KR102481243B1 - Marker for multi-diagnosis of corona virus infection and use thereof - Google Patents

Abstract

The present invention relates to concurrent diagnostic markers for coronavirus infection and uses thereof. The composition for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) according to the present invention targets the spike S1 domain to quickly and accurately verify the current epidemic COVID-19 (SARS-CoV-2) this is possible In addition, the composition for detecting a severe acute respiratory syndrome (SARS) virus or a virus similar to it according to the present invention targets the spike S2 domain and can detect severe acute respiratory syndrome in one reaction even in a small amount of sample. (Severe acute respiratory syndrome; SARS) virus or its similar virus is universally detectable. In addition, the above composition can identify COVID-19 (SARS-CoV-2), severe acute respiratory syndrome (SARS) virus or similar viruses quickly and with high specificity and sensitivity, so that COVID-19 ( SARS-CoV-2), severe acute respiratory syndrome (SARS) virus, or infection diagnosis related to viruses similar thereto, as well as more effective prevention and control can be usefully used.

Description

A simultaneous diagnostic marker for coronavirus infection and its use {Marker for multi-diagnosis of corona virus infection and use thereof}

The present invention relates to a novel coronavirus infection simultaneous diagnostic marker and its use, and more particularly, to the currently prevalent corona 19 (SARS-CoV-2) and the simultaneous SARS-coronavirus and SARS-associated coronavirus including it It relates to a universal marker capable of diagnosis.

The coronavirus (CoV) that causes Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS) and Coronavirus Disease 19 (COVID-19) ) poses a serious threat to public health.

SARS is an infectious disease that occurred in Guangdong Province in southern China in November 2002 and spread to the world via Hong Kong. . The propagation route has not yet been fully elucidated, but it is speculated that it is propagated by fine particles of solid or liquid floating in the atmosphere. SARS was prevalent until July 2003, and a total of 8,096 infected people occurred in 32 countries for about 7 months, of which 774 people died.

MERS was first discovered in Saudi Arabia in 2012, and presents with severe respiratory symptoms such as high fever, cough, and shortness of breath. Although the exact source and route of infection have not been identified, it is highly likely to be infected through contact with camels in the Middle East, and it has been reported that close contact between people is possible. Patients mainly occurred in the Middle East, and in Korea, from May 2015 to the end of December 2015, 186 people were infected across the country, and 38 of them were reported to have died.

COVID-19 is a respiratory infectious disease caused by a new type of coronavirus (SARS-CoV-2) that first emerged in Wuhan, China in December 2019 and has since spread across China and around the world. COVID-19 is transmitted when droplets of an infected person penetrate the respiratory tract or mucous membranes of the eyes, nose, and mouth, and after an incubation period of about 2 to 14 days, fever, respiratory symptoms such as coughing or difficulty breathing, and pneumonia appear as the main symptoms, but cases of asymptomatic infection are rare. Coming out. After the outbreak of COVID-19, the number of confirmed cases around the world continued, and on March 11, 2020, WHO declared COVID-19 a pandemic (global pandemic) for the third time in history, following the Hong Kong flu (1968) and swine flu (2009). , To date, no cure has been prepared, so a huge number of confirmed cases are appearing around the world every day.

SARS, MERS, and Corona 19 are all known to be caused by a variant coronavirus belonging to the genus β-coronavirus. In addition, they are believed to have originated from bats, which are the primary carriers of various coronaviruses, and the exact route of infection has not yet been fully elucidated.

As such, coronavirus-related mutant viruses are continuously occurring, and their rapid detection is an important factor in preventing pandemics. However, the real-time RT-PCR method based on the upE region has a very low detection limit, making precise detection difficult. A difficult problem has arisen, and in the case of RT-PCR for simultaneous detection, the problem of lowering the detection limit and detection specificity due to the increase in non-specific amplification products due to the use of two or more types of primers and probes continues to emerge.

Therefore, it is necessary to develop a new method that can confirm COVID-19 (SARS-CoV-2) infection virus and SARS-coronavirus and SARS-related coronavirus including it with a small sample and a simple method, and detect them simultaneously. The situation is.

Korean Patent Registration No. 10-1916899 Korean Patent Publication No. 10-2007-0044014

The present inventors analyze the nucleotide sequence of coronaviruses generated at home and abroad, and based on this, use specific primers and probes for the spike S1 domain to specifically detect the COVID-19 (SARS-CoV-2) virus. confirmed that it can. In addition, one primer set specific for the spike S2 domain was used and probes specific for pan-SARS-like, SARS-CoV and SARS-CoV-2 were used to treat severe acute respiratory infections. The present invention was completed by confirming that a severe acute respiratory syndrome (SARS) virus or a similar virus thereof could be rapidly detected.

Accordingly, an object of the present invention is to provide a primer set A comprising a pair of primers of SEQ ID NOs: 1 and 2 and a probe of SEQ ID NO: 3 for specifically amplifying a part of a spark gene as a target; and a primer set B comprising at least one probe selected from the group consisting of primer pairs of SEQ ID NOs: 4 and 5 and SEQ ID NOs: 6 to 8 for targeting and specifically amplifying a part of a spark gene; To provide a composition for detecting a severe acute respiratory syndrome (SARS) virus or a virus similar thereto, comprising a.

Another object of the present invention is a primer set A comprising a primer pair of SEQ ID NOs: 1 and 2 and a probe of SEQ ID NO: 3 for specifically amplifying a part of a spark gene as a target; and primer set B comprising primer pairs of SEQ ID NOs: 4 and 5 and probes of SEQ ID NOs: 6 to 8 for targeting and specifically amplifying a part of the spark gene; To provide a composition for detecting severe acute respiratory syndrome (SARS) virus or a similar virus thereof.

Another object of the present invention is (a) a primer pair of SEQ ID NOs: 1 and 2; And (b) the probe of SEQ ID NO: 3; to provide a composition for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) containing.

Another object of the present invention is (a) a primer pair of SEQ ID NOs: 4 and 5; And (b) any one or more probes selected from the group consisting of SEQ ID NOs: 6 to 8; to provide a composition for detecting severe acute respiratory syndrome (SARS) virus or virus similar thereto, including.

Another object of the present invention is a kit for specific detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) comprising the primer pair and probe according to the present invention and severe acute respiratory syndrome (SARS) It is to provide a kit for simultaneous detection of viruses and viruses similar thereto.

Another object of the present invention is to specifically detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using the primer pair and probe according to the present invention using real-time reverse transcription polymerase chain reaction (RT-PCR) To provide a method for detecting and a method for simultaneously detecting severe acute respiratory syndrome (SARS) virus and similar viruses thereof.

In order to achieve the above object, the present invention provides a primer set A comprising a primer pair of SEQ ID NOs: 1 and 2 and a probe of SEQ ID NO: 3 for specifically amplifying a part of a spark gene as a target; and a primer set B comprising at least one probe selected from the group consisting of primer pairs of SEQ ID NOs: 4 and 5 and SEQ ID NOs: 6 to 8 for targeting and specifically amplifying a part of a spark gene; It provides a composition for detecting severe acute respiratory syndrome (Severe acute respiratory syndrome; SARS) virus or a virus similar thereto comprising a.

In the composition for detecting a severe acute respiratory syndrome (SARS) virus or a virus similar thereto of the present invention, the probes of the primer set B preferably include all probes of SEQ ID NOs: 6 to 8.

The present invention provides a primer set A comprising a pair of primers of SEQ ID NOs: 1 and 2 and a probe of SEQ ID NO: 3 for specifically amplifying a part of a spark gene as a target; and primer set B comprising primer pairs of SEQ ID NOs: 4 and 5 and probes of SEQ ID NOs: 6 to 8 for targeting and specifically amplifying a part of the spark gene; Provided is a composition for detecting a severe acute respiratory syndrome (SARS) virus or a virus similar thereto.

In addition, the present invention provides (a) a pair of primers of SEQ ID NOs: 1 and 2; And (b) the probe of SEQ ID NO: 3; provides a composition for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) containing.

In addition, the present invention provides (a) a pair of primers of SEQ ID NOs: 4 and 5; And (b) any one or more probes selected from the group consisting of SEQ ID NOs: 6 to 8; provides a composition for detecting severe acute respiratory syndrome (SARS) virus or virus similar thereto.

In addition, the present invention is a kit for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific detection comprising the primer pair and probe according to the present invention and severe acute respiratory syndrome (SARS) virus and A kit for simultaneous detection of similar viruses is provided.

In addition, the present invention specifically detects severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using the primer pair and probe according to the present invention using real-time reverse transcription polymerase chain reaction (RT-PCR) and a method for simultaneously detecting a severe acute respiratory syndrome (SARS) virus and a similar virus thereof.

The composition for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) according to the present invention targets the spike S1 domain to quickly and accurately verify the current epidemic COVID-19 (SARS-CoV-2) this is possible In addition, the composition for detecting a severe acute respiratory syndrome (SARS) virus or a virus similar to it according to the present invention targets the spike S2 domain and can detect severe acute respiratory syndrome in one reaction even in a small amount of sample. (Severe acute respiratory syndrome; SARS) virus or its similar virus is universally detectable. In addition, the above composition can identify COVID-19 (SARS-CoV-2), severe acute respiratory syndrome (SARS) virus or similar viruses quickly and with high specificity and sensitivity, so that COVID-19 ( SARS-CoV-2), severe acute respiratory syndrome (SARS) virus, or infection diagnosis related to viruses similar thereto, as well as more effective prevention and control can be usefully used.

1 is a schematic diagram showing the target location in the coronavirus of the primer set of the present invention.
2 is a diagram showing the results of sequencing and target selection of primers and probes corresponding to primer set A prepared in the present invention.
3 is a diagram showing the results of sequencing and target selection of primers and probes corresponding to primer set B prepared in the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a primer set A comprising a pair of primers of SEQ ID NOs: 1 and 2 and a probe of SEQ ID NO: 3 for specifically amplifying a part of a spark gene as a target; and a primer set B comprising at least one probe selected from the group consisting of primer pairs of SEQ ID NOs: 4 and 5 and SEQ ID NOs: 6 to 8 for targeting and specifically amplifying a part of a spark gene; It provides a composition for detecting severe acute respiratory syndrome (Severe acute respiratory syndrome; SARS) virus or a virus similar thereto comprising a.

In the composition for detecting a severe acute respiratory syndrome (SARS) virus or a virus similar thereto of the present invention, the probes of the primer set B preferably include all probes of SEQ ID NOs: 6 to 8.

The present invention provides a primer set A comprising a pair of primers of SEQ ID NOs: 1 and 2 and a probe of SEQ ID NO: 3 for specifically amplifying a part of a spark gene as a target; and primer set B comprising primer pairs of SEQ ID NOs: 4 and 5 and probes of SEQ ID NOs: 6 to 8 for targeting and specifically amplifying a part of the spark gene; Provided is a composition for detecting a severe acute respiratory syndrome (SARS) virus or a virus similar thereto.

In the present invention, the term "severe acute respiratory syndrome (SARS) virus or a virus similar thereto" refers to, for example, ptarmigan (bats), pangolin, snake, raccoon, badger, civet, deer, pig , it is derived from cattle, rodents and birds, and refers to a causative virus that can cause acute respiratory syndrome. Representative examples of these viruses in humans include SARS, MERS, and COVID-19.

In particular, the corona virus continuously occurs, its variants are very diverse, and it is fatal to humans, so it is very important to prevent its transmission at an early stage. Therefore, it is essential to develop a diagnostic technique capable of simultaneously detecting them so that measures such as isolation through rapid diagnosis can be taken.

Accordingly, the composition for detecting a severe acute respiratory syndrome (SARS) virus or a virus similar thereto according to the present invention is a pan-SARS-like, SARS-CoV and SARS-CoV-2, respectively. It is characterized by including a probe for.

The primer set was composed of a forward primer and a reverse primer based on the nucleotide sequence of the novel coronavirus that occurred at home and abroad. Specifically, the nucleotide sequence of the forward primer targeting the Spike S1 domain is 5'-CAA TGT TAC TTG GTT CCA TGC-3' (SEQ ID NO: 1), and the nucleotide sequence of the reverse primer is 5'-GTT AGA CTT CTC AGT It consisted of GGA AGC-3' (SEQ ID NO: 2). This primer corresponds to the primer pair of the primer set A of the present invention, and is characterized in that it is included in the composition for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that is, Corona 19 (COVID-19).

In addition, the nucleotide sequence of the forward primer targeting the Spike S2 domain is 5'-ATY AGR GCT GCW GAA ATC AG-3' (SEQ ID NO: 4), and the reverse nucleotide sequence is 5'-CCW TCA TGA CAA ATD GCW GG- 3' (SEQ ID NO: 5). wherein Y is C or T, R is A or G, W is A or T, and D is A, G or T. These primers correspond to the primer pairs of the primer set B of the present invention, and are characterized in that they are included in a composition for detecting a severe acute respiratory syndrome (SARS) virus or a virus similar thereto.

On the other hand, the probe was also constructed based on the nucleotide sequence of the coronavirus that occurred at home and abroad. First, as a probe included in the primer set A of the present invention, SARS-CoV-2 specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that is, Corona 19 (COVID-19), targeting the spike S1 domain 2 probe consists of 5'-CAT GTC TCT GGG ACC AAT GGT-3' (SEQ ID NO: 3).

In addition, as a probe included in the primer set B of the present invention, the pan-SARS-like probe specific for severe acute respiratory syndrome-like virus targeting the spike S2 domain is 5'-GCT TCT GCY AAY CTT GCT GC-3' (SEQ ID NO: 6), and the SARS-CoV probe specific to the SARS-CoV virus is composed of 5'-TTG TGG AAA GGG CTA CCA CCT-3' (SEQ ID NO: 7), and the SARS-CoV-2 virus The specific SARS-CoV-2 probe consists of 5'-ATG AGG TGC TGA CTG AGG GA-3' (SEQ ID NO: 8). Here, Y is C or T, and the probe of the present invention is further conjugated with a label used for detection at the 5' and 3' ends, respectively.

In the present invention, the term "marker" refers to a specific nucleotide sequence capable of distinguishing and diagnosing COVID-19, severe acute respiratory syndrome virus, and severe acute respiratory syndrome-like virus. The composition for detection according to the present invention can accurately identify COVID-19 with high sensitivity according to the configuration of primer set A (primer pairs of SEQ ID NOs: 1 and 2 and probes of SEQ ID NO: 3). In addition, the confirmation and diagnosis of the virus to be tested can be confirmed and diagnosed with high sensitivity depending on whether the probes of SEQ ID NOs 6 to 8 are hybridized with the PCR amplification product obtained by performing PCR amplification using primer set B, that is, the primer pair of SEQ ID NOs: 4 and 5. can be checked accurately.

In the present invention, the term "primer" is a nucleic acid sequence having a short free 3' hydroxyl group, capable of forming a base pair with a complementary template, and a starting point for copying the template strand. Refers to a short nucleic acid sequence that serves as a point. A primer can initiate DNA synthesis in the presence of a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) and different four nucleotide triphosphates in an appropriate buffer and temperature. PCR conditions and lengths of sense and antisense primers can be modified based on those known in the art.

In the present invention, the term "primer set" refers to a set composed of a forward primer and a reverse primer used to amplify a target gene through a polymerase chain reaction, and is compatible with the term "primer pair".

The primer may incorporate additional features that do not change the basic properties of the primer that serves as the starting point of DNA synthesis. In addition, the primer may include a label that can be directly or indirectly detected by spectroscopic, photochemical, biochemical, immunochemical, or chemical means, if necessary. Examples of labels include enzymes (alkaline phosphatase), radioactive isotopes (eg 32P), fluorescent molecules, and chemical groups (eg biotin).

In the present invention, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a few bases to several hundreds of bases as short as possible to achieve specific binding with a gene or mRNA. , It may be manufactured in the form of a single stranded DNA probe, a double stranded DNA probe, an RNA probe, and the like.

The probe may be one in which a label used for detection is further conjugated to the 5' and 3' ends of the corresponding nucleotide sequence, respectively, and specifically, a reporter or a quencher may be conjugated to the 5' and 3' ends, respectively. . Accordingly, when the reporter and the photon quencher are present in the probe in a bonded form, light emission is suppressed due to mutual interference, and when the probe is decomposed through the polymerase chain reaction, the interference between the reporter and the photon quencher labeled on the probe disappears and light emission is suppressed. will do With this reaction, the presence or absence of COVID-19, severe acute respiratory syndrome virus, and severe acute respiratory syndrome-like virus can be detected from the sample, and their infection can be diagnosed.

In the present invention, the probe is preferably further conjugated with a reporter at the 5' end. At this time, the reporter is, for example, FAM (6-carboxyfluorescein), Texas red (texas red), fluorescein (fluorescein), HEX (5-hexachloro-fluorescein), fluorescein chlorotriazinyl (fluorescein chlorotriazinyl), rhoda rhodamine green, rhodamine red, tetramethylrhodamine, fluorescein isothiocyanate (FITC), oregon green, alexa fluor, JOE (6-Carboxy- 4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX (6-Carboxyl-X-Rhodamine), TET (Tetrachloro-Fluorescein), TRITC (tertramethylrodamine isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine), It may be at least one selected from the group consisting of NED (N-(1-Naphthyl) ethylenediamine), cyanine-based dyes, and thiadicarbocyanine.

In the present invention, the probe is preferably further conjugated with a quencher at the 3' end. At this time, the photon is, for example, TAMRA (6-carboxytetramethyl-rhodamine), BHQ1 (black hole quencher 1), BHQ2 (black hole quencher 2), BHQ3 (black hole quencher 3), NFQ (nonfluorescent quencher), dabcyl ), Eclipse, Deep Dark Quencher (DDQ), Blackberry Quencher, and Iowa Black.

Real-time reverse transcription polymerase chain reaction (RT-PCR) using a fluorescently-labeled probe enables real-time monitoring of amplified products, enabling accurate quantification of DNA and RNA and eliminating the need for electrophoresis. , it has the advantage of being able to detect quickly, simply, and accurately. Therefore, the primers of the present invention are preferably for real-time reverse transcription polymerase chain reaction (RT-PCR).

The primers or probes are capable of incorporating additional features that do not alter the basic properties. That is, as long as the simultaneous detection of COVID-19, severe acute respiratory syndrome virus and severe acute respiratory syndrome-like virus, which are the object of the present invention, is possible, the oligonucleotide sequence can be modified using conventional means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of one or more homologues of nucleotides and modifications between nucleotides, such as uncharged linkages (eg methylphosphonates, phosphotriesters, phosphoroamines). dates, carbamates, etc.) or charged linkages (eg phosphorothioates, phosphorodithioates, etc.). In addition, nucleic acids include nucleases, toxins, antibodies, signal peptides, proteins such as poly-L lysine, intercalating agents such as acridine or psoralen, chelating agents such as metals, radioactive metals, metal oxides of iron, and mangolinizing agents, etc. may have one or more additional covalently bound residues of

The composition according to the present invention is characterized in that it enables simultaneous detection as well as specific detection of COVID-19 or severe acute respiratory syndrome virus and severe acute respiratory syndrome-like virus. In particular, the composition is characterized in that it specifically binds to the S1 domain or the S2 domain of the virus, respectively, according to each set.

In one embodiment of the present invention, the COVID-19 virus is specifically detected (primer set A) using the detection composition according to the present invention, and severe acute respiratory syndrome virus and severe acute respiratory syndrome-like virus are specifically detected. (Primer set B), but it was confirmed that other viruses were not detected. That is, it was confirmed that by using the composition for detection according to the present invention, not only the COVID-19 virus, which is currently prevalent worldwide, but also the SARS virus, bats, or SARS-like virus can be simultaneously detected.

In addition, the present invention provides (a) a pair of primers of SEQ ID NOs: 1 and 2; And (b) the probe of SEQ ID NO: 3; provides a composition for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) containing. Primer pairs of SEQ ID NOs: 1 and 2; And the probe of SEQ ID NO: 3; the information about the salpin bar above.

In the case of the above composition alone, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be detected, and in combination with other detection compositions, it can be used for detection of severe acute respiratory syndrome coronavirus 2.

In addition, the present invention provides (a) a pair of primers of SEQ ID NOs: 4 and 5; And (b) any one or more probes selected from the group consisting of SEQ ID NOs: 6 to 8; provides a composition for detecting severe acute respiratory syndrome (SARS) virus or virus similar thereto. Primer pairs of SEQ ID NOs: 4 and 5; And the probes of SEQ ID NOs: 6 to 8; the contents are the same as previously salpin.

In the case of the above composition, it can be used alone to determine the presence or absence of a severe acute respiratory syndrome (SARS) virus or a virus similar to it, and also in combination with a primer pair and probe set to detect a specific type of coronavirus. and can be used.

The present invention also provides a kit for detecting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), that is, Corona 19 virus, comprising the primer pair and probe according to the present invention.

In addition, the present invention provides a kit for simultaneous detection of severe acute respiratory syndrome (SARS) virus and similar viruses thereof, including the primer set and probe according to the present invention described above.

In the present invention, the kit may be characterized in that it is for real-time reverse transcription polymerase chain reaction (RT-PCR).

A kit according to the present invention may further include a composition, solution or device as one or more other components suitable for the assay method. Preferably, the kit may further include essential elements required to perform reverse transcription and RT-PCR.

The amplification means may be a conventional means for carrying out a reverse transcription polymerase chain reaction, and may include, for example, a reaction mixture capable of carrying out a polymerase chain reaction using a reverse transcription polymerase chain reaction.

The kit may further include a user manual describing optimal reaction performance conditions. Instructions include brochures in the form of pamphlets or leaflets, labels affixed to the kits, and instructions on the surface of packages containing the kits.

Further, the description includes information disclosed or provided through an electronic medium such as the Internet.

Since the kit according to the present invention uses the above-described primer pair and probe according to the present invention, the description of the common content is omitted to avoid excessive redundancy of the specification according to repeated description.

On the other hand, the present invention provides a method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using the primer pair and probe according to the present invention described above, and severe acute respiratory syndrome using the primer set and probe according to the present invention. A method for simultaneously detecting a Severe acute respiratory syndrome (SARS) virus and similar viruses thereof is provided.

In the present invention, the method may be characterized by comprising the step of extracting nucleic acid from a sample and amplifying it.

The amplification step is performed at 40 ° C to 50 ° C, preferably 43 ° C to 47 ° C, more preferably 45 ° C for 10 to 20 minutes, preferably 13 to 17 minutes, more preferably 15 minutes and 90 ° C to 100 ° C a first step of cycling preferably at 93° C. to 97° C. more preferably at 95° C. once for 1 to 5 minutes, preferably 1 to 3 minutes, more preferably 1 minute; and 2 cycling 35 to 40 times, preferably 40 times, at 90° C. to 100° C., preferably at 95° C. for 5 seconds, and at 50° C. to 60° C., preferably at 58° C., for 10 to 30 seconds, preferably 20 seconds. A second step may be included. In the present invention, the first step is a reverse transcription process and the second step is a gene amplification process, and the gene amplification process consists of dissociation-coupling-extension steps.

The sample may be a sample isolated from an animal suspected of being infected with SARS or a virus similar thereto, and the animal may be selected from the group consisting of birds, livestock, and humans. In addition, the sample may be a clinical sample or an environmental sample, and for example, the clinical sample may be obtained from tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine.

In the present invention, the reverse transcription polymerase chain reaction is not limited thereto, but may be characterized in that it is a one-step real-time reverse transcription polymerase chain reaction.

In the present invention, detection of amplification products during RT-PCR may be performed through DNA chip, gel electrophoresis, capillary electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto.

According to a preferred embodiment, the diagnostic method of the present invention may include quantifying the amplification of a gene in real time, for example, monitoring in real time when amplifying a gene through real time reverse transcription polymerase chain reaction (Real time RT-PCR). Calculate the Ct value (Cycle threshold) from the amplification curve obtained by doing this, and use it to quantify the presence or absence of the target gene in the gene amplification product or the amplification product.

In one embodiment of the present invention, a Bioline One-Step RT-PCR Kit from Bioline Reagents is used, and a reporter is attached to the 5' end of the probe and a quencher is attached to the 3' end of the probe, and the hybridization reaction between the amplified nucleic acid and the probe is examined. The presence or absence of COVID-19, severe acute respiratory syndrome virus, and severe acute respiratory syndrome-like virus was detected, and their infection was diagnosed.

The detection method using the primer pair and probe according to the present invention can efficiently detect COVID-19, severe acute respiratory syndrome virus, and severe acute respiratory syndrome-like virus with simpler steps than conventional methods.

Terms not defined otherwise in this specification have meanings commonly used in the technical field to which the present invention belongs.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, but the content of the present invention is not limited to the following examples.

Example 1. Preparation of composition for detection according to the present invention

We analyzed the nucleotide sequence of the novel coronavirus that occurred at home and abroad, secured a specific nucleotide sequence, and designed primers and probes for detection. First, SARS including the new coronavirus and 59 protein sequences of bat-derived coronaviruses were obtained. after. The obtained nucleotide sequences were aligned, and based on this, forward/reverse primers and probes for detecting SARS-CoV-2 (COVID-19) were prepared, which were named 'Primer Set A'.

In addition, forward/reverse primers and probes were prepared for diagnosis of SARS-CoV and pan-SARS viruses, and they were named 'Primer Set B'. Figure 1 shows the target position in the coronavirus of the primer set of the present invention, Figure 2 shows the results of sequencing analysis and target selection of primer set A, and Figure 3 shows the results of sequencing analysis and target selection of primer set B. . Probes were prepared to include FAM (5-Carboxyfluorescein), HEX (5-hexachloro-fluorescein), Cy5 (cyanine 5 phosphoramidite), and Texas Red fluorescent dyes. Details of the primers and probes of the primer sets A and B are shown in Table 1 below.

Primer/Probe name Sequence (5'→ Target gene Position Referance sequence number set A SARS-CoV-2
Forward primer CAA TGT TAC TTG GTT CCA TGC Spike S1 domain +207 MN908947 One SARS-CoV-2
Reverse primer GTT AGA CTT CTC AGT GGA AGC -325 2 SARS-CoV-2 Probes Hex-CAT GTC TCT GGG ACC AAT GGT-BHQ1 +232 3 setB pan-SARS-like
Forward primer ATY AGR GCT GCW GAA ATC AG Spike S2 domain +3064 MN908947
FJ882956 4 pan-SARS-like
Reverse primer CCW TCA TGA CAA ATD GCW GG -3281 5 pan-SARS-like probes Texas red-GCT TCT GCY AAY CTT GCT GC-BHQ2 +3085 6 SARS-CoV Probes Cy5-TTG TGG AAA GGG CTA CCA CCT-BHQ2 +3153 7 SARS-CoV-2 Probes FAM-ATG AGG TGC TGA CTG AGG GA-BHQ1 -3201 8 Mixed nucleotides are coded by D(A/G/T), R(A/G), W(A/T) and Y(C/T)

Experimental Example 1. The present invention primer Verification of specificity and sensitivity of the set

To verify the specificity and sensitivity of the primers and probes, real-time reverse transcription quantitative PCR (rRT-qPCR) was performed. As a reagent for this, a Bioline One-Step RT-PCR Kit (Bioline Reagents Ltd., London, UK) was used, and the composition of the reaction for PCR was shown in Table 2 below. The probe mixture in Set A composition contained the SARS-2 probe of SEQ ID NO: 3 alone, and the probe mixture in Set B composition contained SARS-CoV, SARS-CoV-2 and pan-SARS of SEQ ID NOs: 6 to 8. -like (similar) virus probes were included, and the composition probe mixture of Set A + B contained all probes of SEQ ID NO: 3, SEQ ID NO: 6, 7, and 8.

Meanwhile, reaction conditions are shown in Table 3 below. Thermocycling for RT-PCR was performed using the LightCycler 96 System (Roche, USA), and a positive result was estimated according to the analysis of the fluorescence curve from each probe within 40 cycles.

Set A Set B Set A+B Real-time RT- PCR buffer solution 10 μl 10 μl 10 μl RNA template 5 μl 5 μl 5 μl 0.5 pM primer (F+R) 2 μl 2 μl 2 μl 5 μM probe mixture 3 μl 3 μl 3 μl enzyme mixture 0.6 μl 0.6 μl 0.6 μl total amount 20 μl

Temp. (℃) Time Cycle 45 15min One 95 2min 95 5 seconds 40 58 20 seconds

go. using a virus primer / probe specificity validation

Using various viruses possessed by Korea Research Institute of Bioscience and Biotechnology, specificity analysis for primers and probes for detecting SARS-CoV, SARS-CoV-2 and pan-SARS-like viruses prepared in Example 1 was performed. proceeded. To verify the specificity, various virus cultures including human cells were used.

A total of 16 viruses were used in the experiment, specifically Dengue type 1 (Korea/DenKor-07), Dengue type 2 (Korea/DENV-2/KBPV-VR-29), and Dengue type 3 (Korea/DENV-3). /KBPV-VR-30), Dengue type 4 (Korea/DENV-4/KBPV-VR-31), Influenza A (Korea/37/2012, H3N2), Influenza A (California-04/09, H1N1), PRRS (Korea/CP07-401-9), Porcine epidemic diarrhea (Korea/SM98), Parainfluenza virus 1 (Korea/KUMC-44), Human respiratory syncytial virus (HRSV-A/IC688/12), Bat-SARS-related coronavirus (B18-83), Bat-SARS-related coronavirus (B18-92), MERS-Cov (Korea/KNIH/002_5_2015), Coronavirus-NL63 (Korea/CN0601/14), SARS-CoV, SARS-CoV-2 virus (BetaCoV/South Korea/KUMC01/2020) was used.

As shown in Table 4 below, while primer set A detected fluorescence specifically for Corona 19 (COVID-19), it was confirmed that fluorescence was not detected for other viruses.

In addition, it was confirmed that fluorescence was detected specifically for the SARS-CoV-2 virus, SARS virus, and SARS-like virus, whereas fluorescence was not detected for other viruses. Specifically, the SARS-CoV-2 (COVID-19) virus showed specific binding to the SARS-CoV-2 probe ( ct value: 15.35) and pan-SARS-like probe ( ct value: 14.03) of primer set B. , SARS virus showed specific binding to the SARS-CoV probe ( ct value: 12.58) and pan-SARS-like probe ( ct value: 11.75) of primer set B, and Bat-SARS-related coronavirus (B18-83) and Bat-SARS-related coronavirus (B18-92) showed specific binding only to the pan-SARS-like probe of primer set B.

These results show that using the primer set of the present invention can simultaneously detect not only the SARS-CoV-2 (COVID-19) virus that is currently prevalent worldwide, but also SARS-CoV, bats, or SARS-like coronaviruses. will be.

Virus Name Virus Family Host Acession Number Mean ct value (standard Deciation ) Set A set B SARS- CoV -2 SARS- CoV -2 SARS- CoV pan-SARS-like Dengue type 1 Flaviviridae Human KP406803 - - - - Dengue type 2 Flaviviridae Human KP406804 - - - - Dengue type 3 Flaviviridae Human KP406805 - - - - Dengue type 4 Flaviviridae Human KP406806 - - - - Influenza A (H3N2) Orthomyxoviridae Human KT889146 - - - - Influenza A (H1N1)) Orthomyxoviridae Human GQ117044 - - - - PRRS Arteriviridae Swine JX138235 - - - - Porcine epidemic diarrhea Coronaviridae Swine GU937797 - - - - Parainfluenza virus 1 Paramyxoviridae Human MG255129 - - - - Human respiratory syncytial virus (HRSV-A/IC688/12) Pneumoviridae Human KP663728 - - - - Bat-SARS-related coronavirus (B18-83) Coronaviridae Bat MK991935 - - - 22.19 (0.23) Bat-SARS-related coronavirus (B18-92) Coronaviridae Bat MK991936 - - - 17.32 (0.16) MERS-CoV Coronaviridae Human KT029139 - - - - Coronavirus-NL63 Coronaviridae Human MG772808 - - - - SARS-CoV Coronaviridae Human AY278491 - - 12.58 (0.22 11.75 (0.31) SARS-CoV-2 (COVID-19) Coronaviridae Human EPI_ISL_413017 15.29 (0.09) 15.35 (0.41) - 14.03 (0.27)

me. using a virus primer / probe Sensitivity validation

For sensitivity analysis, the SARS-CoV-2 (BetaCoV/South Korea/KUMC01/2020) virus recently isolated in Korea was distributed from the National Pathogen Resource Bank under the National Institutes of Health under the Centers for Disease Control and Prevention, and was used for sensitivity analysis verification.

In order to verify the sensitivity of the primer set of the present invention, the sensitivity of the SARS-CoV-2 diagnostic primer set currently proposed by the World Health Organization (WHO) and the primer set of the present invention were compared and analyzed. During real-time reverse transcription PCR (rRT-PCR), the intensity of fluorescence emitted from hydrolyzed probes (FAM, HEX and Texas Red) is measured every cycle and based on the measured amplification curve. So, the results were shown as quantification cycle values (Cq values).

Detailed information of the SARS-CoV-2 diagnostic primer set currently proposed by the World Health Organization (WHO) is shown in Table 5, and the sensitivity analysis results using the primer set of the present invention are shown in Table 6, and the results of the sensitivity analysis presented by the WHO Table 7 shows the results of sensitivity analysis using primer sets for detecting SARS-CoV-2.

Primer/Probe name Sequence (5'→ Target gene Reference ORF1b-Nsp14F TGG GGY TTT ACR GGT AAC CT ORF1b https://www/who/int/docs/default-source/coronaviruse/peiris-protocol-16-1-20/pdf?sfvrsn=af1aac73_4 Nsp141-Probe FAM-TAG TTG TGA TGC WAT CAT GAC TAG-TAMRA ORF1b-Nsp14R AAC RCG CTT AAC AAA GCA CTC HKU-NF TAA TCA GAC AAG GAA CTG ATT A Nucleocapsid HKU-N-probe FAM-GCA AAT TGT GCA ATT TGC GG-TAMRA HKU-NR CGA AGG TGT GAC TTC CAT G RdRp_SARSr-F GTG ARA TGG TCA TGT GTG GCG G RNA-dependent RNA Polymerase https://www/who/int/docs/default-source/coronaviruse/peiris-protocol-v2-1.pdf?sfvrsn=a9ef618c_2 RdRp_SARSr-P2 FAM-CAG GTG GAA CCT CAT CAG GAG ATG C-BHQ1 RdRp_SARSr-P1 FAM-CCA GGT GGW ACR TCA TCM GGT GAT GC-BHQ1 RdRp_SARSr-R CAR ATG TTA AAS ACA CTA TTA GCA TA E_sarbeco_F ACA GGT ACG TTA ATA GTT AAT AGC GT Envelope E_sarbeco_P1 FAM-ACA CTA GCC ATC CTT ACT GCG CTT CG-BHQ1 E_sarbeco_R ATA TTG CAG CAG TAC GCA CAC A N_sarbeco_F CAC ATT GGC ACC CGC AAT C Nucleocapsid N_sarbeco_P FAM-ACT TCC TCA AGG AAC AAC ATT GCC A-BHQ1 N_sarbeco_R GAG GAA CGA GAA GAG GCT TG

* Mixed nucleotides are coded by D(A/G/T), M(A/C), R(A/G), S(G/C), W(A/T) and Y(C/T)

SARS- CoV -2 RNA serial dilution Ct value S1 target s2 targer s1 + s2 target SARS- CoV -2
(HEX) SARS- CoV -2
(FAM) pan-SARS
(Texas red) SARS- CoV -2
(HEX) SARS- CoV -2
(FAM) pan-SARS
(Texas red) Singleplex Singleplex Multiplex Multiplex Multiplex Multiplex Multiplex 10^-1 18.15 18.25 18.19 16.96 17.93 19.06 16.88 10^-2 20.98 21.95 21.76 20.51 20.71 22.09 20.04 10^-3 24.21 25.05 25.25 23.92 23.96 25.17 23.53 10^-4 27.45 28.24 28.61 27.29 27.35 28.79 27.08 10^-5 30.43 32.32 32.02 30.79 30.72 32.34 30.59 10^-6 34.33 36.22 35.58 34.33 33.52 35.71 34.17 H ₂ O - - - - - - -

SARS- CoV -2 RNA serial dilution Ct value HKU group Germany group ORF1b - Nsp14 HKU -N RdRp _ SARSr E_ sarbeco N_ sarbeco 10^-1 21.14 20.3 20.97 17.68 23.02 10^-2 24.47 23.4 23.7 20.94 25.72 10^-3 27.73 26.46 27.26 24.19 28.87 10^-4 30.82 30.03 30.6 27.36 32.28 10^-5 32.13 31.95 30.78 30.76 35.15 10^-6 32.47 32.39 36.34 34.17 36.91 H ₂ O - - - - -

As shown in Table 6, the Cq values obtained from the diluted viral RNA templates using the primer set of the present invention were significantly correlated with the copy number of each RNA template, with R2 values of 1.0 for each RNA template. appeared to be On the other hand, the negative control (phosphate buffered saline, PBS) did not show any amplification curve.

In addition, when comparing the results shown in Table 5 and Table 6, the primer set of the present invention is SARS-CoV-2 presented by WHO Compared to the detection primer set, it was confirmed to have higher sensitivity. In addition, it was confirmed that the SARS-CoV-2 (COVID-19) virus can be detected with high sensitivity without primer interference even when primer set A and primer set B of the present invention are used together.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Marker for multi-diagnosis of novel corona virus infection disease and its use <130> KRIBB1_60P <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> artificial sequence <220> <223> This is SARS-2 forward primer of primer set A. <400> 1 caatgttact tggttccatg c 21 <210> 2 <211> 21 <212> DNA <213> artificial sequence <220> <223> This is SARS-2 reverse primer of primer set A. <400> 2 gttagacttc tcagtggaag c 21 <210> 3 <211> 21 <212> DNA <213> artificial sequence <220> <223> This is SARS-2 probe of primer set A. <400> 3 catgtctctg ggaccaatgg t 21 <210> 4 <211> 20 <212> DNA <213> artificial sequence <220> <223> This is pan-SARS-like forward primer of primer set B. <400> 4 atyagrgctg cwgaaatcag 20 <210> 5 <211> 20 <212> DNA <213> artificial sequence <220> <223> This is pan-SARS-like reverse primer of primer set B. <400> 5 ccwtcatgac aaatdgcwgg 20 <210> 6 <211> 20 <212> DNA <213> artificial sequence <220> <223> This is pan-SARS-like probe of primer set B. <400> 6 gcttctgcya aycttgctgc 20 <210> 7 <211> 21 <212> DNA <213> artificial sequence <220> <223> This is SARS-1 probe of primer set B. <400> 7 ttgtggaaag ggctaccacc t 21 <210> 8 <211> 20 <212> DNA <213> artificial sequence <220> <223> This is SARS-2 probe of primer set B. <400> 8 atgaggtgct gactgaggga 20

Claims (19)

Primer set A including the primer pair of SEQ ID NOs: 1 and 2 and the probe of SEQ ID NO: 3 for targeting and specifically amplifying a part of a spark gene; and
Primer set B comprising primer pairs of SEQ ID NOs: 4 and 5 and probes of SEQ ID NOs: 6 to 8 for targeting and specifically amplifying a part of the spark gene;
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); and
severe acute respiratory syndrome coronavirus (SARS-CoV) or bat beta corona virus; A composition for simultaneous detection.
delete According to claim 1,
The probe is
Characterized in that a reporter is further spliced at the 5 'end,
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); and
severe acute respiratory syndrome coronavirus (SARS-CoV) or bat beta corona virus; A composition for simultaneous detection.
According to claim 3,
The reporter,
6-carboxyfluorescein (FAM), texas red, fluorescein, 5-hexachloro-fluorescein (HEX), fluorescein chlorotriazinyl, rhodamine green, rhoda rhodamine red, tetramethylrhodamine, fluorescein isothiocyanate (FITC), oregon green, alexa fluor, JOE (6-Carboxy-4',5'-Dichloro- 2',7'-Dimethoxyfluorescein), ROX (6-Carboxyl-X-Rhodamine), TET (Tetrachloro-Fluorescein), TRITC (tertramethylrodamine isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine), NED (N-(1-Naphthyl ) ethylenediamine), cyanine-based dyes, and thiadicarbocyanine, characterized in that at least one selected from the group consisting of,
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); and
severe acute respiratory syndrome coronavirus (SARS-CoV) or bat beta corona virus; A composition for simultaneous detection.
According to claim 1,
The probe is
Characterized in that a quencher is further conjugated to the 3 'end,
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); and
severe acute respiratory syndrome coronavirus (SARS-CoV) or bat beta corona virus; A composition for simultaneous detection.
According to claim 5,
The quencher,
TAMRA (6-carboxytetramethyl-rhodamine), BHQ1 (black hole quencher 1), BHQ2 (black hole quencher 2), BHQ3 (black hole quencher 3), NFQ (nonfluorescent quencher), dabcyl, Eclipse, DDQ (Deep Dark Quencher), blackberry quencher (Blackberry Quencher) and Iowa black (Iowa black) Characterized in that at least one selected from the group consisting of,
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); and
severe acute respiratory syndrome coronavirus (SARS-CoV) or bat beta corona virus; A composition for simultaneous detection.
According to claim 1,
The primer pair,
Characterized in that it is a primer pair for real-time reverse transcription polymerase chain reaction (RT-PCR),
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); and
severe acute respiratory syndrome coronavirus (SARS-CoV) or bat beta corona virus; A composition for simultaneous detection.
According to claim 1,
The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) are, Characterized in that it is derived from at least one selected from the group consisting of pigs, cattle, rodents and birds,
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); and
severe acute respiratory syndrome coronavirus (SARS-CoV) or bat beta corona virus; A composition for simultaneous detection.
delete According to claim 1,
The primer pair of the primer set A,
Characterized in that the spike domain S1 of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is targeted,
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); and
severe acute respiratory syndrome coronavirus (SARS-CoV) or bat beta corona virus; A composition for simultaneous detection.
According to claim 1,
The primer pair of the primer set B,
Characterized in that the target is the spike domain S2 of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV) or bat beta corona virus ,
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); and
severe acute respiratory syndrome coronavirus (SARS-CoV) or bat beta corona virus; A composition for simultaneous detection.
(a) a pair of primers of SEQ ID NOs: 1 and 2; and
(B) a probe of SEQ ID NO: 3; Severe acute respiratory syndrome (SARS) virus 2 (SARS-CoV-2) detection composition comprising a.
(a) a pair of primers of SEQ ID NOs: 4 and 5; and
(b) a primer set B comprising probes of SEQ ID NOs: 6 to 8;
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); and
severe acute respiratory syndrome coronavirus (SARS-CoV) or bat beta corona virus; A composition for simultaneous detection.
Primer sets A and B according to any one of claims 1, 3 to 8, 10 and 11; Or a primer set B according to claim 13;
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); and
severe acute respiratory syndrome coronavirus (SARS-CoV) or bat beta corona virus; Kits for simultaneous detection.
According to claim 14,
The kit,
Characterized in that it is for real-time reverse transcription polymerase chain reaction (RT-PCR),
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); and
severe acute respiratory syndrome coronavirus (SARS-CoV) or bat beta corona virus; Kits for simultaneous detection.
Comprising the primer pair and probe according to claim 12,
Kit for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
According to claim 16,
The kit,
Characterized in that it is for real-time reverse transcription polymerase chain reaction (RT-PCR),
Kit for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
Primer sets A and B according to any one of claims 1, 3 to 8, 10 and 11; Or primer set B according to claim 13; using,
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); and
severe acute respiratory syndrome coronavirus (SARS-CoV) or bat beta corona virus; simultaneous detection method.
A method for detecting severe acute respiratory syndrome (SARS) virus 2 (SARS-CoV-2) using the primer pair and probe according to claim 12.

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