KR20100006282A - Respiratory infectious pathogen differential diagnosis and simultaneous antibiotics resistance analysis, kit and chip comprising same - Google Patents

Respiratory infectious pathogen differential diagnosis and simultaneous antibiotics resistance analysis, kit and chip comprising same Download PDF

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KR20100006282A
KR20100006282A KR1020080066453A KR20080066453A KR20100006282A KR 20100006282 A KR20100006282 A KR 20100006282A KR 1020080066453 A KR1020080066453 A KR 1020080066453A KR 20080066453 A KR20080066453 A KR 20080066453A KR 20100006282 A KR20100006282 A KR 20100006282A
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박민구
이지원
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Abstract

PURPOSE: A multiplex kit and chip for determining pathogen analysis and antibiotics resistance are provided to accurately confirm pathogen of respiratory infection and reduce antibiotics resistance rate. CONSTITUTION: A method for analyzing pathogen diagnosis of infectious diseases and antibiotics resistance comprises: a step of designing a chip in which probe of amino modifier or thiol modifier is spotted; a step of isolating bacteria genomic DNA, virus RNA and DNA from a sample; a step of amplifying probe target site of each pathogen and probe target site for antibiotics resistance analysis through multiplex RT-PCR or multiplex PCR; a step of preparing the target DNA PCR product containing fluorescence dye; a step of hybridizing and washing; and a step of analyzing pathogen diagnosis and antibiotics resistance.

Description

호흡기감염질환 병원체별 감별진단 및 이의 항생제 내성 유무 판별 방법, 키트 그리고 이를 포함하는 칩{Respiratory infectious pathogen differential diagnosis and simultaneous antibiotics resistance analysis, kit and chip comprising same} Respiratory infectious pathogen differential diagnosis and simultaneous antibiotics resistance analysis, kit and chip comprising same}

본 발명은 호흡기감염질환 병원체 감별분석 및 이의 항생제 내성 유무를 동시에 판별하는 조성물에 관한 것으로서, 보다 상세하게는 세균성 병원체 10종, 바이러스성 병원체 13종 그리고 항생제 내성 분석용 19종 유전자들을 특이적으로 판별할 수 있는 프로브(probe)들을 포함하는 판별방법, 멀티플렉스 키트 그리고 칩에 관한 것이다.The present invention relates to a composition for discriminating respiratory infection disease pathogens and simultaneously determining the presence or absence of antibiotic resistance thereof, and more specifically, specifically identifying 10 bacterial pathogens, 13 viral pathogens and 19 genes for antibiotic resistance analysis. The present invention relates to a discrimination method including multiple probes, a multiplex kit, and a chip.

2006년 건강보험 주요 통계에 따르면 호흡기감염질환을 대표하는 폐렴이 노인성 백내장, 뇌경색에 이어 65세 이상 노인이 가장 많이 입원하는 질환으로 분류된 바 있다. 국내 폐렴 사망률은 1994년 17위에서 2004년 10위로 증가 추세에 있다(국민건강보험공단, http://www.nhic.or.kr/). 전 세계 연간 총 사망자는 5,700만 명이며 이 중 27%가량이 감염질환으로 인해 사망한다(U.S Census Bureau, International Data Base, 2007). 폐렴은 미국에서 6번째의 사망원인이며, 감염성 질환 중에서는 제1의 사망원인이다(The American Thoracic Society Am Rev Respir Dis (1993) 148:1426-1478). 국내 한 대학종합병원의 사인(cause of death)별 통계에 의하면 선행 사인별 통계는 악성종양이 1위이지만 직접 사인별 통계는 감염질환이 1위였다(unpublished data). 이는 기저질환이 있는 많은 환자들이 감염에 의해 사망한다는 것을 의미한다.According to the main statistics of health insurance in 2006, pneumonia representing respiratory infections was classified as the most common hospitalization among elderly patients 65 years or older following senile cataract and cerebral infarction. Domestic pneumonia mortality rate is increasing from 17th in 1994 to 10th in 2004 (National Health Insurance Service, http://www.nhic.or.kr/). The world's total annual death toll is 57 million, of which 27% die from infectious diseases (US Census Bureau, International Data Base, 2007). Pneumonia is the sixth leading cause of death in the United States and the leading cause of death among infectious diseases (The American Thoracic Society Am). Rev Respir Dis (1993) 148: 1 426-1478). According to the statistics of the cause of death of a university hospital in Korea, the leading cause of death was malignant tumors, but the direct cause of death was uninfected (inpublished data). This means that many patients with underlying diseases die from infection.

대표적인 호흡기감염질환인 폐렴(pneumonia)은 병원체에 따라 분류하거나, 기존의 호흡기질환의 유무에 따른 이차성 혹은 원발성(primary)폐렴으로 분류할 수 있으며, 흉부 X-선 소견에 따라 폐포성(alveolar)폐렴, 기관지(bronchial)폐렴 및 간질성(interstitial)폐렴으로 분류한다. 원외획득폐렴(community-acquired pneumonia)은 병원획득폐렴(hospital-acquired pneumonia)에 대립되는 용어로 지역사회에서 얻은 폐렴을 뜻한다. 반면에 병원획득폐렴은 입원당시 감염이 존재하지 않았으나 입원 48시간 이후에 발생된 폐렴을 의미한다. 수많은 세균, 바이러스, 진균, 원충 등이 원외획득폐렴을 유발할 수 있다(The American Thoracic Society Am J Respir Crit Care Med (2001) 163:1730-1754, Harrison's online http://www.access-medicine.com/resourceToc.aspx?resourceID=4). Pneumonia, a typical respiratory infection, can be classified according to pathogens or classified as secondary or primary pneumonia according to the presence or absence of existing respiratory diseases, and alveolar pneumonia according to chest X-ray findings. , Bronchial pneumonia and interstitial pneumonia. Community-acquired pneumonia refers to pneumonia acquired in the community as opposed to hospital-acquired pneumonia. Hospital-acquired pneumonia, on the other hand, refers to pneumonia that occurred 48 hours after admission, although no infection was present at admission. Numerous bacteria, viruses, fungi and protozoa can cause outbreaks of pneumonia (The American Thoracic Society Am J Respir Crit Care Med (2001) 163: 1730-1754, Harrison's online http://www.access-medicine.com/resourceToc.aspx?resourceID=4).

폐렴 발생시 감염경로는 비강, 구강, 인두, 후두 등 상기도 근처에 병원체가 정착하여 집락을 이룬 후 미량이 흡인되는 미세흡인이 세균성 폐렴의 주된 경로로 인정되고 있다. 병원체의 흡입(inhalation) 이후, 호흡기질환 원인 바이러스들은 제1형 폐포상피세포를 감염시켜 미만성 폐포손상(diffuse alveolar damage)을 유발한 뒤 간질성 폐렴(interstitial pneumonia)으로 진행되는 반면, 세균들은 급성 염 증반응을 일으키고 폐포강내 염증성 삼출물을 유발한 뒤 대엽성(lobar) 또는 기관지성 폐렴(bronchial pneumonia)으로 진행된다. 그러나 임상의들은 바이러스성 폐렴의 증상과 세균성 폐렴의 증상이 비슷하여, 쉽게 감별진단 할 수 없는 때가 많아 경험에 의한(empirical) 항생제 처방을 통해 호흡기질환을 치료하는 것을 선호하고 있다. 그러나 불필요한 항생제 처방 또는 병원체에 감수성이 떨어지는 비특이적인 항생제 처방으로 인해 항생제 내성균의 증가가 국내처럼 항생제 내성율이 높은 지역에서는 시급한 해결해야할 현안이다. The path of infection during pneumonia, micro-aspiration of microscopic aspiration after colonization of pathogens in the nasal cavity, oral cavity, pharynx, larynx and colonization of the upper respiratory tract is recognized as the main path of bacterial pneumonia. After inhalation of the pathogen, the respiratory disease-causing viruses infect type 1 alveolar epithelial cells, causing diffuse alveolar damage and then progressing to interstitial pneumonia, while bacteria are acute It develops and causes inflammatory exudates in the alveolar cavity and then progresses to lobar or bronchial pneumonia. However, clinicians prefer to treat respiratory diseases through the prescription of empirical antibiotics because the symptoms of viral pneumonia and bacterial pneumonia are similar. However, the increase in antibiotic resistance bacteria is an urgent issue to be solved in regions with high antibiotic resistance rates such as in Korea due to unnecessary antibiotic prescription or nonspecific antibiotic prescription which is less susceptible to pathogens.

세균성 폐렴의 원인균으로는 폐렴구균(Streptococcus pneumoniae)이 가장 흔하며 황색포도구균(Staphylococcus aureus), Klebsiella pneumoniae, Pseudomonas aeruginosa 가 중요한 원인균들 중 하나이다. Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumoniae 는 비정형 폐렴을 일으키는 중요한 원인균이다. 또한 학동에게 급성 기관지염을 성인에겐 만성 기침을 유발하는 Bordetella pertussis, 상기도에 주로 존재하는 Moraxella catarrhalis, 35개의 혈청형이 존재하는 Legionella pneumophila, 인두에 존재하며 공기매개전파되는 Haemophilus influenza 등이 원인균에 속한다.Streptococcus pneumoniae is the most common causative agent of bacterial pneumonia. Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa are one of the most common causative organisms. Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella pneumoniae are important causes of atypical pneumonia. The causative organisms include acute bronchitis in adults, Bordetella pertussis, which causes chronic cough in adults, Moraxella catarrhalis mainly in the upper respiratory tract, Legionella pneumophila in 35 serotypes, and Haemophilus influenza, which is airborne and propagated in the pharynx.

바이러스성 호흡기감염질환의 주요 병원체로 급성 하기도 감염의 주된 원인인 호흡기세포융합바이러스(Respiratory Syncytial Virus) A, B형, 파라인플루엔자바이러스(Parainfluenza Virus) 1, 2, 3형, 인플루엔자바이러스(Influenza Virus) A, B형, 아데노바이러스(Adenovirus) 그리고 최근 소아에서 급성 하기도 감염을 일으키는 원인으로 대두되고 있는 인간메타뉴모바이러스(Human Metapneumovirus) 등 이 속한다. 바이러스성 호흡기 질환은 고유한 임상양상을 뚜렷이 보이지 않으므로 임상적 소견만으로 원인 바이러스를 감별하기 어려운 특징이 있다(Ruest A et al. J Clin Microbiol (2003) 41:3487-3493). 원인 바이러스를 질환 초기에 신속하게 동정하게 되면 적절한 항바이러스제를 즉시 투여할 수 있고 불필요한 항생제 남용을 방지할 수 있게 된다.Respiratory Syncytial Virus A, B, and Parainfluenza Virus Types 1, 2, and 3, Influenza Virus, a major cause of acute respiratory tract infections. A, B, Adenovirus and human metapneumovirus (Human Metapneumovirus) has recently emerged as a cause of acute lower respiratory tract infection in children. Viral respiratory disease is difficult to distinguish the causative virus based on clinical findings because it does not show distinct clinical features (Ruest A et al. J Clin) . Microbiol (2003) 41: 3487-3493). The rapid identification of the causative virus early in the disease can result in immediate administration of the appropriate antiviral agent and prevent unnecessary antibiotic abuse.

1940년대 페니실린 항생제가 도입된 직후부터 항생제 내성균은 발생하였으며 이후 점점 다양한 항생제 내성균들이 증가하고 있다. 항생제 내성의 발현 및 확산 속도는 매우 빠르며 새로운 항생제의 개발 속도를 능가하고 있어 임상에서 많은 문제가 되고 있다. 주요 병원균의 항생제 내성 현황을 살펴보면 폐렴구균(Streptococcus pneumoniae)은 55% 이상이 페니실린 내성 폐렴구균이며, 황색포도구균(Staphylococcus aureus)은 57% 가량이 메티실린 내성균이다. 국내 항생제 사용량을 보면 세팔로스포린(cephalosporin) 계열이 월등히 가장 많고, 페니실린(pecillin)계열, 플로로퀴놀론(fluoroquinolone) 계열 항생제가 다음으로 많이 사용되고 있다(Uh Y et al. Yonsei Med J (2007) 48:773-778).Immediately after the introduction of penicillin antibiotics in the 1940's, antibiotic-resistant bacteria began to develop. The rate of expression and diffusion of antibiotic resistance is very fast and outpaces the development of new antibiotics, which is a problem in clinical practice. According to the current status of antibiotic resistance of major pathogens, more than 55% of Streptococcus pneumoniae is penicillin-resistant pneumococci, and about 57% of Staphylococcus aureus is methicillin-resistant. In terms of the amount of antibiotics used in Korea, cephalosporin is the largest group, and penicillin and fluoroquinolone antibiotics are the next most used (Uh Y et al. Yonsei) . Med J (2007) 48: 773-778).

2008년 3월 미국 식품의약국(FDA)은 12종의 호흡기 바이러스 감염을 판별할 수 있는 진단키트인 xTAG Respiratory Viral Panel(RVP)[Luminex Molecular Diagnostics, Inc., Toronto, Canada)의 판매를 승인했다. 기존 검사법보다는 신속하지만 12종류의 바이러스에만 특이적이라서 세균배양, 흉부X-선 검사, 신속항원 검사 등 다른 진단법과 병용해야 한다. 또한 양성 반응이 나타났어도 세균성 감염이나 복합감염이 반드시 배제되는 것이 아니기에 검출된 바이러스가 환자의 질환이 나 증상의 원인이 아닐 수도 있는 분명한 단점이 존재한다. In March 2008, the US Food and Drug Administration (FDA) approved the sale of the xTAG Respiratory Viral Panel (RVP) (Luminex Molecular Diagnostics, Inc., Toronto, Canada), a diagnostic kit that can detect 12 respiratory viral infections. . Although it is faster than conventional tests, it is specific to only 12 kinds of viruses and should be used in combination with other diagnostic methods such as bacterial culture, chest X-ray, and rapid antigen test. In addition, there is a clear disadvantage that the detected virus may not be the cause of the disease or symptom of the patient even if a positive reaction is not necessarily excluded.

근래까지 호흡기감염질환의 병원체를 확인하는 검사를 반드시 실시해야 하는지에 대해서는 논란이 있었다. 각종 검사법을 동원해도 원인균을 찾는 경우는 50% 정도에 불과했기 때문이다. 경험적인 항생제 치료가 실패할 경우에는 내성균 증가의 부작용이 수반된다(Meehan TP et al. JAMA (1997) 278:2080-4, ). 높은 민감도/특이도의 검사법을 통해 초기에 병원체를 규명하여 대처함으로써 항생제 내성 전파를 억제하고 질환의 치료순응도를 향상시켜 조기 완치를 가능케 하는 신규 검사법이 요구된다. Until recently, there has been controversy over whether tests that identify pathogens of respiratory infections should be performed. Even if various tests are used, only about 50% of the causative organisms are found. Failure of empirical antibiotic therapy is accompanied by side effects of increased resistant bacteria (Meehan TP et al. JAMA (1997) 278: 2080-4,). By identifying and coping with pathogens early through high sensitivity / specificity tests, new tests are needed to suppress the spread of antibiotic resistance, improve the compliance of the disease, and enable early cure.

<객담 배양검사의 한계>Limitations of sputum culture

원외획득폐렴의 원인균을 찾기 위한 검사는 현재까지는 그 필요성에 대한 인식이 낮은 편인데, 주된 이유는 경험적인(empirical) 항생제 치료(매크로리드계 혹은 플로로퀴놀론계)가 대부분 일정 수준의 효과를 나타나기 때문이다. Tests to find out the causative agents of outbreaks of pneumonia are low in awareness of the necessity to date. The main reason is that empirical antibiotic treatment (macrolead or fluoroquinolone) is mostly effective. Because.

그리고 입원한 폐렴환자를 대상으로 혈액배양검사, 객담 그람염색과 배양검사를 대부분 실시하지만 그 유용성에 대해서는 일부 논란이 있다(Gleason PP et al. Arch Intern Med (1999) 159:2562-2572). 이 검사들을 통해 원인균을 발견할 확률이 20% 정도란 보고가 있을 정도로 많은 경우에서 원인균을 밝혀내는데 도움을 주지 못하고 있기 때문이다(원장원 J Korean Acad Fam Med (2008) 29(4):S41-S46).Most blood cultures, sputum gram staining and culture tests are performed in hospitalized patients with pneumonia but there is some controversy about their usefulness (Gleason PP et al. Arch Intern Med (1999) 159: 2562-2572). It is because these tests do not help to identify the causative organism in many cases, with a report that the probability of finding the causative organism is about 20% (J Korean Acad Fam Med (2008) 29 (4): S41-). S46).

폐렴 구균과 헤모필러스 인플루엔자는 흔한 원인균이지만 배양이 까다로워서 배양이 안되는 경우가 흔하며, 반대로 덜 흔한 원인인 포도상구균과 그람음성 간균은 쉽게 배양이 되는 편이다. Streptococcus pneumoniae and Haemophilus influenza are common pathogens, but they are often difficult to culture due to their difficult culture. On the contrary, staphylococcus and gram-negative bacillus, which are less common, are easily cultured.

<바이러스 배양검사의 한계>Limitations of Virus Culture Test

바이러스 배양검사법은 세포변성 효과(cytopathic effect)를 관찰하여 바이러스의 존재를 확인할 수 있으나 genotyping을 위해서는 면역형광염색이나 적혈구흡착(hemadsorption) 등의 방법을 추가로 실시해야 하며 최대 14일의 시간이 소요되는 단점이 있다(Petric M et al. J Infect Dis (2006) 194 Suppl. 2:S98-S110).The virus culture test can confirm the presence of the virus by observing the cytopathic effect, but for genotyping, additional methods such as immunofluorescence staining or hemadsorption are required, which takes up to 14 days. There are disadvantages (Petric M et al. J Infect Dis (2006) 194 Suppl. 2: S98-S110).

<흉부 X-선 검사의 한계>Limitations of chest X-ray examination

폐렴이 의심되면 환자들은 흉부 X-선 사진을 촬영하게 된다. 대엽성 폐렴(lobar pneumonia)은 전형적인 세균에 의해, 간질성 폐렴(interstitial pneumonia)은 바이러스에 의해 잘 발생한다고 알려져 있으나 흉부 X-선 사진 소견만으로 세균성과 비세균성을 확실히 구별하기 어렵다.If pneumonia is suspected, patients will have a chest X-ray. Lobar pneumonia is known to be caused by typical bacteria, and interstitial pneumonia is known to be caused by viruses. However, chest X-ray findings alone make it difficult to distinguish bacterial and non-bacterial properties.

<혈청 검사의 한계>Limitations of Serum Tests

M. pneumoniae, C. pneumoniae, Legionella는 항체 역가가 급성기에 비해 회복기에 4배 이상 증가하는 것으로 진단에 도움이 되지만, 다른 세균에 감염이 되어도 역가가 4배 이상 증가하는 비특이성이 있을 수 있고, genotyping이 불가능하며 초기 진단이 어렵다는 단점이 있다. 신속항원검사(rapid antigen test)는 호흡기 세포융합바이러스(RSV)나 인플루엔자 바이러스를 쉽고 빠르게 검출할 수 있는 검사이나 민감도와 음성예측도가 낮은 단점이 있다. M. pneumoniae, C. pneumoniae, and Legionella may help diagnose the antibody titers by more than fourfold in the recovery phase compared to the acute phase, but may be nonspecific, with titers more than fourfold higher when infected with other bacteria. Genotyping is impossible and early diagnosis is difficult. Rapid antigen test is a test that can quickly and easily detect respiratory syncytial virus (RSV) or influenza virus, but has a low sensitivity and low negative predictive value.

전술한 바와 같이 호흡기감염질환의 진단을 위해 사용되고 있는 배양검사(객담, 바이러스), 흉부 X-선 검사, 그리고 혈청 항체/항원검사는 검사 소요시간이 너무 길거나, 불명확한 결과, 추가 검사의 필요성 발생, 낮은 민감도 등의 단점 또는 한계가 분명히 존재하고 있다(도 1 참조). As mentioned above, the culture test (sputum, virus), chest X-ray, and serum antibody / antigen test used for the diagnosis of respiratory infections may take too long time or are unclear, requiring further tests. There are clearly disadvantages or limitations, such as low sensitivity (see FIG. 1).

로컬의 임상의들이 호흡기감염질환의 진단 및 치료에 있어 경험에 의한(Experience by local clinicians in the diagnosis and treatment of respiratory infections empiricalempirical ) 항생제 처방을 선호함으로써 항생제 내성 병원체들의 발생 및 전파율이 급격히 증가하고 있는 이면에는, 바이러스성 폐렴의 증상과 세균성 폐렴의 증상이 비슷하여 쉽게 감별진단 할 수 없는 때가 많고, 배양검사에 필요한 객담을 폐렴환자의 약 30%는 잘 배출하지 못하며, 배양이 까다롭거나 어려운 원인균이 있 으며, In the case of the rapid increase in the incidence and propagation rate of antibiotic resistant pathogens due to the preference of antibiotics, the symptoms of viral pneumonia and bacterial pneumonia are similar and often cannot be easily diagnosed. About 30% of patients are poorly discharged and have difficult or difficult causative organisms, 그람염색은Gram dye 해석에 오류를 범할 수 있고, 또한 명확한 결과를 위해서는 추가 확진검사가 요구되기 때문에 처음 환자를 치료할 때는 경험적인 치료를  Empirical treatment may be the first time a patient is treated because errors in interpretation can be made, and further confirmation is required for clear results. 할 수 밖에Can only 없다는 의료 현장의 애로가 존재한다( There is a pain in the medical field MoonMoon WKWK KoreanKorean J  J MedMed (1995) 48:719-726). (1995) 48: 719-726.

이에 본 발명은 감염 발생 초기에 고-처리량 칩 분석을 통해 Thus, the present invention provides high throughput chip analysis at the beginning of infection. 호흡기감염증Respiratory infections 병원체를 감별하여 정확한 원인을 규명함으로써 경험에 의한 항생제 처방을 지양할 수 있고 더 나아가 감염 병원체가 항생제 내성을 보유하고 있는지 여부까지 판별함으로써 특이적인 항생제 선택을 통해 항생제  By identifying the exact cause and identifying the exact cause, it is possible to refrain from prescription of antibiotics by experience. Furthermore, by determining whether the infectious agent is resistant to antibiotics, antibiotics can be selected through specific antibiotic selection. 내성율Tolerance 감소에 기여할 수 있다. May contribute to the reduction.

전기 본 발명의 목적을 달성하기 위해, 뇌졸중 발현 예측 유전자 표지자들의 단일염기 다형성(SNP) 고-처리량 분석 및 생화학 표지자 분석을 통해 뇌졸중을 조기진단할 수 있는 분석방법을 제공한다.In order to achieve the object of the present invention, there is provided an analysis method capable of early diagnosis of stroke through single-nucleotide polymorphism (SNP) high-throughput analysis and biochemical marker analysis of stroke expression predictive gene markers.

자세하게는, 호흡기감염질환 발생 초기에 고-처리량 분석을 통해 질환의 정확한 원인을 규명함으로써 경험에 의한(empirical) 항생제 처방을 지양함과 동시에 감염 병원체의 항생제 내성 유무까지 판별함으로써 병원체에 효과적인 항생제 선택을 통해 항생제 내성율을 감소시킬수 있는 분석방법, 멀티플렉스 키트 그리고 칩을 제공한다.In detail, high-throughput analysis at the outset of respiratory infections helps to determine the exact cause of the disease, avoiding antibiotic prescriptions, and determining the presence of antibiotic resistance in infectious pathogens. We offer analytical methods, multiplex kits, and chips to reduce antibiotic resistance.

그리고 더욱 자세하게 상기 분석방법에 있어서, 5'- 또는 3'-말단에 아미노 모디파이어(amino modifier) 또는 티올 모디파이어(thiol modifier)가 수식된 프로브가 스팟팅(spotting)된 칩(chip)을 제작하는 단계;In more detail, in the assay method, a chip in which a probe modified with an amino modifier or a thiol modifier at the 5'- or 3'-end is spotted is manufactured. ;

검체로부터 세균 게노믹(genomic) DNA, 바이러스 RNA 및 DNA를 분리하는 단 계; Separating bacterial genomic DNA, viral RNA, and DNA from the sample;

호흡기감염질환 병원체별 프로브 타겟 부위 및 항생제 내성 분석용 프로브 타겟 부위를 멀티플렉스 RT-PCR(reverse transcription-polymerase chain reaction) 또는 멀티플렉스 PCR을 통해 증폭하면서 형광색소(fluorescent dye)가 포함되도록 타겟(target) DNA 증폭산물을 준비하는 단계;Probe target sites for antibiotic pathogens and probe target sites for antibiotic resistance analysis are amplified by multiplex reverse transcription-polymerase chain reaction (RT-PCR) or multiplex PCR to target fluorescent dyes. ) Preparing a DNA amplification product;

멀티플렉스 반응산물과 칩 표면에 고정화된 프로브간 혼성화(hybridization) 반응을 수행하고 세척(washing)하는 단계;Performing and washing a hybridization reaction between the multiplex reaction product and the probe immobilized on the chip surface;

형광색소에 특이적인 파장의 레이저(laser)로 칩을 스캐닝(scanning)하고 혼성화 반응 결과에 따른 형광강도를 측정함으로써 감염질환 병원체 감별진단 및 이의 항생제 내성 유무 결과를 동시에 분석하는 단계; 그리고Simultaneously analyzing the infectious disease pathogen differential diagnosis and its antibiotic resistance by scanning the chip with a laser of a wavelength specific to the fluorescent dye and measuring the fluorescence intensity according to the hybridization reaction result; And

상기 분석단계 전부를 포함하는 감염질환 병원체 감별진단 및 항생제 내성 동시 분석방법을 제공한다.Provided is an infectious disease pathogen differential diagnosis and antibiotic resistance simultaneous analysis method comprising all of the above analysis step.

그리고, 본 발명의 목적을 바람직하게 실현하기 위해, 서열목록번호 1 내지 79의 염기서열을 갖는 올리고뉴클레오티드(deoxyribonucleic acid oligonucleotide)로 이루어진 그룹에서 선택된 하나 이상의 프로브(probe)를 포함하는 호흡기감염질환 병원체 감별진단 및 항생제 내성 분석용 칩을 제공한다.And, in order to realize the object of the present invention, respiratory infectious disease pathogen differentiation comprising at least one probe selected from the group consisting of oligonucleotides having a nucleotide sequence of SEQ ID NO: 1 to 79 Provides chips for diagnostic and antibiotic resistance analysis.

또한 본 발명의 분석방법을 바람직하게 실현하기 위해, 서열목록번호 1 내지 79의 염기서열을 갖는 프로브와 상보적인 결합이 가능한 타겟 부위를 증폭할 수 있는 멀티플렉스-PCR 키트 그리고 멀티플렉스 RT-PCR 키트를 제공한다.In addition, in order to preferably implement the assay method of the present invention, a multiplex-PCR kit and multiplex RT-PCR kit capable of amplifying a target site capable of complementary binding with a probe having a nucleotide sequence of SEQ ID NOs: 1 to 79 To provide.

호흡기 감염질환은 감염 병원체에 따라 치료기간, 항생제 종류, 합병증 등이 다르기에 병원체의 판별검사가 매우 중요하다. 이에 본 발명은 단 1회의 검체 채취로 4 시간 이내로 신속하게 세균성 또는 바이러스성 호흡기감염질환 병원체 감별진 단 결과를 도출함과 동시에, 해당 병원체의 항생제 그룹별 내성 유무 판별 결과를 제공함으로써, 감염 초기에 항균력이 우수하고 약물동력학 측면에서 우수한 최적의 항생제 선택이 가능한 호흡기감염질환 감별진단 및 항생제 내성 판별방법을 제공할 수 있다. Respiratory infectious diseases are very important because the treatment period, antibiotic type, and complications vary according to the infectious agents. Therefore, the present invention derives the result of differential diagnosis of bacterial or viral respiratory infection disease pathogens within 4 hours by only one sample collection, and provides the result of discrimination of antibiotic group resistance of the corresponding pathogens in the early stage of infection. It is possible to provide a respiratory infectious disease differential diagnosis and antibiotic resistance discrimination method that can select an optimal antibiotic with excellent antibacterial activity and excellent pharmacokinetics .

이하 도면 및 표를 참조하여 본 발명의 바람직한 실시예를 상세히 설명한다. 본 명세서에 기재된 도면, 표 및 실시예에 도시된 구성은 본 발명의 가장 바람직한 일 실시예에 불과할 뿐이고 본 발명의 기술적 사상을 모두 대변하는 것은 아니므로, 본 출원시점에 있어서 이들을 대체할 수 있는 다양한 균등물과 변형예들이 있을 수 있음을 이해하여야 한다. Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings and tables. Configurations shown in the drawings, tables and examples described herein are only one of the most preferred embodiments of the present invention and do not represent all of the technical idea of the present invention, various modifications that can be substituted for them at the time of the present application It should be understood that there may be equivalents and variations.

또한 본 발명이 청구하는 범위내에서 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자에 의해 다양한 변형의 실시가 가능하며 이러한 변형은 본 발명의 범위에 속한다. Also, within the scope of the present invention, various modifications may be made by those skilled in the art to which the present invention pertains, and such modifications are within the scope of the present invention.

<< 실시예Example 1> 호흡기감염질환 병원체 감별진단 및 감염 병원체의 항생제 내성 분석을 위한 멀티플렉스  1> Multiplex for Differential Diagnosis of Respiratory Infectious Disease Pathogens and Analysis of Antibiotic Resistance of Infectious Pathogens PCRPCR 반응에 포함되는  Included in the reaction 프라이머primer (( primerprimer ) 디자인 및 합성A) design and synthesis

호흡기감염질환을 유발하는 병원체별 감별진단(differential diagnosis)을 위한 고-처리량 칩(chip) 반응을 위해 선행하여 실시하는 멀티플렉스 PCR(multiplex polymerase chain reaction) 반응에 포함되는 프라이머(primer)는 미국 NCBI(National Center for Biotechnology Information)의 염기서열 데이터베이스인 GenBank, 일본의 DDBJ(The DNA Data Bank of Japan, 그리고 유럽연합의 EMBL(the European Molecular Biology Laboratory (EMBL) 염기서열 데이터베이스로부터 검색한 10여종의 세균, 10여종 이상의 바이러스 및 20여종에 달하는 항생제 내성관련된 주요 균주들의 타겟 유전자 염기서열들을 분석하여 제작하였다(도 2 참조). The primers included in the multiplex polymerase chain reaction (PCR) reaction that is carried out earlier for high-throughput chip reactions for differential diagnosis of pathogens causing respiratory infections are US NCBI. GenBank, a base sequence database from the National Center for Biotechnology Information, The DNA Data Bank of Japan, and 10 species of bacteria from the European Molecular Biology Laboratory (EMBL) sequence database of the European Union, More than 10 viruses and 20 kinds of antibiotic resistance-related major strains were produced by analyzing the target gene sequences (see Fig. 2).

프라이머(primer)는 병원체별 즉, 세균, 바이러스, 진균 또는 원충별로 유전자-특이적 또는 종(species)-특이적인 증폭이 가능하도록 타겟 부위(target region)를 선별하였다. 또한 필요한 경우 병원체별 타겟 부위 염기서열을 ClustalW method를 이용하여 다중정렬(multiple alignment)을 실시하고 이에 따라 각 병원체별 특이적인(specific) 프라이머 타겟 부위를 선별하였다. 이후, 선별된 프라이머 타겟 부위로부터 프라이머 프리미어(Primer premier version 5, Premier Biosoft International, Palo Alto, CA, USA), 디엔에이시스 맥스 (DNASIS MAX Version 2.7, MiraiBio Group, South San Francisco, CA, USA) 프로그램을 응용하여 종-특이적 또는 유전자-특이적 프라이머를 디자인하였다. 프라이머 선발의 기본 요건은 다음과 같다 ; 프라이머 길이(19 - 24 base pair), 융해온도[Tm(℃), 58 - 62℃], 3' 말단의 GC 함량이 높지 않아야하며, 프라이머 서열내 염기 4개 이상 연속해서 상보적이지 않도록 해서 hairpin 이차구조 형성을 방지하였으며, 3'말단에 동일한 염기가 3개 이상 연속해서 위치하지 않도록 해서 프라이머 이합체(dimer)의 형성을 방지하였다. 그리고 타겟 부위내에 단일염기 다형성(single nucletide polymorphism, SNP)이 포함되지 않도록하여 높은 특이도를 확보하였다. 또한 해당 프라이머 염기서열이 본 발명이 목적하는 칩을 구성하는 타 유전자 또는 병원체 염기서열들과 cross-reactive한 특성을 나타내지 않음을 미국 NCBI 염기서열 데이터베이스 등 다양한 염기서열 검색 툴(tool)을 활용하여 검증하였다. 본 발명에 사용하는 프라이머(primer)는 미국 IDT(Integrated DNA Technologies, Inc., San Jose, CA, USA)를 통해 합성하였다.Primers were selected for target regions to enable gene-specific or species-specific amplification by pathogens, ie bacteria, viruses, fungi or protozoa. In addition, if necessary, the target site sequence for each pathogen was subjected to multiple alignments using the ClustalW method, and thus, specific primer target sites for each pathogen were selected. Subsequently, primer primer (Primer premier version 5, Premier Biosoft International, Palo Alto, Calif., USA) and DNAiSmax (DNASIS MAX Version 2.7, MiraiBio Group, South San Francisco, CA, USA) programs were selected from the selected primer target sites. Application was made to design species-specific or gene-specific primers. The basic requirements for primer selection are as follows; Primer length (19-24 base pair), melting temperature [Tm (℃), 58-62 ℃], GC content at 3 'end should not be high, and hairpin should not be complementary for more than 4 consecutive bases in primer sequence Secondary structure formation was prevented, and the formation of primer dimers was prevented by preventing three or more identical bases from being consecutively located at the 3 ′ end. In addition, single specific polymorphism (SNP) was not included in the target site to ensure high specificity. In addition, the primer sequence is not cross-reactive with other genes or pathogen sequences constituting the target chip of the present invention using various base sequence search tools such as the US NCBI sequence database. It was. Primers used in the present invention were synthesized through US IDT (Integrated DNA Technologies, Inc., San Jose, CA, USA).

본 발명의 바람직한 구현을 통해, 호흡기감염질환의 세균성 병원체 진단을 위한 프라이머는 DNA 염기서열의 5'말단 또는 3'말단에 형광색소가 표지된 프라이머를 사용하거나, 또는 형광색소가 표지되지 않은 일반적인 프라이머를 사용하고 멀티플렉스 PCR 반응 중에 형광색소가 표지된 디옥시리보뉴클레오티드 트리포스페이트가 증폭산물에 삽입되게끔 유도하여 증폭산물을 표지하는 두가지 방법을 모두 확인하였다. 또한 바이러스성 병원체 진단을 위해서는 역전사효소-PCR(reverse transcriptase-PCR, RT-PCR)을 수행하기 때문에 역전사효소 반응 이후 2차 PCR 단계에서 형광색소가 표지된 프라이머를 사용하거나 또는 증폭반응중에 형광색소를 표지하게 된다. 본 발명에 사용하는 형광색소는 6-FAM(6-carboxyfluorescein), Cy5, Cy3, Cy5.5, JOE(6-carboxy-4', 5'-dichloro-2', 7'-dimethoxyfluorescein), Rhodamine Green, TAMRA NHS(N-hydroxysuccinimide) Ester, Texas Red 이다. 프라이머의 농도는 ND-1000 분광광도계(NanoDrop Technologies, Rockland, Maine, USA)를 통해 확인하고 최종 50 - 200 pmole/uL 범위의 농도가 되도록 3차 탈이온 멸균 증류수로 재현탁(resuspension)하고 aliquots으로 -70℃ 냉동보관하였다. According to a preferred embodiment of the present invention, the primer for diagnosing bacterial pathogens of respiratory infectious diseases uses a primer labeled with fluorescent dye at the 5 'end or 3' end of the DNA sequence, or a general primer not labeled with the fluorescent dye. Both methods of labeling amplification products were identified by inducing the insertion of fluorescent dye-labeled deoxyribonucleotide triphosphate into the amplification products during the multiplex PCR reaction. In addition, reverse transcriptase-PCR (RT-PCR) is performed for the diagnosis of viral pathogens. Therefore, fluorescent dye-labeled primers are used in the second PCR step after reverse transcriptase reaction or fluorescent dyes are amplified during amplification reaction. It will be labeled. Fluorescent dyes used in the present invention are 6-FAM (6-carboxyfluorescein), Cy5, Cy3, Cy5.5, JOE (6-carboxy-4 ', 5'-dichloro-2', 7'-dimethoxyfluorescein), Rhodamine Green TAMRA NHS (N-hydroxysuccinimide) Ester, Texas Red. The concentration of the primer was checked with an ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, Maine, USA) and resuspended in tertiary deionized sterile distilled water to a final range of 50-200 pmole / uL and aliquots. Frozen at -70 ° C.

<< 실시예Example 2> 임상검체 채취 및 이로부터  2> Clinical sample collection and from DNADNA 및  And RNARNA 분리 detach

본 발명이 실현하고자 하는 비침습적(non-invasive) 진단을 위해서, 비인두 도말(nasopharyngeal swab) 그리고 이보다 핵산 분리율이 뛰어난 비인두 흡인액(nasopharyngeal aspirate, NPA), 인후 도말(throat swab), 기관지 세척액(bronchoalveolar lavage, BAL), 비강 도말(nasal swab), 유도 객담(induced sputum) 등의 검체로부터 효율적인 DNA 분리가 주요 선행조건 중의 하나였다. 호흡기감염 증상 발현 후 2일 이내에 채취한 검체를 사용하였으며 일반적으로 감염 초기의 검체들의 핵산 분리율이 높았다. 검체는 채취 후 바이러스 수송배지(viral transport media)에 넣어서 냉장조건하에서 검사실로 운송하였다. 채혈된 혈액은 실온에서 운반하였다. 2006년 Clinical and Laboratory Standards Institute의 M41-A 'viral culture' 가이드라인에 따르면, 특히 바이러스 검체는 검체 운송 온도에 주의해야하며 RSV와 CMV의 경우, 실온이상의 온도에서 운반할 경우 바이러스의 titer가 급격히 감소하며 1회 냉동 후 해동만으로도 최대 90%까지 생존율이 감소하는것으로 지적하였다.For non-invasive diagnosis of the present invention, nasopharyngeal swab and nasopharyngeal aspirate (NPA), throat swab, bronchial lavage fluid with superior nucleic acid separation rate Efficient DNA isolation from samples such as bronchoalveolar lavage (BAL), nasal swab, and induced sputum was one of the major prerequisites . Specimens collected within 2 days after onset of symptoms of respiratory infection were used. In general, nucleic acid isolation rates were high in the early stages of infection. Samples were collected and transferred to a laboratory under refrigerated conditions in viral transport media. The collected blood was delivered at room temperature. According to the 2006 Clinical and Laboratory Standards Institute's M41-A 'viral culture' guidelines, viral samples should be particularly careful of the sample transport temperature, and in the case of RSV and CMV, the viral titer decreases dramatically when transported above room temperature. It was pointed out that survival rate decreased up to 90% even after thawing alone.

호흡기감염질환의 세균성 원인균 감별진단을 위해 검체로부터 세균 게노믹 DNA(genomic DNA)를 분리하였으며, 상용화된 DNA 추출 키트(LaboPass™ Tissue kit, 코스모진텍, 서울, 한국)를 사용하였다. 기관지 세척액을 비롯한 체액 검체가 점도가 낮아서 바로 pipetting 이 가능하면 2N 수산화나트륨(NaOH)을 사용하지 않고 검체 0.5 - 1.0 mL을 1.5 mL 에펜도르프 튜브로 옮겼다. 객담의 경우처럼 점도 가 높은 검체의 경우, 2N NaOH를 검체와 동량으로 첨가하여 검체를 액화시켰다. NaOH를 넣어 줄 때 tip으로 저어주면서 검체를 풀어야 하며, pipette으로 0.5 mL을 정확히 취할 수 있을 정도로 풀어 주었다. 시료를 포함하는 에펜도르프 튜브를 덴빌 260D 고속 원심분리기(high-speed centrifuge, Denville Scientific Inc., Metuchen, NJ, USA)를 이용해서 12,000 rpm(revolutions per minute, 분당회전수), 1분 원심분리하였다. 상층액을 제거하고 침전물을 500 uL의 PBS 용액에 풀어준 뒤 다시 동일 조건에서 원심분리하여 세척과정을 거쳤다. 검체의 볼륨(volume)이 적을 경우에는 1X PBS 용액 500 uL를 첨가하여 전기 동일한 조건으로 고속원심분리하였다. 1X PBS 용액은 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4를 3차 멸균 증류수 1L에 최종 pH 7.4가 되도록 조성하였다. 상기 침전물에 시료를 분해하는 TL 완충용액(buffer) 200 uL를 첨가하여 풀어주고 단백분해효소 K(Proteinase K, 100 mg/mL) 20 uL를 첨가하고 약하게 교반(vortex)한 후 56℃, 10분간 반응시켰다. 이후 반응물에 TB 완충용액 400 uL를 첨가하고 교반하여 반응물을 혼합하였다. 이어서 반응물을 스핀 칼럼(spin column)의 상층부에 로딩(loading)하고 8,000 rpm, 1분 원심분리하였다. 컬렉션 튜브(collection tube)를 새로 교체하고 검체로 부터 추출된 DNA를 칼럼의 멤브레인(membrane)에 결합시키는 작용을 하는 BW 완충용액 700 uL를 첨가하고 상기 동일 조건으로 원심분리하였다. 컬렉션 튜브(collection tube)를 새로 교체하고 멤브레인으로부터 불순물을 제거하는 NW 완충용액 500 uL를 첨가하고 13,000 rpm, 2분 원심분리하였다. 이후 스핀 칼럼 상층부를 새로운 에펜도르프 튜브로 옮긴뒤 DNA를 용출시키기 위해 탈이온(de-ionized) 멸균 3차 증류수 100 uL를 로딩하고 실온에서 5분 처리하고 8,000 rpm, 2분 원심분리하여 순수한 DNA를 분리하였다.For differential diagnosis of bacterial causative agents of respiratory infections, bacterial genomic DNA was isolated from the sample, and a commercialized DNA extraction kit (LaboPass ™ Tissue kit, Cosmojintech, Seoul, Korea) was used. If bodily fluid samples, including bronchial lavage fluids, were low in viscosity and were ready for pipetting, 0.5-1.0 mL of the sample was transferred to a 1.5 mL Eppendorf tube without using 2N sodium hydroxide (NaOH). For samples with high viscosity, as in the case of sputum, 2N NaOH was added in the same amount as the sample to liquefy the sample. When NaOH was added, the sample should be released while stirring with a tip. The pipette was released to the point where 0.5 mL was taken accurately. The Eppendorf tube containing the sample was centrifuged at 12,000 rpm (revolutions per minute) using a Denville 260D high-speed centrifuge (Denville Scientific Inc., Metuchen, NJ, USA) for 1 minute. . The supernatant was removed, the precipitate was dissolved in 500 uL of PBS solution, and centrifuged again under the same conditions. When the volume of the sample was small, 500 uL of 1X PBS solution was added and high-speed centrifugation was carried out under the same conditions. The 1 × PBS solution was formulated with 8 g NaCl, 0.2 g KCl, 1.44 g Na 2 HPO 4 , 0.24 g KH 2 PO 4 in 1 L of tertiary sterile distilled water to a final pH of 7.4. After adding 200 uL of TL buffer to decompose the precipitate, add 20 uL of proteinase K (100 mg / mL), gently stir (vortex), and then 56 ° C for 10 minutes. Reacted. Then 400 uL TB buffer was added to the reaction and stirred to mix the reaction. The reaction was then loaded onto the top of the spin column and centrifuged at 8,000 rpm for 1 minute. A new collection tube was replaced and 700 uL of BW buffer, which acts to bind the DNA extracted from the sample to the membrane of the column, was added and centrifuged under the same conditions. The collection tube was freshly replaced and 500 uL of NW buffer was added to remove impurities from the membrane and centrifuged at 13,000 rpm for 2 minutes. The top of the spin column was then transferred to a new Eppendorf tube, loaded with 100 uL of de-ionized sterile tertiary distilled water to elute DNA, treated for 5 minutes at room temperature, centrifuged at 8,000 rpm for 2 minutes, and purified pure DNA. Separated.

호흡기감염질환의 바이러스성 병원체 감별진단을 위해 검체로부터 바이러스 DNA 및 RNA를 분리하였으며, 상용화된 바이러스 핵산 추출 키트(easy-spin total RNA extraction kit, Intron biotechnology, 한국, Cat. No 17221)를 사용하였다. 객담 검체의 경우 전기와 동일한 조건으로 2N NaOH를 사용하여 전처리하였다. 점도가 낮은 기관지 세척액 등은 NaOH를 사용하지 않고, 검체 150 - 200 uL를 RNA 추출버퍼 350 uL가 미리 분주된 1.5 mL 에펜도르프 튜브로 옮긴 뒤 1분간 교반하였다. 실온에서 5분간 방치하고 클로로포름(chloroform) 80 uL를 첨가하여 10초간 교반하여 혼합하였다. 이후 실온에서 3분간 방치한 후에 13,000 rpm으로 실온에서 10분간 원심분리하였다. 맑은 상층액 부분 250 uL 정도를 RNase가 오염되지 않은 멸균 에펜도르프 튜브로 옮겼다. 이후 250 uL의 이소프로판올(isopropanol)을 첨가하고 가볍게 흔들어 혼합하였다. 10분간 방치한 후에 13,000 rpm으로 실온에서 15분간 원심분리를 하고 pellet이 빨려 나가지 않게 조심하여 상층액을 제거하였다. 침전물이 눈에 보이지 않을 경우 없어진 것이 아니므로 상층액을 완전히 제거하지 않고 20 uL 정도 남겨두고 이후 단계를 진행하였다. -20℃ 보관중인 75% 에탄올(ethanol)을 1 mL 첨가하고 13,000 rpm으로 실온에서 5분간 원심분리를 한 뒤, pellet이 빨려 나가지 않게 조심하여 상층액을 제거하고 실온에서 에펜도르프 튜브 캡을 열어 둔 채로 pellet을 자연적으로 건조하였고 곧바로 DNase/RNase가 오염되지 않은 멸균 증류수를 15 - 30 uL를 첨가하여 바이러스 핵산을 추출하였다. Viral DNA and RNA were isolated from the sample for differential diagnosis of viral pathogens of respiratory infections, and a commercially available viral spin kit (Easy-spin total RNA extraction kit, Intron biotechnology, Korea, Cat. No 17221) was used. Sputum samples were pretreated with 2N NaOH under the same conditions as before. The low-viscosity bronchial washing solution was used without NaOH, and 150-200 uL of the sample was transferred to a 1.5 mL Eppendorf tube pre-dispensed with 350 uL of RNA extraction buffer and stirred for 1 minute. It was allowed to stand at room temperature for 5 minutes, and 80 uL of chloroform was added thereto, followed by stirring for 10 seconds and mixing. After standing at room temperature for 3 minutes, the mixture was centrifuged at 13,000 rpm for 10 minutes at room temperature. About 250 uL of clear supernatant portion was transferred to a RNase-free sterile Eppendorf tube. Then 250 uL of isopropanol was added and gently shaken to mix. After standing for 10 minutes, the mixture was centrifuged at 13,000 rpm for 15 minutes at room temperature, and the supernatant was removed by careful not to suck the pellet. If the precipitate is not visible, it does not disappear, so the supernatant was left behind about 20 uL without completely removing the supernatant. Add 1 mL of 75% ethanol stored at -20 ° C, centrifuge at 13,000 rpm for 5 minutes at room temperature, remove supernatant carefully to prevent pellet from being sucked out, and open the Eppendorf tube cap at room temperature. The pellet was dried naturally and immediately extracted 15% of 30 dl of sterile distilled water without DNase / RNase contamination.

<< 실시예Example 3> 멀티플렉스  3> multiplex PCRPCR 을 통한 호흡기감염질환 진단용 칩(Chip for diagnosing respiratory infection disease through chipchip )의 )of 타겟target 유전자 증폭 Gene amplification

고-처리량 칩(chip) 분석에 앞서, 임상 검체로부터 분리한 세균, 진균 DNA 그리고 바이러스 RNA 및 DNA를 주형으로 멀티플렉스 PCR 반응을 통해 칩 반응의 타겟 유전자들을 증폭하였다. 이후, 증폭된 멀티플렉스 앰플리콘들은 칩(chip)표면의 DNA 또는 PNA 프로브들과 듀플렉스(duplex)를 형성함으로써 호흡기감염질환의 병원체별 감별진단 결과가 확인됨과 동시에 해당 병원체의 특정 항생제에 대한 내성 유무 분석결과까지 동시에 도출되게 된다. 본 발명의 바람직한 일 양태에 따른 멀티플렉스 PCR은 내부 대조군(internal control)으로 인간 베타-글로빈(β-globin) 유전자, 세균 16S rRNA 유전자, 또는 바이러스 NS1 유전자를 포함한다. 본 실시예에 바람직하게 기술하는 호흡기감염질환 세균성 병원체 10종 멀티플렉스 PCR의 프리메이드(pre-made) PCR 마스터믹스(PCR mastermix)의 구성 조성 및 PCR 반응조건은 하기 표 1과 같다. Prior to high-throughput chip analysis, target genes of the chip reaction were amplified by multiplex PCR reactions with bacterial, fungal DNA and viral RNA and DNA isolated from clinical samples. Subsequently, the amplified multiplex amplicons form duplexes with DNA or PNA probes on the chip surface to confirm the differential diagnosis results of pathogens of respiratory infections, and at the same time, whether the pathogens are resistant to specific antibiotics. The analysis results can be derived simultaneously. The multiplex PCR according to a preferred embodiment of the present invention includes a human beta-globin gene, a bacterial 16S rRNA gene, or a viral NS1 gene as an internal control. Constitutional composition and PCR reaction conditions of the pre-made PCR mastermix of 10 respiratory infection disease bacterial pathogens multiplex PCR described in this embodiment are shown in Table 1 below.

호흡기감염질환 세균성 병원체 10종 멀티플렉스 Respiratory Tract Infection Bacterial Pathogen 10 Multiplex PCRPCR 마스터믹스Master mix 조성 및  Composition and PCRPCR 조건 Condition PCR 반응액 조성  PCR reaction solution composition 부피(uL) Volume (uL) PCR 반응 조건  PCR reaction conditions 탈이온 3차 멸균 증류수  Deionized Tertiary Sterilized Distilled Water 4.3 4.3 초기변성 (Initial denaturation) Initial denaturation 95℃, 15분 95 ° C, 15 minutes 1회 1 time 10종 프라이머 프리믹스(primer premix)  10 primer premixes 5.7  5.7 변성 (Denaturation)  Denaturation 95℃, 40초  95 ° C, 40 seconds 37회      Episode 37 2X 멀티플렉스 PCR 프리믹스  2X Multiplex PCR Premix 15  15 결합 (Annealing) Annealing 57℃, 40초 57 ° C, 40 seconds 세균 게노믹 DNA(50 ng 이상)  Bacterial Genomic DNA (over 50 ng) 5  5 연장 (Extension) Extension 72℃, 40초 72 ° C., 40 seconds 최종 부피  Final volume 30  30 최종연장 (Extension)  Extension 72℃, 7분 72 ° C., 7 minutes 1회 1 time

그리고 본 실시예에 또한 바람직하게 기술하는 호흡기감염질환 항생제 내성 분석용 19종 멀티플렉스 PCR의 프리메이드(pre-made) PCR 마스터믹스의 구성 조성 및 PCR 반응조건은 하기 표 2와 같다. The composition and PCR reaction conditions of the pre-made PCR mastermix of 19 multiplex PCR for respiratory infection disease antibiotic resistance analysis described in this embodiment are also shown in Table 2 below.

호흡기감염질환 항생제 내성 분석용 19종 멀티플렉스 19 multiplexes for respiratory infections PCRPCR 마스터믹스Master mix 조성 및  Composition and PCRPCR 조건 Condition PCR 반응액 조성  PCR reaction solution composition 부피(uL) Volume (uL) PCR 반응 조건  PCR reaction conditions 탈이온 3차 멸균 증류수  Deionized Tertiary Sterilized Distilled Water 2.5 2.5 초기변성 (Initial denaturation) Initial denaturation 95℃, 15분 95 ° C, 15 minutes 1회 1 time 19종 프라이머 프리믹스(primer premix)  19 primer premixes 7.5  7.5 변성 (Denaturation)  Denaturation 95℃, 45초  95 ° C, 45 seconds 40회      40 times 2X 멀티플렉스 PCR 프리믹스  2X Multiplex PCR Premix 15  15 결합 (Annealing) Annealing 57℃, 60초 57 ° C., 60 seconds 세균 게노믹 DNA(100 ng 이상)  Bacterial Genomic DNA (over 100 ng) 5  5 연장 (Extension) Extension 72℃, 60초 72 ° C., 60 seconds 최종 부피  Final volume 30  30 최종연장 (Extension)  Extension 72℃, 7분 72 ° C., 7 minutes 1회 1 time

멀티플렉스 PCR 반응액 혼합물에 포함되는 PCR 완충용액의 최종농도는 50 mM KCl, 3.5 mM MgCl2, 10mM Tris-HCl, pH 8.2이며, 2.5 unit의 Taq 중합효소, 300 uM dNTPs(Boehringer Mannheim, Mannheim, Germany), 10 mg/mL 소혈청알부민(Bovine serum albumin)을 포함한다. 호흡기감염질환 병원체 감별진단을 위한 멀티플렉스 PCR에 포함되는 개별 유전자들의 정방향(sense) 프라이머는 5'- 또는 3'-말단에 Cy3 또는 Cy5 형광색소를 표지시켜 합성하거나, 또는 양 말단의 변형(modification)없이 nascent 올리고뉴클레오티드로 합성하였다(Integrated DNA Technologies, Inc., Coralville, IA, USA). Final concentration of PCR buffer in the multiplex PCR reaction mixture is 50 mM KCl, 3.5 mM MgCl 2 , 10 mM Tris-HCl, pH 8.2, 2.5 unit Taq polymerase, 300 uM dNTPs (Boehringer Mannheim, Mannheim, Germany), 10 mg / mL Bovine serum albumin. Sense primers of individual genes included in multiplex PCR for differential diagnosis of respiratory infections are synthesized by labeling Cy3 or Cy5 fluorescent dyes at the 5'- or 3'-end, or modification at both ends. ) Was synthesized with nascent oligonucleotides (Integrated DNA Technologies, Inc., Coralville, IA, USA).

본 발명의 또한 바람직한 일 양태에 따른 말단에 형광색소 변형이 없는 프라이머를 포함하는 멀티플렉스 PCR 키트는 PCR 과정중에 Cy3 형광색소가 표지된 디옥시사이토신 트리포스페이트(Cy3-dCTP)가 증폭산물을 구성하는 뉴클레오티드로 첨가되도록 하여 반응의 민감도와 특이도를 향상시킬 수 있었다. Cy3-dCTP를 사용하는 11종 멀티플렉스 PCR 반응에 사용하는 PCR 완충용액은 최종농도 50 mM KCl, 3.5 mM MgCl2, 10mM Tris-HCl, pH 8.2 이였으며, 2.5 unit의 Taq 중합효소, 300 uM dATP, 300 uM dGTP, 300 uM dTTP, 25 uM dCTP, 275 uM Cy3-dCTP(FluoroLink Cy3-dCTP, Amersham Pharmacia Biotech AB, Piscataway, NJ, USA), 10 mg/mL 소혈청알부민(Bovine serum albumin)을 첨가하여 타겟 유전자들을 특이적으로 증폭하였다.In a multiplex PCR kit including a primer having no fluorescent dye modification at the terminal according to a preferred embodiment of the present invention, Cy3 fluorescent dye-labeled deoxycytosine triphosphate (Cy3-dCTP) constitutes an amplification product during PCR. By adding to the nucleotides to improve the sensitivity and specificity of the reaction. The PCR buffer used for 11 multiplex PCR reactions using Cy3-dCTP had a final concentration of 50 mM KCl, 3.5 mM MgCl 2 , 10 mM Tris-HCl, pH 8.2, 2.5 units of Taq polymerase, 300 uM dATP , 300 uM dGTP, 300 uM dTTP, 25 uM dCTP, 275 uM Cy3-dCTP (FluoroLink Cy3-dCTP, Amersham Pharmacia Biotech AB, Piscataway, NJ, USA), 10 mg / mL Bovine serum albumin Target genes were specifically amplified.

그리고 본 실시예에 또한 바람직하게 기술하는 호흡기감염질환 바이러스성 병원체 13종 멀티플렉스 RT(reverse trasnscription)-PCR의 프리메이드(pre-made) RT-PCR 마스터믹스의 구성 조성, 반응조건 및 2차 PCR 반응조건은 하기 표 3, 4와 같다. And compositional composition, reaction conditions and secondary PCR of a pre-made RT-PCR mastermix of 13 respiratory infectious diseases viral pathogens multiplex RT (reverse trasnscription) -PCR, which are also described in the present embodiment. The reaction conditions are shown in Tables 3 and 4 below.

RT-PCR 반응액 조성  RT-PCR reaction solution composition 부피(uL) Volume (uL) RT-PCR 반응 조건  RT-PCR Reaction Conditions 탈이온 3차 멸균 DEPC-증류수  Deionized Tertiary Sterilization DEPC-Distilled Water 1.5  1.5 역전사 효소 반응(reverse transcription reaction)  Reverse transcription reaction 55℃, 3분 55 ℃, 3 minutes 1회 1 time 42℃, 40분 42 ° C, 40 minutes 1회 1 time 95℃, 5분 95 ° C, 5 minutes 1회 1 time RT-PCR 프라이머 프리믹스(primer premix)  RT-PCR Primer premix 1  One 1차 PCR 반응 변성 (Denaturation)Primary PCR Reaction Denaturation 95℃, 30초  95 ° C, 30 seconds 33회      Episode 33 2X RT-PCR 프리믹스  2X RT-PCR Premix 11  11 결합 (Annealing) Annealing 58℃, 40초 58 ° C, 40 seconds 바이러스 RNA 및 DNA(1 mcg 이상)  Viral RNA and DNA (greater than 1 mcg) 8.5  8.5 연장 (Extension) Extension 72℃, 40초 72 ° C., 40 seconds 최종 부피  Final volume 22  22 최종연장 (Extension)  Extension 72℃, 7분 72 ° C., 7 minutes 1회 1 time

바이러스 RNA 및 DNA 산물 8.5 uL와 RNase 저해제와 AMV 역전사 효소(reverse transcriptase)가 포함된 2X RT-PCR 프리믹스 11 uL, 랜덤 헥사머(random hexamer)를 포함하는 RT-PCR 프라이머 프리믹스 1 uL, DEPC(diethylpyrocarbonate)-처리한 3차 멸균 증류수 1.5 uL를 포함하는 RT-PCR 마스터믹스를 제조하고 상기와 같은 반응조건으로 역전사 효소반응을 수행하였다. 이후 RT-PCR 반응산물 3 uL를 이용하여 2차 PCR 반응을 진행하였고 반응 조성 및 조건은 하기와 같다. 8.5 uL of viral RNA and DNA product, 11 uL of 2X RT-PCR premix with RNase inhibitor and AMV reverse transcriptase, 1 uL of RT-PCR primer premix with random hexamer, diethylpyrocarbonate ) -Treated tertiary sterile distilled water was prepared RT-PCR mastermix containing 1.5 uL and reverse transcription enzyme reaction under the same reaction conditions as above. Thereafter, the second PCR reaction was performed using RT-PCR reaction product 3 uL, and the reaction composition and conditions are as follows.

2차 PCR 반응액 조성  2nd PCR reaction solution composition 부피(uL) Volume (uL) 2차 PCR 반응 조건  Secondary PCR Reaction Conditions 탈이온 3차 멸균 증류수  Deionized Tertiary Sterilized Distilled Water 4.9 4.9 초기변성 (Initial denaturation) Initial denaturation 95℃, 15분 95 ° C, 15 minutes 1회 1 time 13종 프라이머 프리믹스(primer premix)  13 primer premixes 7.1  7.1 변성 (Denaturation)  Denaturation 95℃, 30초  95 ° C, 30 seconds 30회      30 times 2X 멀티플렉스 PCR 프리믹스  2X Multiplex PCR Premix 15  15 결합 (Annealing) Annealing 60℃, 40초 60 ℃, 40 seconds 역전사 효소반응 산물  Reverse Transcriptase Product 3  3 연장 (Extension) Extension 72℃, 40초 72 ° C., 40 seconds 최종 부피  Final volume 30  30 최종연장 (Extension)  Extension 72℃, 5분 72 ° C., 5 minutes 1회 1 time

<< 실시예Example 4> 호흡기감염질환 감별진단 및 항생제 내성 분석용 칩( 4> Differential diagnosis for respiratory infection and antibiotic resistance chip ( chipchip )을 구성하는 프로브(Probes that make up probeprobe ) 디자인 및 합성A) design and synthesis

본 발명의 바람직한 실현을 위한 호흡기감염질환 진단 및 항생제 내성 분석 칩(chip)을 제공하기 위하여, 세균성 병원체 10종, 바이러스성 병원체 13종 그리고 항생제 내성 분석용 19종 유전자들을 고-특이도로 판별할 수 있는 프로브(probe)들을 디자인하였다. In order to provide a respiratory infectious disease diagnosis and antibiotic resistance analysis chip for the preferred embodiment of the present invention, 10 bacterial pathogens, 13 viral pathogens and 19 genes for antibiotic resistance analysis can be discriminated with high specificity. Probes were designed.

상세하게는, 본 발명의 바람직한 양태에 따른 뇌졸중 DNA 또는 PNA 칩(chip)이 포함하는 프로브들은, 멀티플렉스 PCR 앰플리콘(amplicon)의 타겟 유전자에 특이적으로 결합하도록 고안하기 위하여 프라이머 프리미어(Primer premier version 5, Premier Biosoft International, Palo Alto, CA, USA), 디엔에이시스 맥스 (DNASIS MAX Version 2.7, MiraiBio Group, South San Francisco, CA, USA), 또는 올리고어레이(OligoArray 2.0, http://cbr-rbc.nrc-cnrc.gc.ca) 프로그램을 응용하여 디자인하였고 그 세부정보는 표 5과 같다. 목적하는 DNA 또는 PNA 프로브 염기서열의 5'말단 또는 3'말단에 아민(amine) 또는 티올(thiol) 그룹이 위치하도록 변형(modification)된 프로브를 합성하였다(MWG-Biotech AG, Ebersberg, Germany). 프로브의 5'말단에 아미노 모디파이어를 수식할 경우 탄소 6 - 9개 또는 티민(thymine) 염기 12 - 15개의 스페이서(spacer)를 첨가하여 반응 효율을 촉진하였다. 프로브의 3'말단에 아미노 모디파이어를 수식할 경우에는 스페이서를 사용하지 않았다. 프로브의 아민기는 1차 아민기의 성질을 지니며, 알데히드-활성화된 또는 카르복실(carboxyl)-활성화된 칩(chip) 표면에 부착하였다. 프로브의 농도는 260nm에서 흡광도 수치를 통해 환산하였고 몰디-토프(MALDI-TOF, Matrix Assisted Laser Desorption/Ionization Time-of-Flight)를 통해 불순물 함유 여부를 확인하고 고성능 액체 크로마토그래피(HPLC, High-performance liquid chromatography)를 통해 순수 정제한 후 멸균 3차 증류수에 최종농도가 100-250 pM 되도록 제조하였다.Specifically, probes included in the stroke DNA or PNA chip according to a preferred embodiment of the present invention, Primer premier (Designer Premier) to be designed to specifically bind to the target gene of the multiplex PCR amplicon (amplicon) version 5, Premier Biosoft International, Palo Alto, Calif., USA, DNASIS MAX Version 2.7, MiraiBio Group, South San Francisco, Calif., USA, or OligoArray 2.0, http: // cbr-rbc .nrc-cnrc.gc.ca) program is applied and the details are shown in Table 5. Probes modified to place amine or thiol groups at the 5 'or 3' end of the DNA or PNA probe sequence of interest were synthesized (MWG-Biotech AG, Ebersberg, Germany). When modifying the amino modifier at the 5 'end of the probe, spacers of 6 to 9 carbons or 12 to 15 thymine bases were added to promote the reaction efficiency. No spacer was used when modifying the amino modifier at the 3 'end of the probe. The amine groups of the probe have the properties of primary amine groups and are attached to the aldehyde-activated or carboxyl-activated chip surface. The concentration of the probe was converted to absorbance values at 260 nm, and it was checked for impurities by Maldi-TOF (MALDI-TOF, Matrix Assisted Laser Desorption / Ionization Time-of-Flight), and high performance liquid chromatography (HPLC) After pure purification through liquid chromatography) was prepared so that the final concentration in sterile tertiary distilled water 100-250 pM.

호흡기감염질환 감별진단 및 항생제 분석 칩(Respiratory infectious disease differential diagnosis and antibiotic analysis chip ( chipchip )을 구성하는 Make up) 프로브Probe (( probeprobe ) 조합) Combination 서열 목록 번호Sequence listing number 프로브 타겟 구분, (반응 균주 명), 타겟 유전자 명 Probe target classification, (response strain name), target gene name 프로브 방향, (야생형 또는 변이형), 염기서열(5'-3')Probe orientation, (wild or variant), nucleotide sequence (5'-3 ') 길이Length 1One 항생제 내성, TEM Antibiotic resistance, TEM 정방향, GAATAAGGGCGACACGGAAAForward, GAATAAGGGCGACACGGAAA 2020 22 역방향, TTTCCGTGTCGCCCTTATTCReverse, TTTCCGTGTCGCCCTTATTC 2020 33 항생제 내성, SHVAntibiotic resistance, SHV 역방향, CAGCACGGAGCGGATCAACGReverse, CAGCACGGAGCGGATCAACG 2020 44 정방향, CGCCCTGCTTGGCCCGAATAForward, CGCCCTGCTTGGCCCGAATA 2020 55 항생제 내성, CTX-MAntibiotic resistance, CTX-M 역방향, AGTTCTGCCAGCGTCATTGTGCCReverse, AGTTCTGCCAGCGTCATTGTGCC 2323 66 역방향, GCGCCGGTCGTATTGCCTTTGAReverse, GCGCCGGTCGTATTGCCTTTGA 2222 77 항생제 내성, OXA-1Antibiotic Resistance, OXA-1 정방향, TTGCGATGCTCTATGAGTGGCTForward, TTGCGATGCTCTATGAGTGGCT 2222 88 정방향, GGCGATGAGCGAAATGTAGTGCForward, GGCGATGAGCGAAATGTAGTGC 2222 99 항생제 내성, AmpCAntibiotic Resistance, AmpC 정방향, GACCGTTACGCCGCTGATGAAForward, GACCGTTACGCCGCTGATGAA 2121 1010 정방향, TGACAGGCAAGCAGTGGCAGGForward, TGACAGGCAAGCAGTGGCAGG 2121 1111 항생제 내성, PBP2BAntibiotic resistance, PBP2B 정방향, (야생형), TTGTTCCAGGTTCGGTTGTCAAForward, (wild type), TTGTTCCAGGTTCGGTTGTCAA 2222 1212 정방향, (변이형), AATTGGCATATGGATCTTTTCCTATForward, (variable), AATTGGCATATGGATCTTTTCCTAT 2525 1313 항생제 내성, PBP1AAntibiotic resistance, PBP1A 정방향, (야생형), TAACTGGGATAGGGGCTACTTTGGCForward, (wild type), TAACTGGGATAGGGGCTACTTTGGC 2525 1414 정방향, (변이형), GGACGATGCCAGACGGACTTForward, (variant), GGACGATGCCAGACGGACTT 2020 1515 항생제내성, gyrAAntibiotic resistance, gyrA 정방향, (야생형), GGTATTTACCCATGACATCCCCTForward, (wild type), GGTATTTACCCATGACATCCCCT 2323 1616 정방향, (변이형), GGTATTTACCCATGACATTCCCTForward, (variable), GGTATTTACCCATGACATTCCCT 2323 1717 항생제내성, parCAntibiotic resistance, parC 정방향, (야생형), TCCACCCACACGGGGATTCTTCForward, (wild type), TCCACCCACACGGGGATTCTTC 2222 1818 정방향, (변이형1), TCCACCCACACGGGGATTTTTCForward, (variant 1), TCCACCCACACGGGGATTTTTC 2222 1919 정방향, (변이형2), TCCACCCACACGGGGATTATTCForward, (variant 2), TCCACCCACACGGGGATTATTC 2222 2020 항생제내성, parEAntibiotic resistance, parE 정방향, (야생형), AAGGCCAAGATGGCGGATATCCTCForward, (wild type), AAGGCCAAGATGGCGGATATCCTC 2424 2121 정방향, (변이형), AAGGCCAAGATGGCGGATGTCCTCForward, (variant), AAGGCCAAGATGGCGGATGTCCTC 2424 2222 항생제내성, qnrAAntibiotic resistance, qnrA 정방향, GGGGCTGTGACCTAACCTTTGCForward, GGGGCTGTGACCTAACCTTTGC 2222 2323 역방향, ATCGGCAAAGGTTAGGTCACAGCReverse, ATCGGCAAAGGTTAGGTCACAGC 2323 2424 항생제내성, qnrBAntibiotic resistance, qnrB 역방향, AGTCGTGCGATGCTGAAAGATGReverse, AGTCGTGCGATGCTGAAAGATG 2222 2525 정방향, TTTTCGCCAACGAGTGCCAGForward, TTTTCGCCAACGAGTGCCAG 2020 2626 항생제내성, qnrSAntibiotic resistance, qnrS 정방향, TGCCCATCAAGTGAGTAATCGTATForward, TGCCCATCAAGTGAGTAATCGTAT 2424 2727 역방향, AGGTTCGTTCCTATCCAGCGReverse, AGGTTCGTTCCTATCCAGCG 2020 2828 항생제내성, ermAAntibiotic resistance, ermA 정방향, AAATGAGTCAACGGGTGAATGCTAForward, AAATGAGTCAACGGGTGAATGCTA 2424 2929 역방향, ATTTTTCGGGTTTTTCTGTTTCATReverse, ATTTTTCGGGTTTTTCTGTTTCAT 2424 3030 항생제내성, ermBAntibiotic resistance, ermB 정방향, CATCAAGCAATGAAACACGCCAForward, CATCAAGCAATGAAACACGCCA 2222 3131 정방향, TCAAGCAATGAAACACGCCAATGForward, TCAAGCAATGAAACACGCCAATG 2323 3232 항생제내성, ermCAntibiotic resistance, ermC 정방향, TCGTGGAATACGGGTTTGCTAAForward, TCGTGGAATACGGGTTTGCTAA 2222 3333 역방향, CTTTTAGCAAACCCGTATTCCACReverse, CTTTTAGCAAACCCGTATTCCAC 2323 3434 항생제내성, mefAntibiotic resistance, mef 정방향, ATGGGCAGGGCAAGCAGTATForward, ATGGGCAGGGCAAGCAGTAT 2020 3535 역방향, GCAATCACAGCACCCAATACGReverse, GCAATCACAGCACCCAATACG 2121 3636 항생제 내성, mecAAntibiotic resistance, mecA 역방향, CGTTGCGATCAATGTTACCGTAGTReverse, CGTTGCGATCAATGTTACCGTAGT 2424 3737 정방향, ACTACGGTAACATTGATCGCAACGForward, ACTACGGTAACATTGATCGCAACG 2424 3838 세균성, S. pneumoniae, lytABacterial, S. pneumoniae, lytA 정방향, GAAGCGGATTATCACTGGCGGForward, GAAGCGGATTATCACTGGCGG 2121 3939 정방향, GGCGGTTGGAATGCTGAGACForward, GGCGGTTGGAATGCTGAGAC 2020 4040 세균성, S. aureus, entC1Bacterial, S. aureus, entC1 정방향, GCCAACCAGACCCTACGCCAGForward, GCCAACCAGACCCTACGCCAG 2121 4141 역방향, CTGGCGTAGGGTCTGGTTGGCTCReverse, CTGGCGTAGGGTCTGGTTGGCTC 2323 4242 세균성, H. influenzae, P6Bacterial, H. influenzae, P6 정방향, TTGGCGGATACTCTGTTGCTGATForward, TTGGCGGATACTCTGTTGCTGAT 2323 4343 역방향, CCTAATGCGATGTTGTATTCTGGTGReverse, CCTAATGCGATGTTGTATTCTGGTG 2525 4444 세균성, B. pertussis, IS481Bacterial, B. pertussis, IS481 정방향, TAGACGACGCCTACCGCACCCTForward, TAGACGACGCCTACCGCACCCT 2222 4545 정방향, ACGACGCCTACCGCACCCTGATForward, ACGACGCCTACCGCACCCTGAT 2222 4646 세균성, M. catarrhalis, CopBBacterial, M. catarrhalis, CopB 정방향, CCAAAGATGTGCCCAAAGAGATAForward, CCAAAGATGTGCCCAAAGAGATA 2323 4747 역방향, ACCACATCATTGCCCAACAGAReverse, ACCACATCATTGCCCAACAGA 2121 4848 세균성, L. pneumophila, mipBacterial, L. pneumophila, mip 정방향, AGCGTTGGCGGACCTATTGGForward, AGCGTTGGCGGACCTATTGG 2020 4949 역방향, CCACTTGGTAATACAACAACGCCReverse, CCACTTGGTAATACAACAACGCC 2323 5050 세균성, M. pneumoniae, P1Bacterial, M. pneumoniae, P1 정방향, CACCACCGCAACAAGGGACGForward, CACCACCGCAACAAGGGACG 2020 5151 역방향, CGAACCGCCTGTAATCATCGTCTReverse, CGAACCGCCTGTAATCATCGTCT 2323 5252 세균성, C. pneumoniae, ompABacterial, C. pneumoniae, ompA 정방향, CATTTGCTGGTTCTGTCGGCTCCForward, CATTTGCTGGTTCTGTCGGCTCC 2323 5353 정방향, ACTGCCGTAGATAGACCTAACCCGForward, ACTGCCGTAGATAGACCTAACCCG 2424 5454 세균성, K. pneumoniae, 16S rRNABacterial, K. pneumoniae, 16S rRNA 정방향, GGACGGGTGAGTAATGTCTGGGAForward, GGACGGGTGAGTAATGTCTGGGA 2323 5555 정방향, CGGCGGACGGGTGAGTAATGTForward, CGGCGGACGGGTGAGTAATGT 2121 5656 바이러스성, Influenza A, M2Viral, Influenza A, M2 정방향, CCCTTGTTGTTGCTGCGAGTATCForward, CCCTTGTTGTTGCTGCGAGTATC 2323 5757 역방향, GATACTCGCAGCAACAACAAGGGReverse, GATACTCGCAGCAACAACAAGGG 2323 5858 바이러스성, Influenza B, M1Viral, Influenza B, M1 역방향, TCTTTCCTGGTCTTTGGGCTTReverse, TCTTTCCTGGTCTTTGGGCTT 2121 5959 역방향, GTTTCTCGCACAAAGCACAGAGCReverse, GTTTCTCGCACAAAGCACAGAGC 2323 6060 바이러스성, PIV1, neuraminidaseViral, PIV1, neuraminidase 정방향, TATGCTCCTTGCCCACTGTGAATGForward, TATGCTCCTTGCCCACTGTGAATG 2424 6161 정방향, AACTCCGCTCCAAGGCGACACTAAForward, AACTCCGCTCCAAGGCGACACTAA 2424 6262 바이러스성, PIV2, neuraminidaseViral, PIV2, neuraminidase 역방향, CACCCGTTGGGAGATGTTGCTGAReverse, CACCCGTTGGGAGATGTTGCTGA 2323 6363 정방향, ATGAGTCCAACCGAACCAACCCCAForward, ATGAGTCCAACCGAACCAACCCCA 2424 6464 바이러스성, PIV3, neuraminidaseViral, PIV3, neuraminidase 역방향, CTCATTGCCAGCATCCTTTCCGTReverse, CTCATTGCCAGCATCCTTTCCGT 2323 6565 역방향, AAGTAACCTTCCTTCTGACCCCCAReverse, AAGTAACCTTCCTTCTGACCCCCA 2424 6666 바이러스성, RSV A, fusion proteinViral, RSV A, fusion protein 역방향, ACGGCCTTGTTTGTGGATAGTAGAGReverse, ACGGCCTTGTTTGTGGATAGTAGAG 2525 6767 정방향, GTGCTCTACTATCCACAAACAAGGCForward, GTGCTCTACTATCCACAAACAAGGC 2525 6868 바이러스성, RSV B, fusion proteinViral, RSV B, fusion protein 정방향, TTCTGGGCTTCTTGTTAGGTGTAGGForward, TTCTGGGCTTCTTGTTAGGTGTAGG 2525 6969 정방향, ATTCCAGCAGAAGAACAGCAGATTGForward, ATTCCAGCAGAAGAACAGCAGATTG 2525 7070 바이러스성, hMPV, matrix proteinViral, hMPV, matrix protein 역방향, GATCATAATCAATCCCGCATAAGGTReverse, GATCATAATCAATCCCGCATAAGGT 2525 7171 역방향, CATCCCGTATGGTTTCATGGTTGTReverse, CATCCCGTATGGTTTCATGGTTGT 2424 7272 바이러스성, Adenovirus, hexon geneViral, Adenovirus, hexon gene 정방향, AAACTCCCTCMCTCGGCTCGGForward, AAACTCCCTCMCTCGGCTCGG 2121 7373 역방향, ATGGTRGGTGCGAGGTAGCCGReverse, ATGGTRGGTGCGAGGTAGCCG 2121 7474 바이러스성, CMV, IE geneViral, CMV, IE gene 정방향, AGTTYTGTCGGGTGCTGTGCTGCForward, AGTTYTGTCGGGTGCTGTGCTGC 2323 7575 정방향, CGGGTGCTGTGCTGCTATRTCTTForward, CGGGTGCTGTGCTGCTATRTCTT 2323 7676 바이러스성, HSV-1 & -2, glycoproteinViral, HSV-1 & -2, glycoprotein 정방향, CACATCAAGGTGGGCCAGCCGForward, CACATCAAGGTGGGCCAGCCG 2121 7777 역방향, CTGCGGCTGGCCCACCTTGATReverse, CTGCGGCTGGCCCACCTTGAT 2121 7878 바이러스성, HSV-2, glycoproteinViral, HSV-2, glycoprotein 정방향, TGGGACTGGGTGCCGAAGCGAForward, TGGGACTGGGTGCCGAAGCGA 2121 7979 역방향, CGGTCGCTTCGGCACCCAGTCReverse, CGGTCGCTTCGGCACCCAGTC 2121

프로브의 변별력을 향상시키기 위하여 프로브 길이, 칩 세척과정의 스트린전시(stringency), 칩 혼성화 반응 온도 등의 다양한 조건들을 확인하였고, 위양성과 위음성 결과를 방지하기 위해 칩 혼성화 반응 온도 및 형광 시그널을 조절 가능한 첨가제(additive)를 통하여 우수한 변별 조건을 확인할 수 있었다. In order to improve the discrimination ability of the probe, various conditions such as probe length, stringency of chip cleaning process and chip hybridization reaction temperature were checked, and chip hybridization reaction temperature and fluorescence signal were controlled to prevent false positive and false negative results. A good discrimination condition could be confirmed through possible additives.

<< 실시예Example 5> 호흡기감염질환 감별진단 및 항생제 내성 분석 칩( 5> Differential diagnosis of respiratory infection disease and antibiotic resistance analysis chip ( chipchip ) 제작Production

본 발명의 바람직한 양태로 제공하는 호흡기감염질환 병원체별 감별진단 및 항생제 내성 분석을 위한 DNA 또는 PNA 칩(chip)은 세균성 병원체 10종, 바이러스성 병원체 13종 그리고 항생제 내성 분석용 19종 유전자들을 고-특이도로 판별할 수 있는 총 42종의 프로브(probe)가 집적되어 있다. 내부 대조군(internal control)으로 인간 베타-글로빈(β-globin) 유전자, 세균 16S rRNA 유전자, 또는 바이러스 NS1 유전자에 대한 프로브가 고정화되어있다. 하나의 칩 기판위에 총 42종의 유전자 고-처리량 분석이 가능한 그리드(grid)가 8개씩 포함되도록 칩 레이아웃을 고안하였으며, 8 웰 혼성화 반응 챔버(8 well hybridization reaction chamber)를 통해 8개의 개별 검체에 대한 분석을 동시에 진행할 수 있도록 고안하였다(도 3 참조). 영하 70℃ 보관중인 프로브 스탁(stock)을 실온에서 해동한 후, 탈이온 멸균 3차 증류수에 최종농도 100 μM이 되도록 희석하여 워킹 스탁(working stock)으로 사용하였다. 워킹 스탁을 50배 희석한 각각의 프로브들을 3X SSC 스포팅 용액(500 mM NaCl, 3 mM sodium citrate, 1.5 M N,N,N-trimethylglycine, pH 6.8)과 1 : 5 - 10 비율(v/v)로 혼합하여 최종 96 웰 플레이트(well plate)에 분주되는 프로브의 농도범위가 20 - 30 pmole/uL 가 되도록 하였다. 상기 플레이트를 Microssys 5100 microarrayer(Cartesian Technologies, Ann Arbor, MI, USA)에 장착하고 이로부터 알데하이드(aldehyde)-, 티오이소시아네이트(thioisocyanate)-활성화된 글라스 슬라이드(CEL associates Inc., Houston, TX, USA), 에폭시(epoxy)-활성화된 플라스틱 칩, 또는 골드 필름 표면에 프로브들을 순서에 따라 2개씩 duplicate으로 스포팅하였다. 스팟(spot)의 평균 크기(diameter)는 80 - 140 마이크로미터(micrometer)이며 스팟간 크로스-토크(cross-talk)효과를 최소화하기 위해 스팟간의 거리는 350 - 500 마이크로미터를 유지하였다. 칩 제작은 75% 습도(humidity)를 유지하는 클래스 10,000 룸에서 실시하였다. 프로브가 스팟팅된 칩은 120℃, 1시간 베이킹(baking)한 후, 0.25% SDS(Sodium dodecyl sulfate)용액에서 3분간 세척하고 멸균 3차 증류수로 다시 세척하였다. 이후 칩을 0.2% 소디움 보로하이드라이드(NaBH4)를 포함하는 용액에 반응시켜 프로브를 블럭킹(blocking)하였다. 이후 3차 증류수로 2회 세척하고 물기를 제거한 후 사용시점까지 데시케이터(dessicator)에 보관하였다.DNA or PNA chip for the differential diagnosis and antibiotic resistance analysis of respiratory infectious disease pathogens provided as a preferred embodiment of the present invention is a high-quality 10 genes, 13 viral pathogens and 19 genes for antibiotic resistance analysis. There are a total of 42 probes that can be determined by their specificity. As an internal control, probes for human beta-globin gene, bacterial 16S rRNA gene, or viral NS1 gene are immobilized. The chip layout was designed to include eight grids for a total of 42 genes for high-throughput analysis on a single chip substrate, and eight individual specimens were placed in an eight well hybridization reaction chamber. It was designed to proceed with the analysis at the same time (see Figure 3). After thawing at 70 ° C., the probe stock was stored at room temperature, and then diluted with deionized sterile tertiary distilled water to a final concentration of 100 μM and used as a working stock. Each probe with 50-fold dilution of working stock was subjected to a 1: 5-10 ratio (v / v) with 3X SSC spotting solution (500 mM NaCl, 3 mM sodium citrate, 1.5 MN, N, N-trimethylglycine, pH 6.8). By mixing, the concentration range of the probe dispensed into the final 96 well plate was 20-30 pmole / uL. The plate was mounted on a Microssys 5100 microarrayer (Cartesian Technologies, Ann Arbor, MI, USA), from which aldehyde-, thioisocyanate-activated glass slides (CEL associates Inc., Houston, TX, USA) The probes were spotted in duplicate on the surface of the epoxy-activated plastic chip or gold film. The average diameter of the spots is 80-140 micrometers and the distance between spots was maintained at 350-500 micrometers to minimize cross-talk effects between spots. Chip fabrication was carried out in a class 10,000 room maintaining 75% humidity. The chip spotted with the probe was baked at 120 ° C. for 1 hour, and then washed for 3 minutes in 0.25% SDS (Sodium dodecyl sulfate) solution, and then again washed with sterile tertiary distilled water. The probe was then blocked by reacting the chip with a solution containing 0.2% sodium borohydride (NaBH 4 ). After washing twice with 3 distilled water, the water was removed and stored in a desiccator (dessicator) until the point of use.

<< 실시예Example 6> 호흡기감염질환 감별진단 및 항생제 내성 분석 칩( 6> Differential diagnosis of respiratory infectious disease and antibiotic resistance analysis chip ( chipchip ) 혼성화() Hybridization ( hybridizationhybridization ) 반응 및 결과 분석) Response and result analysis

본 발명의 바람직한 일실시예에 따른 세균성 병원체 10종 및 바이러스성 병원체 13종 멀티플렉스 PCR 증폭산물과 감염된 병원체의 항생제 내성 보유 여부를 분석하기 위한 19종 멀티플렉스 PCR 증폭산물을 각각 10 - 20 uL씩 총 30 - 60 uL를 이와 동일한 volume의 탈이온 3차 멸균 증류수가 미리 첨가된 1.5 mL 에펜도르프 튜브에 첨가하였다. 이 mixture를 softly vortex하고, 95℃, 5분간 열변성시킨 후 얼음위에 5분간 보존하고 원심분리기로 스핀다운(spin down)하였다. 칩(chip) 표면에는 8 웰 혼성화 반응 챔버(8 well hybridization chamber)를 위치시키고 웰 커버(cover)로 웰 상층부를 덮어두었다. 이후 반응시키고자하는 웰(well)에 상기 반응 혼합용액에 혼성화 반응 온도인 56℃로 미리 가열해둔 60 - 80uL의 혼성화 반응 용액(3X SSC, 0.1% SDS, 0.2 mg/mL 소혈청알부민, pH 7)을 첨가하여 혼합한 후 웰 커버의 구멍(hole)을 통해 반응액 혼합물을 주입하고 버블(bubble)이 발생하지 않도록 주의하였다. 챔버 리드(lid)를 고정시킨 후 56℃, 30분간 혼성화 반응을 통해 칩 표면의 프로브와 멀티플렉스 PCR 반응산물간 특이적인 뉴클레오티드 상보적(complementary) 결합을 유도하였다. 혼성화 반응이 종료된 칩 표면의 웰 커버를 제거하고 칩을 세척버퍼 1(0.1X SSC, 0.05% SDS)용액에 담그고 2분간 2,000 rpm에서 교반하면서 세척(washing)하고 이를 반복하였다. 이후 세척용액 2(2X SSC, 0.1% SDS)용액에 2분간 2,000 rpm에서 교반하면서 세척(washing)하고 이를 반복하였다. 이후 탈이온 3차 멸균 증류수에 담가 2회 세척하고 1,000 rpm에서 원심분리하여 칩을 건조하였다. 상기 칩을 스캔어래이 라이트(ScanArray Lite, Packard Instrument Co., Meriden, CT, USA) 스캐너를 이용하여 판독하였고, 분석 소프트웨어(QuantArray 2.0)를 이용하여 양성 대조군 스팟들의 평균 형광강도(fluorescence intensity) 및 표준오차를 스팟 주변의 값들과 비교하여 signal-to-noise(S/R) 비율을 구한 뒤 그 값이 4 이상일 경우 양성 값으로 스코어링(scoring) 처리하였다(도 5, 6, 7 참조). 10-20 uL each of 10 bacterial and 10 viral pathogens according to a preferred embodiment of the present invention and 19 multiplex PCR amplification products for analyzing the antibiotic resistance of infected pathogens A total of 30-60 uL was added to this volume of 1.5 mL eppendorf tubes pre-added with deionized tertiary sterile distilled water. The mixture was softly vortexed, thermally denatured at 95 ° C. for 5 minutes, preserved on ice for 5 minutes, and spun down with a centrifuge. An 8 well hybridization chamber was placed on the chip surface and the well top was covered with a well cover. Thereafter, 60-80 uL of a hybridization reaction solution (3X SSC, 0.1% SDS, 0.2 mg / mL bovine serum albumin, pH 7) was preheated to the reaction mixture solution at 56 ° C. in the reaction mixture solution. ) Was added and mixed, and then the reaction mixture was injected through the hole of the well cover, and care was taken not to generate bubbles. After fixing the chamber lid, hybridization reaction was performed at 56 ° C. for 30 minutes to induce specific nucleotide complementary binding between the probe on the chip surface and the multiplex PCR reaction product. The well cover of the chip surface where the hybridization reaction was completed was removed, the chip was immersed in the washing buffer 1 (0.1X SSC, 0.05% SDS) solution, washed with stirring at 2,000 rpm for 2 minutes, and repeated. After washing with washing solution 2 (2X SSC, 0.1% SDS) solution at 2,000 rpm for 2 minutes (washing) was repeated. Thereafter, the chips were dried by immersion in deionized tertiary sterile distilled water twice and centrifuged at 1,000 rpm. The chip was read using a ScanArray Lite (Packard Instrument Co., Meriden, CT, USA) scanner, and the mean fluorescence intensity and standard of the positive control spots using the analysis software (QuantArray 2.0). Signal-to-noise (S / R) ratio was obtained by comparing the error with the values around the spot, and when the value was 4 or more, scoring was performed as a positive value (see FIGS. 5, 6, and 7).

도 1은 본 발명에 따른 호흡기감염질환 병원체 진단 및 항생제 내성 분석 시간(4시간)과 종래의 gold standard 검사법인 배양검사를 비롯한 흉부 X-선, 그람 염색 검사, 항생제 내성 검사에 소요되는 시간(4일)을 비교한 모식도이다. 1 is a time required for diagnosis of respiratory infection pathogens and antibiotic resistance analysis time according to the present invention (4 hours) and chest X-ray, gram staining test, antibiotic resistance test, including culture test, which is a conventional gold standard test method (4). It is a schematic diagram comparing work).

도 2는 본 발명이 구성하는 호흡기감염질환 감별진단 및 항생제 내성 분석 칩(chip)을 통해 분석가능한 감염성 병원체와 항생제 그룹별 분석 대상인 내성 유전자를 나타내었다. 10여종의 세균성 병원체와 12종의 바이러스성 병원체 그리고 베타-락탐계, 페니실린계, 퀴놀론계, 매크로리드계 및 메티실린계 항생제 내성 분석을 위한 타겟 유전자 명을 나타내었다.Figure 2 shows the resistance genes analyzed by infectious agents and antibiotics groups that can be analyzed through the respiratory infection disease differential diagnosis and antibiotic resistance analysis chip (chip) constituting the present invention. The target gene names for the analysis of 10 bacterial and 12 viral pathogens and beta-lactam, penicillin, quinolone, macrolide and methicillin antibiotic resistance were shown.

도 3은 본 발명에 따라 제작된 호흡기감염질환 병원체별 감별진단 및 이의 항생제 내성 분석이 가능한 칩의 모식도이다. 항생제 내성 분석용 그리고 세균성 병원체 및 바이러스성 병원체의 타겟 프로브(probe)가 배열된 칩 그리드(grid)를 나타내었으며, 형광물질이 표지된 타겟 DNA와 칩 위에 배열된 프로브간 혼성화(hybridization)반응이 이루어지는 8웰 혼성화 반응 챔버를 모식하여 나타냈다.Figure 3 is a schematic diagram of a chip capable of differential diagnosis and antibiotic resistance analysis of each respiratory infection disease pathogens prepared according to the present invention. The chip grid shows the array of target probes for antibiotic resistance analysis and the bacterial and viral pathogens, and the hybridization reaction between the fluorescent DNA-labeled target DNA and the probes arranged on the chip is performed. An 8 well hybridization reaction chamber was schematically shown.

도 4는 본 발명에 따라 프로브가 고정화된 칩 제작 이후, 검체로부터 분리한 DNA 또는 바이러스 RNA로부터 역전사한 cDNA(complementary DNA)를 주형으로 타겟 유전자를 멀티플렉스 PCR 또는 RT-PCR 반응을 통해 증폭한 뒤, 칩 위의 선택적 프로브들과 혼성화(hybridization) 반응을 실시함으로써 호흡기감염질환 병원체 감별진단 및 이의 항생제 내성 유무를 판별하는 칩 분석과정의 주요 단계를 나타낸 흐 름도이다.4 is amplified by a multiplex PCR or RT-PCR reaction of the target gene as a template after the preparation of the chip immobilized probe according to the present invention, a template of reverse-transcribed cDNA (complementary DNA) from DNA or viral RNA isolated from the sample This is a flow chart showing the main steps of the chip analysis process to discriminate between respiratory infection pathogens and determine their antibiotic resistance by performing hybridization with selective probes on the chip.

도 5는 본 발명에 따라 호흡기감염질환 병원체 감별진단 및 이의 항생제 내성 유무를 분석한 결과를 나타내는 칩 스캔 이미지(좌측 그림)와 칩 그리드(우측 그림)를 나타낸다. 세균성 호흡기감염이 발생한 검체로, 폐렴구균과 H. 인플루엔자, M. 뉴모니애가 감염되었으며, CTX-M, OXA-1 양성으로 플라스미드성 베타-락탐계 항생제 내성이 존재하며, PBP2B 돌연변이형에 따른 페니실린계 내성이 예상되며, gyrA 돌연변이형 및 qnrA, qnrB 양성에 따른 퀴놀론계 내성 및 ermA 양성에 따른 매크로리드계 내성이 존재하는 분석결과를 나타낸다.Figure 5 shows a chip scan image (left picture) and chip grid (right picture) showing the results of analyzing the differential diagnosis of respiratory infection pathogens and their antibiotic resistance according to the present invention. Bacterial respiratory infections were infected with pneumococci, H. influenza, and M. pneumoniae, and plasmid beta-lactam antibiotic resistance was positive for CTX-M and OXA-1, and penicillin according to PBP2B mutant System resistance is expected, and the analysis results show that there are gyrA mutants and quinolone resistance according to qnrA and qnrB positive and macrolide resistance according to ermA positive.

도 6은 본 발명에 따라 호흡기감염질환 병원체 감별진단 및 이의 항생제 내성 유무를 분석한 결과를 나타내는 또 다른 칩 스캔 이미지(좌측 그림)와 칩 그리드(우측 그림)를 나타낸다. 세균성 및 바이러스성 호흡기복합감염이 발생한 검체로, H. 인플루엔자, M. 뉴모니애 및 K. 뉴모니애가 세균성감염되었으며, 인플루엔자 B, 파라인플루엔자 2, 3형 및 호흡기세포융합바이러스 A형이 바이러스성감염되었다. TEM, CTX-M 양성으로 플라스미드성 베타-락탐계 항생제 내성이 존재하며, PBP2B 및 PBP1A 돌연변이형에 따른 페니실린계 내성이 예상되며, gyrA 및 parC 돌연변이형 그리고 qnrA 양성에 따른 퀴놀론계 내성이 존재하고, mecA 양성에 따른 메티실린계 내성을 확인한 분석결과를 나타낸다.Figure 6 shows another chip scan image (left picture) and chip grid (right picture) showing the results of the differential diagnosis of respiratory infection pathogens and their antibiotic resistance according to the present invention. Bacterial and viral respiratory infections occurred in H. influenza, M. pneumoniae and K. pneumoniae, and influenza B, parainfluenza 2, 3 and respiratory syncytial virus type A were viral. Infected. TEM, CTX-M positive plasmid beta-lactam antibiotic resistance is present, penicillin resistance according to PBP2B and PBP1A mutant is expected, quinolone resistance according to gyrA and parC mutant and qnrA positive, Analysis results confirming methicillin resistance according to mecA positive.

도 7은 본 발명의 바람직한 구현에 따른 호흡기감염질환 병원체 감별진단 및 이의 항생제 내성 유무를 분석한 결과를 나타내는 또 다른 칩 스캔 이미지(좌측 그림)와 칩 그리드(우측 그림)를 나타낸다. 바이러스성 호흡기감염이 발생한 검체로, 인플루엔자 A, B형, 호흡기세포융합바이러스 A, B형, 아데노바이러스 그리고 세포거대바이러스가 감염되었다.Figure 7 shows another chip scan image (left picture) and chip grid (right picture) showing the results of the differential diagnosis of respiratory infection pathogens and their antibiotic resistance according to a preferred embodiment of the present invention. Viral respiratory infections include influenza A, B, respiratory syncytial virus A, B, adenovirus and cytomegalovirus.

<110> PARK, MinKoo <120> Respiratory infectious pathogen differential diagnosis and simultaneous antibiotics resistance analysis, kit and chip comprising same <160> 79 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for TEM gene, beta-lactam antibiotics resistance <400> 1 gaataagggc gacacggaaa 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for TEM gene, beta-lactam antibiotics resistance <400> 2 tttccgtgtc gcccttattc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for SHV gene, beta-lactam antibiotics resistance <400> 3 cagcacggag cggatcaacg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for SHV gene, beta-lactam antibiotics resistance <400> 4 cgccctgctt ggcccgaata 20 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for CTX-M gene, beta-lactam antibiotics resistance <400> 5 agttctgcca gcgtcattgt gcc 23 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for CTX-M gene, beta-lactam antibiotics resistance <400> 6 gcgccggtcg tattgccttt ga 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for OXA-1 gene, beta-lactam antibiotics resistance <400> 7 ttgcgatgct ctatgagtgg ct 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for OXA-1 gene, beta-lactam antibiotics resistance <400> 8 ggcgatgagc gaaatgtagt gc 22 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for AmpC gene, beta-lactam antibiotics resistance <400> 9 gaccgttacg ccgctgatga a 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for AmpC gene, beta-lactam antibiotics resistance <400> 10 tgacaggcaa gcagtggcag g 21 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for PBP2B gene, wild type, penicillins resistance <400> 11 ttgttccagg ttcggttgtc aa 22 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for PBP2B gene, mutant type, penicillins resistance <400> 12 aattggcata tggatctttt cctat 25 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for PBP1A gene, wild type, penicillins resistance <400> 13 taactgggat aggggctact ttggc 25 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for PBP1A gene, mutant type, penicillins resistance <400> 14 ggacgatgcc agacggactt 20 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for gyrA gene, wild type, quinolone resistance <400> 15 ggtatttacc catgacatcc cct 23 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for gyrA gene, mutant type, quinolone resistance <400> 16 ggtatttacc catgacattc cct 23 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for parC gene, wild type, quinolone resistance <400> 17 tccacccaca cggggattct tc 22 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for parC gene, mutant type, quinolone resistance <400> 18 tccacccaca cggggatttt tc 22 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for parC gene, mutant type, quinolone resistance <400> 19 tccacccaca cggggattat tc 22 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for parE gene, wild type, quinolone resistance <400> 20 aaggccaaga tggcggatat cctc 24 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for parE gene, mutant type, quinolone resistance <400> 21 aaggccaaga tggcggatgt cctc 24 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for qnrA gene, quinolone resistance <400> 22 ggggctgtga cctaaccttt gc 22 <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for qnrA gene, quinolone resistance <400> 23 atcggcaaag gttaggtcac agc 23 <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for qnrB gene, quinolone resistance <400> 24 agtcgtgcga tgctgaaaga tg 22 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for qnrB gene, quinolone resistance <400> 25 ttttcgccaa cgagtgccag 20 <210> 26 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for qnrS gene, quinolone resistance <400> 26 tgcccatcaa gtgagtaatc gtat 24 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for qnrS gene, quinolone resistance <400> 27 aggttcgttc ctatccagcg 20 <210> 28 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for ermA gene, macrolides resistance <400> 28 aaatgagtca acgggtgaat gcta 24 <210> 29 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for ermA gene, macrolides resistance <400> 29 atttttcggg tttttctgtt tcat 24 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for ermB gene, macrolides resistance <400> 30 catcaagcaa tgaaacacgc ca 22 <210> 31 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for ermB gene, macrolides resistance <400> 31 tcaagcaatg aaacacgcca atg 23 <210> 32 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for ermC gene, macrolides resistance <400> 32 tcgtggaata cgggtttgct aa 22 <210> 33 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for ermC gene, macrolides resistance <400> 33 cttttagcaa acccgtattc cac 23 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for mef gene, macrolides resistance <400> 34 atgggcaggg caagcagtat 20 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for mef gene, macrolides resistance <400> 35 gcaatcacag cacccaatac g 21 <210> 36 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for mecA gene, methicillin resistance <400> 36 cgttgcgatc aatgttaccg tagt 24 <210> 37 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for mecA gene, methicillin resistance <400> 37 actacggtaa cattgatcgc aacg 24 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Streptococcus pneumoniae <400> 38 gaagcggatt atcactggcg g 21 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for Streptococcus pneumoniae <400> 39 ggcggttgga atgctgagac 20 <210> 40 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Staphylococcus aureus <400> 40 gccaaccaga ccctacgcca g 21 <210> 41 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Staphylococcus aureus <400> 41 ctggcgtagg gtctggttgg ctc 23 <210> 42 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Haemophilus influenzae <400> 42 ttggcggata ctctgttgct gat 23 <210> 43 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for Haemophilus influenzae <400> 43 cctaatgcga tgttgtattc tggtg 25 <210> 44 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for Bordetella pertussis <400> 44 tagacgacgc ctaccgcacc ct 22 <210> 45 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for Bordetella pertussis <400> 45 acgacgccta ccgcaccctg at 22 <210> 46 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Moraxella catarrhalis <400> 46 ccaaagatgt gcccaaagag ata 23 <210> 47 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Bordetella pertussis <400> 47 accacatcat tgcccaacag a 21 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for Legionella pneumophila <400> 48 agcgttggcg gacctattgg 20 <210> 49 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Legionella pneumophila <400> 49 ccacttggta atacaacaac gcc 23 <210> 50 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for Mycoplasma pneumoniae <400> 50 caccaccgca acaagggacg 20 <210> 51 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Mycoplasma pneumoniae <400> 51 cgaaccgcct gtaatcatcg tct 23 <210> 52 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Chlamydiae pneumoniae <400> 52 catttgctgg ttctgtcggc tcc 23 <210> 53 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for Chlamydiae pneumoniae <400> 53 actgccgtag atagacctaa cccg 24 <210> 54 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Klebsiella pneumoniae <400> 54 ggacgggtga gtaatgtctg gga 23 <210> 55 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Klebsiella pneumoniae <400> 55 cggcggacgg gtgagtaatg t 21 <210> 56 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Influenza A <400> 56 cccttgttgt tgctgcgagt atc 23 <210> 57 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Influenza A <400> 57 gatactcgca gcaacaacaa ggg 23 <210> 58 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Influenza B <400> 58 tctttcctgg tctttgggct t 21 <210> 59 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Influenza B <400> 59 gtttctcgca caaagcacag agc 23 <210> 60 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for Parainfluenza virus 1 <400> 60 tatgctcctt gcccactgtg aatg 24 <210> 61 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for Parainfluenza virus 1 <400> 61 aactccgctc caaggcgaca ctaa 24 <210> 62 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Parainfluneza virus 2 <400> 62 cacccgttgg gagatgttgc tga 23 <210> 63 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for Parainfluenza virus 2 <400> 63 atgagtccaa ccgaaccaac ccca 24 <210> 64 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Parainfluenza virus 3 <400> 64 ctcattgcca gcatcctttc cgt 23 <210> 65 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for Parainfluenza virus 3 <400> 65 aagtaacctt ccttctgacc ccca 24 <210> 66 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for Respiratory syncytial virus A <400> 66 acggccttgt ttgtggatag tagag 25 <210> 67 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for Respiratory syncytial virus A <400> 67 gtgctctact atccacaaac aaggc 25 <210> 68 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for Respiratory syncytial virus B <400> 68 ttctgggctt cttgttaggt gtagg 25 <210> 69 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for Respiratory syncytial virus B <400> 69 attccagcag aagaacagca gattg 25 <210> 70 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for human metapneumovirus <400> 70 gatcataatc aatcccgcat aaggt 25 <210> 71 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for human metapneumovirus <400> 71 catcccgtat ggtttcatgg ttgt 24 <210> 72 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Adenovirus <400> 72 aaactccctc mctcggctcg g 21 <210> 73 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Adenovirus <400> 73 atggtrggtg cgaggtagcc g 21 <210> 74 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Cytomegalovirus <400> 74 agttytgtcg ggtgctgtgc tgc 23 <210> 75 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Cytomegalovirus <400> 75 cgggtgctgt gctgctatrt ctt 23 <210> 76 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Herpes simplex virus 1 & 2 <400> 76 cacatcaagg tgggccagcc g 21 <210> 77 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Herpes simplex virus 1 & 2 <400> 77 ctgcggctgg cccaccttga t 21 <210> 78 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Herpes simplex virus 2 <400> 78 tgggactggg tgccgaagcg a 21 <210> 79 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Herpes simplex virus 2 <400> 79 cggtcgcttc ggcacccagt c 21 <110> PARK, MinKoo <120> Respiratory infectious pathogen differential diagnosis and          simultaneous antibiotics resistance analysis, kit and chip          configure same <160> 79 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for TEM gene, beta-lactam antibiotics resistance <400> 1 gaataagggc gacacggaaa 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for TEM gene, beta-lactam antibiotics resistance <400> 2 tttccgtgtc gcccttattc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for SHV gene, beta-lactam antibiotics resistance <400> 3 cagcacggag cggatcaacg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for SHV gene, beta-lactam antibiotics resistance <400> 4 cgccctgctt ggcccgaata 20 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for CTX-M gene, beta-lactam antibiotics resistance <400> 5 agttctgcca gcgtcattgt gcc 23 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for CTX-M gene, beta-lactam antibiotics resistance <400> 6 gcgccggtcg tattgccttt ga 22 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for OXA-1 gene, beta-lactam antibiotics resistance <400> 7 ttgcgatgct ctatgagtgg ct 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for OXA-1 gene, beta-lactam antibiotics resistance <400> 8 ggcgatgagc gaaatgtagt gc 22 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for AmpC gene, beta-lactam antibiotics resistance <400> 9 gaccgttacg ccgctgatga a 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for AmpC gene, beta-lactam antibiotics resistance <400> 10 tgacaggcaa gcagtggcag g 21 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for PBP2B gene, wild type, penicillins resistance <400> 11 ttgttccagg ttcggttgtc aa 22 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for PBP2B gene, mutant type, penicillins resistance <400> 12 aattggcata tggatctttt cctat 25 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for PBP1A gene, wild type, penicillins resistance <400> 13 taactgggat aggggctact ttggc 25 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for PBP1A gene, mutant type, penicillins resistance <400> 14 ggacgatgcc agacggactt 20 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for gyrA gene, wild type, quinolone resistance <400> 15 ggtatttacc catgacatcc cct 23 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for gyrA gene, mutant type, quinolone resistance <400> 16 ggtatttacc catgacattc cct 23 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for parC gene, wild type, quinolone resistance <400> 17 tccacccaca cggggattct tc 22 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for parC gene, mutant type, quinolone resistance <400> 18 tccacccaca cggggatttt tc 22 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for parC gene, mutant type, quinolone resistance <400> 19 tccacccaca cggggattat tc 22 <210> 20 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for parE gene, wild type, quinolone resistance <400> 20 aaggccaaga tggcggatat cctc 24 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for parE gene, mutant type, quinolone resistance <400> 21 aaggccaaga tggcggatgt cctc 24 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for qnrA gene, quinolone resistance <400> 22 ggggctgtga cctaaccttt gc 22 <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for qnrA gene, quinolone resistance <400> 23 atcggcaaag gttaggtcac agc 23 <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for qnrB gene, quinolone resistance <400> 24 agtcgtgcga tgctgaaaga tg 22 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for qnrB gene, quinolone resistance <400> 25 ttttcgccaa cgagtgccag 20 <210> 26 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for qnrS gene, quinolone resistance <400> 26 tgcccatcaa gtgagtaatc gtat 24 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for qnrS gene, quinolone resistance <400> 27 aggttcgttc ctatccagcg 20 <210> 28 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for ermA gene, macrolides resistance <400> 28 aaatgagtca acgggtgaat gcta 24 <210> 29 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for ermA gene, macrolides resistance <400> 29 atttttcggg tttttctgtt tcat 24 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for ermB gene, macrolides resistance <400> 30 catcaagcaa tgaaacacgc ca 22 <210> 31 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for ermB gene, macrolides resistance <400> 31 tcaagcaatg aaacacgcca atg 23 <210> 32 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for ermC gene, macrolides resistance <400> 32 tcgtggaata cgggtttgct aa 22 <210> 33 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for ermC gene, macrolides resistance <400> 33 cttttagcaa acccgtattc cac 23 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for mef gene, macrolides resistance <400> 34 atgggcaggg caagcagtat 20 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for mef gene, macrolides resistance <400> 35 gcaatcacag cacccaatac g 21 <210> 36 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for mecA gene, methicillin resistance <400> 36 cgttgcgatc aatgttaccg tagt 24 <210> 37 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for mecA gene, methicillin resistance <400> 37 actacggtaa cattgatcgc aacg 24 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Streptococcus pneumoniae <400> 38 gaagcggatt atcactggcg g 21 <210> 39 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for Streptococcus pneumoniae <400> 39 ggcggttgga atgctgagac 20 <210> 40 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Staphylococcus aureus <400> 40 gccaaccaga ccctacgcca g 21 <210> 41 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Staphylococcus aureus <400> 41 ctggcgtagg gtctggttgg ctc 23 <210> 42 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Haemophilus influenzae <400> 42 ttggcggata ctctgttgct gat 23 <210> 43 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for Haemophilus influenzae <400> 43 cctaatgcga tgttgtattc tggtg 25 <210> 44 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for Bordetella pertussis <400> 44 tagacgacgc ctaccgcacc ct 22 <210> 45 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> probe for Bordetella pertussis <400> 45 acgacgccta ccgcaccctg at 22 <210> 46 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Moraxella catarrhalis <400> 46 ccaaagatgt gcccaaagag ata 23 <210> 47 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Bordetella pertussis <400> 47 accacatcat tgcccaacag a 21 <210> 48 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for Legionella pneumophila <400> 48 agcgttggcg gacctattgg 20 <210> 49 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Legionella pneumophila <400> 49 ccacttggta atacaacaac gcc 23 <210> 50 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> probe for Mycoplasma pneumoniae <400> 50 caccaccgca acaagggacg 20 <210> 51 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Mycoplasma pneumoniae <400> 51 cgaaccgcct gtaatcatcg tct 23 <210> 52 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Chlamydiae pneumoniae <400> 52 catttgctgg ttctgtcggc tcc 23 <210> 53 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for Chlamydiae pneumoniae <400> 53 actgccgtag atagacctaa cccg 24 <210> 54 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Klebsiella pneumoniae <400> 54 ggacgggtga gtaatgtctg gga 23 <210> 55 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Klebsiella pneumoniae <400> 55 cggcggacgg gtgagtaatg t 21 <210> 56 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Influenza A <400> 56 cccttgttgt tgctgcgagt atc 23 <210> 57 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Influenza A <400> 57 gatactcgca gcaacaacaa ggg 23 <210> 58 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Influenza B <400> 58 tctttcctgg tctttgggct t 21 <210> 59 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Influenza B <400> 59 gtttctcgca caaagcacag agc 23 <210> 60 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for Parainfluenza virus 1 <400> 60 tatgctcctt gcccactgtg aatg 24 <210> 61 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for Parainfluenza virus 1 <400> 61 aactccgctc caaggcgaca ctaa 24 <210> 62 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Parainfluneza virus 2 <400> 62 cacccgttgg gagatgttgc tga 23 <210> 63 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for Parainfluenza virus 2 <400> 63 atgagtccaa ccgaaccaac ccca 24 <210> 64 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Parainfluenza virus 3 <400> 64 ctcattgcca gcatcctttc cgt 23 <210> 65 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for Parainfluenza virus 3 <400> 65 aagtaacctt ccttctgacc ccca 24 <210> 66 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for Respiratory syncytial virus A <400> 66 acggccttgt ttgtggatag tagag 25 <210> 67 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for Respiratory syncytial virus A <400> 67 gtgctctact atccacaaac aaggc 25 <210> 68 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for Respiratory syncytial virus B <400> 68 ttctgggctt cttgttaggt gtagg 25 <210> 69 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for Respiratory syncytial virus B <400> 69 attccagcag aagaacagca gattg 25 <210> 70 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> probe for human metapneumovirus <400> 70 gatcataatc aatcccgcat aaggt 25 <210> 71 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> probe for human metapneumovirus <400> 71 catcccgtat ggtttcatgg ttgt 24 <210> 72 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Adenovirus <400> 72 aaactccctc mctcggctcg g 21 <210> 73 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Adenovirus <400> 73 atggtrggtg cgaggtagcc g 21 <210> 74 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Cytomegalovirus <400> 74 agttytgtcg ggtgctgtgc tgc 23 <210> 75 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> probe for Cytomegalovirus <400> 75 cgggtgctgt gctgctatrt ctt 23 <210> 76 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Herpes simplex virus 1 & 2 <400> 76 cacatcaagg tgggccagcc g 21 <210> 77 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Herpes simplex virus 1 & 2 <400> 77 ctgcggctgg cccaccttga t 21 <210> 78 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Herpes simplex virus 2 <400> 78 tgggactggg tgccgaagcg a 21 <210> 79 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> probe for Herpes simplex virus 2 <400> 79 cggtcgcttc ggcacccagt c 21  

Claims (13)

감염질환 병원체 감별과 항생제 내성 유무 분석을 동시에 고-처리량 방식으로 수행하는 분석방법.Analytical method to perform infectious disease pathogen discrimination and antibiotic resistance analysis at the same time in high-throughput method. 제 1항에 있어서, 5'- 또는 3'-말단에 아미노 모디파이어(amino modifier) 또는 티올 모디파이어(thiol modifier)가 수식된 프로브가 스팟팅(spotting)된 칩(chip)을 제작하는 단계;The method of claim 1, further comprising: manufacturing a chip in which a probe modified with an amino modifier or a thiol modifier at the 5′- or 3′-end is spotted; 검체로부터 세균 게노믹(genomic) DNA, 바이러스 RNA 및 DNA를 분리하는 단계; Separating bacterial genomic DNA, viral RNA, and DNA from the sample; 호흡기감염질환 병원체별 프로브 타겟 부위 및 항생제 내성 분석용 프로브 타겟 부위를 멀티플렉스 RT-PCR(reverse transcription-polymerase chain reaction) 또는 멀티플렉스 PCR을 통해 증폭하면서 형광색소(fluorescent dye)가 포함되도록 타겟(target) DNA 증폭산물을 준비하는 단계;Probe target sites for antibiotic pathogens and probe target sites for antibiotic resistance analysis are amplified by multiplex reverse transcription-polymerase chain reaction (RT-PCR) or multiplex PCR to target fluorescent dyes. ) Preparing a DNA amplification product; 상기 멀티플렉스 반응산물과 칩 표면에 고정화된 프로브간 혼성화(hybridization) 반응을 수행하고 세척(washing)하는 단계;Performing and washing a hybridization reaction between the multiplex reaction product and the probe immobilized on the chip surface; 형광색소에 특이적인 파장의 레이저(laser)로 칩을 스캐닝(scanning)하고 혼성화 반응 결과에 따른 형광강도를 측정함으로써 감염질환 병원체 감별진단 및 이의 항생제 내성 유무 결과를 동시에 분석하는 단계; 그리고Simultaneously analyzing the infectious disease pathogen differential diagnosis and its antibiotic resistance by scanning the chip with a laser of a wavelength specific to the fluorescent dye and measuring the fluorescence intensity according to the hybridization reaction result; And 상기 분석단계 전부를 포함하는 감염질환 병원체 감별진단 및 항생제 내성 동시 분석방법.Simultaneous diagnosis of infectious disease pathogens and antibiotic resistance analysis comprising all of the above analysis steps. 제 1항에 있어서, 세균, 바이러스, 진균, 원충 등에 유발되는 코감기, 인두염, 후두염의 상기도 감염과 이로 인한 부비동염, 중이염 합병증 그리고 기관지염, 폐렴의 하기도 감염을 포함하는 호흡기질환군의 병원체를 종(species)- 또는 타입(type)-특이적으로 감별진단함과 동시에 병원체의 항생제 내성 유무를 판별하는 것을 특징으로하는 분석방법.The pathogen of the respiratory disease group according to claim 1, which includes upper respiratory tract infections of nasal cold, pharyngitis, and laryngitis caused by bacteria, viruses, fungi, protozoa, and associated sinusitis, otitis media complications and lower respiratory tract infections of bronchitis and pneumonia. (Species)-or type (type) -specific analysis and characterized in that at the same time to determine the presence or absence of antibiotic resistance of the pathogen. 제 1항 내지 3항에 있어서, 세균성 호흡기감염질환 병원체 감별진단 분석방법은, According to claim 1 to 3, bacterial respiratory infection pathogen differential diagnosis analysis method, 폐렴구균(Streptococcus pneumoniae);Pneumococcal (Streptococcus pneumoniae); 황색포도구균(Staphylococcus aureus);Staphylococcus aureus; 클렙시엘라 뉴모니애(Klebsiella pneumoniae);Klebsiella pneumoniae; 슈도모나스 애루지노사(Pseudomonas aeruginosa);Pseudomonas aeruginosa; 마이코플라즈마 뉴모니애(Mycoplasma pneumoniae);Mycoplasma pneumoniae; 클라미디아 뉴모니애(Chlamydia pneumoniae);Chlamydia pneumoniae; 레지오넬라 뉴모필라(Legionella pneumophila);Legionella pneumophila; 보드텔라 퍼투시스(Bordetella pertussis);Bodetella pertussis; 모락셀라 카타랄리스(Moraxella catarrhalis);Moraxella catarrhalis; 헤모필러스 인플루엔자(Haemophilus influenza);로 구성된 그룹으로부터 선 택되는 하나 이상의 타겟 유전자를 종-특이적으로 증폭하는 것을 특징으로 하는 분석방법.Hemophilus influenza (Haemophilus influenza); characterized in that the species-specific amplification of one or more target genes selected from the group consisting of. 제 1항 내지 3항에 있어서, 바이러스성 호흡기감염질환 병원체 감별진단 분석방법은,According to claim 1 to 3, Viral respiratory infection disease pathogen differential diagnosis analysis method, 호흡기세포융합바이러스(Respiratory Syncytial Virus) A 타입;Respiratory Syncytial Virus A type; 호흡기세포융합바이러스(Respiratory Syncytial Virus) B 타입;Respiratory Syncytial Virus B type; 파라인플루엔자바이러스(Parainfluenza Virus) 1 타입;Parainfluenza Virus 1 type; 파라인플루엔자바이러스(Parainfluenza Virus) 2 타입;Parainfluenza Virus 2 type; 파라인플루엔자바이러스(Parainfluenza Virus) 3 타입;Parainfluenza virus 3 type; 인플루엔자바이러스(Influenza Virus) A 타입;Influenza Virus A type; 인플루엔자바이러스(Influenza Virus) B 타입;Influenza Virus B type; 아데노바이러스(Adenovirus);Adenovirus; 인간메타뉴모바이러스(Human Metapneumovirus);Human metapneumovirus; 세포거대바이러스(Cytomegalovirus);Cytomegalovirus; 허피스심플렉스바이러스(Herpes Simplex Virus) 1 타입;Herpes Simplex Virus 1 type; 허피스심플렉스바이러스(Herpes Simplex Virus) 2 타입;으로 구성된 그룹으로부터 선택되는 하나 이상의 타겟 유전자를 타입(type)-특이적으로 증폭하는 것을 특징으로 하는 분석방법.Herpes Simplex Virus 2 type; an analysis method characterized in that the type-specific amplification of one or more target genes selected from the group consisting of. 제 1항 내지 3항에 있어서, 호흡기감염질환 병원체의 항생제 내성 유무 분석방법은, The method of claim 1, wherein the method for analyzing antibiotic resistance of the pathogen for respiratory infections is 베타-락탐계 항생제 내성 분석을 위해,For beta-lactam antibiotic resistance assays, TEM, TEM, SHV, SHV, CTX-M, CTX-M, OXA-1, OXA-1, AmpC 타겟 유전자; 그리고AmpC target genes; And 페니실린계 항생제 내성 분석을 위해,For penicillin antibiotic resistance analysis, PBP2B, PBP2B, PBP1A, PBP1A, mecA 타겟 유전자; 그리고mecA target gene; And 퀴놀론계 항생제 내성 분석을 위해, For quinolone antibiotic resistance analysis, gyrA, gyrA, parC, parC, parE, parE, qnrA, qnrA, qnrB, qnrB, qnrS 타겟 유전자; 그리고qnrS target gene; And 매크로리드계 항생제 내성 분석을 위해, For macrolide antibiotic resistance analysis, ermA, ermA, ermB, ermB, ermC, mef 타겟 유전자; 를 포함하는 그룹으로부터 선택되는 하나 이상의 타겟 유전자를 특이적으로 증폭하는 것을 특징으로 하는 분석방법.ermC, mef target gene; Analytical method characterized in that the amplification of one or more target genes selected from the group comprising a. 제 1항 내지 6항에 있어서, 서열목록번호 1 내지 79의 염기서열을 갖는 DNA 올리고뉴클레오티드(deoxyribonucleic acid oligonucleotide)로 이루어진 그룹에서 선택된 하나 이상의 DNA 프로브(probe)를 포함하는 호흡기감염질환 병원체 감별진단 및 항생제 내성 분석용 DNA 칩.According to claim 1 to 6, Respiratory infection pathogenic differential diagnosis comprising at least one DNA probe (probe) selected from the group consisting of DNA oligonucleotides (deoxyribonucleic acid oligonucleotide) having a nucleotide sequence of SEQ ID NO: 1 to 79 and DNA chip for antibiotic resistance analysis. 제 1항 내지 6항에 있어서, 서열목록번호 1 내지 79의 염기서열을 갖는 PNA 올리고뉴클레오티드(peptide nucleic acid oligonucleotide)로 이루어진 그룹에서 선택된 하나 이상의 PNA 프로브(probe)를 포함하는 호흡기감염질환 병원체 감별진단 및 항생제 내성 분석용 PNA 칩.According to claim 1 to 6, Respiratory infectious disease pathogenic differential diagnosis comprising at least one PNA probe (probe) selected from the group consisting of PNA oligonucleotides (Pptide nucleic acid oligonucleotide) having a nucleotide sequence of SEQ ID NO: 1 to 79 And PNA chips for antibiotic resistance analysis. 제 1항 내지 8항에 있어서, 호흡기감염질환 병원체 감별진단 및 항생제 내성 유무 분석을 위해, 서열목록번호 1 내지 79의 염기서열을 갖는 프로브와 상보적인 결합이 가능한 타겟 부위를 증폭할 수 있는 멀티플렉스-PCR 및 멀티플렉스 RT-PCR 키트.The method of claim 1, wherein the multiplexes capable of amplifying a target site complementary to a probe having a nucleotide sequence of SEQ ID NOs: 1 to 79 for differential diagnosis of respiratory infection pathogens and analysis of antibiotic resistance. PCR and multiplex RT-PCR kits. 제 9항에 있어서, 정방향(sense) 또는 역방향(anti-sense) 프라이머의 5' 또는 3' 말단에 형광색소(Fluorescent dye)가 표지된 프라이머(primer)를 포함하는 키트.10. The kit of claim 9, comprising a primer labeled with a fluorescent dye at either the 5 'or 3' end of a forward or anti-sense primer. 제 9항에 있어서, PCR 증폭 과정중에 형광색소가 표지된 디옥시시티딘 트리포스페이트(Deoxycytidine triphosphate, dCTP) 또는 디옥시유리딘 트리포스페이트(Deoxyuridine triphosphate, dUTP)을 사용하여 증폭산물의 염기서열내 무작위적으로(randomly) 형광색소가 표지되는 반응원리를 갖는 키트.10. The method of claim 9, wherein a randomized sequence of amplification products using deoxycytidine triphosphate (dCTP) or deoxyuridine triphosphate (dUTP) labeled with fluorescent dyes during PCR amplification. A kit having the principle of reaction in which fluorescent dyes are labeled randomly. 제 10항 내지 11항에 있어서, 형광색소는 비오틴(Biotin), 로다민(Rhodamine), Cy3, Cy5, Cy5.5, 6-FAM(6-carboxyfluorescein), JOE(6-carboxy-4', 5'-dichloro-2', 7'-dimethoxyfluorescein), Rhodamine Green, TAMRA NHS(N-hydroxysuccinimide) Ester, Texas Red 그룹으로부터 선택되는 형광색소를 사용하는 것을 특징으로 하는 키트. The method of claim 10 to 11, wherein the fluorescent pigments (Biotin), Rhodamine (Rhodamine), Cy3, Cy5, Cy5.5, 6-FAM (6-carboxyfluorescein), JOE (6-carboxy-4 ', 5 A kit comprising fluorescent dyes selected from the group '-dichloro-2', 7'-dimethoxyfluorescein), Rhodamine Green, TAMRA NHS (N-hydroxysuccinimide) Ester, and Texas Red. 제 7항 내지 9항에 있어서, 호흡기감염질환 발생 초기에 고-처리량 분석을 통해 질환의 정확한 원인을 규명함으로써 경험에 의한(empirical) 항생제 처방을 지양함과 동시에 감염 병원체의 항생제 내성 유무까지 판별함으로써 병원체에 효과적인 항생제 선택을 통해 항생제 내성율을 감소시킬수 있는 것을 특징으로 하는 분석방법.10. The method according to claim 7 to 9, by identifying the exact cause of the disease through high-throughput analysis at the beginning of respiratory infections, avoiding empirical antibiotic prescriptions and determining the presence of antibiotic resistance of the infectious agent. An analysis method characterized in that the antibiotic resistance rate can be reduced by selecting an effective antibiotic for the pathogen.
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