CN107475421A

CN107475421A - Pneumonia pathogenic bacteria drug resistant gene quickly identifies genetic chip

Info

Publication number: CN107475421A
Application number: CN201710868656.9A
Authority: CN
Inventors: 陈良安; 马秀清; 王升启; 刘琪琦; 李春笋; 梁志欣; 杨震; 赵微
Original assignee: Chinese PLA General Hospital
Current assignee: Academy of military medicine, PLA Academy of Military Sciences; Chinese PLA General Hospital
Priority date: 2017-09-08
Filing date: 2017-09-22
Publication date: 2017-12-15
Anticipated expiration: 2037-09-22
Also published as: CN107475421B; CN107475422B; CN107475422A

Abstract

The application is related to the genetic chip that can detect 24 kinds of pneumonia pathogenic bacteria drug resistant genes.This 24 kinds of pneumonia pathogenic bacteria drug resistant genes are the closest with Relationship with Clinical from aminoglycoside, quinolones, extended spectrum β lactamases class (ESBLs), cephalosporins (AmpC), Carbapenems, vancomycin resistance, methicillin-resistant and the gene for causing membrane permeability to change.The genetic chip is by designing and screening repeatedly, and most sensitivity is good at last, and the high probe of specificity is fixed on chip, clinical culture and drug sensitive test need not be carried out, simple and quick, largely the reasonable employment for antibiotic has striven for valuable time, clinic popularization and application.

Description

Pneumonia pathogenic bacteria drug resistant gene quickly identifies genetic chip

Technical field

The present invention relates to biochip technology, more particularly to pneumonia pathogenic bacteria drug resistant gene quickly to identify genetic chip.

Background technology

In recent years, because the aggravation of air pollution and the generation of public health event, breathing problem cause increasing pass Note.Pneumonia refers to include last air flue eventually, and the pulmonary parenchyma inflammation including alveolar and interstitial lung etc., is that global infectious diseases is dead First cause^[1].Counted according to the World Health Organization (WHO), there are about 3,500,000 people every year and die from ALRI, shelter has extremely Die reason the 3rd^[2].In the U.S., pneumonia and influenza are located at the 9th of all causes of the death, have within 2010 and 2011 People dies of pneumonia more than 50000^[3-5], and the statistics not by the cancer of septicemia caused by pneumonia and the complication that dies of pneumonia, The cases such as Parkinson include pneumonia group, so the actual number dead because of pneumonia is more taller than this number^[2].In China, every year extremely Rare 2,500,000 people suffers from pneumonia, and rural area is more than city, wherein less than 5 years old Death-pneumonia of Children rate be 184,/10 ten thousand -1,223 ten thousand/ 100000^[6], Pneumonia in Older Patients case fatality rate do not have a definite data, but auspicious according to yellow China^[7]Pneumonia in Older Patients is apparently higher than other ages Section.It can be seen that pneumonia serious threat the life and health of the mankind.

Pneumonia it is pathogenic it is original a lot, including bacterium, virus, fungi, parasite etc., wherein most important most commonly bacterium. It is well known that pneumonia can be divided into community acquired pneumonia (CAP) and Nosocomial Pneumonia (HAP) according to onset environment.Big portion CAP patient is divided only to need outpatient clinic, but still the patient for having about 20% still needs to hospitalization^[8].CAP pathogenic bacteria are with gram Based on positive bacteria, wherein again using streptococcus pneumonia as first, 35-80% is accounted for, next is haemophilus influenzae, Shi Fei legions Bacterium, mycoplasma pneumoniae, CPN, staphylococcus aureus etc.^[9].HAP pathogenic bacteria are than wide, with Gram-negative bacteria Based on, wherein commonly pseudomonas aeruginosa, EHEC, Klebsiella Pneumoniae, Acinetobacter bauamnnii are other also thermophilic Stenotrophomonas maltophilia, Burkholderia cepacia, enterococcus faecalis, VREF, enterobacter cloacae etc., in addition Gram-positive Bacterium S. aureus L-forms also see HAP, especially Methicillin-resistant Staphylococcus aureus (MRSA)^[10].Antibiotic is to treat bacterial pneumonia most Effective method.The discovery of nineteen forty-one penicillin makes infectious diseases obtain effective control, and then new antibiotic is continuous It is found and produces, and is applied to many fields, including aquaculture^[11]And planting industry^[12], the mankind rapidly enter antibiotic Golden age.As antibiotic is widely applied, antibody-resistant bacterium but continues to bring out, and is no longer limited to a kind of antibiotic Resistance, but Multiple Classes of Antibiotics can be resisted simultaneously, such as some clinical strains VREF, S. aureus L-forms, Klebsiella Pneumoniae, Baos Graceful acinetobacter calcoaceticus, pseudomonas aeruginosa and enterobacteria are to the equal resistance of most antibiotic^[12].Multiple antibiotic resistant strain and general resistance to The super antibody-resistant bacterium such as medicine bacterial strain, such as the streptococcus pneumonia (PRSP) of penicillin resistant^[13], MRSA^[14], the intestines ball of vancomycin resistance Bacterium (VRE)^[15]Deng eruption and prevalence, once caused global fear, resistance problems have almost been absorbed in runaway condition^[12].Antibiotic Infected patient case fatality rate is significantly increased in Endodontic failure, especially seriously ill patient.According to CDC of the U.S. (CDC) Analysis is annual to have 2,000,000 patients to infect drug-fast bacteria, wherein 23000 patients are therefore dead^[16].2008,35,000,000,000 dollars of the U.S. There are 20,000,000,000 dollars to be used to control and treat infection caused by bacterial resistance in medical treatment cost^[16].Therefore, clearly cause a disease as early as possible The resistant characterization of bacterium, it is the key for reducing pneumonia case fatality rate and the death rate using targetedly remedy measures.

Bacterial resistance sex chromosome mosaicism produces along with the discovery of antibiotic, from single resistance to multidrug resistant, multidrug resistance and general Resistance, effective antibiotic increasingly finite sum are expensive.In May, 2014, WHO announced a whole world resistance resistance plan, the same year 6 Month British government 10,000,000 pounds of bacterial drug resistance problems to be solved of budget, September U.S. government require that relevant departments make pin The concrete measure threatened bacterial resistance^[12], the antibacterium resistance action plan appearance in 5 years by a definite date March one in 2015, and 1,200,000,000 dollars are appropriated funds to be used to tackle bacterial resistance in the financial budget of 2016, it is seen that bacterial resistance problem is that the whole world is urgently to be resolved hurrily Problem.

Specifying the species of pneumonia pathogenic bacteria and its resistant characterization is pneumonia early diagnosis, takes effective remedy measures in time, reduces lung The important step of scorching case fatality rate.The drug sensitive test of detection resistant characterization includes disk diffusion method (K-B methods), agar or broth dilution Method, antibiotic concentration gradient method (E-test methods), wherein conventional golden allusion quotation method is K-B methods.K-B methods need to cultivate and separated, About 48-72 hours, and influence factor is more, such as the concentration of bacterium solution, bacterial load, culture medium, the size of tablet etc.^[18]。 Drug sensitive test also has the operating system of automation, is a kind of discontinuous dilution process used, alleviates the burden of operator, But automation Analysis of Drug Susceptibility system can not flexibly select the species of antibiotic, still can not substitute K-B methods completely.Infect in the U.S. Disease association (IDSA) guide once suggested giving patients with pulmonary infection antibiosis extract for treating in 8 hours, and this time is 2003 Year shortens to 4 hours^[19,20], it is seen then that drug sensitive test at present can not reach clinical quick diagnosis, take immunotherapy targeted autoantibody to arrange in time The demand applied, the treatment of pneumonia early stage can only rely on empirical treatment, so further exacerbate the generation of antibody-resistant bacterium, because This, a kind of method of accurate, quick detection pneumonia pathogenic bacteria resistant characterization of urgent clinical needs.

The content of the invention

In view of the above problems, the present invention is directed to propose a kind of side for the drug resistance that can quickly and accurately detect pneumonia pathogenic bacteria Method.

The pneumonia pathogenic bacteria drug resistant gene of the present invention quickly identifies genetic chip, and it includes corresponding to pneumonia pathogenic bacteria drug resistant gene Probe；Wherein, the pneumonia pathogenic bacteria drug resistant gene comprises at least metal beta-lactam class drug resistant gene, acyl in the metal β Amine drug resistant gene includes at least one of NDM1, IMP, VIM；

Wherein, it is corresponding to the sequence of NDM1 probe：

ATGGAAACTGGCGACCAACGGTTTGGCGATCTTTTTTTTTTTT；

Sequence corresponding to IMP probe is：

AGATAACGTAGTGGTTTGGTTTTTTTTTTTT；

Sequence corresponding to VIM probe is：

TCTAGAAGGACTCTCATCGATTTTTTTTTTTT。

Preferably, the pneumonia pathogenic bacteria drug resistant gene also includes Carbapenem-resistant gene, the Carbapenem-resistant Gene includes at least one of OXA2, OXA10, OXA23, OXA48, KPC；

Wherein, it is corresponding to the sequence of OXA2 probe：

TTGCAGGCCACAATCAAGACCAAGATTTTTTTTTTTTT；

Sequence corresponding to OXA10 probe is：

TCAAATGGGACGGAAAGCCAAGAGCCATGAATTTTTTTTTTTT；

Sequence corresponding to OXA23 probe is：

GAGAGTAATGGCTACAAAATTTTTTTTTTTTTTT；

Sequence corresponding to OXA48 probe is：

AGTTTCTGGCTCGACGGTGGTATTCTTTTTTTTTTTT；

Sequence corresponding to KPC probe is：

TGACGGCCTTCATGCGCTCTATCGGCGATACTTTTTTTTTTTT。

Preferably, the pneumonia pathogenic bacteria drug resistant gene also includes aminoglycoside resistant gene, the aminoglycoside resistant Gene includes aac (3)-II, aac (6') at least one of-I, ant (2 ")-I, ant (3 ")-I；

Wherein, it is corresponding to the sequence of aac (3)-II probe：

GTGGGTTCGGCCTGCTGAATCAATTTCTGGTTTTTTTTTTTT；

Sequence corresponding to aac (6')-I probes is：

AGGTCACCAAGATCCAAACGGACTTTTTTTTTTTT；

Sequence corresponding to ant (2 ")-I probe is：

CACGCAAGCACGATGATATTGATCTGACGTTTTTTTTTTTT；

Sequence corresponding to ant (3 ")-I probe is：

TCTCCGCGCTGTAGAAGTCACCATTGTTTTTTTTTTTTTT。

Preferably, the pneumonia pathogenic bacteria drug resistant gene also includes quinolones drug resistant gene, and quinolones drug resistant gene includes At least one of qnrA, qnrB, qnrS；

Wherein, it is corresponding to the sequence of qnrA probe：

ATGTACTTCTGCTCGGCTTATATCTCAGGTTTTTTTTTTTTT；

Sequence corresponding to qnrB probe is：

GTAGCGCATATATCACGAATACCAATCTAAGCTACGCCTTTTTTTTTTTT；

Sequence corresponding to qnrS probe is：

TCGCTGGATAGGAACGAACCTAGCTTTTTTTTTTTT。

Preferably, the pneumonia pathogenic bacteria drug resistant gene also includes ESBLs class drug resistant genes, the ESBLs classes drug resistant gene bag Include at least one of CTX-M-14, CTX-M-15, SHV, TEM；

Wherein, it is corresponding to the sequence of CTX-M-14 probe：

CAGCCTGTCGAGATCAAGCCTGTTTTTTTTTTTT；

Sequence corresponding to CTX-M-15 probe is：

AGACTGGGTGTGGCATTGATTATTTTTTTTTTTT；

Sequence corresponding to SHV probe is：

CTCCAGCTGTTCGTCACCGGCATTTTTTTTTTTT；

Sequence corresponding to TEM probe is：

GTGCTGCCATAACCATGAGTGATAACTTTTTTTTTTTT。

Preferably, the pneumonia pathogenic bacteria drug resistant gene also includes cephalosporins drug resistant gene, the cephalosporins resistance Gene is DHA；

Wherein, it is corresponding to the sequence of DHA probe：

GCTTGATGCGGAATCTTACGGTTTTTTTTTTTT。

Preferably, the pneumonia pathogenic bacteria drug resistant gene also includes vancomycin resistance class drug resistant gene, the vancomycin resistance class Drug resistant gene includes at least one of vanA, vanB；

Wherein, it is corresponding to the sequence of vanA probe：

CCGCATTGTACTGAACGAAGTCTTTTTTTTTTTT；

Sequence corresponding to vanB probe is：

GCTTACCTACCCTGTCTTTGTGATTTTTTTTTTTT。

Preferably, the pneumonia pathogenic bacteria drug resistant gene also includes methicillin-resistant class drug resistant gene, the methicillin-resistant class Drug resistant gene is mecA；

Wherein, it is corresponding to the sequence of mecA probe：

GTAAGCACACCTTCATATGACGTCTATCCTTTTTTTTTTTT。

Preferably, the pneumonia pathogenic bacteria drug resistant gene also includes porin class drug resistant gene, the porin class resistance Gene is oprD2；

Wherein it is corresponding to the sequence of oprD2 probe：

TGTTCCCGCAGACCGCGATTTTTTTTTTTT。

Preferably, the genetic chip further comprises 3 negative probes；Three negative probes are respectively： TCAGAGCCTGTGTTTCTACCAATTTTTTTTTTTT、CATCAATAGGGTCCGATATTTTTTTTTTTT、 CGAACGCAAATCAATCTTTTTCCAGGTTTTTTTTTTTTT。

The application to aminoglycoside, quinolones, extended spectrum β lactamases class (ESBLs), cephalosporins (AmpC), Carbapenems, vancomycin resistance, methicillin-resistant and cause in the gene that membrane permeability changes, have chosen 24 and closed with clinical System's drug resistant gene the closest has carried out the development of the biological chip of pneumonia pathogenic bacteria drug resistant gene as target gene.Should Genetic chip can be directly used for clinical samples, it is not necessary to culture and drug sensitive test, it is simple and quick, and it is largely anti- The reasonable employment of raw element has striven for valuable time, clinic popularization and application.

Brief description of the drawings

Fig. 1 is that PCR primer cuts glue purification reclaimer operation flow chart；

Fig. 2 is plasmid extraction operational flowchart；

Fig. 3 is gene chip hybridization step；

Fig. 4 is drug resistant gene agarose gel electrophoresis figure；

Fig. 5 is 3 synthesis drug resistant gene fragment Blast comparison results；

Fig. 6 is 21 drug resistant gene purpose fragment plasmid construction sequence charts；

Fig. 7 is part probe sensitivity results；

Fig. 8 is drug resistant gene chip specificity experimental result

Fig. 9 is drug resistant gene chip repeated experiment.

Embodiment

Materials and methods

First, experiment material

1st, antibody-resistant bacterium

The involved antibody-resistant bacterium of this experiment comes from antibody-resistant bacterium that this seminar collects early stage (including 21 kerekou pneumonias primary Bacillus and pseudomonas aeruginosa, 14 Acinetobacter bauamnniis and 8 Escherichia coli), in addition by Military Medical Science Institute radiation with Radiation medicine research institute present inactivation antibody-resistant bacterium (including production KPC Klebsiella Pneumoniae strain, produce NDM1 enterobacteria, MRSA bacterial strains and VRE bacterial strains)

2nd, key instrument and reagent

Instrument is used in table 1-2 experiments

Experiment reagent

LB culture mediums：Fluid nutrient medium --- 10g peptones (TRYPTONE), 10g NaCl, 5g yeast extracts (YEAST EXTRACT) plus distilled water is to 1L, standby after high pressure.Solid medium --- 10g peptones (TRYPTONE), 10g NaCl, 5g Yeast extract (YEAST EXTRACT), 15g agar (AGAR) plus distilled water to 1L are standby after high pressure.

Sample treatment liquid：25mmol/L NaOH, 0.1nmol/L EDTA, 10mmol/L Tris-HCl, 1%NP-40,2% Chelex-100,1%Triton X-100；

Electrophoresis liquid：50 × TAE storing liquids --- glacial acetic acid 28.5ml, Tris 121g, 0.5ml/L Na2EDTA50ml is mixed, fixed Hold to 500ml and store.1 × TAE storing liquids --- 50 × TAE storing liquid 10ml, add distilled water to be settled to 500ml, it is standby.

2% Ago-Gel：1g agar Icing Sugar is weighed, is dissolved in 50ml 1 × TAE solution, micro-wave oven is heated to melting completely Change, taking-up shakes up, and in cooling a moment, adds 5 μ l EB solution, shakes up, slowly pour into electrophoresis tank, treats that agarose gel solidifies.

Sampling liquid：0.1%SDS, 6 × SSC, 5% glycerine, 2% (mass/volume) Ficoll400；

Hybridization solution：0.6%SDS, 8 × SSC, 10 × Denhardt solution, 10% formamide；

Pre- washing lotion：0.2%SDS (25ml 50 × SDS solution+2475ml distilled water)；

Washing lotion A：1 × SSC, 0.2%SDS (125ml 20 × SSC solution+50ml 50 × SDS solution+2325ml distilled water)；

Washing lotion B：0.2%SDS (25ml 50 × SDS solution+2475ml distilled water)；

Washing lotion C：0.1%SSC (12.5ml 20 × SSC solution+2487.5ml distilled water)；

PBST solution：2500ml PBS solution+5ml Tween-20, adjust PH to 7.0-7.2；

Marking fluid：Streptavidin-horseradish peroxidase；

Luminescent solution A：MILLIPORE companies；

Luminescent solution B：MILLIPORE companies；

2×Gold Star Best Master Mix：Beijing health is century biology Co., Ltd

Multiplex PCR 5×Master Mix：The U.S. New England Biolabs (NEB) company；

The small extraction reagent kit of plasmid：Beijing Tiangeng biochemical technology Co., Ltd；

Ago-Gel QIAquick Gel Extraction Kit：Beijing Tiangeng biochemical technology Co., Ltd；

DH5 α competent cells：Beijing Tiangeng biochemical technology Co., Ltd；

PMDTM18-T Vector Cloning Kits：TaKaRa companies；

DL2000 DNA marker：TaKaRa companies；

Chip chip base：Baiao Science and Technology Co. Ltd., Shanghai.

2nd, experimental method

1st, the selection of drug resistant gene

24 drug resistant genes are finally determined by consulting literatures, are aminoglycoside resistant gene ant (3 ")-I, aac respectively (3)-II、aac(6')-I、ant(2")-I；Quinolones drug resistant gene qnrA, qnrB, qnrS；Carbapenem-resistant gene OXA2、OXA10、OXA23、OXA48、KPC；ESBLs class drug resistant genes CTX-M-14, CTX-M-15, TEM, SHV；Cynnematin Class drug resistant gene DHA；Metallo-β-lactamase class drug resistant gene NDM1, IMP, VIM；Resistance to vanA, vanB through the ages；Resistance to methoxy west The mecA and hole path albumen oprD2 of woods.Wherein oprD2 normal expressions in sensitive strain, the structure hair of film when it is lacked It is raw to change, resistance can be produced.

2nd, the design and synthesis of drug resistant gene primer and probe

The sequence of each drug resistant gene is first searched from ncbi database, each drug resistant gene at least downloads 3 different ID numbers That is the sequence of GenBank sequence numbers, to prevent the change of drug resistant gene Individual base in different strains, in design primer and spy These bases are avoided during pin as far as possible.The design of primer and probe uses DNAMAN softwares and Oligo7 softwares.Specific design principle It is as follows：

Design of primers principle：(1) G/C content is between 40% to 60%；(2) length is 18-30bp；(3) 3 ' ends can not occur The continuous base (such as TTT or GGG) of 3 or more than 3；(4) primer itself should avoid hairpin structure or dimer；(5) product Secondary structure should be avoided；(6) between primer or itself there can not be 4 continuous base complementrities.The length of PCR primer preferably exists Within 300bp, it need to also be analyzed according to the designed primer of the principle on NCBI websites by BLAST, preliminary identification primer Specificity.

Probe design principle：(1) probe itself should avoid producing secondary structure；(2) probe length is typically in 17-50nt；(3) visit Pin sequence is in the conservative region between upstream and downstream primer, not including upstream and downstream primer sequence；(4) probe sequence avoids continuous 4 The appearance of identical base more than individual；(5) probe sequence will have specificity, can not have between other pathogen PCR products compared with Long continuous matched sequence.Each gene can design 2-3 bar probes and be screened, and equally, these are designed on principle Probe needs to carry out BLAST analyses, in addition, also needing to compare probe and probe, probe and other pathogenic bacteria using DNAMAN softwares With the presence or absence of pairing phenomenon, the specificity of Preliminary detection probe between PCR primer.

The synthesis of primed probe

During the screening of primer, the synthesis of common PCR primers is radiated by Academy of Military Medicine, PLA cures with radiating Learn synthesized by research.Screen successful primer to be synthesized by Shanghai Sheng Gong engineering services Co., Ltd, during synthesis, under primer The end mark biotin of trip 5 ' (Biotin).The synthesis of probe is mainly：(1) T-sequence of 12 repetitions is connected in the end of probe 3 ', Purpose is to increase the spatial flexible of probe；(2) while with the end of amino (- NH2) mark 3 '.

3rd, the screening of the structure of drug resistant gene plasmid and drug resistant gene primer

Except MRSA and VRE, remaining bacterial strain does not know specific drug resistant gene Carriage, and this experiment is chosen in 4 kinds of bacterial strains every time Select 3 kinds of antibody-resistant bacterium to carry out the screening of target gene, while be also the screening to primer.For the resistance base not screened Cause, redesign primer and screened.The drug resistant gene not filtered out by testing repeatedly is by Beijing Bo Maide gene technology Co., Ltd carries out fragment synthesis, and fragment is building up in Escherichia coli, is prepared into plasmid bacterial.

4th, DNA of bacteria extracting method, regular-PCR and its product purification, the preparation of plasmid reference material and extraction, the preparation of chip, Chip hybridization step is as follows.

DNA of bacteria extracting method

Using direct boiling method, i.e., bacterium solution is taken and be put into right amount in EP pipes, covered tightly lid, 10min is boiled in boiling water, it is rapid after taking-up Place on ice, 30min, then 12000 leave heart 10min, take supernatant standby.

Regular-PCR and its product purification

Regular-PCR system and condition, it is shown in Table 1-3 and table 1-4

Table 1-3 common PCR reaction systems

Table 1-4 common PCR reaction conditions

PCR primer is added in Ago-Gel hole, electrophoresis is carried out, then coagulates the agarose containing single purpose band Glue is cut, it is not too big also should not be too small, be just advisable comprising purpose band, be put into clean EP centrifuge tubes, its recovery is adopted With plain agar sugar gel DNA QIAquick Gel Extraction Kits, operated according to specification, key step such as Fig. 1.

The preparation and extraction of plasmid reference material

The pathogenic bacteria for having reference culture are expanded by the method for regular-PCR to target gene, without the pathogenic bacteria of reference culture By Beijing, Bo Maide companies directly synthesize target-gene sequence.Then objective gene sequence is cloned by pMDTM18-T Vector Kit carries out carrier T clone, and specific experiment step is as follows：

Carrier T and Solution I are placed on and melted on ice, according to table 1-5 addition linked system, carrier T and PCR cut glue and returned The mol ratio for receiving purified product DNA is generally：1:2-10, gently mix, PCR pipe is placed in 16 DEG C of constant-temperature metal bath overnight It is attached.

Table 1-5 pMD18-T support agents box clones system

DH5 α competent cells are placed on and dissolved on ice, after dissolving, are rapidly added thereto 10 μ l connection products, gently rotate from Heart pipe makes mixing, stands 30min on ice.

Centrifuge tube is put into 42 DEG C of water-baths, stands 60-90s, centrifuge tube is then moved into ice bath (this step is fast), mesh Be make cell it is rapid it is cold go, time 2-3min, the process can not rock centrifuge tube.

The μ l of LB fluid nutrient mediums 900 without antibiotic are added in centrifuge tube, 37 DEG C of shaking tables, 150rpm trainings are put into after mixing Support 45min.

By the liquid blending in centrifuge tube, the competent cell that 100 μ l of absorption have been converted to (Amp, 100 μ g/ containing ampicillin Ml it is with sterile elbow glass rod with gentle that cell is uniformly spreadable on LB solid mediums), close the lid, room temperature is placed about 1h, flat board is inverted, is placed in 12-16h in 37 DEG C of incubators.

Single bacterium colony on picking culture medium, it is inoculated into the LB fluid nutrient mediums that 5ml contains Amp (100 μ g/ml), is placed in 37 DEG C shaking table, 200rpm shaken cultivations 12-16h.

Bacterium is carried out using the special primer of bacterial target genes and universal primer M13F (- 47)/M13R (- 48) of pMD18-T carriers Liquid PCR, through agargel electrophoresis, choose the correct bacterium solution of pillar location and be sequenced and preserved.Sequencing is by Beijing Bo Maidesheng Thing Technology Co., Ltd. is carried out；During preservation, take the μ l of bacterium solution 500 to be saved to be mixed with 30% isometric glycerine, be placed in -70 DEG C Refrigerator preserves.

Sequencing result carries out plasmid extraction to the bacterium solution containing purpose fragment, operated according to plasmid extraction reagent through sequence alignment The specification of box is carried out, specific steps such as Fig. 2.

At 260nm wavelength, the concentration of measure extraction DNA, according to formula：Plasmid copy number (Copies/ μ l)=Avobenzene Jia Deluo constants × plasmid concentration (ng/ μ l) × 10^-9/ (660 × plasmid length bp) g/mol, the copy number of plasmid is calculated, so 10 times of gradient dilution is carried out afterwards, and it is respectively 10 to obtain copy number¹,10²,10³,10⁴,10⁵,10⁶Copies/ μ l plasmid reference Product, -20 DEG C of refrigerators preserve after packing, standby (multigelation is avoided, in order to avoid influence plasmid concentration).

The preparation of chip

First die bonding film is firmly attached in chip base, it is impossible to have bubble, a chip base is divided into 10 microarray conversion zones；

Utilize ddH₂O into final concentration of 100 μM of solution, vibrates the probe dilution of synthesis, mixing will on compact centrifuge The liquid of wall built-up is thrown to bottom, then by 1:1 ratio, 5 μ l probe solutions and 5 μ l chip sampling liquids are added to 384 orifice plates In, mix, avoid the generation of bubble；

Identity column should be also added in 384 orifice plates simultaneously, i.e., 20 repetition T-sequences of 3 ' mark amino, the final concentration of identity column is 1 μM, it is not necessary to it is too obvious, it is mainly used in the positioning of probe and monitors the appearance of chip hybridization false negative.Identity column and sampling liquid Ratio is equally 1:1；

, need to be by spotting needle ultrasound 10min or so, it is ensured that the cleaning of spotting needle before point sample；

During point sample, distance >=7mm of point and point, then repeated according to the size of microarray, every probe of how much determinations of probe Number, it is general 2-3 times.The temperature and humidity of lime light specimen chamber is further needed exist for, temperature room temperature, humidity is 30% or so Proper, humidity is too big, and deposition probe is not easy to assemble, and humidity is too small, and point of sample is small and non-round.

After point sample terminates, need also exist for, by spotting needle ultrasound 10min or so, keeping the cleaning of spotting needle, preventing loading wells from blocking And cross pollution.Then chip is placed in chip cartridges, puts drier into together with chip cartridges, 24h rears can be used.

The step of chip hybridization, as shown in Figure 3.

5th, chip sensitivity experiment

5.1 chip probe arrays design

Selection specificity is good, the probe of high sensitivity as final probe, and select 3 respectively from the mankind, virus and The probe (its sequence and all probe sequences are not complementary) of fungal gene sequence, as negative probes, to monitor in crossover process The appearance of false positive.

5.2 chip sensitivity experiments

With the plasmid reference material of gradient dilution early stage, concentration is respectively 10²,10³,10⁴,10⁵,10⁶Copies/ μ l plasmid is made For template, multiple asymmetric PCR reaction is carried out, hybridizes, the sensitivity of probe is then determined according to the result of chip.

6th, chip specificity is tested

Using concentration as 10⁴Copies/ μ l dilution plasmid is template, is reacted by multiple asymmetric PCR, chip hybridization, detection The specificity of chip.

As a result

1st, the screening of drug resistant gene

Successfully filter out 21 drug resistant genes from all antibody-resistant bacterium, 3 drug resistant genes OXA48, OXA2 and qnrA of residue by Beijing Bo Maide gene technology Co., Ltd synthesizes, and is shown in Table 2-1.

The source of table 2-1 drug resistant genes

2nd, primer screening result

The primer of 24 drug resistant genes is successfully filtered out, its sequence is shown in Table 2-2.The agarose gel electrophoresis of 24 pairs of primer screenings As a result Fig. 4 is seen.

Table 2-2 drug resistant gene primer sequences

Note：F, forward primer；R, reverse primer

3rd, plasmid construction result

After primer screening success, the correct PCR primer of piece fragment position is cut into glue purification through agarose gel electrophoresis, passes through pMD18 Carrier is gone in bacillus coli DH 5 alpha, and extraction plasmid is sequenced with primer M13F (- 47) and M13R (+48), then DNAMAN Software carries out sequence alignment, and selection is sequenced correct plasmid and carries out the preparation of reference material and follow-up experiment.This experiment totally 24 Drug resistant gene, wherein 3 are synthesized due to a lack of template by company, sequence alignment result is shown in Fig. 5, in addition the purpose of 21 drug resistant genes Fragment is successfully building up in Escherichia coli, and all drug resistant gene purpose fragment sequencing results are correct, as a result see Fig. 6.

4th, the optimization of multiple asymmetric PCR

This experiment shares 24 pairs of primers, by the optimization repeatedly of concentration between the collocation between primer, primer, finally determines to be divided into 4 pipes PCR, often pipe 6 is to primer.Multi-PRC reaction reagent is that Beijing CoWin Bioscience Co., Ltd. produces 2 × Gold Star Best Master Mix, specific reaction condition are shown in Table 2-3, and reaction system is shown in Table 2-4 to table 2-7.

The multiple asymmetric PCR reaction conditions of table 2-3

First group of multi-PRC reaction system of table 2-4

Second group of multi-PRC reaction system of table 2-5

The 3rd group of multi-PRC reaction body of table 2-6

The 4th group of multi-PRC reaction system of table 2-7

5th, probe the selection result

It is used to screen for drug resistant gene purpose fragment design at least 3 probes every time, it is underproof to need to redesign, finally It is most bright to choose probe signals, is used for subsequent experimental with probe that other purposes fragment does not have hybridization reaction.In chip sensitivity and In specificity experiment, if probe sensitivity is low, poor specificity, then probe screening is re-started, by checking repeatedly, finally Following 24 probes are determined, are shown in Table 2-8.

Table 2-8 resistance gene probe the selection results

6th, the determination of drug resistant gene chip array

After all probe sequences determine, subsequent experimental is carried out according to following array of figure, is shown in Table 2-9.According to the size of chip and spy The quantity of pin, it is 0.075cm to determine the distance between probe and probe, and every probe repeats 2 points.

Table 2-9 drug resistant gene chip array figures

7th, drug resistant gene chip sensitivity results

According to the result of the quick identification chip sensitivity of pneumonia pathogenic bacteria strain early stage, the minimum plasmid concentration of probe in detecting exists 10²More than copies/ μ l, therefore the concentration that we choose plasmid reference material for the chip is 10²、10³、10⁴、10⁵、 10⁶copies/μl.Chip hybridization results show that all probe sensitivity are up to 10³Copies/ μ l, partial results are shown in Fig. 7.

8th, drug resistant gene chip specificity result

With the plasmid reference material 10 of 24 drug resistant genes⁴Copies/ μ l are that template carries out chip specificity checking, and as a result display is every One probe can detect respective purpose fragment, and not have non-specific hybridization with other purposes fragment, as a result see Fig. 8.

9th, drug resistant gene chip repeatability result

Randomly select several resistance gene probes and carry out repeated experiment, template is corresponding 10⁴Copies/ μ l plasmids refer to Product.Repeated experiment has 2 kinds, first, with a collection of chip, same template and reaction system and condition, is repeated 3 times PCR reactions, its Reaction product and chip hybridization；Second, same PCR reactions and different batches chip hybridization.

Drug resistant gene chip repeated experiment result is shown in Fig. 9.

24 drug resistant genes have respective fashion trend and resistance feature in the application.

CTX-M drug resistant genes are one kind of ESBLs class drug resistant genes, it is considered to be nearly 20 years most heavy in terms of bacterial resistance progress The drug resistant gene wanted^[52].The gene is present on the chromosome of three kinds of Ke Lvwo Pseudomonas or surrounding plant roots environmental bacteria, and one Denier is induced to be moved on the plasmid with insetion sequence ISEcp1 and IS903, can between Enterobacter bacteria turn Move^[53].CTX-M is to be found in the Escherichia coli separation strains of a cancer patient for 1989, resistance to third generation cephalosporin Medicine^[54].CTX-M is different according to heredity and genotype is identical is divided into 4 main types, i.e., 1,2,9 and 25, wherein again with CTX- M-14 and CTX-M-15 genotype is most common.CTX-M-15 is mainly smaller than CTX-M-14 in India's generally existing, Epidemic Scope. China CTX-M-14 in 1998 has found in the enterobacteriaceae persister of Guangzhou separation first, while result of study shows that Guangzhou has Escherichia coli production ESBLs more than 35%, and CTX was in many hospital's extensive uses in Guangzhou at that time^[55].Hawkey etc. pairs The investigation of antibody-resistant bacterium that traveller carries show, the traveller from India carry most of CTX-M1 types, its genotype with CTX-M-15 is similar, from China traveller carry most of CTX-M9 types, its genotype it is similar with CTX-M-14, from angstrom And carry this two groups of genes simultaneously with the traveller of Thailand, and the relatively good Switzerland's CTX-M carrying rates of sanitary condition very it is low only 3%^[56]。

TEM and SHV is that earliest discovery is same by plasmid and the ESBLs class drug resistant genes of transposon-mediated propagation, both genes Also there is Multi-genotype, TEM there are 219 kinds of genotype, and SHV there are 186 kinds of genotype, and indivedual alkali may be only had by having between genotype The difference of base.For TEM-1 in addition to plasmid-mediated, pseudomonas aeruginosa, haemophilus influenzae also have production TEM-1 ability. TEM-1 types ESBLs is mainly to penicillins and cephalosporin analog antibiotic resistance, but the intensity of resistance is inconsistent, to penicillins The very strong drug resistance of performance, but to cephalosporin analog antibiotic, the drug resistance such as CTX and cefotaxime is weaker^[57].Greatly The verification and measurement ratio of TEM genes is very high in enterobacteria, reaches 85.4%, wherein TEM-1 genes are more common than TEM-2 gene^[58]。SHV-1 The generally existing in klebsiella pneumoniae, its variant are the genes on Klebsiella chromosome, are turned by some changes Move on in plasmid, transmitted in various enterobacterias, the also high expression in Escherichia coli.SHV type ESBLs and TEM types ESBLs is right The Carbapenems medicaments insensitive such as Imipenem.

IMP and VIM is the type of pan category extended spectrum β lactamases (MBLs) in ESBLs class drug resistant genes, by plasmid-mediated biography Broadcast, nineteen ninety, IMP appeared in Japan first^[59].As CTX-M types ESBLs, IMP and VIM also have many genotype, these Genotype some only has the difference of 1 base.Chip in this experiment can be detected simultaneously by IMP1, IMP2, IMP4, IMP6, IMP10 and VIM1, VIM4, VIM26, VIM33, VIM34, VIM35, VIM39, VIM42, the sequence differences of this several genotype are not Too.The main reason for VIM2 is pseudomonas aeruginosa production MBLs, there is important clinical meaning.IMP1 is that most occur early in Japan Genotype, it is then false single in different enterobacteriaceae-Acinetobacter bauamnniis and verdigris in the various IMP genotype of 1995-2001 Spread rapidly in born of the same parents bacterium.IMP4 is found in Guangdong Province of China Young citric acid bacillus, is compiled by the class integron of plasmid 1 Code, is transferred in Escherichia coli.It is worth noting that the metal beta-lactam enzyme drug resistant gene IMP of 1 class mediated by integron and VIM can not only be shifted between bacterial strain and bacterial strain between strain and strain, and remain very high expression quantity^[60].Wide East finds generation IMP4 soon, expresses IMP4 Acinetobacter bauamnnii in Hong Kong eruption and prevalence^[61].2005, carry IMP4's Antibody-resistant bacterium is exploded prevalence again, specifically mainly in Melbourne, Australia and Sydney, and persister be no longer Bao Man not All Enterobacter bacterias such as lever bacterium, also pseudomonas aeruginosa, klebsiella pneumoniae.

2008, a kind of new ESBLs was produced, i.e. New Delhi metal beta lactamase NDM (New Delhi metallo- β- Lactamase), from India, hence it is evident that different from IMP and VIM metal beta lactamases, to Carbapenems Drug-resistant, claimed For super drug resistant gene.Report has cases that some are dispersed in carry NDM1 genes later for Britain, and these cases have and surpass 50% Patient is in India, Bangladesh or Pakistani Zhu Guo institutes^[62].Also compare in the data of Southern Asia distribution situation on NDM Lack, but can speculate that NDM is probably to be distributed most commonly used carbapenem enzyme in this area, be often accompanied by OXA48 genes Expression, therefore be difficult to be detected by the phenotype of drug-fast bacteria.NDM is primarily present in the kerekou pneumonia of Carbapenem-resistant class antibiotic In primary bacillus and Escherichia coli.Although current NDM is confined to spread in the range of India, generation simply is dispersed in other areas, NDM is not controlled in the Spreading and diffusion trend of India, and is risen year by year.Saleem etc.^[63]Report 2010 23% (6/26) Carbapenem-resistant Klebsiella Pneumoniae separates from newborn intensive care unit, and this data rapidly rises within 2011 72% (13/18).

KPC is Klebsiella Pneumoniae carbapenem enzyme, is reported first in the U.S. within 1996, and New York is reported again within 2000, and 2 areas in the U.S. cause eruption and prevalence^[64].KPC1 and KPC2 is same gene, and both have one due to sequencing mistake The sequence of the difference of base, wherein KPC2 is correct sequence.KPC also has other genotype, but all be not as common as KPC2.KPC3 pairs The drug resistance of cefotaxime is stronger than CTX.This experiment can detect KPC2 and KPC3 simultaneously.KPC is to rely on serine hair The beta lactamase of effect is waved, except the temocillin of high concentration, almost to all beta-lactam class Drug-resistants, in addition in β Lactamase inhibitor is also invalid to KPC, but new beta lactamase restrainer AVM hereinafter Batan joint cefotaxime can control its production It is raw.KPC can not only be produced by klebsiella pneumoniae, and other kinds of enterobacteriaceae can also produce, and threaten the mankind Life and health.KPC is rapid in the obvious propagation in the area of the abuse of carbapenem antibiotic.In China, KPC is often with IMP4 Exist together, but not generally existing, and be accredited for single expression KPC clinical separation strain.Qi etc.^[65]Collect me Totally 95 plants of the klebsiella pneumoniae of 13 hospitals of province of state 5, detection show that all separation strains bacterium contain KPC-2 genes, and more Number contains ST-11 sequences (variant in mono- site of ST-258), and other sequence types come through PFGE identification From different host bacterias, prompt plasmid-mediated KPC to transmit and may be from different backgrounds.

OXA type carbapenem enzymes are D class beta lactamases, are produced by Gram-negative bacteria, especially enterobacteria and P. aeruginosa Bacterium.OXA type carbapenem enzyme energy hydrolyzing penicillin class antibiotic, such as OXA, methicillin.OXA equally has many bases Because of type, wherein OXA2 and OXA10 are mainly in various enterobacterias especially Acinetobacter bauamnnii and pseudomonas aeruginosa；OXA23 Genotype is primarily present in China's Acinetobacter bauamnnii^[66], with carbapenem antibiotic drug-resistant phenotype correlation highest； OXA48 is also a kind of common drug resistant gene for producing carbapenem enzyme, mainly causes cross-infection, particularly holds in ICU It is also easy to produce cross-infection.It can be seen that specifying the expression of these four OXA genes, to instructing clinical application, its popularity is monitored Etc. there is important clinical meaning.

DHA is the drug resistant gene of cynnematin enzyme, by plasmid or Chromosome-encoded, is easily propagated or popular.Some hospital DHA Mainly expressed in klebsiella pneumoniae, some hospitals mainly in expression in escherichia coli, belong to Enterobacter.DHA is not It can be suppressed by clavulanic acid, therefore to antibiotics resistances such as cephalosporins, monocyclic classes, such as ampicillin, cephalo-type, AZT Etc. resistance, to forth generation cephalo-type (Cefepime), Carbapenems (Imipenem), quinolones, (ring third is husky in children Star) etc. sensitivity, wherein QNS seldom used in children due to its toxic action to children's Bones and joints, so To childhood infection caused by DHA type AmpC enzymes, Cefepime or Imipenem can be selected^[67].In China, popular DHA hypotypes are DHA1, this experiment design mainly for the DHA1 primer and probes carried out.

Qnr genes were found in 1998, were produced by Klebsiella Pneumoniae, plasmid-mediated propagation and prevalence, and qnr has 5 hypotypes, bag Include qnrA, qnrB, qnrS, qnrC and qnrD, it can be common that qnrA1, qnrB1, qnrS1.This experiment is exactly to be directed to these three bases Because the chip of progress designs.Qnr albumen is to resistance caused by QNS by Reverse transcriptase.QNS Bacterium for degrading needs the combination of DNA gyrases and Topoisomerase Ⅳ and bacterium target DNA molecule, and qnr albumen divides with the target DNA Minor structure is similar, can also be combined with both enzymes, so as to reduce degraded of the Du-6859a to bacterium.Due to competitive relation, The bacterial strain for carrying qnr genes is sensitiveness reduction to FQNS^[68].Nevertheless, carry the bacterial strain of qnr genes Caused infection still can cause length of patient stay's extension, medical expense increase, Endodontic failure etc., therefore by qnr genes Caused fluoroquinolones sensitiveness is reduced and can not be ignored, and the detection to qnr genes is very necessary.

The generation of aminoglycoside antibiotics modification enzyme is considered as the antibody-resistant bacterium such as Acinetobacter bauamnnii and pseudomonas aeruginosa The main reason for aminoglycoside antibiotics resistance, this enzyme can be to the amino and hydroxyl on aminoglycoside antibiotics side chain Base is modified, and so as to cause the combination of antibiotic and target gene to fail, antibiotic can not play its antibacterial activity.Aminoglycoside Class antibiotics modification enzyme includes aminoglycoside acetyltransferase and aminoglycoside nucleotidyltransferase.Aac (3)-II is amino sugar The genotype of glycosides acetyltransferase, ant (2 ")-I and ant (3 ")-I are the genotype of aminoglycoside nucleotidyltransferase, by Plasmid-mediated propagation and transfer.In Iran, (6')-II (36%) mainly in antibiotic resitance of P. aeruginosa strain, is secondly aac Ant (2 ")-I and aac (6')-I^[69], China ant (3 ")-I and aac (6')-I gene is in Acinetobacter bauamnnii persister Height expression, ant (2 ")-I and aac (6')-I gene high expression in antibiotic resitance of P. aeruginosa strain, aac (3)-II are in large intestine bar Recall rate in bacterium may be up to 88.5%, it is seen then that drug resistant gene caused by different areas is also different, and this may be with regional antibiosis Plain service condition difference is relevant.

Early in 1986, Quinn etc.^[70]Just find in the pseudomonas aeruginosa to imipenem-resistant, its outer membrane protein molecule Amount significantly reduces, thus it is speculated that Imipenem enters pseudomonas aeruginosa and outer membrane protein is closely related.Hancock etc.^[71]Will be this Outer membrane protein is divided into D class 1 outer-membrane proteins, and D class 1 outer-membrane proteins have D1 and the classes of D2 two.Nineteen ninety, Trains etc.^[72]Confirm and Asia Amine training southing enter pseudomonas aeruginosa specific channel be outer membrane protein D2 classes (outer membrane protein D2, oprD2).Different from other drug resistant genes, oprD2 genes are the genes of normal expression on outer membrane protein, are produced when it is lacked Resistance.OprD2 deletion mutants have 2 kinds, and a kind of missing for being encoded to 11 bases between 395-405 positions, another kind is to open - 519 missings to 685 1204 bases in mover upstream^[73,74], this experiment with reference to text when designing primer and probe Offer^[74]In primer, lacking in 2 can detect, but cannot distinguish between the size of deletion fragment.

The principal causative of MRSA not still hospital infections now is former, and the pathogenic original of one kind of Nosocomial Infections.MecA genes are MRSA crucial drug resistant gene, on chromosome, Renicillin binding protein PBP2a.PBP2a can be with beta-lactam class Antibiotic combines, and so as to prevent the normal combination of beta-lactam class antibiotic and PBPs, prevents beta-lactam class antibiotic from playing Effect, bacterium normal growth, form resistance.Research is found^[75]Nosocomial Infections MRSA (CA-MRSA) and hospital infection MRSA (HA- MRSA antibiotic resistance power) is inconsistent, and CA-MRSA is mainly to beta-lactam class antibiotics resistance, and to many non-beta-lactams Class antibiotic sensitive, it is not easy to form multidrug resistant, and HA-MRSA is not only to beta-lactam class antibiotics resistance, to a lot of other The antibiotic of type also resistance, often shows as multidrug resistant, alternative medicine is seldom.The generation of this species diversity is main Relevant with SCCmec box genes, SCCmec box genes are made up of mec gene complexs and chromosome box recombinase gene complex, It is divided into 5 types, i.e. I type, II type, III type, IV type and V type^[76], wherein I type, II type and III type are primarily present in HA-MRSA, IV type and V type are primarily present in CA-MRSA, are the main reason for causing MRSA drug resistance differences^[77]。

VanA and vanB genes are VRE two Main Drug-Resistant Genes, and both are 3 kinds VRE points, i.e. vanA genotype is shown VanA phenotypes, vanB genotype show that vanB phenotypes and vanA genotype show vanB phenotypes^[78].The resistance of these three Characteristic is inconsistent, and former shows different journeys to vancomycin to vancomycin and teicoplanin height resistance, latter two The resistance of degree, and it is sensitive to teicoplanin.Current research is shown^[79], in Canadian 1927 parts of enterococcus spp separation strains, 4.2% (80) be VRE, infection rate from 2007 1.6% rise to 2013 6%, and all VRE are VREF, wherein 90% carries vanA, shows as multidrug resistant, but sensitive in various degree to Doxycycline, Linezolid and Daptomycin.At me State VRE is also mainly based on vanA genotype VREFs^[80].VanA and vanB genes can also be transferred to it by plasmid-mediated His Gram-positive strain, such as MRSA^[81], so as to form the stronger antibody-resistant bacterium of virulence.

In above drug resistant gene, have it is every kind of there are many genotype, this experiment have chosen it is wherein most common, it is anti-in bacterial resistance 24 genes to be played an important role in answering are detected.Pneumonia pathogenic bacteria drug resistant gene detection chip susceptibility is high, specificity and It is reproducible.The application is studied for 24 drug resistant genes, when the maximum advantage of the drug resistant gene chip is its detection Between it is short, blank phase of 2-3 days can be made up before drug sensitive experiment result comes out, preliminary antibiotic usage guidance is provided for clinic.

English list of abbreviations

Bibliography

Niederman MS,Mandell LA,Anzueto A,et al.Guidelines for the management of adults with community-acquired pneumonia.Diagnosis,assessment of severity, antimicrobial therapy,and prevention.Am J Respir Crit Care Med,2001,163(7): 1730-1754.

Wunderink RG,Waterer GW.Clinical practice.Community-acquired pneumonia.N Engl J Med,2014,370(6):543-551.

Murphy SL,Xu J,Kochanek KD.Deaths:final data for 2010.Natl Vital Stat Rep,2013,61(4):1-117.

Kochanek KD,Murphy SL,Xu J et al.Mortality in the United States 2013.NCHS Data Brief,2014(178):1-8.

Hoyert DL,Xu J.Deaths:preliminary data for2011.Natl Vital Stat Rep, 2012,61(6):1-51.

Official's rising sun China, Benjamin J Silk, Wenkai Li etc., China's Mainland pneumonia incidence and mortality:1985- The network analysis of Chinese and English document in 2008.Public Health and Preventive Medicine, 2011,22 (1):14-19.

The preventing and treating internal medicine Severe acute disease magazines of the auspicious Pneumonia in Older Patients of Huang Hua, 2001,7 (1):42-44.

Weiss K,Tillotson GS.The controversy of combination vs monotherapy in the treatment of hospitalized community-acquired pneumonia.Chest,2005,128(2): 940-946.

Garau J,Calbo E.Community-acquired pneumonia.Lancet.2008,371(9611): 455-458.

American Thoracic Society；Infectious Diseases Society of America.Guidelines for the management of adults with hospital-acquired, ventilator-associated,and healthcare-associated pneumonia.Am J Respir Crit Care Med,2005,171(4):388-416.

Jiang HX,Tang D,Liu YH,et al.Prevalence and characteristics of beta- lactamase and plasmid-mediated quinolone resistance genes in Escherichia coli isolated from farmed fish in China.J Antimicrob Chemother 2012,67:2350e2353.

Nathan C,Cars O.Antibiotic resistance--problems,progress,and prospects.N Engl J Med,2014,371(19):1761-1763.

Stanek RJ,Maher MB,Norton NB,et al.Emergence of a unique penicillin- resistant Streptococcus pneumoniae serogroup 35strain.J Clin Microbiol,2011, 49(1):400-404.

Centers for Disease Control and prevention.Four pediatric deaths from community-acquired methicillin-resistant Staphylococcus aureus-Minnesota and North Dakota,1997-1999.Morb Mortal Wkly Rep,1999,48(32):707-710.

Bhatt P,Patel A,Sahni AK,et al.Emergence of multidrug resistant enterococci at a tertiary care centre.Med J Armed Forces India.2015,71(2): 139-144.

Catherine M.Oliphant,Kathryn Eroschenko.Antibiotic Resistance,Part 1: Gram-positive Pathogens.The Journal for Nurse Practitioners,2015,11(1):70-78.

Hsieh SY,Tseng CL,Lee YS,et al.Highly efficient classification and identification of human pathogenic bacteria by MALDI-TOF MS.Mol Cell Proteomics,2008,7(2):448-456.

The .K-B such as Tan Yao, Zhao Qing, Shu Weiqun Method of paper diffusion laboratory medicines are with clinical, 2010,7 (20): 2290-2291.

Bartlett JG,Breiman RF,Mandell LA,et al.Community-acquired pneumonia in adults:guidelines for management.Clin Infect Dis 1998,26:811–838.

Mandell LA,Bartlett JG,Dowell SF,et al.Update of practice guidelines for the management of communityacquired pneumonia in immunocompetent adults.Clin Infect Dis 2003；37:1405–33.

You Y,Fu C,Zeng X,et al.A novel DNA microarray for rapid diagnosis of enteropathogentic bacteria in stool specimens of patients with diarrhea.J Microbiol Methods,2008,75(3):566-571.

DeRisi J,van den Hazel B,Marc P,et al.Genome microarray analysis of transcriptional activation in multidrug resistance yeast mutants.FEBS Lett, 2000,470(2):156-160.

Crameri A,Marfurt J,Mugittu K,et al.Rapid microarray-based method for monitoring of all currently known single-nucleotide polymorphisms associated with parasite resistance to antimalaria drugs.J Clin Microbiol,2007,45(11): 3685-3691.

Avarre JC,de Lajudie P,Béna G.Hybridization of genomic DNA to microarrays:a challenge for the analysis of environmental samples.J Microbiol Methods,2007,69(2):242-248.

Young RA.Biomedical discovery with DNA arrays.Cell,2000,102(1):9-15.

Kolquist KA,Schultz RA,Furrow A,et al.Microarray-based comparative genomic hybridization of cancer targets reveals novel,recurrent genetic aberrations in the myelodysplastic syndromes.Cancer Genet,2011,204(11):603- 628

Zhou J.Microarrays for bacterial detection and microbial community analysis.Curr Opin Microbiol,2003,6(3):288-294.

Ye RW,Wang T,Bedzyk L,et al.Applications of DNA microarrays in microbial systems.J Microbiol Methods,2001,47(3):257-272.

Jin D,Qi H,Chen S,et al.Simultaneous detection of six human diarrheal pathogens by using DNA microarray combined with tyramidesignal amplification.J Microbiol Methods,2008,75(2):365-368.

Rhoads DD,Cox SB,Rees EJ,et al.Clinical identification of bacteria in human chronic wound infections:culturing vs.16S ribosomal DNAsequencing.BMC Infect Dis,2012,24；12:321.

Woo PC,Lau SK,Teng JL,et al.Then and now:use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories.Clin Microbiol Infect.2008,14(10):908-934

Sontakke S,Cadenas MB,Maggi RG,et al.Use of broad range 16S rDNA PCR in clinical microbiology.J Microbiol Methods,2009,76(3):217-225.

Carvalho Mda G,Tondella ML,McCaustland K,et al.Evaluation and improvement of real-time PCR assays targeting lytA,ply and psaA genes for detection of pneumococcal DNA.J Clin Microbiol,2007,45(8):2460-2466.

Brakstad OG,Aasbakk K,Maeland JA.Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene.J Clin Microbiol, 1992,30(7):1654-1660.

A,Lantz P,P,et al.Evaluation of an extended diagnostic PCR assay for detection and verification of the common causes ofbacterial meningitis in CSF and other biological samples.Mol Cell Probes, 1999,13(1):49-60.

Thong KL,Lai MY,Teh C SJ,et al.Simultaneous detection of methicillin- resistant Staphylococcus aureus,Acinetobacter baumannii,Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa by multiplex PCR.Trop Biomed,2011,28(1):21-31.

Curran B,Jonas D,Grundmann H,et al.Development of a multilocus sequence typing scheme for the opportunistic pathogen Pseudomonas aeruginosa.J Clin Microbiol,2004,42(12):5644-5649.

Pavlovic M,Konrad R,Iwobi AN,et al.A dual approach employing MALDI- TOF MS and real-time PCR for fast species identification within the Enterobacter cloacae complex.FEMS Microbiol Lett,2012,328(1):46-53.

Ginevra C,Barranger C,Ros A,et al.Development and evaluation of Chlamylege,a new commercial test allowing simultaneous detection and identification of Legionella,Chlamydophila pneumoniae,and Mycoplasma pneumoniae in clinical respiratoryspecimens by multiplex PCR.J Clin Microbiol,2005,43(7):3247-3254.

Apfalter P,Barousch W,Nehr M,et al.Comparison of a new quantitative ompA-based real-Time PCR TaqMan assay for detection of Chlamydia pneumoniae DNA in respiratory specimens with four conventional PCR assays.J Clin Microbiol,2003,41(2):592-600.

Naserpour Farivar T,Najafipour R,Johari P,et al.Development and evaluation of a Quadruplex Taq Man real-time PCR assay for simultaneous detection of clinical isolates of Enterococcus faecalis,Enterococcus faecium and their vanA and vanB genotypes.Iran J Microbiol,2014,6(5):335-340.

da Silva Filho LV,Tateno AF,Velloso Lde F,et al.Identification of Pseudomonas aeruginosa,Burkholderia cepacia complex,and Stenotrophomonas maltophilia inrespiratory samples from cystic fibrosis patients using multiplex P CR.Pediatr Pulmonol,2004,37(6):537-547.

Wright C,Herbert G,Pilkington R,et al.Real-time PCR method for the quantification of Burkholderia cepacia complex attached to lung epithelial cells and inhibition of that attachment.Lett Appl Microbiol,2010,50(5):500- 506.

Al-Marzooq F,Imad MA,How SH,et al.Development ofmultiplex real-time PCR for the rapid detection of five bacterial causes of community acquired pnuemonia.Trop Biomed,2011,28(3):545-556.

Zhang Haiyan, Marvin's is beautiful, and the application asymmetric PCRs such as Li Ling technology improves oligonucleotide gene chip hybridization efficiency armies Cure college of continuing education's journal, 2005,26 (4):266-268.

Zhang Y,Liu Q,Wang D,et al.Simultaneous detection of oseltamivir-and amantadine-resistant influenza by oligonucleotide microarray visualization.PLoS One,2013,8(2):e57154.

National Nosocomial Infections Surveillance System.National Nosocomial Infections Surveillance(NNIS)System Report,data summary from January 1992through June 2004,issued October 2004.Am J Infect Control,2004,32 (8):470-485.

Ramazanzadeh R,Chitsaz M,Bahmani N.Prevalence and antimicrobial susceptibility of extended-spectrum beta-lactamase-prod ucing bacteria in intensive units of Sanandaj general hospitals(Kurdistan,Iran).Chemotherapy, 2009,55(4):287-292.

Boucher HW,Talbot GH,Bradley JS,et al.Bad bugs,no drugs:no ESKAPE！An update from in Infectious Disease Society of America.Cin Infect Dis,2009,48 (1):1-12.

Wang HY,Kim S,Kim J,et al.Multiplex real-time PCR assay for rapid detection of methicillin-resistant staphylococci directly from positive blood cultures.J Clin Microbiol.2014,52(6):1911-1920.

Lucier TS,Heitzman K,Liu SK,et al.Transition mutations in the 23S rRNA of erythromycin-resistant isolates of Mycoplasma pneumoniae.Antimicrob Agents Chemother,1995,39(12):2770-2773.

Hawkey PM,Jones AM.The changing epidemiology of resistance.J Antimicrob Chemother 2009；64(Suppl.1):i3-i10.

Humeniuk C,Arlet G,Gautier V,et al.Beta-lactamases of Kluyvera ascorbata,probable progenitors of some plasmid-encoded CTX-M types.Antimicrob Agents Chemother,2002；46(9):3045-3049.

Bernard H,Tancrede C,Livrelli V,et al.A novel plasmid-mediated extended-spectrum betalactamase not derived from TEM-or SHV-type enzymes.J Antimicrob Chemother,1992；29(5):590-592.

Chanawong A,M’Zali FH,Heritage J,et al.Three cefotaximases,CTX-M-9, CTX-M-13,and CTX-M-14,among Enterobacteriaceae in the People’s Republic of China.Antimicrob Agents Chemother,2002；46(3):630-637.

Tham J,Odenholt I,Walder M,et al.Extended-spectrum beta-lactamase- producing Escherichia coli in patients with travellers’diarrhoea.Scand J Infect Dis,2010,42(4):275-280.

Stiffler MA,Hekstra DR,Ranganathan R,et al.Evolvability as a function of purifying selection in TEM-1β-lactamase.Cell.2015,160(5):882-892.

TEM yielding characteristicses study in the red Extended-spectrum β-lactamases EHEC of Chen Canfeng, Zhong Liuzhen, Chen Xiu Heilungkiang medical science, 2013,37 (12):1197-1198.

Ito H,Arakawa Y,Ohsuka S,et al.Plasmid-mediated dissemination of the metallo-beta-lactamase gene blaIMP among clinically isolated strains of Serratia marcescens.Antimicrob Agents Chemother 1995；39(4):824-829.

Nordmann P.Carbapenemase-producing Enterobacteriaceae:overview of a major public health challenge.Med Mal Infect,2014；44(2):51-56.

Chu YW,Afzal-Shah M,Houang ET,et al.IMP-4,a novel metallo-blactamase from nosocomial Acinetobacter spp.collected in Hong Kong between 1994 and 1998.Antimicrob Agents Chemother,2001；45(3):710-714.

Kumarasamy KK,Toleman MA,Walsh TR,et al.Emergence of a new antibiotic resistance mechanism in India,Pakistan,and the UK:a molecular,biological,and epidemiological study.Lancet Infect Dis,2010；10(9):597-602.

Saleem AF,Qamar FN,Shahzad H,et al.Trends in antibiotic susceptibility and incidence of late-onset Klebsiella pneumoniae neonatal sepsis over a six-year period in a neonatal intensive care unit in Karachi, Pakistan.Int J Infect Dis,2013；17(11):e961-e965.

Chen L,Mathema B,Chavda KD,et al.Carbapenemase-producing Klebsiella pneumoniae:molecular and genetic decoding.Trends Microbiol,2014(12)；22:686- 696.

Qi Y,Wei Z,Ji S,et al.ST11,the dominant clone of KPC-producing Klebsiella pneumoniae in China.J Antimicrob Chemother,2011；66(2):307-312.

The multidrug resistances Escherichia coli OXA genetic tests such as Hu Haiying, Wang Dongguo, Wei Zhiying and cluster analysis medical researches Magazine, 2012,41 (9):129-133.

The such as Liu Jianlong, Mo Liya, Song Chunrong children's ALRI Klebsiella pneumoniae Resistances and dha genes are ground Study carefully practicality preventive medicine, 2014,21 (3):363-365.

Strahilevitz J,Jacoby GA,Hooper DC,et al.Plasmid-mediated quinolone resistance:a multifaced threat.Clin Microbial Rev,2009,22(4):664-689.

Vaziri F,Peerayeh SN,Nejad QB,et al.The prevalence of aminoglycoside- modifying enzyme genes(aac(6')-I,aac(6')-II,ant(2")-I,aph(3')-VI)in Pseudomonas aeruginosa.Clinics(Sao Paulo),2011；66(9):1519-1522.

Quinn,J.P.,C.A.Dudek,C.A.diVincenzo,et al.Emergence of resistance to imipenem during therapy for Pseudomonas aeruginosa infections.J.Infect.Dis.1986,154(2):289-294.

Hancock,R.E.W.,A.M.Carey.Outermembrane of Pseudomonas aeruginosa: heat-and 2-mercaptoethanol-modifiable proteins.J.Bacteriol,1979,140(3):902- 910.

Trias J,Nikaido H.Outer membrane protein D2 catalyzes facilitated diffusion of carbapenems and penems through the outer membrane of Pseudomonas aeruginosa.Antimicrob Agents Chemother,1990,34(1):52-57.

The carbapenem antibiotic tolerant Pseudomonas aeruginosa Outer membrane proteins such as Shen Jilu, Zhu Demei, Wu Weihong OprD₂Research China infection with chemotherapy magazine, 2011,11 (4):281-286.

The such as Song Shiduo, Zheng Dongjun, Zhang Yuwen are clinically separated imipenem-resistant Pseudomonas aeruginosa chromosome oprD genes and dashed forward The detection China infectious disease magazine of change, 2003,21 (2):144-145.

Naimi TS,LeDell KH,Como-Sabetti K,et al.Comparison of community-and health care-associated methicillin-resistant Staphylococcus aureus infection.JAMA.2003,290(22):2976-2984.

Ito T,Ma XX,Takeuchi F,et al.Novel type V staphylococcal cassette chromosome mec driven by a novel cassette chromosome recombinase, ccrC.Antimicrob Agents Chemother,2004,48(7):2637-2651.

Chongtrakool P,Ito T,MaXX,et al.Staphylococcal cassette chromosome mec (SCCmec)typing of methicillin-resistant Staphylococcus aureusstrains isolated in 11 Asian countries:a proposal for a new nomenclature for SCCmec elements.Antimicrob Agents Chemother,2006,50(3):1001-1012.

Strateva T,Atanasova D,Mitov I,et al.Emergence of VanB phenotype-vanA genotype Enterococcus faecium clinical isolate in Bulgaria.Braz J Infect Dis, 2014,18(6):693-695.

Simner PJ,Adam H,Baxter M,et al.Epidemiology of Vancomycin-Resistant Enterococci(VRE)in Canadian Hospitals:CANWARD 2007-2013.Antimicrob Agents Chemother.2015,pii:AAC.00384-15.[Epub ahead of print].

Wang Lipeng, He Yunyan, sternly vertical etc., the detection of Vancomycin-resistant Enterococcus drug resistant gene and molecular epidemiology are adjusted Look into clinical examination magazines, 2014,32 (2):136-143.

Palazzo IC,Araujo ML,Darini AL.First report of vancomycin-resistant staphylococci isolated from healthy carriers in Brazil.J Clin Microbiol,2005, 43(1):179-185.

Claims

1. a kind of pneumonia pathogenic bacteria drug resistant gene quickly identifies genetic chip, it is included corresponding to pneumonia pathogenic bacteria drug resistant gene Probe；Wherein, the pneumonia pathogenic bacteria drug resistant gene comprises at least metal beta-lactam class drug resistant gene, the metal beta-lactam Class drug resistant gene includes at least one of NDM1, IMP, VIM；

Wherein, it is corresponding to the sequence of NDM1 probe：

ATGGAAACTGGCGACCAACGGTTTGGCGATCTTTTTTTTTTTT；

Sequence corresponding to IMP probe is：

AGATAACGTAGTGGTTTGGTTTTTTTTTTTT；

Sequence corresponding to VIM probe is：

TCTAGAAGGACTCTCATCGATTTTTTTTTTTT。

2. pneumonia pathogenic bacteria drug resistant gene as claimed in claim 1 quickly identifies genetic chip, it is characterised in that：The pneumonia Pathogenic bacteria drug resistant gene also includes Carbapenem-resistant gene, the Carbapenem-resistant gene include OXA2, OXA10, At least one of OXA23, OXA48, KPC；

Wherein, it is corresponding to the sequence of OXA2 probe：

TTGCAGGCCACAATCAAGACCAAGATTTTTTTTTTTTT；

Sequence corresponding to OXA10 probe is：

TCAAATGGGACGGAAAGCCAAGAGCCATGAATTTTTTTTTTTT；

Sequence corresponding to OXA23 probe is：

GAGAGTAATGGCTACAAAATTTTTTTTTTTTTTT；

Sequence corresponding to OXA48 probe is：

AGTTTCTGGCTCGACGGTGGTATTCTTTTTTTTTTTT；

Sequence corresponding to KPC probe is：

TGACGGCCTTCATGCGCTCTATCGGCGATACTTTTTTTTTTTT。

3. pneumonia pathogenic bacteria drug resistant gene as claimed in claim 2 quickly identifies genetic chip, it is characterised in that：The pneumonia Pathogenic bacteria drug resistant gene also includes aminoglycoside resistant gene, the aminoglycoside resistant gene include aac (3)-II, Aac (6') at least one of-I, ant (2 ")-I, ant (3 ")-I；

Wherein, it is corresponding to the sequence of aac (3)-II probe：

GTGGGTTCGGCCTGCTGAATCAATTTCTGGTTTTTTTTTTTT；

Sequence corresponding to aac (6')-I probes is：

AGGTCACCAAGATCCAAACGGACTTTTTTTTTTTT；

Sequence corresponding to ant (2 ")-I probe is：

CACGCAAGCACGATGATATTGATCTGACGTTTTTTTTTTTT；

Sequence corresponding to ant (3 ")-I probe is：

TCTCCGCGCTGTAGAAGTCACCATTGTTTTTTTTTTTTTT。

4. pneumonia pathogenic bacteria drug resistant gene as claimed in claim 3 quickly identifies genetic chip, it is characterised in that：The pneumonia Pathogenic bacteria drug resistant gene also includes quinolones drug resistant gene, and quinolones drug resistant gene is included in qnrA, qnrB, qnrS extremely Few one kind；

Wherein, it is corresponding to the sequence of qnrA probe：

ATGTACTTCTGCTCGGCTTATATCTCAGGTTTTTTTTTTTTT；

Sequence corresponding to qnrB probe is：

GTAGCGCATATATCACGAATACCAATCTAAGCTACGCCTTTTTTTTTTTT；

Sequence corresponding to qnrS probe is：

TCGCTGGATAGGAACGAACCTAGCTTTTTTTTTTTT。

5. pneumonia pathogenic bacteria drug resistant gene as claimed in claim 4 quickly identifies genetic chip, it is characterised in that：The pneumonia Pathogenic bacteria drug resistant gene also includes ESBLs class drug resistant genes, the ESBLs classes drug resistant gene include CTX-M-14, CTX-M-15, At least one of SHV, TEM；

Wherein, it is corresponding to the sequence of CTX-M-14 probe：

CAGCCTGTCGAGATCAAGCCTGTTTTTTTTTTTT；

Sequence corresponding to CTX-M-15 probe is：

AGACTGGGTGTGGCATTGATTATTTTTTTTTTTT；

Sequence corresponding to SHV probe is：

CTCCAGCTGTTCGTCACCGGCATTTTTTTTTTTT；

Sequence corresponding to TEM probe is：

GTGCTGCCATAACCATGAGTGATAACTTTTTTTTTTTT。

6. pneumonia pathogenic bacteria drug resistant gene as claimed in claim 5 quickly identifies genetic chip, it is characterised in that：The pneumonia Pathogenic bacteria drug resistant gene also includes cephalosporins drug resistant gene, and the cephalosporins drug resistant gene is DHA；

Wherein, it is corresponding to the sequence of DHA probe：

GCTTGATGCGGAATCTTACGGTTTTTTTTTTTT。

7. pneumonia pathogenic bacteria drug resistant gene as claimed in claim 6 quickly identifies genetic chip, it is characterised in that：The pneumonia Pathogenic bacteria drug resistant gene also includes vancomycin resistance class drug resistant gene, the vancomycin resistance class drug resistant gene include vanA, At least one of vanB；

Wherein, it is corresponding to the sequence of vanA probe：

CCGCATTGTACTGAACGAAGTCTTTTTTTTTTTT；

Sequence corresponding to vanB probe is：

GCTTACCTACCCTGTCTTTGTGATTTTTTTTTTTT。

8. pneumonia pathogenic bacteria drug resistant gene as claimed in claim 7 quickly identifies genetic chip, it is characterised in that：The pneumonia Pathogenic bacteria drug resistant gene also includes methicillin-resistant class drug resistant gene, and the methicillin-resistant class drug resistant gene is mecA；

Wherein, it is corresponding to the sequence of mecA probe：

GTAAGCACACCTTCATATGACGTCTATCCTTTTTTTTTTTT。

9. pneumonia pathogenic bacteria drug resistant gene as claimed in claim 8 quickly identifies genetic chip, it is characterised in that：The pneumonia Pathogenic bacteria drug resistant gene also includes porin class drug resistant gene, and the porin class drug resistant gene is oprD2；

Wherein it is corresponding to the sequence of oprD2 probe：

TGTTCCCGCAGACCGCGATTTTTTTTTTTT。

10. the pneumonia pathogenic bacteria drug resistant gene as described at least one in claim 1-9 quickly identifies genetic chip, its feature It is：The genetic chip further comprises 3 negative probes；Three negative probes are respectively： TCAGAGCCTGTGTTTCTACCAATTTTTTTTTTTT、CATCAATAGGGTCCGATATTTTTTTTTTTT、 CGAACGCAAATCAATCTTTTTCCAGGTTTTTTTTTTTTT。