WO2018074762A1 - Quantamatrix assay platform-based diagnostic method and kit capable of detecting and identifying gram-positive and gram-negative bacteria and candida species and determining resistance to antibiotics simultaneously - Google Patents

Abstract

The present invention relates to a method for rapidly discriminating Gram-positive and negative bacteria and Candida species and determining whether the bacteria are resistant like MRSA, VRE, and bacteria having genes corresponding to ESBLs (TEM-, CTX-M-, SHV-type), AmpC (ACT, CMY2, DHA, CMY-1 like/MOX, ACC-1, FOX), and carbapenemases (OXA-48 like, IMP, VIM, NDM, KPC, SPM), and a primer and a probe for use in the method.

Description

Diagnosis and its kit for detecting and identifying Gram-positive, Gram-negative bacteria, and Candida based on Quanta Matrix Assay Platform

The present invention relates to a diagnostic method and a kit for detecting and identifying Gram-positive, Gram-negative bacteria, and Candida based on Quanta Matrix Assay platform, and whether antibiotics can be simultaneously identified.

Sepsis is a serious disease with a mortality rate of 23% to 46%, depending on when it is found, and is a problem that increases the economic burden. Sepsis is the most common cause of death in non-cardiovascular intensive care units, with at least 750,000 new cases annually in the United States, of which 50% progress to septic shock and half of those die 200,000.

In recent decades, the incidence of septic shock has been steadily increasing, while the mortality rate for it has remained unchanged or slightly reduced.

The clinical symptoms of sepsis are complicated and diverse because the infection of pathogenic microorganisms affects various functional systems of the body such as the immune system, coagulation system, and neurohormonal system of the host, resulting in systemic reactions. Thus, both the degree of response of the host and the characteristics of the causative agent of infection have a significant effect on the prognosis of sepsis.

Since the mid-1980s, Gram-positive bacteria accounted for more sepsis than the traditional Gram-negative bacteria. Since 1990, the incidence of sepsis caused by fungi has also increased. Changes in the strains that cause sepsis are accompanied by an increase in the number of older patients with comorbid diseases, and the increase of diseases caused by human immunodeficiency virus and the existing antibiotics, together with improved and aggressive medical and surgical treatments compared to the past. It is thought to be due to the expression of resistant strains resistant to.

Many yeast fungi, which have been known to be non-pathogenic, have recently emerged as important opportunistic infections in immunocompromised patients. This opportunistic fungal disease is a common complication of many medical and surgical inpatients, including malignancy, acquired immunodeficiency, major surgery, severe burns, transplantation of bone marrow or organs, intravascular catheterization, long-term antibiotic administration and chemotherapy. Is generated. In addition to Candidal albicans , a common causative agent, Candida spp. For recent among Candida tropicalis and Candida parapsilosis are on the rise, and prophylactic administration of fluconazole in patients with bone marrow transplantation is resistant to Candida. krusei and Candida There is an increasing number of reports of opportunistic fungal diseases caused by glabrata .

Since penicillin was first introduced into clinical medicine in 1940, many antibiotics have been developed and used in the past 70 years to make a decisive contribution to saving the lives of many patients from infectious diseases. 'S antibiotic resistance has evolved much faster than antibiotic development, leading to a worldwide decline in the effectiveness of most antibiotics in just 70 years. Given the frequency and clinical significance of bacterial infections, antibiotic resistance is a global health crisis.

The main bacterium for which antibiotic resistance is a problem is Enterococcus faecium , Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. Examples of resistance to Gram-positive bacteria include methicillin (including community MRSA) and Vancomycin resistance from Staphylococcus aureus, vancomycin resistance from Enterococcus faecium, and pneumococcal bacteria, the major bacteria in the community. Macrolide and multidrug resistance of Streptococcus pneumoniae.

MRSA is one of the most important pathogens in hospitals around the world. In particular, in Korea, Japan, Taiwan, Hong Kong, Singapore, Sri Lanka and some US hospitals, more than 50% of staphylococcus aureus isolates are reported to be MRSA.

The incidence of MRSA in domestic hospitals was 83.7% in 1996 in 15 hospitals in Korea, and the frequency of MRSA reported through multicenter research from 1997 to 2006 was 64-72%. As the most common cause of hospital infection in domestic hospitals, it was identified.

Enterococcus was known as a major infectious agent of endocarditis, but as the use of third-generation cephalosporin antibiotics increased in the mid-1970s, enterococcus began to be recognized as an important cause of infection in hospitals. Enterococci have inherent resistance to most antibiotics and can easily acquire antibiotic resistance by delivery of plasmids and transposons. Vancomycin-resistant enterococci (VRE) were first reported in 1988 in the United Kingdom and France.

Since then, VRE has tended to increase rapidly in the United States, suggesting that transposon transfer of VanA gene is the main mechanism.

In Korea, VRE infection was reported for the first time in 1992, and the percentage of VRE among E. faecium isolates in Korea was 4% in 1997, but it increased continuously to 29% in 2009. Among the E. faecium that caused hospital infections in 2009-2010, the percentage of VRE was 38.9%.

Antibiotic resistance of pathogens is a common problem in both developed and underdeveloped countries, which WHO defines as a serious threat to global public health, but Asian countries, including Korea, have higher overall bacterial resistance rates than Western countries. have. Recently, the most problematic antibiotic resistance in Pneumococcal and Escherichia coli is the production of extended-spectrum β-lactamase (ESBL), which is resistant to a wide range of cephalosporin antibiotics.

ESBL is an enzyme that inactivates extended-spectrum β-lactam antimicrobial agents such as cefotaxime, ceftazidime and aztreonam. Recently, the frequency of separation of fungi in Enterobacteriaceae producing this enzyme is increasing. These strains are also difficult to detect, mainly Klebsiella spp. It is common in and E. coli and in addition to these two species, Citrobacter spp., Enterobacter spp., Serratia spp., Morganella spp., Pseudomonas spp., Salmonella spp. Gram-negative bacillus has been reported to have ESBL-producing bacteria, and the isolation frequency varies by country and region, and 1.3-8.6% of K. pneumoniae and E. coli isolates in the United States produce ESBL. It has been reported that 7.5-15% and 22.8-38% of E. coli and K. pneumoniae are ESBL-producing strains.

In Korea, studies of bacteremia caused by ESBL-producing pneumonia were reported to have a 23.3% mortality rate for ESBL-producing bacteria and 20.0% for non-ESBL infection.

In the case of mild and moderate infections caused by ESBL-producing bacteria, β-lactam / β-lactamase inhibitors and fluoroquinolone may be used depending on the susceptibility results. It is known that it is highly likely to fail cephalosporin treatment and is resistant to other antimicrobial agents such as aminoglycoside and cotrimoxazole. . However, when carbapenem resistance occurs, there is practically no antibiotic that can be treated safely and effectively, and carbapenem-resistant Enterobacteriaceae (CRE) has emerged, which is a global problem. For this reason, the detection of ESBL-producing strains and the rapid determination of resistance to drugs such as β-lactamase and carbapenem may be helpful in minimizing the risk of indiscriminate use and antibiotic resistance.

In particular, bacteremia patients are very serious condition, so the rapid results will play a big role in saving the patient's life. Since sepsis can be accompanied by various infectious diseases and caused by various species, blood cultures are mainly used to identify the causative organisms.The results of the blood cultures are analyzed to examine the trends of changes and isolates of different species and antimicrobial susceptibility. Grasping has provided important information for the treatment of the patient. Blood cultures are a very important method of bacteremia testing and are essential for determining the diagnosis, treatment guidelines and prognosis.

However, blood culture takes more than 5 days, and when culture positive signal is given, it is subcultured to perform gram staining, bacteriological identification, and antibiotic susceptibility test. It takes 10 days or more, and is Gram-positive, Gram-negative, or Candida. And the like, especially for gram positive S. aureus methicillin resistance, Enterococcus spp. In the species, the presence of Vancomycin resistance and ESBL resistance are detected when bacteria in Enterobacteriaceae are detected.

Recently, methods for detecting genes related to Gram-positive and negative bacteria have been introduced using molecular diagnostics such as real-time PCR, microarray, and MALDI-TOF MS. However, these assays can only detect genes related to ESBL, AmpC, or carbapenemase, but not all related genes simultaneously. In domestic hospitals, ESBL is tested, but resistance of antibiotics such as AmpC β-lactamases and carbapenems is low, and the tests to detect them are complicated. A fast detection method is required.

The present invention has been made in view of the above necessity, and an object of the present invention is to provide a diagnostic composition capable of simulating simple and rapid Gram-positive, Gram-negative bacteria detection and identification and antibiotic resistance.

It is another object of the present invention to provide a diagnostic method capable of simplicity and rapid detection of Gram-positive bacteria, Gram-negative bacteria, and identification and antibiotic resistance.

In order to achieve the above object, the present invention provides a method for amplifying DNA from a sample, comprising: a) separating DNA from a sample sample; b) PCR amplifying from the DNA using primers of SEQ ID NOs: 1 to 56; and c) SEQ ID NO: 57 Gram-positive, Gram-negative bacteria comprising hybridizing the disks to which the oligomeric probes of 117 to the PCR amplification products obtained in step b) are measured, and then measuring the images of the disks through the quanta matrix assay platform software. It also provides a method for detecting and identifying Candida and for determining antibiotic resistance.

In one embodiment of the invention, the detection and confirmation strain Enterococcus ( Staphylococcus ), 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.

In another embodiment of the present invention, the antibiotic is preferably at least one antibiotic selected from the group consisting of methicillin (methcillin), vancomycin (Vancomycin), and cephalosporin-based antibiotics, but is not limited thereto.

In another aspect, the present invention for the detection and identification of Gram-positive, Gram-negative bacteria, Candida, including a disk coupled to the primers of SEQ ID NO: 1 to SEQ ID NO: 56 and the oligomer probe of SEQ ID NO: 57 to 117 and to determine whether the antibiotic resistance Provide the kit.

The kit is a reagent for performing a PCR amplification reaction, and preferably further includes DNA polymerase, dNTPs, and a buffer, but is not limited thereto.

In another aspect, the present invention provides a composition for detecting and identifying Gram-positive, Gram-negative bacteria, Candida, including the primers of SEQ ID NO: 1 to SEQ ID NO: 56 and the oligomer probes of SEQ ID NO: 57 to 117, and confirming antibiotic resistance.

Hereinafter, the present invention will be described.

The present invention distinguishes Gram-negative-negative and Candida species, and is characterized by MRSA and VRE and ESBLs (TEM-, CTX-M-, SHV-type), AmpC (ACT, CMY2, DHA, CMY-1 like / MOX, ACC-1 We have developed a QuantaMatrix assay Platform (QMAP) -based molecular diagnostic test that can confirm the resistance of each gene to the corresponding carbapenemases (OXA-48 like, IMP, VIM, NDM, KPC, SPM).

QMAP system is based on suspension array technology based on Quanta Matrix's original technology (Patent Registration No. 1011013100000 (2011.12.26) / 1015823840000 (2015.12.28)) and combines the probe to 50μm magnetic disk After reacting with the PCR product, it is possible to check whether the mutation is detected by fluorescence. The biggest feature of QMAP is to inscribe a unique code on the disc, which distinguishes the disc without interference, and technically 1024 codes are possible. It is possible to multi-test using 1024 kinds of disks with unique codes engraved and high throughput is possible because all processes are performed on 96 well plates (Fig. 1).

QMAP-based Sepsis-ID amplifies 16S rRNA gene sites with species-specific polymorphisms with primers with biotin groups, and reacts the resulting PCR products with microdisks with species-specific probes to determine gram-positive, gram-negative, and cantida-equality. 2 genes, 12 species, 19 bacteria, and antibiotic resistance 16 genes designed to be detected. It can be automated by molecular diagnostic test, DNA extraction (30 minutes), PCR progress (1 hour), TB / NTM detection during 96 tests And identification and rifampin resistance (1 hour 30 minutes) a total of 3 hours can be confirmed that the big advantage (Table 1).

In the present invention, using the QMAP-based dual-ID developed in Korea, the usefulness of the solid and liquid culture solution was evaluated.

QMAP Sepsis-ID Gram-negative bacteria Gram-positive bacteria Candida spp. Gram-negative Gram-positive Candida albicans Esherichia coli Of Shigella spp . Enterococcus spp . C. tropicalis Shigella spp . E. faecalis C. glabrata Salmonella spp . Of Enterobacter spp. Streptococcus spp . C. parapsilosis Salmonella spp . S pneumoniae C. krusei Klebsiella pneumoniae S. agalactiae K. oxytoca S. pyogenes Citrobacter spp . Staphylococcus spp . Proteus mirabilis S. aureus Serratia spp . Micrococcus  spp./ Corynebacterium spp./ Propionibacterium spp . Morganella spp . Acinetobacter spp . Psedomonas spp . Acinetobacter baumannii Psedomonas aeruginosa Resistance gene Resistance gene CTX -M groups (M1 and M9 types) mecA AmpC  β- lactamase  ( DHA , CMY2 , ACT, MOX , ACC -1, FOX) vanA Carbapenemase  ( NDM , KPC , OXA48 -like, IMP, VIM, SPM ) vanB

As can be seen from the present invention, the present invention quickly distinguishes Gram-positive-negative and Candida species, MRSA and VRE and ESBLs (TEM-, CTX-M-, SHV-type), AmpC (ACT, CMY2, DHA). , CMY-1 like / MOX, ACC-1, FOX), and carbapenemases (OXA-48 like, IMP, VIM, NDM, KPC, SPM) can be confirmed whether each gene is resistant.

1 is a diagram for a QMAP system.

Hereinafter, the present invention will be described in more detail with reference to non-limiting examples. However, the following examples are intended to illustrate the present invention and the scope of the present invention is not to be construed as limited by the following examples.

The present invention was carried out on blood culture specimens commissioned by the Department of Diagnostic Medicine, Wonju Christian Hospital, and the specimens with antibiotic susceptibility.

Blood cultures were inoculated with 5-10 ml of blood in a pair of BACTEC Standard 10 Aerobic / F bottles and BACTEC PLUS Anaerobic / F bottles in patients suspected of bacteremia. All blood culture bottles were received 24 hours and within two hours, two blood culture automation equipment were used: the BACTEC 9240 system (BD, Franklin Lakes, NJ, USA) and the BacT / Alert 3D system (BioMerieux, Durham, NC, * SA). Incubated at 37 ° C. for 5 days. When culture positive signal appeared, gram staining, species identification and antibiotic sensitivity test were performed. Bacterial identification was performed using a biochemical method using the Vitek II system (BioMerieux, Marcy, 1'Eltoile, France). Candida albicans was identified as positive after the germination tube test, and germ-negative bacteria were identified using ATB ID 32C (bioMerieux SA, Marcy-1'Etoile, France).

Example 1: Standard strain In clinical isolates genomic  DNA extraction

Take a portion of the culture cultured through the BACTEC 9240 system (Becton Dickinson Microbiology System, Sparks, Md, USA), add 100 μl of DNA extraction solution, vortex for 1 minute, heat at 100 ° C. for 10 minutes, and then at 13,000 at room temperature. Nucleic acid was separated using a method of taking the supernatant obtained by centrifugation for 3 minutes at rpm. The isolated nucleic acid was used as a template for PCR to perform REBA sepsis-ID.

Example 2: REBA  Gene Collection and Analysis of Sepsis-ID

PCR primers and oligonucleotide probes targeted by the Genbank of the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov ) are used to identify Gram positive bacteria for accurate bacterial identification. 16s rRNA gene for Gram negative bacteria, internal transcribed sequence (ITS) gene between 18S rRNA and 5.8S rRNA for fungus, MecA gene for MRSA antibiotic resistance, VRE VanA and VanB genes to determine whether they are antibiotic resistant, TEM, CTX-M1, CTX-M9, SHV, genes to know whether antibiotics are resistant, ACT, CMY2, DHA, CMY The nucleotide sequence was collected by searching for -1 like / MOX, ACC-1, and FOX genes, OXA-48 like, IMP, VIM, NDM, KPC, and SPM genes to determine whether carbapenem antibiotic resistance. After performing multi-alignment ( http://multalin.toulouse.inra.fr/multalin ), we designed two pairs of primers at the nucleotide sequence of each target gene. Biotin was attached at the 5 'end for the REBA method. We designed an oligonucleotide probe that can detect each target species at the bacteria-specific sites in the two pairs of primers, and each oligonucleotide probe has an amine group at the 5 'end to enable a thin membrane and peptide bond. Attached. The designed primers and oligonucleotide probes were submitted to Bioneer (Daejeon, Korea) for synthesis.

Example 3: PCR

PCR was performed using Prime Taq Premix (2X) (Genet Bio, Nonsan, Korea) commercialized genomic DNA of each strain as a template. The composition of Prime Taq Premix (2X) is 1unit / 10µl of primer Taq polymerase, 2X reaction buffer, 4mM MgCl2, enzyme stabilizer, sedment, loading dye, pH 9.0, 0.5mM of each dATP, dCTP, dGTP, dTTP. Each composition for PCR was 10 μl of Prime Taq premix (2X), 1 μl of a pair of 10 pmole primers, 3 μl of ultrapure water, and 5 μl of genomic DNA of each strain. PCR reaction was repeated 95 times 5 minutes for the initial denaturation process, 95 ℃ 30 seconds for the first amplification, 65 ℃ 30 seconds reaction 10 times, 95 ℃ 30 seconds for the second amplification, 60 ℃ 30 seconds reaction 40 times After repeating, the mixture was reacted for 10 minutes at 72 ° C. for complete extension. After completion of the PCR reaction, the PCR product was electrophoresed for 2 minutes at 2% TBE (Tris-borate-ethylenediaminetetraacetic acid disodium salt dihydrate) agarose gene (W / V ratio) at 290 volts for 10 minutes and then stained with ethidium bromide for 10 minutes. It was confirmed.

Example 4: QMAP  Based Sepsis-ID

In order to perform QMAP-based Sepsis-ID, the nucleic acid isolated from the sample was subjected to PCR using a biotin-labeled primer, and then the same amount of Denaturation solution (0.2N NaOH, 0.2mM EDTA) was mixed with the amplified PCR product at room temperature. After leaving for 5 minutes, the mixture was diluted in a hybridization buffer and placed in a coupled disk (Quantamatrix, Seoul, Korea) and reacted at 40 ° C. for 30 minutes, and washed three times at 25 ° C. for 1 minute using a WS (washing solution). After treatment with streptavidin R-phycoerythrin conjugate (Prozyme, San Leandro, CA) diluted 1: 2000 (v / v) and reacted for 10 minutes at room temperature. Wash the reaction microdisk with washing buffer 3 times at room temperature for 1 minute, and automatically detect the gram-positive, gram-negative or Candida by measuring the image of the disk through the provided QMAP software. MRSA, VanA, VanB, ESBLs Tolerance was confirmed. The cutoff values of the fluorescence intensity of all the microdisks in the images were positive for more than 500.

The result of the said Example is as follows.

QMAP  system probe availability  evaluation

In order to confirm the reaction of the bacterium-specific oligonucleotide probe prepared to confirm the usefulness of the probe used for QMAP Sepsis-ID, the test was performed through the standard strain. As a result, each DNA sample responded only to the probe of the corresponding target. And it was confirmed that no other cross-reaction (Table 2 to Table 8)

Table 2 shows probe test results for Gram-positive bacteria

Table 3 shows probe test results for Gram-negative bacteria

Table 4 shows probe test results for Candida

Table 5 shows the test results of resistance-related probes of Gram-negative bacteria (CTX-M1)

Table 6 shows the results of probe tests related to resistance of Gram-negative bacteria (CTX-M9).

Table 7 shows the results of probe tests related to resistance of Gram-negative bacteria (TEM, SHV).

Table 8 shows the results of probe tests related to resistance of Gram-negative bacteria (AmpC β-lactamase, Carbapenemase).

In solid culture QMAP  Evaluation of the usefulness of the system

In order to confirm the usefulness of QMAP-based Sepsis-ID, gram-positive, gram-negative, and Candida bacteria were tested using 244 continuous-monitoring blood culture systems (CMBCS). As a result, 50 Gram-positive bacteria, 175 Gram-negative bacteria and 19 fungi 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 ], this result is in full agreement with the results of CMBCS (Table 9).

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

Table 3 compares the results of QMAP Sepsis-ID for isolates from 244 cultures.

2 strains are Saccharomyces Separated into cerevisiae .

In addition, antibiotic resistance was shown in 149 cultured strains by traditional culture method, and resistance to antibiotics such as methodillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), and extended spectrum beta-lactamase (ESBL) by the QMAP system. For gene detection, 9 strains were MRSA in 14 S. aureus, 8 of 9 coagulase-negative staphylococci (CoNS) strains were resistant to mecA, and 12 Enterococcus spp. Seven strains of the strains were resistant to vancomycin. On the other hand, 113 strains from 114 Enterobacteriaceae appeared to be ESBL-producing bacteria, but not from one strain of K. pneumonia e (Table 10).

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

Table 10 compares the results of QMAP Sepsis-ID for detection of resistance genes in 149 antibiotic resistant cultures.

In blood cultures QMAP  Evaluation of the usefulness of the system

To evaluate the usefulness of the QMAP system in blood cultures, we analyzed the blood cultures isolated from 619 patients with suspected sepsis, and compared the results with the traditional culture separation system. In 619 blood cultures, 419 (67.7%) were blood cultured and 200 (32.3%) were blood cultured negative. In 419 blood cultured bacteria, 266 (63.5%) Gram-positive bacteria, 116 (27.7%) Gram-negative bacteria, and 30 (7.2%) Candida were isolated. In addition, CoNS, including Staphylococcus epidermidis , accounted for 58.7% (n = 156) in 266 Gram-positive bacteria. S. aureus (n = 42) and Streptococcus spp. (n = 23), Enterococcus spp. (n = 15), Micrococcus spp. (n = 11), and Bacillus spp. (n = 10) accounted for respectively. 116 Gram-negative bacteria accounted for 50% (n = 58) of 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). 30 in Candida, 15 C. albicans , 8 Candida glabrata , 4 Candida parapsilosis and 1 Candida tropicalis were isolated (Table 11).

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

Table 11 compares the results of the QMAP system for the detection of infectious species in blood cultured bacteria.

Culture method for the detection and detection of antibiotic resistance QMAP  Compare results between systems

In traditional culture results, a total of 144 strains [26 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 )] were resistant to oxacillin, and the mecA gene was detected in all strains except one. See also 3 Enterococcus spp. (2 E. faecium and 1 E. gallinarum ) were resistant to vancomycin, and all three of them detected vanA gene in the QMAP system.

Twelve strains (11 E. coli and 1 K. pneumoniae ) were identified as ESBL-producing bacteria among 92 Enterobacteriaceae hematopoiesis cultures, and 11 E. coli (8 CTX-M1, 2 CTX-M9, 1 CTX-M1 / M9 mix) and 1 K. pneumoniae showed resistance in XTX-M9, respectively (Table 12).

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

Table 12 compares the results of QMAP system for the detection and detection of antibiotic resistance in blood cultured bacteria.

Gene Sequence SEQ ID NO: Size Modification 16s rRNA 16S-F T AAY ACA TGC AAG TCG ARC G One 280R * TGT GGC YGR TCR YCC TCT CAG 2 280 bp Biotin 120F AGYKGCGRACGGGTGAGTAA 3 280R * TGT GGC YGR TCR YCC TCT CAG 4 210 bp Biotin 407F CTACGGGAGGCAGCAGTRGGGAAT 5 608R * TATTACCGCGGCTGCTGGCA 6 170 bp Biotin ITS MF3 AACGCANMTTGCRCYCHHTG 7 CR3 * CAGCGGGTADYCCYACCTGA 8 230 bp Biotin Nuc nuc-251F TAAAGCGATTGATGGTGATACGGT 9 nuc-394R * TTTCGTAAATGCACTTGCTTCAGG 10 143bp Biotin MecA MecA-1456F CTAGGTGTTGGTGAAGATATACCAAGTG 11 MecA-1600R * TTGAAAGGATCTGTACTGGGTTAATCAG 12 144bp Biotin vanA vanA555-F AGTCAATAGCGCGGACGAATTG 13 vanA791-R * GCGGGAACGGTTATAACTGCGTTTTC 14 150 bp Biotin vanB vanB-548F CTTACCTACCCTGTCTTTGTGAAGCC 15 vanB-746R * CGCCGACAATCAAATCATCCTCGTT 16 198 bp Biotin invA sal-1828F TCTGGCAGTACCTTCCTCAGCC 17 sal-1961R * AATCGACAGACGTAAGGAGGACAAG 18 133bp Biotin ipaH Shi-1243F GCAGTTGCAGTCTCCTAGGTAAAGGG 19 Shi-1337R * ACTTGAGGTTAGGAACAACGAACTGCA 20 100bp Biotin spn-cpsA 297F GACCAATCGTTTAAATGCGACTTCT 21 130 bp 416R * GTCCCAGTCGGTGCTGTCACACT 22 Biotin ESBLs TEMGlu104Lys-716F CAACTCGGTCGCCGCATACACTATTC 23 269 bp TEMGlu104Lys-985R * GTCACGCTCGTCGTTTGGTATG 24 Biotin TEMGly238-668F AACGGCAGCTGCTGCAGTG 25 102 bp TEMGly238-770R * ACGATACGGGAGGGCTTACCAT 26 Biotin SHV238-827F AGCTGCTGCAGTGGATGGT 27 151 bp SHVS238-978R * CTCTGCTTTGTTATTCGGGCCAA 28 Biotin CTXM1-209-F CTGATGTGCAGCACCAGTAAAGTGATG 29 124bp CTXM1-333-R * AATCGGATTATAGTTAACMARGTCAGATTT 30 Biotin CTXM9-209-F GGTGTGCAGTACCAGTAAAGTTATGGCG 31 120 bp CTXM9-329-R * GGATTGTAGTTAACCAGATCGGCAGGC 32 Biotin AmpC DHA-259-F ACTTTCACAGGTGTGCTGGGTGC 33 140 bp DHA-399-R * TGCGGTATAGGTAGCCAGATCCAGCAATG 34 Biotin CMY2-261-F AAGACGTTTAACGGCGTGTTGG 35 140 bp CMY2-401-R * GCCGTATAGGTGGCTAAGTGCAG 36 Biotin ACT-MIR-416-F TACAGGTRCCGGATGAGGTCACG 37 141 bp ACT-MIR-557-R * GAAGGTTTGACCGCCAGCGC 38 Biotin ACC-1-4067F TGCACGT AGCGTAACAAGTCGCTGGAG 39 132 bp ACC-1-538R * GCTGACCAGATGGAAAACTATGCGTG 40 Biotin CMY1 / MOX-126F CGCTGCTCAAGGAGCACMGGATC 41 132 bp CMY1 / MOX-258R * TATCTCGAACAGGGTCTGCTCGCT 42 Biotin Fox-180F CTATTTCAACTATGGGGTTGCCAACC 43 138 bp Fox-318R * TGCTGGCTCACCTTGTCATCCAGC 44 Biotin Carbapenemases IMP-261F AATAAAAGGCAGTATYTCCTCWCATTT 45 128 bp IMP-389R * ACCTTACCGTCTTTTTTAAGMAGTTCA 46 Biotin VIM-134F GGCTTTACCAGATTGCCGATGGTGTT 47 132 bp VIM-266R * GCACCCCACGCTGTATCAATCAAAAG 48 Biotin KPC-267F GCTGGACACACCCATCCGTTACG 49 131bp KPC-398R * GCGGCGTTATCACTGTATTGCACGG 50 Biotin NDM-327F AGATCCTCAACTGGATCAAGCAGG 51 134 bp NDM-461R * ACGCATTGGCATAAGTCGCAAT 52 Biotin OXA48-129F AGTTGTGCTCTGGAATGAGAATAAGCAG 53 123bp OXA48-252R * GCCAAATCGAGGGCGATCAAGCT 54 Biotin SPM-47-F GTTCGGATCATGTCGACTTGCCCT 55 152 bp SPM-199R * TTTCAAACGGCGAAGAGACAATGAC 56 Biotin

 Table 13 shows the primer sequences used in the present invention.

Name SEQ ID NO: Sequence (5'-3 ') Species GN-1 57 TTT TTT TTT TTT TTT AAA ACGATCCCTAGCTGGT Gram negative GN-3 58 TTT TTT TTT TTT TTT AAG ACGATCCCTAGCTGGT Gram Negative Eco-7 59  TTT TTT TTT TTT TTT AGTAAAGTTAATACCTTTGCTC E. coli Eco-9 60  TTT TTT TTT TTT TTT GTAAAGTTAATACCTTTGCTC E. coli shi-2 61  TTT TTT TTT TTT TTT GTAAGATGGTTGTGCGCAACCTTAAGTATC Shigella spp. sal-1863 62   TTT TTT TTT TTT TTT CTCCGCTAATTTGATGGATCTCATTACACT Salmonella spp. Eclo-523-1-3 63 TTT TTT TTT TTT TTT AAGA AG GTGTTGTGGTTAATAACCG E. cloacae K.pn-14-3 64 TTT TTT TTT TTT TTT AA GGAAG GC GGTGAGGTTAATAACCTCATCGA K. pneumoniae Koxy-523 65 TTT TTT TTT TTT TTT GAGT GAGGTTAATAACCTTAT K. oxytoca Koxy-523-2 66 TTT TTT TTT TTT TTT AAA G GAGT GAGGTTAATAACCTTAT K. oxytoca cit-110 67 TTT TTT TTT TTT TTT AA TAGCGCAGAGGAGCTTGCTCCTT Citrobacter spp. Pmir-517-3 68 TTT TTT TTT TTT TTT AAA GGTGATAA G GTTAATACC C TT G Proteus mirabilis serr-118 69 TTT TTT TTT TTT TTT GCACAGGGGAGCTTGCTCCCT Serratia spp. Mor-2 70 TTT TTT TTT TTT TTT GG AAG GTG TCA AGG TTA ATA ACC TTG GC Morganella spp. Mor523-1 71 TTT TTT TTT TTT TTT AAAG GTG TCA AGG TTA ATA ACC TTG Morganella spp. Aba-Pae-255-2 72 TTT TTT TTT TTT TTT AAGAAAGYRGGGGATCTTCGGA Acinetobacter spp. Pseudomonas spp. Aba-278 73 TTT TTT TTT TTT TTT ACCTTGCGCTAATAGATGAGCCTAAGTC A. baumannii Pae-3-2 74 TTT TTT TTT TTT TTT TCTGCC TGGTAGTGGGGGATAACGTCC P.aeruginosa GP-0 75 TTT TTT TTT TTT TTT CVACGATRCRTAGCCGAC Gram positive GP350M2 76 TTT TTT TTT TTT TTT AA A CGACGGGTAGCCGGC Micrococcus spp./Corynebacterium spp./Propionibacterium spp. Ent-195-1 77 TTT TTT TTT TTT TTT GGATAA CACTTGGAAACAGGTG Enterococcus spp. Efae-234 78 TTT TTT TTT TTT TTT CATAACAGTTTATGCCGCATGGCATAAG E. faecalis Strep-7 79 TTT TTT TTT TTT TTT GAACGAGTGTGAGAGTGGAAAGTTCACACTG Streptococcus spp. Strep-246 80 TTT TTT TTT TTT TTT GATGTTGCATGACATTTGCTTAAAAGGTGCA Streptococcus spp. Saga-239 81 TTT TTT TTT TTT TTT GAGTAATTAACACATGTTAGTTATTTAAAAGGA Streptococcus agalactiae Spyo-254 82 TTT TTT TTT TTT TTT ATGTTAGTAATTTAAAAGGGGCAATTGCTC S. pyogenes spn356-P 83 TTT TTT TTT TTT TTT TAGCAGATAGTGAGATCGAAAATGTTAC S.pneumoniae sta-216 84 TTT TTT TTT TTT TTT AAACCGGAGCTAATACCGGATAATATTTTGA Staphylococcus spp. staphyl-479-1 85 TTT TTT TTT TTT TTT CAWAYGTGTAAGTAACTRTGCACAT Staphylococcus spp. Pacnes-185-1 86 TTT TTT TTT TTT TTT AAA CTTGACTTTGGGATAACTTCA Propionibacterium acnes cory-233 87 TTT TTT TTT TTT TTT GATAGGACCATCGTTTAGTGTC Corynebacterium spp. alb-3 88 TTT TTT TTT TTT TTT TGCTTGCGGCGGTAACGT CCACCACGTAT Candida albicans tro 89 TTT TTT TTT TTT TTT GAATTTAACG TGGAAACTTATTTT AAGCGA C.tropicalis gla 90 TTT TTT TTT TTT TTT GACACGAGCGCAAGCTTCTCTATTAATCTG C.glabrata para 91 TTT TTT TTT TTT TTT GAA AGGCG GA GTATAAACTAATGGATAGGT C.parapsilosis kru 92 TTT TTT TTT TTT TTT GGAGCGGAGCGGACGACGTGTAAAGAGC C.krusei IC.util-610 93 TTT TTT TTT TTT TTT CTGTGTTAACTTGAAATACTCTAGGCAGAGCT Candida utilis IC.lusi-598 94 TTT TTT TTT TTT TTT AACCGCGCTGTCAAACACGTTTACAGCA C. lusitaniae IC.hae-593 95 TTT TTT TTT TTT TTT ATATCATGCCACAGTGAAGTCTACGCT Candida haemulonis IC.auris-617 96 TTT TTT TTT TTT TTT GCATTCACAAAATTACAGCTTGCACGAAAA C. auris

Table 14 shows probe identification related probe sequences

Name SEQ ID NO: Sequence (5 'amine-3') resistance gene nuc 97 TTT TTT TTT TTT TTT TTGGTTGATACACCTGAAACAAAG S.aureus MecA 98 TTT TTT TTT TTT TTT AGCTGATTCAGGTTACGGACAAGGT MecA Vana 99 TTT TTT TTT TTT TTT AATCGTATTCATCAGGAAGTCGAG Vana VanB 100 TTT TTT TTT TTT TTT AATCGTCCTTTGGCGTAACCAA VanB M15-258-1 101 TTT TTT TTT TTT TTT AAGTGAAAGCGAACCGAATCT G ESBL CTXM9-261-6 102 TTT TTT TTT TTT TTT AGT GAAACGCAAAAGCAG CTGCTT AATCAG CCTG TEMGlu104Lys-1 103 TTT TTT TTT TTT TTT GACTTGGTT A AGTACTCACCAGTCA TEMGly238Ser-an 104 TTT TTT TTT TTT TTT GAGA T CCA T GCTCACCGGCTC SHVGly238Ser-an 105 TTT TTT TTT TTT TTT CGCTCGCTAGCTCCGGTCT DHA-299-P 106 TTT TTT TTT TTT TTT AAGAGATGGCGCTGAATGATCC AmpC β-lactamase CMY2-328-P 107 TTT TTT TTT TTT TTT ACGAAATACTGGCCAGAACTGACA ACT-MIR-464-P 108 TTT TTT TTT TTT TTT ATCAAAACTGGCAGCCGCAG CMY1 / MOX-172-1 109 TTT TTT TTT TTT TTT A A GATGGCAAGGCCCACTA ACC-1-439 110 TTT TTT TTT TTT TTT GCTTCGTTACCCAAAATCTCCATATTC SPM-98 111 TTT TTT TTT TTT TTT CGGACGTTT TCG TCGTCACAGACCGCGA IMP-307P 112 TTT TTT TTT TTT TTT AATAGAGTGGCTTAATTCTCRATCTAT Carbapenemase VIM-198P 113 TTT TTT TTT TTT TTT CTACCCGTCCAATGGTCTCATTGT OXA48-186P 114 TTT TTT TTT TTT TTT GAACCAAGCATTTTTACCCGCA KPC-329-1P 115 TTT TTT TTT TTT TTT AATATCTGACAACAGGCATGACGGT NDM-385-2 116 TTT TTT TTT TTT TTT ACTCACGCGCATCAGGACAAGATG Fox-238 117 TTT TTT TTT TTT TTT CTGTTCGAGATTGGCTCGGTCAG

Table 15 shows resistance related probe sequences

Claims (6)

a) separating DNA from the sample sample; b) PCR amplification from the DNA using primers SEQ ID NO: 1 to SEQ ID NO: 56; and c) hybridizing the disk to which the oligomer probes of SEQ ID NOS: 57 to 117 are coupled with the PCR amplification product obtained in step b), and then measuring the image of the disk through the quanta matrix assay platform software. Method to detect and identify Gram-positive, Gram-negative bacteria, and Candida and to determine whether antibiotics are resistant. The method of claim 1, wherein the detection and confirmation strains are Enterococcus , Staphylococcus , Keulrep when Ella (Klebsiella), Acinetobacter (Acinetobacter), Pseudomonas (Pseudomonas), At least one strain selected from the group consisting of Enterobacter , Staphylococcus aureus, Shigella, Escherichia coli, or Candida species Method to detect and identify Gram-positive, Gram-negative bacteria, and Candida and to determine whether antibiotics are resistant. The method according to claim 1 or 2, wherein the antibiotic is Gram-positive, Gram, characterized in that at least one antibiotic selected from the group consisting of methicillin (Vancomycin), and cephalosporin-based antibiotics A method for detecting and identifying negative bacteria and Candida and for determining antibiotic resistance. Primers of SEQ ID NO: 1 to SEQ ID NO: 56 and A kit for detecting and identifying Gram-positive, Gram-negative bacteria, and Candida, including a disk to which oligomer probes of SEQ ID NOs: 57 to 117 are coupled, and simultaneously confirming antibiotic resistance. The method of claim 4, wherein the detection and confirmation strain Enterococcus (Enterococcus), Staphylococcus (Staphylococcus), Keulrep when Ella (Klebsiella), Acinetobacter (Acinetobacter), Pseudomonas (Pseudomonas), One or more strains selected from the group consisting of Enterobacter , Staphylococcus aureus, Shigella, Escherichia coli, or Candida species, wherein the antibiotics are methicillin, vancomycin, and cephalosporin-based antibiotics At least one antibiotic selected from the group consisting of Kit for the detection and identification of Gram-positive, Gram-negative bacteria, Candida, and antibiotic resistance. Primers of SEQ ID NO: 1 to SEQ ID NO: 56 and A composition for detecting and identifying Gram-positive, Gram-negative bacteria and Candida, including the oligomer probes of SEQ ID NOs: 57 to 117, and simultaneously confirming antibiotic resistance.

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