KR101850854B1 - A method for simultaneously detecting and identifying Gram positive bacteria, Gram negative bacteria, Candida species and antibiotics-resistance, and a kit therefor based QuantaMatrix assay Platform - Google Patents

A method for simultaneously detecting and identifying Gram positive bacteria, Gram negative bacteria, Candida species and antibiotics-resistance, and a kit therefor based QuantaMatrix assay Platform Download PDF

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KR101850854B1
KR101850854B1 KR1020160134370A KR20160134370A KR101850854B1 KR 101850854 B1 KR101850854 B1 KR 101850854B1 KR 1020160134370 A KR1020160134370 A KR 1020160134370A KR 20160134370 A KR20160134370 A KR 20160134370A KR 101850854 B1 KR101850854 B1 KR 101850854B1
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이혜영
왕혜영
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연세대학교 원주산학협력단
주식회사 옵티팜
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Abstract

The present invention relates to a method capable of rapidly distinguishing gram-positive bacteria, gram-negative bacteria, and Candida species and identifying resistance of each gene corresponding to MRSA and VRE, ESBLs (TEM-, CTX-M-, and SHV-type), AmpC (ACT, CMY2, DHA, CMY-1 like/MOX, ACC-1, and FOX), and carbapenemases (OXA-48 like, IMP, VIM, NDM, KPC, and SPM) and a primer and a probe used therefor. The method comprises the steps of: a) separating DNA from a specimen sample; b) performing PCR amplification from the DNA using the primer of a sequence number 1-56; and c) hybridizing PCR amplification a disk to which oligomer probe of a sequence number 57-117 coupled with a product obtained in the step b) and then measuring an image of the disk through a Quanta Matrix assay Platform software.

Description

퀀타매트릭스 어세이 플랫폼 기반 그람양성, 그람음성균 및 캔디다의 검출 및 동정과 항생제 내성여부를 동시 확인할 수 있는 진단법 및 그 키트{A method for simultaneously detecting and identifying Gram positive bacteria, Gram negative bacteria, Candida species and antibiotics-resistance, and a kit therefor based QuantaMatrix assay Platform}FIELD OF THE INVENTION [0001] The present invention relates to a kit for detecting and identifying Gram-positive, Gram-negative bacteria and candida, based on Quanta matrix assay platforms, and a kit for identifying the Gram positive bacteria, Candida species and antibiotics -resistance, and a kit therefor based on QuantaMatrix assay Platform}

본 발명은 퀀타매트릭스 어세이 플랫폼 기반 그람양성, 그람음성균, 캔디다의 검출 및 동정과 항생제 내성여부를 동시 확인할 수 있는 진단법 및 그 키트에 관한 것이다.The present invention relates to a diagnostic method and a kit for simultaneously detecting the detection and identification of Gram-positive, Gram-negative, Candida, and antibiotic resistance based on a Quanta matrix assay platform.

패혈증은 발견되는 시기에 따라서 그 사망률이 23%에서 46%까지 보고되는 심각한 질환이며 많은 경제적 부담을 증가시키는 질환으로 보건정책의 난제로 알려져 있다. 패혈증은 비 심혈관계 중환자실 사망의 가장 흔한 원인으로 미국에서는 최소한 연평균 75만 명의 새로운 환자가 발생하며, 그 중 50%가 패혈성 쇼크로 진행하고 그 절반 정도인 20만명이 사망한다.Sepsis is a serious disease with a reported mortality rate ranging from 23% to 46% depending on the time of its discovery, and it is known as a health policy problem because it increases the economic burden. Sepsis is the most common cause of death among noncardiatic ICUs. In the United States, at least an average of 750,000 new patients develop annually, of which 50% progress to septic shock and about half of the 20,000 deaths occur.

최근 수십년 동안 패혈성 쇼크의 발생빈도는 꾸준히 증가해온 반면 그에 대한 사망률은 거의 변함이 없거나 약간 감소한 상태이다.In recent decades, the incidence of septic shock has steadily increased, while the mortality rate for it has remained almost unchanged or slightly decreased.

패혈증의 임상증상은 병원성 미생물의 감염이 숙주의 면역계통, 응고계통, 신경호르몬계통 등 신체의 다양한 기능계통에 영향을 주어 이와 관련된 전신반응으로 나타나기 때문에 복잡하고 다양한 임상 양상을 보인다. 따라서 숙주의 반응 정도와 감염 원인균의 특성 모두 패혈증의 예후에 중대한 영향을 미친다.Clinical manifestations of sepsis are complicated and diverse clinical manifestations because the infection of pathogenic microorganisms affects the various functional systems of the body such as immune system, coagulation system and neurohormone system of the host, resulting in systemic reaction related to the system. Therefore, both the degree of host reaction and the characteristics of infectious agents have a significant impact on the prognosis of sepsis.

1980년대 중반 이후부터는 그람 양성균이 전통적인 그람 음성균에 비하여 패혈증의 원인균으로 더 많은 부분을 차지하였고 1990년대 이후 진균에 의한 패혈증의 발생 빈도 역시 증가하고 있다. 이러한 패혈증의 원인이 되는 균주의 변화는 동반질환이 있는 고령환자의 증가와 함께 과거에 비하여 향상되고 적극적인 내과적, 외과적 치료와 더불어 사람면역결핍바이러스(human immunodeficiency virus)에 의한 질환 증가와 기존 항생제에 대해 내성을 보이는 내성균주의 발현에 기인한 것으로 생각된다.Since the mid - 1980s, Gram - positive bacteria have become more common cause of sepsis than traditional Gram - negative bacteria, and the incidence of sepsis due to fungi has also increased since the 1990s. The major cause of this sepsis was the increase in the number of elderly patients with concomitant illnesses and the increase in the number of diseases caused by the human immunodeficiency virus, along with the active and medical and surgical treatment, Of the resistant bacteria.

비병원성으로 알려져 왔던 많은 효모양 진균들이 최근에 와서 면역 저하 환자들에게서 중요한 기회 감염균으로 부각되고 있다. 이런 기회 감염 진균증은 악성종양, 후천성 면역 결핍증, 주요 수술, 심한 화상, 골수나 장기의 이식, 혈관내 도관 삽입, 장기간의 항생제 투여 및 화학요법을 받는 환자 등 많은 내과 및 외과 입원 환자들의 흔한 합병증으로 발생된다. 흔한 원인균으로 알려져 왔던 Candidal albicans외에 Candida spp. 중에서 최근 들어 Candida tropicalis와 Candida parapsilosis가 증가하는 추세에 있으며, 골수이식 환자 등에서 fluconazole을 예방적으로 투여한 결과 이에 내성을 지닌 Candida krusei 및 Candida glabrata에 의한 기회감염 진균증의 보고가 증가되고 있다.Many hyphae fungi, which have been known to be non - pathogenic, have recently emerged as an important opportunistic infectious disease in immunocompromised patients. These opportunistic infections are common complications of many medical and surgical inpatients, including malignant tumors, acquired immunodeficiency syndromes, major surgery, severe burns, bone marrow or organ transplantation, intravascular catheterization, long-term antibiotic administration and chemotherapy . Besides Candidal albicans it has been known as a common cause Candida spp. Recently, Candida tropicalis and Candida parapsilosis, and for a tendency to increase, with a tolerance The results of administration of fluconazole prophylactically, etc. Bone marrow transplant patients Candida krusei and Candida Reports of opportunistic infections caused by glabrata are increasing.

1940년에 페니실린이 임상의학에 처음 도입된 이후 지난 70년간 많은 항생제가 개발되고 임상에서 사용되어 감염질환으로부터 수많은 환자의 생명을 구하는데 결정적인 기여를 하였다 그러나 항생제의 사용과 더불어 급속하게 발현하기 시작한 세균의 항생제 내성은 항생제 개발보다 훨씬 빠른 속도로 진화하여 불과 70년만에 대부분의 항생제가 치료효과가 감소하는 현상이 범세계적으로 나타나고 있다. 세균감염질환의 빈도 및 임상적 중요성을 감안할 때 항생제 내성은 세계적인 보건상의 위기라고 할 수 있다. Since the first introduction of penicillin in clinical medicine in 1940, many antibiotics have been developed and used clinically in the past 70 years, making a decisive contribution to saving the lives of numerous patients from infectious diseases. However, the use of antibiotics, Antibiotic resistance has evolved much faster than the development of antibiotics, and the effect of most antibiotics has decreased in only 70 years. Given the frequency and clinical significance of bacterial infections, antibiotic resistance is a global health crisis.

항생제 내성이 문제가 되는 주요 세균은 Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , Enterobacter spp. 라고 할 수 있는데, 이중 그람 양성균으로 대표적인 내성의 예는 포도알균(Staphylococcus aureus)의 methicillin(지역사회 MRSA 포함) 및 Vancomycin 내성, 장알균(Enterococcus faecium)의 vancomycin 내성 그리고 지역사회의 주요세균인 폐렴알균(Streptococcus pneumoniae)의 macrolide 및 다제 내성을 들 수 있다.The major bacteria in which antibiotic resistance is a problem are Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , Pseudomonas aeruginosa , Enterobacter spp. The most common examples of Gram-positive bacteria are Staphylococcus aureus methicillin (including community MRSA) and vancomycin resistance, vancomycin resistance of Enterococcus faecium, and pneumococcus bacteria, Macrolide and multidrug resistance of Streptococcus pneumoniae.

MRSA는 전세계 각 병원에서 가장 중요한 병원감염균으로 자리잡고 있다. 특히 한국, 일본, 대만, 홍콩, 싱가포르, 스리랑카 및 일부 미국 병원에서는 병원에서 분리되는 포도알균의 50% 이상이 MRSA인 것으로 보고되고 있다.MRSA is one of the most important hospital infections in hospitals worldwide. In Korea, Japan, Taiwan, Hong Kong, Singapore, Sri Lanka and some US hospitals, more than 50% of the staphylococci isolated from hospitals are reported to be MRSA.

국내병원의 MRSA 발생율은 1996년에 국내 15개 병원에서 전향적으로 시행한 연구의 결과 83.7%인 것으로 나타났으며, 1997년부터 2006년까지 다기관 연구를 통하여 보고된 MRSA의 빈도는 64-72%로서 국내 병원에서 가장 흔한 병원 감염의 원인균으로 확인되었다.The incidence of MRSA in domestic hospitals was 83.7% as a result of a prospective study in 15 hospitals in 1996. The frequency of MRSA reported from multicenter studies from 1997 to 2006 was 64-72% Which is the most common cause of hospital infection in domestic hospitals.

장알균(Enterococcus)은 심내막염의 주요 감염균으로 알려져 있었으나 1970년대 중반부다 3세대 세팔로스포린계항생제의 사용이 증가하면서 병원내 감염의 중요한 원인균으로 인식되기 시작하였다. 장알균은 대부분의 항생제에 고유내성을 보유한 데다 plasmid 및 transposon의 전달에 의해 쉽게 항생제 내성을 획득할 수 있다. Vancomycin 내성 장내알균(VRE)은 1988년에 영국과 프랑스에서 처음으로 보고되었다.Enterococcus was known to be the major infectious organism of endocarditis, but by the mid-1970s, the use of third-generation cephalosporin antibiotics began to be recognized as an important pathogen in hospital infections. Ganoderma lucidum possesses intrinsic resistance to most antibiotics, and can easily acquire antibiotic resistance by the transfer of plasmids and transposons. Vancomycin resistant intestinal bacteria (VRE) were first reported in England and France in 1988.

이후 VRE는 미국에서 급속히 증가하는 경향을 보였으며 이는 transposon에 의한 VanA gene의 전달이 주요기전임이 밝혀졌다.Thereafter, VRE tended to increase rapidly in the United States, indicating that transposon-mediated delivery of the VanA gene was predominant.

국내에서는 1992년에 처음으로 VRE 감염이 보고된 바 있으며 국내에서 분리되는 E.faecium 중 VRE 비율은 1997년 4%였으나 지속적으로 증가하여 2009년에는 29%에 증가하였고 전국적인 다기간 연구의 결과에서는 2009-2010년에 병원 감염을 일으킨 E. faecium 중 VRE의 비율이 38.9%이었다.In Korea, VRE infection was reported for the first time in 1992, and the proportion of VFA among E. faecium isolated from Korea increased from 4% in 1997 to 29% in 2009, The proportion of VRE among E. faecium causing hospital infections in 2009-2010 was 38.9%.

병원균의 항생제 내성은 WHO에서 세계 공공 보건의 심각한 위협으로 규정할 정도로 선진국이나 후진국 모두의 공통된 문제이기는 하나, 특히 한국을 포함한 아시아 국가들이 주요 세균의 내성률이 서구 국가들에 비하여 전반적으로 높은 양상을 보이고 있다. 최근 폐렴간균과 대장균에서 가장 문제가 되고 있는 항생제 내성은 광범위 cephalosporin 계열 항생제에 내성을 나타내는 extended-spectrum β-lactamase (ESBL)의 생성이다.Antimicrobial resistance of pathogens is a common problem in both advanced and underdeveloped countries, as it is defined as a serious threat to public health by WHO. However, Asian countries including Korea have a higher overall resistance rate of major bacteria than Western countries have. Recently, antibiotic resistance, which is the most problematic in pneumococcal bacteria and Escherichia coli, is the generation of extended-spectrum β-lactamase (ESBL), which is resistant to broad-spectrum cephalosporin antibiotics.

ESBL은 cefotaxime, ceftazidime 및 aztreonam 등의 extended-spectrum β-lactam 항균제를 불활화하는 효소로서, 최근 이 효소를 생성하는 Enterobacteriaceae 내의 균종의 분리 빈도가 증가하고 있어 문제가 되고 있다. 이러한 균주는 검출에 있어서도 어려움이 있고, 주로 Klebsiella spp. 와 E. coli에서 흔하며 이 두 균종 외에도 Citrobacter spp., Enterobacter spp., Serratia spp., Morganella spp., Pseudomonas spp., Salmonella spp. 등의 그람음성 간균에서도 ESBL을 생성하는 세균이 있음이 보고되고 있으며, 분리빈도는 나라 및 지역에 따라 달라서 미국에서 분리되는 K. pneumoniae 와 E. coli 의 1.3-8.6%가 ESBL 을 생성하며 국내에서는 E. coli 및 K. pneumoniae 의 7.5-15%, 22.8-38%가 ESBL 생성 균주임이 보고된 바 있다. ESBL is an enzyme that inactivates the extended-spectrum β-lactam antibiotics such as cefotaxime, ceftazidime, and aztreonam. Recently, the frequency of isolating Enterobacteriaceae in this enzyme has been increasing. These strains are also difficult to detect, and mainly Klebsiella spp. , Enterobacter spp., Serratia spp., Morganella spp., Pseudomonas spp., Salmonella spp., And E. coli in addition to these two species. , And ESBLs are produced in the Gram-negative bacilli. The isolation frequency varies depending on the country and region. K. pneumoniae isolated from the USA and 1.3-8.6% of E. coli produce ESBL It has been reported that 7.5-15% and 22.8-38% of E. coli and K. pneumoniae are ESBL producing strains.

국내에서 시행된 ESBL 생성폐렴 간균에 의한 균혈증에 대한 연구에서 ESBL 생성균 감염의 경우 23.3%의 사망률을 보였고, non-ESBL 균주 감염의 경우 20.0%의 사망률을 보이는 것으로 보고 되고 있다.In Korea, ESBL-producing pneumococcal bacteremia has been reported to have a mortality rate of 23.3% in the case of ESBL-producing bacteria infection and 20.0% in the case of non-ESBL strain infection.

ESBL 생성균에 의한 경증 및 중등증 감염의 경우 감수성 결과에 따라 β-lactam/β-lactamase inhibitor, fluoroquinolone을 사용하기도 하지만, cephalosporin 및 aztreonam에 감수성을 보이는 경우라고 하더라도 임상적으로는 이들 항균제에 내성인 것으로 알려져 있어, cephalosporin 치료에 실패할 가능성이 높으며, aminoglycoside, cotrimoxazole 등의 다른 항균제에도 내성이기 때문에 그 감염증의 치료를 위한 항균제 선택이 쉽지 않으나, carbapenem이 이 효소에 가장 안정하기 때문에 선택약제로 권고되고 있다. 하지만 carbapenem에 내성이 발생하는 경우 안전하고 효과적으로 치료할 수 있는 항생제가 실제적으로 없는 상황이며 carbapenem-resistant Enterobacteriaceae (CRE)가 출현하여 증가하는 추세로 이는 전 세계적인 문제가 되고 있다. 이런 이유로 ESBL 생성 균주에 대한 검출 및 사용되는 β-lactamase, carbapenem등의 약제에 대한 내성여부를 빠르게 파악하는 것은 무분별한 약제의 사용과 항생제 내성의 위험을 최소화시키는데 도움이 되리라 보여진다. In the case of mild to moderate infections caused by ESBL-producing bacteria, β-lactam / β-lactamase inhibitor and fluoroquinolone may be used depending on susceptibility results. However, even if they are susceptible to cephalosporin and aztreonam, they are clinically resistant to these antimicrobial agents It is well known that cephalosporin treatment is unsuccessful and is resistant to other antimicrobial agents such as aminoglycoside and cotrimoxazole. Therefore, it is not easy to select antimicrobial agents for the treatment of infectious diseases, but carbapenem is recommended as a selective agent because it is most stable in this enzyme . However, when carbapenem resistance occurs, there is virtually no antibiotic that can be safely and effectively treated, and carbapenem-resistant Enterobacteriaceae (CRE) have emerged and this is becoming a global problem. For this reason, rapid detection of ESBL-producing strains and resistance to β-lactamase and carbapenem drugs will help to minimize the risks of antibiotic resistance and the use of indiscriminate drugs.

특히 균혈증 환자는 매우 위중한 상태이므로 신속한 결과가 환자의 생명을 구하는데 큰 역할을 하게 된다. 패혈증은 여러 가지 감염증에 동반될 수 있고 다양한 균종에 의해 야기되기 때문에 그 원인균을 규명하기 위해 혈액배양이 주로 이용되며, 혈액배양 결과를 분석하여 그 변화 추이를 조사하고 분리되는 균종과 항균제 감수성 양상을 파악하는 일은 환자의 치료에 중요한 정보를 제공해 왔다. 혈액배양은 균혈증 검사의 아주 중요한 방법으로 질병의 진단, 치료의 지침 및 예후를 결정하는데 필수적인 검사법이다. In particular, bacteremia patients are in a very critical condition, so rapid results play a major role in saving the patient's life. Because sepsis can be accompanied by various infectious diseases and is caused by various kinds of bacteria, blood cultures are mainly used to identify causative organisms. Analysis of blood culture results, Identification has provided important information for the treatment of patients. Blood culture is a very important method of bacteremia testing and is an indispensable test to determine the diagnosis, treatment guidelines and prognosis of disease.

하지만 혈액배양은 5일 이상의 시간이 소요되며 배양양성 신호가 나오면 계대배양하여 그람염색과 균종동정 및 항생제 감수성 검사를 시행하게 되는데 10일 이상의 시간이 소요하게 되며, 그람 양성, 그람음성균, 또는 캔디다균 등이 검출될 경우, 특히 그람 양성의 경우 S. aureus methicillin 내성유무, Enterococcus spp. 균종에서는 Vancomycin 내성 유무 및, Enterobacteriaceae 내의 세균이 검출될 경우 ESBL 내성 유무를 확인하게 된다. However, blood culture takes more than 5 days, and if the positive signal of culture is obtained, it will take more than 10 days to perform Gram stain, identification of the species, and antibiotic susceptibility test. Gram positive, gram negative, or Candida , S. aureus methicillin resistance, Enterococcus spp. The presence of vancomycin resistance in bacteria and the presence of ESBL resistance in Enterobacteriaceae will be confirmed.

최근에는 Real-time PCR, Microarray, MALDI-TOF MS 등의 분자진단법을 이용하여 그람 양성 및 음성균과 내성 관련된 유전자를 검출하는 방법이 소개되고 있다. 하지만 이들 검사법은 ESBL, AmpC, 또는 carbapenemase에 관련한 유전자만 각각 검출할 수 있으며 모든 관련된 유전자를 동시에 검출하지는 않는다. 국내의 병원에서도 ESBL에 대한 검사는 진행하지만, AmpC β-lactamases 나 카바페넴과 같은 항생제의 내성은 빈도도 낮고 검출하기 위한 테스트가 복잡하여 내성검사를 추가로 진행하지 않고 있어 점점 발생빈도가 높아지는 이들의 빠른 검출법이 요구되어지고 있다. Recently, a method of detecting genes associated with Gram-positive and negative bacteria using molecular diagnostic methods such as Real-time PCR, Microarray, and MALDI-TOF MS has been introduced. However, these tests can only detect genes associated with ESBL, AmpC, or carbapenemase, and do not detect all related genes at the same time. Although ESBL tests are available at hospitals in Korea, resistance to antibiotics such as AmpC β-lactamases and carbapenems is low and the tests for detecting them are complex, A rapid detection method is required.

본 발명은 상기의 필요성에 의하여 안출된 것으로서 본 발명의 목적은 간단하고 신속한 그람양성, 그람음성균의 검출 및 동정과 항생제 내성여부를 동시 확인할 수 있는 진단 조성물을 제공하는 것이다.The present invention has been made in view of the above needs, and it is an object of the present invention to provide a diagnostic composition capable of simultaneously and easily detecting Gram-positive, Gram-negative bacteria and identification and antibiotic resistance.

본 발명의 다른 목적은 간단하고 신속한 그람양성, 그람음성균의 검출 및 동정과 항생제 내성여부를 동시 확인할 수 있는 진단 방법물을 제공하는 것이다.It is another object of the present invention to provide a diagnostic method capable of simultaneously and quickly confirming the detection and identification of Gram-positive, Gram-negative bacteria, and the resistance to antibiotics.

상기의 목적을 달성하기 위하여 본 발명은 a)검체 시료로부터 DNA를 분리하는 단계;b)서열번호 1 내지 서열번호 56의 프라이머를 사용하여, 상기 DNA로부터 PCR 증폭하는 단계;및 c) 서열번호 57 내지 117의 올리고머 프로브가 커플링된 디스크와 상기 단계 b)에서 얻어진 PCR 증폭산물을 하이브리드형성시키는 후,퀀타매트릭스 어세이 플랫폼 소프트웨어를 통하여 상기 디스크의 이미지를 측정하는 단계를 포함하는 그람양성, 그람음성균, 캔디다의 검출 및 동정과 항생제 내성여부를 동시에 확인하는 방법을 제공한다.B) PCR amplification from the DNA using primers of SEQ ID NO: 1 to SEQ ID NO: 56; and c) PCR amplification of the DNA of SEQ ID NO: Comprising the step of hybridizing the oligonucleotide probe-coupled disk to the PCR amplification product obtained in step b), followed by measuring the image of the disk through a Quanta Matrix Assay platform software. , A method for simultaneously detecting the detection and identification of candida, and antibiotic resistance.

본 발명의 일 구현예에 있어서, 상기 검출 및 확인 균주는 엔테로코커스(Enterococcus), 스타필로코커스(Staphylococcus), 클렙시엘라(Klebsiella), 아시네토박터(Acinetobacter), 슈도모나스(Pseudomonas), 엔테로박터(Enterobacter), 포도상구균, 시겔라, 대장균, 또는 캔디다 종으로 구성된 군으로부터 선택된 하나 이상의 균주인 것이 바람직하나 이에 한정되지 아니한다.In one embodiment of the invention, the detection and identification strain is selected from the group consisting of Enterococcus, Staphylococcus , Keulrep when Ella (Klebsiella), Acinetobacter (Acinetobacter), Pseudomonas (Pseudomonas), Enterobacter (Enterobacter), Staphylococcus aureus, Shigella, E. coli, Candida species or with one preferably selected from the group consisting of one or more strains is not limited to this.

본 발명의 다른 구현예에 있어서, 상기 항생제는 메티실린(methcillin), 반코마이신(Vancomycin), 및 세팔로스포린(cephalosporin) 계열 항생제로 구성된 군으로부터 선택된 하나 이상의 항생제인 것이 바람직하나 이에 한정되지 아니한다.In another embodiment of the present invention, the antibiotic is preferably but not limited to one or more antibiotics selected from the group consisting of methicillin, vancomycin, and cephalosporin antibiotics.

또 본 발명은 서열번호 1 내지 서열번호 56의 프라이머 및 서열번호 57 내지 117의 올리고머 프로브가 커플링된 디스크를 포함하는 그람양성, 그람음성균, 캔디다의 검출 및 동정과 항생제 내성여부를 동시에 확인하기 위한 키트를 제공한다.The present invention also relates to a method for simultaneously detecting the detection and identification of Gram-positive, Gram-negative, and Candida including the primers of SEQ ID NOS: 1 to 56 and the oligomer probe of SEQ ID NOS: 57 to 117 Provide a kit.

상기 키트는 PCR 증폭 반응을 수행하기 위한 시약으로, DNA 폴리머라제, dNTPs 및 버퍼를 더욱 포함하는 것이 바람직하나 이에 한정되지 아니한다.The kit is preferably a reagent for carrying out a PCR amplification reaction, but it is preferably, but not limited to, a DNA polymerase, dNTPs and a buffer.

또한 본 발명은 서열번호 1 내지 서열번호 56의 프라이머 및 서열번호 57 내지 117의 올리고머 프로브를 포함하는 그람양성, 그람음성균, 캔디다의 검출 및 동정과 항생제 내성여부를 동시에 확인용 조성물을 제공한다.The present invention also provides a composition for simultaneously detecting the detection and identification of Gram-positive, Gram-negative, Candida and the antibiotic resistance including primers of SEQ ID NOS: 1 to 56 and oligomer probes of SEQ ID NOS: 57 to 117.

이하 본 발명을 설명한다.Hereinafter, the present invention will be described.

본 발명은 그람 양성-음성균 및 캔디다 균종을 구별하고 MRSA와 VRE 및 ESBLs (TEM-, CTX-M-, SHV-type), AmpC (ACT, CMY2, DHA, CMY-1 like/MOX, ACC-1, FOX), 및 carbapenemases (OXA-48 like, IMP, VIM, NDM, KPC, SPM) 에 해당되는 각각의 유전자의 내성여부를 확인할 수 있는 QuantaMatrix assay Platform (QMAP) 기반 분자진단검사법을 개발하였다. The present invention distinguishes between Gram-positive-negative bacteria and Candida species and isolates MRSA and VRE and ESBLs (TEM-, CTX-M-, SHV-type), AmpC (ACT, CMY2, DHA, CMY- (QMAP) -based molecular diagnostic test to confirm the resistance of each of the genes to the corresponding genes, FOX, and carbapenemases (OXA-48 like, IMP, VIM, NDM, KPC and SPM).

QMAP system은 퀀타매트릭스사의 원천기술 (특허 등록번호 1011013100000 (2011.12.26) / 1015823840000 (2015.12.28) 로 suspension array technology를 기반으로 하고 있으며 50μm 크기의 자성을 띠고 있는 디스크 (Microdisk)에 프로브를 결합시키고 PCR산물과 반응시킨 후, 돌연변이를 여부를 형광으로 확인할 수 있는 검사법으로 QMAP의 가장 큰 특징은 디스크에 고유의 코드를 새겨 이 코드로 간섭현상 없이 디스크를 구별하며 기술적으로 1024개의 코드가 가능하므로 각각 고유의 코드가 새겨진 1024종의 디스크를 이용한 다중검사가 가능하며 모든 과정이 96 well plate에서 진행되기 때문에 high throughput이 가능한 시스템이다(도 1).The QMAP system is based on a suspension array technology based on Quanta Matrix's proprietary technology (Patent Registration No. 1011013100000 (Dec. 26, 2011) / 1015823840000 (Dec. 28, 2015), which combines a probe with a magnetic disk of 50 μm in size PCR product, and mutation can be confirmed by fluorescence. The most important feature of QMAP is to inscribe a unique code on the disc, distinguish the disc without interference, and technically it can code 1024 It is possible to perform multiple tests using 1024 kinds of discs each having a unique code, and the whole process is performed in a 96-well plate, thereby enabling high throughput (FIG. 1).

QMAP 기반 Sepsis-ID는 종특이 다형성이 존재하는 16S rRNA 유전자 부위를 biotin group이 부착된 프라이머로 증폭하고 얻어진 PCR산물을 종특이 프로브가 부착되어있는 microdisk와 반응시켜 그람양성, 그람음성, 캔티다균등의2속, 12종, 19균 및 항생제 내성 16 유전자 검출가능하도록 고안된 되었다 분자진단 검사법으로 자동화가 가능하여 96테스트를 진행시 DNA 추출 (30분), PCR 진행 (1시간), TB/NTM 검출 및 동정과 리팜핀 내성여부 (1시간 30분) 총 3시간이면 확인이 가능한 부분이 큰 장점이다 (표 1). The QMAP-based Sepsis-ID was generated by amplifying a 16S rRNA gene region with species specific polymorphism with a biotin group-attached primer and reacting the resulting PCR product with a microdisk with a species-specific probe to generate gram-positive, gram- DNA extraction (30 min), PCR (1 hr), TB / NTM detection in the 96 test, which can be automated by the molecular diagnostic test method, was designed to detect 16 genes of 2 genus, 12 species, 19 bacteria and antibiotics. And identification with rifampin resistance (1 hour and 30 minutes).

본 발명에서는 국내에서 개발된 QMAP기반 dual-ID를 이용하여 고체 및 액체배양액을 가지고 유용성을 평가해 보았다.In the present invention, using QMAP-based dual-ID developed in Korea, the usefulness of solid and liquid culture media was evaluated.

QMAPQMAP Sepsis-ID Sepsis-ID Gram-negative bacteriaGram-negative bacteria Gram-positive bacteriaGram-positive bacteria CandidaCandida sppspp .. Gram-negative Gram-negative Gram-positiveGram-positive CandidaCandida albicansalbicans EscherichiaEscherichia colicoli // ShigellaShigella sppspp . . EnterococcusEnterococcus sppspp .. C. C. tropicalistropicalis ShigellaShigella sppspp . . E. faecalisE. faecalis C. C. glabrataglabrata Salmonella Salmonella sppspp . . // EnterobacterEnterobacter sppspp .. Streptococcus Streptococcus sppspp .. C. C. parapsilosisparapsilosis Salmonella Salmonella sppspp . . S S pneumoniaepneumoniae C. C. kruseikrusei KlebsiellaKlebsiella pneumoniaepneumoniae S.S. agalactiaeagalactiae K. K. oxytocaoxytoca S. S. pyogenespyogenes CitrobacterCitrobacter sppspp .. StaphylococcusStaphylococcus sppspp . . ProteusProteus mirabilismirabilis S. S. aureusaureus SerratiaSerratia sppspp . . MicrococcusMicrococcus sppspp ././ CorynebacteriumCorynebacterium spp./spp./ PropionibacteriumPropionibacterium sppspp .. MorganellaMorganella sppspp .. Acinetobacter sppAcinetobacter spp ..
PsedomonasPsedomonas sppspp .. AcinetobacterAcinetobacter baumanniibaumannii PsedomonasPsedomonas aeruginosaaeruginosa Resistance geneResistance gene Resistance geneResistance gene CTXCTX -M groups (M1 and M9 types)-M groups (M1 and M9 types) mecAmecA AmpCAmpC β- β- lactamaselactamase ( ( DHADHA , , CMY2CMY2 , ACT, , ACT, MOXMOX , ACC-1, FOX), ACC-1, FOX) vanAvanA CarbapenemaseCarbapenemase ( ( NDMNDM , , KPCKPC , , OXA48OXA48 -like, IMP, VIM, -like, IMP, VIM, SPMSPM )) vanBvanB

본 발명을 통하여 알 수 있는 바와 같이 본 발명은 신속하게 그람 양성-음성균 및 캔디다 균종을 구별하고 MRSA와 VRE 및 ESBLs (TEM-, CTX-M-, SHV-type), AmpC (ACT, CMY2, DHA, CMY-1 like/MOX, ACC-1, FOX), 및 carbapenemases (OXA-48 like, IMP, VIM, NDM, KPC, SPM)에 해당되는 각각의 유전자의 내성여부를 확인할 수 있다. As can be seen from the present invention, the present invention rapidly differentiates Gram-positive-negative bacteria and Candida species from MRSA, VRE and ESBLs (TEM-, CTX-M-, SHV-type), AmpC (ACT, CMY2, DHA , CMY-1 like / MOX, ACC-1 and FOX) and carbapenemases (OXA-48 like IMP, VIM, NDM, KPC and SPM).

도 1은 QMAP 시스템에 대한 그림.Figure 1 is a diagram of a QMAP system.

이하 비한정적인 실시예를 통하여 본 발명을 더욱 상세하게 설명한다. 단 하기 실시예는 본 발명을 예시하기 위한 의도로 기재한 것으로서 본 발명의 범위는 하기 실시예에 의하여 제한되는 것으로 해석되지 아니한다.The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the present invention and the scope of the present invention is not to be construed as being limited by the following examples.

본 발명은 원주 기독병원 진단검사의학과에 의뢰된 혈액배양 검체를 대상으로 혈액배양에서 양성으로 나온 균종들과 항생제 감수성 결과가 있는 검체를 대상으로 실시하였다.The present invention was carried out on blood culture specimens submitted to Wonju Christian Hospital Department of Diagnostic Imaging, and specimens with positive results in blood culture and samples with antibiotic susceptibility results.

혈액배양은 균혈증이 의심되는 환자에서 BACTEC Standard 10 Aerobic/F병과 BACTEC PLUS Anaerobic/ F병에 한 쌍으로 이루어진 혈액배양 병에 각각 5~10ml의 혈액을 접종하였다. 모든 혈액배양 병은 24시간 내내 접수되었으며 3시간이내 BACTEC 9240 시스템(BD, Franklin Lakes, NJ, USA)과 BacT/Alert 3D system (BioMerieux, Durham, NC, *SA)의 두가지 혈액배양 자동화 장비를 이용하여 37℃에서 5일 동안 배양되었다. 배양양성 신호가 나오면 계대배양하여 그람염색과 균종 동정 및 항생제 감수성 검사를 시행하였다. 세균의 동정은 Vitek II system을 (BioMerieux, Marcy, 1'Eltoile, France) 이용한 생화학적인 방법을 이용하였다. 칸디다 균종의 동정은 발아관 시험을 시행한 후 양성이면 Candida albicans로 동정하였고 발아관 음성인 균은 ATB ID 32C (bioMerieux SA, Marcy-1'Etoile, France)를 이용하여 동정하였다. Blood cultures were inoculated with 5 to 10 ml of blood each in a pair of BACTEC Standard 10 Aerobic / F and BACTEC PLUS Anaerobic / F bottles in patients suspected of bacteremia. All blood culture bottles were received 24 hours a day, and two blood culture automation equipment, BACTEC 9240 system (BD, Franklin Lakes, NJ, USA) and BacT / Alert 3D system (BioMerieux, Durham, NC, * SA) Lt; RTI ID = 0.0 > 37 C < / RTI > Gram stain, identification of isolates, and antimicrobial susceptibility test were carried out by subculture when the culture positive signal was detected. Biochemical methods using the Vitek II system (BioMerieux, Marcy, 1'Eltoile, France) were used to identify bacteria. Candida albicans was identified as Candida albicans after positive germination test and identified by ATB ID 32C (bioMerieux SA, Marcy-1'Etoile, France).

실시예Example 1:표준균주와1: standard strain and 임상분리균주에서의Of clinical isolates genomicgenomic DNA 추출 DNA extraction

BACTEC 9240 system (Becton Dickinson Microbiology System, Sparks, Md, USA)를 통해 배양된, 배양액의 일부를 취하여 DNA extraction solution 100 ㎕를 넣은 후 1분간 vortex하고, 100℃에서 10분간 가열한 후, 실온에서 13,000 rpm으로 3분간 원심분리하여 얻은 상층액을 취하는 방법을 이용하여 핵산을 분리하였다. 분리된 핵산은 REBA sepsis-ID를 수행하기 위한 PCR의 주형으로 사용되었다.A portion of the culture was cultured on a Bactec 9240 system (Becton Dickinson Microbiology System, Sparks, Md., USA). 100 μl of the DNA extraction solution was added, vortexed for 1 minute, heated at 100 ° C. for 10 minutes, and the supernatant obtained by centrifugation for 3 minutes at rpm was used to isolate the nucleic acid. The isolated nucleic acid was used as a template for PCR to perform REBA sepsis-ID.

실시예Example 2:REBA2: REBA Sepsis-ID의 유전자 수집 및 분석 Gene collection and analysis of Sepsis-ID

미국 국립생물정보센터 ( National center for biotechnology information, NCBI, http://www.ncbi.nlm.nih.gov) Genbank에서 표적으로 하는 PCR 프라이머와 올리고뉴클레오타이드 프로브는 Sepsis를 정확한 균 동정을 위해 Gram positive bacteria와 Gram negative bacteria를 위한 16s rRNA 유전자를, 진균(Fungus)를 위한 18S rRNA와 5.8S rRNA의 사이에 존재하는 internal transcribed sequence (ITS) 유전자를, MRSA의 항생제 내성여부를 알수 있는 MecA 유전자를, VRE의 항생제 내성여부를 알 수 있는 VanA와 VanB 유전자, ESBL항생제 내성여부를 알 수 있는 TEM, CTX-M1, CTX-M9, SHV, 유전자를, AmpC 내성 여부를 알 수 있는 ACT, CMY2, DHA, CMY-1 like/MOX, ACC-1, FOX 유전자를, 카바페넴 항생제 내성여부를 알 수 있는 OXA-48 like, IMP, VIM, NDM, KPC, SPM 유전자를 검색하여 염기서열을 수집하였다. 멀티얼라인 (http://multalin.toulouse.inra.fr/multalin)을 수행한 후 표적으로 하는 각각의 유전자들이 공통으로 가지는 염기서열 부위에서 2쌍의 primer를 디자인하였고, 그 중 reverse primer에는 PCR-REBA법을 위해 biotin을 5‘말단에 붙였다. 그리고 2쌍의 primer 내부에 존재하는 균특이 부위에서 각각의 표적 균종을 검출할 수 있는 올리고뉴클레오타이드 프로브를 디자인 하였고 각 올리고뉴클레오타이드 프로브에는 얇은 막과 펩타이드 결합을 할 수 있도록 하기 위해 5’말단에 amine기를 붙였다. 디자인한 primer와 올리고뉴클레오타이드 프로브는 바이오니아(대전, 한국)에 합성을 의뢰하였다.US National Biological Information Center (National center for biotechnology information, NCBI , http://www.ncbi.nlm.nih.gov) oligonucleotides and PCR primers that target in Genbank nucleotide probe Gram positive bacteria for accurate identification of bacteria Sepsis And the 16s rRNA gene for Gram negative bacteria, the internal transcribed sequence (ITS) gene existing between 18S rRNA and 5.8S rRNA for fungus, the MecA gene for detecting the antibiotic resistance of MRSA, the VRE CMV, CMV, CMV, CMV, CMV, CMV, CMV, CMV, CMV, CMV, The sequences of OXA-48 like, IMP, VIM, NDM, KPC, and SPM genes, which can detect the -1 like / MOX, ACC-1 and FOX genes and carbapenem antibiotic resistance, were collected and sequenced. Two pairs of primers were designed in the nucleotide sequence common to each of the target genes after performing multi-lane ( http://multalin.toulouse.inra.fr/multalin ), and the reverse primer contained PCR Biotin was attached to the 5 'end for the -REBA method. An oligonucleotide probe was designed to detect each target species in two different primer pairs. Each oligonucleotide probe was designed with an amine group at the 5 'end to make a thin membrane and peptide bond. . Designed primers and oligonucleotide probes were submitted for synthesis to Bioneer (Daejeon, Korea).

실시예Example 3:PCR3: PCR

추출한 각 균주의 genomic DNA를 주형으로 상용화된 Prime Taq Premix (2X) (Genet Bio, 논산, 한국)를 이용하여 PCR을 수행하였다. Prime Taq Premix (2X)의 조성은 primer Taq polymerase 1unit/10㎕, 2X reaction buffer, 4mM MgCl2, enzyme stabilizer, sedment, loading dye, pH 9.0, 0.5mM of each dATP, dCTP, dGTP, dTTP이다. PCR을 위한 각 조성은 Prime Taq premix (2X) 10㎕, 한 쌍의 10 pmole primer를 각각 1㎕, ultrapure water 3㎕, 각 균주의 genomic DNA 5㎕로 총량을 20㎕로 하였다. PCR 반응은 초기 변성과정을 위해 95℃ 5분, 일차 증폭을 위해 95℃ 30초, 65℃ 30초 반응을 10번 반복, 2차 증폭을 위해 95℃ 30초, 60℃ 30초 반응을 40번 반복 후 완전한 신장반응을 위해 72℃ 10분간 반응시켰다. PCR 반응이 완료된 PCR 산물은 2% TBE (Tris-borate-ethylenediaminetetraacetic acid disodium salt dihydrate) agarose gene (W/V ratio)에서 290 bolt로 20분간 전기영동하여 ethidium bromide에 10분간 염색 후 자외선 투과조명기로 증폭여부를 확인하였다.PCR was performed using Prime Taq Premix (2X) (Genet Bio, Nonsan, Korea), which was used as a template for the genomic DNA of each strain. The composition of Prime Taq Premix (2X) is 1 unit / 10 μl of primer Taq polymerase, 2X reaction buffer, 4 mM MgCl2, enzyme stabilizer, sediment, loading dye, pH 9.0, 0.5 mM each dATP, dCTP, dGTP and dTTP. For each PCR, 10 μl of Prime Taq premix (2X), 1 μl each of a pair of 10 pmole primers, 3 μl of ultrapure water, and 5 μl of genomic DNA of each strain were used to make a total amount of 20 μl. The PCR reaction was repeated 10 times at 95 ° C for 5 minutes for the first denaturation, 30 seconds at 95 ° C for 30 seconds at 65 ° C for 30 seconds, 30 seconds at 95 ° C for the second amplification, 40 times After repeating, the reaction was carried out at 72 ° C for 10 minutes for a complete elongation reaction. After PCR, the PCR product was electrophoresed in 290 bolts at 2% TBE (Tris-borate-ethylenediaminetetraacetic acid disodium salt dihydrate) agarose gene (W / V ratio) for 20 minutes. After staining with ethidium bromide for 10 minutes, Respectively.

실시예Example 4:QMAP4: QMAP 기반 Sepsis-ID 수행 Performing a Sepsis-ID based

QMAP 기반 Sepsis-ID 를 수행하기 위해서 검체로부터 분리된 핵산을 biotin이 표지된 primer를 이용하여 PCR을 수행한 후 증폭한 PCR 산물에 동량의 Denaturation solution (0.2N NaOH, 0.2mM EDTA)을 섞어 실온에 5분간 방치한 후 hybridization buffer에 희석시켜 준비된 커플링 된 disk (Quantamatrix, Seoul, Korea)에 넣은 후 40℃ 에서 30분간 반응시키고, WS (washing solution)을 이용하여 25℃에서 1분간 3회 씻어준 후 1:2000 (v/v)으로 희석한 streptavidin R-phycoerythrin conjugate (Prozyme, San Leandro, CA)을 처리하여 실온에서 10분간 반응시켰다. 반응이 끝난 microdisk를 washing buffer로 실온에 1분간 3회 세척하고, 제공되는 QMAP software를 통해 자동으로 disk의 이미지를 측정하여 그람양성, 그람음성균, 또는 캔디다균 을 검출하고 MRSA, VanA, VanB, ESBLs 내성여부를 확인하였다. 이미지에 나타나는 모든 microdisk의 fluorescence intensity 의 cutoff값은 500이상은 양성으로 표기하였다.In order to perform QMAP-based Sepsis-ID, the nucleic acid isolated from the specimen was subjected to PCR using a biotin-labeled primer, and the amplified PCR product was mixed with an equal amount of denaturation solution (0.2N NaOH, 0.2 mM EDTA) After incubation for 5 minutes, the mixture was diluted in hybridization buffer and placed in a prepared coupled disk (Quantamatrix, Seoul, Korea), followed by reaction at 40 ° C for 30 minutes and washing with WS (washing solution) for 3 minutes at 25 ° C The cells were treated with streptavidin R-phycoerythrin conjugate (Prozyme, San Leandro, CA) diluted 1: 2000 (v / v) for 10 min at room temperature. After the reaction, the microdisks were washed with washing buffer for 3 minutes at room temperature, and the image of the disk was automatically measured using the QMAP software provided. The Gram-positive, Gram-negative, or Candida bacteria were detected and MRSA, VanA, VanB, ESBLs Resistant. The cutoff value of the fluorescence intensity of all the microdisks in the image was more than 500 positive.

상기 실시예의 결과는 하기와 같다.The results of the above embodiment are as follows.

QMAPQMAP system의  system probe유용성probe availability 평가 evaluation

QMAP Sepsis-ID에 사용된 probe의 유용성을 확인하기 위해 제작된 균특이적인 올리고 뉴클레오타이드 프로브의 반응여부를 확인하기 위하여 표준균주를 통해 테스트를 진행해봤으며, 그 결과 각 DNA 샘플들은 해당 타겟의 프로브에만 반응을 하였으며 다른 교차반응이 일어나지 않음을 확인할 수 있었다(표 2 내지 표 8)In order to confirm the usefulness of the probe used in the QMAP Sepsis-ID, the test was carried out through a standard strain to confirm whether or not the prepared specific oligonucleotide probe was reacted. As a result, each DNA sample was only reacted to the target probe And no other cross-reaction occurred (Table 2 to Table 8)

Figure 112016100335306-pat00001
Figure 112016100335306-pat00001

표 2는 그람양성균에 대한 프로브 테스트 결과Table 2 shows the results of probe test on Gram-positive bacteria

Figure 112016100335306-pat00002
Figure 112016100335306-pat00002

표 3은 그람음성균에 대한 프로브 테스트 결과Table 3 shows the results of the probe test on Gram-

Figure 112016100335306-pat00003
Figure 112016100335306-pat00003

표 4는 Candida균에 대한 프로브 테스트 결과Table 4 shows the results of probe test on Candida

Figure 112016100335306-pat00004
Figure 112016100335306-pat00004

표 5는 그람음성균의 내성관련 프로브 테스트 결과(CTX-M1)Table 5 shows the resistance-related probe test results (CTX-M1) of Gram-

Figure 112016100335306-pat00005
Figure 112016100335306-pat00005

표 6은 그람음성균의 내성관련 프로브 테스트 결과(CTX-M9)Table 6 shows the resistance-related probe test results (CTX-M9) of Gram-

Figure 112016100335306-pat00006
Figure 112016100335306-pat00006

표 7은 그람음성균의 내성관련 프로브 테스트 결과(TEM, SHV)Table 7 shows the resistance-related probe test results (TEM, SHV) of Gram-

Figure 112016100335306-pat00007
Figure 112016100335306-pat00007

표 8은 그람음성균의 내성관련 프로브 테스트 결과(AmpC β-lactamase, Carbapenemase)Table 8 shows the resistance-related probe test results of Gram-negative bacteria (AmpC β-lactamase, Carbapenemase)

고체배양균에서의In a solid culture QMAPQMAP system의 유용성 평가 Evaluation of system's usefulness

QMAP기반 Sepsis-ID의 유용성 평가를 확인하기 위해서 244 전통 배양기반[continuous-monitoring blood culture systems (CMBCS)]인 고체배양균을 이용하여 그람양성, 그람음성, 캔디다 균의 검출과 동정을 테스트하였다. 그 결과 50 그람양성균, 175 그람음성균과 19 곰팡이균이 분리되었으며 [Staphylococcus spp., Streptococcus spp., Enterococcus spp., Escherichia coli , Klebsiella pneumonia, Pseudomonas aeruginosa , Acinetobacter baumannii , Proteus mirabilis, Citrobacter spp., Enterobacter spp., Serratia marcescens, Morganella morganii, Staphylococcus aureus , Streptococcus pneumonia, Streptococcus agalactiae , Candida albicans , Candida tropicalis , Candida glabrata, Candida parapsilosis, Saccharomyces cerevisiae], 이 결과는 CMBCS의 결과와 모두 일치하였다 (표 9). In order to confirm the usefulness evaluation of QMAP-based Sepsis-ID, the detection and identification of Gram-positive, Gram-negative, Candida spp. Were tested using a solid culture of 244 traditional culture-based blood culture systems (CMBCS). As a result, 50 gram positive bacteria, 175 gram negative bacteria and 19 mold bacteria were isolated [ Staphylococcus spp., Streptococcus spp., Enterococcus spp., Escherichia coli , Klebsiella pneumonia, Pseudomonas aeruginosa , Acinetobacter baumannii , Proteus mirabilis , Citrobacter spp., Enterobacter spp., Serratia Marcescens , Morganella morganii , Staphylococcus aureus , Streptococcus pneumonia, Streptococcus agalactiae , Candida albicans , Candida tropicalis , Candida glabrata , Candida parapsilosis, Saccharomyces cerevisiae ], the results were all consistent with the results of CMBCS (Table 9).

Conventional methodsConventional methods QMAPQMAP Sepsis, n ( Sepsis, n ( %% )) Gram-positive bacteria (n=50)Gram-positive bacteria (n = 50) 50 (100)50 (100) Gram-negative bacteria (n=175)Gram-negative bacteria (n = 175) 175 (100)175 (100) Fungi (n=19)** Fungi (n = 19 ) ** 17 (89.5)17 (89.5) Total (n=244)Total (n = 244) 242 (99.2)242 (99.2)

표 3는 244 배양균주에서 균종분리를 위한QMAP Sepsis-ID 결과비교Table 3 compares the results of QMAP Sepsis-ID for 244 culture isolates.

**2균주는 Saccharomyces cerevisiae로 분리됨.** 2 strains were Saccharomyces cerevisiae .

또한 전통배양법에 의해 총 149배양균주에서 항생제 내성을 보였으며, QMAP system에 의한methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), and extended spectrum beta-lactamase (ESBL)와 같은 항생제 내성 유전자 검출의 경우 14 S. aureus 에서 9균주가 MRSA로, 9 coagulase-negative staphylococci (CoNS) 균주 중 8개가 mecA에 내성을 나타냈으며, 12 Enterococcus spp. 균주 중 7균주가 vancomycin에 내성을 보였다. 한편 114 Enterobacteriaceae 에서 113 균주가 ESBL생성균으로 나타났으나 K. pneumoniae 한 균종에서는 검출되지 않았다 (표 10). In addition, antibiotic resistance was detected in a total of 149 strains by the conventional culture method. Antibiotic resistance such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE) and extended spectrum beta-lactamase (ESBL) In the case of gene detection, 9 out of 14 S. aureus strains were MRSA and 8 out of 9 coagulase-negative staphylococci (CoNS) strains were resistant to mecA, 12 Enterococcus spp. Seven isolates showed resistance to vancomycin. In 114 Enterobacteriaceae , 113 isolates were ESBL-producing bacteria but not K. pneumoniae (Table 10).

Conventional methodsConventional methods QMAPQMAP Sepsis, n ( Sepsis, n ( %% )) MRSA (n=9)MRSA (n = 9) 9 (100)9 (100) MSSA (n=5)MSSA (n = 5) 5 (100)5 (100) MRCoNS (n=8)MRCoNS (n = 8) 8 (100)8 (100) MSCoNS (n=1)MSCoNS (n = 1) 1 (100)1 (100) VRE (n=7)VRE (n = 7) 7 (100)7 (100) VSE (n=5)VSE (n = 5) 5 (100)5 (100) ESBL producing (n=114)ESBL producing (n = 114) 113 (99.1)113 (99.1) Total (n=149)Total (n = 149) 148 (99.3)148 (99.3)

표 10은 149 항생제 내성 배양균주에서의 내성 유전자 검출을 위한 QMAP Sepsis-ID의 결과비교Table 10 shows the results of QMAP Sepsis-ID for the detection of resistant genes in 149 antibiotic-resistant strains

혈액배양균에서의In the blood culture QMAPQMAP system의 유용성 평가 Evaluation of system's usefulness

혈액배양균에서 QMAP system의 유용성을 평가하기 위하여 패혈증이 의심되는 619 환자에서 분리 배양한 혈액배양균을 이용하여 분석하였으며, 그 결과는 전통배양분리시스템을 통하여 비교하였다. 619 혈액배양균에서 419 (67.7%)는 혈액배양양성균으로 200(32.3%)는 혈액배양음성균으로 분리되었다. 419 혈액배양양성균에서는 266 (63.5%) 그람양성균, 116 (27.7%) 그람음성균, 30 (7.2%) 캔디다균으로 각각 분리되었으며 이 결과는 배양결과와 모두 일치하였다. 또한 266 그람양성균에서 Staphylococcus epidermidis 을 포함한 CoNS가 전체 58.7% (n=156)을 차지하였으며, S. aureus (n=42), Streptococcus spp. (n = 23), Enterococcus spp. (n=15), Micrococcus spp. (n = 11), 와 Bacillus spp. (n = 10)이 각각 차지하였다. 116 그람음성균은 E. coli가 전체 50% (n=58)이 차지하였으며, Klebsiella pneumoniae (19.8%, n = 23), Acinetobacter spp. (9.5%, n = 11), Pseudomonas aeruginosa (7.8%, n = 9), Enterobacter spp. (4.3%, n = 5) 순으로 나타났다. 30 캔디다균에서는 15 C. albicans, 8 Candida glabrata, 4 Candida parapsilosis, 1 Candida tropicalis이 각각 분리되었다 (표 11).To evaluate the utility of the QMAP system in blood cultures, 619 patients suspected of sepsis were analyzed by blood cultures isolated from different cultures. The results were compared using a conventional culture separation system. In 619 blood cultures, 419 (67.7%) were blood culture positive and 200 (32.3%) were blood culturing negative bacteria. 419 blood culture positive bacteria, 266 (63.5%) gram positive bacteria, 116 (27.7%) gram negative bacteria and 30 (7.2%) Candida bacteria, respectively. Among 266 Gram-positive bacteria, CoNS including Staphylococcus epidermidis accounted for 58.7% (n = 156), while S. aureus (n = 42), Streptococcus spp. (n = 23), Enterococcus spp. (n = 15), Micrococcus spp. (n = 11), and Bacillus spp. (n = 10), respectively. The total number of gram-negative bacteria was 50% (n = 58) in E. coli, and Klebsiella pneumoniae (19.8%, n = 23), Acinetobacter spp . (9.5%, n = 11), Pseudomonas aeruginosa (7.8%, n = 9), Enterobacter spp. (4.3%, n = 5), respectively. 30 In Candida, 15 C. albicans , 8 Candida glabrata , 4 Candida parapsilosis , and 1 Candida tropicalis were isolated (Table 11).

Conventional methodsConventional methods Total no. (%) of samplesTotal no. (%) of samples QMAP systemQMAP system Positive (%)Positive (%) Negative (%)Negative (%) Monomicrobial bacteremiaMonomicrobial bacteremia 419 (67.7)419 (67.7) 418 (99.8)418 (99.8) 1 (0.2)1 (0.2) Gram-positive bacteriaGram-positive bacteria 266 (63.5)266 (63.5) 265 (99.6)265 (99.6) 1 (0.4)1 (0.4) Gram-negative bacteriaGram-negative bacteria 116 (27.7)116 (27.7) 116 (100)116 (100) 0 (0)0 (0) Candida species Candida species 30 (7.2)30 (7.2) 30 (100)30 (100) 0 (0)0 (0) Polymicrobial bacteremiaPolymicrobial bacteremia 7 (1.7)7 (1.7) 7 (100)7 (100) 0 (0)0 (0) Blood culture negativeBlood culture negative 200 (32.3)200 (32.3) 0 (0)0 (0) 200 (100)200 (100) TotalTotal 619 (100)619 (100) 418 (67.5)418 (67.5) 201 (32.5)201 (32.5)

표 11은 혈액배양양성균에서 감염균종의 검출을 위한 QMAP system의 결과비교Table 11 shows the results of the QMAP system for detection of infectious organisms in blood culture positive bacteria

항생제 내성 분리검출을 위한 배양법과 A culture method for antibiotic resistance separation detection QMAPQMAP system 사이의 결과 비교 Compare results between systems

전통적인 배양결과에서 총 144 균주 [43 S. aureus 중 26 균주가, 118 CoNS(62 S. epidermidis cases, 27 Staphylococcus hominis, 17 Staphylococcus capitis, 6 Staphylococcus haemolyticus, 2 Staphylococcus saprophyticus, and 1 Staphylococcus cohnii, 1Staphylococcus caprae, 1Staphylococcus auricularis, 1Staphylococcus lugdunensis)]가 oxacillin에 내성을 보였으며, QMAP system 에 의해 검출된 항생제 내성 결과는 하나를 제외한 모든 균주에서 mecA유전자가 검출되었다. 또한 3 Enterococcus spp. (2 E. faecium and 1 E. gallinarum)는 vancomycin에 내성을 보였으며, 3개 모두가 QMAP system에서 vanA유전자가 검출되었다.A total of 144 strains [26 strains of 43 S. aureus , 118 CoNS (62 S. epidermidis cases, 27 Staphylococcus hominis , 17 Staphylococcus capitis , 6 Staphylococcus haemolyticus , 2 Staphylococcus saprophyticus , and 1 Staphylococcus cohnii , 1 Staphylococcus caprae , 1 staphylococcus auricularis , 1 Staphylococcus lugdunensis ] showed resistance to oxacillin, and the mecA gene was detected in all strains except one except for the antibiotic resistance results detected by the QMAP system. In addition, 3 Enterococcus spp. (2 E. faecium and 1 E. gallinarum ) were resistant to vancomycin, and all three vanA genes were detected in the QMAP system.

92 Enterobacteriaceae 혈액배양양성균 사이에서 12 균주가 (11 E. coli and 1 K . pneumoniae) ESBL생성균으로 확인되었으며, QMAP system에서 11 E. coli (8 CTX-M1, 2 CTX-M9, 1 CTX-M1/M9 mix)와 1 K . pneumoniae 는 XTX-M9에서 각각 내성을 나타냈다 (표 12).In the QMAP system, 11 E. coli (8 CTX-M1, 2 CTX-M9, 1 CTX-M1 / 2) were identified as 12 E. coli and 1 K. pneumoniae strains among 92 Enterobacteriaceae blood culture positive bacteria . M9 mix) and 1K . pneumoniae were respectively resistant to XTX-M9 (Table 12).

Blood cultureBlood culture Total no.Total no. QMAP system, No. (%) of isolatesQMAP system, No. (%) of isolates Genus and speciesGenus and species of samples of samples Consistent results (%)Consistent results (%) Not detectedNot detected DiscrepantDiscrepant Fluorescence intensityFluorescence intensity (%)(%) results (%)results (%) Range Range Mean (± SD) valueMean (± SD) value Drug resistanceDrug resistance 159 (100)159 (100) 158 (99.4)158 (99.4) 1 (0.6)1 (0.6) -- 697-6,088697-6,088 4,072 (± 1,202)4,072 (± 1,202) Oxacillin resistanceOxacillin resistance 144 (90.6)144 (90.6) 143 (99.3)143 (99.3) 1 (0.7)1 (0.7) -- 743-6,088743-6,088 4,288 (± 978)4,288 (+ -978) Vancomycin resistanceVancomycin resistance 3 (1.9)3 (1.9) 3 (100)3 (100) -- -- 2,848-3,4192,848-3,419 3,219 (± 321)3,219 (± 321) ESBL productionESBL production 12 (7.5)12 (7.5) 12 (100)12 (100) -- -- 697-4,737697-4,737 1,755 (± 1,233)1,755 (± 1,233)

표 12는 혈액배양양성균에서 항생제 내성 분리검출을 위한 QMAP system의 결과비교Table 12 shows the results of the QMAP system for antibiotic resistance detection in blood culture positive bacteria

GeneGene 　 SequenceSequence 서열번호SEQ ID NO: SizeSize ModificationModification 16s rRNA16s rRNA 16S-F16S-F T AAY ACA TGC AAG TCG ARC GT AAY ACA TGC AAG TCG ARC G 1One 　 　 　 280R*280R * TGT GGC YGR TCR YCC TCT CAGTGT GGC YGR TCR YCC TCT CAG 22 280bp280bp BiotinBiotin 　 120F120F AGYKGCGRACGGGTGAGTAAAGYKGCGRACGGGTGAGTAA 33 　 　 　 280R*280R * TGT GGC YGR TCR YCC TCT CAGTGT GGC YGR TCR YCC TCT CAG 44 210bp210bp 　BiotinBiotin 　 407F407F CTACGGGAGGCAGCAGTRGGGAAT CTACGGGAGGCAGCAGTRGGGAAT 55 　 　 　 608R*608R * TATTACCGCGGCTGCTGGCA TATTACCGCGGCTGCTGGCA 66 170bp170bp 　BiotinBiotin ITSITS MF3MF3 AACGCANMTTGCRCYCHHTG AACGCANMTTGCRCYCHHTG 77 　 　 　 CR3*CR3 * CAGCGGGTADYCCYACCTGACAGCGGGTADYCCYACCTGA 88 230bp230bp BiotinBiotin NucNuc nuc-251Fnuc-251F TAAAGCGATTGATGGTGATACGGTTAAAGCGATTGATGGTGATACGGT 99 　 　 　 nuc-394R*nuc-394R * TTTCGTAAATGCACTTGCTTCAGGTTTCGTAAATGCACTTGCTTCAGG 1010 143bp143bp BiotinBiotin 　 　 　 　 　 MecAMeca MecA-1456FMecA-1456F CTAGGTGTTGGTGAAGATATACCAAGTGCTAGGTGTTGGTGAAGATATACCAAGTG 1111 　 　 　 MecA-1600R*MecA-1600R * TTGAAAGGATCTGTACTGGGTTAATCAGTTGAAAGGATCTGTACTGGGTTAATCAG 1212 144bp144bp BiotinBiotin 　 　 　 　 　 vanAvanA vanA555-FvanA555-F AGTCAATAGCGCGGACGAATTGAGTCAATAGCGCGGACGAATTG 1313 　 　 　 vanA791-R*vanA791-R * GCGGGAACGGTTATAACTGCGTTTTCGCGGGAACGGTTATAACTGCGTTTTC 1414 150bp150bp BiotinBiotin 　 　 　 　 　 vanBvanB vanB-548FvanB-548F CTTACCTACCCTGTCTTTGTGAAGCCCTTACCTACCCTGTCTTTGTGAAGCC 1515 　 　 　 vanB-746R*vanB-746R * CGCCGACAATCAAATCATCCTCGTTCGCCGACAATCAAATCATCCTCGTT 1616 198bp198bp BiotinBiotin 　 　 　 　 　 invAinvA sal-1828Fsal-1828F TCTGGCAGTACCTTCCTCAGCCTCTGGCAGTACCTTCCTCAGCC 1717 　 　 　 sal-1961R*sal-1961R * AATCGACAGACGTAAGGAGGACAAGAATCGACAGACGTAAGGAGGACAAG 1818 133bp133bp BiotinBiotin 　 　 　 　 　 ipaHipaH Shi-1243FShi-1243F GCAGTTGCAGTCTCCTAGGTAAAGGGGCAGTTGCAGTCTCCTAGGTAAAGGG 1919 　 　 　 Shi-1337R*Shi-1337R * ACTTGAGGTTAGGAACAACGAACTGCAACTTGAGGTTAGGAACAACGAACTGCA 2020 100bp100bp BiotinBiotin 　 　 　 　 　 spn-cpsAspn-cpsA 297F297F GACCAATCGTTTAAATGCGACTTCTGACCAATCGTTTAAATGCGACTTCT 2121 130bp130bp 　 　 416R*416R * GTCCCAGTCGGTGCTGTCACACTGTCCCAGTCGGTGCTGTCACACT 2222 BiotinBiotin ESBLsESBLs TEMGlu104Lys-716FTEMGlu104Lys-716F CAACTCGGTCGCCGCATACACTATTCCAACTCGGTCGCCGCATACACTATTC 2323 269bp269 bp 　 　 TEMGlu104Lys-985R*TEMGlu104Lys-985R * GTCACGCTCGTCGTTTGGTATGGTCACGCTCGTCGTTTGGTATG 2424 　 BiotinBiotin 　 TEMGly238-668FTEMGly238-668F AACGGCAGCTGCTGCAGTGAACGGCAGCTGCTGCAGTG 2525 102bp102bp 　 　 TEMGly238-770R*TEMGly238-770R * ACGATACGGGAGGGCTTACCATACGATACGGGAGGGCTTACCAT 2626 　 BiotinBiotin 　 SHV238-827FSHV238-827F AGCTGCTGCAGTGGATGGTAGCTGCTGCAGTGGATGGT 2727 151bp151bp 　 　 SHVS238-978R*SHVS238-978R * CTCTGCTTTGTTATTCGGGCCAACTCTGCTTTGTTATTCGGGCCAA 2828 　 BiotinBiotin 　 CTXM1-209-FCTXM1-209-F CTGATGTGCAGCACCAGTAAAGTGATGCTGATGTGCAGCACCAGTAAAGTGATG 2929 124bp124bp 　 　 CTXM1-333-R*CTXM1-333-R * AATCGGATTATAGTTAACMARGTCAGATTTAATCGGATTATAGTTAACMARGTCAGATTT 3030 　 BiotinBiotin 　 CTXM9-209-FCTXM9-209-F GGTGTGCAGTACCAGTAAAGTTATGGCGGGTGTGCAGTACCAGTAAAGTTATGGCG 3131 120bp120bp 　 　 CTXM9-329-R*CTXM9-329-R * GGATTGTAGTTAACCAGATCGGCAGGCGGATTGTAGTTAACCAGATCGGCAGGC 3232 　 BiotinBiotin AmpCAmpC DHA-259-FDHA-259-F ACTTTCACAGGTGTGCTGGGTGCACTTTCACAGGTGTGCTGGGTGC 3333 140bp140bp 　 　 DHA-399-R*DHA-399-R * TGCGGTATAGGTAGCCAGATCCAGCAATGTGCGGTATAGGTAGCCAGATCCAGCAATG 3434 　 BiotinBiotin 　 CMY2-261-FCMY2-261-F AAGACGTTTAACGGCGTGTTGGAAGACGTTTAACGGCGTGTTGG 3535 140bp140bp 　 　 CMY2-401-R*CMY2-401-R * GCCGTATAGGTGGCTAAGTGCAGGCCGTATAGGTGGCTAAGTGCAG 3636 　 BiotinBiotin 　 ACT-MIR-416-FACT-MIR-416-F TACAGGTRCCGGATGAGGTCACGTACAGGTRCCGGATGAGGTCACG 3737 141bp141bp 　 　 ACT-MIR-557-R*ACT-MIR-557-R * GAAGGTTTGACCGCCAGCGCGAAGGTTTGACCGCCAGCGC 3838 　 BiotinBiotin 　 ACC-1-4067FACC-1-4067F TGCACGT AGCGTAACAAGTCGCTGGAGTGCACGT AGCGTAACAAGTCGCTGGAG 3939 132bp132bp 　 　 ACC-1-538R*ACC-1-538R * GCTGACCAGATGGAAAACTATGCGTGGCTGACCAGATGGAAAACTATGCGTG 4040 　 BiotinBiotin 　 CMY1/MOX-126FCMY1 / MOX-126F CGCTGCTCAAGGAGCACMGGATCCGCTGCTCAAGGAGCACMGGATC 4141 132bp132bp 　 　 CMY1/MOX-258R*CMY1 / MOX-258R * TATCTCGAACAGGGTCTGCTCGCTTATCTCGAACAGGGTCTGCTCGCT 4242 　 BiotinBiotin 　 Fox-180FFox-180F CTATTTCAACTATGGGGTTGCCAACCCTATTTCAACTATGGGGTTGCCAACC 4343 138bp138bp 　 　 Fox-318R*Fox-318R * TGCTGGCTCACCTTGTCATCCAGCTGCTGGCTCACCTTGTCATCCAGC 4444 　 BiotinBiotin CarbapenemasesCarbapenemases IMP-261FIMP-261F AATAAAAGGCAGTATYTCCTCWCATTTAATAAAAGGCAGTATYTCCTCWCATTT 4545 128bp128bp 　 　 IMP-389R*IMP-389R * ACCTTACCGTCTTTTTTAAGMAGTTCAACCTTACCGTCTTTTTTAAGMAGTTCA 4646 　 BiotinBiotin 　 VIM-134FVIM-134F GGCTTTACCAGATTGCCGATGGTGTTGGCTTTACCAGATTGCCGATGGTGTT 4747 132bp132bp 　 　 VIM-266R*VIM-266R * GCACCCCACGCTGTATCAATCAAAAGGCACCCCACGCTGTATCAATCAAAAG 4848 　 BiotinBiotin 　 KPC-267FKPC-267F GCTGGACACACCCATCCGTTACGGCTGGACACACCCATCCGTTACG 4949 131bp131bp 　 　 KPC-398R*KPC-398R * GCGGCGTTATCACTGTATTGCACGGGCGGCGTTATCACTGTATTGCACGG 5050 　 BiotinBiotin 　 NDM-327FNDM-327F AGATCCTCAACTGGATCAAGCAGGAGATCCTCAACTGGATCAAGCAGG 5151 134bp134bp 　 　 NDM-461R*NDM-461R * ACGCATTGGCATAAGTCGCAATACGCATTGGCATAAGTCGCAAT 5252 　 BiotinBiotin 　 OXA48-129FOXA48-129F AGTTGTGCTCTGGAATGAGAATAAGCAGAGTTGTGCTCTGGAATGAGAATAAGCAG 5353 123bp123bp 　 　 OXA48-252R*OXA48-252R * GCCAAATCGAGGGCGATCAAGCTGCCAAATCGAGGGCGATCAAGCT 5454 　 BiotinBiotin 　 SPM-47-FSPM-47-F GTTCGGATCATGTCGACTTGCCCTGTTCGGATCATGTCGACTTGCCCT 5555 152bp152bp 　 　 SPM-199R*SPM-199R * TTTCAAACGGCGAAGAGACAATGACTTTCAAACGGCGAAGAGACAATGAC 5656 　 BiotinBiotin

표 13은 본 발명에서 사용된 프라이머 서열 Table 13 lists the primer sequences used in the present invention

NameName 서열번호SEQ ID NO: Sequence (5'-3')Sequence (5'-3 ') SpeciesSpecies GN-1GN-1 5757 TTT TTT TTT TTT TTT AAA ACGATCCCTAGCTGGTTTT TTT TTT TTT TTT AAA ACGATCCCTAGCTGGT Gram NegativeGram Negative GN-3GN-3 5858 TTT TTT TTT TTT TTT AAG ACGATCCCTAGCTGGTTTT TTT TTT TTT TTT AAG ACGATCCCTAGCTGGT Gram NegativeGram Negative Eco-7Eco-7 5959 TTT TTT TTT TTT TTT AGTAAAGTTAATACCTTTGCTC TTT TTT TTT TTT TTT AGTAAAGTTAATACCTTTGCTC E. coliE. coli Eco-9Eco-9 6060 TTT TTT TTT TTT TTT GTAAAGTTAATACCTTTGCTC TTT TTT TTT TTT TTT GTAAAGTTAATACCTTTGCTC E. coliE. coli shi-2shi-2 6161 TTT TTT TTT TTT TTT GTAAGATGGTTGTGCGCAACCTTAAGTATC TTT TTT TTT TTT TTT GTAAGATGGTTGTGCGCAACCTTAAGTATC Shigella spp.Shigella spp. sal-1863sal-1863 6262 TTT TTT TTT TTT TTT CTCCGCTAATTTGATGGATCTCATTACACT  TTT TTT TTT TTT TTT CTCCGCTAATTTGATGGATCTCATTACACT Salmonella spp.Salmonella spp. Eclo-523-1-3Eclo-523-1-3 6363 TTT TTT TTT TTT TTT AAGA AG GTGTTGTGGTTAATAACCG TTT TTT TTT TTT TTT AAGA AG GTGTTGTGGTTAATAACCG E. cloacaeE. cloacae K.pn-14-3K.pn-14-3 6464 TTT TTT TTT TTT TTT AA GGAAG GC GGTGAGGTTAATAACCTCATCGATTT TTT TTT TTT TTT AA GGAAG GC GGTGAGGTTAATAACCTCATCGA K. pneumoniaeK. pneumoniae Koxy-523Koxy-523 6565 TTT TTT TTT TTT TTT GAGT GAGGTTAATAACCTTATTTT TTT TTT TTT TTT GAGT GAGGTTAATAACCTTAT K. oxytocaK. oxytoca Koxy-523-2Koxy-523-2 6666 TTT TTT TTT TTT TTT AAA G GAGT GAGGTTAATAACCTTATTTT TTT TTT TTT TTT AAA G GAGT GAGGTTAATAACCTTAT K. oxytocaK. oxytoca cit-110cit-110 6767 TTT TTT TTT TTT TTT AA TAGCGCAGAGGAGCTTGCTCCTTTTT TTT TTT TTT TTT AA TAGCGCAGAGGAGCTTGCTCCTT Citrobacter spp.Citrobacter spp. Pmir-517-3Pmir-517-3 6868 TTT TTT TTT TTT TTT AAA GGTGATAAGGTTAATACCCTTG TTT TTT TTT TTT TTT AAA GGTGATAA G GTTAATACC C TT G Proteus mirabilisProteus mirabilis serr-118serr-118 6969 TTT TTT TTT TTT TTT GCACAGGGGAGCTTGCTCCCTTTT TTT TTT TTT TTT GCACAGGGGAGCTTGCTCCCT Serratia spp.Serratia spp. Mor-2Mor-2 7070 TTT TTT TTT TTT TTT GG AAG GTG TCA AGG TTA ATA ACC TTG GCTTT TTT TTT TTT TTT GG AAG GTG TCA AGG TTA ATA ACC TTG GC Morganella spp.Morganella spp. Mor523-1Mor523-1 7171 TTT TTT TTT TTT TTT AAAG GTG TCA AGG TTA ATA ACC TTGTTT TTT TTT TTT TTT AAAG GTG TCA AGG TTA ATA ACC TTG Morganella spp.Morganella spp. Aba-Pae-255-2Aba-Pae-255-2 7272 TTT TTT TTT TTT TTT AAGAAAGYRGGGGATCTTCGGATTT TTT TTT TTT TTT AAGAAAGYRGGGGATCTTCGGA Acinetobacter spp.
Pseudomonas spp.Acinetobacter spp.
Pseudomonas spp. Aba-278Aba-278 7373 TTT TTT TTT TTT TTT ACCTTGCGCTAATAGATGAGCCTAAGTCTTT TTT TTT TTT TTT ACCTTGCGCTAATAGATGAGCCTAAGTC A. baumanniiA. baumannii Pae-3-2Pae-3-2 7474 TTT TTT TTT TTT TTT TCTGCC TGGTAGTGGGGGATAACGTCCTTT TTT TTT TTT TTT TCTGCC TGGTAGTGGGGGATAACGTCC P.aeruginosaP.aeruginosa GP-0GP-0 7575 TTT TTT TTT TTT TTT CVACGATRCRTAGCCGACTTT TTT TTT TTT TTT CVACGATRCRTAGCCGAC Gram positiveGram positive GP350M2GP350M2 7676 TTT TTT TTT TTT TTT AA A CGACGGGTAGCCGGCTTT TTT TTT TTT TTT AAA CGACGGGTAGCCGGC Micrococcus spp./Corynebacterium spp./Propionibacterium spp.Micrococcus spp./Corynebacterium spp./Propionibacterium spp. Ent-195-1Ent-195-1 7777 TTT TTT TTT TTT TTT GGATAA CACTTGGAAACAGGTGTTT TTT TTT TTT TTT GGATAA CACTTGGAAACAGGTG Enterococcus spp.Enterococcus spp. Efae-234Efae-234 7878 TTT TTT TTT TTT TTT CATAACAGTTTATGCCGCATGGCATAAGTTT TTT TTT TTT TTT CATAACAGTTTATGCCGCATGGCATAAG E. faecalisE. faecalis Strep-7Strep-7 7979 TTT TTT TTT TTT TTT GAACGAGTGTGAGAGTGGAAAGTTCACACTG TTT TTT TTT TTT TTT GAACGAGTGTGAGAGTGGAAAGTTCACACTG Streptococcus spp.Streptococcus spp. Strep-246Strep-246 8080 TTT TTT TTT TTT TTT GATGTTGCATGACATTTGCTTAAAAGGTGCATTT TTT TTT TTT TTT GATGTTGCATGACATTTGCTTAAAAGGTGCA Streptococcus spp.Streptococcus spp. Saga-239Saga-239 8181 TTT TTT TTT TTT TTT GAGTAATTAACACATGTTAGTTATTTAAAAGGA TTT TTT TTT TTT TTT GAGTAATTAACACATGTTAGTTATTTAAAAGGA Streptococcus agalactiae Streptococcus agalactiae Spyo-254Spyo-254 8282 TTT TTT TTT TTT TTT ATGTTAGTAATTTAAAAGGGGCAATTGCTCTTT TTT TTT TTT TTT ATGTTAGTAATTTAAAAGGGGCAATTGCTC S. pyogenesS. pyogenes spn356-PSPN356-P 8383 TTT TTT TTT TTT TTT TAGCAGATAGTGAGATCGAAAATGTTAC TTT TTT TTT TTT TTT TAGCAGATAGTGAGATCGAAAATGTTAC S.pneumoniaeS.pneumoniae sta-216sta-216 8484 TTT TTT TTT TTT TTT AAACCGGAGCTAATACCGGATAATATTTTGATTT TTT TTT TTT TTT AAACCGGAGCTAATACCGGATAATATTTTGA Staphylococcus spp.Staphylococcus spp. staphyl-479-1staphyl-479-1 8585 TTT TTT TTT TTT TTT CAWAYGTGTAAGTAACTRTGCACATTTT TTT TTT TTT TTT CAWAYGTGTAAGTAACTRTGCACAT Staphylococcus spp.Staphylococcus spp. Pacnes-185-1Pacnes-185-1 8686 TTT TTT TTT TTT TTT AAA CTTGACTTTGGGATAACTTCATTT TTT TTT TTT TTT AAA CTTGACTTTGGGATAACTTCA Propionibacterium acnes　Propionibacterium acnes cory-233cory-233 8787 TTT TTT TTT TTT TTT GATAGGACCATCGTTTAGTGTCTTT TTT TTT TTT TTT GATAGGACCATCGTTTAGTGTC Corynebacterium　spp.Corynebacterium spp. alb-3alb-3 8888 TTT TTT TTT TTT TTT TGCTTGCGGCGGTAACGT CCACCACGTATTTT TTT TTT TTT TTT TGCTTGCGGCGGTAACGT CCACCACGTAT Candida albicansCandida albicans trotro 8989 TTT TTT TTT TTT TTT GAATTTAACG TGGAAACTTATTTT AAGCGATTT TTT TTT TTT TTT GAATTTAACG TGGAAACTTATTTT AAGCGA C.tropicalisC.tropicalis glagla 9090 TTT TTT TTT TTT TTT GACACGAGCGCAAGCTTCTCTATTAATCTGTTT TTT TTT TTT TTT GACACGAGCGCAAGCTTCTCTATTAATCTG C.glabrataC.glabrata paramoney 9191 TTT TTT TTT TTT TTT GAA AGGCG GA GTATAAACTAATGGATAGGTTTT TTT TTT TTT TTT GAA AGGCG GA GTATAAACTAATGGATAGGT C.parapsilosisC.parapsilosis krukru 9292 TTT TTT TTT TTT TTT GGAGCGGAGCGGACGACGTGTAAAGAGCTTT TTT TTT TTT TTT GGAGCGGAGCGGACGACGTGTAAAGAGC C.kruseiC.krusei IC.util-610IC.util-610 9393 TTT TTT TTT TTT TTT CTGTGTTAACTTGAAATACTCTAGGCAGAGCTTTT TTT TTT TTT TTT CTGTGTTAACTTGAAATACTCTAGGCAGAGCT Candida utilisCandida utilis IC.lusi-598IC.lusi-598 9494 TTT TTT TTT TTT TTT AACCGCGCTGTCAAACACGTTTACAGCATTT TTT TTT TTT TTT AACCGCGCTGTCAAACACGTTTACAGCA C. lusitaniaeC. lusitaniae IC.hae-593IC.hae-593 9595 TTT TTT TTT TTT TTT ATATCATGCCACAGTGAAGTCTACGCTTTT TTT TTT TTT TTT ATATCATGCCACAGTGAAGTCTACGCT Candida haemulonisCandida haemulonis IC.auris-617IC.auris-617 9696 TTT TTT TTT TTT TTT GCATTCACAAAATTACAGCTTGCACGAAAATTT TTT TTT TTT TTT GCATTCACAAAATTACAGCTTGCACGAAAA C. aurisC. auris

표 14는 균종 동정 관련 프로브 서열Table 14 shows the relationship between the species identification

NameName 서열번호SEQ ID NO: Sequence (5' amine-3')Sequence (5 'amine-3') resistance generesistance gene nucnuc 9797 TTT TTT TTT TTT TTT TTGGTTGATACACCTGAAACAAAGTTT TTT TTT TTT TTT TTGGTTGATACACCTGAAACAAAG S.aureusS.aureus MecAMeca 9898 TTT TTT TTT TTT TTT AGCTGATTCAGGTTACGGACAAGGTTTT TTT TTT TTT TTT AGCTGATTCAGGTTACGGACAAGGT MecAMeca VanAVanA 9999 TTT TTT TTT TTT TTT AATCGTATTCATCAGGAAGTCGAGTTT TTT TTT TTT TTT AATCGTATTCATCAGGAAGTCGAG VanAVanA VanBVanB 100100 TTT TTT TTT TTT TTT AATCGTCCTTTGGCGTAACCAATTT TTT TTT TTT TTT AATCGTCCTTTGGCGTAACCAA VanBVanB M15-258-1M15-258-1 101101 TTT TTT TTT TTT TTT AAGTGAAAGCGAACCGAATCTGTTT TTT TTT TTT TTT AAGTGAAAGCGAACCGAATCTG ESBLESBL CTXM9-261-6CTXM9-261-6 102102 TTT TTT TTT TTT TTT AGT GAAACGCAAAAGCAG CTGCTT AATCAG CCTGTTT TTT TTT TTT TTT AGT GAAACGCAAAAGCAG CTGCTT AATCAG CCTG 　 TEMGlu104Lys-1TEMGlu104Lys-1 103103 TTT TTT TTT TTT TTT GACTTGGTTAAGTACTCACCAGTCATTT TTT TTT TTT TTT GACTTGGTT A AGTACTCACCAGTCA 　 TEMGly238Ser-anTEMGly238Ser-an 104104 TTT TTT TTT TTT TTT GAGATCCATGCTCACCGGCTCTTT TTT TTT TTT TTT GAGA T CCA T GCTCACCGGCTC 　 SHVGly238Ser-anSHVGly238Ser-an 105105 TTT TTT TTT TTT TTT CGCTCGCTAGCTCCGGTCTTTT TTT TTT TTT TTT CGCTCGCTAGCTCCGGTCT 　 DHA-299-PDHA-299-P 106106 TTT TTT TTT TTT TTT AAGAGATGGCGCTGAATGATCCTTT TTT TTT TTT TTT AAGAGATGGCGCTGAATGATCC AmpC β-lactamaseAmpC? -Lactamase CMY2-328-PCMY2-328-P 107107 TTT TTT TTT TTT TTT ACGAAATACTGGCCAGAACTGACATTT TTT TTT TTT TTT ACGAAATACTGGCCAGAACTGACA 　 ACT-MIR-464-PACT-MIR-464-P 108108 TTT TTT TTT TTT TTT ATCAAAACTGGCAGCCGCAGTTT TTT TTT TTT TTT ATCAAAACTGGCAGCCGCAG 　 CMY1/MOX-172-1CMY1 / MOX-172-1 109109 TTT TTT TTT TTT TTT A A GATGGCAAGGCCCACTATTT TTT TTT TTT TTT A A GATGGCAAGGCCCACTA 　 ACC-1-439ACC-1-439 110110 TTT TTT TTT TTT TTT GCTTCGTTACCCAAAATCTCCATATTCTTT TTT TTT TTT TTT GCTTCGTTACCCAAAATCTCCATATTC 　 SPM-98SPM-98 111111 TTT TTT TTT TTT TTT CGGACGTTT TCG TCGTCACAGACCGCGATTT TTT TTT TTT TTT CGGACGTTT TCG TCGTCACAGACCGCGA 　 IMP-307PIMP-307P 112112 TTT TTT TTT TTT TTT AATAGAGTGGCTTAATTCTCRATCTATTTT TTT TTT TTT TTT AATAGAGTGGCTTAATTCTCRATCTAT CarbapenemaseCarbapenemase VIM-198PVIM-198P 113113 TTT TTT TTT TTT TTT CTACCCGTCCAATGGTCTCATTGTTTT TTT TTT TTT TTT CTACCCGTCCAATGGTCTCATTGT 　 OXA48-186POXA48-186P 114114 TTT TTT TTT TTT TTT GAACCAAGCATTTTTACCCGCATTT TTT TTT TTT TTT GAACCAAGCATTTTTACCCGCA 　 KPC-329-1PKPC-329-1P 115115 TTT TTT TTT TTT TTT AATATCTGACAACAGGCATGACGGTTTT TTT TTT TTT TTT AATATCTGACAACAGGCATGACGGT 　 NDM-385-2NDM-385-2 116116 TTT TTT TTT TTT TTT ACTCACGCGCATCAGGACAAGATGTTT TTT TTT TTT TTT ACTCACGCGCATCAGGACAAGATG 　 Fox-238Fox-238 117117 TTT TTT TTT TTT TTT CTGTTCGAGATTGGCTCGGTCAGTTT TTT TTT TTT TTT CTGTTCGAGATTGGCTCGGTCAG 　

표 15는 내성 관련 프로브 서열Table 15 shows the resistance-related probe sequences

<110> Optipharm.CO.,LTD UNIVERSITY INDUSTRY FOUNDATION, YONSEI UNIVERSITY WONJU CAMPUS <120> A method for simultaneously detecting and identifying Gram positive bacteria, Gram negative bacteria, Candida and antibotics-resistance, and a kit therefor based QuantaMatrix assay Platform <130> P16-0288HS <160> 117 <170> KopatentIn 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 taayacatgc aagtcgarcg 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tgtggcygrt crycctctca g 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 agykgcgrac gggtgagtaa 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tgtggcygrt crycctctca g 21 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ctacgggagg cagcagtrgg gaat 24 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 tattaccgcg gctgctggca 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 aacgcanmtt gcrcychhtg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 cagcgggtad yccyacctga 20 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 taaagcgatt gatggtgata cggt 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 tttcgtaaat gcacttgctt cagg 24 <210> 11 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ctaggtgttg gtgaagatat accaagtg 28 <210> 12 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 ttgaaaggat ctgtactggg ttaatcag 28 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 agtcaatagc gcggacgaat tg 22 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gcgggaacgg ttataactgc gttttc 26 <210> 15 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 cttacctacc ctgtctttgt gaagcc 26 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 cgccgacaat caaatcatcc tcgtt 25 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 tctggcagta ccttcctcag cc 22 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 aatcgacaga cgtaaggagg acaag 25 <210> 19 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 gcagttgcag tctcctaggt aaaggg 26 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 acttgaggtt aggaacaacg aactgca 27 <210> 21 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 gaccaatcgt ttaaatgcga cttct 25 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 gtcccagtcg gtgctgtcac act 23 <210> 23 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 caactcggtc gccgcataca ctattc 26 <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 gtcacgctcg tcgtttggta tg 22 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 aacggcagct gctgcagtg 19 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 acgatacggg agggcttacc at 22 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 agctgctgca gtggatggt 19 <210> 28 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 ctctgctttg ttattcgggc caa 23 <210> 29 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 ctgatgtgca gcaccagtaa agtgatg 27 <210> 30 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 aatcggatta tagttaacma rgtcagattt 30 <210> 31 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 ggtgtgcagt accagtaaag ttatggcg 28 <210> 32 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 ggattgtagt taaccagatc ggcaggc 27 <210> 33 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 actttcacag gtgtgctggg tgc 23 <210> 34 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 tgcggtatag gtagccagat ccagcaatg 29 <210> 35 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 aagacgttta acggcgtgtt gg 22 <210> 36 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 gccgtatagg tggctaagtg cag 23 <210> 37 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 tacaggtrcc ggatgaggtc acg 23 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 38 gaaggtttga ccgccagcgc 20 <210> 39 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 39 tgcacgtagc gtaacaagtc gctggag 27 <210> 40 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 40 gctgaccaga tggaaaacta tgcgtg 26 <210> 41 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 41 cgctgctcaa ggagcacmgg atc 23 <210> 42 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 42 tatctcgaac agggtctgct cgct 24 <210> 43 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 43 ctatttcaac tatggggttg ccaacc 26 <210> 44 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 44 tgctggctca ccttgtcatc cagc 24 <210> 45 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 45 aataaaaggc agtatytcct cwcattt 27 <210> 46 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 46 accttaccgt cttttttaag magttca 27 <210> 47 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 47 ggctttacca gattgccgat ggtgtt 26 <210> 48 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 48 gcaccccacg ctgtatcaat caaaag 26 <210> 49 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 49 gctggacaca cccatccgtt acg 23 <210> 50 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 50 gcggcgttat cactgtattg cacgg 25 <210> 51 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 51 agatcctcaa ctggatcaag cagg 24 <210> 52 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 52 acgcattggc ataagtcgca at 22 <210> 53 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 53 agttgtgctc tggaatgaga ataagcag 28 <210> 54 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 54 gccaaatcga gggcgatcaa gct 23 <210> 55 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 55 gttcggatca tgtcgacttg ccct 24 <210> 56 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 56 tttcaaacgg cgaagagaca atgac 25 <210> 57 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 57 tttttttttt tttttaaaac gatccctagc tggt 34 <210> 58 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 58 tttttttttt tttttaagac gatccctagc tggt 34 <210> 59 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 59 tttttttttt tttttagtaa agttaatacc tttgctc 37 <210> 60 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 60 tttttttttt tttttgtaaa gttaatacct ttgctc 36 <210> 61 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 61 tttttttttt tttttgtaag atggttgtgc gcaaccttaa gtatc 45 <210> 62 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 62 tttttttttt tttttctccg ctaatttgat ggatctcatt acact 45 <210> 63 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 63 tttttttttt tttttaagaa ggtgttgtgg ttaataaccg 40 <210> 64 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 64 tttttttttt tttttaagga aggcggtgag gttaataacc tcatcga 47 <210> 65 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 65 tttttttttt tttttgagtg aggttaataa ccttat 36 <210> 66 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 66 tttttttttt tttttaaagg agtgaggtta ataaccttat 40 <210> 67 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 67 tttttttttt tttttaatag cgcagaggag cttgctcctt 40 <210> 68 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 68 tttttttttt tttttaaagg tgataaggtt aatacccttg 40 <210> 69 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 69 tttttttttt tttttgcaca ggggagcttg ctccct 36 <210> 70 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 70 tttttttttt tttttggaag gtgtcaaggt taataacctt ggc 43 <210> 71 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 71 tttttttttt tttttaaagg tgtcaaggtt aataaccttg 40 <210> 72 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 72 tttttttttt tttttaagaa agyrggggat cttcgga 37 <210> 73 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 73 tttttttttt tttttacctt gcgctaatag atgagcctaa gtc 43 <210> 74 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 74 tttttttttt ttttttctgc ctggtagtgg gggataacgt cc 42 <210> 75 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 75 tttttttttt tttttcvacg atrcrtagcc gac 33 <210> 76 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 76 tttttttttt tttttaaacg acgggtagcc ggc 33 <210> 77 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 77 tttttttttt tttttggata acacttggaa acaggtg 37 <210> 78 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 78 tttttttttt tttttcataa cagtttatgc cgcatggcat aag 43 <210> 79 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 79 tttttttttt tttttgaacg agtgtgagag tggaaagttc acactg 46 <210> 80 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 80 tttttttttt tttttgatgt tgcatgacat ttgcttaaaa ggtgca 46 <210> 81 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 81 tttttttttt tttttgagta attaacacat gttagttatt taaaagga 48 <210> 82 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 82 tttttttttt tttttatgtt agtaatttaa aaggggcaat tgctc 45 <210> 83 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 83 tttttttttt ttttttagca gatagtgaga tcgaaaatgt tac 43 <210> 84 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 84 tttttttttt tttttaaacc ggagctaata ccggataata ttttga 46 <210> 85 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 85 tttttttttt tttttcaway gtgtaagtaa ctrtgcacat 40 <210> 86 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 86 tttttttttt tttttaaact tgactttggg ataacttca 39 <210> 87 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 87 tttttttttt tttttgatag gaccatcgtt tagtgtc 37 <210> 88 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 88 tttttttttt ttttttgctt gcggcggtaa cgtccaccac gtat 44 <210> 89 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 89 tttttttttt tttttgaatt taacgtggaa acttatttta agcga 45 <210> 90 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 90 tttttttttt tttttgacac gagcgcaagc ttctctatta atctg 45 <210> 91 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 91 tttttttttt tttttgaaag gcggagtata aactaatgga taggt 45 <210> 92 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 92 tttttttttt tttttggagc ggagcggacg acgtgtaaag agc 43 <210> 93 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 93 tttttttttt tttttctgtg ttaacttgaa atactctagg cagagct 47 <210> 94 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 94 tttttttttt tttttaaccg cgctgtcaaa cacgtttaca gca 43 <210> 95 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 95 tttttttttt tttttatatc atgccacagt gaagtctacg ct 42 <210> 96 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 96 tttttttttt tttttgcatt cacaaaatta cagcttgcac gaaaa 45 <210> 97 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 97 tttttttttt tttttttggt tgatacacct gaaacaaag 39 <210> 98 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 98 tttttttttt tttttagctg attcaggtta cggacaaggt 40 <210> 99 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 99 tttttttttt tttttaatcg tattcatcag gaagtcgag 39 <210> 100 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 100 tttttttttt tttttaatcg tcctttggcg taaccaa 37 <210> 101 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 101 tttttttttt tttttaagtg aaagcgaacc gaatctg 37 <210> 102 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 102 tttttttttt tttttagtga aacgcaaaag cagctgctta atcagcctg 49 <210> 103 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 103 tttttttttt tttttgactt ggttaagtac tcaccagtca 40 <210> 104 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 104 tttttttttt tttttgagat ccatgctcac cggctc 36 <210> 105 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 105 tttttttttt tttttcgctc gctagctccg gtct 34 <210> 106 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 106 tttttttttt tttttaagag atggcgctga atgatcc 37 <210> 107 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 107 tttttttttt tttttacgaa atactggcca gaactgaca 39 <210> 108 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 108 tttttttttt tttttatcaa aactggcagc cgcag 35 <210> 109 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 109 tttttttttt tttttaagat ggcaaggccc acta 34 <210> 110 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 110 tttttttttt tttttgcttc gttacccaaa atctccatat tc 42 <210> 111 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 111 tttttttttt tttttcggac gttttcgtcg tcacagaccg cga 43 <210> 112 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 112 tttttttttt tttttaatag agtggcttaa ttctcratct at 42 <210> 113 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 113 tttttttttt tttttctacc cgtccaatgg tctcattgt 39 <210> 114 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 114 tttttttttt tttttgaacc aagcattttt acccgca 37 <210> 115 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 115 tttttttttt tttttaatat ctgacaacag gcatgacggt 40 <210> 116 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 116 tttttttttt tttttactca cgcgcatcag gacaagatg 39 <210> 117 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 117 tttttttttt tttttctgtt cgagattggc tcggtcag 38 <110> Optipharm.CO., LTD          UNIVERSITY INDUSTRY FOUNDATION, YONSEI UNIVERSITY WONJU CAMPUS <120> A method for detecting and identifying Gram          positive bacteria, Gram negative bacteria, Candida and          antibotics-resistance, and a kit therefor based on QuantaMatrix          assay Platform <130> P16-0288HS <160> 117 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 taayacatgc aagtcgarcg 20 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tgtggcygrt crycctctca g 21 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 agykgcgrac gggtgagtaa 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tgtggcygrt crycctctca g 21 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ctacgggagg cagcagtrgg gaat 24 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 tattaccgcg gctgctggca 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 aacgcanmtt gcrcychhtg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 cagcgggtad yccyacctga 20 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 taaagcgatt gatggtgata cggt 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 tttcgtaaat gcacttgctt cagg 24 <210> 11 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ctaggtgttg gtgaagatat accaagtg 28 <210> 12 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 ttgaaaggat ctgtactggg ttaatcag 28 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 agtcaatagc gcggacgaat tg 22 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gcgggaacgg ttataactgc gttttc 26 <210> 15 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 cttacctacc ctgtctttgt gaagcc 26 <210> 16 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 cgccgacaat caaatcatcc tcgtt 25 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 tctggcagta ccttcctcag cc 22 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 aatcgacaga cgtaaggagg acaag 25 <210> 19 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 gcagttgcag tctcctaggt aaaggg 26 <210> 20 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 acttgaggtt aggaacaacg aactgca 27 <210> 21 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 gaccaatcgt ttaaatgcga cttct 25 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 gtcccagtcg gtgctgtcac act 23 <210> 23 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 caactcggtc gccgcataca ctattc 26 <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 gtcacgctcg tcgtttggta tg 22 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 aacggcagct gctgcagtg 19 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 acgatacggg agggcttacc at 22 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 agctgctgca gtggatggt 19 <210> 28 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 ctctgctttg ttattcgggc caa 23 <210> 29 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 ctgatgtgca gcaccagtaa agtgatg 27 <210> 30 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 aatcggatta tagttaacma rgtcagattt 30 <210> 31 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 ggtgtgcagt accagtaaag ttatggcg 28 <210> 32 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 ggattgtagt taaccagatc ggcaggc 27 <210> 33 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 33 actttcacag gtgtgctggg tgc 23 <210> 34 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 34 tgcggtatag gtagccagat ccagcaatg 29 <210> 35 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 35 aagacgttta acggcgtgtt gg 22 <210> 36 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 36 gccgtatagg tggctaagtg cag 23 <210> 37 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 37 tacaggtrcc ggatgaggtc acg 23 <210> 38 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 38 gaaggtttga ccgccagcgc 20 <210> 39 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 39 tgcacgtagc gtaacaagtc gctggag 27 <210> 40 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 40 gctgaccaga tggaaaacta tgcgtg 26 <210> 41 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 41 cgctgctcaa ggagcacmgg atc 23 <210> 42 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 42 tatctcgaac agggtctgct cgct 24 <210> 43 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 43 ctatttcaac tatggggttg ccaacc 26 <210> 44 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 44 tgctggctca ccttgtcatc cagc 24 <210> 45 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 45 aataaaaggc agtatytcct cwcattt 27 <210> 46 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 46 accttaccgt cttttttaag magttca 27 <210> 47 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 47 ggctttacca gattgccgat ggtgtt 26 <210> 48 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 48 gcaccccacg ctgtatcaat caaaag 26 <210> 49 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 49 gctggacaca cccatccgtt acg 23 <210> 50 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 50 gcggcgttat cactgtattg cacgg 25 <210> 51 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 51 agatcctcaa ctggatcaag cagg 24 <210> 52 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 52 acgcattggc ataagtcgca at 22 <210> 53 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 53 agttgtgctc tggaatgaga ataagcag 28 <210> 54 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 54 gccaaatcga gggcgatcaa gct 23 <210> 55 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 55 gttcggatca tgtcgacttg ccct 24 <210> 56 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 56 tttcaaacgg cgaagagaca atgac 25 <210> 57 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 57 tttttttttt tttttaaaac gatccctagc tggt 34 <210> 58 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 58 tttttttttt tttttaagac gatccctagc tggt 34 <210> 59 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 59 tttttttttt tttttagtaa agttaatacc tttgctc 37 <210> 60 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 60 tttttttttt tttttgtaaa gttaatacct ttgctc 36 <210> 61 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 61 tttttttttt tttttgtaag atggttgtgc gcaaccttaa gtatc 45 <210> 62 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 62 tttttttttt tttttctccg ctaatttgat ggatctcatt acact 45 <210> 63 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 63 tttttttttt tttttaagaa ggtgttgtgg ttaataaccg 40 <210> 64 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 64 tttttttttt tttttaagga aggcggtgag gttaataacc tcatcga 47 <210> 65 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 65 tttttttttt tttttgagtg aggttaataa ccttat 36 <210> 66 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 66 tttttttttt tttttaaagg agtgaggtta ataaccttat 40 <210> 67 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 67 tttttttttt tttttaatag cgcagaggag cttgctcctt 40 <210> 68 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 68 tttttttttt tttttaaagg tgataaggtt aatacccttg 40 <210> 69 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 69 tttttttttt tttttgcaca ggggagcttg ctccct 36 <210> 70 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 70 tttttttttt tttttggaag gtgtcaaggt taataacctt ggc 43 <210> 71 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 71 tttttttttt tttttaaagg tgtcaaggtt aataaccttg 40 <210> 72 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 72 tttttttttt tttttaagaa agyrggggat cttcgga 37 <210> 73 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 73 tttttttttt tttttacctt gcgctaatag atgagcctaa gtc 43 <210> 74 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 74 tttttttttt ttttttctgc ctggtagtgg gggataacgt cc 42 <210> 75 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 75 tttttttttt tttttcvacg atrcrtagcc gac 33 <210> 76 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 76 tttttttttt tttttaaacg acgggtagcc ggc 33 <210> 77 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 77 tttttttttt tttttggata acacttggaa acaggtg 37 <210> 78 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 78 tttttttttt tttttcataa cagtttatgc cgcatggcat aag 43 <210> 79 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 79 tttttttttt tttttgaacg agtgtgagag tggaaagttc acactg 46 <210> 80 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 80 tttttttttt tttttgatgt tgcatgacat ttgcttaaaa ggtgca 46 <210> 81 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 81 tttttttttt tttttgagta attaacacat gttagttatt taaaagga 48 <210> 82 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 82 tttttttttt tttttatgtt agtaatttaa aaggggcaat tgctc 45 <210> 83 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 83 tttttttttt ttttttagca gatagtgaga tcgaaaatgt tac 43 <210> 84 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 84 tttttttttt tttttaaacc ggagctaata ccggataata ttttga 46 <210> 85 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 85 tttttttttt tttttcaway gtgtaagtaa ctrtgcacat 40 <210> 86 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 86 tttttttttt tttttaaact tgactttggg ataacttca 39 <210> 87 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 87 tttttttttt tttttgatag gaccatcgtt tagtgtc 37 <210> 88 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 88 tttttttttt ttttttgctt gcggcggtaa cgtccaccac gtat 44 <210> 89 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 89 tttttttttt tttttgaatt taacgtggaa acttatttta agcga 45 <210> 90 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 90 tttttttttt tttttgacac gagcgcaagc ttctctatta atctg 45 <210> 91 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 91 tttttttttt tttttgaaag gcggagtata aactaatgga taggt 45 <210> 92 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 92 tttttttttt tttttggagc ggagcggacg acgtgtaaag agc 43 <210> 93 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 93 tttttttttt tttttctgtg ttaacttgaa atactctagg cagagct 47 <210> 94 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 94 tttttttttt tttttaaccg cgctgtcaaa cacgtttaca gca 43 <210> 95 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 95 tttttttttt tttttatatc atgccacagt gaagtctacg ct 42 <210> 96 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 96 tttttttttt tttttgcatt cacaaaatta cagcttgcac gaaaa 45 <210> 97 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 97 tttttttttt tttttttggt tgatacacct gaaacaaag 39 <210> 98 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 98 tttttttttt tttttagctg attcaggtta cggacaaggt 40 <210> 99 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 99 tttttttttt tttttaatcg tattcatcag gaagtcgag 39 <210> 100 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 100 tttttttttt tttttaatcg tcctttggcg taaccaa 37 <210> 101 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 101 tttttttttt tttttaagtg aaagcgaacc gaatctg 37 <210> 102 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 102 tttttttttt tttttagtga aacgcaaaag cagctgctta atcagcctg 49 <210> 103 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 103 tttttttttt tttttgactt ggttaagtac tcaccagtca 40 <210> 104 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 104 tttttttttt tttttgagat ccatgctcac cggctc 36 <210> 105 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 105 tttttttttt tttttcgctc gctagctccg gtct 34 <210> 106 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 106 tttttttttt tttttaagag atggcgctga atgatcc 37 <210> 107 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 107 tttttttttt tttttacgaa atactggcca gaactgaca 39 <210> 108 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 108 tttttttttt tttttatcaa aactggcagc cgcag 35 <210> 109 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 109 tttttttttt tttttaagat ggcaaggccc acta 34 <210> 110 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 110 tttttttttt tttttgcttc gttacccaaa atctccatat tc 42 <210> 111 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 111 tttttttttt tttttcggac gttttcgtcg tcacagaccg cga 43 <210> 112 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 112 tttttttttt tttttaatag agtggcttaa ttctcratct at 42 <210> 113 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 113 tttttttttt tttttctacc cgtccaatgg tctcattgt 39 <210> 114 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 114 tttttttttt tttttgaacc aagcattttt acccgca 37 <210> 115 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 115 tttttttttt tttttaatat ctgacaacag gcatgacggt 40 <210> 116 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 116 tttttttttt tttttactca cgcgcatcag gacaagatg 39 <210> 117 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> probe <400> 117 tttttttttt tttttctgtt cgagattggc tcggtcag 38

Claims (6)

삭제delete 삭제delete 삭제delete 서열번호 1 내지 서열번호 56의 프라이머 및
서열번호 57 내지 117의 올리고머 프로브가 커플링된 디스크를 포함하는 그람양성, 그람음성균, 캔디다의 검출 및 동정과 항생제 내성여부를 동시에 확인하기 위한 키트;
여기서, 상기 검출 및 확인 균주는 엔테로코커스(Enterococcus), 스타필로코커스(Staphylococcus), 클렙시엘라(Klebsiella), 아시네토박터(Acinetobacter), 슈도모나스(Pseudomonas), 엔테로박터(Enterobacter), 포도상구균, 시겔라, 대장균, 또는 캔디다 종으로 구성된 군으로부터 선택된 하나 이상의 균주이고, 상기 항생제는 메티실린(methcillin), 반코마이신(Vancomycin), 및 세팔로스포린(cephalosporin) 계열 항생제로 구성된 군으로부터 선택된 하나 이상의 항생제인 것을 특징으로 하는 그람양성, 그람음성균, 캔디다의 검출 및 동정과 항생제 내성여부를 동시에 확인하기 위한 키트.The primers of SEQ ID NOS: 1 to 56 and
A kit for simultaneously detecting the detection and identification of Gram-positive, Gram-negative, Candida and an antibiotic resistance including a disk to which oligomer probes of SEQ ID Nos: 57 to 117 are coupled;
Here, the detection and identification strains are selected from the group consisting of Enterococcus, Staphylococcus , Keulrep when Ella (Klebsiella), Acinetobacter (Acinetobacter), Pseudomonas (Pseudomonas), Wherein said antibiotic is one or more strains selected from the group consisting of Enterobacter , Staphylococcus, Shigella, Escherichia coli, or Candida species, said antibiotic being selected from the group consisting of methcillin, vancomycin, and cephalosporin antibiotics Lt; RTI ID = 0.0 &gt; antibiotics &lt; / RTI &gt; A kit for simultaneous detection of gram-positive, gram-negative bacteria, candida and identification and antibiotic resistance. 삭제delete 서열번호 1 내지 서열번호 56의 프라이머 및
서열번호 57 내지 117의 올리고머 프로브를 포함하는 그람양성, 그람음성균, 캔디다의 검출 및 동정과 항생제 내성여부 동시 확인용 조성물;
상기 검출 및 확인 균주는 엔테로코커스(Enterococcus), 스타필로코커스(Staphylococcus), 클렙시엘라(Klebsiella), 아시네토박터(Acinetobacter), 슈도모나스(Pseudomonas), 엔테로박터(Enterobacter), 포도상구균, 시겔라, 대장균, 또는 캔디다 종으로 구성된 군으로부터 선택된 하나 이상의 균주이고, 상기 항생제는 메티실린(methcillin), 반코마이신(Vancomycin), 및 세팔로스포린(cephalosporin) 계열 항생제로 구성된 군으로부터 선택된 하나 이상의 항생제인 것을 특징으로 하는 그람양성, 그람음성균, 캔디다의 검출 및 동정과 항생제 내성여부 동시 확인용 조성물.

The primers of SEQ ID NOS: 1 to 56 and
A composition for simultaneous detection of gram-positive, gram-negative, and Candida including an oligomer probe of SEQ ID NOS: 57 to 117 and for identification and antibiotic resistance;
The detection and validation strains may be selected from the group consisting of Enterococcus, Staphylococcus , Keulrep when Ella (Klebsiella), Acinetobacter (Acinetobacter), Pseudomonas (Pseudomonas), Wherein said antibiotic is one or more strains selected from the group consisting of Enterobacter , Staphylococcus, Shigella, Escherichia coli, or Candida species, said antibiotic being selected from the group consisting of methcillin, vancomycin, and cephalosporin antibiotics Lt; RTI ID = 0.0 &gt; antibiotics &lt; / RTI &gt; A composition for simultaneous detection of gram-positive, gram-negative bacteria, candida, and identification and antibiotic resistance.

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