequences of SEQ ID NOs: 25 and 26 capable of amplifying the hexon protein gene of adenovirus, rhino Primers having the nucleotide sequences of SEQ ID NOs: 27 and 31 capable of amplifying the 5'UTR of viruses A, B, C, and nucleotide sequences of SEQ ID NOs: 23 and 24 capable of amplifying the nuclear protein (N) gene of metapneumovirus Primer having a, Boca virus nucleoc Primers having nucleotide sequences of SEQ ID NOs: 32 and 33 capable of amplifying the apsid protein (NP) gene and the Viral protein (VP) gene were each constructed using a conventional method (see Table 1).

Primer sequence name Base sequence Length Sequence number 229E-F CAGTCAAATGGGCTGATGCA 20 One 229E-R AAAGGGCTATAAAGAGAATAAGGTATTCT 29 2 OC43-F AYGAGGCTATTCCGACTAGGT 21 3 OC43-R CTTCCTGAGCCTTCAATATAGTAACC 26 4 NL63-F ACGTACTTCTATTATGAAGCATGATATTAA 30 5 NL63-R AGCAGATTTAATGTTATACTTAAAACTACG 30 6 PIV1-F GTTGTCAATGTCTTAATYCGTATCAATAATT 31 7 PIV1-R TAGCCTMCCYTCGGCACCTAA 21 8 PIV2-F TTTCCAATYTTCAGGACTATGAA 25 9 PIV2-R TCCTGGTATRGCAGTGACTGAA 26 10 PIV3-2-F CAGGATATAGGAAAATCATATCAAGT 26 11 PIV3-2-R ACATGACTTYCTATTGTCATTTATGTT 27 12 IfA-F AGACCAATYYTGTCACCTCT 20 13 IfA-R TGGACAAAKCGTCTACGCT 19 14 RNaseP-F AGATTTGGACCTGCGAGCG 19 15 RNaseP-R GAGCGGCTGTCTCCACAAGT 20 16 IfB-F AARTACGGTGGATTAAAYAAAAGCAA 26 17 IfB-R AATAGTTTTGCAGGMGGTCTATATTTGG 28 18 RSV A-1-F ATTGTTATCATTAATTGCTGTTGGA 25 19 RSV A-1-R CTAAATGCAATATTATTTATACCACTCAG 29 20 RSV B-1-F TGCAGTRACAGAATTACAGCTACTT 25 21 RSV B-1-R TTAGTGGTATTGATTGTRTAGTTCA 25 22 MPV-2-F TCATCAGGYAAYATYCCACA 20 23 MPV-2-R ACTTCTATDGTTGATGCTAGYTT 23 24 AdV-F CACNGTGGGGTTTCTRAACTT 21 25 AdV-R CARTGGKCWTACATGCAYATC 21 26 RV-1-F TGTGAAGAGCCSCRTGTG 18 27 RV-2-F TGTGAAGACTCGCATGTGCT 20 28 RV-3-F GTGYGAAGAGYCTANTGTGCT 21 29 RV-R GGACRCCCAAAGTAGTYGGTYC 22 30 RV-2-R GGACAYCCAAAGTAGTYGGTYC 22 31 BoV-1-F GAAATGCTTTCTGCTGYTGAAAG 23 32 BoV-1-R GGTTCACCGTTWTCAAGWGGATT 23 33

Example 1-2 : Fabrication of respiratory virus-specific probe

The present inventors identified a probe having a nucleotide sequence of SEQ ID NO: 34 capable of detecting the nuclear protein (N) gene of coronavirus 229E, and a nucleotide sequence of SEQ ID NO: 35 capable of detecting a nuclear protein (N) gene of corona virus OC43. Eggplant probe, a probe having a nucleotide sequence of SEQ ID NO: 36 that can detect the polyprotein (1a) gene of coronavirus NL63, can detect the hemeglutinin-neuramidase (HN) gene of parainfluenza virus 1. A probe having a nucleotide sequence of SEQ ID NO: 37, a probe having a nucleotide sequence of SEQ ID NO: 38 capable of detecting the hemeglutinin-neuraminidase (HN) gene of parainfluenza virus 2, all hemeglutinin- A probe having a nucleotide sequence of SEQ ID NO: 39 capable of detecting a neuraminidase (HN) gene, a probe having a nucleotide sequence of SEQ ID NO: 40 capable of detecting a matrix protein (M) gene of influenza A virus, an internal control As a probe having the nucleotide sequence of SEQ ID NO: 41 capable of detecting RNase P, a probe having the nucleotide sequence of SEQ ID NO: 42 capable of detecting the hemeglutinin (HA) gene of influenza B virus, fusion of Rs virus A A probe having a nucleotide sequence of SEQ ID NO: 43 capable of detecting a protein gene, a probe having a nucleotide sequence of SEQ ID NO: 44 capable of detecting a fusion protein gene of Rs virus B, and a nuclear protein (N) gene of metapneumovirus A probe having a detectable nucleotide sequence of SEQ ID NO: 45, a probe having a nucleotide sequence of SEQ ID NO: 46-49 capable of detecting a hexon protein gene of adenovirus, and detecting 5'UTR of rhinovirus A, B, C A probe having a nucleotide sequence of SEQ ID NO: 50-51, a probe having a nucleotide sequence of SEQ ID NO: 52 capable of detecting the nucleocapsid protein (NP) gene and Viral protein (VP) gene of the Boca virus, is used in a conventional method. Each was prepared using (see Table 2).

The base sequence of the probe name 5 Base sequence 3 Length Sequence number 229E-P-rev FAM CCCTGACGACCACGTTGTGGTTCA TAMRA 24 34 OC43-P HEX CGCCTGGCACGGTACTCCCTC TAMRA 21 35 NL63-P Cy5 ATTGCCAAGGCTCCTAAACGTACAGGTGTT BHQ3 30 36 PIV1-P FAM AGGCCAAAGATTGTTGTCGAGACWATTCCAAT TAMRA 32 37 PIV2-P HEX CYATTTACCTAAGTGATGGAATCAATCGCAAA TAMRA 31 38 PIV3-2-P Cy5 CAGACTTGGTACCTGACTTAAATCCYAGGA BHQ3 30 39 IfA-P FAM ACGCTCACCGTGCCCAGT TAMRA 18 40 RNaseP-P HEX TTCTGACCTGAAGGCTCTGCGCG TAMRA 23 41 IfB-P Cy5 TGCAAARGCMATAGGRAATTGCCCA BHQ3 25 42 RSV A-1-P FAM CTGTAAGGCCAGAAGCACACCARTCACAC TAMRA 29 43 RSV B-1-P Cy5 CGGGCCAGAAGAGAAGCACCACAGTA BHQ3 26 44 MPV-2-P HEX CAGAGRCCYTCAGCACCAGACACAC TAMRA 25 45 AdV-1-P FAM TGCACCAGCCCGGGGCTCAGGTACT TAMRA 25 46 AdV-2-P FAM TGCACCAGACCSGGACTCAGGTACT TAMRA 25 47 AdV-3-P FAM TGCACCAGGCCCGGGCTCAGRTACT TAMRA 25 48 AdV-4-P FAM TGCACCAGCCCGGKACTCAGGTAYT TAMRA 25 49 RV-P HEX CCGGCCCCTGAATGYGGCTAAYC TAMRA 23 50 RV-2-P HEX CCGGCYCCYGAATGTGGCTAACC TAMRA 23 51 BoV-1-P Cy5 CCTRGAGGGTGGGTGCTKCCT BHQ3 21 52

Example 2 : Diagnosis of respiratory virus

Example 2-1 : Preparation of necessary solution

The reagents of Table 3 were prepared. These were left at room temperature to thaw before use, and it is preferable not to stand at room temperature for more than 30 minutes.

Composition and Ingredients number Configuration Ingredients Sequence number One RT-PCR Premix One-step RT-PCR premix 2 Primer/probe mixture 1
(primer/probe
Mixture1) Corona Virus primer/probe mix
1.229E primer probe mix (FAM)
2.OC43 primer probe mix (HEX)
3.NL63 primer probe mix (Cy5)
1-2, 34
3-4, 35
5-6, 36 3 Primer/probe mixture 2
(primer/probe
Mixture2) Parainfluenza Virus (PIV) primer/probe mix
1.PIV1 primer/probe mix (FAM)
2. PIV2 primer/probe mix (HEX)
3. PIV3 primer/probe mix (Cy5)
7-8, 37
9-10, 38
11-12, 39 4 Primer/probe mixture 3
(primer/probe
Mixture3) Influenza Virus/ RNase P primer/probe mix
1.Influenza A pirmer/probe mix (FAM)
2. RNase P primer/probe mix (HEX)
3. Influenza B primer/probe mix (Cy5)
13-14, 40
15-16, 41
17-18, 42 5 Primer/probe mixture 4
(primer/probe
Mixture4) Respiratory syncytial Virus (RSV) primer/probe mix
1.RSV A primer/probe mix (FAM)
2. RSV B primer/probe mix (Cy5)
3.Metapneumovirus primer/probe mix (HEX)

19-20, 43
21-22, 44
23-24, 45 6 Primer/probe mixture 5
(primer/probe
Mixture5) Adeno Virus / Rhino Virus primer probe mix
1.Adeno Virus primer/probe mix (FAM)
2. Rhino Virus A,B,C primer/probe mix (HEX)
3. Boca Virus primer/probe mix (Cy5)
25-26, 46-49
27-31, 50-51
32-33, 52 7 Positive control
(Positive Control) Positive control containing 12 types of Respiratory Virus, RNase P primer/probe binding sequences 8 Negative control
(Negative Control) Negative control containing RNase P primer/probe binding sequences

Example 2-2 : Preparation of specimen

Nasopharyngeal aspirates or swab samples (Nasopharyngeal swabs (NPS), Nasal swabs (NS), Throat swabs (TS), Nasal aspirates (NA)) were used.

Example 2-3 : Respiratory virus diagnosis method

RNA was extracted from the sample prepared in Example 2-2 using an RNA extraction kit. A 200 µl PCR tube in which 10 µl of RT-PCR Premix was dispensed was prepared as much as the sample, plus the positive control and the negative control. To each RT-PCR Premix tube dispensed in advance, 5 µl of the RVP primer/probe mixture and 5 µl of the extracted sample or positive and negative controls were added each (see Table 4).

Sample and composition Name of sample and composition Amount used per 1 test (µl) RT-PCR Premix 10 RV primer/probe mixture 5 Input sample amount Extracted sample, positive control solution or negative control solution 5 Sum 20

The lid was closed, centrifuged, and mixed well. After centrifugation, the reaction was carried out by putting it in a real-time PCR cycler. Specific real-time PCR reaction conditions are as follows (see Table 5).

Real-time PCR reaction conditions Progress Temperature time Number of repetitions (times) Reverse-transcription Step 1 45℃ 10 minutes 1 time Step 2 94℃ 5 minutes PCR reaction Step 3 94℃ 5 seconds 40 times ( * Signal detection response interval ) Step 4 53℃ 30 seconds Step 5* 72℃ 30 seconds

The PCR reaction result was measured in real time by a real-time PCR detection system, and after the reaction was completed, a cut-off value (device name: SLAN, FAM: 0.06, HEX: 0.06, CY5: 0.04) was entered.

Example 2-4 : Result judgment

(1) Ct value for each control group

The Ct value (threshold cycle: number of cycles passing the limit value) when reacted to the control group is as follows (see Table 6 and Table 7), and the Ct value of the sample is each item (FAM, HEX, and CY5). All were determined as positive when they were 37 or less (see Tables 6 and 7).

Criteria for determining positive control amount Positive control FAM HEX CY5 Primer/probe mixture 1 30 ± 3 30 ± 3 30 ± 3 Primer/probe mixture 2 30 ± 3 30 ± 3 30 ± 3 Primer/probe mixture 3 30 ± 3 30 ± 3 30 ± 3 Primer/probe mixture 4 30 ± 3 30 ± 3 30 ± 3 Primer/probe mixture 5 30 ± 3 30 ± 3 30 ± 3

Criteria for determining the negative control amount Negative control FAM HEX CY5 Primer/probe mixture 1 No Ct No Ct No Ct Primer/probe mixture 2 No Ct No Ct No Ct Primer/probe mixture 3 No Ct 30 ± 3 No Ct Primer/probe mixture 4 No Ct No Ct No Ct Primer/probe mixture 5 No Ct No Ct No Ct

As shown in Tables 6 and 7, in the case of the positive control solution, the Ct value should be within the range of 30 ± 3 for each wavelength, and in the case of the negative control solution, only the internal control RNase P shows the Ct value, and the rest of the respiratory viruses have the Ct value. It could be seen that the result is valid only when is not displayed (No Ct).

(2) Analysis of results

For each reaction, the signal formation of FAM, HEX, and CY5 wavelengths was confirmed, and reaction effectiveness, positive, and negative were analyzed as shown in the following table (see Table 8).

Response effectiveness and positive and negative analysis criteria Item FAM HEX CY5 Interpret the results Primer probe mixture 1 + - - 229E - + - OC43 - - + NL63 - - - Corona Virus negative Primer probe mixture 2 + - - PIV1 - + - PIV2 - - + PIV3 - - - Parainfluenza Virus negative Primer Probe Mixture 3 + + - Influenza A - + + Influenza B - + - Influenza A/B negative - - - condition Primer probe mixture 4 + - - RSV A - - + RSV B - - + Metapneumovirus - - - RSV A/B/Metapneumovirus negative Primer probe mixture 5 + - - Adeno Virus - + - Rhino Virus A,B,C - - + Boca Virus - - - Adeno/Rhino/Boca Virus negative

Table 8 is a result analysis table for each type showing the determination of negative and positive respiratory viruses. For example, if the Ct value is less than 37 only in the FAM item using Primer Probe Mixture 1 (positive), it means that you are infected with Coronavirus 229E. Outlet, marked as retest in the above table means that no internal control was detected and should be retested.

<110> LG CHEM, LTD. <120> Composition for Detecting Virus Relating Respiratory Disease and Kit for Detecting Virus Relating Respiratory Disease Comprising the Same <130> PA100856-KR-D2 <160> 52 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer 229E-F <400> 1 cagtcaaatg ggctgatgca 20 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer 229E-R <400> 2 aaagggctat aaagagaata aggtattct 29 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer OC43-F <400> 3 aygaggctat tccgactagg t 21 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer OC43-R <400> 4 cttcctgagc cttcaatata gtaacc 26 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer NL63-F <400> 5 acgtacttct attatgaagc atgatattaa 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer NL63-R <400> 6 agcagattta atgttatact taaaactacg 30 <210> 7 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer PIV1-F <400> 7 gttgtcaatg tcttaatycg tatcaataat t 31 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer PIV1-R <400> 8 tagcctmccy tcggcaccta a 21 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer PIV2-F <400> 9 tttccaatyt tcaggactat gaa 23 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer PIV2-R <400> 10 tcctggtatr gcagtgactg aa 22 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer PIV3-2-F <400> 11 caggatatag gaaaatcata tcaagt 26 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer PIV3-2-R <400> 12 acatgactty ctattgtcat ttatgtt 27 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer IfA-F <400> 13 agaccaatyy tgtcacctct 20 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer IfA-R <400> 14 tggacaaakc gtctacgct 19 <210> 15 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer RNaseP-F <400> 15 agatttggac ctgcgagcg 19 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer RNaseP-R <400> 16 gagcggctgt ctccacaagt 20 <210> 17 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer IfB-F <400> 17 aartacggtg gattaaayaa aagcaa 26 <210> 18 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer IfB-R <400> 18 aatagttttg caggmggtct atatttgg 28 <210> 19 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer RSV A-1-F <400> 19 attgttatca ttaattgctg ttgga 25 <210> 20 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer RSV A-1-R <400> 20 ctaaatgcaa tattatttat accactcag 29 <210> 21 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer RSV B-1-F <400> 21 tgcagtraca gaattacagc tactt 25 <210> 22 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer RSV B-1-R <400> 22 ttagtggtat tgattgtrta gttca 25 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer MPV-2-F <400> 23 tcatcaggya ayatyccaca 20 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer MPV-2-R <400> 24 acttctatdg ttgatgctag ytt 23 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer AdV-F <400> 25 cacngtgggg tttctraact t 21 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer AdV-R <400> 26 cartggkcwt acatgcayat c 21 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer RV-1-F <400> 27 tgtgaagagc cscrtgtg 18 <210> 28 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer RV-2-F <400> 28 tgtgaagact cgcatgtgct 20 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer RV-3-F <400> 29 gtgygaagag yctantgtgc t 21 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer RV-R <400> 30 ggacrcccaa agtagtyggt yc 22 <210> 31 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer RV-2-R <400> 31 ggacayccaa agtagtyggt yc 22 <210> 32 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer BoV-1-F <400> 32 gaaatgcttt ctgctgytga aag 23 <210> 33 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer BoV-1-R <400> 33 ggttcaccgt twtcaagwgg att 23 <210> 34 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe 229E-P-rev <400> 34 ccctgacgac cacgttgtgg ttca 24 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe OC43-P <400> 35 cgcctggcac ggtactccct c 21 <210> 36 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> probe NL63-P <400> 36 attgccaagg ctcctaaacg tacaggtgtt 30 <210> 37 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> probe PIV1-P <400> 37 aggccaaaga ttgttgtcga gacwattcca at 32 <210> 38 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> probe PIV2-P <400> 38 cyatttacct aagtgatgga atcaatcgca aa 32 <210> 39 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> probe PIV3-2-P <400> 39 cagacttggt acctgactta aatccyagga 30 <210> 40 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> probe IfA-P <400> 40 acgctcaccg tgcccagt 18 <210> 41 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe RNaseP-P <400> 41 ttctgacctg aaggctctgc gcg 23 <210> 42 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe IfB-P <400> 42 tgcaaargcm ataggraatt gccca 25 <210> 43 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> probe RSV A-1-P <400> 43 ctgtaaggcc agaagcacac cartcacac 29 <210> 44 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> probe RSV B-1-P <400> 44 cgggccagaa gagaagcacc acagta 26 <210> 45 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe MPV-2-P <400> 45 cagagrccyt cagcaccaga cacac 25 <210> 46 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe AdV-1-P <400> 46 tgcaccagcc cggggctcag gtact 25 <210> 47 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe AdV-2-P <400> 47 tgcaccagac csggactcag gtact 25 <210> 48 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe AdV-3-P <400> 48 tgcaccaggc ccgggctcag rtact 25 <210> 49 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe AdV-4-P <400> 49 tgcaccagcc cggkactcag gtayt 25 <210> 50 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe RV-P <400> 50 ccggcccctg aatgyggcta ayc 23 <210> 51 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe RV-2-P <400> 51 ccggcyccyg aatgtggcta acc 23 <210> 52 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe BoV-1-P <400> 52 cctrgagggt gggtgctkcc t 21

Claims (10)

(i) a primer set comprising a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 19 and 20, and a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 21 and 22; And
(ii) a probe consisting of the nucleotide sequence of SEQ ID NO: 43, and a probe consisting of the nucleotide sequence of SEQ ID NO: 44,
As a composition for simultaneous diagnosis of respiratory virus, suitable for real-time multiplex PCR (Polymerase Chain Reaction),
The composition further comprises a primer pair consisting of the nucleotide sequences of SEQ ID NO: 45 and a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 23 and 24; Or a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 32 and 33 and a probe consisting of the nucleotide sequences of SEQ ID NO: 52.
The composition of claim 1, wherein the composition further comprises one or more sets of primers and probes selected from (a) and (b):
(a) a primer set comprising a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 1 and 2, a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 3, and 4, and a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 5 and 6; And a probe consisting of the nucleotide sequence of SEQ ID NO: 34, the probe consisting of the nucleotide sequence of SEQ ID NO: 35, and the probe consisting of the nucleotide sequence of SEQ ID NO: 36; And
(b) a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 25 and 26, and a forward primer consisting of the nucleotide sequences of any one of SEQ ID NOs: 27 to 29, and a reverse primer consisting of the nucleotide sequences of SEQ ID NOs: 30 or 31, A primer set comprising a primer pair; And a probe consisting of a nucleotide sequence of SEQ ID NO: 46, a probe consisting of a nucleotide sequence of SEQ ID NO: 47, a probe consisting of a nucleotide sequence of SEQ ID NO: 48, a probe consisting of a nucleotide sequence of SEQ ID NO: 49, a probe consisting of a nucleotide sequence of SEQ ID NO: 50 , And a probe consisting of the nucleotide sequence of SEQ ID NO: 51.
delete The method of claim 2, wherein the set (b) further comprises a primer pair consisting of a nucleotide sequence of SEQ ID NOs: 23 and 24 and a probe consisting of a nucleotide sequence of SEQ ID NO: 45; Or a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 32 and 33 and a probe consisting of the nucleotide sequences of SEQ ID NO: 52.
The composition of claim 1, further comprising a hot start Taq DNA polymerase and a reverse transcriptase.
According to claim 1, A primer pair consisting of the nucleotide sequence of SEQ ID NO: 15 and 16 for amplifying the internal control RNase P; A probe consisting of the nucleotide sequence of SEQ ID NO: 41 for detecting an internal control RNase P; Or both of them further comprising, simultaneous respiratory virus diagnostic composition.
Respiratory virus simultaneous diagnosis kit comprising the composition of claim 1.
The kit for simultaneous diagnosis of respiratory virus according to claim 7, wherein the kit further comprises one or more sets of primers and probes selected from (a) and (b):
(a) a primer set comprising a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 1 and 2, a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 3, and 4, and a primer pair consisting of the nucleotide sequences of SEQ ID NOs: 5 and 6; And a probe consisting of the nucleotide sequence of SEQ ID NO: 34, the probe consisting of the nucleotide sequence of SEQ ID NO: 35, and the probe consisting of the nucleotide sequence of SEQ ID NO: 36; And
(b) a pair of primers consisting of the nucleotide sequences of SEQ ID NOs: 25 and 26, and a forward primer consisting of the nucleotide sequences of any one of SEQ ID NOs: 27 to 29, and a reverse primer consisting of the nucleotide sequences of SEQ ID NOs: 30 or 31, A primer set comprising a primer pair; And a probe consisting of a nucleotide sequence of SEQ ID NO: 46, a probe consisting of a nucleotide sequence of SEQ ID NO: 47, a probe consisting of a nucleotide sequence of SEQ ID NO: 48, a probe consisting of a nucleotide sequence of SEQ ID NO: 49, a probe consisting of a nucleotide sequence of SEQ ID NO: 50 , And a probe consisting of the nucleotide sequence of SEQ ID NO: 51.
a) mixing an enzyme composition comprising a hot start Taq DNA polymerase and a reverse transcriptase to the composition of claim 1;
b) adding the sample DNA or RNA to the mixture prepared in step a); And,
c) performing a reverse transcription reaction and a real-time PCR reaction with a reaction solution containing the sample DNA or RNA prepared in step b).
Method of providing information for simultaneous diagnosis of respiratory virus.
10. The method of claim 9, wherein the sample is saliva or phlegm.