CN104328167B - Can the genetic chip of ten kinds of Main Pathogenic Bacterias of parallel detection mastitis for milk cows and detection method - Google Patents
Can the genetic chip of ten kinds of Main Pathogenic Bacterias of parallel detection mastitis for milk cows and detection method Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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Abstract
The present invention disclose a kind of can the genetic chip of ten kinds of Main Pathogenic Bacterias of parallel detection mastitis for milk cows and detection method, described genetic chip includes chip carrier, chip carrier is fixed with the nucleotide probe for detecting proteus mirabilis, streptococcus dysgalactiae, Streptococcusagalactiae, MRSE, streptococcus uberis, Klebsiella Pneumoniae, Pseudomonas aeruginosa, staphylococcus aureus, Escherichia coli and salmonella typhi, is further fixed on fixing positive control, hybridization positive control and hybridization negative control.This genetic chip devises a plurality of probe for the different loci of gyrB and fliC, the detection leakage phenomenon that effectively prevent minority base mutation in the difference of different subtype bacterial classification nucleotide sequence and bacterial classification genome and cause, realize in mastitis for milk cows ten kinds of high fluxs of the main pathogenic fungi, high specific, high sensitivity, detections of efficient quick, for monitoring and the early warning of pathogen in milk cattle cultivating, mastitis for milk cows, there is practical significance.
Description
Technical field:
The invention belongs to technical field of microbial detection, being specifically related to one can parallel detection mastitis for milk cows ten
Plant genetic chip and the detection method of Main Pathogenic Bacteria.
Background technology:
Biochip technology (Gene chip) is that a kind of gene grown up phase early 1990s divides
The High biotechnology of analysis, is that nucleotide fragments (probe) point of the genes of interest of Prof. Du Yucang is placed in one
On fixed carrier, then extract the complete genome DNA of tested microorganism, expand this genes of interest by PCR
Fragment, and in these genetic fragments after mark fluorescent material, make under suitable conditions on itself and chip
Probe hybridizes, and the fragment that homology is high can be by hybridization complementary pairing, and cleaned scanning can be at corresponding square
Battle array probe site display fluorescence signal.The purpose fragment of genechip detection can be PCR primer, genome
DNA, total serum IgE, cDNA, DNA or oligonucleotides etc. (Schena etal, 2000).At present
Combine method for gene chip with multiplex PCR in the detection method of report, universal primer combines method for gene chip
Etc. for main.Biochip technology application in microbial identification, has high specificity, highly sensitive spy
Point, especially when tackling a large amount of, complicated sample, advantage becomes apparent from, convenient and efficient, with low cost,
Error is little.Its prominent feature be integrated, be miniaturized, automate, high flux etc., can with high flux,
Parallelization carries out precise Identification to multiple target gene, can be Modern microbiological bacterial classification identification, virulence factor determine,
Resistance detection etc. provide perfect technology platform (Call etal, 2001).Therefore, necessary in a short time
High-new Protocols in Molecular Biology is utilized to set up the high flux of multi-infection pathogenic bacteria, supervise fast and accurately
Survey system, to guarantee that the various pathogens the most correctly diagnosing such as mastitis for milk cows etc. infects
Property disease, with the ability improving the prevention of China many Infectious diseases bacterium, diagnose and control.
Pathogens from dairy cattle affected smastitis infection ability, route of transmission are wide, simultaneously because antibiotic a large amount of use and
The problem of medicament residue makes people when in the face of the occurred frequently of mastitis for milk cows and mass-sending, often feels simply helpless, many
Kind pathogenic bacteria are fallen ill by sporadic, group and milk industry cause great loss, and then give the dairy of people
Product consumption makes a big impact, to aspect bands such as the normal life quality of consumer, social economy's sustainable developments
Carry out immeasurable loss, therefore, the Main Pathogenic Bacteria causing mastitis for milk cows is realized fast and parallel detection
Extremely the most urgent.Cause the pathogenic bacteria huge number of mastitis for milk cows, the pathogenic bacteria being currently known about 220
Multiple the most common just have a kind more than 20, and 90% pathogens from dairy cattle affected smastitis is mainly Escherichia coli, golden yellow
Look staphylococcus, MRSE, Friedlander's bacillus, proteus mirabilis, streptococcus dysgalactiae,
Streptococcus uberis, Streptococcusagalactiae, salmonella typhi, Pseudomonas aeruginosa, Shigella, waxy brood cell's bar
Bacterium etc..Prior art has main pathogen of bovine mastitis is detected grinding of genetic chip and detection method
Study carefully, the detection gene of a kind of main pathogen of bovine mastitis as disclosed in patent of invention CN 101492739A
Chip and detection method, its be to 6 kinds of main pathogen of bovine mastitis (Streptococcusagalactiae, stop breast hammer
Bacterium, Escherichia coli, Friedlander's bacillus, proteus mirabilis and staphylococcus aureus) genetic chip
The research of detection method, described genetic chip includes solid phase carrier and the specific inspection being fixed on solid phase carrier
Probing pin, specificity detection probe comprises be substrate sequences Design with Escherichia coli 16S rDNA conserved region logical
With the specific few core of design in the Variable Area of 6 kinds of main pathogen of bovine mastitis between primer sequence
Thuja acid hybridization probe, and define degenerate temperature that PCR reacts, optimize PCR amplification nucleic acid fragment and
Genetic chip carries out the condition hybridized, it is thus achieved that well crossbreeding effect, finally gives testing result accurately.
The method utilizes 16S rDNA conserved region for substrate sequences Design universal primer, reflects as Bacteria Detection box
Fixed target molecule, but owing to 16S rRNA is inadequate for the bacterium resolution ratio that affiliation is nearer and different sub-
In the difference of type bacterial classification nucleotide sequence and bacterial classification genome, minority base mutation all can cause detection leakage phenomenon.
Summary of the invention:
It is an object of the invention to overcome the deficiencies in the prior art, it is provided that one can parallel detection mastitis for milk cows ten
Plant Main Pathogenic Bacteria proteus mirabilis, streptococcus dysgalactiae, Streptococcusagalactiae, MRSE, breast
Streptococcus, Klebsiella Pneumoniae, Pseudomonas aeruginosa, staphylococcus aureus, Escherichia coli and Salmonella typhimurium
The genetic chip of bacterium and detection method, it is achieved to the parallel quick and high-flux of complex samples, high specific,
Highly sensitive detection.
The genetic chip of existing main pathogen of bovine mastitis is to utilize 16S rRNA conserved region as base
End sequences Design universal primer, as Bacteria Detection box identify target molecule, but 16S rRNA for
The nearer bacterium resolution ratio of affiliation is inadequate, and applicant finds through long-term research, uses gyrB conduct
Bacteria Identification and the target gene of classification, gyrB gene is the gene of coding DNA unwindase B subunit, this base
Because evolutionary rate is very fast, base replacement frequency is higher, can better discriminate between sibling species than 16srRNA gene,
Disclosure satisfy that proteus mirabilis, streptococcus dysgalactiae, Streptococcusagalactiae, MRSE, breast hammer
The qualification of bacterium, Klebsiella Pneumoniae, Pseudomonas aeruginosa, staphylococcus aureus and nine kinds of bacteriums of Escherichia coli needs
Ask.Further, since the name of current salmonella is more chaotic, by 16s rRNA and gyrB sequence
Conservative Analysis, according to two kinds of gene orders design probes, it may only be possible to identify Salmonella, and
Cannot ensure that the probe specificity of design is just for salmonella typhimurium.Through substantial amounts of literature survey, Shen
Ask someone to use fliC as the target gene identifying salmonella typhi.Therefore, often the present invention is directed to above-mentioned ten kinds
See pathogens from dairy cattle affected smastitis, choose specific probe respectively according to two kinds of genes of gyrB and fliC, to ensure
The bacterial classification generation specific signals that each bacterial classification probe sum is corresponding, is simultaneous for described ten kind bacterium
Two kinds of genes of gyrB and fliC, have separately designed a plurality of probe, effectively prevent due to different subtype bacterial classification core
Minority base mutation in the difference of acid sequence and bacterial classification genome and the detection leakage phenomenon that causes.
The present invention is to take techniques below scheme to be achieved:
A kind of can the genetic chip of ten kinds of Main Pathogenic Bacterias of parallel detection mastitis for milk cows, include chip and carry
Body, chip carrier is fixed with for detect proteus mirabilis, streptococcus dysgalactiae, Streptococcusagalactiae,
MRSE, streptococcus uberis, Klebsiella Pneumoniae, Pseudomonas aeruginosa, staphylococcus aureus, big
Enterobacteria and the nucleotide probe of salmonella typhi.
Described be used for detecting proteus mirabilis, streptococcus dysgalactiae, Streptococcusagalactiae, MRSE,
Streptococcus uberis, Klebsiella Pneumoniae, Pseudomonas aeruginosa, staphylococcus aureus and colibacillary probe are
Design from gyrB gene.
Described design from gyrB and fliC gene for detecting salmonella typhi probe.
Described is used for detecting the nucleotide probe of proteus mirabilis (pro), and its sequence is SEQ ID NO:1.
Described is used for detecting the nucleotide probe of streptococcus dysgalactiae (sdy), and its sequence is SEQ ID NO:2.
Described is used for detecting the nucleotide probe of Streptococcusagalactiae (sag), and its sequence is SEQ ID NO:3.
Described is used for detecting the nucleotide probe of MRSE (sep), and its sequence is SEQ ID NO:4.
Described is used for detecting the nucleotide probe of streptococcus uberis (supi), and its sequence is SEQ ID NO:5.
Described is used for detecting the nucleotide probe of Klebsiella Pneumoniae (kpn), and its sequence is SEQ ID NO:6.
Described is used for detecting the nucleotide probe of Pseudomonas aeruginosa (pae), and its sequence is SEQ ID NO:7.
Described is used for detecting the nucleotide probe of staphylococcus aureus (sau), and its sequence is SEQ ID
NO:8。
Described is used for detecting the nucleotide probe of Escherichia coli (eco), and its sequence is SEQ ID NO:9.
Described is used for detecting the nucleotide probe of salmonella typhi (sen), and its sequence is SEQ ID
NO:10。
Table 1: the probe sequence information table used in the present invention
Fixing positive control (HEX), hybridization positive control (PC) and miscellaneous it is further fixed on the chip of the present invention
Handing over negative control (NC), its nucleotide sequence is followed successively by SEQ ID NO:11, SEQ ID NO:12 and SEQ
ID NO:13。
The above-mentioned positive quality control fragment with the hybridization of positive quality control probe is hybridization PC-TAMRA, its nucleotides sequence
It is classified as SEQ ID NO:14.(sequence information of quality control probes and positive quality control fragment such as table 2).
Table 2: quality control probes and the sequence information of positive quality control fragment
The genetic chip preparation process of the present invention is:
After nucleotide probe is diluted to debita spissitudo, at brilliant 16 people's point sample instruments of core PersonalArrayer
(CapitalBio) on, according to actual conditions respectively to 384 hole point templates, slide, punch block, dot matrix, sample
Product and cleaning parameters are configured, by running brilliant 16 people's point sample instruments of core PersonalArrayer by probe points
It is formed on solid phase amino chip；Then by rich brilliant core LuxScan10K scanner difficult to understand, detection chip point feelings
Condition.
Above-mentioned steps nucleotide probe is diluted to debita spissitudo, be use sterilized water dissolve each probe to 40 μMs,
With brilliant core@The ratio of gene chip sampling liquid 1:1 mixes, and makes final concentration of 20 μMs of each probe；Then
By probe according to the probe arrangement table designed, add to successively in 384 hole point templates.
Of the present invention can the detection side of genetic chip of ten kinds of Main Pathogenic Bacterias of parallel detection mastitis for milk cows
Method, comprises the following steps successively:
(1) extraction of sample nucleic acid to be checked: separate from the mastitis for milk cows disease milk sample collected and obtain carefully
Bacterium；Then the genomic DNA with CTAB/NaCl method extraction gained bacterium is standby as template；
(2) common or multiplexed PCR amplification reaction: common or multiplexed PCR amplification system is: 2 μ L 10 ×
PCR reaction buffer, 0.4 μ L 10mM dNTPs, 1U Taq archaeal dna polymerase, on 0.5 μ L 10 μMs
Trip primer and 10 μMs of downstream primers of 0.5 μ L, 1-2 μ L (about 100ng) measuring samples DNA, distilled water
Supply 20 μ L cumulative volumes.And the condition of described PCR reaction is: 94 DEG C of denaturations 5min；94 DEG C of changes
Property 30s, 55 DEG C (according to primer annealing temperature adjust) annealing 30s, 72 DEG C extend 1min30s, 35
Circulation；72 DEG C extend 10min, 4 DEG C of preservations；
(3) fluorescence labeling PCR reaction: 1. pretreatment: directly take 5 μ L PCR primer as template to nothing
In the 0.2ml centrifuge tube that bacterium is clean, in each sample cell, then add 3 μ L random primers, and moisturizing is extremely
19 μ L, vibration mixing, brief centrifugation；Sample cell after being centrifuged again puts into PCR instrument, 95 DEG C of sex change 3min,
Ice bath 3min immediately, brief centrifugation；2. fluorescence labeling: take a new 0.2ml centrifuge tube and be placed on ice,
The most inwardly add 2.5 μ L5 × Klenow Buffer, 1 μ L Klenow enzyme, 2.5 μ L2.5mM dNTPs；
Then fluorescence labeling reaction system Mix of 6 μ L is joined in the sample cell of pretreatment, vibration mixing,
Brief centrifugation；Finally sample cell is put in PCR instrument, first 37 DEG C of reaction 90min, then 72 DEG C of reactions
10min, 4 DEG C of preservations；
(4) hybridization: 1. preparing hybrid system: melt hybridization buffer at 42 DEG C, take an aseptic
0.2ml centrifuge tube, the most inwardly adds 5 μ L hybridization buffers, 15 μ L fluorescent markers, is placed in the most standby
With；2. hybridization: open chip hybridization box, kept flat by hybridizing box on desktop, adds in hybridization and bottom groove
Enter about 80 μ L sterilized waters, to prevent hybridization solution in crossover process from volatilizing in a large number；By chip front side (mark upward
Sign towards operator) put between two alignment pins of hybridizing box；Put chip cover plate and (have the one side of boss
Towards chip), upper end elder generation contact chip, more slowly lid under；Slowly noted by cover glass well with pipettor
Enter the hybridization solution after 20 μ L sex change, hybridization solution can by surface tension of liquid boss below cover glass and
Forming one liquid film between chip surface, vibrations cover plate or chip do not avoid damage to liquid film；By hybridizing box
Cover plate is buckled, it is ensured that the alignment pin at frid two ends inserts in the dowel hole at cover plate two ends respectively；Buckle cover plate
After, respectively two metal clips are sticked into both sides, it is to note that action wants mild, does not allow hybridizing box have big shake
Dynamic, to prevent hybridization solution from spilling dot matrix region；It addition, metal clip needs to stick into completely, to guarantee hybridizing box
Sealing；Hybridizing box is put into rich difficult to understand BioMixerTM II hybridization instrument, 42 DEG C, reaction 2h or overnight.
(5) wash-out: useChip is carried out by SlibeWasherTM8 chip cleaning device: 1. two
Plant the configuration of eluent: eluent I is 2 × sodium citrate salting liquid and 0.2% sodium dodecyl sulfate solution
Mixture, eluent II is 0.2 × sodium citrate salting liquid: 2. elution action: at 42 DEG C, first with washing
De-liquid I cleans twice, each 120s；Then clean three times with eluent II, each 80s, 1500rpm
Centrifugal 1min dries；
(6) scanner detection is used, analysis result: use scanner scanning to go out result, use and analyze software
Quantify the signal value of each point.
The present invention through a series of optimizing research, does not count and prepares gene on the basis of existing genetic chip
The time of chip, the whole sample detection time can complete in 6 hours, and due to for gyrB and fliC
Different loci devise multiple probe, therefore on the one hand this genetic chip can be prevented effectively from single probe at mirror
Determine the situation of missing inspection during the microorganism of different subtype, on the other hand can effectively reduce single probe and identify micro-life
The missing inspection situation that during thing, gene mutation causes；The Standard Operating Procedure optimized avoids the shadow of manual operation as far as possible
Ringing, stability is more preferable.In general, the present invention can break through all drawbacks of existing microorganism detection technology,
Realize in mastitis for milk cows ten kinds of high fluxs of the main pathogenic fungi, high specific, high sensitivity, the fastest
Prompt detection, has practical significance for monitoring and the early warning of pathogen in milk cattle cultivating, mastitis for milk cows.
Accompanying drawing illustrates:
Fig. 1 is the result figure of the unrelated bacterium of genetic chip specific detection of the present invention test；
Fig. 2 is the result figure of genetic chip specific detection standard bacteria of the present invention test；
Fig. 3 is the result figure of genetic chip specific detection two strain standard bacteria of the present invention test；
Fig. 4 is the result figure of genetic chip sensitivity technique Streptococcusagalactiae of the present invention.
Detailed description of the invention:
Below in conjunction with specific embodiment and accompanying drawing, the present invention is described in more detail.
1. the design of probe
The genetic chip of the present invention is used for detecting ten kinds of common pathogens in mastitis for milk cows disease milk sample, bag
Include proteus mirabilis, streptococcus dysgalactiae, Streptococcusagalactiae, MRSE, streptococcus uberis, lung
Scorching klebsiella, Pseudomonas aeruginosa, staphylococcus aureus, Escherichia coli and salmonella typhi.It uses
Probe design all in accordance with gyrB and the fliC gene order of bacterial classification, ensure it through BLAST contrast
Specifically, probe used, primer sequence information are shown in Table 3.Probe used, primer are closed by special company's industry
Become.
Table 3: the sequence information of probe, primer used by the present invention
2. the structure of genetic chip described in:
(1) oligonucleotide probe being commercially synthesized is diluted to debita spissitudo: use sterilized water to dissolve each probe extremely
40 μMs, with brilliant core@The ratio of gene chip sampling liquid 1:1 mixes, and makes final concentration of 20 μMs of each probe.
Then, respectively 384 hole point templates, slide, punch block, dot matrix, sample and cleaning are joined according to actual conditions
Number is configured, and by running brilliant 16 people's point sample instruments of core PersonalArrayer, probe points is formed on solid phase ammonia
On base chip.A kind of sample application array of the gene chip probes of above-mentioned structure such as table 4.
Table 4: a kind of sample application array of said gene chip probe
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
| 1 | HEX | NC | NC | PC | PC | NC | NC | HEX |
| 2 | pro_1 | pro_2 | pro_3 | pro_4 | pro_5 | pro_6 | pro_7 | pro_8 |
| 3 | pro_9 | pro_10 | pro_11 | |||||
| 4 | sdy_1 | sdy_2 | sdy_3 | sdy_4 | sdy_5 | sdy_6 | sdy_7 | sdy_8 |
| 5 | sdy_9 | sdy_10 | sdy_11 | sdy_12 | sdy_13 | sdy_14 | ||
| 6 | sag_1 | sag_2 | sag_3 | sag_4 | sag_5 | sag_6 | ||
| 7 | sag_7 | sag_8 | sag_9 | sag_10 | sag_11 | sag_12 | sag_13 | sag_14 |
| 8 | sep_1 | sep_2 | sep_3 | sep_4 | sep_5 | sep_6 | sep_7 | sep_8 |
| 9 | sep_9 | sep_10 | sep_11 | sep_12 | ||||
| 10 | supi_1 | supi_2 | supi_3 | supi_4 | supi_5 | supi_6 | supi_7 | supi_8 |
| 11 | supi_9 | supi_10 | supi_11 | supi_12 | supi_13 | supi_14 | ||
| 12 | kpn_1 | kpn_2 | kpn_3 | kpn_4 | kpn_5 | kpn_6 | kpn_7 | kpn_8 |
| 13 | kpn_9 | |||||||
| 14 | pae_1 | pae_2 | pae_3 | pae_4 | pae_5 | pae_6 | pae_7 | pae_8 |
| 15 | pae_9 | pae_10 | ||||||
| 16 | sau_1 | sau_2 | sau_3 | sau_4 | sau_5 | sau_6 | sau_7 | sau_8 |
| 17 | sau_9 | sau_10 | ||||||
| 18 | sen_1 | sen_2 | sen_3 | sen_4 | sen_5 | sen_6 | ||
| 19 | sen_7 | sen_8 | sen_9 | sen_10 | sen_11 | sen_12 | sen_13 | sen_14 |
| 20 | sen_15 | sen_16 | sen_17 | sen_18 | sen_19 | sen_20 | ||
| 21 | eco_1 | eco_2 | eco_3 | eco_4 | eco_5 | eco_6 | ||
| 22 | HEX | NC | NC | PC | PC | PC | NC | HEX |
Note: HEX is point sample Quality Control probe；NC is the negative Quality Control probe of hybridization；PC is hybridization positive quality control
Probe.
(2) probe and chip hydrated cross-linked: need water and hybridization 12h in 37 DEG C of wet boxes after some system.
(3) after hydration, chip processes: using distilled water to clean 3 times, each 2 minutes, wash-out went to be not connected with
, connect untight probe.Use 0.2%NaBH4Close: make chip be totally submerged, stand 5min
After, 150pm vibrates 5min, then stands 5min；Finally clean 3 times with distilled water, each 2min.
2000rpm is centrifuged 2min and removes chip surface water mark, and 4 DEG C keep in Dark Place standby.
3. the detection method of genetic chip described in
(1) extraction of sample nucleic acid to be detected: first, divides from the mastitis for milk cows disease milk sample collected
From obtaining bacterium；Then the genomic DNA with CTAB/NaCl method extraction gained bacterium is standby as template；
(2) PCR amplification: 1. common or multiplexed PCR amplification system: 2 μ L 10 × PCR reaction buffer,
0.4 μ L 10mM dNTPs, 1U Taq DNA Polymerase, 0.5 μ L 10 μMs upstream primer and 0.5 μ L
10 μMs of downstream primers, 1-2 μ L (about 100ng) measuring samples DNA, it is overall that distilled water supplies 20 μ L
Long-pending.2. PCR reaction condition: 94 DEG C of denaturations 5min；94 DEG C of sex change 30s, 55 DEG C (are moved back according to primer
Fire temperature adjusts) annealing 30s, 72 DEG C extend 1min 30s, 35 circulations；72 DEG C extend 10min,
4 DEG C of preservations；
(3) fluorescence labeling PCR primer: 1. pretreatment: directly take 5 μ L PCR primer as template to nothing
In the 0.2ml centrifuge tube that bacterium is clean, in each sample cell, then add 3 μ L random primers, and moisturizing is extremely
19 μ L, vibration mixing, brief centrifugation；Sample cell after being centrifuged again puts into PCR instrument, 95 DEG C of sex change 3min,
Ice bath 3min immediately, brief centrifugation；2. fluorescence labeling: take a new 0.2ml centrifuge tube and be placed on ice,
The most inwardly add 2.5 μ L5 × Klenow Buffer, 1 μ L Klenow enzyme, 2.5 μ L2.5mM dNTPs；
Then fluorescence labeling reaction system Mix of 6 μ L is joined in the sample cell of pretreatment, vibration mixing,
Brief centrifugation；Finally sample cell is put in PCR instrument, first 37 DEG C of reaction 90min, then 72 DEG C of reactions
10min, 4 DEG C of preservations；
(4) hybridization: 1. preparing hybrid system: melt hybridization buffer at 42 DEG C, take the 0.2 of an aseptic
Ml centrifuge tube, the most inwardly adds 5 μ L and hybridizes buffer, 15 μ L fluorescent markers, be placed in the most standby；
2. hybridization: open chip hybridization box, kept flat by hybridizing box on desktop, adds about in hybridization and bottom groove
80 μ L sterilized waters, to prevent hybridization solution in crossover process from volatilizing in a large number；By chip front side upward (label towards
Operator) put between two alignment pins of hybridizing box；Put chip cover plate and (have the one of boss facing to core
Sheet), upper end elder generation contact chip, more slowly lid under；It is slowly injected into 20 μ L by cover glass well with pipettor
Hybridization solution after sex change, hybridization solution can be by surface tension of liquid boss below cover glass and chip surface
Between form one liquid film, vibrations cover plate or chip avoid damage to liquid film；The cover plate of hybridizing box is buckled,
Guarantee in the dowel hole that the alignment pin at frid two ends inserts cover plate two ends respectively；After buckling cover plate, respectively will
Two metal clips stick into both sides, it is to note that action wants mild, does not allow hybridizing box have big vibrations, to prevent
Hybridization solution spills dot matrix region；It addition, metal clip needs to stick into completely, to guarantee the sealing of hybridizing box；
Hybridizing box is put into rich difficult to understand BioMixerTM II hybridization instrument, 42 DEG C, reaction 2h or overnight；
(5) wash-out: useChip is carried out by SlibeWasherTM8 chip cleaning device.1. two
Plant the configuration of eluent: eluent I is 2 × sodium citrate salting liquid and 0.2% sodium dodecyl sulfate solution
Mixture, eluent II is 0.2 × sodium citrate salting liquid: 2. elution action: at 42 DEG C, first with washing
De-liquid I cleans twice, each 120s；Then clean three times with eluent II, each 80s, 1500rpm
Centrifugal 1min dries；
(6) scanning chip, analysis result: use scanner scanning chip, export image.Use analysis soft
Part quantifies the signal value of each point.Positive findings has hybridization signal clearly.
4. use genechip detection proteus mirabilis
(1) extraction of sample nucleic acid to be detected: extract the base of proteus mirabilis according to CTAB/NaCl method
Because group DNA is standby as template；
(2) PCR amplification: 1. common or multiplexed PCR amplification system: 2 μ L 10 × PCR reaction buffer,
0.4 μ L 10mM dNTPs, 1U Taq DNA Polymerase, 0.5 μ L 10 μMs upstream primer and 0.5 μ L
10 μMs of downstream primers, 1-2 μ L (about 100ng) measuring samples DNA, it is overall that distilled water supplies 20 μ L
Long-pending.2. PCR reaction condition: 94 DEG C of denaturations 5min；94 DEG C of sex change 30s, 55 DEG C (are moved back according to primer
Fire temperature adjusts) annealing 30s, 72 DEG C extend 1min30s, 35 circulations；72 DEG C extend 10min,
4 DEG C of preservations；
(3) fluorescence labeling PCR primer: 1. pretreatment: directly take 5 μ L PCR primer as template to nothing
In the 0.2ml centrifuge tube that bacterium is clean, in each sample cell, then add 3 μ L random primers, and moisturizing is extremely
19 μ L, vibration mixing, brief centrifugation；Sample cell after being centrifuged again puts into PCR instrument, 95 DEG C of sex change 3min,
Ice bath 3min immediately, brief centrifugation；2. fluorescence labeling: take a new 0.2ml centrifuge tube and be placed on ice,
The most inwardly add 2.5 μ L5 × Klenow Buffer, 1 μ L Klenow enzyme, 2.5 μ L2.5mM dNTPs；
Then fluorescence labeling reaction system Mix of 6 μ L is joined in the sample cell of pretreatment, vibration mixing,
Brief centrifugation；Finally sample cell is put in PCR instrument, first 37 DEG C of reaction 90min, then 72 DEG C of reactions
10min, 4 DEG C of preservations；
(4) hybridization: 1. preparing hybrid system: melt hybridization buffer at 42 DEG C, take the 0.2 of an aseptic
Ml centrifuge tube, the most inwardly adds 5 μ L and hybridizes buffer, 15 μ L fluorescent markers, be placed in the most standby；
2. hybridization: open chip hybridization box, kept flat by hybridizing box on desktop, adds about in hybridization and bottom groove
80 μ L sterilized waters, to prevent hybridization solution in crossover process from volatilizing in a large number；By chip front side upward (label towards
Operator) put between two alignment pins of hybridizing box；Put chip cover plate and (have the one of boss facing to core
Sheet), upper end elder generation contact chip, more slowly lid under；It is slowly injected into 20 μ L by cover glass well with pipettor
Hybridization solution after sex change, hybridization solution can be by surface tension of liquid boss below cover glass and chip surface
Between form one liquid film, vibrations cover plate or chip avoid damage to liquid film；The cover plate of hybridizing box is buckled,
Guarantee in the dowel hole that the alignment pin at frid two ends inserts cover plate two ends respectively.After buckling cover plate, respectively will
Two metal clips stick into both sides, it is to note that action wants mild, does not allow hybridizing box have big vibrations, to prevent
Hybridization solution spills dot matrix region；It addition, metal clip needs to stick into completely, to guarantee the sealing of hybridizing box；
Hybridizing box is put into rich difficult to understand BioMixerTM II hybridization instrument, 42 DEG C, reaction 2h or overnight；
(5) wash-out: useChip is carried out by SlibeWasherTM8 chip cleaning device.1. two
Plant the configuration of eluent: eluent I is 2 × sodium citrate salting liquid and 0.2% sodium dodecyl sulfate solution
Mixture, eluent II is 0.2 × sodium citrate salting liquid: 2. elution action: at 42 DEG C, first with washing
De-liquid I cleans twice, each 120s；Then clean three times with eluent II, each 80s, 1500rpm
Centrifugal 1min dries；
(6) scanning chip, analysis result: use scanner scanning chip, export image.Use analysis soft
Part quantifies the signal value of each point.Aggregation point array information and scanning information thus clear and definite testing result；
(7) testing result of the present embodiment method shows, genetic chip only gyrB to proteus mirabilis
Genetic test goes out stronger signal value, false negative situation does not occur, and the best.
5. use genechip detection streptococcus dysgalactiae
Detection method shows with embodiment 4, testing result scan image, and genetic chip is only to streptococcus dysgalactiae
GyrB genetic test go out stronger signal value, false negative situation does not occur, and the best.
6. use genechip detection Streptococcusagalactiae
Detection method shows with embodiment 4, testing result scan image, and genetic chip is only to Streptococcusagalactiae
GyrB genetic test go out stronger signal value, false negative situation does not occur；And it is the best.
7. use genechip detection MRSE
Detection method shows with embodiment 4, testing result scan image, and genetic chip is only to epidermis grape ball
The gyrB genetic test of bacterium goes out stronger signal value, false negative situation does not occur, and the best.
8. use genechip detection streptococcus uberis
Detection method shows with embodiment 4, testing result scan image, and genetic chip is only to streptococcus uberis
GyrB genetic test go out stronger signal value, false negative situation does not occur, and the best.
9. use genechip detection Klebsiella Pneumoniae
Detection method shows with embodiment 4, testing result scan image, and genetic chip is only to kerekou pneumonia primary
The gyrB genetic test of bacterium goes out stronger signal value, false negative situation does not occur, and the best.
10. use genechip detection Pseudomonas aeruginosa
Detection method shows with embodiment 4, testing result scan image, and genetic chip is only to Pseudomonas aeruginosa
GyrB genetic test goes out stronger signal value, false negative situation does not occur, and the best.
11. use genechip detection staphylococcus aureus
Detection method shows with embodiment 4, testing result scan image, and genetic chip is only to golden yellow grape
The gyrB genetic test of coccus goes out stronger signal value, false negative situation does not occur, and the best.
12. use genechip detection Escherichia coli
Detection method shows with embodiment 4, testing result scan image, and genetic chip is only to colibacillary
GyrB genetic test goes out stronger signal value, false negative situation does not occur, and the best.
13. use genechip detection salmonella typhi
Detection method shows with embodiment 4, testing result scan image, and genetic chip is only to Salmonella typhimurium
The gyrB genetic test of bacterium goes out stronger signal value, false negative situation does not occur, and the best.
The specific detection that the present embodiment genetic chip is carried out, including unrelated bacterium test, standard bacteria test and
Two strain standard bacteria tests, as shown in Figures 1 to 3, its testing result is as follows:
(1) genetic chip specific detection unrelated bacterium test: unrelated bacterium be A-streptococcus pneumonia 5-D,
B-proteus vulgaris 1527, C-MRSH CN4, as shown in Figure 1: 1 is PCR amplification knot
Really, 2 is gene chip hybridization result, and PCR amplification is when expanding tri-kinds of bacteriums of A, B and C, and primer is special
The opposite sex is good, and equal amixia signal between PCR primer and genetic chip, and testing result shows chip
Specific good.
(2) genetic chip specific detection standard bacteria test: to ten kinds of pathogenic bacteria: proteus mirabilis,
Streptococcus dysgalactiae, Streptococcusagalactiae, MRSE, streptococcus uberis, Klebsiella Pneumoniae, green pus
Bacillus, staphylococcus aureus, Escherichia coli and salmonella typhi be the most single, and to carry out genetic chip special
Property detection, as shown in Figure 2, A figure is PCR amplification, and B figure is gene chip hybridization result, result
Display: equal amixia signal between PCR primer and genetic chip, testing result shows all without there is false negative
Situation is the best.
(3) genetic chip specific detection two strain standard bacteria test: to proteus mirabilis type strain and
The hybridization of MRSE type strain carries out the specific detection of genetic chip, as shown in Figure 3,2,3 row:
Proteus mirabilis hybridization signal, 8,9 row: MRSE hybridization signal, 1,22 row: Quality Control is visited
Pin, result shows: positive findings has hybridization signal clearly, the best, and flux is high.
The sensitivity technique of 14. genetic chips
(1) extraction of sample nucleic acid to be detected: extract the gene of Streptococcusagalactiae according to CTAB/NaCl method
DNA is as template for group, then accurately measures Streptococcusagalactiae nucleic acid concentration with Qubit2.0, dense according to nucleic acid
Degree calculates contained genome copy numbers in 1 μ l sample, is 1 × 10 by copy number gradient dilution sample5、1
×104、5×103、1×103、5×102、1×102、5×101With 1 × 101Standby.
(2) PCR amplification body: 1. common or multiplexed PCR amplification system: 2 μ L 10 × PCR reaction
Buffer, 0.4 μ L10mM dNTPs, 1U Taq DNA Polymerase, 0.5 μ L 10 μMs upstream is drawn
Thing and 10 μMs of downstream primers of 0.5 μ L, 1-2 μ L (about 100ng) each gradient sample DNA, distilled water is mended
Foot is to 20 μ L cumulative volumes.2. PCR reaction condition: 94 DEG C of denaturations 5min；94 DEG C of sex change 30s, 55 DEG C
(adjusting according to primer annealing temperature) annealing 30s, 72 DEG C extend 1min30s, 35 circulations；72℃
Extend 10min, 4 DEG C of preservations.
(3) fluorescence labeling PCR primer: 1. pretreatment: directly take 5 μ L PCR primer as template to nothing
In the 0.2ml centrifuge tube that bacterium is clean, in each sample cell, then add 3 μ L random primers, and moisturizing is extremely
19 μ L, vibration mixing, brief centrifugation；Sample cell after being centrifuged again puts into PCR instrument, 95 DEG C of sex change 3min,
Ice bath 3min immediately, brief centrifugation；2. fluorescence labeling: take a new 0.2ml centrifuge tube and be placed on ice,
The most inwardly add 2.5 μ L5 × Klenow Buffer, 1 μ L Klenow enzyme, 2.5 μ L 2.5mM dNTPs；
Then fluorescence labeling reaction system Mix of 6 μ L is joined in the sample cell of pretreatment, vibration mixing,
Brief centrifugation；Finally sample cell is put in PCR instrument, first 37 DEG C of reaction 90min, then 72 DEG C of reactions
10min, 4 DEG C of preservations.
(4) hybridization: 1. preparing hybrid system: melt hybridization buffer at 42 DEG C, take the 0.2 of an aseptic
Ml centrifuge tube, the most inwardly adds 5 μ L and hybridizes buffer, 15 μ L fluorescent markers, be placed in the most standby；
2. hybridization: open chip hybridization box, kept flat by hybridizing box on desktop, adds about in hybridization and bottom groove
80 μ L sterilized waters, to prevent hybridization solution in crossover process from volatilizing in a large number；By chip front side upward (label towards
Operator) put between two alignment pins of hybridizing box；Put chip cover plate and (have the one of boss facing to core
Sheet), upper end elder generation contact chip, more slowly lid under；It is slowly injected into 20 μ L by cover glass well with pipettor
Hybridization solution after sex change, hybridization solution can be by surface tension of liquid boss below cover glass and chip surface
Between form one liquid film, vibrations cover plate or chip avoid damage to liquid film；The cover plate of hybridizing box is buckled,
Guarantee in the dowel hole that the alignment pin at frid two ends inserts cover plate two ends respectively.After buckling cover plate, respectively will
Two metal clips stick into both sides, it is to note that action wants mild, does not allow hybridizing box have big vibrations, to prevent
Hybridization solution spills dot matrix region；It addition, metal clip needs to stick into completely, to guarantee the sealing of hybridizing box；
Hybridizing box is put into rich difficult to understand BioMixerTM II hybridization instrument, 42 DEG C, reaction 2h or overnight.
(5) wash-out: useChip is carried out by SlibeWasherTM8 chip cleaning device.1. two
Plant the configuration of eluent: eluent I is 2 × sodium citrate salting liquid and 0.2% sodium dodecyl sulfate solution
Mixture, eluent II is 0.2 × sodium citrate salting liquid: 2. elution action: at 42 DEG C, first with washing
De-liquid I cleans twice, each 120s；Then clean three times with eluent II, each 80s, 1500rpm
Centrifugal 1min dries.
(6) scanning chip, analysis result: use scanner scanning chip, export image.Use analysis soft
Part quantifies the signal value of each point.Positive findings has hybridization signal clearly.
The present embodiment method is with Streptococcusagalactiae as representative strain, and the sensitivity to this genetic chip detects
Analyze, the gradient concentration of Streptococcusagalactiae complete genome DNA in test: 1-1 × 105, 2-1 × 104, 3-5
×103, 4-1 × 103, 5-5 × 102, 6-1 × 102, 7-5 × 101, 8-1 × 101, 9-NC, 1 × 105～1
×101Representing the genome copies of PCR amplification 1ul template, as shown in Figure 4, result shows: this gene
Chip is able to detect that as little as 5 × 102The pathogen nucleic acid of copy, therefore result shows the spirit of this genetic chip
Sensitivity is higher.Similarly, to other nine kinds of pathogenic bacteria proteus mirabilises, streptococcus dysgalactiae, epidermis grape
Coccus, streptococcus uberis, Klebsiella Pneumoniae, Pseudomonas aeruginosa, staphylococcus aureus, Escherichia coli,
Salmonella typhi uses this embodiment method, carries out the sensitivity technique of genetic chip, and result all shows this
The sensitivity of genetic chip is higher.
Claims (1)
1. can the genetic chip of ten kinds of Main Pathogenic Bacterias of parallel detection mastitis for milk cows, including chip carrier,
It is fixed with on chip carrier for detecting proteus mirabilis, streptococcus dysgalactiae, Streptococcusagalactiae, epidermis Portugal
Grape coccus, streptococcus uberis, Klebsiella Pneumoniae, Pseudomonas aeruginosa, staphylococcus aureus, Escherichia coli
Nucleotide probe with salmonella typhi, it is characterised in that:
For detecting the nucleotide probe of proteus mirabilis (pro), its sequence is SEQ ID NO:1；
For detecting the nucleotide probe of streptococcus dysgalactiae (sdy), its sequence is SEQ ID NO:2；
For detecting the nucleotide probe of Streptococcusagalactiae (sag), its sequence is SEQ ID NO:3；
For detecting the nucleotide probe of MRSE (sep), its sequence is SEQ ID NO:4；
For detecting the nucleotide probe of streptococcus uberis (supi), its sequence is SEQ ID NO:5；
For detecting the nucleotide probe of Klebsiella Pneumoniae (kpn), its sequence is SEQ ID NO:6；
For detecting the nucleotide probe of Pseudomonas aeruginosa (pae), its sequence is SEQ ID NO:7；
For detecting the nucleotide probe of staphylococcus aureus (sau), its sequence is SEQ ID NO:8；
For detecting the nucleotide probe of Escherichia coli (eco), its sequence is SEQ ID NO:9；
For detecting the nucleotide probe of salmonella typhi (sen), its sequence is SEQ ID NO:10；
Wherein, be used for detecting proteus mirabilis, streptococcus dysgalactiae, Streptococcusagalactiae, MRSE,
Streptococcus uberis, Klebsiella Pneumoniae, Pseudomonas aeruginosa, staphylococcus aureus and colibacillary probe are
Design from gyrB gene；
Design from gyrB and fliC gene for detecting salmonella typhi probe.
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| CN105331688B (en) * | 2015-10-22 | 2019-04-09 | 大连民族大学 | The five weight PCR detection methods and its detection kit of pathogenic bacteria in a kind of detection fresh and live agricultural product |
| CN105695584B (en) * | 2016-03-11 | 2019-03-05 | 集美大学 | The gyrB primer of test experience animal staphylococcus aureus combines |
| CN105950731A (en) * | 2016-05-25 | 2016-09-21 | 江南大学 | Method for analyzing and researching butanol tolerance key genes of Escherichia coli on basis of gene chips |
| CN107541567B (en) * | 2016-06-29 | 2021-02-02 | 博奥生物集团有限公司 | LAMP primer combination for detecting 8 environmental pathogens of cow mastitis and application thereof |
| CN107022629A (en) * | 2017-05-16 | 2017-08-08 | 广东海大畜牧兽医研究院有限公司 | A kind of multiple PCR detection primer group of mastitis for milk cows pathogen and its application |
| CN107898440A (en) * | 2017-11-07 | 2018-04-13 | 广西小草信息产业有限责任公司 | A kind of cow mammary gland monitors system and method |
| EP4130272A4 (en) * | 2020-03-31 | 2024-05-22 | Denka Company Limited | Primer set and probe for detecting klebsiella bacteria |
| CN112226526A (en) * | 2020-10-12 | 2021-01-15 | 河南省奶牛生产性能测定有限公司 | Milk cow mastitis Klebsiella pneumoniae detection kit and detection method |
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| CN101492739A (en) * | 2009-01-09 | 2009-07-29 | 华南农业大学 | Detection gene chip and method for main pathogen of bovine mastitis |
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| CN101492739A (en) * | 2009-01-09 | 2009-07-29 | 华南农业大学 | Detection gene chip and method for main pathogen of bovine mastitis |
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| Detection and identification of Escherichia coli,Shigella,and Salmonella by Microarrays Using the gyrB Gene;Kenichi Kakinuma et al;《Biotechnology and Bioengineering》;20030130;第83卷(第6期);摘要以及table1及其相关部分 * |
| Improved PCR for identification of Pseudomonas aeruginosa;Hyeon Jin Choi et al;《Appl Microbiol Biotechnol》;20131231;第97卷;全文 * |
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