KR20220132418A - Primer set for detection and identification of influenza virus type A and type B and use thereof - Google Patents
Primer set for detection and identification of influenza virus type A and type B and use thereof Download PDFInfo
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- KR20220132418A KR20220132418A KR1020220020349A KR20220020349A KR20220132418A KR 20220132418 A KR20220132418 A KR 20220132418A KR 1020220020349 A KR1020220020349 A KR 1020220020349A KR 20220020349 A KR20220020349 A KR 20220020349A KR 20220132418 A KR20220132418 A KR 20220132418A
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- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
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Abstract
Description
본 발명은 인플루엔자 바이러스(influenza virus) 감염을 신속하고 정확하게 진단하기 위한 방법으로서, Rea-time PCR 기법에 사용되는 인플루엔자 바이러스 A형 및 B형 검출용 프라이머 세트 등에 관한 것이다. 본 발명은 A형 및 B형 인플루엔이자 바이러스 각각에 특이적으로 결합하는 프로브를 이용하여 이를 동시에 감별하여 검출할 수 있다. The present invention relates to a primer set for detecting influenza virus type A and type B used in a ready-time PCR technique as a method for rapidly and accurately diagnosing influenza virus infection. In the present invention, using a probe that specifically binds to type A and type B influenza and viruses, respectively, it can be detected and differentiated at the same time.
인플루엔자 바이러스는 감염성 호흡기 질환의 원인 병원체로 단백질(예를 들어, NP (nuleocapsid), 및 M (matrix) 등)의 항원성 차이로 인해 A, B, C, D형으로 구분된다. Influenza A virus는 인간의 모든 연령과 동물에서 중간 또는 심각한 독감을 일으키며, influenza B virus는 오직 인간에게만 영향을 미친다고 알려져 있다. 독감은 상부 호흡기계 (코, 목)나 하부 호흡기계 (폐)를 침범하며 갑작스러운 고열, 두통, 근육통, 전신 쇠약감과 같은 전반적인 신체 증상을 동반한다. 이러한 독감은 매년 겨울철에 유행하여 건강인에서 업무상의 차질을 일으키고 노인, 만성질환자, 영유아, 임산부 등 고위험군에서 이환률 및 사망률의 증가를 초래해 막대한 사회, 경제적 손실을 유발하는 질환이다. 또한 일부 지역에 한정된 발병이 아닌, 새로운 종류의 독감 바이러스에 의해 짧은 시간에 넓은 지역에 유행하게 되면 젊은 사람도 많이 사망할 수 있다. Influenza viruses are causative agents of infectious respiratory diseases and are classified into A, B, C, and D types due to differences in antigenicity of proteins (eg, NP (nuleocapsid), and M (matrix), etc.). Influenza A virus causes moderate to severe influenza in all ages and animals in humans, and influenza B virus is known to only affect humans. Influenza affects the upper respiratory tract (nose, throat) or lower respiratory tract (lungs) and is accompanied by general physical symptoms such as sudden high fever, headache, muscle aches, and general weakness. The flu is a disease that causes enormous social and economic loss by causing an increase in morbidity and mortality in high-risk groups such as the elderly, chronically ill, infants, pregnant women, etc. In addition, if a new type of influenza virus spreads over a large area in a short period of time, rather than an outbreak limited to a certain area, many young people may die.
호흡기 바이러스 관련 질병의 정확한 원인은 증상과 징후만으론 파악하기 어려우므로 호흡기 감염의 원인 병원체를 정확하게 규명할 수 있는 진단법의 개발이 필요하다. 현재까지 알려진 호흡기 바이러스 관련 질병의 표준 진단방법은 배양을 통한 균의 동정, 혈청학적 검사, 및 중합효소반응(PCR)을 통한 유전자 검사가 있다. 배양을 통한 균의 동정의 경우, 균을 동정하는데 7∼21일이 걸리며 40∼90%에서만 배양되기 때문에 임상 현장에서 실제적으로 이용하는데 한계가 있다. 그리고 바이러스 감염질환의 혈청학적 진단은 가장 널리 이용되고 있는 방법이지만, 이들 보체결합법(CF test, complement fixation test)은 시간이 오래 걸리고 낮은 민감도를 나타내는 등 문제가 있어 최근에는 젤라틴 입자응집법(예, Fujirebio 사의 SeroDia MycoII)이나 효소면역측정법(ELISA; 예, Bio-Rad 사의Platelia Myco IgM/IgG)이 보편적으로 사용되고 있다. 하지만, 상기 두 면역측정법들 역시 방법이 복잡하며, 검사 소요시간이 3~4 시간으로 오래 걸리고, 고가의 장비를 사용하여야 하므로 현장에서 검사를 실시할 수 없는 단점이 있다. Since it is difficult to determine the exact cause of respiratory virus-related diseases only with symptoms and signs, it is necessary to develop a diagnostic method that can accurately identify the pathogen causing respiratory infections. Standard diagnostic methods for respiratory virus-related diseases known to date include identification of bacteria through culture, serological testing, and genetic testing through polymerase reaction (PCR). In the case of the identification of bacteria through culture, it takes 7 to 21 days to identify the bacteria, and since only 40 to 90% of the bacteria are cultured, there is a limit to practical use in the clinical field. And although the serological diagnosis of viral infectious diseases is the most widely used method, these complement fixation tests (CF test) have problems such as taking a long time and showing low sensitivity. Fujirebio's SeroDia MycoII) or enzyme immunoassay (ELISA; eg, Bio-Rad's Platelia Myco IgM/IgG) are commonly used. However, these two immunoassays also have disadvantages in that they are complex, take a long time (3 to 4 hours), and use expensive equipment, making it impossible to conduct the test in the field.
상술한 면역측정법의 단점들을 보안하기 위해 핵산증폭법 분자진단을 활용한 방법이 개발되고 있는데 현재의 호흡기 바이러스의 분자진단 검출은 probe-based real-time PCR을 통한 수행이 주를 이루고 있다. Real-time PCR은 PCR 반응으로 증폭된 증폭산물을 실시간으로 모니터링하는 방법으로 정확도 및 검출 감도가 높고 신속한 정량이 가능하다.In order to secure the disadvantages of the above-described immunoassay method, a method using molecular diagnosis by nucleic acid amplification is being developed. Currently, molecular diagnosis detection of respiratory viruses is mainly performed through probe-based real-time PCR. Real-time PCR is a method of monitoring amplified products amplified by PCR reaction in real time.
본 발명이 이루고자 하는 기술적 과제는 호흡기 질환 원인균을 신속하게 진단할 수 있는 조성물을 제공하는 것으로서, 본 발명은 인플루엔자 바이러스 A형 검출용 조성물과 인플루엔자 바이러스 B형 검출용 조성물을 제공하는 것을 목적으로 한다. An object of the present invention is to provide a composition capable of rapidly diagnosing a respiratory disease causative agent, and the present invention aims to provide a composition for detecting influenza virus type A and a composition for detecting influenza virus type B.
또한, 본 발명은 상기 양 바이러스 검출용 조성물을 모두 포함하여 A형 및 인플루엔자 바이러스 B형을 동시에 감별하여 검출할 수 있는 조성물 제공을 목적으로 한다.In addition, an object of the present invention is to provide a composition capable of simultaneously discriminating and detecting type A and influenza virus B, including both of the above-mentioned compositions for detecting viruses.
또한, 본 발명은 상기 조성물을 포함하는 인플루엔자 바이러스 검출용 PCR 키트와 A형 및 인플루엔자 바이러스 B형 동시 감별 검출용 PCR 키트를 제공하는 것을 목적으로 한다. In addition, an object of the present invention is to provide a PCR kit for detecting influenza virus and a PCR kit for simultaneous differential detection of type A and influenza virus B comprising the composition.
또한, 본 발명은 A형 및 인플루엔자 바이러스 B형 동시 검출 방법 제공을 목적으로 한다. Another object of the present invention is to provide a method for simultaneous detection of type A and influenza virus type B.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당해 기술분야의 통상의 기술자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.
상기 과제를 해결하기 위하여, 본 발명은 서열번호 1 내지 3의 프라이머 및 프로브를 포함하는 인플루엔자 바이러스 A형 검출용 조성물을 제공한다.In order to solve the above problems, the present invention provides a composition for detecting influenza virus type A comprising the primers and probes of SEQ ID NOs: 1 to 3.
또한, 본 발명은 서열번호 4 내지 6의 프라이머 및 프로브를 포함하는 인플루엔자 바이러스 B형 검출용 조성물을 제공한다. In addition, the present invention provides a composition for detecting influenza virus type B comprising the primers and probes of SEQ ID NOs: 4 to 6.
또한, 본 발명은 서열번호 1 내지 6의 프라이머 및 프로브를 포함하는 A형 및 인플루엔자 바이러스 B형 감별 검출용 조성물을 제공한다. In addition, the present invention provides a composition for differential detection of type A and influenza virus B comprising the primers and probes of SEQ ID NOs: 1 to 6.
본 발명의 조성물은 각각 A형 및/또는 인플루엔자 바이러스 B형 검출을 위한 PCR 키트로 제공될 수 있다. The composition of the present invention may be provided as a PCR kit for detecting type A and/or influenza virus type B, respectively.
또한, 본 발명의 조성물은 각각 A형 및/또는 인플루엔자 바이러스 B형 감염 진단을 위한 정보제공방법에 제공될 수 있다. In addition, the composition of the present invention may be provided in an information providing method for diagnosing type A and/or influenza virus type B infection, respectively.
구체적으로, 인플루엔자 바이러스 감염 진단을 위한 정보제공방법은 하기 단계를 포함한다:Specifically, the information providing method for diagnosing influenza virus infection includes the following steps:
(1) 생물학적 시료로부터 RNA를 추출하는 단계; 및(1) extracting RNA from the biological sample; and
(2) 상기 추출된 RNA를 상기 인플루엔자 바이러스 검출용 조성물을 이용하여 PCR을 수행하는 단계.(2) performing PCR on the extracted RNA using the composition for detecting influenza virus.
본 발명의 일 구현예로서, 상기 프로브의 5' 말단은 형광물질로 표지된 것일 수 있으며, 상기 프로브의 3'-말단은 소광자(quencher)가 추가로 표지된 것일 수 있다. In one embodiment of the present invention, the 5' end of the probe may be labeled with a fluorescent material, and the 3'-end of the probe may be additionally labeled with a quencher.
본 발명의 다른 구현예로서, 상기 형광물질은 FAM(6-carboxyfluorescein), 텍사스 레드(texas red), 플루오레신(fluorescein), HEX(2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), 플루오레신 클로로트리아지닐(fluorescein chlorotriazinyl), 로다민 그린(rhodamine green), 로다민 레드(rhodamine red), 테트라메틸 로다민(tetramethyl rhodamine), FITC(fluorescein isothiocyanate), 오레곤 그린(oregon green), 알렉사 플루오로(alexa fluor), JOE(6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX(6-Carboxyl-XRhodamine), TET(Tetrachloro-Fluorescein), TRITC(tertramethylrodamine isothiocyanate), TAMRA(6-carboxytetramethyl-rhodamine), NED(N-(1-Naphthyl) ethylenediamine), 시아닌(Cyanine) 계열 염료 및 씨아디카르보시아닌(thiadicarbocyanine)으로 구성된 군으로부터 선택되는 어느 하나일 수 있으며, 상기 서열번호 3 및 6의 프로브는 서로 상이한 형광물질로 표지된 것일 수 있다. In another embodiment of the present invention, the fluorescent material is FAM (6-carboxyfluorescein), Texas red (texas red), fluorescein (fluorescein), HEX (2',4',5',7'-tetrachloro-6 -carboxy-4,7-dichlorofluorescein), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethyl rhodamine, FITC (fluorescein isothiocyanate) ), oregon green, alexa fluor, JOE (6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX (6-Carboxyl-XRhodamine), TET (Tetrachloro-Fluorescein), TRITC (tertramethylrodamine isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine), NED (N-(1-Naphthyl) ethylenediamine), cyanine-based dyes and thiadicarbocyanine It may be any one selected from the group, and the probes of SEQ ID NOs: 3 and 6 may be labeled with different fluorescent substances.
구체적으로 서열번호 3의 프로브는 FAM으로 표지된 것일 수 있으며, 서열번호 6의 프로브는 ROX로 표지된 것일 수 있다. Specifically, the probe of SEQ ID NO: 3 may be labeled with FAM, and the probe of SEQ ID NO: 6 may be labeled with ROX.
본 발명의 조성물은 호흡기 질환의 원인균의 template 유전자를 증폭하여 이를 검출할 수 있는바, 극소량의 유전자가 포함된 샘플에서도 인플루엔자를 정확하고 신속하게 검출할 수 있으다. 또한 PCR 반응 후, 개별적인 확인 작업이 필요했던 기존 conventional PCR 기술과 달리 real-time PCR은 실시간으로 검출 결과의 확인이 가능하며, 동시에 절대 정량(표준 곡선법)을 통해 미확인 샘플의 정량 분석이 가능하다. 아울러, 혈청형에 따른 인플루엔자의 감별하여 검출할 수 있는바, 호흡기 질환 원인균의 빠른 진단이 필요한 현장에서 임상진단을 위해 이용될 수 있다.The composition of the present invention can detect and amplify the template gene of the causative agent of respiratory disease, and can accurately and quickly detect influenza even in a sample containing a very small amount of gene. In addition, unlike conventional PCR technology, which requires individual confirmation after PCR reaction, real-time PCR enables real-time confirmation of detection results and at the same time, quantitative analysis of unidentified samples through absolute quantification (standard curve method). . In addition, since influenza can be differentially detected according to serotypes, it can be used for clinical diagnosis in a field requiring rapid diagnosis of respiratory disease causative bacteria.
도 1은 influenza A virus 및 influenza B virus 검출 기술의 원리에 관한 도면이다. 각 바이러스의 유전자 서열에 특이적인 primer와 probe를 합성해주며, probe에서 influenza A virus 검출 probe에는 FAM 형광을, inlfuenza B virus 검출 probe에는 ROX 형광을 붙여준다. 본 primer와 probe를 이용하여 virus 샘플의 Real-time PCR 결과 분석 시, FAM 형광이 나오면 influenza A virus 검출, ROX 형광이 나오면 influenza B virus 검출, 두 개의 형광이 동시에 나오면 influenza A, B virus의 동시 검출인 것으로 해석된다.
도 2은 influenza A virus 및 influenza B virus 검출 기술의 실제 적용에 관한 도면이다. Tube에 PCR master mix와 internal control mix 또는 influenza virus oligo mix를 혼합한 후, 각 샘플이 들어있는 tube에 이를 분주해준 뒤, real-time PCR 장비 전용 chip의 각 well에 혼합물을 10ul씩 분주한다. Real-time PCR 반응 후, 검출 결과를 분석한다.
도 3은 본 발명의 합성 샘플을 이용한 프라이머 세트의 민감도 측정 테스트 결과이다.1 is a diagram relating to the principle of influenza A virus and influenza B virus detection technology. Primers and probes specific to the gene sequence of each virus are synthesized, and FAM fluorescence is applied to the influenza A virus detection probe and ROX fluorescence is applied to the influenza A virus detection probe. When analyzing real-time PCR results of virus samples using this primer and probe, FAM fluorescence detects influenza A virus, ROX fluorescence detects influenza B virus, and simultaneous detection of influenza A and B virus if two fluorescence is interpreted as being
2 is a diagram related to the practical application of influenza A virus and influenza B virus detection technology. After mixing PCR master mix and internal control mix or influenza virus oligo mix in a tube, dispense it to the tube containing each sample, and then dispense 10ul of the mixture into each well of the chip dedicated to real-time PCR equipment. After real-time PCR reaction, the detection result is analyzed.
3 is a sensitivity measurement test result of a primer set using a synthetic sample of the present invention.
본 발명자들은 현장에서 신속하고 빠른 호흡기 질환의 원인균 동정 방법에 대해 예의 연구하여 본 발명을 완성하였다. The present inventors have completed the present invention by intensively researching a method for rapidly and rapidly identifying the causative agent of respiratory disease in the field.
본 발명은 Real-time RT PCR 방법을 이용함으로써 종래에 conventional RT-PCR에 비해 인플루엔자 검출 시간을 단축시켰다. 본 발명은 기존의 일반 PCR법으로 발생될 수 있는 결과 분석의 에러와 소요 시간을 줄이고, 1회 실험으로 혈청형에 따른 인플루엔자 바이러스의 감별 검출이 가능하다.The present invention shortens the influenza detection time compared to conventional RT-PCR by using the real-time RT PCR method. The present invention reduces the error and time required for analysis of results that may occur with the existing general PCR method, and enables differential detection of influenza viruses according to serotypes in one experiment.
본 발명자들은 A형 및 인플루엔자 바이러스 B형의 전장 염기서열 데이터베이스를 이용하여 상호간 또는 타종간에 간섭이 없고 타겟 유전자에 민감하게 반응하여 정확한 검사결과를 제공할 수 있는 것으로 예측되는 복수개의 프라이머 및 프로브의 타겟 영역을 설정하고, 가장 효과적인 영역을 확인하여 본 발명을 제공한다. The present inventors use the full-length nucleotide sequence database of type A and influenza virus type B. A plurality of primers and probes predicted to be capable of providing accurate test results by sensitively reacting to the target gene without interference between each other or other species. The present invention is provided by setting the area and identifying the most effective area.
구체적으로, 본 발명의 인플루엔자 바이러스 A형 검출용 조성물은 서열번호 1의 정방향 프라이머 및 서열번호 2의 역방향 프라이머로 구성된 프라이머 세트, 및 서열번호 3의 프로브를 포함한다. Specifically, the composition for detecting influenza virus type A of the present invention includes a primer set consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2, and a probe of SEQ ID NO: 3.
또한, 본 발명의 인플루엔자 바이러스 B형 검출용 조성물은 서열번호 4의 정방향 프라이머 및 서열번호 4의 역방향 프라이머로 구성된 프라이머 세트, 및 서열번호 6의 프로브를 포함한다. In addition, the composition for detecting influenza virus type B of the present invention includes a primer set consisting of a forward primer of SEQ ID NO: 4 and a reverse primer of SEQ ID NO: 4, and a probe of SEQ ID NO: 6.
한편, 본 발명은 서열번호 1 내지 6의 프라이머 및 프로브를 포함하는 조성물을이용하여 A형 및 B형 인플루엔자를 동시에 검출할 수 있다. Meanwhile, in the present invention, type A and type B influenza can be simultaneously detected using a composition comprising the primers and probes of SEQ ID NOs: 1 to 6.
본 발명에 있어서, 서열번호 3 및 6의 프로브는 각각 형광물질로 표지되어 인플루엔자의 검출에 이용될 수 있으며, 형광의 강도 또는 형광의 파장을 통해 샘플 내에 인플루엔자 바이러스의 존부와 A형 및 B형의 감별이 가능하다. 본 발명의 서열번호 3 및 6의 프로브는 바람직하게는 서로 다른 파장을 발하는 형광물질로 표지될 수 있으며, 이 경우 혈청형에 따른 인플루엔자 바이러스의 감별 검출이 보다 용이할 수 있다. In the present invention, the probes of SEQ ID NOs: 3 and 6 are each labeled with a fluorescent substance and can be used to detect influenza, and the presence or absence of influenza virus in the sample and the presence or absence of type A and type B in the sample through the intensity of fluorescence or the wavelength of fluorescence. discrimination is possible. The probes of SEQ ID NOs: 3 and 6 of the present invention may be preferably labeled with fluorescent substances emitting different wavelengths, and in this case, differential detection of influenza viruses according to serotypes may be easier.
본 발명에 있어서, "프라이머 (primer)"란 적절한 완충용액 중의 적절한 조건(예를 들면, 4개의 다른 뉴클레오시드 트리포스페이트 및 DNA, RNA 폴리머라제 또는 역전사 효소와 같은 중합제) 및 적당한 온도 하에서 주형-지시 DNA 합성의 시작점으로서 작용할 수 있는 단일가닥 올리고뉴클레오티드를 말한다. 상기 프라이머의 적절한 길이는 사용 목적에 따라 달라질 수 있으나, 통상 15 내지 30 뉴클레오티드이다. 짧은 프라이머 분자는 일반적으로 주형과 안정한 혼성체를 형성하기 위해서는 더 낮은 온도를 필요로 한다. 프라이머 서열은 주형과 완전하게 상보적일 필요는 없으나, 주형과 혼성화할 정도로 충분히 상보적이어야 한다. In the present invention, the term "primer" means a template under suitable conditions (for example, four different nucleoside triphosphates and a polymerization agent such as DNA, RNA polymerase or reverse transcriptase) in an appropriate buffer and at an appropriate temperature. - refers to single-stranded oligonucleotides that can serve as a starting point for directing DNA synthesis. The appropriate length of the primer may vary depending on the intended use, but is usually 15 to 30 nucleotides. Short primer molecules generally require lower temperatures to form stable hybrids with the template. The primer sequence need not be completely complementary to the template, but must be sufficiently complementary to hybridize to the template.
본 발명의 프라이머는 예를 들면, 포스포르아미다이트 고체 지지체 방법과 같은 당 분야에 공지된 방법을 이용하여 화학적으로 합성할 수 있다. 또한, 공지된 방법으로 메틸화, 캡화 등으로 변형시킬 수 있다. 또한, 본 발명의 프라이머는 필요한 경우, 분광학적, 광화학적, 생화학적, 면역화학적 또는 화학적 수단에 의해 직접적으로 또는 간접적으로 검출 가능한 표지를 포함할 수 있다. 표지의 예로는, 효소(예를 들어, 호스래디쉬 퍼옥시다제, 알칼린 포스파타아제), 방사성 동위원소(예를 들어, 32P), 형광성 분자, 화학그룹(예를 들어, 바이오틴) 등이 있다.The primer of the present invention can be chemically synthesized using methods known in the art, such as, for example, a phosphoramidite solid support method. In addition, it can be modified by methylation, capping, etc. by a known method. In addition, if necessary, the primer of the present invention may include a label detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of labels include enzymes (eg, horseradish peroxidase, alkaline phosphatase), radioactive isotopes (eg, 32P), fluorescent molecules, chemical groups (eg, biotin), and the like. have.
본 발명에서 “프로브”란 염기서열과 특이적으로 결합할 수 있는 수개 내지 수백 개의 염기로 이루어진 핵산단편을 의미하며 라벨링되어 있어 특정 핵산 존재여부를 확인할 수 있다. 프로브는 올리고뉴클레오타이드 프로브, 단쇄 DNA 프로브, 이중쇄 DNA 프로브, RNA 프로브 등의 형태로 제작될 수 있고 비오틴, FITC, 로다민, DIG 등으로 표지되거나 방사선 동위 원소등으로 표지될 수 있다.In the present invention, the term “probe” refers to a nucleic acid fragment consisting of several to hundreds of bases capable of specifically binding to a nucleotide sequence, and is labeled to confirm the presence of a specific nucleic acid. 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, etc., and may be labeled with biotin, FITC, rhodamine, DIG, or the like, or labeled with a radioisotope.
본 발명의 프로브는 바람직하게는 5'-말단에 형광물질로 표지될 수 있으며, 3'-말단은 소광자(quencher)가 접합된 것일 수 있다. 본 발명에서 상기 형광물질은 FAM(6-carboxyfluorescein), 텍사스 레드(texas red), 플루오레신(fluorescein), HEX(2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), 플루오레신 클로로트리아지닐(fluorescein chlorotriazinyl), 로다민 그린(rhodamine green), 로다민 레드(rhodamine red), 테트라메틸 로다민(tetramethyl rhodamine), FITC(fluorescein isothiocyanate), 오레곤 그린(oregon green), 알렉사 플루오로(alexa fluor), JOE(6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX(6-Carboxyl-XRhodamine), TET(Tetrachloro-Fluorescein), TRITC(tertramethylrodamine isothiocyanate), TAMRA(6-carboxytetramethyl-rhodamine), NED(N-(1-Naphthyl) ethylenediamine), 시아닌(Cyanine) 계열 염료 및 씨아디카르보시아닌(thiadicarbocyanine) 등일 수 있으며, 또한 상기 소광자는 TAMRA(6-carboxytetramethyl-rhodamine), BHQ1(black hole quencher 1), BHQ2(black hole quencher 2), BHQ3(black hole quencher 3), NFQ(nonfluorescent quencher), 답실(dabcyl), Eclipse, DDQ(Deep Dark Quencher), 블랙베리 퀸처(Blackberry Quencher), 아이오와 블랙(Iowa black) 등일 수 있다. Preferably, the probe of the present invention may be labeled with a fluorescent material at the 5'-end, and a quencher may be conjugated at the 3'-end. In the present invention, the fluorescent material is FAM (6-carboxyfluorescein), Texas red (texas red), fluorescein, HEX (2',4',5',7'-tetrachloro-6-carboxy-4, 7-dichlorofluorescein), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethyl rhodamine, FITC (fluorescein isothiocyanate), Oregon green ( oregon green), Alexa fluor, JOE (6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX (6-Carboxyl-XRhodamine), Tetrachloro-Fluorescein (TET) , TRITC (tertramethylrodamine isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine), NED (N-(1-Naphthyl) ethylenediamine), cyanine-based dyes and thiadicarbocyanine, etc. 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, Iowa black, and the like.
본 발명에서 생물학적 시료로서 혈액, 혈청, 혈장, 말초혈 단핵세포, 말초혈 백혈구, 타액, 소변, 분변, 인두 면봉 스와브(throat swabs), 피부 상처 스와브(dermal lesion swabs), 뇌척수액(cerebrospinal fluids), 경부 도말 표본(cervical smears), 농즙 샘플, 식품 기질(food matrices), 및 뇌, 비장 및 간을 포함하는 신체의 다양한 부분의 세포 또는 조직을 포함할 수 있다. As a biological sample in the present invention, blood, serum, plasma, peripheral blood mononuclear cells, peripheral blood leukocytes, saliva, urine, feces, throat swabs, dermal lesion swabs, cerebrospinal fluids ), cervical smears, juice samples, food matrices, and cells or tissues of various parts of the body including the brain, spleen and liver.
본 발명의 조성물은 A형 및 인플루엔자 바이러스 B형 검출을 위한 PCR에 이용될 수 있으며 상기 PCR에는 멀티플렉스 PCR (multiplex PCR), 실시간 PCR (realtime PCR) 또는 이들의 조합이 포함될 수 있다.The composition of the present invention may be used for PCR for detecting type A and influenza virus type B, and the PCR may include multiplex PCR, realtime PCR, or a combination thereof.
본 발명은 다양한 변환을 가할 수 있고 여러 가지 실시예를 가질 수 있는 바, 이하 특정 실시예들을 도면에 예시하고 상세한 설명에 상세하게 설명하고자 한다. 그러나, 이는 본 발명을 특정한 실시 형태에 대해 한정하려는 것이 아니며, 본 발명의 사상 및 기술 범위에 포함되는 모든 변환, 균등물 내지 대체물을 포함하는 것으로 이해되어야 한다. 본 발명을 설명함에 있어서 관련된 공지 기술에 대한 구체적인 설명이 본 발명의 요지를 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.The present invention can apply various transformations and can have various embodiments. Hereinafter, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.
[실시예][Example]
실시예 1. 프라이머 및 프로브 설계Example 1. Primer and Probe Design
Primer와 probe는 influenza A, B virus의 유전자를 Genebank NCBI(Nation Center for Biotechnology Information) 웹 사이트 (http;//www.ncbi.nlm.nih.gov/) 에서 확보하여 비교 분석하였다. 이후 Bioedit 프로그램을 이용하여 primer 및 probe를 제작하였고 NCBI BLAST 웹 사이트(https://blast.ncbi.nlm.nih.gov/Blast.cgi) 를 이용하여 제작한 primer 및 probe와 각 바이러스에 대한 특이도를 확인하였다. Primer 및 probe 제작에 참고하였던 NCBI의 accession number는 아래와 같다 (Influenza A virus, 5000개 이상; Influenza B virus, 5000개 이상).Primer and probe were compared and analyzed by obtaining the genes of influenza A and B virus from the Genebank NCBI (Nation Center for Biotechnology Information) website (http;// www.ncbi.nlm.nih.gov/) . Afterwards, primers and probes were prepared using the Bioedit program, and primers and probes prepared using the NCBI BLAST website ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ) and specificity for each virus was confirmed. The accession numbers of NCBI that were referenced in the preparation of primers and probes are as follows (Influenza A virus, more than 5000; Influenza B virus, more than 5000).
...MT477772.1 MT439898.1 MT407138.1 MT407098.1 MT407058.1 MT407050.1 MT407034.1 MT407018.1 MT090543.1 MT090539.1 MT090417.1 MT090342.1 MT090329.1 MT090212.1 MT090208.1 LC496347.1 LC496339. 1 MT020281.1 MT020273.1 MT020241.1 MT020225.1 MT020209.1 MT020185.1 MT020153.1 MN759718.1 MN700162.1 MN700154.1 MN700146.1 MN700138.1 KX156653.1 KX156651.1 KX156647.1 MH991790.1 MH991782 .1 MH991774.1 MH991758.1 MH283040.1 MN703050.1 MN703038.1 MN703008.1 MN702993.1 MN695039.1 MN588297.1 MN588289.1 MN584917.1 MG063443.1 MG063442.1 MK453351.1 MK453345.1 MK453339.1 MN493054.1 MN483235.
...
...MT270033.1 MT270025.1 MT270018.1 MT269978.1 MT269962.1 MT269948.1 MN754245.1 MN589202.1 MN589194.1 MN589162.1 MN589146.1 MN589138.1 MN589106.1 MN588994.1 MN588978.1 MN588930.1 MK961884. 1 MK961860.1 MK961804.1 MK961677.1 MK961359.1 MK911845.1 MK676290.1 MK630435.1 MG002414.1 MK469432.1 MH727013.1 MH726810.1 MH706689.1 MH636855.1 MH607108.1 MH606828.1 MH604839.1 MH604807 .1 MH584685.1 MH584646.1 MG925828.1 CY263965.1 CY263941.1 CY263925.1 CY263901.1 CY263861.1 CY263828.1 CY263065.1 CY231372.1 CY229785.1 CY224442.1 CY224434.1 KX270004.1 KY509873.1 KY509872.1 KY509871.1
...
제작된 프라이머와 프로브의 구체적인 서열정보는 하기 표 2에 나타내었다. 각 프로브는 형광물질(FAM 또는 ROX)로 표지하였다.Specific sequence information of the prepared primers and probes is shown in Table 2 below. Each probe was labeled with a fluorescent substance (FAM or ROX).
(Matrix gene)Influenza A virus
(Matrix gene)
(Nuclear export protein
& nonstructural protein gene)Influenza B virus
(Nuclear export protein
& nonstructural protein gene)
실시예 2. PCR 수행Example 2. Perform PCR
본 발명 기술에 사용되는 장비는 channel이 10개인 단일 chip을 사용한다. Influenza A virus 및 influenza B virus 검출을 위한 oligo mixed set를 제작하며 단일 chip 내에서 2가지 바이러스를 검출할 수 있다. Oligo mixed set는 tube 형태로 제작되며, internal control을 위한 oligo mix 1 set와 이중 바이러스를 검출하기 위한 oligo mix 1 set로 구성된다. 10 strips 맨 왼쪽에 PCR premix 6ul와 internal control oligo mix 6ul를 분주 후 혼합한다. PCR premix 55ul와 이중 호흡기 바이러스 검출용 oligo mix 27.5ul를 혼합 후 남은 9 well에 7.5ul씩 분주한다. 10strips에 있는 혼합물을 chip 내 channel에 10ul씩 순서대로 분주한다. Real-time RT-PCR 조건은 Reverse transcription 45℃ 10분, Pre-denaturation 95℃ 30초, Denaturation 95℃ 5초, Annealing 및 Extension 60℃ 30초로 Denaturation과 Annealing 및 Extension 과정을 40cycle 반복해주며 과정 종료 후 결과를 확인한다. 해당 내용들은 가안이며, oligo mixed set의 개수, 제공되는 oligo mixed set의 형태, channel당 시료 volume 등은 변경될 수 있다. 하기 표 3은 PCR 수행에 이용된 조성물이고, 표 4는 반응 조건이다. The equipment used in the present invention uses a single chip having 10 channels. An oligo mixed set is prepared for the detection of influenza A virus and influenza B virus, and two viruses can be detected within a single chip. Oligo mixed set is produced in tube form and consists of oligo mix 1 set for internal control and oligo mix 1 set for double virus detection. Dispense 6ul of PCR premix and 6ul of internal control oligo mix on the far left of the 10 strips and mix. After mixing 55ul of PCR premix and 27.5ul of oligo mix for double respiratory virus detection, dispense 7.5ul each to the remaining 9 wells. Dispense the mixture in 10 strips in order of 10ul to each channel in the chip. Real-time RT-PCR conditions are
실시예 3. 민감도 테스트Example 3. Sensitivity Test
아래의 방법을 이용하여 우선적으로 합성 샘플 (DNA)을 1x105 ~ 1x101 copy number로 희석한 후 sensitivity를 측정해본 결과, 각각의 검출한계는 influenza A virus가 1x101 copy number, influenza B virus는 1x101 copy number로 높은 민감도를 가지는 것을 알 수 있었다.As a result of measuring the sensitivity after first diluting the synthetic sample (DNA) to 1x10 5 ~ 1x10 1 copy number using the method below, each detection limit is 1x10 1 copy number for influenza A virus, 1x10 1 copy number for influenza B virus It was found to have high sensitivity with 1 copy number.
Influenza A virus 검출용 프라이머에 대한 보다 세밀한 검출 한계 확인을 위해 추가적인 실험을 진행하였다. 합성 샘플 (DNA)을 1x105 ~ 1x100, 2x105 ~ 2x100 및 3x105 ~ 3x100 copy number로 각각 희석한 후 sensitivity를 측정해본 결과, influenza virus의 검출한계가 3x100 copy number로 높은 민감도를 가지는 것을 알 수 있었다. Additional experiments were carried out to confirm more detailed detection limits for the primers for detecting Influenza A virus. After diluting the synthetic sample (DNA) with 1x10 5 ~ 1x10 0 , 2x10 5 ~ 2x10 0 and 3x10 5 ~ 3x10 0 copy number, respectively, the sensitivity was measured . knew to have
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
<110> Industry-University Cooperation Foundation Hanyang University ERICA Campus <120> Primer set for detection and identification of influenza virus type A and type B and use thereof <130> APC-2022-0225 <150> KR 10-2021-0037249 <151> 2021-03-23 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IAV_primer F <400> 1 acaagaccaa tcctgtcacc t 21 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> IAV_primer R <400> 2 tggacaaagc gtctacgct 19 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> IAV_probe <400> 3 ctcaccgtgc ccagtgagc 19 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IBV_primer F <400> 4 ggatcctcaa ctcactcttc ga 22 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IBV_primer R <400> 5 cggtgctctt gaccaaattg g 21 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> IBV_probe <400> 6 caattcgagc agctgaaact gcg 23 <110> Industry-University Cooperation Foundation Hanyang University ERICA Campus <120> Primer set for detection and identification of influenza virus type A and type B and use thereof <130> APC-2022-0225 <150> KR 10-2021-0037249 <151> 2021-03-23 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IAV_primer F <400> 1 acaagaccaa tcctgtcacc t 21 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> IAV_primer R <400> 2 tggacaaagc gtctacgct 19 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> IAV_probe <400> 3 ctcaccgtgc ccagtgagc 19 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> IBV_primer F <400> 4 ggatcctcaa ctcactcttc ga 22 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> IBV_primer R <400> 5 cggtgctctt gaccaaattg g 21 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> IBV_probe <400> 6 caattcgagc agctgaaact gcg 23
Claims (15)
서열번호 3의 프로브를 포함하는, 인플루엔자 바이러스 A형 검출용 조성물로서,
상기 프로브의 5' 말단은 형광물질로 표지된 것을 특징으로 하는, 조성물.Primer sets of SEQ ID NOs: 1 and 2 and
A composition for detecting influenza virus type A, comprising the probe of SEQ ID NO: 3,
The composition, characterized in that the 5' end of the probe is labeled with a fluorescent material.
상기 형광물질은 FAM(6-carboxyfluorescein), 텍사스 레드(texas red), 플루오레신(fluorescein), HEX(2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), 플루오레신 클로로트리아지닐(fluorescein chlorotriazinyl), 로다민 그린(rhodamine green), 로다민 레드(rhodamine red), 테트라메틸 로다민(tetramethyl rhodamine), FITC(fluorescein isothiocyanate), 오레곤 그린(oregon green), 알렉사 플루오로(alexa fluor), JOE(6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX(6-Carboxyl-XRhodamine), TET(Tetrachloro-Fluorescein), TRITC(tertramethylrodamine isothiocyanate), TAMRA(6-carboxytetramethyl-rhodamine), NED(N-(1-Naphthyl) ethylenediamine), 시아닌(Cyanine) 계열 염료 및 씨아디카르보시아닌(thiadicarbocyanine)으로 구성된 군으로부터 선택되는 어느 하나인 것을 특징으로 하는, 인플루엔자 바이러스 A형 검출용 조성물. The method of claim 1,
The fluorescent material is FAM (6-carboxyfluorescein), Texas red (texas red), fluorescein (fluorescein), HEX (2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein) ), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethyl rhodamine, FITC (fluorescein isothiocyanate), oregon green , Alexa fluor, JOE (6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX (6-Carboxyl-XRhodamine), TET (Tetrachloro-Fluorescein), TRITC ( tertramethylrodamine isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine), NED (N-(1-Naphthyl) ethylenediamine), cyanine-based dyes and thiadicarbocyanine A composition for detecting influenza virus type A, characterized in that.
상기 프로브의 3'-말단은 소광자(quencher)가 추가로 표지된 것을 특징으로 하는, 인플루엔자 바이러스 A형 검출용 조성물.According to claim 1,
A composition for detecting influenza virus type A, characterized in that the 3'-end of the probe is further labeled with a quencher.
(2) 상기 추출된 RNA를 서열번호 1 및 2의 프라이머와 서열번호 3의 프로브를 이용하여 PCR을 수행하는 단계;를 포함하는 인플루엔자 바이러스 A형 감염 진단을 위한 정보제공방법.(1) extracting RNA from the biological sample; and
(2) performing PCR on the extracted RNA using the primers of SEQ ID NOs: 1 and 2 and the probe of SEQ ID NO: 3; An information providing method for diagnosing influenza virus type A infection comprising a.
서열번호 6의 프로브를 포함하는, 인플루엔자 바이러스 B형 검출용 조성물로서,
상기 프로브의 5' 말단은 형광물질로 표지된 것을 특징으로 하는, 조성물.the primer sets of SEQ ID NOs: 4 and 5;
A composition for detecting influenza virus type B, comprising the probe of SEQ ID NO: 6,
The composition, characterized in that the 5' end of the probe is labeled with a fluorescent material.
상기 형광물질은 FAM(6-carboxyfluorescein), 텍사스 레드(texas red), 플루오레신(fluorescein), HEX(2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein), 플루오레신 클로로트리아지닐(fluorescein chlorotriazinyl), 로다민 그린(rhodamine green), 로다민 레드(rhodamine red), 테트라메틸 로다민(tetramethyl rhodamine), FITC(fluorescein isothiocyanate), 오레곤 그린(oregon green), 알렉사 플루오로(alexa fluor), JOE(6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX(6-Carboxyl-XRhodamine), TET(Tetrachloro-Fluorescein), TRITC(tertramethylrodamine isothiocyanate), TAMRA(6-carboxytetramethyl-rhodamine), NED(N-(1-Naphthyl) ethylenediamine), 시아닌(Cyanine) 계열 염료 및 씨아디카르보시아닌(thiadicarbocyanine)으로 구성된 군으로부터 선택되는 어느 하나인 것을 특징으로 하는, 인플루엔자 바이러스 B형 검출용 조성물.7. The method of claim 6,
The fluorescent material is FAM (6-carboxyfluorescein), Texas red (texas red), fluorescein (fluorescein), HEX (2',4',5',7'-tetrachloro-6-carboxy-4,7-dichlorofluorescein) ), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethyl rhodamine, FITC (fluorescein isothiocyanate), oregon green , Alexa fluor, JOE (6-Carboxy-4',5'-Dichloro-2',7'-Dimethoxyfluorescein), ROX (6-Carboxyl-XRhodamine), TET (Tetrachloro-Fluorescein), TRITC ( tertramethylrodamine isothiocyanate), TAMRA (6-carboxytetramethyl-rhodamine), NED (N-(1-Naphthyl) ethylenediamine), cyanine-based dyes and thiadicarbocyanine A composition for detecting influenza virus type B, characterized in that.
상기 프로브의 3'-말단은 소광자(quencher)가 추가로 표지된 것을 특징으로 하는, 인플루엔자 바이러스 B형 검출용 조성물.7. The method of claim 6,
A composition for detecting influenza virus type B, characterized in that the 3'-end of the probe is further labeled with a quencher.
(2) 상기 추출된 RNA를 서열번호 4 및 5의 프라이머와 서열번호 6의 프로브를 이용하여 PCR을 수행하는 단계;를 포함하는 인플루엔자 바이러스 B형 감염 진단을 위한 정보제공방법.(1) extracting RNA from the biological sample; and
(2) performing PCR on the extracted RNA using the primers of SEQ ID NOs: 4 and 5 and the probe of SEQ ID NO: 6; Information providing method for diagnosing influenza virus type B infection comprising a.
서열번호 4 내지 6의 프라이머 및 프로브 세트를 포함하는, A형 및 제3항 인플루엔자 바이러스 감별 검출용 조성물로서,
상기 서열번호 3 및 서열번호 6의 프로브는 5' 말단에 서로 다른 파장을 발하는 형광물질로 표지된 것을 특징으로 하는, 조성물. Primer and probe sets of SEQ ID NOs: 1 to 3 and
A composition for differential detection of type A and claim 3 influenza virus, comprising the primers and probe sets of SEQ ID NOs: 4 to 6,
The composition, characterized in that the probes of SEQ ID NO: 3 and SEQ ID NO: 6 are labeled with fluorescent materials emitting different wavelengths at the 5' end.
상기 프로브의 3'-말단은 소광자(quencher)가 추가로 표지된 것을 특징으로 하는, A형 및 제3항 인플루엔자 바이러스 감별 검출용 조성물.10. The method of claim 9,
The 3'-end of the probe is characterized in that the quencher (quencher) is additionally labeled, the composition for differential detection of influenza virus type A and claim 3.
(2) 상기 추출된 RNA를 제9항의 조성물을 이용하여 PCR을 수행하는 단계;를 포함하는 A형 및 인플루엔자 바이러스 B형 감염의 감별 진단을 위한 정보제공방법.(1) extracting RNA from the biological sample; and
(2) performing PCR using the composition of claim 9 on the extracted RNA; Information providing method for differential diagnosis of type A and influenza virus type B infection comprising a.
상기 생물학적 시료는 혈액, 혈청, 혈장, 말초혈 단핵세포, 말초혈 백혈구, 타액, 소변, 분변, 및 인두 면봉 스와브(throat swabs)로 이루어진 군으로부터 선택되는 하나 이상인 것을 특징으로 하는, 정보제공방법.15. The method of claim 14,
The biological sample is characterized in that at least one selected from the group consisting of blood, serum, plasma, peripheral blood mononuclear cells, peripheral blood leukocytes, saliva, urine, feces, and throat swabs, information providing method .
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