CN102321763B

CN102321763B - Detection chip for drug resistance gene of bacteria, and application thereof

Info

Publication number: CN102321763B
Application number: CN 201110276409
Authority: CN
Inventors: 李越希; 潘英; 傅雅丽; 潘明洁
Original assignee: Individual
Current assignee: Individual
Priority date: 2011-09-19
Filing date: 2011-09-19
Publication date: 2013-04-10
Anticipated expiration: 2031-09-19
Also published as: CN102321763A

Abstract

The present invention relates to a high-throughput detection chip for drug resistance gene of bacteria, and an application thereof. The detection chip comprises 117 gene probes, the drug resistance gene probes are selected from 17 categories of drug resistance genes, which comprise extended spectrum beta-lactamase, cephalosporinase, carbapenemase, integrase gene, tetracycline resistance gene, aminoglycoside resistance gene, disinfectant resistance gene, erythromycin resistance gene, macrolide efflux gene, vancomycin resistance gene, multidrug resistance efflux pump gene, mupirocin resistance gene, sulfanilamide resistance gene, tylosin resistance gene, fluoroquinolone resistance gene, chloramphenicol acetyltransferase and commonly-used genetic engineering vector resistance gene. The chip is adopted for detecting the resistance gene of the pathogenic bacteria. The chip is characterized in that: the chip comprises (1) 117-oligonucleotide probe composition and quality control probes of 17 categories of the drug resistance genes; (2) probe arrays, wherein the oligonucleotide probes are solidified on the vector material through arm molecules to form the probe arrays.

Description

Detection chip for drug resistance gene of bacteria and application thereof

Technical field

What the present invention relates to is a kind of high-throughout detection chip for drug resistance gene of bacteria and application thereof.The probe of gene chip selects to comprise extended spectrum β lactamases; cephalosporinase; the carbapenem enzyme; the Integrases gene; tsiklomitsin family drug resistant gene; the aminoglycoside drug drug resistant gene; Disinfectant resistant gene; the erythromycin-resistant genes involved; Macrolide effluxes gene; the vancomycin resistance drug resistant gene; multidrug resistance efflux pump gene; the gene of anti-the mupirocin; Sulfonamides-resistant genes; the Tylosin drug resistant gene; the fluoroquinolones drug resistant gene; the paraxin acyltransferase; the 17 large class drug resistant genes such as engineering carrier drug resistant gene commonly used; totally 117 gene probes can be used for the detection of pathogenic bacteria drug resistant gene.The technical field that the present invention and application relate to is medical test and biochip technology.

Technical background

In recent decades, along with the extensive unreasonable use of Broad spectrum antibiotics, cause most of bacteriums that multi-drug resistant is arranged, superbacteria occurred especially in recent years, very easily cause hospital acquired infections.It is reported, the whole world 6,000,000,000 populations, annual 300000000 people are in hospital, and 1,500 ten thousand people suffer from hospital infection, and 1,500,000 people die from hospital infection.Inland of China ward infection incidence is about 8%, and rate of failing to report is very high, causes very large difficulty to clinical treatment, has also consisted of serious public health problem.Therefore these pathogenic bacteria rapid detection are determined its resistance, not only instructing clinical rational to select antimicrobial drug that important value is arranged, also for prevention with the control drug-resistant bacteria infects and propagation provides foundation, for resistance epidemic situation monitoring and supervision and inspection provide reliable technique means, have important practical significance

The present situation that bacterial drug resistance is serious has caused the attention of a lot of countries, and in order to prevent, control the propagation of pathogenic bacteria, national governments all increase input and develop fast and effectively that diagnostic method detects, diagnose and control pathogenic bacteria.At present, the chemical sproof method of general detection has: drug sensitivity test, plasmid Elimination test, plasmid fingerprinting graphical spectrum technology, molecular hybridization, PCR etc., but these detection methods are difficult to carry out early etiological diagnosis and drug clinically, can't satisfy clinical treatment of severe the infected's needs.Such as the cultivation of bacterium, do not wait from 18h to a couple of days according to different pathogenic bacteria type needs, need the longer time for individual bacteria (such as mycobacterium tuberculosis), even need special laboratory growth conditions.Bacterial drug resistance detects the main drug sensitive test of using clinically, and its advantage is conveniently, simple and direct, easy judgement, but can only detect one to one analysis to a small amount of sample, also can't reflect source and the mechanism of transmission of bacterial resistance.Molecular hybridization and PCR also have been widely used in the detection of bacterial drug resistance in recent years, remedied the deficiency of drug sensitive test, the molecular hybridization specificity is better, but complex operation step, length consuming time, and conventional round pcr often only be limited to single drug resistant gene detection, detect a large amount of work of need to doing to the numerous drug resistant gene of bacterium, even be difficult to realize.

Biochip technology (Gene chip) grew up the nineties in last century, multiple oligonucleotide molecules to be fixed in form dna microarray on the solid phase carrier, sample nucleic acid is used the method marks such as isotropic substance and fluorescence, with the multiple specific DNA sequence in the chip probe hybridization check sample, by computer system analysis, obtain rapidly obtaining lots of genes sequence signature or allelic expression information in the sample.

Gene chip provides strong instrument for the clinical diagnosis of infectious diseases, has compared multiple advantage with traditional detection method: can carry out simultaneously high throughput analysis; Need not the immune response reaction, can early detection diagnose; Few to the test sample requirement, efficient is high; Can be used for large-scale pathogenic bacteria classification and identify resistance and virulence factor detection etc.Therefore, gene chip has had in medical field widely and has used.

The genome sequence of multiple pathogenic microorganisms is illustrated, and various drug resistant genes are cloned, check order, and lays a good foundation for setting up detection chip for drug resistance gene of bacteria.The development detection chip for drug resistance gene of bacteria can provide foundation for resistance, the clinical antimicrobial drug of correctly selecting of determining fast pathogenic bacteria, to prevention with the control drug-resistant bacteria infects and propagation is of great importance.

Summary of the invention

The present invention seeks to provides a kind of detection chip for drug resistance gene of bacteria and application thereof for above-mentioned weak point; it is the gene chip of setting up a kind of detection 17 large class drug resistant genes; can carry out the high-throughput rapid detection to pathogenic bacteria, 17 large class drug resistant genes comprise extended spectrum β lactamases; cephalosporinase; the carbapenem enzyme; the Integrases gene; tsiklomitsin family drug resistant gene; the aminoglycoside drug drug resistant gene; Disinfectant resistant gene; the erythromycin-resistant genes involved; Macrolide effluxes gene; the vancomycin resistance drug resistant gene; multidrug resistance efflux pump gene; the gene of anti-the mupirocin; Sulfonamides-resistant genes; the Tylosin drug resistant gene; the fluoroquinolones drug resistant gene; the paraxin acyltransferase; engineering carrier drug resistant gene commonly used etc.According to the drug resistant gene sequence that ncbi database provides, from 17 large class drug resistant genes, screened 117 gene probes, for the preparation of gene chip, set up detection and the analytical procedure of gene chip, and use.This gene chip can be used for containing the sick monitoring of detection of pathogens, somatotype, rational use of drug, infection and the epidemiology survey of 17 large class drug resistant genes.

A kind of detection chip for drug resistance gene of bacteria and application thereof are to take following technical scheme to realize:

A kind of detection chip for drug resistance gene of bacteria comprises that 117 oligonucleotide probes of (1) 17 large class drug resistant gene make up and quality control probes; (2) oligonucleotide probe is solidificated in the probe array that forms on the solid support material by arm molecule;

Described oligonucleotide probe refers to the complementary oligonucleotide of high conservative region of the multidrug resistant gene sequence of chemosynthesis, and length is ten to 50 bases;

Described detection chip for drug resistance gene of bacteria refers to detect the oligonucleotide arrays of 17 large class drug resistant genes, and 17 large class drug resistant genes comprise extended spectrum β lactamases, cephalosporinase, the carbapenem enzyme, the Integrases gene, tsiklomitsin family drug resistant gene, the aminoglycoside drug drug resistant gene, Disinfectant resistant gene, the erythromycin-resistant genes involved, Macrolide effluxes gene, the vancomycin resistance drug resistant gene, multidrug resistance efflux pump gene, the gene of anti-the mupirocin, Sulfonamides-resistant genes, the Tylosin drug resistant gene, the fluoroquinolones drug resistant gene, the paraxin acyltransferase, engineering carrier drug resistant gene commonly used;

Described quality control probes comprises two kinds of probes, i.e. positive control and negative control at least; Negative control is for detection of hybridization signal mistake or pollution, and normally whether positive control for detection of hybridization and accurate location;

Described arm molecule refers to have the long-chain organic compound of double-active group;

The end that described oligonucleotide is fixed as oligonucleotide has a special groups, when being solidificated in the solid support material stromal surface and the end group of the arm molecule of finishing form covalent linkage;

Described 117 gene probes, any combination of its middle probe is used for the preparation of gene chip etc., and solid support material includes but are not limited to slide, tinsel, silicon chip, ceramic plate, plastic sheet, nitrocellulose membrane, nylon membrane etc.

Described detection chip for drug resistance gene of bacteria is used for containing the sick monitoring of detection of pathogens, somatotype, rational use of drug, infection and the epidemiology survey of 17 large class drug resistant genes.

The solid support material of these probes comprises film that slide, tinsel, silicon chip, ceramic plate, plastic sheet, nitrocellulose membrane, nylon membrane and other high molecular polymer that several different methods is processed are made and sheet etc.

These surface of solid phase carriers are modified with the long-chain organic compound with double-active group, commonly used has: glutaraldehyde, trishydroxymethyl aminosilane (APTES), N, N-diethoxy aminopropyltriethoxywerene werene, poly-lysine can adopt a kind of or two kinds combination wherein wherein.

117 gene probes of 17 large class drug resistant genes are selected and principle of design:

According to the sequence of 17 large class drug resistant genes in the ncbi database, select probe length at 40～50nt, 5 ' end carries out amination and modifies and add 10 base T.Other parameter such as annealing temperature Tm and GC% content etc. are strictly controlled according to principle of design.Every kind of probe is all through two or more biological software screenings, and again with artificial selected and adjustment, Blast verifies that final the selection has the representational sequence of all kinds of drug resistant genes as detection probes.The principle of probe design is as follows:

(1) specificity and the susceptibility of height

The probe that filters out should be positioned at the high conserved region of gene order, to guarantee specificity and the susceptibility of probe; Simultaneously, the probe of screening should be in the genome sequence of human genome sequence, other bacteriums and microorganism, relevant animal, environment the frequently seen plants gene order homology is low as far as possible, to avoid false positive results.It is vital seeking and filtering out the high degree of specificity probe.

The Tm value of (2) 17 large class resistance gene probes is consistent as far as possible

Because be the hybridization that carries out simultaneously many probes at a chip, at a certain temperature, this moment, each probe Tm value was most important, if the Tm value is inconsistent, gap is excessive, and the fluorescent signal power after the hybridization can be subject to suitable impact.It is more suitable less than three degree that general Tm value differs.

(3) various probes have approximate base length

The length of chip probe has certain impact to susceptibility and the specificity that detects.Each probe base length is consistent, can eliminate to a certain extent the difference that bring the rear chip scanner scanning of hybridization focal length location, and the probe base number of our design is at 40～50nt.

(4) probe of screening is avoided secondary structure as far as possible

Probe should preferably not have hairpin structure, self dimer, mispairing etc.Otherwise form easily error result.

(5) sequence of avoiding choosing G, C rich region is made probe

Do not form dimer and mispairing between the complementary sequence of (6) 17 large class resistance gene probes

The method of probe screening:

Log in U.S.'s biotechnology center (National Center for Biotechnology Information, NCBI) homepage, from ncbi database, retrieved the sequence of 17 large class drug resistant genes, more same classification drug resistant gene sequence has or not the variation situation that exists, according to above-mentioned probe design principle, use Primer Primers5.0, the biological software such as Oligo6.0 are found out all possible probe at the base alignment figure of drug resistant gene, look for the high conservative region of the specific drug resistant gene target gene dna sequence dna in place in conjunction with homology analysis software such as Clustal W, select the Blast of nucleotide sequence, carry out class database like retrieval (retrieving simultaneously the GenBank+EMBL+DDBJ+PDB database), the probe of screening is in known human genomic sequence, other common bacteria and microorganism, the genome sequence of relevant animal, homology is low as far as possible for the frequently seen plants gene order in the environment, about the several 40～50nt of base, between average 83～84 ℃ of the Tm value, G+C content is about 50%, according to Blast result, in conjunction with demand and experience, by hand the probe that sifts out is finely tuned and rebuild again.Then this 17 large class resistance gene probe that obtains is analyzed with RNA Structure software, to get rid of the secondary structure that forms impact between the different probe complementary sequence and disturb hybridization.What positive internal reference probe adopted is and the plant genetic sequences fragment of antibiotics resistance gene without homology.During actual chip hybridization detects in the back, according to overall needs and the result of detection, adjusted the probe that partly uses.

117 gene probes of 17 large class drug resistant genes of design screening

117 oligonucleotide gene probes of described detection 17 large class drug resistant genes are as follows:

The Integrases gene probe is selected from I class integrase gene (IntI1) and II class integrase gene (IntI2), and each gene is chosen each one of positive minus strand probe, totally 4 probes:

IntI1 gene normal chain probe 5'-NH2-T (10) CCAGTGGACATAAGCCTGTTCGGTTCGTAAACTGTAATGCAAGTAGC-3'

IntI1 gene minus strand probe 5'-NH2-T (10) CTCTACGACGATGATTTACACGCATGTGCTGAAAGTTGGCG-3'

IntI2 gene normal chain probe 5'-NH2-T (10) GGGCATTTAAAGCGATTTTCTGCGTGTTTATGGCTACATGTCTG-3'

IntI2 gene minus strand probe 5'-NH2-T (10) ATGCTTGCGTTTGCGGGTTAAAGATTTTGATTTTGATAATGGCTG-3'

Tsiklomitsin family resistance gene probe is selected the tetM gene, designs each one of positive minus strand probe, and Tet (M) is the rrna protected protein of studying at most, has the GTP hydrolytic enzyme activities that rrna relies on, totally 2 probes:

TetM gene normal chain probe 5'-NH2-T (10) AGTGGGAAAATACGAAGGTGAACATCATAGACACGCCAGGACA-3'

TetM gene minus strand probe 5'-NH2-T (10) AAGCGGATCACTATCTGAGATTTCCAAAAGGGCATCAAGCAA-3'

The aminoglycoside drug resistance gene probe, chosen to be easy to propagate and to endanger larger aminoglycosides modification gene aac (3)-I, aac (3)-II, aac (3)-III, aac (3)-IV, aac (6')-I, aac (6')-II, aphA6 arranged, totally 9 kinds of ant (3')-I and ant (2'')-I, each gene is chosen each one of positive minus strand probe, totally 18 probes:

Aac (3)-I gene normal chain probe 5'-NH2-T (10) GGTGCTTATGTGATCTACGTGCAAGCAGATTACGGTGACGATC-3'

Aac (3)-I gene minus strand probe 5'-NH2-T (10) GAAAAGATCAAGAGCAGCCCTCATGGATTTGACTTGGTCAGG-3'

Aac (3)-II gene normal chain probe 5'-NH2-T (10) GTCGAAACTATAGCAAATGCTTACGTGAAGCTCGGTCGCCAT-3'

Aac (3)-II gene minus strand probe 5'-NH2-T (10) AATCGAGAATGCCGTTTGAATCGTATTCTGATGCCGTTTTCC-3'

Aac (3)-III gene normal chain probe 5'-NH2-T (10) TGGCTAAACTGGTGGCAATAGAAGGATACGTGCTGATGCTTG-3'

Aac (3)-III gene minus strand probe 5'-NH2-T (10) CTATCCGTATGACGCTGAGTCACCGAACCGTGATTCAAGC-3'

Aac (3)-IV gene normal chain probe 5'-NH2-T (10) CTCAAGGAGAAGAGCCTTCAGAAGGAAGGTCCAGTCGGTCAT-3'

Aac (3)-IV gene minus strand probe 5'-NH2-T (10) GTACCAACTTGCCATCCTGAAGAATGGTGCAGTGTCTCGG-3'

Aac (6')-Ib gene normal chain probe 5'-NH2-T (10) ACTTGCTGACGTACAGGAACAGTACTTGCCAAGCGTTTTAGCG-3'

Aac (6')-Ib gene minus strand probe 5'-NH2-T (10) ACTGGTCTATTCCGCGTACTCCTGGATCGGTTTCTTCTTCCC-3'

Aac (6')-II gene normal chain probe 5'-NH2-T (10) GGTGGGAAGATGAAACTGATCCAGGAGTGCGAGGAATAGACC-3'

Aac (6')-II gene minus strand probe 5'-NH2-T (10) GTAGTGTTCCAGCACTTCATCAAGAGTCGGTCGCTCTTCGTC-3'

AphA6 gene normal chain probe 5'-NH2-T (10) TTGCCCAATATTATTCAACAATTTATCGGAAACAGCGTTTTAGAGCCA-3'

AphA6 gene minus strand probe 5'-NH2-T (10) CGATTAAAAGAATAAACATCCGATGGCGACTGACCAATTTTATTTGGC-3'

Ant (3')-I gene normal chain probe 5'-NH2-T (10) TTGATCCGGTTCCTGAACAGGATCTATTTGAGGCGCTAAATG-3'

Ant (3')-I gene minus strand probe 5'-NH2-T (10) ACGGAATGATGTCGTCGTGCACAACAATGGTGACTTCTACAG-3'

Ant (2'')-I gene normal chain probe 5'-NH2-T (10) TACAAAGCACATAGAGTCCTACAGGCTCGCATGCACCTCACTC-3'

Ant (2'')-I gene minus strand probe 5'-NH2-T (10) CATCGGCATAGTAAAAGTAATCCCAGATGATCGCCTCCCAGC-3'

The qacA/B of anti-sterilizing agent family gene probe, design each one of positive minus strand probe, totally 2 probes:

QacA/B gene normal chain probe 5'-NH2-T (10) GATTTAGCTCATGTAGCTGAAGAATCTGTAGTGGGCGCTGTCGAA-3'

QacA/B gene minus strand probe 5'-NH2-T (10) TGCCATGAAAATTGCTCAAGTAAAGCTCCTCCGATAATTGGTCC-3'

Erythromycin-resistant genes involved probe has found that the Erm gene family has more than 20 to plant gene hypotype, and with ErmA, ErmB and ErmC three types are main, and each gene is chosen each one of positive minus strand probe, totally 6 probes:

ErmA gene normal chain probe 5'-NH2-T (10) GATCCCCTACGGCATCACCTCCGCCATCGTCGACTGGT-3'

ErmA gene minus strand probe 5'-NH2-T (10) ACCCGTCGAGGAGCTGGAACAGCGTGATCCACTGGTCG-3'

ErmB gene normal chain probe 5'-NH2-T (10) TTGAAAGCCATGCGTCTGACATCTATCTGATTGTTGAAGAAGGATTC-3'

ErmB gene minus strand probe 5'-NH2-T (10) GCAAGAGCAACCCTAGTGTTCGGTGAATATCCAAGGTACGCTT-3'

ErmC gene normal chain probe 5'-NH2-T (10) CGTGGAATACGGGTTTGCTAAAAGATTATTAAATACAAAACGCTCATTG

GC-3'

ErmC gene minus strand probe 5'-NH2-T (10) GGGTAAAATGCCCTTTTCCTGAGCCGATTTCAAAGATATTATCATGTTC-3'

Macrolide effluxes (mefA) gene probe, designs each one of positive minus strand probe, totally 2 probes:

MefA gene normal chain probe 5'-NH2-T (10) TACCCCAGCACTCAATGCGGTTACACCACTTTTAGTACCAGAAGAA-3'

MefA gene minus strand probe 5'-NH2-T (10) TGCAATCACAGCACCCAATACGTCGATGGCAATAATAGCATTTAA-3'

The vancomycin resistance resistance gene probe has been selected VanA, VanB, VanC1, VanC2, VanD by different drug resistant genes bunch coding, VanE6 kind phenotype, and each gene is chosen each one of positive minus strand probe, totally 12 probes:

VanA gene normal chain probe 5'-NH2-T (10) AAAATCTTAATTGAGCAGGCTGTTTTGGGCTGTGAGGTCGGT-3'

VanA gene minus strand probe 5'-NH2-T (10) TACAAATCGCTGAGCTTTGAATATCGCAGCCTACAAAGGGGA-3'

VanB gene normal chain probe 5'-NH2-T (10) ACGGAAGAACTTAACGCTGCGATAGAAGCGGCAGGACAATAT-3'

VanB gene minus strand probe 5'-NH2-T (10) CCGTATCAATGTTCGCAGCAATTTCTATTGCGGATTTTACCGA-3'

VanC1 gene normal chain probe 5'-NH2-T (10) CTTGAACTAATGAACCTGCCTTATGTTGGTTGCCATGTCGCTG-3'

VanC1 gene minus strand probe 5'-NH2-T (10) CACAGTAGAACCGTAAGCAAAAGCAGTCGTTAATGCAGATTGGAGC-3'

VanC2 gene normal chain probe 5'-NH2-T (10) AAATCAATACTATGCCGGGCTTTACGAGTCACTCCCGCTATCC-3'

VanC2 gene minus strand probe 5'-NH2-T (10) CGTCTACTAATGAAATGGCGTCACAAGCACCGACAGTCAAAGA-3'

VanD gene normal chain probe 5'-NH2-T (10) AGGAACATGATGTTTCAGTGAAATCTGCGATGGAGGTTGCA-3'

VanD gene minus strand probe 5'-NH2-T (10) ATGCGTGGATAACGGCTATAGGAAGTAAATCCAGGCATGGTGTTC-3'

VanE gene normal chain probe 5'-NH2-T (10) CATGGAGGTTATGGTGAGAATGGTGCTATGCAGGGAGTATTTGAG-3'

VanE gene minus strand probe 5'-NH2-T (10) TGTCGTTCCTTCAAATAGATACCAATGACCTTCTTCGGTGATCCCTA-3'

Multidrug resistance efflux pump gene probe, select to comprise that AcrAB-TolC, OprM and Sme DEF efflux pump gene, each gene choose each one of positive minus strand probe, totally 6 probes:

AcrAB-TolC gene normal chain probe 5'-NH2-T (10) GCAGAAGTTCGTCCTCAAGTTAGCGGGATTATCCTGAAGCGT-3'

AcrAB-TolC gene minus strand probe 5'-NH2-T (10) TTCAGCAGGATTTTGCCGAACTCTTCAGTAGAGGTCAGACGC-3'

OprM gene normal chain probe 5'-NH2-T (10) CCAAAAGAGGGCGGGATAGGCTAGAGCCCCTATAGCACTAGG-3'

OprM gene minus strand probe 5'-NH2-T (10) GTAGCTGCGCTGGGTCAGGTCGAAACTCTTCTGGTAGGTG-3'

Sme DEF gene normal chain probe 5'-NH2-T (10) AGTACCGATGGAAGTGATCCCCATGAAAAGTGCATCCCTGTT-3'

Sme DEF gene minus strand probe 5'-NH2-T (10) GTTGGACGAGCTGTTGGAGGAGAAGTAGATCAGGCCATCAAG-3'

Anti-mupirocin (ileS) gene probe, design each one of positive minus strand probe, totally 2 probes:

IleS gene normal chain probe 5'-NH2-T (10) GAGCCGATTCTTTAAGATGGGCCTTAATTTCGGATAGTGCTCCA-3'

IleS gene minus strand probe 5'-NH2-T (10) TTTCTGGTTATCAAAAGGATAATGATGCTGAGCAAACGGCATAGAGC-3'

The Sulfonamides-resistant genes probe has been selected dfrA and dfrD amphitypy, and each gene is chosen each one of positive minus strand probe, totally 4 probes:

DfrA gene normal chain probe 5'-NH2-T (10) GGAAACCATTGCCAAATAGACGTAACGTCGTTCTCACTAACCAAGCT-3'

DfrA gene minus strand probe 5'-NH2-T (10) CGTCCCATTACAAGTGTATTCCCAGTGGTCAGTTGTTTAACATGCTTT-3'

DfrD gene normal chain probe 5'-NH2-T (10) TTGTTGCGATGGATAAGAAAAGAGTAATCGGCAAGGATAACGACATTC-3'

DfrD gene minus strand probe 5'-NH2-T (10) CCCTTCCGATTGATTGAAGGTTCTTTCTACCTAATATGATTGCATGTCCT-3'

Tylosin (tlrB) resistance gene probe, design each one of positive minus strand probe, totally 2 probes:

TlrB gene normal chain probe 5'-NH2-T (10) CTACGGTCATGCGGAAGAACGTCGTGCGATATCTGCGCTGTC-3'

TlrB gene minus strand probe 5'-NH2-T (10) AGCTTCGTCGGGCGTCTGAGCAGATTCACATAGCCCTGCC-3'

Fluoroquinolones (norA) Drug-resistant gene probe, the resistance of norA gene mediated is quite common in golden Portugal bacterium, designs each one of positive minus strand probe, totally 2 probes:

NorA gene normal chain probe 5'-NH2-T (10) TCCTCACAAAGCAACTACTGATGGATTCCACCAATATCAACCTGAA-3'

NorA gene minus strand probe 5'-NH2-T (10) TCGTCCAATAACCGTTTGCAAGCACTAACATAACGAGAACAATGG-3'

Beta-lactam enzyme (BLA) resistance gene probe is divided into extended spectrum β lactamases, cephalosporin enzyme, carbapenem enzyme, streptococcus aureus (MRSA) gold standard mecA gene.

Extended spectrum β lactamases (ESBLs) drug resistant gene wherein, be divided into TEM type, SHV type, CTX-M type (because CDS nucleic acid gene sequence difference is larger between each hypotype by the different coding dna homolog, divide 3 groups according to the homology of its sequence), PER type and VEB type, each gene is chosen each one of positive minus strand probe, totally 14 probes:

TEM gene normal chain probe 5'-NH2-T (10) TTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAA-3'

TEM gene minus strand probe 5'-NH2-T (10) GCGTCAACACGGGATAATACCGCACCACATAGCAGAACTTTAA-3'

SHV gene normal chain probe 5'-NH2-T (10) TAACAAAGCAGAGCGCATCGTGGTGATTTATCTGCGGGATA-3'

SHV gene minus strand probe 5'-NH2-T (10) AGTAGTCCACCAGATCCTGCTGGCGATAGTGGATCTTTCGC-3'

CTX-M1 gene normal chain probe 5'-NH2-T (10) ATGAGACGTTTCGTCTGGATCGCACTGAACCTACGCTGAATA-3'

CTX-M1 gene minus strand probe 5'-NH2-T (10) CCGCCATAACTTTACTGGTACTGCACATTGGAAAGCGTTCATC-3'

CTX-M2 gene normal chain probe 5'-NH2-T (10) AAGAAGAGCGACCTGGTTAACTACAATCCCATTGCGGAGAAACA-3'

CTX-M2 gene minus strand probe 5'-NH2-T (10) CCCAGATGGGCAATCAGCTTATTCATGGCAGTATTGTCGCTAT-3'

CTX-M3 gene normal chain probe 5'-NH2-T (10) GCGGTGCTGAAGAAAAGTGAAAGCGAACCGAATCTGTTAAATC-3'

CTX-M3 gene minus strand probe 5'-NH2-T (10) CGTGAGCAATCAGCTTATTCATCGCCACGTTATCGCTGTACT-3'

PER gene normal chain probe 5'-NH2-T (10) CGGCCACTAATGATTTAGGTATCATTCTGTTGCCTGATGGACG-3'

PER gene minus strand probe 5'-NH2-T (10) GCACTGGAACACTAAACTCGTCTCCCTGATACGCTTTCATTATCGG-3'

VEB gene normal chain probe 5'-NH2-T (10) AGATTACCCCTCAAGACCTTTTGCCTAAAACGTGGAGTCCGATTAAA-3'

VEB gene minus strand probe 5'-NH2-T (10) TTTGATATTGGGTATTCCAATCCTTGTGCATTTGTTCTTCGTTTGCT-3'

Cephalosporinase (AmpC) resistance gene probe is divided into 6 groups, has almost contained all plasmid AmpC gene orders, designs each six of positive minus strand probes, totally 12 probes:

AmpC gene normal chain probe article one 5'-NH2-T (10) AGCATCCAGCCGCTGCTCAAGGAGCACAGGATC-3'

AmpC gene minus strand probe article one 5'-NH2-T (10) GCCTGCTTCGGCACATTGACATAGGTGTGGTGCAT-3'

AmpC gene normal chain probe second 5'-NH2-T (10) CCAGAACTGACAGGCAAAAAGTGGCAGGGTATCCGC-3'

AmpC gene minus strand probe second 5'-NH2-T (10) GTTTTCTCCTGAACGTGGCTGGCATCCATGTTGGC-3'

AmpC gene normal chain the 3rd 5'-NH2-T of probe (10) CTTTCACAGGTGTGCTGGGTGCGGTTTCTGTGGC-3'

AmpC gene minus strand the 3rd 5'-NH2-T of probe (10) GTACGCATACTGGCTTTGCGCACTTTCCGGCACAGTAATAAA-3'

AmpC gene normal chain the 4th 5'-NH2-T of probe (10) TATGGGTTAGCGGCAAAACAGCCTCAGCAGCCGGTTA-3'

AmpC gene minus strand the 4th 5'-NH2-T of probe (10) AGACTTTTCGCCGCAATCATCCCTAGCAAACCAGTACCGATA-3'

AmpC gene normal chain the 5th 5'-NH2-T of probe (10) CGCTTTTATCAAAACTGGCAGCCGCAGTGGAAGCC-3'

AmpC gene minus strand the 5th 5'-NH2-T of probe (10) CGAGCTGCTTTTCAGGAATAAATGCCACGTAGCTGCCAAAC-3'

AmpC gene normal chain the 6th 5'-NH2-T of probe (10) CATGGCGAACTATGCCTACGGCTATTCGAAGGAAGATAAGCC-3'

AmpC gene minus strand the 6th 5'-NH2-T of probe (10) AACCCCATAGTTGAAATAGTGGGCCTTGCCATCTTTCAGCAC-3'

Carbapenem enzyme resistance gene probe has been chosen common genotype IMP1 type and universal, OXA23 type, OXA24 type, and VIM type and GES2 type, each gene are chosen each one of positive minus strand probe, totally 11 probes:

The general normal chain probe of IMP gene 5'-NH2-T (10) CACTCCATTTACGGCTAAAGATACTGAAAAGTTAGTCACTTGGTTTG

TGG-3'

IMP1 gene normal chain probe 5'-NH2-T (10) CATTTTCATAGCGACAGCACGGGCGGAATAGAGTGGCTTAAT-3'

IMP1 gene minus strand probe 5'-NH2-T (10) CCGCCTGCTCTAATGTAAGTTTCAAGAGTGATGCGTCTCCAAC-3'

OXA23 gene normal chain probe 5'-NH2-T (10) TTAAAATGTTGAATGCCCTGATCGGATTGGAGAACCAGAAAGC-3'

OXA23 gene minus strand probe 5'-NH2-T (10) TGATGAATCACCTGATTATGTCCTTGAACAATCTGACTCGGGGTTT-3'

OXA24 gene normal chain probe 5'-NH2-T (10) AATGGGTGTTACTCCACAGGTAGGTTGGTTGACTGGTTGGGT-3'

OXA24 gene minus strand probe 5'-NH2-T (10) GCTGACAATGCCATTGCCTCACCTAAAGTCATATCTTTCTCCCAC-3'

VIM gene normal chain probe 5'-NH2-T (10) GTGATGGTGATGAGTTGCTTTTGATTGATACAGCGTGGGGTG-3'

VIM gene minus strand probe 5'-NH2-T (10) GTTGCGATATGCGACCAAACACCATCAGCAATCTGGTAAAGC-3'

GES2 gene normal chain probe 5'-NH2-T (10) CTGCGGTGCAGCTTAGCGACAATGGGGCTACTAACCTCTTAC-3'

GES2 gene minus strand probe 5'-NH2-T (10) CCGCCATAGAGGACTTTAGCCACAGTACGTGCCATAGCAATAGG-3'

MRSA gold standard mecA gene probe, choose each one of positive minus strand probe, totally 2 probes:

MecA gene normal chain probe 5'-NH2-T (10) GAACTCAAAATGAAACAAGGAGAAACTGGCAGACAAATTGGGTGG-3'

MecA gene minus strand probe 5'-NH2-T (10) TGGTCTTTCTGCATTCCTGGAATAATGACGCTATGATCCCAATCTAACT-3'

Engineering carrier resistance gene probe commonly used; comprise paraxin acyltransferase (Cat) gene, bleomycin (Zeocin) gene, blasticidin (Bsr) gene, tetracycline (Puromycin) gene, kantlex (Kana) gene; ammonia benzyl mycin (Amp) gene and tsiklomitsin (Tet) gene; each gene is chosen each one at least of positive minus strand probe, totally 16 probes:

Cat gene normal chain probe 5'-NH2-T (10) CCTTGCAGCTTCATCATGCTGTATGTGATGGTTACCATGCTTC-3'

Cat gene minus strand probe 5'-NH2-T (10) CCAAGGAATCATTGAAATCGGTAGGGTGTTTTCAGGTATCGGTTT-3'

Zeocin gene normal chain probe 5'-NH2-T (10) GAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTT-3'

Zeocin gene minus strand probe 5'-NH2-T (10) TCAGTCCTGCTCCTCGGCCACGAAGTGCACGCAGTTG-3'

Bsr gene normal chain probe 5'-NH2-T (10) CCATTCATCTCAATGAGCACAAAGCAGTCAGGAGCATAGTCAGA-3'

Bsr gene minus strand probe 5'-NH2-T (10) AGGTCGCCACTGAGAAGATCACCATGCTCTATGAGGACAACA-3'

Puromycin gene normal chain probe 5'-NH2-T (10) GCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCT-3'

Puromycin gene minus strand probe 5'-NH2-T (10) GGTGACGGTGAAGCCGAGCCGCTCGTAGAAGGGGAGGTT-3'

Kana gene normal chain probe 5'-NH2-T (10) GAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAG-3'

Kana gene minus strand probe 5'-NH2-T (10) CGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTAT-3'

Tet gene normal chain probe 5'-NH2-T (10) CACCGTCACCCTGGATGCTGTAGGCATAGGCTTGG-3'

Tet gene minus strand probe 5'-NH2-T (10) CGTAGTCGATAGTGGCTCCAAGTAGCGAAGCGAGCAGGA-3'

Amp gene normal chain probe article one 5'-NH2-T (10) CACGACGGGGAGTCAGGCAACTATGGATGAACGA-3'

Amp gene minus strand probe article one 5'-NH2-T (10) CGGGCAAGAGCAACTCGGTCGCCGCATACACTA-3'

Amp gene normal chain probe second 5'-NH2-T (10) GCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCC-3'

Amp gene minus strand probe second 5'-NH2-T (10) CGCGAGACCCACGCTCACCGGCTCCAGATTTAT-3'

Described detection chip for drug resistance gene of bacteria, its corner is levied and is described 117 gene probes, any combination of its middle probe, be used for the preparation of gene chip etc., solid support material includes but are not limited to slide, tinsel, silicon chip, ceramic plate, plastic sheet, nitrocellulose membrane, nylon membrane etc.

The making of detection chip for drug resistance gene of bacteria of the present invention:

Selecting slide glass is the carrier of chip, and slide is used the chromic acid lotion soaked overnight available from Fisher Scientific company, then cleans with distilled water, spends the night in the ammoniacal liquor of immersion 25%, cleans with distilled water.Dry the middle 20min of 95% ethanol (pH4.5) that rear immersion contains aminopropyl trimethoxysilane, use again 95% ethanol ultrasonic cleaning, change the distilled water ultrasonic cleaning, 115 ℃ of oven dry.The slide of silanization is immersed in 50min in 5% the glutaraldehyde solution, ultrasonic 10min, washing twice is dried for subsequent use.

The gene probe that filters out carries out chemosynthesis by commercial company.Synthetic probe is dissolved with 3 * SSC in 96 orifice plates, use Calligrapher ^TMMiniArrayer point sample instrument MCP360 point needle is by the scheme point sample of design.117 probes with 17 large class drug resistant genes, article 1, blank probe, article 1, positive confidential reference items probe design forms a sample application array, every probe repeats 4 times, right side and the corresponding detection probes of downside at array are put respectively positive confidential reference items probe, positive in playing simultaneously positioning action, blank monitoring chip background signal to noise ratio.Point sample is complete, 37 ℃ of aquation 2h, and 4 ℃ save backup.

In order to seal the aldehyde radical of not being combined with oligonucleotide on the slide, according to following program slide is processed: 0.2%SDS washes twice, each 5min; Distilled water washes twice, each 5min; Slide is put into sealing (reduction) liquid 20min.0.2%SDS washes three times, each 1min; Distilled water washes twice, each 1min; The slide seasoning can be come into operation.

The extraction of sample nucleic acid and mark:

We collect sample, test and relatively optimized the treatment process of tested sample, have set up the Standard operation procedure SOP of the complete genome DNA of the high-quality all kinds of pathogenic bacterias of quick extracting.Identical sample is taked identical treatment process as far as possible.For the bacterium complete genome DNA random priming fluorescent mark that extracts, complementary in conjunction with (annealing), so that 3 ' hydroxyl terminal to be provided at random with random primer and the denatured double stranded dna of 6mer.Then without 5 ' → 3, under the effect of the Klenow fragment of 5 prime excision enzyme activity, add one by one the Nucleotide that contains the Cy3 mark at 3 ' C-terminal of primer, progressively form the DNA of mark.

The detection of gene chip and analysis:

With fluorescently-labeled target dna and hybridization solution (fellatio ginseng) the abundant mixing of 1:1,98 ℃ of sex change 5min ice baths.Point of draw covers sizeable cover glass through silanization in the probe array zone, and chip is put into hybridizing box (box has 2 suitable * SSC to keep humidity), hybridizes 4 hours for 42 ℃.Take out chip, use successively the hybridization washing lotion of 42 ℃ of preheatings

, , (0.2 * SSC) respectively washes 5min, uses at last ddH ₂O washes 2min, naturally dries (above process all requires lucifuge), uses LuxScan ^TMThe image analysis of 10K Version3.0 micro-array chip scanner scanner uni.

Interpretation:

Experimental result shows, 17 large class drug resistant gene detection chip of preparation are there are no specific cross reaction, and can distinguish different genotype.Multiple sample is detected, all reality preferably specificity and susceptibility.The sensitivity of genechip detection and 17 large class drug resistant genes is to be limited by the amplification label method, the sensitivity detected result shows that random primering does not have the pcr amplification marking sensitivity high, this is that target dna proportion in complete genome DNA is limited because we directly carry out mark and non-PCR amplification to complete genome DNA.Yet, along with whole genome amplification technology (WGA), (MDA) progressively is applied in the gene chip such as multiple displacement amplification, it is can be highly loyal copies whole genomic dna, amplify the fragment of 10～100 kb sizes, can provide a large amount of homogeneous complete whole genome sequence, for chip mark hybridization provides enough nucleic acid amounts.

The advantage that the present invention compared with prior art has:

(1) high specific: the specificity of chip detection is through Double Selection;

(2) high-throughput: to the high-throughput Parallel testing of a plurality of genes;

(3) lack detection time, consumption sample amount is few, and reaction volume is little;

(4) arbitrary combination probe according to actual needs;

(5) be easy to automatization and detection.

Description of drawings

The invention will be further described below with reference to accompanying drawing:

Fig. 1 is the whole chip manufacturing of the present invention and testing process.

Fig. 2 is the present invention's resistant gene chip probe sample application array synoptic diagram of recombinating.

Fig. 3 is the present invention's resistant gene chip sensitivity detected result of recombinating, and wherein the corresponding DNA concentration of A, B, C and D is respectively 80ng/ μ l, 20ng/ μ l, 5ng/ μ l.

Fig. 4 is detection chip for drug resistance gene of bacteria point sample matrix synoptic diagram of the present invention.

Fig. 5 is the detected result of extended spectrumβ-lactamase resistant organism of the present invention.

Fig. 6 is the detected result of cynnematin resistant organism of the present invention.

Embodiment

With reference to accompanying drawing 1～6, gene detecting chip comprises that 117 oligonucleotide probes of (1) 17 large class drug resistant gene make up and quality control probes; (2) oligonucleotide probe is solidificated in the probe array that forms on the solid support material by arm molecule;

GC-3'

TGG-3'

The detection gene chip of a kind of detection 17 large class drug resistant genes has comprised extraction and the amplification label of complete genome DNA; Carrier such as slide, silicon chip etc.; The modification of carrier surface arm molecule; Oligonucleotide probe (comprise and detect oligonucleotide probe and quality control probes) and curing thereof; Determination and analysis etc.

Described detection chip for drug resistance gene of bacteria, it is characterized in that described 117 gene probes, any combination of its middle probe, be used for the preparation of gene chip etc., solid support material includes but are not limited to slide, tinsel, silicon chip, ceramic plate, plastic sheet, nitrocellulose membrane, nylon membrane etc.

Embodiment one. drug resistant gene chip detection Kana, Amp resistance intestinal bacteria

Prepare gene chip with synthetic gene probe, detect the genetically engineered recombinant escherichia coli that contains Kana, Amp drug resistant gene.

1. the extraction of sample nucleic acid and mark

Bacterial Genomic DNA Extraction kit test kit is adopted in the preparation of bacterium complete genome DNA, and by specification requires to operate.DNA is adjusted to 1 μ g/ μ l, 4 times of gradient dilutions then, the DNA concentration of ultimate sensitivity experiment is respectively 80ng/ μ l, 20ng/ μ l, 5ng/ μ l, 0.8ng/ μ l, 0.2ng/ μ l, random primering mixes DNA with Cy3 dCTP, and reaction system is seen lower

Complete genome DNA 2 μ g

Random primer (2nmol/ μ l) 2 μ l

Add ddH2O cumulative volume to 10 μ l, 97 ℃, ice bath 3min behind the 3min50s.

Place on ice, add:

1M Cy3 dCTP 1.5μl

10×dUTPs 2.5μl

Klenow fragment Buffer 2.5μl

ddH2O 4.5μl

Room temperature is placed 2min

Klenow fragment 4μl

Total 25μl

After room temperature is placed 5min, 37 ℃ of reaction 2.5h.Add 2 μ l klenow fragment, 37 ℃ of reaction 2.5h, 65 ℃ of reaction 5min.In-20 ℃ of preservations.

2. the preparation of chip

Selecting the slide of processing through silanization is chip carrier, soaks 50min with 5% glutaraldehyde, and ddH2O ultrasonic cleaning 2 times is put drying and located for subsequent use.

Selected Tet S1, Kana A2, Kana S1 and four probes of Amp A2, synthetic probe has been dissolved in 3 * SSC.Carry out point sample according to Fig. 2 array, other is provided with positive confidential reference items and blank probe.Behind the point sample, 37 ℃ of hydrations, 2h.During use, wash twice with 0.2%SDS first, each 5min; Distilled water washes twice, each 5min; Slide is put into sealing (reduction) liquid 20min.0.2%SDS washes three times, each 1min; Distilled water washes twice, each 1min; The slide seasoning is stand-by.

3. the hybridization check of chip

Get target dna and the abundant mixing of 5 μ l hybridization solutions (fellatio ginseng) of 5 μ l marks, 98 ℃ of sex change 5min ice baths, point are hybridized 4h for 42 ℃ in the probe array zone.Take out chip, use successively the hybridization washing lotion of 42 ℃ of preheatings ,

,

Respectively wash 5min, use at last ddH ₂O washes 2min, naturally dries (above process all requires lucifuge), uses LuxScan ^TMThe image analysis of 10K Version3.0 micro-array chip scanner scanner uni.Whole chip manufacturing and testing process are seen Fig. 1.

4. detected result

The hybridization signal value of every probe is 4 mean values that repeat signal value a little, adopts signal to noise ratio (SNR) value to judge as standard.We are lower than 1.5 negative (-) with the signal to noise ratio average of target gene probe hybridization signal, are higher than 1.5 positive (+), and signal value difference is higher than 3 times to any point signal value less than hybrid context or between repeating a little, and it is invalid all to be considered to.As shown in Figure 3, although when target dna concentration is 5ng/ μ l, can detect the gene chip hybridization signal, but adopt LuxScan ^TMSignal to noise ratio all＜1.5 did not reach effective standard after the 10K analysis software was processed.When target dna concentration was 20ng/ μ l, Kana A2 and Kana S1 each point signal to noise ratio average were respectively 4 and 7, and namely the detection sensitivity of chip is the DNA of 20ng/ μ l.

Embodiment two. bacterial resistance gene chip detection extended spectrum β lactamases Resistant strain

Clinically 3 generation antibiotic being widely used of extended spectrum β lactamases (ESBLs) such as cephalosporin, rise year by year so that produce the recall rate of ESBLs bacterial strain, it is mainly produced by SHV, CTX-M gene mediated at home, shows as multidrug resistant, for clinical treatment brings difficulty.Gene chip is compared traditional detection method has unique advantage in infectious diseases detects, detection chip for drug resistance gene of bacteria has been set up in this experiment, optimizes and determines the chip detection condition, and part sample resistance is detected.

1. the preparation of chip

117 synthetic probes, with 3 * SSC dissolving, concentration is 40 pmol/ μ l in 96 orifice plates.Use Calligrapher ^TMMiniArrayer point sample instrument MCP360 point needle, by the scheme point (see figure 4) of design, point sample is complete, 37 ℃ of aquation 2h, 4 ℃ save backup.During use, wash twice with 0.2%SDS first, each 5min; Distilled water washes twice, each 5min; Slide is put into sealing (reduction) liquid 20min.0.2%SDS washes three times, each 1min; Distilled water washes twice, each 1min; The slide seasoning is stand-by.

2. nucleic acid extraction and mark

After each bacterial strain increased bacterium and cultivate, the bacterium complete genome DNA extracted according to the working specification in the Bacterial Genomic DNA Extraction kit test kit operation instruction, and agarose gel electrophoresis is estimated quality and the integrity of DNA.Klenow fragment random primering mixes DNA with Cy3 dCTP, reaction system: complete genome DNA 2 μ g, random primer 2 μ l add ddH ₂O to 10 μ l, 97 ℃ of reaction 3min50s, ice bath 3min.Place on ice, add Cy3-dCTP 1.5 μ l, 10 * dNTPs, 2.5 μ l, Klenow fragment Buffer 2.5 μ l, Klenow fragment 4 μ l add ddH ₂O to 25 μ l.Room temperature leaves standstill 5min, and 37 ℃ of reaction 2.5h add 4 μ l klenow fragment, continue reaction 2.5h, 65 ℃ of reaction 5min.In-20 ℃ of preservations.

3. hybridize and chip detection

,

4. detected result

Show that through the resistance test 4 strains are the extended spectrum β lactamases resistant organism.Chip Preliminary Applications detected result shows that the clinical bacterium of 4 strains all detects ESBL-CTXM1-3 totally 6 drug resistant genes, and wherein 1,2 strains detect the TEM gene, and 4 strains detect the PER gene, see Fig. 5.The chip hybridization scan image signal is clear, has lower whole background and higher signal to noise ratio (S/N〉10), and drug resistant gene type detected result is consistent with the bacterial resistance phenotype, and can distinguish different genotype.

Embodiment three. bacterial resistance gene chip detection cynnematin Resistant strain

In the resistance Gram-negative bacteria, cynnematin resistant organism (AmpC) is very large to health threat, and AmpC has than the wider substrate of ESBL spectrum and insensitive to enzyme inhibitors, can cause the serious resistance of bacterium, the treatment difficulty, and case fatality rate is high.Fast, accurately, high-throughput diagnostic pathogenic bacteria and detection drug resistant gene are the key points of prevention and control infectious diseases.We have set up detection chip for drug resistance gene of bacteria, and the part sample has been carried out detecting relatively.

1. the full genome of bacterium extracts and mark

2. the preparation of chip

117 synthetic probes, with 3 * SSC dissolving, concentration is 40 pmol/ μ l in 96 orifice plates.Use Calligrapher ^TMMiniArrayer point sample instrument MCP360 point needle is by the scheme point sample (see figure 4) of design.Afterwards, 37 ℃ of aquation 2h, 4 ℃ save backup.During use, wash twice with 0.2%SDS first, each 5min; Distilled water washes twice, each 5min; Slide is put into sealing (reduction) liquid 20min.0.2%SDS washes three times, each 1min; Distilled water washes twice, each 1min; The slide seasoning is stand-by.

3. chip hybridization

With fluorescently-labeled target dna and hybridization solution (fellatio ginseng) the abundant mixing of 1:1,98 ℃ of sex change 5min ice baths.Point of draw was hybridized 4 hours for 42 ℃ in the probe array zone.Take out chip, use successively the hybridization washing lotion of 42 ℃ of preheatings

,

Respectively wash 5min, use at last ddH ₂O washes 2min, naturally dries (above process all requires lucifuge), and whole chip manufacturing and testing process are seen Fig. 1.

4. detected result

The pathogenic bacteria of known detection is the cynnematin resistant organism.Use LuxScan ^TM10K Version3.0 micro-array chip scanner scanner uni image analysis bacterial resistance chip, detected result shows, 3 pathogen strain bacterium all detect totally 12 AmpC drug resistant genes, conform to conventional resistance test-results, chip background and hybridization signal value are all more stable, there are no non-specific hybridization signal, the result meets experimental design, sees Fig. 6.

Claims

1. detection chip for drug resistance gene of bacteria is characterized in that gene detecting chip comprises 117 oligonucleotide probes and the quality control probes of (1) 17 large class drug resistant gene; (2) oligonucleotide probe is solidificated in the probe array that forms on the solid support material by arm molecule;

GC-3'

Beta-lactam enzyme (BLA) resistance gene probe is divided into extended spectrum β lactamases, cephalosporin enzyme, carbapenem enzyme, streptococcus aureus (MRSA) gold standard mecA gene;

TGG-3'

Amp gene minus strand probe second 5'-NH2-T (10) CGCGAGACCCACGCTCACCGGCTCCAGATTTAT-3'.

2. detection chip for drug resistance gene of bacteria according to claim 1, it is characterized in that described 117 gene probes, be used for the preparation of gene chip, solid support material includes but are not limited to slide, tinsel, silicon chip, ceramic plate, plastic sheet, nitrocellulose membrane, nylon membrane.

3. detection chip for drug resistance gene of bacteria claimed in claim 1 is used for containing the sick monitoring of detection of pathogens, somatotype, rational use of drug, infection and the epidemiology survey of 17 large class drug resistant genes.