CN1231597C - Septicaemia detection chip, its preparation process and application method - Google Patents

Abstract

The present invention relates to the medicinal technology of in vitro diagnosis, more specifically a gene chip-a septicaemia detecting chip, preparing technology thereof and a using method thereof. A plurality of micro arrays in different zones are distributed on a glass substrate, and specific probes of common pathogenic microorganisms of meningitis are respectively fixed in each of the micro arrays. Corresponding probes are hybridized with the DNA of amplified pathogenic microorganisms added with fluorescence labels, the chip is scanned by a scanner, and then hybridized signals are treated and analyzed to obtain relevant information. The chip can simultaneously detect a plurality of microorganisms, such as bacteria, viruses, fungi, protozoa, etc., and the chip has the characteristics of rapid and accurate diagnosis, high specificity and large information quantity and is very helpful to clinical diagnosis and epidemiology screening.

Description

A kind of septicemia detection chip and preparation technology and using method

One, technical field: the present invention relates to the medical science vitro diagnostic techniques, is a kind of gene chip concretely---a kind of septicemia detection chip and preparation technology and using method.

Two, background technology: septicemia is common clinical disease, almost all may occur in each section office.Though medical level improves at present, the novel drugs new therapy constantly occurs, and the sickness rate of septicemia significantly reduces, and serious septicemia is in case occur fatal often.Thereby can obtain appropriate treatment in time and depend on to a great extent whether accurately rapidly diagnosis.The result of treatment that multiple factor affecting septicemia is arranged can generalizedly be divided into: the virulence of pathogenic bacterium, patient's self immunizing power and medical treatment and nursing situation.The antibiotic use of Zeng Jiaing makes clinical treatment have to face pathogenic bacterium resistance problem day by day, and the waste of the human and material resources that caused thus.The pathogenic bacterium kind is varied, and the physico-chemical property of each kind is different with resistant characterization, even all very big with the virulence difference of causing a disease in the kind, between different subtype, causes very burden to clinical treatment equally.

The method of traditional detection septicemia pathogenic bacterium comprises: gramstaining, detect and microbial culture etc. based on the immunization of antibody.Microscopy is simply quick, but needs a considerable amount of bacteriums.Though antibody test speed is very fast, once can only detect a kind of antibody, efficient is low.Microbial culture has improved sensitivity, and incubation time but becomes impassable obstacle.And antibiotic application can also cause the false negative result of gramstaining and microbial culture.Invent a kind of efficient highly sensitive detection method become essential.

Three, summary of the invention:

1, goal of the invention: the invention provides a kind of septicemia detection chip and preparation technology and using method, its purpose is to solve the problem that aspects such as the efficient that exists in the septicemia detection is low, diagnosis is inaccurate exist.

2, technical scheme: the present invention comes to be realized by the following technical programs:

A kind of septicemia detection chip is characterized in that: this chip is the microarray that is distributed with a plurality of different zones on the glass substrate, in each microarray zone, is fixed with the specific probe of common septicemia pathogenic bacterium respectively;

60 of probe design, designed probe sequence sees below;

Preparation technology of the present invention carries out as follows:

(1), design septicemia specific probe and primer;

From database, obtain reliable septicemia pathogenic bacterium gene order, according to described probe sequence designing probe;

From database, obtain reliable septicemia pathogenic bacterium virus gene sequence, select with using primers F 482:

5 '-CGGCTAACTC TGTGCCAGC; R789:ATCTATGGGA CCATCAGGTACGG-5 ' increases at 16s portion gene sequence, and amplification is the mark fluorescent element simultaneously;

(2), synthesising probing needle, carry out synthesising probing needle according to the above-mentioned probe sequence that designs;

(3), preparation chip;

With the dissolving of synthetic probe, concentration is 200mmol/l, carries out point sample on glass substrate with deionized water; The good chip of point is in 37 ℃ of following aquations 3 days, then 80 ℃ of oven dry 2 hours down; With probe damping fluid and distilled water flushing slide, dry up;

With confining liquid sealing slide, wash afterwards 3 times, dry up standby.

In the preprocessing process to blood sample to be measured, pcr amplification primer sequence sees below;

Probe 5 ' end adds amino the modification.

Point sample from tens to thousands of points, the size of point is the 50-100 micron.

The probe damping fluid is 1xSSC, 0.1%SDS; Confining liquid is 1%BSA, PH=7 0.01mol/1PB.

Using method is carried out as follows:

(1) get venous patient blood 3-5 milliliter, extract DNA respectively, standby; Place for a long time as need, frozen down at-20 ℃;

(2) .PCR amplification

In reaction tubes, add amplification reaction mixture---Taq enzyme, sample of handling well accordingly and primer, the dUTP of the dNTP of capacity and fluorescein Cy3 mark; Amplification condition is as follows:

94℃?5min

94℃?30sec

56℃?30sec

72℃?1min

72℃?5min

Above step is 30 circulations;

(3). hybridization;

Probe on PCR product and the chip was hybridized 2 hours down at 60 ℃, and (10xSSC, 0.1%SDS) ratio with the PCR product is 1: 1 to hybridization buffer, washs 3 times.

(4). detect;

Chip with after the scanner scanning hybridization obtains image and exports the result;

(5). data analysis

The chip analysis result is exported.

3, advantage and effect:

Gene chip is one of great science and technology progress of the tool characteristics of the times that occurred in high-tech area in recent years, and it is the high-tech of physics, chemistry, microtronics, precision optical machinery and life science cross-synthesis.What gene chip was integrated is not electronic devices and components, adopt combinatorial chemistry on the throne, microelectronic chip photoetching technique, perhaps utilize other method to be fixed on the dna probe of a large amount of particular sequences on the substrate in an orderly manner, with testing sample DNA effect after, a large amount of life-informations be can detect, gene recognition, transgenation and genetic expression etc. comprised.Utilize gene chip can obtain or handle a large amount of life-informations fast and efficiently, it has revolutionary pushing effect to life science, medical diagnosis, new medicament screen and judicial expertise etc.

The invention provides a kind of septicemia detection chip.Synthetic specific probe matrix is fixed in substrate surface, by chip and sample to be tested DNA hybridization, can obtain a large amount of biological informations relevant with septicemia.Utilize this chip, just can finish the detection of septicemia pathogenic bacterium simultaneously by single job.This gene chip has the advantages that diagnosis is accurate, specificity is high, contain much information.If this chip is successfully applied to clinical, will bring great economic benefit and social benefit.

Four, embodiment: concrete steps of the invention process are:

1. design septicemia specific probe and primer

Obtain reliable septicemia pathogenic bacterium gene order and drug-resistance characteristics sequence etc. from databases such as NCBI, select septicemia pathogenic bacterium 16s gene order, utilize information biology software design primer and specific probe.Select with using primers F 482:5 '-CGGCTAACTC TGTGCCAGC; R789:ATCTATGGGA CCATCAGGTACGG-5 ' increases at 16s portion gene sequence, and amplification is with tense marker Cy3 fluorescein.On gene chip, fix 61 kinds of various pathogenic bacteria specific probes, differentiate Acinetobacter in the mode of point sample, Actinomyces, Borrelia burgdorferi, Burkholderia cepacia, Bacteroides fragilis, Burkholderia pseudomallei, Bartonella sp., Borrelia afzelii, Borrelia garinii, Clostridium difficile, Clostridium perfringens, Capnocytophaga ochracea, Chlamydia pneumoniae, Clostridium septicum, Coxiella burnetii strainlM, Escherichia coli, Enterobacter aerogenes, Enterococcus dispar[duran], Enterococcus faecalis strainK4, Enterococcus faecium, Flavimonasoryzihabitans, Flavobacterium sp., Fusobacterium necrophorum, Haemophilus influenzae, Klebsiellaoxytoca-16, Klebsiella pneumoniae, Leptospira interrogans, Lactobacillus sp.B5406, Legionellasp.strain LLAP9, Mycobacterium tuberculosis, Moraxella sp., Mycoplasma pneumoniae strainATCC 15531, Neisseria meningitidis, Nocardia asteroides, p-mor, p-pro, Peptococcus niger, Peptostreptococcus sp., Prevotella intermedia ATCC 25611, Proteus vulgaris, Pseudomonas sp., Rhodococcus sp., Rickettsia typhi strain Wilmington, Staphylococcus epidermidis, Staphylococcushaemolyticus, s-aea, Streptococcus agalactiae, Streptococcus bovis, Streptococcus milleri, Streptococcus pyogenes, Staphylococcus haemolyticus, Salmonella, Serratia sp., Staphylococcusaureus, Staphylococcus saccharolyticus, Stenotrophomonas sp., Treponema pallidum, V.cholerae, V.vulnificus, bacillus subtilis pathogenic micro-organism.

2. synthesising probing needle

Synthetic by Shanghai biotechnology company limited.Probe 5 ' end adds amino the modification.

3. preparation chip

Set the point sample program as required, matrix distribution requires according to probe hybridization kinetics and whether point sample makes things convenient for and arrange.With the dissolving of synthetic probe, concentration is 200mmol/l with deionized water.The glass substrate that uses CEL company to produce carries out point sample with the point sample instrument of BioRobotics company by pre-set program.Require from tens according to difference that the size of point is about the 50-100 micron to thousands of points, dot spacing is decided according to the quantity of point.The good chip of point is in 37 ℃ of following aquations 3 days, then 80 ℃ of oven dry 2 hours down.(1xSSC 0.1%SDS) and the distilled water flushing slide, dries up with the probe damping fluid.With confining liquid (1%BSA, PH=7 0.01mol/l PB) sealing slide, wash afterwards 3 times.Dry up standby.

The application method of above-mentioned septicemia detection chip may further comprise the steps:

(1). handle sample

Get venous patient blood 3-5 milliliter, with N,O-Diacetylmuramidase dissolving, chloroform extracting, ethanol precipitation extracts DNA of bacteria, and is standby.Place for a long time as need, frozen down at-20 ℃.

(2) .PCR amplification

In reaction tubes, add amplification reaction mixture, Taq enzyme, sample of handling well and primer, the dUTP of the dNTP of capacity and fluorescein (Cy3) mark.Amplification condition is as follows:

94℃?5min

94℃?30sec

30 circulations of 56 ℃ of 30sec

72℃?1min

72℃?5min

Above step is 30 circulations;

(3). hybridization

Probe on PCR product and the chip was hybridized 2 hours down at 60 ℃, and (10xSSC, 0.1%SDS) ratio with the PCR product is 1: 1 to hybridization buffer.Wash 3 times.

(4). detect

Chip with after the scanner scanning hybridization of Genomic Solutions company obtains image and exports the result.

(5). data analysis

The supporting analysis software of the scanner of Genomic Solutions company is exported the chip analysis result.

Be further described below in conjunction with the technical scheme of specific embodiment invention:

Embodiment:

60 of design septicemia primer and common pathogen specific probes.The size of probe is a 15-20 base.Synthetic by Shanghai biotechnology company limited.Probe 5 ' end adds amino the modification.

The preparation gene chip:

With the glass substrate that CEL company produces, use the point sample instrument of BioRobotics company to carry out point sample by pre-set program.With the dissolving of synthetic probe, concentration is 200mmol/l with deionized water.The good chip of point is in 37 ℃ of following aquations 3 days, then 80 ℃ of oven dry 2 hours down.(1xSSC 0.1%SDS) and the distilled water flushing slide, dries up with the probe damping fluid.With confining liquid (1%BSA, PH=7 0.01mol/l PB) sealing slide, wash afterwards 3 times.Dry up standby.

Handle sample: patient's venous blood of getting clinical cultivation results and being infection of staphylococcus aureus is a small amount of, with the extracting of phenol chloroform, ethanol precipitation precipitate DNA, standby.

Pcr amplification: the primer sequence of employing is: F482:5 '-CGGCTAACTC TGTGCCAGC; R789:ATCTATGGGA CCATCAGGTACGG-5 '..

The reaction cumulative volume is 20 μ l, adds 10X buffer 2 μ l in reaction tubes, Taq enzyme 0.2 μ l, and each 2 μ l of sample 0.8 μ l that handles well and upstream and downstream primer, the dUTP1.2 μ l of dNTP0.8 μ l and Cy3 mark, remaining uses H ₂O.Amplification condition is as follows:

94℃?5min

94℃?30sec

56℃?40sec

72℃?1min

72℃?5min

Above step is 30 circulations;

Hybridization: the probe on PCR product and the chip was hybridized 2 hours down at 60 ℃, and (10xSSC, 0.1%SDS) ratio with the PCR product is 1: 1 to hybridization buffer.Wash 3 times.

Detect: the chip with after the scanner scanning hybridization of Genomic Solutions company obtains image and exports the result.Data analysis: the supporting analysis software of the scanner of Genomic Solutions company is exported chip results.Compare coincidence rate 100% with the Clinical Laboratory result.

Septicemia detection chip primer and probe sequence table

Sepsis

primer Sequence

F482 5′-CGGCTAACTC?TGTGCCAGC

R789 ATCTATGGGA?CCATCAGGTACGG-5′

Probe Sequence

Acinetobacter F577 5′-CAAATGTGAA?ATCCCCGAGC

Actinomyces F577 5′-CCGTGAAATC?CCCTGGCT

Borrelia?burgdorferi F577 5′-AAGTCTATGC?ATAAAATACC?ACAGCTC

Burkholderia?cepacia F577 5′-TGAAATCCCC?GGGCTCA

Bacteroides?fragilis F577 5′-TTGTGAAAGT?TTGCGGCTCA

Burkholderia?pseudomallei F557 5′-CTAAGACCGA?TGTGAAATCC?CC

Bartonella?sp. F557 5′-TAAGTCAGAG?GTGAAATCCC?AGG

Borrelia?afzelii F557 5′-TCTATGCATA?AAATACCACA?GCTCA

Borrelia?garinii F557 5′-TGCATAAAAT?ACCACGGCTC?A

Clostridium?difficile F557 5′-AGTCAGGAGT?GAAAGGCTAC?GG

Clostridium?perfringens F557 5′-GATGATTAAG?TGGGATGTGA?AATACC

Capnocytophaga?ochracea F557 5′-TATTAAGTCA?GGGGTGAAAG?GTTTC

Chlamydia?pneumoniae F557 5′-AAGGAAAGTT?AGATGTTAAA?TTTTGGG

Clostridium?septicum F557 5′-GCGGACTTTT?AAGTGAGATG?TGA

5′-ATGTGAAATA?CCCGGGCTCA

Coxiella?burnetii?strain1M F557 5′-TCTAAGTCGG?ATGTGAAAGC?CC

Escherichia?coli F557 5′-TGTTAAGTCA?GATGTGAAAT?CCCC

Enterobacter?aerogenes F557 5′-AGGCGGTCTG?TTAAGTCAGA?TGT

Enterococcus?dispar[duran] F557 5′-AGGCGGTTTC?TTAAGTCTGA?TGT

e-dis，?5′-CAGGCGGTTT?CTTAAGTCTG?ATG

Enterococcus?faecalis?strainK4 F557 5′-AGGCGGTTTC?TTAAGTCTGA?TGT

e-faeci?5′-GCAGGCGGTT?CTTAAGTCTG?A

Enterococcus?faecium F557 5′-AGGCGGTTCT?TAAGTCTGAT?GTG

Flavimonas?oryzihabitans F557 5′-GGGTGGTTTG?TTAAGTTGGA?TGT

Flavobacterium?sp. F557 5′-TTAAGTCAGT?AGTGAAATCT?TGCAGC

Fusobacterium?necrophorum F557 5′-GCGGGTTCAT?AAGTCTGATG?TTAA

Haemophilus?influenzae F557 5′-AGGCGGTTAT?TTAAGTGAGG?TGT

Klebsiella?oxytoca-16 F557 5′-CGGTCTGTCA?AGTCGGATGT?G

k-oxy 5′-GATCTGGAGG?AATACCGGTG?G

Klebsiella?pneumoniae F557 5′-CGGTCTGTCA?AGTCGGATGT?G

Leptospira?interrogans F557 5′-TGTGAAAACT?GCGGGCTCA

Lactobacillus?sp.B5406 F557 5′-AAGTCTGATG?TGAAAGCCCT?CG

Legionella?sp.strain?LLAP9 F557 5′-AGGTGGTTTG?GTAAGTTATC?TGTGA

Mycobacterium?tuberculosis F557 5′-GTTTGTCGCG?TTGTTCGTGA

Moraxella?sp. F557 5′-TGACTTTTAA?GTCAGGGGTG?AAAT

Mycoplasma?pneumoniae?strain?ATCC?15531 F557 5′-GGATTGAAAA?GTCTGGTGTT?AAAGG

Neisseria?meningitidis F557 5′-AGACGGTTAC?TTAAGCAGGA?TGTG

Nocardia?asteroides F557 5′-GTCGTTTGTG?AAAACTTGGG?G

p-mor F557 5′-GGTTGATTGA?GTCAGATGTG?AAATC

p-pro F557 5′-GCGGTTGATT?AAGTTAGATG?TGAAA

Peptococcus?niger F557 5′-AGGCGGTAAA?TTAAGTCAGG?TGT

Peptostreptococcus?sp. F557 5′-GTGGTCTTTC?AAGTCGGTGG?TT

Prevotella?intermedia?ATCC?25611 F557 5′-ATGATTAAGC?GTGACGTGAA?ATG

Proteus?vulgaris F557 5′-CAATTAAGTC?AGATGTGAAA?GCCC

Pseudomonas?sp.strain?Circle F557 5′-AGGTGGTTAG?TTAAGTTGGATGTGA

Rhodococcus?sp F557 5′-CGTTTGTGAA?AACCAGCAGC

Rickettsia?typhi?strain?Wilmington F557 5′-TTTAGTAAGT?TGGAAGTGAAAGCCC

Staphylococcus?epidermidis F557 5′-AGTCTGATGT?GAAAGCCCAC?G

epi-hae?5′-AACTGGAAAA?CTTGAGTTCA?GAAGAG

Staphylococcus?haemolyticus F557 5′-AGTCTGATGT?GAAAGCCCAC?G

hae-epi?5′-ATTGGAAACT?GTAAAACTTG?AGTGC

s-aea F557 5′-AGGCGGTTCT?TTAAGTCTGA?AGTT

Streptococcus?agalactiae F557 5′-AGGCGGTTAG?ATAAGTCTGA?AGTT

Streptococcus?bovis F557 5′-AGGCGGTTTA?ATAAGTCTGA?AGTTAA

Streptococcus?milleri F557 5′-GCGGTTAGAA?AAGTCTGAAG?TGAA

Streptococcus?pyogenes F557 5′-TCTGAAGTTA?AAGGCATTGG?CTC

Staphylococcus?haemolyticus F557 5′-TCAAGTCGGA?TGTGAAGTCC?C

Salmonella F557 5′-TGAAATCCCC?GGGCTCA

Serratia?sp. F557 5′-GTGAAATCC?CCGCGCTTA

Staphylococcus?aureus F557 5′-AGTCTGATGT?GAAAGCCCAC?G

Staphylococcus?saccharolyticus F557 5′-AGTCTGATGT?GAAAGCCCAC?G

Stenotrophomonas?sp. F557 5′-AGGTGGTCGT?TTAAGTCTGT?TGTG

Treponema?pallidum F557 5′-GCGGACTGGT?AAGCCTGGT

V.cholerae F557 5′-TCAGATGTGA?AAGCCCTGGG

V.vulnificus F557 5′-GAAAGCCCGG?GGCTCA

bacillus?subtilis F557 CTGATGTGAAAGCCCCCG

Pcr amplification primer sequence is as follows:

Sepsis

primer Sequence

F482 5′-CGGCTAACTC?TGTGCCAGC

R789 ATCTATGGGA?CCATCAGGTACGG-5′

Claims (6)

1. septicemia detection chip, it is characterized in that: this chip is the microarray that is distributed with a plurality of different zones on the glass substrate, in each microarray zone, is fixed with the specific probe of common septicemia pathogenic bacterium respectively; Probe design is 60, and designed probe sequence is as follows:

Sepsis

Probe Sequence

Acinetobacter F577 5`-CAAATGTGAA?ATCCCCGAGC

Actinomyces F577 5`-CCGTGAAATC?CCCTGGCT

Borrelia?burgdorferi F577 5`-AAGTCTATGC?ATAAAATACC?ACAGCTC

Burkholderia?cepacia F577 5`-TGAAATCCCC?GGGCTCA

Bacteroides?fragilis F577 5`-TTGTGAAAGT?TTGCGGCTCA

Burkholderia?pseudomallei F557 5`-CTAAGACCGA?TGTGAAATCC?CC

Bartonella?sp. F557 5`-TAAGTCAGAG?GTGAAATCCC?AGG

Borrelia?afzelii F557 5`-TCTATGCATA?AAATACCACA?GCTCA

Borrelia?garinii F557 5`-TGCATAAAAT?ACCACGGCTC?A

Clostridium?difficile F557 5`-AGTCAGGAGT?GAAAGGCTAC?GG

Clostridium?perfringens F557 5`-GATGATTAAG?TGGGATGTGA?AATACC

Capnocytophaga?ochracea F557 5`-TATTAAGTCA?GGGGTGAAAG?GTTTC

Chlamydia?pneumoniae F557 5`-AAGGAAAGTT?AGATGTTAAA?TTTTGGG

Clostridium?septicum F557 5`-GCGGACTTTT?AAGTGAGATG?TGA

5`-ATGTGAAATA?CCCGGGCTCA

Coxiella?burnetii?strainlM F557 5`-TCTAAGTCGG?ATGTGAAAGC?CC

Escherichia?coli F557 5`-TGTTAAGTCA?GATGTGAAAT?CCCC

Enterobacter?aerogenes F557 5`-AGGCGGTCTG?TTAAGTCAGA?TGT

Enterococcus?dispar[duran] F557 5`-AGGCGGTTTC?TTAAGTCTGA?TGT

e-dis，?5`-CAGGCGGTTT?CTTAAGTCTG?ATG

Enterococcus?faecalis?strainK4 F557 5`-AGGCGGTTTC?TTAAGTCTGA?TGT

e-faeci?5`-GCAGGCGGTT?CTTAAGTCTG?A

Enterococcus?faecium F557 5`-AGGCGGTTCT?TAAGTCTGAT?GTG

Flavimonas?oryzihabitans F557 5`-GGGTGGTTTG?TTAAGTTGGA?TGT

Flavobacterium?sp. F557 5`-TTAAGTCAGT?AGTGAAATCT?TGCAGC

Fusobacterium?necrophorum F557 5`-GCGGGTTCAT?AAGTCTGATG?TTAA

Haemophilus?influenzae F557 5`-AGGCGGTTAT?TTAAGTGAGG?TGT

Klebsiella?oxytoca-16 F557 5`-CGGTCTGTCA?AGTCGGATGT?G

k-oxy 5`-GATCTGGAGG?AATACCGGTG?G

Klebsiella?pneumoniae F557 5`-CGGTCTGTCA?AGTCGGATGT?G

Leptospira?interrogans F557 5`-TGTGAAAACT?GCGGGCTCA

Lactobacillus?sp.B5406 F557 5`-AAGTCTGATG?TGAAAGCCCT?CG

Legionella?sp.strain?LLAP9 F557 5`-AGGTGGTTTG?GTAAGTTATC?TGTGA

Mycobacterium?tuberculosis F557 5`-GTTTGTCGCG?TTGTTCGTGA

Moraxella?sp. F557 5`-TGACTTTTAA?GTCAGGGGTG?AAAT

Mycoplasma?pneumoniae?strain?ATCC?15531 F557 5`-GGATTGAAAA?GTCTGGTGTT?AAAGG

Neisseria?meningitidis F557 5`-AGACGGTTAC?TTAAGCAGGA?TGTG

Nocardia?asteroides F557 5`-GTCGTTTGTG?AAAACTTGGG?G

p-mor F557 5`-GGTTGATTGA?GTCAGATGTG?AAATC

p-pro F557 5`-GCGGTTGATT?AAGTTAGATG?TGAAA

Peptococcus?niger F557 5`-AGGCGGTAAA?TTAAGTCAGG?TGT

Peptostreptococcus?sp. F557 5`-GTGGTCTTTC?AAGTCGGTGG?TT

Prevotella?intermedia?ATCC?25611 F557 5`-ATGATTAAGC?GTGACGTGAA?ATG

Proteus?vulgaris F557 5`-CAATTAAGTC?AGATGTGAAA?GCCC

Pseudomonas?sp.strain?Circle F557 5`-AGGTGGTTAG?TTAAGTTGGATGTGA

Rhodococcus?sp F557 5`-CGTTTGTGAA?AACCAGCAGC

Rickettsia?typhi?strain?Wilmington F557 5`-TTTAGTAAGT?TGGAAGTGAA?AGCCC

Staphylococcus?epidermidis F557 5`-AGTCTGATGT?GAAAGCCCAC?G

epi-hae?5`-AACTGGAAAA?CTTGAGTTCA?GAAGAG

Staphylococcus?haemolyticus F557 5`-AGTCTGATGT?GAAAGCCCAC?G

hae-epi?5`-ATTGGAAACT?GTAAAACTTG?AGTGC

s-aea F557 5`-AGGCGGTTCT?TTAAGTCTGA?AGTT

Streptococcus?agalactiae F557 5`-AGGCGGTTAG?ATAAGTCTGA?AGTT

Streptococcus?bovis F557 5`-AGGCGGTTTA?ATAAGTCTGA?AGTTAA

Streptococcus?milleri F557 5`-GCGGTTAGAA?AAGTCTGAAG?TGAA

Streptococcus?pyogenes F557 5`-TCTGAAGTTA?AAGGCATTGG?CTC

Staphylococcus?haemolyticus F557 5`-TCAAGTCGGA?TGTGAAGTCC?C

Salmonella F557 5`-TGAAATCCCC?GGGCTCA

Serratia?sp. F557 5`-GTGAAATCC?CCGCGCTTA

Staphylococcus?aureus F557 5`-AGTCTGATGT?GAAAGCCCAC?G

Staphylococcus?saccharolyticus F557 5`-AGTCTGATGT?GAAAGCCCAC?G

Stenotrophomonas?sp. F557 5`-AGGTGGTCGT?TTAAGTCTGT?TGTG

Treponema?pallidum F557 5`-GCGGACTGGT?AAGCCTGGT

V.cholerae F557 5`-TCAGATGTGA?AAGCCCTGGG

V.vulnificus F557 5`-GAAAGCCCGG?GGCTCA

bacillus?subtilis F557 CTGATGTGAAAGCCCCCG。

2. the preparation technology of a septicemia detection chip, it is characterized in that: preparation technology of the present invention carries out as follows:

(1), design septicemia specific probe and primer;

From database, obtain reliable septicemia pathogenic bacterium gene order, according to described probe sequence designing probe; From database, obtain reliable septicemia pathogenic bacterium virus gene sequence, select with using primers F 482:5`-CGGCTAACTC TGTGCCAGC; R789:ATCTATGGGA CCATCAGGTACGG-5` increases at 16s portion gene sequence, mark fluorescent element in the time of amplification;

(2), synthesising probing needle, the probe sequence that designs according to claim 1 carries out synthesising probing needle;

(3), preparation chip;

With the dissolving of synthetic probe, obtain the solution that concentration is 200mmol/l with deionized water, on glass substrate, carry out point sample; The good chip of point is in 37 ℃ of following aquations 3 days, then 80 ℃ of oven dry 2 hours down; With probe damping fluid and distilled water flushing slide, dry up; With confining liquid sealing slide, wash afterwards 3 times, dry up standby.

3. the preparation technology of septicemia detection chip according to claim 2 is characterized in that: in the preprocessing process to blood sample to be measured, pcr amplification primer sequence is as follows:

Sepsis

primer Sequence

F482?5`-CGGCTAACTC?TGTGCCAGC

R789?ATCTATGGGA?CCATCAGGTACGG-5`。

4. the preparation technology of septicemia detection chip according to claim 2 is characterized in that: in the step of synthesising probing needle, probe 5 ' end adds amino the modification.

5. the preparation technology of septicemia detection chip according to claim 2 is characterized in that: point sample from tens to thousands of points, the size of point is the 50-100 micron.

6. the preparation technology of septicemia detection chip according to claim 2 is characterized in that: the probe damping fluid is 1xSSC, 0.1%SDS; Confining liquid is 1%BSA, PH=7 0.01mol/l PB.