KR101038519B1 - Human infectious diseases-related pathogen differential diagnosis and simultaneous antibiotics resistance analysis, multiplex kit and chip comprising same - Google Patents

Abstract

Conventional infectious disease pathogen screening methods are constantly being improved and further developed due to relatively low accuracy, high cost, long-term analysis disadvantages, and limitation of the number of pathogens that can be detected once.

The present invention relates to a method for differential diagnosis of major pathogens of representative human infectious diseases. Using a nucleic acid isolated from a non-invasive sample as a template, 64 pathogens of infectious diseases are amplified by multiplex-PCR for each disease group, followed by high-throughput chip. The hybridization reaction provides analytical methods, kits, and chips that determine not only the pathogen infection, but also the pathogen genotype and its antibiotic resistance.

The present invention defines human genitourinary tract, nasopharyngeal and oral infectious diseases, which are increasingly correlated in the pathogenesis, as a single infectious disease group in a comprehensive range, rather than individual diseases, and simultaneously implements differential diagnosis of pathogens. Effective disease control in the early stages of infection, such as reducing the antibiotic resistance rate, shortening the treatment period, and reducing medical costs by enabling effective prescription of alternative antibiotics, if the pathogen is resistant to a specific antibiotic through the determination of antibiotic resistance of the infected pathogen. Becomes possible.

Infectious diseases, differential diagnosis, multiplex-PCR, DNA chip, antibiotics resistance, sexually transmitted diseases, urinary tract infections urinary tract infection, human papillomavirus, prostatitis, mycobacteria.

Description

Human infectious diseases-related pathogen differential diagnosis and simultaneous antibiotics resistance analysis, multiplex kit and chip comprising same}

The present invention relates to a composition for differentially diagnosing human infectious disease pathogens and determining their genotype and antibiotic resistance at the same time.
In detail, the nucleic acid separated from the sample is subjected to a template, and a multiplex-polymerase chain reaction (hereinafter referred to as "multiplex-PCR") for each infectious disease disease group is performed. In the chip reaction well immobilized, the specifically binding probe undergoes a hybridization reaction, a washing process, and finally, whether a specific pathogen is infected through fluorescence signal scanning and analysis. Rather, it relates to analytical methods, multiplex kits, and chips that can collectively identify infected pathogen genotypes and their antibiotic resistance.

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The culture test, a gold standard of traditional pathogen detection methods, is a concept that selects infectious bacteria by culturing bacteria and viruses present in a sample using special media suitable for the characteristics of each pathogen. However, some pathogenic microorganisms and viruses have been reported to be culture-resistant, resulting in the low sensitivity of the culture test, which requires an average culture period of one week, making it difficult to perform a rapid test. In addition, antibiotic susceptibility testing may be performed separately to confirm resistance, but there is a possibility of error due to contamination because it is analyzed through bacterial culture (Thiemo Rudolph et al., Acta Ophthalmol. Scandinavica. , 82, 463-). 467, 2004, Van Dyck E. et.al., J of Clin.Microbiology, 39, 1751-1756, 2001, Anthony J. Schaeffer et al., The J of Urology , 163, 127-130, 2000, John N Krieger et al., J of Clin.Microbiology, 36, 1646-1652, 1998, MA Pfaller et al., Emerging Infectious Diseases , 7, 312-318, 2001).

When determining whether a pathogen is not easy or impossible, a test for analyzing bacterial or viral nucleic acids (NAT, Nucleic Acid Test) has emerged. The sensitivity of the immunoassay based on the existing antigen-antibody response, and This was because the window period, which was infected but not detected by the test, could be shortened. Nucleic acid testing (NAT) is the fastest growing field of in vitro diagnostics (IVD), and the market in 2008, according to the IVD Technology data (May 2006, Canon Communications LLC, Los Angeles, CA, USA). Its market share is estimated to be 13% of the entire diagnostic market and reach $ 4.9 billion. Most nucleic acid tests detect nucleic acids using amplification methods. Target amplification is a method of amplifying fragments of target target nucleic acids by enzymatic reaction using Taq polymerase and deoxyribonucleotide triphosphate (dNTPs). In addition to Roche Molecular Systems 'PCR method, Gen-Probe's Transcription-mediated Amplification (TMA), BioMerieux's Nucleic Acid Sequence-based Amplification (BSA), Becton Dickinson's Strand Displacement Amplification (SDA), and Innogenetics Diagnostics' LiPA (Line-probe assay) (HY Park et al., J of Clin. Microbiology , 38, 4080-4085, 2000, P. Wattiau et al., Appl. Microbiol. Biotechnol. , 56, 816-819, 2001, K. Rantakokko-Jalava et al., J of Clin.Microbiology, 38, 32-39, 2000, Boddinghaus.B. Et al., J of Clin.Microbiology, 28, 1751-1759, 1990, WE Hill et al. , Bacteriological Analytical Manual 8th ed. , 1998).

Immediately after the introduction of penicillin antibiotics in the 1940's, antibiotic-resistant bacteria were already developed. The rate of expression and diffusion of antibiotic resistance is very fast and outpaces the development of new antibiotics, which is a problem in clinical practice. In the case of urinary tract infections, for example, because E. Coli is the main pathogen, drugs prescribed as an empirical antibiotic in the clinic may also contain TMP / SMX (Trimethoprim-Sulfamethoxazole, Bactrim) or ciprofloxacin antibiotics, which can treat Enterobacteriaciae. Although it has been used as a tea drug, the rate of TMP / SMX resistant strains in Korea is 38.7%, more than double that of the US, and the resistance to ciprofloxacin is reported to be 20% (Lee SJ et al., Korean J Urol. 44, 697-701, 2003, Song HJ et al., Korean J Urol. 46, 68-73, 2005). In the case of differential diagnosis of infectious disease pathogens and determination of antibiotic resistance at the same time through the preferred embodiment of the present invention, when TMP / SMX or ciprofloxacin-resistant strains are read as infectious agents, prescription antibiotics, fluoroquinolone, fosfomycin, nitrofurantoin, etc., are prescribed. By doing so, it is possible to realize early cure of infectious diseases and to suppress the transmission of antibiotic resistant strains. Antimicrobial resistance of major pathogens in humans shows that more than 55% of Streptococcus pneumoniae is penicillin-resistant, and approximately 57% of Staphylococcus aureus is methicillin-resistant. Immunity is on the rise. In terms of the amount of antibiotics used in Korea, cephalosporin is the most frequently used, and penicillin and fluoroquinolone antibiotics are the next most used (Uh Y et al., Yonsei Med J , 48, 773-778, 2007).
Recently, there is an increasing demand for a highly sensitive and reproducible multiplex assay platform. The most recent DNA chip test has the main advantage of being able to detect pathogens with high-throughput. After amplifying a nucleic acid fragment of a desired bacterium or virus from the sample, the nucleic acid probes that can be bound thereto are immobilized on a chip substrate, and hybridization reaction between the nucleic acid fragment amplified at a specific annealing temperature and the nucleic acid probe specific thereto is performed. After induction, washing is performed to remove the nonspecific hybridization signal, and the result is a test method that scans a signal of a specific fluorescent marker and reads the result (SV Tillib et al., Curr. Opin. In Biotechnology , 12 , 53-58, 2001, JG Hacia et al., Nature Genetics supplement, 21, 42-47, 1999, DG Wang et al., Science , 280, 1077-1082, 1998).

The present invention is to break down the conventional test method for topically identifying a few pathogens of a single infectious disease, and to integrate the infectious disease group by integrating six or more infectious diseases closely related to the site of infection, the path of infection, and the transition path. By identifying pathogens and determining their genotype and antibiotic resistance, the accuracy and usefulness of diagnosis of infectious diseases in humans can be greatly improved. For example, if the genitourinary infection spreads to the respiratory tract, such as the throat, oral cavity, and exhibits symptoms specific to the genitourinary infection, the conventional respiratory infection test does not include the analysis of the genitourinary infection, and the clinical analysis results are false negatives. As a result, nonspecific antibiotics are likely to reduce the cure rate. Recently, as the frequency of oral sex increases due to changes in sexual life patterns, the number of clinical cases of the detection of nasopharyngeal diseases, such as Neisseria gonorrhoeae and Ureaplasma urealyticum, which are the causes of urethritis, is increasing.Trichomonas, which causes vaginitis or urethritis and is parasitic to the genitourinary system vaginalis has been transmitted to the oral or nasopharyngeal respiratory system and lungs through oral sex (Osborne PT et al., Acta Cytol. 28, 136-138, 1984). Cases have also been reported (Hersh SM et al., J Med Microbiol. 20, 1-10, 1985). The present invention differentially diagnoses infectious diseases with the same or similar pathogens, lesions, etiologies, infections, or paths of transition to a comprehensive disease group, thereby eliminating the possibility of non-specific antibiotics according to idiopathic diagnosis and prescribing specific antibiotics according to accurate infectious pathogens. The purpose of this study is to support a regimen that can be cured early.
Of the molecular diagnostic tests approved by the US FDA as of 2007, the multiplex diagnostic kit is the only HC2 HR and LR product from Digene (http://www.fda.gov/cdrh/oivd/index.html). Digene's product can analyze 18 human papillomaviruses by Hybrid Capture method, which reacts RNA probe to HPV DNA isolated from the sample, not target amplification method. There is a disadvantage that the stability problem and contamination potential of the RNA probe is present. By PCR, Roche Molecular Diagnostics' AMPLICOR CT / NG Test product can simultaneously identify only two bacteria, Chlamydia trachomatis and Neisseria gonorrhoeae.
This product analyzes infectious disease pathogens based on DNA chips. As of 2008, four domestic companies (Mijin, Biomedlab, Goodzen, Biocoa) can read the infection of human papillomavirus single disease from Korea Food & Drug Administration. It has been approved to manufacture HPV DNA chips.

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However, due to the nature of the pathogenesis of a single infectious disease, additional tests are often required for the accurate diagnosis and prescription of the analysis of the single infectious disease itself. The present invention integrates six or more infectious diseases closely related to the site of infection, path of infection, and transition path into an infectious disease group to identify infectious pathogens, and to determine the presence of genotypes and antibiotic resistance. We offer analytical methods, multiplex kits and chips that greatly improve the accuracy and usefulness of prescriptions.

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Recently, microbial analysis is a common method of screening for pathogens in a sample through culture assay, enzyme immunoassay (EIA) based on antigen-antibody reaction or PCR-sequencing. . The culture test has the disadvantage of low sensitivity and long test period (average 1 week) in the case of difficult or difficult pathogens. Most enzyme immunoassay methods for analyzing proteins of bacteria or viruses have consistently reported literatures with high specificity but low sensitivity, and above all, fluorescence generated by biochemical or immunological reactions. The disadvantage is that it only measures the fluorescence level to identify a limited number of microorganisms. PCR-sequencing analysis amplifies 16S rRNA (ribosomal RNA) genes of all bacteria and analyzes the sequencing of these genes to identify pathogens. Compared to bacterial culture test, PCR sensitivity is superior and specific. Figures are commonly reported as similar. However, due to the technical limitation that only a few of the various pathogens of infectious diseases can be tested, it is difficult to test dozens of pathogens at a time. In addition, the possibility of misuse of antibiotics remains because there is no information on whether the pathogens possess antibiotic resistance.

Therefore, the present invention is to provide a new test method that can overcome the disadvantages of the lack of accuracy, long time, high cost, the number of microorganisms that can be detected at one time, the conventional infectious disease pathogen test method.
Due to the nature of the pathogenesis of multi-faceted infectious diseases, additional tests are often required for accurate diagnosis and prescription as a result of analysis of the infection of a single infectious disease itself. By defining six or more related infectious diseases as a single infectious disease group and identifying infectious pathogens and combining their genotype and antibiotic resistance, an analysis method that greatly improves the accuracy of diagnosis and the usefulness of prescription with only one analysis. , Multiplex kits and chips. In addition, to provide a highly reproducible and highly sensitive test method for non-invasive samples containing body fluid (fluid).

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In order to achieve the object of the present invention, not only to differentially diagnose infectious disease pathogens, but also to provide a composition that performs genotyping and antibiotic resistance analysis simultaneously in a high-throughput manner.
In detail, by differentially diagnosing major pathogens of representative human infectious diseases, genotype analysis, which is very important for prognosis and treatment direction, and comprehensive antibiotic analysis, whether or not antibiotic resistance is a major indicator in antibiotic prescription, is provided. In addition, we provide analytical methods, multiplex kits, and chips to reduce the duration of treatment, reduce medical costs, and reduce antibiotic resistance rates by early diagnosis of infectious diseases and effective antibiotic treatment.
And in more detail in the above analysis method, DNA or PNA probe having a nucleotide sequence of SEQ ID NO: 91 to 216 specifically binding to the target amplification products of a total of 64 bacteria or viruses for analysis of the presence of infectious diseases pathogens and antibiotic resistance Spotting the chip to fabricate the chip;
Extracting bacterial and viral genomic DNA from the sample;
Preparing a target DNA amplification product such that a fluorescent dye is included while amplifying a probe target site for each infectious disease pathogen and a probe target site for antibiotic resistance analysis through multiplex-PCR;
Injecting a hybridization solution with a target DNA amplification product into a reaction chamber cover on a chip substrate to induce and wash a hybridization reaction with a probe immobilized on the chip surface;
Scanning the chip with a laser of a wavelength specific to the fluorescent dye and measuring the fluorescence intensity according to the hybridization reaction to collectively read the results of differential diagnosis of infectious diseases, pathogen genotyping and the presence of antibiotic resistance thereof; And
The present invention provides an analysis method for simultaneously identifying a human infectious disease pathogen differential diagnosis including all the above analysis steps and determining genotype and antibiotic resistance thereof.
And in order to realize preferably the analysis method of the present invention, multiplex-PCR kit comprising at least one oligonucleotide primer (primer) selected from the group consisting of oligonucleotides having a nucleotide sequence of SEQ ID NO: 1 to 90 To provide.
In addition, in order to achieve the object of the present invention preferably 64 different infectious diseases differential diagnosis, genotyping and antibiotics comprising at least one probe selected from the group consisting of oligonucleotides having a nucleotide sequence of SEQ ID NOs: 91 to 216 Provides a chip for tolerance analysis.

Through the preferred embodiment of the present invention, it is possible to determine whether the representative pathogens of major human infectious diseases, as well as their genotype and antibiotic resistance.
Specifically, high-throughput pathogen differential diagnosis of 64 bacteria and viruses is performed in a single analysis, genotype analysis and antibiotics are very important for prognostic and therapeutic direction. It can provide a comprehensive analysis of antibiotic resistance, a key indicator in prescriptions.
In addition to early diagnosis of major human infectious disease pathogens, it is possible to prescribe antibiotics specific to infectious strains, thereby improving disease recovery and cure rates by reducing abuse of antibiotics, shortening treatment periods, and reducing costs. In addition, non-invasive collectible samples can be used to improve the sensitivity and specificity while pursuing ease of examination.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings and tables. The configurations shown in the figures, tables, and examples described herein are intended to illustrate the present invention most preferably, and the scope of the present invention is not limited by these examples, and may be substituted for them in the present application. It should be understood that there may be various equivalents and variations.
Also, within the scope of the present invention, various modifications may be made by those skilled in the art to which the present invention pertains, and such modifications are within the scope of the present invention.
Example 1 Primer Design and Synthesis in Multiplex PCR Reaction for Diagnostic Chip Analysis

Prior to the analysis of diagnostic chips that differentially analyze 64 pathogens (including bacteria and viruses) of representative human infectious diseases, primers included in the preceding multiplex-PCR reaction are US National Center for NCBI. The sequences of 64 pathogens retrieved from GenBank, the DNA database of Biotechnology Information (DDBJ), The DNA Data Bank of Japan (DDBJ) of Japan, and the European Molecular Biology Laboratory (EMBL) of the European Union, were analyzed. Target genes of 64 pathogens used in primer design include structural genes such as 5.8S, 16S, 18S, 23S, 28S ribosomal RNA (RNA), 16S rRNA-ITS (Internal transcribed spacer) -23S rRNA, Alternatively, individual genes capable of species-specific amplification were used as target regions of the primer design. The target site sequences for each infectious disease group were subjected to multiple alignments using the Clustal method, and thus, specific primer target sites for each pathogen were selected. Subsequently, primer primer (Primer premier version 5, Premier Biosoft International, Palo Alto, Calif., USA) and DNAiSmax (DNASIS MAX Version 2.7, MiraiBio Group, South San Francisco, CA, USA) programs were selected from the selected primer target sites. Application was made to design strain-specific primers. Primer selection conditions are as follows; Primer length is 18-25 base pair, melting temperature Tm (℃) is 58-63 ℃, GC content of 3 'end should not be high, and secondary structure formation is made by not complementary 4 or more bases in primer sequence consecutively. The formation of primer dimers was prevented by avoiding three or more identical bases consecutively located at the 3 'end. In addition, it was verified using a variety of sequencing tools commonly used by those skilled in the art that the primer sequence does not exhibit cross-reactive characteristics with other pathogens constituting the chip of the present invention. Table 1 shows the details of the primers used in the diagnostic chip for differential analysis of 64 pathogens of representative human infectious diseases. It was synthesized through the primer US IDT (Integrated DNA Technologies, Inc., San Jose, Calif., USA) used in the present invention.
The amplification product resulting from the multiplex-PCR reaction should be labeled with a fluorescent dye for chip hybridization. For this purpose, a primer labeled with a fluorescent dye at the 5 'or 3' end or fluorescent Both methods of labeling amplification products were identified by using a non-pigmented general primer and inducing the insertion of fluorescent dye-labeled deoxyribonucleotide triphosphate into the amplification product during the multiplex-PCR reaction. Fluorescent dyes used in the present invention are 6-FAM (6-carboxyfluorescein), Cy5, Cy3, Cy5.5, JOE (6-carboxy-4 ', 5'-dichloro-2', 7'-dimethoxyfluorescein), Rhodamine Green TAMRA NHS (N-hydroxysuccinimide) Ester, Texas Red. In the primer synthesis step, MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time-of-Flight) checks for impurities and purifies pure water by high-performance liquid chromatography (HPLC). It was. The concentration of the primer was checked through an ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, Maine, USA) and resuspended in tertiary deionized sterile distilled water to a final range of 50-200 pmole / uL and -70 ° C as aliquots. Cryopreservation.

Primer combination to perform 64 infectious diseases multiplex-PCR by disease group SEQ ID NO: Reaction target
Strain name Primer direction, base sequence (5'-3 ') Length One Fungus sp.
general Forward, GCATCGATGAAGAACGCAGC 20 2 Reverse, TCCTCCGCTTATTGATATGC 20 3 Rhizopus sp.
general Forward, ATTACCATGAGCAAATCAGA 20 4 Reverse, CAATCCAAGAATTTCACCTCTAG 23 5 Aspergillus sp.
general Forward, CGGCCCTTAAATAGCCCG 18 6 Reverse, GACCGGGTTTGACCAACTTT 20 7 Candida sp.
general Forward, GCATCGATGAAGAACGCAGC 20 8 Reverse, TCCTCCGCTTATTGATATGC 20 9 Mycobacterium
tuberculosis Forward, TTTCGCTGTTGTGGTTCTCA 20 10 Reverse, GGGCACTGGACCTGTATGAG 20 11 Mycobacterium sp.
general Forward, DCCKCYTTTCTAAGGWGCACC 21 12 Reverse, GATGCTCGCAACCACTATCCA 21 13 Human papilloma
Virus Forward, TTTBTHACHGTDGTDGAYACHAC 23 14 Reverse, GAAAAATAAACTGTAAATCATATTC 25 15 Proteus mirabilis Forward, GCGGTTTATCACGAAGGG 18 16 Reverse, GCTTGGCGAGATTGAGTGC 19 17 Enterobacter sp.
Forward, CCTGGACGAAGACTGACGC 19 18 Reverse, CGGACTACGACGCACTTTATG 21 19 Escherichia Coli
sp. Forward, AGCGTCGCAGAACATTACAT 20 20 Reverse, GGGCAACAAGCCGAAAGA 18 21 Enterococcus
faecalis Forward, AGTTTCTGCTGCTGATGGT 19 22 Reverse, TAACAACGCCTGAACCTAC 19 23 Staphylococcus sp.
general Forward, AGTATCTGCTGCTGACGGTCC 21 24 Reverse, GTAGCAACAGTACCACGACCA 21 25 Staphylococcus
aureus Forward, AATGGACGGCGGTATCTT 18 26 Reverse, TCAACACGGCCTGTAGCA 18 27 Streptococcus
agalactiae Forward, TGCGGTAACGAACGAAAT 18 28 Reverse, TTCACAAGGCGCTCACTCA 19 29 Streptococcus
pneumoniae Forward, TCGTTTCATCAAAGAGGGTAA 21 30 Reverse, CCGCAAGAAGAGTGGGATT 19 31 Corynebacterium sp.
Forward, CCGCAAGGCTAAAACTCAAAGGAAT 25 32 Reverse, ACCGACCACAAGGGAAAGACT 21 33 Pseudomonas
aeruginosa Forward, TGAAGGGTGACAACGAGGAG 20 34 Reverse, GCCCGCACTGAGGAATAAA 19 35 Veillonella sp.
Forward, TGAAAGGTGGCCTCTATTTAT 21 36 Reverse, CAATCCTTCTAACTGTTCGCAAG 23 37 Leptotrichia sp.
Forward, CAATTCTGTGTGTGTGAAGAAG 22 38 Reverse, ACAGTTTTGTAGGCAAGCCTAT 22 39 Lactobacillus sp.
Forward, TCTGCCTTGAAGATCGGAGTGC 22 40 Reverse, ACAGTTGATAGGCATCATCTG 21 41 Enterobacteriaceae
sp. Forward, TTTCCGTGTCGCCCTTAT 18 42 Reverse, CGACCGAGTTGCTCTTGC 18 43 Enterobacteriaceae
sp. Forward, CCGCTGGGAAACGGAACT 18 44 Reverse, CCCGCAGATAAATCACCACAAT 22 45 Enterobacteriaceae
sp. Forward, TGCCGCACCTCAACAAATC 19 46 Reverse, CAATAGCGTCGCCACCAA 18 47 Bacteria
general Forward, TCATAGACACGCCAGGACATA 21 48 Reverse, CAGATTCGGTAAAGTTCGTCA 21 49 Bacteria
general Forward, TGCTGTCCAGGCAGGTAGAT 20 50 Reverse, GGCATAAATCGCCGTGAAG 19 51 Bacteria
general Forward, GAAAAGGTACTCAACCAA 18 52 Reverse, ATAAGTAACGGTACTTAAATTG 22 53 Herpes Simplex
Virus general Forward, CCGAGTACGGCGGCTCCTTC 20 54 Reverse, TGCAGCTCGCACCACGCG 18 55 Herpes Simplex Virus, type 2 Forward, CGACAAGATTAACGCCAAGGG 21 56 Reverse, CGTCGCCAGCACAAACTCA 19 57 Haemophilus
ducreyi Forward, AGCGTGGGTGCCAGTAAAT 19 58 Reverse, GAAAGGTAGGCGTGAGAGAATC 22 59 Treponema
pallidum Forward, GGTATGAAGTTTGTCCCAGTTGC 23 60 Reverse, GCGTCATCACCGTAGTAGTCGT 22 61 Mycoplasma
hominis Forward, AATGGCTAATGCCGGATACGC 21 62 Reverse, AGGTACCGTCAGTCTGCAATCAT 23 63 Gardnerella
vaginalis Forward, GGGCGTATTGGTTGGATGC 19 64 Reverse, CCCCGAATACGCAACACCT 19 65 Candida
albicans Forward, CGACCAATAGAGGCGTTACAA 21 66 Reverse, ACGGATTTAGGTGGCAAGG 19 67 Trichomonas
vaginalis Forward, CTCAGTTCGCAAAGGCAGTC 20 68 Reverse, ATGCGATTGGCTGCTTGA 18 69 Ureaplasma
urealyticum Forward, CAGCATTAAAAATACTGGTGACCG 24 70 Reverse, ATTCCCTAACTTGTCGTCTAACTTC 25 71 Mycoplasma
genitalium Forward, AGTTGATGAAACCTTAACCCCTT 23 72 Reverse, TGAGGGGTTTTCCATTTTTGC 21 73 Chlamydiae
trachomatis Forward, TGGAGTGCTGGCTCGTATAAA 21 74 Reverse, GACCGCTGTCTCGCAAATC 19 75 Neisseria
gonorrhoeae Forward, CGGTTTCCGTGCGTTACG 18 76 Reverse, ACTGGTTTCATCTGATTACTTTCCA 25 77 Actinobacillus
actinomycetemcomitans Forward, GGGGCTTTCTACTACGGGAC 20 78 Reverse, AGCATCTGCGATCCCTGTAT 20 79 Porphyromonas
gingivalis Forward, GAATAACGGGCGATACGAG 19 80 Forward, GCTGACTTACCGAACAACCT 20 81 Treponema
denticola Reverse, AGAATAAGAAGAAGAGGGAATGCT 24 82 Forward, GCTTACCTAACCGCCTACATACC 23 83 Tannerella
forsythensis Reverse, CGGGCTGCAATGGAACTA 18 84 Forward, GCTTCTCAGGTCCCAGCAA 19 85 Prevotella
intermedia Reverse, CCAACCTTCCCTCCACTCG 19 86 Forward, CGTCAATCCTGCACGCTACTT 21 87 Fusobacterium
nucleatum Forward, CATTTATTGTGATGGAGGGACG 22 88 Reverse, CCTCTTCACTGCGACCCTC 19 89 Bacteria
general Forward, TCCTACGGGAGGCAGCAGT 19 90 Reverse, GGACTACCAGGGTATCTAATCCTGTT 26
Example 2 Specimen Collection and DNA Separation Therefrom

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For the non-invasive diagnosis which the present invention intends to realize, the same sensitivity and sensitivity can be obtained from almost all kinds of body fluids in addition to conventional tissue, swab, and urine samples. A test method showing specificity is required, and efficient DNA separation from the sample is essential. Specimen used in the present invention are VB1 (first-voided urine), VB3 (third-voided urine), prostate secretion (EPS, expressed prostatic secretion), semen (semen), urethral swab (urethral swab), ulcer swap (ulcer) swabs, urethral discharges, cervical swabs, blood, tissues, sputum, buccal swabs, nasal swabs. The collected samples were analyzed in a fresh state within 24 hours as much as possible. Swap samples were collected using a container filled with 3-5 mL of 1 × PBS (phosphate-buffered solution). The 1 × PBS solution was formulated with 8 g NaCl, 0.2 g KCl, 1.44 g Na ₂ HPO ₄ , 0.24 g KH ₂ PO ₄ in 1 L of tertiary sterile distilled water to a final pH of 7.4. Urine samples contain 0.5-2% (w / v) sodium azide, 1-3% (v / v) toluene or 1-2% (w / v) during transport. ) Was taken from the test vessel containing boric acid of Transported.

A commercially available DNA extraction kit (LaboPass ™ Tissue kit, Cosmojin Tech, Seoul, Korea) was used to separate genomic DNA from the sample. For urine specimens, take 1.5 mL of urine sample from the collection vessel and use a Denville 260D high-speed centrifuge (Denville Scientific Inc., Metuchen, NJ, USA) at 12,000 rpm (revolutions per minute). Water), and centrifuged for 1 minute. The supernatant was removed and the pellet was dissolved in 500 uL of 1X PBS solution and then washed again by centrifugation under the same conditions. Other low-volume samples were transferred to a 1.5 mL Eppendorf tube, centrifuged at 12,000 rpm for 2 minutes in a high-speed centrifuge, and the precipitate was freed with 500 uL of 1X PBS solution. The supernatant was removed by centrifugation. Add 200 uL of TL buffer to decompose sample to precipitate, add 20 uL of proteinase K (Proteinase K, 100 mg / mL), stir gently, and react at 56 ℃ for 10 minutes I was. Then 400 uL TB buffer was added to the reaction and stirred to mix the reaction. The reaction was then loaded onto the top of the spin column and centrifuged at 8,000 rpm for 1 minute. A new collection tube was replaced and 700 uL of BW buffer, which acts to bind the DNA extracted from the sample to the membrane of the column, was added and centrifuged under the same conditions. The collection tube was freshly replaced and 500 uL of NW buffer was added to remove impurities from the membrane and centrifuged at 13,000 rpm for 2 minutes. The top of the spin column was then transferred to a new Eppendorf tube, loaded with 100 uL of de-ionized sterile tertiary distilled water to elute DNA, treated for 5 minutes at room temperature, centrifuged at 8,000 rpm for 2 minutes, and purified pure DNA. Separated.

Example 3 Preparation of Target DNA of Differential Diagnosis of Infectious Diseases of 64 Human Pathogens by Multiplex-PCR

The present invention performs a multiplex-PCR for each infectious disease group, and at the same time performs a multiplex-PCR for antibiotic resistance determination, and then performs a hybridization reaction with a selective probe immobilized on the chip. It provides an analytical method that can determine not only the infection of species pathogens, but also the pathogen genotype and its antibiotic resistance. In the present embodiment will be described as a representative example of the multiplex-PCR process for determining the periodontitis (periodontitis) causing bacteria included in the analysis method. The multiplex-PCR reaction composition of other infectious diseases also differs in the volume of deionized tertiary sterile distilled water added according to the difference in the volume of the primer premix, and the other PCR reaction compositions and conditions are the same. Periodontitis which uses DNA isolated from various specimens as a template to amplify primer premixes capable of selectively amplifying six periodontal causative bacteria and two internal controls in a single reaction in the same PCR tube. Eight multiplex-PCRs were performed. Internal controls included in the reaction are primer sets (forward and reverse primers) that amplify human betaclobin genes and bacterial 16S ribosomal ribonucleic acid (ribosomal RNA). The reaction composition and reaction conditions of the multiplex PCR are shown in Table 2.

Periodontitis Multiplex-PCR Reaction Composition and Conditions
PCR reaction solution composition

Volume (uL)
PCR reaction conditions

Deionized Tertiary Sterilized Distilled Water

7

Initial denaturation
(Initial denaturation)
95 ° C., 15 minutes
1 time
Periodontitis 8 primers
Premix

3


denaturalization
(Denaturation)
95 ℃, 50 seconds



Episode 37





2X PCR Premix

15

Combination
(Annealing)
58 ° C., 60 seconds
Template DNA (40 ng or more)

5

extension
(Extension)
72 ° C., 60 seconds
Final volume

30

Last extension
(Extension)
72 ° C., 10 minutes
1 time

The final concentration of the PCR buffer in the multiplex-PCR reaction mixture was 50 mM KCl, 3.5 mM MgCl ₂ , 10 mM Tris-HCl, pH 8.2, 2.5 unit Taq polymerase, 300 uM dNTPs (Boehringer Mannheim, Mannheim) , Germany), 10 mg / mL Bovine serum albumin. Sense primers of periodontitis species-specific primers included in the periodontitis 8 primer premix were synthesized by labeling Cy3 fluorescent dyes at their 5 'ends (Integrated DNA Technologies, Inc., Coralville, IA, USA), 8 The composition ratio (unit is pmole / uL each) of the longitudinal forward / reverse primers is as follows. A. Actinomycetem Comitans 5 pmole each, P. Seriously 5 pmole each, T. Denticola 16 pmole each, T. Posiddensis 8 pmole each, P. Intermediate 5 pmole each, F. Nuclei Atom 7 pmole each, bacteria 16S rRNA 11 pmole each, human beta globin 7 pmole each was prepared.

In addition, the multiplex PCR kit of the present invention improves the sensitivity and specificity of the reaction by allowing Cy3 fluorescent dye-labeled deoxycytosine triphosphate (Cy3-dCTP) to be added as nucleotides constituting the amplification product during the PCR process. I could make it. In the periodontitis 8 multiplex-PCR reaction using Cy3-dCTP, a primer premix without fluorescent dye was used. The final concentrations of the reactants constituting the PCR buffer were 50 mM KCl, 3.5 mM MgCl ₂ , and 10 mM Tris-. HCl, pH 8.2, 2.5 unit Taq polymerase, 300 uM dATP, 300 uM dGTP, 300 uM dTTP, 25 uM dCTP, 275 uM Cy3-dCTP (FluoroLink Cy3-dCTP, Amersham Pharmacia Biotech AB, Piscataway, NJ, USA ), 10 mg / mL bovine serum albumin.

<Example 4> Design and synthesis of probes constituting chips for differential diagnosis of 64 pathogens of human infectious diseases

The high-specificity probes used for the 64 pathogen differential diagnosis chips of representative human infectious diseases are 64 species searched from the US NCBI sequencing database GenBank, Japan's DDBJ and the European Union's EMBL sequencing database. The base sequence of the pathogen was analyzed and produced. Target genes of 64 pathogens used in probe design target structural genes such as 5.8S, 16S, 18S, 23S, 28S ribosomal RNA (RNA), 16S rRNA-ITS (Internal transcribed spacer) -23S rRNA, or Individual genes such as plasmids, pseudogenes, and repeated elements were used as target sites for probe design.
Multiple alignments of target site sequences by the Clustal method for each infectious disease group are performed, and accordingly, specific PCR primer target sites are selected for each pathogen, and then the selected primer target sites are targeted. Primer Premier, dienex max, or oligoarray programs were used to design probe sequences that bind with high specificity within strain-specific amplification products that are amplified from primer sets. The selection conditions for the probe design are as follows; The probe length was limited to 18-30 base pairs, melting temperature Tm (℃) was 60-70 ℃, GC content was 40-65% range, and secondary structure formation, self hybridization or cross hybridization formation was examined. In addition, it was verified that the designed probe does not exhibit cross-reactive characteristics with other pathogen probes constituting the chip using various sequencing tools commonly used in the art. Details of the probes used in the 64 differentiation chips for representative human infectious diseases are described in Table 3. A species-specific probe was synthesized by labeling amino modifiers at the 5 'end or 3' end of the DNA or PNA probe sequence of interest (MWG-Biotech AG, Ebersberg, Germany). When modifying the amino modifier at the 5 'end of the probe, 6-12 carbon spacers were added to promote chip reaction efficiency. No spacer was used when modifying the amino modifier at the 3 'end of the probe. The amine groups of the probe have the properties of primary amine groups and are attached to the aldehyde-activated or carboxyl-activated chip surface. The concentration of the probe was converted to absorbance value at 260 nm, and it was checked for impurities by MALDI-TOF, purified by high performance liquid chromatography (HPLC), and then the final concentration was 100 in sterile tertiary distilled water. Prepared to be -300 pM.

Probe combinations that make up 64 different pathogens for human infectious diseases order
List
number Reaction target
Strain name Probe orientation, nucleotide sequence (5'-3 ') Length
91 Fungus sp.
general
Reverse, GCATCGATGAAGAACGCAGC
20
92 Fungus sp.
general Forward, TTGACCTCRRATCAGGTAGGRATACCCGCTGAACTTAA
38
93 Rhizopus sp.
general
Reverse, CTAGCGGCCAAATACAAATGC
21
94 Rhizopus sp.
general
Reverse, TTCACCTCTAGCGGCCAAAT
20
95 Aspergillus sp.
general
Reverse, GGCTTGAGCCGATAGTCCCC
20
96 Aspergillus sp.
general
Forward, TCAAGCCGATGGAAGTGCG
19
97 Candida sp.
general
Reverse, GAAGGCAACACCAAACCCG
19
98 Candida sp.
general
Reverse, TCCTACCTGATTTGAGGGCGA
21
99 Mycobacterium
avium
Forward, GACCGAGTGTTGTCTCAGGGC
21
100 Mycobacterium
chelonae
Forward, ATTTCCCAGCCGAATGAGC
19
101 Mycobacterium
flavescens
Forward, GGTCTGGTGTCGCCCTGTCTT
21
102 Mycobacterium
gordonae
Forward, CTCGGGTGCTGTCCCTCCA
19
103 Mycobacterium
kansasii
Forward, GAGGCAACACTCGGGCTCTG
20
104 Mycobacterium
simiae
Forward, TTCGGTTGAAGTGGTGTCCCTC
22
105 Mycobacterium
szulgai
Forward, CGGCAACGAACAAGCCAGACA
21
106 Mycobacterium
vaccae
Forward, CGGCGAGGGAAATCATCAGACA
22
107 Mycobacterium
fortuitum
Forward, GTCTTACCCGAGCCGTGAGGA
21
108 Mycobacterium
intracellulare
Forward, CCCTGAGACAACACTCGGTCG
21
109 Mycobacterium
tuberculosis
Forward, TTGGGTCCTGAGGCAACACTCG
22
110 Mycobacterium
abscessus
Forward, TTGGGTCCTGAGGCAACACG
20
111 Mycobacterium
bovis
Forward, TTGGGTCCTGAGGCAACACTCG
22
112 Human Papilloma Virus
type 16
Forward, ACCTCCAGCACCTAAAGAAGAT
22
113 Human Papilloma Virus type 18
Reverse, GGACCCGTGTATACAGGCACAT
23
114 Human Papilloma Virus type 31
Reverse, ATGGATCTTCCTTGGGCTTTT
21
115 Human Papilloma Virus
type 33
Forward, CAGGCTATTACGTGTCAAAAAAC
23
116 Human Papilloma Virus
type 35
Reverse, CCAGAAGGCGGTGGTGTAAG
20
117 Human Papilloma Virus
type 39
Reverse, CAAACTGGCAGATGGTGGAG
20
118 Human Papilloma Virus
type 52
Forward, CCCCACCACCGTCTGCATC
19
119 Human Papilloma Virus
type 56
Forward, GGGTTATCCCCGCCAGTG
18
120 Human Papilloma Virus
type 58
Reverse, GTCCTGTAAACTGGCAGACGG
21
121 Human Papilloma Virus
type 6
Forward, CCCCAAATGGTACATTAGAAGATA
24
122 Human Papilloma Virus
type 11
Reverse, CCATTTGGTGGAGGCGATA
19
123 Human Papilloma Virus
type 30
Forward, AACTCCACTTTACTTGAGGGCTG
23
124 Human Papilloma Virus
type 54
Reverse, TGCAGGGGCATTATTCTTTTG
21
125 Human Papilloma Virus
type 62
Forward, TCACTATTTGCAGTCTCGGGCTA
23
126 Proteus
mirabilis
Forward, CGCACTCAATCTCGCCAAG
19
127 Proteus
mirabilis
Reverse, ATGGCATTTAGAGGATGTAGCA
22
128 Proteus
mirabilis
Forward, GCGGTTTATCACGAAGGGGT
20
129 Enterobacter sp.
general
Reverse, GACATCGTTTACGGCGTGGACT
22
130 Enterobacter sp.
general
Reverse, CCTCAAGGGCACAACCTCCAAG
22
131 Enterobacter sp.
general
Reverse, TCAGGTGCGAAAGCGTGGG
19
132 Escherichia Coli sp.
general
Reverse, CGTCCGATCACCTGCGTCAA
20
133 Escherichia Coli sp.
general
Reverse, GCGAAGAGGCAGTCAACGGG
20
134 Escherichia Coli sp.
general
Reverse, GGGCAACAAGCCGAAAGAACTG
22
135 Enterococcus
faecalis
Reverse, GGAACATCATCGCCTGGGAA
20
136 Enterococcus
faecalis
Reverse, GATAACTGGAACATCATCGCC
21
137 Enterococcus
faecalis
Reverse, ATGATGTTCCAGTTATCGCAGG
22
138 Staphylococcus sp.
general
Reverse, CGTATTGAGCATCGCCTTCTA
21
139 Staphylococcus sp.
general
Reverse, CGTATTGAGCATCGCCTTC
19
140 Staphylococcus sp.
general
Reverse, TTAGAAGGCGATGCTCAATAC
21
141 Staphylococcus
aureus
Reverse, TCGTATTGAGCATCGCCTT
19
142 Staphylococcus
aureus
Reverse, CTTCGTATTGAGCATCGCC
19
143 Staphylococcus
aureus
Forward, AAGGCGATGCTCAATACGA
19
144 Streptococcus agalactiae
Forward, GAGTATCAAGCAGCCCACG
19
145 Streptococcus agalactiae
Forward, ATCAAGCAGCCCACGATTC
19
146 Streptococcus agalactiae
Reverse, AAGGAATACATGCTGTTGCG
20
147 Streptococcus pneumoniae
Forward, GCTACCCGATGAGTTTGTTGTT
22
148 Streptococcus pneumoniae
Forward, AGCTACCCGATGAGTTTGTTGTT
23
149 Streptococcus pneumoniae
Reverse, CGATAACAACAAACTCATCGGGT
23
150 Corynebacterium sp.
general
Forward, GCACAAGCGGCGGAGCAT
18
151 Corynebacterium sp.
general
Reverse, ATGCTCCGCCGCTTGTGC
18
152 Corynebacterium sp.
general
Forward, TGCAACGCGAAGAACCTTACCT
22
153 Pseudomonas
aeruginosa
Reverse, CCGTACACGCCGGTAGCA
18
154 Pseudomonas
aeruginosa
Reverse, GCCGGGTCCAGGATGCCC
18
155 Veillonella sp.
general
Reverse, CCACATTGGGACTGAGACACGG
22
156 Veillonella sp.
general
Forward, TCCTACGGGAGGCAGCAGTG
20
157 Veillonella sp.
general
Forward, CTACGGGAGGCAGCAGTGGG
20
158 Leptotrichia sp.
general
Reverse, CGGATAACGCTCGCAACATA
20
159 Leptotrichia sp.
general
Forward, TATGTTGCGAGCGTTATCCG
20
160 Leptotrichia sp.
general
Forward, AGGCGGTAAGACAAGTTGAAGG
22
161 Lactobacillus sp.
general
Reverse, CGTGTTACTCACCCGTCCGC
20
162 Lactobacillus sp.
general
Forward, CGGCGGACGGGTGAGTAA
18
163 Lactobacillus sp.
general
Forward, AGCGGCGGACGGGTGAGT
18
164 Enterobacteriaceae
sp.
Reverse, GAATAAGGGCGACACGGAAA
20
165 Enterobacteriaceae
sp.
Forward, TTTCCGTGTCGCCCTTATTC
20
166 Enterobacteriaceae
sp.
Reverse, CAGCACGGAGCGGATCAACG
20
167 Enterobacteriaceae
sp.
Forward, CGCCCTGCTTGGCCCGAATA
20
168 Enterobacteriaceae
sp.
Reverse, CGTTGATTTGTTGAGGTGCGG
21
169 Enterobacteriaceae
sp.
Reverse, TACCGCCACCGCCATACC
18
170 Bacteria
general
Reverse, CGAGTTTGTGCTTGTACGCCAT
22
171 Bacteria
general
Forward, AAAGATGGCGTACAAGCACAAAC
23
172 Bacteria
general
Forward, TGATCTTCACGGCGATTTATGC
22
173 Bacteria
general
Forward, CATTGGACCGCTGATCTTCACG
22
174 Bacteria
general
Reverse, TTGGCGTGTTTCATTGCTTG
20
175 Bacteria
general
Reverse, ATCAAGCAATGAAACACGCCAA
22
176 Herpes Simplex Virus
general
Forward, CACATCAAGGTGGGCCAGCCGC
22
177 Herpes Simplex Virus
general
Reverse, TGCGGCTGGCCCACCTTGATG
21
178 Herpes Simplex Virus
general
Reverse, CCAGGTAGTACTGCGGCTGGCC
22
179 Herpes Simplex Virus
type 2
Reverse, CCGTGGAGCGGCAGACCCC
19
180 Herpes Simplex Virus
type 2
Reverse, GCCGTGGAGCGGCAGACC
18
181 Herpes Simplex Virus
type 2
Reverse, TGGCCGTGGAGCGGCAGACC
20
182 Haemophilus
ducreyi
Forward, GCGCCGTATCGGTTGGGT
18
183 Haemophilus
ducreyi
Reverse, AAGGTAGGCGTGAGAGAATCAAAAA
25
184 Haemophilus
ducreyi
Reverse, CGTAGGCATCAAGAAGGTAAAGCG
24
185 Treponema
pallidum
Reverse, AGGAACCGCAACTGGGACAAA
21
186 Treponema
pallidum
Reverse, GAGGAACCGCAACTGGGACA
20
187 Treponema
pallidum
Forward, TGAAGTTTGTCCCAGTTGCGGT
22
188 Mycoplasma
hominis
Reverse, ACTAATGTTCCGCACCCTCATCT
23
189 Mycoplasma
hominis
Forward, AGATGAGGGTGCGGAACATTAGT
23
190 Gardnerella
vaginalis
Forward, GCTGCCGAGTGGGCTTTG
18
191 Gardnerella
vaginalis
Forward, GTCAGGTGTTGCGTATTCGGG
21
192 Candida
albicans
Reverse, GCATCTCCAATCATTCGCCTA
21
193 Candida
albicans
Forward, AGATGCCTTGCCACCTAAATCC
22
194 Trichomonas
vaginalis
Reverse, GGACTGCCTTTGCGAACTGA
20
195 Trichomonas
vaginalis
Reverse, GGCTGCTTGACCATCCGAAA
20
196 Ureaplasma
urealyticum
Forward, GGGGATGAACTCTACTATGAAGTTA
25
197 Ureaplasma
urealyticum
Reverse, GTTAACTAAGCCGTTTACACCTCAA
25
198 Mycoplasma
genitalium
Reverse, ATATTTAAGTTGTCATTTTGGCTTC
25
199 Mycoplasma
genitalium
Forward, AAGAAGCCAAAATGACAACTTAAAT
25
200 Chlamydiae
trachomatis
Reverse, GAGATAGGAAACCAACTCTACGCTG
25
201 Chlamydiae
trachomatis
Forward, CAGCGTAGAGTTGGTTTCCTATCTC
25
202 Neisseria
gonorrhoeae
Reverse, GCAGGCGTATAGGCGGACTTG
21
203 Neisseria
gonorrhoeae
Reverse, GGGAATCGTAACGCACGGAAA
21
204 Actinobacillus actinomycetemcomitans
Forward, GGGGCTTTCTACTACGGGACCT
22
205 Actinobacillus actinomycetemcomitans
Reverse, CAGCATCTGCGATCCCTGTAT
21
206 Porphyromonas gingivalis
Reverse, TACCGAACAACCTACGCACCCT
22
207 Porphyromonas gingivalis
Forward, GCGGTAATACGGAGGATGCG
20
208 Treponema
denticola
Reverse, GCCTACATACCCTTTACGCCCA
22
209 Treponema
denticola
Reverse, GGGCTTATTCGCATGACTACCG
22
210 Tannerella forsythensis
Reverse, CGGGCGTGGGATTGGTGATG
20
211 Tannerella forsythensis
Reverse, TGTATCGGGCGTGGGATTGGT
21
212 Prevotella
intermedia
Forward, ATGGCATCTGACGTGGACCAAA
22
213 Prevotella
intermedia
Reverse, CGTAGCCTTGGTGGGCCGTTA
21
214 Fusobacterium nucleatum
Reverse, TTCTGCGTCCCTCCATCACA
20
215 Fusobacterium nucleatum
Reverse, ACTTCCGTTCGTCCGTGC
18
216 Bacteria
general
Reverse, CGTATTACCGCGGCTGCTGGCAC
23

<Example 5> Chips for differential diagnosis of 64 pathogens for human infectious diseases

Representative human infectious disease pathogen probe constituting the present invention for the ease of analysis of the analysis results were divided into a group of diseases, and a chip grid was prepared in a total of seven groups including the antibiotic resistance analysis group (see Figure 1). Eight grids were prepared on one chip substrate and designed to perform simultaneous analysis of eight different samples through an 8 well hybridization reaction chamber. After thawing at 70 ° C., the probe stock was stored at room temperature, and then diluted with deionized sterile tertiary distilled water to a final concentration of 10 nM and used as a working stock. A 50-fold dilution of the working stock was mixed with 3X SSC spotting solution (500 mM NaCl, 3 mM sodium citrate, 1.5 MN, N, N-trimethylglycine, pH 6.8) in a 1: 5-10 ratio (v / v). The concentration range of the probe dispensed into the final 96 well plate was 20-40 pmole / uL. The plate was mounted on a Microssys 5100 microarrayer (Cartesian Technologies, Ann Arbor, MI, USA) and from this to an aldehyde-, thioisocyanate-, or carboxyl-activated glass slide (CEL associates Inc., Houston, TX, USA). Or, spotted duplicates of the probes in sequence on the epoxy-activated plastic chip surface. The average diameter of the spots is 80-150 micrometers and the distance between spots was maintained at 400-500 micrometers to minimize cross-talk effects between spots. Chip fabrication was carried out in a class 10,000 room maintaining 74% humidity. The probe spotted chips were baked at 120 ° C. for 1 hour, and then washed for 3 minutes in 0.25% SDS (sodium dodecyl sulfate) solution and again with sterile tertiary distilled water. The probe was then blocked by reacting the chip with a solution containing 0.2% sodium borohydride (NaBH ₄ ). After washing twice with 3 distilled water, the water was removed and stored in a desiccator (dessicator) until the point of use.

<Example 6> Chip hybridization reaction and results analysis for differential diagnosis of 64 pathogens for human infectious diseases

For differential diagnosis of 64 pathogens of representative human infectious diseases in accordance with one preferred embodiment of the present invention, each PCR reaction product 6 uL after each of the six major infectious diseases and for the analysis of antibiotic resistance 42 uL of deionized tertiary sterile distilled water was added to 42 uL, which was thermally denatured at 95 ° C. for 5 minutes, and then preserved on ice for 5 minutes and spun down with a centrifuge. An 8 well hybridization reaction chamber was placed on the chip surface and the well top layer was covered with a well cover. Thereafter, 80 uL of the hybridization reaction solution (3X SSC, 0.1% SDS, 0.2 mg / mL bovine serum albumin, pH 7), which was previously heated to 56 ° C., the hybridization temperature of the reaction mixture solution, in the well to be reacted. After the addition and mixing, the reaction mixture was injected through the hole of the well cover, and care was taken not to generate bubbles. After fixing the chamber lid, hybridization reaction was performed at 56 ° C. for 30 minutes to induce specific nucleotide complementary bonds between the probe on the chip surface and the multiplex-PCR reaction product. The well cover of the chip surface where the hybridization reaction was completed was removed, the chip was immersed in the washing buffer 1 (0.1X SSC, 0.05% SDS) solution, washed with stirring at 2,000 rpm for 2 minutes, and this was repeated once. After washing with washing buffer 2 (2X SSC, 0.1% SDS) solution at 2,000 rpm for 2 minutes and this was repeated once. Thereafter, the chips were dried by immersion in deionized tertiary sterile distilled water twice and centrifuged at 1,000 rpm. The chip was read using a ScanArray Lite, Packard Instrument Co., Meriden, CT, USA scanner and the mean fluorescence intensity and standard of positive control spots using analytical software (QuantArray 2.0). The signal-to-noise (S / R) ratio was obtained by comparing the error with the values around the spot, and when this value was 5 or more, it was scored as a positive value.

<Example 7> Performance verification through comparison test between 64 types of pathogen differential diagnosis chip of human infectious disease

The present invention provides a composition that differentially analyzes not only the main pathogen infection of human infectious diseases, but also the infected pathogen genotype and antibiotic resistance. This example confirmed the performance of the present invention by comparing the results of the chip analysis of the urinary tract infection pathogen group among the six major infectious diseases constituting the present invention with the PCR methodology, which is the state of the art. Prior to comparative testing with clinical specimens, screening between the two methods using standard strains obtained from the Korea Institute of Bioscience and Biotechnology Center (KCTC, Daejeon, Korea) or US ATCC (American Tissue Type Collection, Manassas, VA, USA) Sensitivity and specificity were compared. Standard strains used for comparison were Pseudomonas aeruginosa (KCTC1637), Shigella flexneri (KCTC2008), Salmonella typhimurium (KCTC2057), E. coli K12 (Escherichibacter Co., Ltd. Camphylobacter jejuni (ATCC43431), Enterococcus faecalis (ATCC10741), Enterobacter cloacae (ATCC10699), Staphylococcus aureus (ATCC10390), Streptococcus streptococcus agalactiae, ATCC14364), Klebsiella oxytoca (ATCC13182), Haemophilus influenzae (ATCC10211), Lactobacillus sp. (ATCC10746), Acinetobacter (Acinetobacter 71 sp.) It's a species. The comparative method is added to the final culture of the body fluid (negatively determined) 3 mL of the strain is added to the final 1 X 10 ^{1-10 5} CFU (colony forming unit) / mL ² single strain, double infection by strain And a total of 130 samples were compared by simulating multiple infections. The comparison results are summarized in Table 4.

Comparison of chips and prior art (PCR) for urinary tract infectious bacteria group
Compare group or condition
Diagnostic chip
(percentage,%)
Multiplex-PCR
(percentage,%)
13 individual single infection mock groups
(N = 130)

126
(96.9)
121
(93.1)
13 kinds of duplicate infection group
(N = 65)

64
(96.9)

53
(81.5)

13 multiple mono-infection mock groups
(N = 45)

43
(95.6)

34
(75.6)

Positive control
(N = 10)

10
(100)

9
(90)

Negative control
(N = 10)
10
(100)

10
(100)

DNA Separation from Specimen

same
same
Inspection time

3.5 hours
2.5 hours
Number of samples available for one analysis

Less than 1,000
Less than 100
Readable target strain

60 to 90 species

6-8 types

 In case of chip analysis for differential diagnosis of infectious disease pathogen, DNA was separated from body fluid-derived sample in the same manner as in Example 2, and then the multiplex-PCR reaction was performed in the same manner as described in Example 3. It was. However, the used 10 urinary infection primer premix volume is 4 uL and PCR reaction conditions are shown in Table 5. Thereafter, a chip was manufactured in the same manner as in Example 5, and hybridization, washing, and chip scanning were performed in the same process as in Example 6.

Urinary Tract Infection Multiplex-PCR Reaction Composition and Conditions
PCR reaction solution composition

Volume (uL)
PCR reaction conditions

Deionized Tertiary Sterilized Distilled Water

6

Initial denaturation
(Initial denaturation)
95 ° C., 15 minutes
1 time
Urinary Tract Infection 10 Primers
Premix

4


denaturalization
(Denaturation)
95 ℃, 50 seconds



Episode 37





2X PCR Premix

15

Combination
(Annealing)
58 ° C., 60 seconds
Template DNA (40 ng or more)

5

extension
(Extension)
72 ° C., 60 seconds
Final volume

30

Last extension
(Extension)
72 ° C., 10 minutes
1 time

 In the case of multiplex-PCR analysis, the results are read by performing the above 10 urinary tract infection multiplex-PCR reaction steps and checking the size of the reaction product through agarose gel electrophoresis. Or, if necessary, the nucleotide sequence was confirmed through a very common DNA automatic base sequence analyzer (ABI3130xL genetic analyzer, Applied Biosystems Inc., Foster city, CA, USA). The comparison test was conducted in a duplicate blind method in which two different examiners duplicated the same specimen individually and read the results, and classified the test failures when the results were not identical.

Figure 1 shows a chip (chip) arranged in groups of 64 infectious diseases pathogens and antibiotic resistance analysis according to the present invention, grouped by group, hybridization between the target DNA labeled with the fluorescent material and the probe arranged on the chip ( An 8 well hybridization reaction chamber in which a hybridization reaction is performed is shown, and a grid arrangement for each disease group of probes spotted on a chip is shown.

FIG. 2 is a flowchart illustrating a process of designing a probe having high specificity and having no cross-talk with other bacteria or viruses except for the pathogen.

3 is a chip for amplifying target DNA through a multiplex-PCR reaction by separating DNA from various samples according to the present invention, and analyzing the presence, infection, genotype and antibiotic resistance of 64 infectious diseases pathogens (chip) A flow chart showing the major steps in the analysis process.

4 is a diagram comparing DNA concentrations by sample type by agarose gel electrophoresis after separating DNA from various samples according to Example 2 of the present invention. Well numbers 1-19 are cervical swaps, numbers 20-39 are blood, numbers 39-49 are VB1 urine, numbers 50-69 are oral swabs, numbers 70-79 are prostate secretions, numbers 80-96 are tissues DNA isolated from can be identified.

FIG. 5 shows the target DNA distribution of three clinical specimens isolated from oral swab samples according to the present invention and amplified by periodontitis eight multiplex-PCR reactions. Three specimens of S1 and two of S2 could identify infections of the periodontal causative organism, and sample S3 could confirm negative results.

Figure 6 shows a chip scan image and chip grid showing the analysis results of 64 infectious diseases pathogens and antibiotic resistance analysis in accordance with the present invention. The subjects were human papilloma virus type 35, Herpes simplex virus (HSV) type 2 in mysterious disease group, Mycoplasma hominis, ureaplasma urea lyticum, Chlamydia trachomatis, Neisseria gonorrea, and E. coli in UTI. Streptococcus agalactia and Pseudomonas aeruginosa were infected and these bacteria were resistant to cephalosporin / penicillin, tetracycline and macrolide antibiotics.

Figure 7 shows another chip scan image and chip grid showing the analysis results of 64 infectious diseases pathogens and antibiotic resistance analysis in accordance with the present invention. The subjects were Staphylococcus aureus, Enterococcus faecalis in the urinary tract infection group, Mycobacterium fortuitum in the mycobacteria group, Mycobacterium intracellular and periodontitis group in the group of treponima dentica, Fusobacter Fungal and Candida bacteria were infected in the Leeum nucleatum and sinusitis groups, and these bacteria were resistant to cephalosporin / penicillin, tetracycline, and macrolide antibiotics.
8 is a schematic diagram comparing the main diagnosis of infectious disease differential diagnosis chip and the conventional single infectious disease diagnosis chip according to a preferred embodiment of the present invention.

<110> PARK, MinKoo <120> Novel probes, multiplex-PCR kit, DNA chip, PNA chip required for          multiplex-PCR and antibiotics resistance analysis to detect          infectious diseases-related microorganisms and method <160> 216 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Fungus spp. <400> 1 gcatcgatga agaacgcagc 20 <210> 2 <211> 20 <212> DNA <213> Fungus spp. <400> 2 tcctccgctt attgatatgc 20 <210> 3 <211> 20 <212> DNA <213> Rhizopus spp. <400> 3 attaccatga gcaaatcaga 20 <210> 4 <211> 23 <212> DNA <213> Rhizopus spp. <400> 4 caatccaaga atttcacctc tag 23 <210> 5 <211> 18 <212> DNA <213> Aspergillus spp. <400> 5 cggcccttaa atagcccg 18 <210> 6 <211> 20 <212> DNA <213> Aspergillus spp. <400> 6 gaccgggttt gaccaacttt 20 <210> 7 <211> 20 <212> DNA Candida spp. <400> 7 gcatcgatga agaacgcagc 20 <210> 8 <211> 20 <212> DNA Candida spp. <400> 8 tcctccgctt attgatatgc 20 <210> 9 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <400> 9 tttcgctgtt gtggttctca 20 <210> 10 <211> 20 <212> DNA <213> Mycobacterium tuberculosis <400> 10 gggcactgga cctgtatgag 20 <210> 11 <211> 21 <212> DNA <213> Mycobacterium spp. <400> 11 dcckcytttc taaggwgcac c 21 <210> 12 <211> 21 <212> DNA <213> Mycobacterium spp. <400> 12 gatgctcgca accactatcc a 21 <210> 13 <211> 23 <212> DNA <213> Human Papilloma Virus L1 region <400> 13 tttbthachg tdgtdgayac hac 23 <210> 14 <211> 25 <212> DNA <213> Human Papilloma Virus L1 region <400> 14 gaaaaataaa ctgtaaatca tattc 25 <210> 15 <211> 18 <212> DNA <213> Proteus mirabilis <400> 15 gcggtttatc acgaaggg 18 <210> 16 <211> 19 <212> DNA <213> Proteus mirabilis <400> 16 gcttggcgag attgagtgc 19 <210> 17 <211> 19 <212> DNA <213> Enterobacter sp. <400> 17 cctggacgaa gactgacgc 19 <210> 18 <211> 21 <212> DNA <213> Enterobacter sp. <400> 18 cggactacga cgcactttat g 21 <210> 19 <211> 20 <212> DNA Escherichia coli sp. <400> 19 agcgtcgcag aacattacat 20 <210> 20 <211> 18 <212> DNA Escherichia coli sp. <400> 20 gggcaacaag ccgaaaga 18 <210> 21 <211> 19 <212> DNA <213> Enterococcus faecalis <400> 21 agtttctgct gctgatggt 19 <210> 22 <211> 19 <212> DNA <213> Enterococcus faecalis <400> 22 taacaacgcc tgaacctac 19 <210> 23 <211> 21 <212> DNA <213> Staphylococcus sp. <400> 23 agtatctgct gctgacggtc c 21 <210> 24 <211> 21 <212> DNA <213> Staphylococcus sp. <400> 24 gtagcaacag taccacgacc a 21 <210> 25 <211> 18 <212> DNA <213> Staphylococcus aureus <400> 25 aatggacggc ggtatctt 18 <210> 26 <211> 18 <212> DNA <213> Staphylococcus aureus <400> 26 tcaacacggc ctgtagca 18 <210> 27 <211> 18 <212> DNA <213> Streptococcus agalactiae <400> 27 tgcggtaacg aacgaaat 18 <210> 28 <211> 19 <212> DNA <213> Streptococcus agalactiae <400> 28 ttcacaaggc gctcactca 19 <210> 29 <211> 21 <212> DNA <213> Streptococcus pneumoniae <400> 29 tcgtttcatc aaagagggta a 21 <210> 30 <211> 19 <212> DNA <213> Streptococcus pneumoniae <400> 30 ccgcaagaag agtgggatt 19 <210> 31 <211> 25 <212> DNA <213> Corynebacterium sp. <400> 31 ccgcaaggct aaaactcaaa ggaat 25 <210> 32 <211> 21 <212> DNA <213> Corynebacterium sp. <400> 32 accgaccaca agggaaagac t 21 <210> 33 <211> 20 <212> DNA <213> Pseudomonas aeruginosa <400> 33 tgaagggtga caacgaggag 20 <210> 34 <211> 19 <212> DNA <213> Pseudomonas aeruginosa <400> 34 gcccgcactg aggaataaa 19 <210> 35 <211> 21 <212> DNA <213> Veillonella sp. <400> 35 tgaaaggtgg cctctattta t 21 <210> 36 <211> 23 <212> DNA <213> Veillonella sp. <400> 36 caatccttct aactgttcgc aag 23 <210> 37 <211> 22 <212> DNA <213> Leptotrichia sp. <400> 37 caattctgtg tgtgtgaaga ag 22 <210> 38 <211> 22 <212> DNA <213> Leptotrichia sp. <400> 38 acagttttgt aggcaagcct at 22 <210> 39 <211> 22 <212> DNA <213> Lactobacillus sp. <400> 39 tctgccttga agatcggagt gc 22 <210> 40 <211> 21 <212> DNA <213> Lactobacillus sp. <400> 40 acagttgata ggcatcatct g 21 <210> 41 <211> 18 <212> DNA <213> Enterobacteriaceae sp. <400> 41 tttccgtgtc gcccttat 18 <210> 42 <211> 18 <212> DNA <213> Enterobacteriaceae sp. <400> 42 cgaccgagtt gctcttgc 18 <210> 43 <211> 18 <212> DNA <213> Enterobacteriaceae sp. <400> 43 ccgctgggaa acggaact 18 <210> 44 <211> 22 <212> DNA <213> Enterobacteriaceae sp. <400> 44 cccgcagata aatcaccaca at 22 <210> 45 <211> 19 <212> DNA <213> Enterobacteriaceae sp. <400> 45 tgccgcacct caacaaatc 19 <210> 46 <211> 18 <212> DNA <213> Enterobacteriaceae sp. <400> 46 caatagcgtc gccaccaa 18 <210> 47 <211> 21 <212> DNA <213> Bacteria general <400> 47 tcatagacac gccaggacat a 21 <210> 48 <211> 21 <212> DNA <213> Bacteria general <400> 48 cagattcggt aaagttcgtc a 21 <210> 49 <211> 20 <212> DNA <213> Bacteria general <400> 49 tgctgtccag gcaggtagat 20 <210> 50 <211> 19 <212> DNA <213> Bacteria general <400> 50 ggcataaatc gccgtgaag 19 <210> 51 <211> 18 <212> DNA <213> Bacteria general <400> 51 gaaaaggtac tcaaccaa 18 <210> 52 <211> 22 <212> DNA <213> Bacteria general <400> 52 ataagtaacg gtacttaaat tg 22 <210> 53 <211> 20 <212> DNA <213> Herpes Simples Virus <400> 53 ccgagtacgg cggctccttc 20 <210> 54 <211> 18 <212> DNA <213> Herpes Simplex Virus <400> 54 tgcagctcgc accacgcg 18 <210> 55 <211> 21 <212> DNA <213> Herpes Simplex Virus type 2 <400> 55 cgacaagatt aacgccaagg g 21 <210> 56 <211> 19 <212> DNA <213> Herpes Simplex Virus type 2 <400> 56 cgtcgccagc acaaactca 19 <210> 57 <211> 19 <212> DNA <213> Haemophilus ducreyi <400> 57 agcgtgggtg ccagtaaat 19 <210> 58 <211> 22 <212> DNA <213> Haemophilus ducreyi <400> 58 gaaaggtagg cgtgagagaa tc 22 <210> 59 <211> 23 <212> DNA <213> Treponema pallidum <400> 59 ggtatgaagt ttgtcccagt tgc 23 <210> 60 <211> 22 <212> DNA <213> Treponema pallidum <400> 60 gcgtcatcac cgtagtagtc gt 22 <210> 61 <211> 21 <212> DNA <213> Mycoplasma hominis <400> 61 aatggctaat gccggatacg c 21 <210> 62 <211> 23 <212> DNA <213> Mycoplasma hominis <400> 62 aggtaccgtc agtctgcaat cat 23 <210> 63 <211> 19 <212> DNA <213> Gardnerella vaginalis <400> 63 gggcgtattg gttggatgc 19 <210> 64 <211> 19 <212> DNA <213> Gardnerella vaginalis <400> 64 ccccgaatac gcaacacct 19 <210> 65 <211> 21 <212> DNA Candida albicans <400> 65 cgaccaatag aggcgttaca a 21 <210> 66 <211> 19 <212> DNA Candida albicans <400> 66 acggatttag gtggcaagg 19 <210> 67 <211> 20 <212> DNA <213> Trichomonas vaginalis <400> 67 ctcagttcgc aaaggcagtc 20 <210> 68 <211> 18 <212> DNA <213> Trichomonas vaginalis <400> 68 atgcgattgg ctgcttga 18 <210> 69 <211> 24 <212> DNA <213> Ureaplasma urealyticum <400> 69 cagcattaaa aatactggtg accg 24 <210> 70 <211> 25 <212> DNA <213> Ureaplasma urealyticum <400> 70 attccctaac ttgtcgtcta acttc 25 <210> 71 <211> 23 <212> DNA <213> Mycoplasma genitalium <400> 71 agttgatgaa accttaaccc ctt 23 <210> 72 <211> 21 <212> DNA <213> Mycoplasma genitalium <400> 72 tgaggggttt tccatttttg c 21 <210> 73 <211> 21 <212> DNA <213> Chlamydiae trachomatis <400> 73 tgaggggttt tccatttttg c 21 <210> 74 <211> 19 <212> DNA <213> Chlamydiae trachomatis <400> 74 gaccgctgtc tcgcaaatc 19 <210> 75 <211> 18 <212> DNA <213> Neisseria gonorrhoeae <400> 75 cggtttccgt gcgttacg 18 <210> 76 <211> 25 <212> DNA <213> Neisseria gonorrhoeae <400> 76 actggtttca tctgattact ttcca 25 <210> 77 <211> 20 <212> DNA <213> Actonobacillus actinomycetem comitans <400> 77 ggggctttct actacgggac 20 <210> 78 <211> 20 <212> DNA <213> Actinobacillus actinomycetem comitans <400> 78 agcatctgcg atccctgtat 20 <210> 79 <211> 19 <212> DNA <213> Porphyromonas gingivalis <400> 79 gaataacggg cgatacgag 19 <210> 80 <211> 20 <212> DNA <213> Porphyromonas gingivalis <400> 80 gctgacttac cgaacaacct 20 <210> 81 <211> 24 <212> DNA <213> Treponema denticola <400> 81 agaataagaa gaagagggaa tgct 24 <210> 82 <211> 23 <212> DNA <213> Treponema denticola <400> 82 gcttacctaa ccgcctacat acc 23 <210> 83 <211> 18 <212> DNA <213> Tannerella forsythensis <400> 83 cgggctgcaa tggaacta 18 <210> 84 <211> 19 <212> DNA <213> Tannerella forsythensis <400> 84 gcttctcagg tcccagcaa 19 <210> 85 <211> 19 <212> DNA <213> Prevotella intermedia <400> 85 ccaaccttcc ctccactcg 19 <210> 86 <211> 21 <212> DNA <213> Prevotella intermedia <400> 86 cgtcaatcct gcacgctact t 21 <210> 87 <211> 22 <212> DNA <213> Fusobacterium nucleatum <400> 87 catttattgt gatggaggga cg 22 <210> 88 <211> 19 <212> DNA <213> Fusobacterium nucleatum <400> 88 cctcttcact gcgaccctc 19 <210> 89 <211> 19 <212> DNA <213> Bacteria general <400> 89 tcctacggga ggcagcagt 19 <210> 90 <211> 26 <212> DNA <213> Bacteria general <400> 90 ggactaccag ggtatctaat cctgtt 26 <210> 91 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Fungus spp. <400> 91 gcatcgatga agaacgcagc 20 <210> 92 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Fungus spp. <400> 92 ttgacctcrr atcaggtagg ratacccgct gaacttaa 38 <210> 93 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Rhizopus spp. <400> 93 ctagcggcca aatacaaatg c 21 <210> 94 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Rhizopus spp. <400> 94 ttcacctcta gcggccaaat 20 <210> 95 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Aspergillus spp. <400> 95 ggcttgagcc gatagtcccc 20 <210> 96 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Aspergillus spp. <400> 96 tcaagccgat ggaagtgcg 19 <210> 97 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Candida spp. <400> 97 gaaggcaaca ccaaacccg 19 <210> 98 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Candida spp. <400> 98 tcctacctga tttgagggcg a 21 <210> 99 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Mycobacterium avium <400> 99 gaccgagtgt tgtctcaggg c 21 <210> 100 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Mycobacterium chelonae <400> 100 atttcccagc cgaatgagc 19 <210> 101 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Mycobacterium flavescens <400> 101 ggtctggtgt cgccctgtct t 21 <210> 102 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Mycobacterium gordonae <400> 102 ctcgggtgct gtccctcca 19 <210> 103 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Mycobacterium kansasii <400> 103 gaggcaacac tcgggctctg 20 <210> 104 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Mycobacterium simiae <400> 104 ttcggttgaa gtggtgtccc tc 22 <210> 105 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Mycobacterium szulgai <400> 105 cggcaacgaa caagccagac a 21 <210> 106 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Mycobacterium vaccae <400> 106 cggcgaggga aatcatcaga ca 22 <210> 107 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Mycobacterium fortuitum <400> 107 gtcttacccg agccgtgagg a 21 <210> 108 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Mycobacterium intracellulare <400> 108 ccctgagaca acactcggtc g 21 <210> 109 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for Mycobacterium tuberculosis <400> 109 ttgggtcctg aggcaacact cg 22 <210> 110 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Mycobacterium abscessus <400> 110 ttgggtcctg aggcaacacg 20 <210> 111 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Mycobacterium bovis <400> 111 ttgggtcctg aggcaacact cg 22 <210> 112 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for HPV type 16 <400> 112 acctccagca cctaaagaag at 22 <210> 113 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for HPV type 18 <400> 113 ggacccgtgt atacaggcac at 22 <210> 114 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for HPV type 31 <400> 114 atggatcttc cttgggcttt t 21 <210> 115 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for HPV type 33 <400> 115 caggctatta cgtgtcaaaa aac 23 <210> 116 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for HPV type 35 <400> 116 ccagaaggcg gtggtgtaag 20 <210> 117 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for HPV type 39 <400> 117 caaactggca gatggtggag 20 <210> 118 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for HPV type 52 <400> 118 ccccaccacc gtctgcatc 19 <210> 119 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for HPV type 56 <400> 119 gggttatccc cgccagtg 18 <210> 120 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for HPV type 58 <400> 120 gtcctgtaaa ctggcagacg g 21 <210> 121 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for HPV type 6 <400> 121 ccccaaatgg tacattagaa gata 24 <210> 122 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for HPV type 11 <400> 122 ccatttggtg gaggcgata 19 <210> 123 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for HPV type 30 <400> 123 aactccactt tacttgaggg ctg 23 <210> 124 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for HPV type 54 <400> 124 tgcaggggca ttattctttt g 21 <210> 125 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for HPV type 62 <400> 125 tcactatttg cagtctcggg cta 23 <210> 126 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for Proteus mirabilis <400> 126 cgcactcaat ctcgccaag 19 <210> 127 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Proteus mirabilis <400> 127 atggcattta gaggatgtag ca 22 <210> 128 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for Proteus mirabilis <400> 128 gcggtttatc acgaaggggt 20 <210> 129 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for Enterobacteriaceae spp. <400> 129 gacatcgttt acggcgtgga ct 22 <210> 130 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for Enterobacteriaceae spp. <400> 130 cctcaagggc acaacctcca ag 22 <210> 131 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for Enterobacteriaceae spp. <400> 131 tcaggtgcga aagcgtggg 19 <210> 132 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for Enterobacteriaceae spp. <400> 132 cgtccgatca cctgcgtcaa 20 <210> 133 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for Escherichia coli sp. <133> 133 gcgaagaggc agtcaacggg 20 <210> 134 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for Escherichia coli sp. <400> 134 gggcaacaag ccgaaagaac tg 22 <210> 135 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Enterococcus faecalis <400> 135 ggaacatcat cgcctgggaa 20 <210> 136 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for Enterococcus faecalis <400> 136 gataactgga acatcatcgc c 21 <210> 137 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for Enterococcus faecalis <400> 137 atgatgttcc agttatcgca gg 22 <210> 138 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for Staphylococcus spp. <400> 138 cgtattgagc atcgccttct a 21 <139> <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe seqeunce for Staphylococcus spp. <400> 139 cgtattgagc atcgccttc 19 <210> 140 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Staphylococcus spp. <400> 140 ttagaaggcg atgctcaata c 21 <210> 141 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Staphylococcus aureus <400> 141 tcgtattgag catcgcctt 19 <210> 142 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Staphylococcus aureus <400> 142 cttcgtattg agcatcgcc 19 <210> 143 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Staphylococcus aureus <400> 143 aaggcgatgc tcaatacga 19 <210> 144 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Streptococcus agalactiae <400> 144 gagtatcaag cagcccacg 19 <210> 145 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Streptococcus agalactiae <400> 145 atcaagcagc ccacgattc 19 <210> 146 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Streptococcus agalactiae <400> 146 aaggaataca tgctgttgcg 20 <210> 147 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Streptococcus pneumoniae <400> 147 gctacccgat gagtttgttg tt 22 <210> 148 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Streptococcus pneumoniae <400> 148 agctacccga tgagtttgtt gtt 23 <210> 149 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Streptococcus pneumoniae <400> 149 cgataacaac aaactcatcg ggt 23 <210> 150 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Corynebacterium sp. <400> 150 gcacaagcgg cggagcat 18 <210> 151 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Corynebacterium sp. <400> 151 atgctccgcc gcttgtgc 18 <210> 152 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Corynebacterium sp. <400> 152 tgcaacgcga agaaccttac ct 22 <210> 153 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Pseudomonas aeruginosa <400> 153 ccgtacacgc cggtagca 18 <210> 154 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Pseudomonas aeruginosa <400> 154 gccgggtcca ggatgccc 18 <210> 155 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Veillonella sp. <400> 155 ccacattggg actgagacac gg 22 <210> 156 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Veillonella sp. <400> 156 tcctacggga ggcagcagtg 20 <210> 157 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Veillonella sp. <400> 157 ctacgggagg cagcagtggg 20 <210> 158 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Leptotrichia sp. <400> 158 cggataacgc tcgcaacata 20 <210> 159 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Leptotrichia sp. <400> 159 tatgttgcga gcgttatccg 20 <210> 160 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Leptotrichia sp. <400> 160 aggcggtaag acaagttgaa gg 22 <210> 161 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Lactobacillus sp. <400> 161 cgtgttactc acccgtccgc 20 <210> 162 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Lactobacillus sp. <400> 162 cggcggacgg gtgagtaa 18 <210> 163 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Lactobacillus sp. <400> 163 agcggcggac gggtgagt 18 <210> 164 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for plasmid-borne beta-lactamase TEM gene <400> 164 gaataagggc gacacggaaa 20 <210> 165 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for plasmid-borne beta-lactamase TEM gene <400> 165 tttccgtgtc gcccttattc 20 <210> 166 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for plasmid-borne beta-lactamase SHV gene <400> 166 cagcacggag cggatcaacg 20 <210> 167 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for plasmid-borne beta-lactamase SHV gene <400> 167 cgccctgctt ggcccgaata 20 <210> 168 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for chromosomal beta-lactamase AmpC gene <400> 168 cgttgatttg ttgaggtgcg g 21 <210> 169 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for chromosomal beta-lactamase AmpC gene <400> 169 taccgccacc gccatacc 18 <210> 170 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for TetM gene <400> 170 cgagtttgtg cttgtacgcc at 22 <210> 171 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for TetM gene <400> 171 aaagatggcg tacaagcaca aac 23 <210> 172 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for TetC gene <400> 172 tgatcttcac ggcgatttat gc 22 <210> 173 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for TetC gene <400> 173 cattggaccg ctgatcttca cg 22 <210> 174 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for ermB gene <400> 174 ttggcgtgtt tcattgcttg 20 <175> 175 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for emrB gene <400> 175 atcaagcaat gaaacacgcc aa 22 <210> 176 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for HSV <400> 176 cacatcaagg tgggccagcc gc 22 <210> 177 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for HSV <400> 177 tgcggctggc ccaccttgat g 21 <210> 178 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for HSV <400> 178 ccaggtagta ctgcggctgg cc 22 <210> 179 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for HSV type 2 <400> 179 ccgtggagcg gcagacccc 19 <210> 180 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for HSV type 2 <400> 180 gccgtggagc ggcagacc 18 <210> 181 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for HSV type 2 <400> 181 tggccgtgga gcggcagacc 20 <210> 182 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Haemophilus ducreyi <400> 182 gcgccgtatc ggttgggt 18 <210> 183 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Haemophilus ducreyi <400> 183 aaggtaggcg tgagagaatc aaaaa 25 <210> 184 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Haemophilus ducreyi <400> 184 cgtaggcatc aagaaggtaa agcg 24 <210> 185 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Treponema pallidum <400> 185 aggaaccgca actgggacaa a 21 <210> 186 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Treponema pallidum <400> 186 gaggaaccgc aactgggaca 20 <210> 187 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Treponema pallidum <400> 187 tgaagtttgt cccagttgcg gt 22 <210> 188 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Mycoplasma hominis <400> 188 actaatgttc cgcaccctca tct 23 <210> 189 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Mycoplasma hominis <400> 189 agatgagggt gcggaacatt agt 23 <210> 190 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Gardnerella vaginalis <400> 190 gctgccgagt gggctttg 18 <210> 191 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Gardnerella vaginalis <400> 191 gtcaggtgtt gcgtattcgg g 21 <210> 192 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Candida albicans <400> 192 gcatctccaa tcattcgcct a 21 <210> 193 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Candida albicans <400> 193 agatgccttg ccacctaaat cc 22 <210> 194 <211> 20 <212> DNA <213> Artificial Sequence <220> Trichomonas vaginalis <400> 194 ggactgcctt tgcgaactga 20 <210> 195 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Trichomonas vaginalis <400> 195 ggctgcttga ccatccgaaa 20 <210> 196 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Ureaplasma urealyticum <400> 196 ggggatgaac tctactatga agtta 25 <210> 197 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Ureaplasma urealyticum <400> 197 gttaactaag ccgtttacac ctcaa 25 <210> 198 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Mycoplasma genitalium <400> 198 atatttaagt tgtcattttg gcttc 25 <210> 199 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Mycoplasma genitalium <400> 199 aagaagccaa aatgacaact taaat 25 <210> 200 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Chlamydiae trachomatis <400> 200 gagataggaa accaactcta cgctg 25 <210> 201 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Chlamydiae trachomatis <400> 201 cagcgtagag ttggtttcct atctc 25 <210> 202 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Neisseria gonorrhoeae <400> 202 gcaggcgtat aggcggactt g 21 <210> 203 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Neisseria gonorrhoeae <400> 203 gggaatcgta acgcacggaa a 21 <210> 204 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Actinobacillus actinomycetem comitans <400> 204 ggggctttct actacgggac ct 22 <210> 205 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Actinobacillus actinomycetem comitans <400> 205 cagcatctgc gatccctgta t 21 <206> 206 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Porphyromonas gingivalis <400> 206 taccgaacaa cctacgcacc ct 22 <210> 207 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Porphyromonas gingivalis <400> 207 gcggtaatac ggaggatgcg 20 <210> 208 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Treponema denticola <400> 208 gcctacatac cctttacgcc ca 22 <210> 209 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Treponema denticola <400> 209 gggcttattc gcatgactac cg 22 <210> 210 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Tannerella forsythensis <400> 210 cgggcgtggg attggtgatg 20 <210> 211 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Tannerella forsythensis <400> 211 tgtatcgggc gtgggattgg t 21 <210> 212 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Prevotella intermedia <400> 212 atggcatctg acgtggacca aa 22 <210> 213 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Prevotella intermedia <400> 213 cgtagccttg gtgggccgtt a 21 <210> 214 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Fusobacterium nucleatum <400> 214 ttctgcgtcc ctccatcaca 20 <210> 215 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for Fusobacterium nucleatum <400> 215 acttccgttc gtccgtgc 18 <210> 216 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Probe sequence for bacterial 16S ribosomal RNA <400> 216 cgtattaccg cggctgctgg cac 23  

Claims (13)

As a chip for the detection of infectious diseases, genotyping and antibiotic resistance genotyping, the chip includes all oligonucleotide probes having a nucleotide sequence of SEQ ID NOs: 91 to 216, and the infectious disease includes urinary tract infection ), Rhinosinusitis (Rhinosinusitis), periodontitis (Periodontitis), sexually transmitted disease (Sexually transmitted disease), human papilloma virus infection or mycobacterial infection. The chip of claim 1, wherein the probe comprises DNA (deoxyribonucleic acid) or PNA (peptide nucleic acid). The chip of claim 1, wherein the chip is divided into eight wells in which the probe can be integrated. 4. The detection of a causative agent of infectious disease, comprising a chip according to any one of claims 1 to 3, a primer set for amplifying a nucleic acid derived from the causative agent of infectious disease, and a labeling means for detecting the amplified nucleic acid. Kit for genotyping and analysis of antibiotic resistance genotypes. The kit according to claim 4, wherein the primer set is an oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1 to 90. Selected from Urinary tract infection, Rhinosinusitis, Periodontitis, Sexually transmitted disease, Human papilloma virus infection, or Mycobacteria infection, including the following steps: Detection of genotypes of infectious diseases, genotyping and analysis of antibiotic resistance genotypes: (a) amplifying the nucleic acid by a multiplex PCR amplification method using a nucleic acid amplification primer of the causative agent of infectious disease; (b) hybridizing the amplified nucleic acid fragments to a chip according to any one of claims 1 to 3; And (c) detecting the hybridization obtained by the hybridization. The method of claim 6, wherein the primer of step (a) is characterized in that the oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to 90. 8. The method of claim 7, wherein the multiple PCR amplification of step (a), 6-FAM (6-carboxyfluorescein), Cy5, Cy3, Cy5.5, JOE (6'-carboxy-4 ', 5'-dichloro-2 Deoxycytidine triphosphate (dCTP) or deoxyuridine labeled with fluorescent dyes selected from the group consisting of ', 7'-dimethoxyfluorescein), Rhodamine Green, TAMRA NHS (N-hydroxysuccinimide) Ester and Texas Red A method for labeling fluorescent dyes randomly in amplified nucleic acid fragments using triphosphate (deoxyuridine triphosphate, dUTP). The method of claim 8, wherein the step (c) is to measure the fluorescence intensity by scanning the chip with a laser of a wavelength specific to the fluorescent dye. delete delete delete delete

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