KR102428504B1 - RT-LAMP pirmer set for detecting SARS-CoV-2 omicron variant and uses thereof - Google Patents

Abstract

The present invention relates to a reverse transcription loop-mediated isothermal amplification (RT-LAMP) primer set for detecting SARS-CoV-2 Omicron variant and a use thereof. By using an oligonucleotide primer set consisting of the nucleotide sequences of SEQ ID NOs: 1 to 5 and an oligonucleotide probe consisting of the nucleotide sequence of SEQ ID NO: 6 according to the present invention, it is expected that SARS-CoV-2 Omicron variant can be rapidly detected through a point of care testing using a simple real-time isothermal amplification device.

Description

RT-LAMP primer set for detecting SARS-CoV-2 omicron variant and uses thereof

The present invention relates to a set of RT-LAMP primers for detecting SARS-CoV-2 micron mutation and uses thereof.

SARS-CoV-2/Omicron mutant virus (B.1.1.529) is a mutation of SARS-CoV-2 that was newly discovered in Botswana on November 9, 2021. On November 26, the WHO officially named it Omicron and classified it as a Variant of Concern (VOC).

Omicron mutant virus is the first mutant strain overlapped with a purine cleavage mutation (P681H), and there are P681H mutations that cause infectious enhancement in alpha mutations and N679K mutations observed in C.1.2 at the same time. Moreover, there are at least four vaccine avoidance mutations (E484A, S477N, N440K, Q493R), and there is also an amplifying mutation K417N. Although less than lambda mutations, mutations occurred in two nucleocapsid proteins (N:R203K, N:G204R). In addition, A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N in the spike protein used when SARS-CoV-2 penetrates into human cells. , N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K and L981F mutations were observed. The mutation in the spike protein of the Omicron virus is known to be more than 6 times greater than the existing delta mutation (B.1.617.2).

Omicron mutant virus is likely to be the most drug-resistant of the mutations discovered so far. It is estimated to have a minimum of five-fold mutation and a maximum of nine-fold mutation. According to the research results of the Ministry of Health of South Africa, 58 RNA blueprints were different from the existing ones, and 32 mutations in the spike protein were identified.

Meanwhile, Korean Patent Application Laid-Open No. 2021-0113932 discloses a 'high-sensitivity multi-loop-mediated isothermal amplification primer set for detecting novel coronaviruses', and Korean Patent Application Publication No. 2021-0132628 discloses 'a novel guide RNA and a coronavirus using the same'. Although a method for diagnosing infection 2019 is disclosed, there is no description of the 'RT-LAMP primer set for detecting SARS-CoV-2 micron mutation and its use' of the present invention.

The present invention has been derived from the above needs, and the present inventors have developed a diagnostic method capable of discriminating SARS-CoV-2 micron mutations, the S (spike) gene of SARS-CoV-2 micron virus. A primer set for loop-mediated isothermal amplification (LAMP) including an omicron mutation-specific forward inner primer (FIP) and an omicron non-specific FIP was prepared by targeting the region.

Thereafter, as a result of performing an amplification reaction using the primer set for LAMP prepared in the present invention for clinical samples containing omicron mutations and clinical specimens containing delta mutations, SARS-CoV-2 omicron mutations were By confirming that it was detected clearly, this invention was completed.

In order to solve the above problems, the present invention provides an oligonucleotide primer set consisting of the nucleotide sequence of SEQ ID NOs: 1 to 5 and an oligonucleotide probe consisting of the nucleotide sequence of SEQ ID NO: 6, SARS-CoV-2 micron mutant virus detection It provides a composition for Reverse Transcriptional Loop-mediated isothermal amplification for

In addition, the present invention provides a kit for detecting SARS-CoV-2 micron mutant virus comprising the composition for reverse cycle-mediated isothermal amplification.

In addition, the present invention comprises the steps of isolating total RNA from a biological sample isolated from a subject suspected of SARS-CoV-2 infection; using the isolated total RNA as a template and amplifying a target sequence by a reverse cycle-mediated isothermal amplification reaction using the composition for reverse cycle-mediated isothermal amplification; and detecting the product of the amplification step.

Using the primer set according to the present invention, it is possible to quickly distinguish SARS-CoV-2 micron mutations in the field with only a simple real-time isothermal amplification device, so it will be usefully used for response management of COVID-19.

1 shows the nucleotide sequence information used in the preparation of the LAMP primer for distinguishing the SARS-CoV-2 micron mutation used in the present invention, and information on the LAMP primer set and probe.
2 is a LAMP reaction result performed in a mobile isothermal amplification device using the Omicron-specific/non-specific primer set according to the present invention for a clinical sample containing a clinical evaluation material from the Korea Centers for Disease Control and Prevention and a delta mutation.
3 is a LAMP reaction result performed on an ABI7500 Real-Time PCR System equipment using an Omicron-specific/non-specific primer set according to the present invention for a clinical sample containing a clinical evaluation material from the Korea Centers for Disease Control and Prevention and a delta mutation.

In order to achieve the object of the present invention, the present invention provides an oligonucleotide primer set consisting of the nucleotide sequence of SEQ ID NOs: 1 to 5 and an oligonucleotide probe consisting of the nucleotide sequence of SEQ ID NO: 6, SARS-CoV-2 micron mutation Provided is a composition for Reverse Transcriptional Loop-mediated isothermal amplification (RT-LAMP) for virus detection.

In addition, the composition of the present invention may further include an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 7, but is not limited thereto.

In the composition for RT-LAMP for detecting SARS-CoV-2 micron mutant virus according to the present invention, the primer set consisting of the nucleotide sequences of SEQ ID NOs: 1 to 5 is GenBank accession no. Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/BEL/rega-20174/2021, OL672836.1, a gene encoding a spike protein in the complete genome (bases at positions 21,497 to 25,309 in the genome sequence) ) is specific for The oligonucleotide primer of SEQ ID NO: 3 according to the present invention is designed based on the Δ69-70 mutation (ΔTACATG) of the spike protein coding region specifically observed in the micron mutation, and consists of the oligonucleotide sequence of SEQ ID NOs: 1 to 5 If the nucleotide primer set is used, an amplification reaction occurs specifically for SARS-CoV-2 micron mutation. On the other hand, the oligonucleotide primer of SEQ ID NO: 7 is designed based on the base (TACATG) present in SARS-CoV-2 including delta mutation, and consists of the nucleotide sequence of SEQ ID NO: 1, 2, 4, 5, 7 When the nucleotide primer set is used, the amplification reaction occurs specifically for SARS-CoV-2 except for the micron mutation.

That is, the oligonucleotide primer set consisting of the nucleotide sequences of SEQ ID NOs: 1 to 5 is specific for SARS-CoV-2 micron mutation, and the oligonucleotide primer set consisting of the nucleotide sequences of SEQ ID NOs: 1, 2, 4, 5 and 7 is non-specific for SARS-CoV-2 micron mutation.

The primer set according to the present invention extracts and purifies a nucleic acid from a sample (specimen), and mixes a reverse transcriptase and a polymerase using the isolated nucleic acid, particularly RNA, as a template in one tube to perform a reverse transcription reaction and It can be used in the reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) method, which is performed so that the loop-mediated isothermal amplification reaction occurs simultaneously. It can also be used for a method of performing a ring-mediated isothermal amplification reaction alone by using it, but is not limited thereto.

As used herein, the term "Loop-mediated isothermal amplification (LAMP)" refers to a method capable of performing an amplification reaction under isothermal conditions, unlike the existing polymerase chain reaction (PCR) method. Four types of primers (F3, B3, FIP, BIP) are basically required for the LAMP reaction, and two types of primers (LoopF, LoopB) can be added to improve the reaction rate.

The four basic primers are composed of two types of outer primers and two types of inner primers, and the outer primers are a forward outer (F3) primer and two types of backward outer (B3) primers. It is composed of and serves to unwind the DNA double strand during the non-cyclic step of the reaction. The inner primer consists of two types: a forward inner primer (FIP) and a backward inner primer (BIP). It consists of the corresponding nucleotides. The additional two types of primers are composed of a forward loop (LoopF) primer and a backward loop (LoopB) primer and are attached to a nucleotide sequence to which the inner primer does not bind to perform a loop-mediated isothermal amplification reaction. accelerate

In the oligonucleotide primer set consisting of the nucleotide sequences of SEQ ID NOs: 1 to 5 according to the present invention, the primer of SEQ ID NO: 1 is F3 as a forward external primer, the primer of SEQ ID NO: 2 is B3 as a reverse external primer, and SEQ ID NO: 3 The primer of SEQ ID NO: 4 is a forward internal primer, FIP, the primer of SEQ ID NO: 4 is a reverse internal primer, BIP, and the primer of SEQ ID NO: 5 is a reverse loop primer, LoopB.

In addition, the primer of SEQ ID NO: 7 is a forward internal primer, FIP.

The primer of the present invention may include an oligonucleotide consisting of a fragment of 15 or more, 16 or more, 17 or more consecutive nucleotides in SEQ ID NOs: 1, 2 and 5, depending on the sequence length of each primer, and SEQ ID NO: oligonucleotides consisting of segments of at least 32, at least 33, at least 34, at least 35, at least 36, at least 37 contiguous nucleotides within 3, 4 and 7. For example, the primer of SEQ ID NO: 1 (22 oligonucleotides) is 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more nucleotides in the sequence of SEQ ID NO: 1 It may include an oligonucleotide consisting of a fragment of

In addition, in the composition for reverse cycle-mediated isothermal amplification for detecting SARS-CoV-2 micron mutant virus of the present invention, the oligonucleotide probe consisting of the nucleotide sequence of SEQ ID NO: 6 is one of the 5' end and the 3' end. may be labeled with a fluorescent dye, and a quencher may be labeled on the other end, preferably a fluorescent dye is labeled on the 5' end, and a quencher labeled on the 3' end, , a probe that specifically binds to a product amplified by a set of primers (omicron-specific and non-specific).

In one embodiment of the present invention, the oligonucleotide probe consisting of the nucleotide sequence of SEQ ID NO: 6 is labeled with FAM (Fluorescein amidites) at the 5' end and BHQ ₁ (Black Hole Quencher-1) at the 3' end, The 1st to 7th bases ('CTGGGAC') form a stem-loop structure so that FAM fluorescence does not appear by BHQ ₁ , but the stem-loop structure is broken as the temperature rises in the isothermal amplification reaction step and the primer set It is designed to bind to the amplicon amplified by and to show FAM fluorescence.

As used herein, the term “primer” refers to a single-stranded oligonucleotide sequence complementary to a nucleic acid strand to be replicated, and may serve as a starting point for synthesis of a primer extension product. The length and sequence of the primers should allow synthesis of the extension product to begin. The specific length and sequence of the primer will depend on the conditions of use of the primer, such as temperature and ionic strength, as well as the complexity of the DNA or RNA target required.

In the present invention, the oligonucleotide used as a primer may also contain a nucleotide analogue, for example, a phosphorothioate, an alkylphosphorothioate or a peptide nucleic acid or An intercalating agent may be included.

The primer set according to the present invention is preferably for reverse cycle mediated isothermal amplification (RT-LAMP), in which the reverse transcription reaction and the LAMP reaction occur simultaneously by mixing a reverse transcriptase and a polymerase with RNA as a template in one tube. have.

The present invention also provides a kit for detecting SARS-CoV-2 micron mutant virus comprising the composition for reverse cycle-mediated isothermal amplification of the present invention.

In the kit of the present invention, the composition for multiplex reverse cycle mediated isothermal amplification includes a primer set and probe for reverse cycle mediated isothermal amplification, and the specific details are the same as described above.

In addition, the kit according to the present invention includes a reagent for performing a reverse cycle-mediated isothermal amplification reaction, and the reagent for performing the reverse cycle-mediated isothermal amplification reaction may include a reverse transcriptase, a DNA polymerase, dNTPs, and a buffer. can DNA polymerase according to an embodiment of the present invention may be Bst polymerase, but is not limited thereto, and for reverse cycle-mediated isothermal amplification, faster amplification instead of Bst polymerase mainly used in the existing loop-mediated isothermal amplification method GspSSD DNA polymerase having the ability and strong reverse transcriptase ability may be used, but is not limited thereto.

In addition, the kit of the present invention may further include a user's guide describing optimal conditions for performing the reaction. A handbook is a printout that explains how to use the kit, eg, how to prepare buffers, and suggested reaction conditions. Instructions include a brochure in the form of a pamphlet or leaflet, a label affixed to the kit, and instructions on the surface of the package containing the kit. In addition, the guide includes information published or provided through an electronic medium such as the Internet.

The present invention also

Isolating total RNA from a biological sample isolated from a subject suspected of SARS-CoV-2 infection;

using the isolated total RNA as a template and amplifying a target sequence by a reverse cycle-mediated isothermal amplification reaction using the composition for reverse cycle-mediated isothermal amplification according to the present invention; and

It provides a method for detecting a SARS-CoV-2 micron mutant virus comprising the step of detecting the product of the amplification step.

The detection method of the present invention includes isolating total RNA from a biological sample isolated from a subject suspected of SARS-CoV-2 infection. The biological sample may be, for example, a collected human sample (nasal swab or isolated human blood), but is not limited thereto, and is a biological sample isolated from a suspected SARS-CoV-2 infection. not limited A method for isolating total RNA from the sample may use any method known in the art, for example, a phenol extraction method may be used. Alternatively, total RNA can be isolated by reacting the isolated biological sample with a lysis buffer consisting of 30-500 mM Tris of pH 6.0-7.0, containing 0.1-1.5% (v/v) Triton X-100, It is not limited thereto.

In addition, in the method for detecting SARS-CoV-2 micron mutation according to the present invention, the reverse cycle-mediated isothermal amplification reaction is performed in a single tube with an omicron-specific primer set of SARS-CoV-2 (SEQ ID NOs: 1 to 5) Alternatively, after mixing the oligonucleotide probe consisting of the Omicron non-specific primer set (SEQ ID NO: 1, 2, 4, 5, 7) and the nucleotide sequence of SEQ ID NO: 6 at a specific concentration, at 60 to 68 ° C for 10 to 50 minutes It may be carried out, preferably it may be carried out for 15 minutes at 67 ℃, but is not limited thereto.

In the method for detecting SARS-CoV-2 micron mutation according to the present invention, the oligonucleotide probe of SEQ ID NO: 6 labeled with a FAM fluorescent dye specifically binds to an amplicon amplified by a primer set, and fluorescence according to the fluorescent dye It is characterized by being able to check the result of nucleic acid amplification in real time through level measurement.

Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

Example 1. Design of a LAMP primer set for the discrimination of SARS-CoV-2 micron mutations

In order to prepare a primer set for LAMP for discriminating SARS-CoV-2 micron mutations, GenBank accession no. Severe acute respiratory syndrome coronavirus 2 isolate SARS-CoV-2/human/BEL/rega-20174/2021, OL672836.1, a gene encoding a spike protein in the complete genome (bases at positions 21,497 to 25,309 in the genome sequence) ) specific primer sequences were designed (see FIG. 1 ). The primer for LAMP was designed using OptiGene's LAMP Design software. In particular, the FIP primer (SEQ ID NO: 3) according to the present invention was designed not to bind to other SARS-CoV-2 viruses including delta mutations, and was designed not to bind to other SARS-CoV-2 viruses, and Omicron non-specific. A FIP primer consisting of the nucleotide sequence of SEQ ID NO: 7 was additionally prepared. In addition, a fluorescently-labeled probe (SEQ ID NO: 6) capable of specifically binding to the amplicon amplified by the primer set was prepared.

Example 2. RT-LAMP assay evaluation

To analyze the SARS-CoV-2 micron mutation detection ability using the prepared LAMP primer set, nucleic acids were extracted from clinical specimens using the QIAamp Viral RNA Mini Kit, and isothermal amplification was performed using the extracted nucleic acids. The SARS-CoV-2 micron mutation clinical sample was co-operated by the Korea Centers for Disease Control and Prevention, and the delta mutation clinical sample was provided by the Public Health Department of the Armed Forces Medical Research Institute, and the extracted nucleic acid was diluted with DEPC-treated water.

For RT-LAMP reaction, 2 μl of nucleic acid was taken, Bst polymerase 1 μl (8U), 10×buffer 2.5 μl, 25mM dNTP 1.5 μl, 10mM MgSO ₄ 1 μl, 5M betaine 5 μl, Reverse Transcriptase 0.1 μl (10U), and 1 μl of a primer-probe mixture (5 pmol of each of F3 and B3, 40 pmol of FIP-1 or FIP-2, 20 pmol of BIP, 20 pmol of LoopB and 5 pmol of FAM-BHQ ₁ probe). In order to confirm the result of the isothermal amplification product in real time, an isothermal amplification reaction was performed at 67°C for 15 minutes using a portable isothermal fluorescence PCR device, Gene-8C (ALLSHENG). The fluorescence signal was measured every 20 seconds.

As a result, as shown in FIG. 2 , in the case of the omicron mutation-specific primer set (combination of SEQ ID NOs: 1 to 5), the amplification of the fluorescence signal was specifically confirmed only in clinical specimens containing SARS-CoV-2 omicron mutation. , no amplification of the fluorescence signal was observed in clinical samples containing delta mutations until the end of the reaction. In addition, in the case of using the non-specific primer set of Omicron (a combination of SEQ ID NOs: 1, 2, 4, 5, and 7), amplification of the fluorescence signal was not confirmed in clinical samples containing SARS-CoV-2 Omicron mutations, and delta mutations were included. Amplification of the fluorescence signal was confirmed only in clinical specimen samples. In particular, it was found that the detection of micron mutations within 15 minutes was possible.

In addition, the present inventors performed the isothermal amplification reaction under the same conditions as described above using the ABI7500 Real-Time PCR System equipment. It was reconfirmed that only the sample reacted specifically ( FIG. 3 ). From the above results, the use of the primer set (SEQ ID NOs: 1 to 5) and the probe of the present invention can quickly and accurately detect an micron-mutated virus, so it can be very usefully utilized in on-site diagnosis using portable equipment. was expected

<110> Republic of Korea (Armed Forces Medical Command) <120> RT-LAMP pirmer set for detecting SARS-CoV-2 omicron variant and uses it <130> PN21502 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 acatcaact caggacttgt tc 22 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ctgaactcac tttccatcca a 21 <210> 3 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 ccatcattaa atggtaggac aggggttact tggttccatg ctatct 46 <210> 4 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tgcttccact gagaagtcta acaaagtagg gactgggtct tc 42 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 gaggctggat ttttggtact act 23 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 6 ctgggactct tagtaccatt ggtcccag 28 <210> 7 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ccatcattaa atggtaggac agggggttcc atgctataca tgtct 45

Claims (6)

Having a mutation in which amino acids 69 to 70 of the spike protein of SARS-CoV-2 are deleted, including an oligonucleotide primer set consisting of the nucleotide sequence of SEQ ID NOs: 1 to 5 and an oligonucleotide probe consisting of the nucleotide sequence of SEQ ID NO: 6 A composition for Reverse Transcriptional Loop-mediated isothermal amplification for distinguishing SARS-CoV-2 micron mutated and delta mutant viruses. According to claim 1, wherein the oligonucleotide probe consisting of the nucleotide sequence of SEQ ID NO: 6 is characterized in that one end of the 5' end and the 3' end is labeled with a fluorescent dye, and the other end is labeled with a quencher. A composition for reverse cycle-mediated isothermal amplification for distinguishing between SARS-CoV-2 micron mutant and delta mutant viruses having mutations in which amino acids 69 to 70 of SARS-CoV-2 spike protein are deleted. According to claim 1, wherein the composition further comprises the oligonucleotide primer of SEQ ID NO: 7, SARS-CoV-2 having a mutation in which amino acids 69 to 70 of the spike protein of SARS-CoV-2 are deleted. 2 A composition for reverse cycle-mediated isothermal amplification for distinguishing between micron- and delta-mutated viruses. The SARS-CoV-2 omicron mutation and A kit for the identification of a delta mutant virus. isolating total RNA from a biological sample isolated from a subject suspected of SARS-CoV-2 infection;
using the isolated total RNA as a template and amplifying the target sequence by a reverse cycle-mediated isothermal amplification reaction using the composition for reverse cycle-mediated isothermal amplification of any one of claims 1 to 3; and
A method for distinguishing between SARS-CoV-2 micron mutant and delta mutant viruses having a mutation in which amino acids 69 to 70 of SARS-CoV-2 spike protein are deleted, comprising: detecting the product of the amplification step. The method according to claim 5, wherein the detection of the product of the amplification step is performed through fluorescence measurement.

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