CN103451310B - Gene chip capable of simultaneously detecting various vibrios and method for detecting vibrios - Google Patents

Abstract

The invention relates to a gene chip capable of simultaneously detecting various vibrios and a method for detecting the vibrios, and provides species-specific genes of four common pathogenic vibrios in the aquatic environment to encode an oligonucleotide probe sequence of an hsp60 gene and an oligonucleotide probe sequence of a toxR gene, wherein the four common pathogenic vibrios include vibrio alginolyticus, vibrio parahaemolyticus, vibrio vulnificus and vibrio haeveyi. On the basis of the base complementary and pairing principle, by combining the method with an optimized ordinary PCR and fluorescence labeled PCR reaction system, the aim of detection is achieved through oligonucleotide probes fixed to a solid-phase aldehyde chip. The invention provides the detecting method which is high in flux, specificity and sensitivity, rapid and efficient for the bottleneck of a current method for detecting pathogenic microorganisms.

Description

A kind of can the gene chip of parallel detection Vibrio and detection method thereof

Technical field

The invention belongs to technical field of microbial detection, be specifically related to a kind of can the gene chip of parallel detection Vibrio and detection method thereof.

Background technology

Method for microorganism detection has multiple, and more widely used at present have traditional bio-chemical characteristics method, immune antibody detection technique, PCR detection technique etc.Traditional bio-chemical characteristics needs the Isolation and Culture to sample to be tested, bacterial classification is screened by the feature of a series of loaded down with trivial details bio-chemical characteristics, result, although the method is a kind of conventional, classical discrimination method in Microbiological Lab, its process often needs time a couple of days, needs expend larger manpower and materials, test the phenotypic characteristic obtained may be affected by the external environment and make mistakes, for some atypia strains and close on that kind is more difficult to be differentiated according to phenotype.Particularly some are in the bacterium of " live can not cultivation conditions " (VBNC), traditional cultural method is difficult to cultivate, screen, and this can leave major hidden danger in aquaculture and field of food safety.

PCR detection technique carrys out a large amount of amplifying target genes fragment by designing specific primer, genes involved information according to bacterial classification to be detected identifies bacterial classification, highly sensitive and the quickness and high efficiency of round pcr, particularly along with the great-leap-forward development of Nucleic acid sequencing techniques, make expending of round pcr lower.But its sensitivity detected often is limited by the sensitivity of sepharose, and detect the limited in one's ability of great amount of samples simultaneously, on the basis of regular-PCR technology, derive again multiple PCR technique in recent years can address this problem to a certain extent, but still be limited to the gene groups number that can increase.

Immune antibody detection technique specificity and sensitivity higher, but often single can only check single culture, needs to prepare antiserum(antisera).

Biochip technology has been in progress rapidly since the nineties in last century, it acts on huge in functional genome research, along with the development in the fields such as materialogy, mechanical automation, genome sequencing, gene chip is made to have also been obtained increasing application in microorganism detection field.The ultimate principle of gene chip is the nucleotide fragments by synthetic goal gene, and point is formed on (as solid phase aldehyde radical chip) on certain carrier; Extract the complete genome DNA of sample to be tested, by mode amplifying target genes fragments such as PCR, and mark fluorescent substance in these gene fragments; It added on chip and hybridize under suitable conditions, the fragment that homology is high can be combined by base pair complementarity, through cleaning scanning, can occur fluorescent signal at corresponding some place.

Detection chip for microorganism detection field is also improved on the basis of original chip: the oligonucleotide probe of the synthetic of use, then in conjunction with other improve make its specificity and sensitivity high; Fluorescence labeling method mode is various, mostly uses the materials such as single base of fluorescein Cy3 mark or random primer, easy to operate, expend lower; Automatization, the intelligent level of chip point system, scanner are also improving gradually, significantly can shorten detection time, reduce personal errors; The raising that the high flux property of chip is embodied in chip print density (can reach every square centimeter at present and print 10 ⁴individual probe), this context of detection that is a large amount of, complex sample in environmental monitoring, food inspection is with the obvious advantage.

The genechip detection chip of vibrio alginolyticus of the present invention, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio vulnificus can realize parallel quick, high-throughput, high specific, high-sensitivity detection to complex samples, on the basis of these four kinds of pathogenic vibrios, it is also with good expansibility, and this monitoring and detection for briny environment pathogenic micro-organism has practical significance.

Summary of the invention

The object of this invention is to provide a kind of can parallel detection vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, the gene chip of Vibrio vulnificus and detection method thereof, thus make up the deficiencies in the prior art.

Existing gene chip usually utilizes the hsp60 gene of encoding heat shock proteins to come as bacterial strain Identification of Species target gene.This gene belongs to house-keeping gene, and it extensively exists and has species specificity difference in bacterium, is usually used as the target of Identification of Species, Phylogenetic analysis in current systematic bacteriology.But the qualification probe that house-keeping gene is set up can only identify the kind of pathogenic strains, in the culture environment of aquatic products of complexity, it is pathogenic also variant with or without, power different strains often in same bacterium, and therefore whether decorrelation virulence gene exists and have practical significance.And applicant finds in long-term research, the toxR gene of encode transmembrane transcriptional regulation protein can as the molecular target of qualification virulence gene, this gene has critical regulatory function for the generation of the toxin of pathogenic strains, also extensively exists in pathogenic strains.Therefore, the present invention is directed to vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio vulnificus these two kinds of genes choose specific fragment designing probe respectively, with ensure each bacterial classification probe only and corresponding bacterial classification produce specific signals.

Of the present invention a kind of can the gene chip of parallel detection Vibrio, include chip carrier, chip carrier be fixed with the nucleotide probe for detecting vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi and Vibrio vulnificus;

Described is design from the hsp60 gene and/or toxR gene of vibrio alginolyticus for detecting the probe of vibrio alginolyticus;

Described is design from the hsp60 gene and/or toxR gene of vibrio alginolyticus for detecting the probe of Vibrio parahaemolyticus;

Described is design from the toxR gene of vibrio alginolyticus for detecting the probe of Vibrio harveyi;

Described is design from the hsp60 gene of vibrio alginolyticus for detecting the probe of Vibrio vulnificus.

The described nucleotide probe for detecting vibrio alginolyticus (Vibrio alginolyticus), its sequence is SEQ ID NO:1 or SEQ ID NO:2.

The described nucleotide probe for detecting Vibrio parahaemolyticus (Vibrio parahaemolyticus), its sequence is SEQ ID NO:3 or SEQ ID NO:4.

The described nucleotide probe for detecting Vibrio vulnificus (Vibrio vulnificus), its sequence is SEQ ID NO:5.

The described nucleotide probe for detecting Vibrio harveyi (Vibrio haeveyi), its sequence is SEQ ID NO:6.

Table 1: the probe sequence information used by the present invention

Chip of the present invention is also fixed with the positive quality control probe of display probe coordinate position, its nucleotides sequence is classified as SEQ ID NO:7; And be classified as SEQ ID NO:8(table 2 with the nucleotides sequence of the positive quality control fragment of positive quality control probe hybridization).

Table 2: the sequence information of positive quality control probe and positive quality control fragment

5 ' end of probes all above all adds that amino is modified.

The preparation process of gene chip of the present invention is:

After nucleotide probe is diluted to proper concn, under suitable temperature, humidity condition, use some instrument point manufacturing probe processed on solid phase aldehyde radical chip; Then by probe and chip hydrated cross-linked; That wash-out goes not connect, connect untight probe, closed preparation.

Described step nucleotide probe is diluted to proper concn, is to use damping fluid to be diluted to concentration for 4-10 μm of ol/L, with 0.3M Na ₂hPO ₄solution (pH=8.5) carries out a system according to after the ratio mixing of 1:1.

The described some instrument point manufacturing probe processed that uses under suitable temperature, humidity condition is on solid phase aldehyde radical chip: point sample carries out under the condition of room temperature (20 DEG C), humidity 60%-70%.

Gene chip of the present invention is for detecting vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio vulnificus.

The detection method of gene chip of the present invention, comprises the following steps successively: the extraction of (1) sample nucleic acid to be detected; (2) common or multiplexed PCR amplification reaction; (3) the recovery purifying of PCR primer; (4) fluorescent mark PCR reacts; (5) prehybridization; (6) hybridize; (7) wash-out; (8) scanner scanning is used, analytical results.

The extraction of described step (1) sample nucleic acid to be detected: according to DNA extraction method or to use test kit to extract the complete genome DNA of sample to be detected for subsequent use as template.

Common or the multiplexed PCR amplification system of described step (2) is: 10 × PCR reaction buffer 2.5 μ L, 25mM MgCl ₂2 μ L, 2.5mM dNTPs mixture 4 μ L, Taq archaeal dna polymerase 2.5U, 10 μMs of forward primers and 10 μMs of reverse primer each 1 μ L, measuring samples DNA 1-2 μ L, distilled water supplies 25 μ L.And the condition of described PCR reaction is: 94 DEG C 7 minutes; 94 DEG C 45 seconds, 55 DEG C 45 seconds, 72 DEG C 30 seconds, 35 circulations; 72 DEG C 7 minutes, 4 DEG C of preservations.

The recovery purifying of described step (3) PCR primer: reclaim according to the operating process of the recovery purification kit of PCR primer.

Described step (4) fluorescent mark PCR system and step are: (1) pre-treatment: PCR reclaims purify DNA product 5 μ L, the random primer 0.75-1.5 μ L of 100 μMs of Cy3 marks, distilled water supplies 19 μ L, said mixture through 95 DEG C of sex change 3 minutes, is placed in cold shock on ice 3 minutes in PCR instrument; (2) fluorescent mark: add Klenow enzyme 1 μ L again, Klenow buffer2.5 μ L, 2.5mM dNTPs mixture 2.5 μ L, the condition of fluorescent mark PCR is: 37 DEG C 90 minutes, 72 DEG C 10 minutes.

Described step (5), (6) prehybridization and hybridization: the compound method of prehybridization mixture is deionized formamide 500mL, 50 × standard saline citrate 100mL, 50 × Denhardt ' s solution 100mL, 10% sodium lauryl sulphate 50mL, sterilizing distilled water is settled to 1000mL.100 μ g/mL sex change salmon sperm DNAs are added according to 1:10 ratio before using.The preparation of hybrid mixed liquid adds T 500 100g in the formula of prehybridization solution.100 μ g/mL sex change salmon sperm DNAs are added according to 1:10 ratio before using.And the adding proportion of fluorescent mark product described in the same step of hybridization solution (4) must approximate 1:1, separately need add 10 μMs of positive quality control 1 μ L.

The three kinds of elutriant preparations used in described step (7) wash-out and working method: elutriant I is 1 × Trisodium Citrate salts solution and 0.2% sodium dodecyl sulfate solution mixture; Elutriant II is 0.2 × Trisodium Citrate salts solution; Elutriant III is 0.1 × Trisodium Citrate salts solution.And must 60 DEG C of each elutriants of preheating before wash-out, 42 DEG C, use above-mentioned three kinds of elutriants concussion wash-out 15 minutes successively under the condition of 150rpm.

Described step (8) uses scanner scanning, analytical results: use scanner scanning to go out result, can check whether the point of synthesis has signal by naked eyes, and analysis software also can be used to quantize the signal value of each point.

The present invention explores through a series of optimization on the basis of traditional gene chip, the whole sample detection time was completed in 24 hours, if do not count the time of preparing gene chip, can complete detection in 12 hours; And probe specificity can reach 100%; Chip can reach the level of sensitivity identifying 3.5fg bacterium complete genome DNA (or 4 CFU/mL thalline), this exceeds PCR detection technique, immune antibody detection technique two orders of magnitude, and has reached the sensitivity highest level at microorganism detection other field in the research of domestic and international gene chip; The Standard Operating Procedure optimized avoids the impact of manual operation as far as possible, and stability is better.In general, the present invention can break through all drawbacks of existing microorganism detection technology, realize the detection that can realize high-throughput, high specific, highly sensitive, efficient quick to the pathogenic strains in briny environment, for disease monitoring early warning in fishery cultivating, fishery products, there is practical significance.

Accompanying drawing explanation

Fig. 1: the sensitivity curve of genechip detection vibrio alginolyticus complete genome DNA gradient concentration of the present invention;

Fig. 2: the sensitivity curve of genechip detection vibrio alginolyticus thalline gradient concentration of the present invention.

Embodiment

1. the design of probe

Gene chip of the present invention is used for detecting vibrio alginolyticus in fishery cultivating environment, fishery products, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio vulnificus.Its probe used all designs according to the hypervariable region sequence of hsp60 and the toxR gene of bacterial classification, ensures its specificity through BLAST comparison, probe sequence with the sequence alignment information of other proximal probes of publication in table 3.Use primer, probe to be synthesized by special company trade.

Table 3. the present invention apply for the sequence alignment information of probe with other proximal probes of publication

Note: proximal probes patent publication No.: 1. CN101475986A; 2. CN101475986A; 3.CN101967510A; 4. CN101967510A; 5. CN101113476A.

2. the structure of described gene chip:

(1) oligonucleotide probe of business synthesis is diluted to proper concn: using 1 × TE damping fluid to be diluted to concentration is 4-10 μm of ol/L, with 0.3M Na ₂hPO ₄solution (pH=8.5) is formed on solid-state aldehyde radical chip according to point after the ratio mixing of 1:1.At room temperature (20 DEG C), under the condition of humidity 60%-70%, use some instrument point manufacturing probe processed on aldehyde radical chip.

A kind of sample application array of table 4. gene chip probes of the present invention

SEQ ID NO.7

SEQ ID NO.1

SEQ ID NO.1

SEQ ID NO.1

SEQ ID NO.2

SEQ ID NO.2

SEQ ID NO.2

SEQ ID NO.7

SEQ ID NO.3

SEQ ID NO.3

SEQ ID NO.3

SEQ ID NO.4

SEQ ID NO.4

SEQ ID NO.4

SEQ ID NO.7

SEQ ID NO.5

SEQ ID NO.5

SEQ ID NO.5

SEQ ID NO.6

SEQ ID NO.6

SEQ ID NO.6

SEQ ID NO.7

SEQ ID NO.7

SEQ ID NO.7

SEQ ID NO.7

SEQ ID NO.7

SEQ ID NO.7

SEQ ID NO.7

(2) probe and chip hydrated cross-linked: point needs hydration in 37 DEG C of wet boxes to hybridize 12 hours after making.

(3) chip process after hydration: use distilled water to clean 3 times, each 2 minutes, that wash-out goes not connect, connect untight probe.Use 0.2% NaBH ₄close: make the complete submergence of chip, leave standstill after 5 minutes, 150 rpm shake 5 minutes, then leave standstill 5 minutes; Finally clean 3 times with distilled water, each 2 minutes.Centrifugal segregation chip surface water mark, parameter of noncentricity: 2000rpm, 2 minutes.4 DEG C keep in Dark Place for subsequent use.

3. the detection method of the gene chip described in

(1) extraction of sample nucleic acid to be detected: according to DNA extraction method or to use test kit to extract the complete genome DNA of sample to be detected (as seawater fish) for subsequent use as template.

(2) pcr amplification: common or multiplexed PCR amplification system is: 10 × PCR reaction buffer 2.5 μ L, 25mM MgCl ₂2 μ L, 2.5mM dNTPs mixture 4 μ L, Taq archaeal dna polymerase 2.5U, 10 μMs of forward primers and 10 μMs of reverse primer each 1 μ L, measuring samples DNA 1-2 μ L, distilled water supplies 25 μ L.And PCR reaction condition be: 94 DEG C 7 minutes; 94 DEG C 45 seconds, 55 DEG C 45 seconds, 72 DEG C 30 seconds, 35 circulations; 72 DEG C 7 minutes, 4 DEG C of preservations.

(3) the recovery purifying of PCR primer: the operating process reclaiming purification kit according to business-like PCR primer reclaims.

(4) fluorescent mark PCR:PCR reclaims purify DNA product 5 μ L, the random primer 0.75-1.5 μ L of 100 μMs of Cy3 marks, and distilled water supplies 19 μ L, and said mixture through 95 DEG C of sex change 3 minutes, is placed in cold shock on ice 3 minutes in PCR instrument; Add Klenow enzyme 1 μ L again, Klenow buffer 2.5 μ L, 2.5mM dNTPs mixture 2.5 μ L, PCR mark condition be: 37 DEG C 90 minutes, 72 DEG C 10 minutes.

(5) prehybridization: the compound method of prehybridization solution is deionized formamide 500mL, 50 × standard saline citrate 100mL, 50 × Denhardt ' s solution 100mL, 10% sodium lauryl sulphate 50mL, sterilizing distilled water is settled to 1000mL.100 μ g/mL sex change salmon sperm DNAs are added according to 1:10 ratio before using.According to 10 μ L/cm ²ratio add prehybridization mixture in the point sample district of the gene chip prepared, cover cover plate, be placed in 60 DEG C of wet box prehybridizations 1 hour.Then take out centrifugal (2000rpm, 2 minutes) for subsequent use.

(6) hybridize: the preparation of hybridization solution adds T 500 100g in the formula of prehybridization solution.100 μ g/mL sex change salmon sperm DNAs are added according to 1:10 ratio before using.Fluorescent mark product described in same for hybridization solution step (4) is mixed according to the ratio of approximate 1:1, separately need add 10 μMs of positive quality control 1 μ L.According to 10 μ L/cm ²ratio add hybrid mixed liquid in the point sample district of the good gene chip of prehybridization, cover cover plate, be placed in 60 DEG C of wet boxes hybridization 2 hours.

(7) wash-out: three kinds of elutriants of use carry out wash-out: elutriant I is 1 × Trisodium Citrate salts solution and 0.2% sodium dodecyl sulfate solution mixture; Elutriant II is 0.2 × Trisodium Citrate salts solution; Elutriant III is 0.1 × Trisodium Citrate salts solution.And must 60 DEG C of each elutriants of preheating before wash-out, 42 DEG C, use above-mentioned three kinds of elutriants concussion wash-out 15 minutes successively under the condition of 150rpm.Centrifuge dripping moisture (2000rpm, 2 minutes) subsequently.

(8) chip is scanned, analytical results: use scanner scanning chip, output image.Analysis software is used to quantize the signal value of each point.Positive findings has hybridization signal clearly.

4. use genechip detection vibrio alginolyticus

(1). the extraction of sample nucleic acid to be detected: according to DNA extraction method or to use test kit to extract the complete genome DNA of vibrio alginolyticus for subsequent use as template.

(2) .PCR amplification: common or multiplexed PCR amplification system is: 10 × PCR reaction buffer 2.5 μ L, 25mM MgCl ₂2 μ L, 2.5mM dNTPs mixture 4 μ L, Taq archaeal dna polymerase 2.5U, 10 μMs of forward primers and 10 μMs of reverse primer each 1 μ L, measuring samples DNA 1-2 μ L, distilled water supplies 25 μ L.And PCR reaction condition be: 94 DEG C 7 minutes; 94 DEG C 45 seconds, 55 DEG C 45 seconds, 72 DEG C 30 seconds, 35 circulations; 72 DEG C 7 minutes, 4 DEG C of preservations.

(3) the recovery purifying of .PCR product: the operating process reclaiming purification kit according to business-like PCR primer reclaims.

(4). fluorescent mark PCR:PCR reclaims purify DNA product 5 μ L, the random primer 0.75-1.5 μ L of 100 μMs of Cy3 marks, and distilled water supplies 19 μ L, and said mixture through 95 DEG C of sex change 3 minutes, is placed in cold shock on ice 3 minutes in PCR instrument; Add Klenow enzyme 1 μ L again, Klenow buffer 2.5 μ L, 2.5mM dNTPs mixture 2.5 μ L, PCR mark condition be: 37 DEG C 90 minutes, 72 DEG C 10 minutes.

(5). prehybridization: the compound method of prehybridization solution is deionized formamide 500mL, 50 × standard saline citrate 100mL, 50 × Denhardt ' s solution 100mL, 10% sodium lauryl sulphate 50mL, sterilizing distilled water is settled to 1000mL.100 μ g/mL sex change salmon sperm DNAs are added according to 1:10 ratio before using.According to 10 μ L/cm ²ratio add prehybridization mixture in the point sample district of the gene chip prepared, cover cover plate, be placed in 60 DEG C of wet box prehybridizations 1 hour.Then take out centrifugal (2000rpm, 2 minutes) for subsequent use.

(6). hybridization: the preparation of hybridization solution adds T 500 100g in the formula of prehybridization solution.100 μ g/mL sex change salmon sperm DNAs are added according to 1:10 ratio before using.Fluorescent mark product described in same for hybridization solution step (4) is mixed according to the ratio of approximate 1:1, separately need add 10 μMs of positive quality control 1 μ L.According to 10 μ L/cm ²ratio add hybrid mixed liquid in the point sample district of the good gene chip of prehybridization, cover cover plate, be placed in 60 DEG C of wet boxes hybridization 2 hours.

(7). wash-out: use three kinds of elutriants to carry out wash-out: elutriant I is 1 × Trisodium Citrate salts solution and 0.2% sodium dodecyl sulfate solution mixture; Elutriant II is 0.2 × Trisodium Citrate salts solution; Elutriant III is 0.1 × Trisodium Citrate salts solution.And each elutriant must 60 DEG C of preheatings before wash-out, 42 DEG C, use above-mentioned three kinds of elutriants concussion wash-out 15 minutes successively under the condition of 150rpm.Centrifuge dripping moisture (2000rpm, 2 minutes) subsequently.

(8). scanning chip, analytical results: use scanner scanning chip, output image.Use analysis software to quantize the signal value of each point, also with the naked eye can check whether the point of printing has signal.Aggregation point array information and scanning information thus determine detected result.

(9) detected result of the present embodiment method shows, gene chip only goes out stronger signal value to the hsp60 gene of vibrio alginolyticus and toxR gene test, does not occur false negative situation; Specificity is good, does not also occur non-specific signals at the probe place of other vibrios.

5. use genechip detection Vibrio parahaemolyticus

Detection method is with embodiment 4, and detected result scan image shows, gene chip only goes out stronger signal value to the hsp60 gene of Vibrio parahaemolyticus and toxR gene test, does not occur false negative situation; Specificity is good, does not also occur non-specific signals at the probe place of other vibrios.

6. use genechip detection Vibrio vulnificus

Detection method is with embodiment 4, and detected result shows, gene chip only goes out stronger signal value to the hsp60 gene test of Vibrio vulnificus, does not occur false negative situation; Specificity is good, does not also occur non-specific signals at the probe place of other vibrios.

7. use genechip detection Vibrio harveyi

Detection method is with embodiment 4, and detected result shows, gene chip only goes out stronger signal value to the toxR gene test of Vibrio harveyi, does not occur false negative situation; Specificity is good, does not also occur non-specific signals at the probe place of other vibrios.

8. the sensitivity technique of gene chip

(1). cell concentration dilutes: will be 4.0 × 10 through pure culture of spending the night, cell concentration ¹²the vibrio alginolyticus of CFU/mL in 1 × PBS solution, is diluted to 4.0 CFU/mL with 100 times of gradient dilutions successively, and arranges blank one group.

(2). the extraction of sample nucleic acid to be detected: get 1.5 mL bacterium liquid from each dilution group, according to DNA extraction method or to use test kit to extract the complete genome DNA of each concentration gradient vibrio alginolyticus for subsequent use as template; Simultaneously Example 4(1) in the complete genome DNA of vibrio alginolyticus, measure its complete genome DNA concentration (3.5 × 10 ⁸fg/ μ l), and with 100 times of gradient dilutions in 1 × TE solution, be diluted to 3.5fg/ μ l successively, the complete genome DNA of each weaker concn gradient vibrio alginolyticus is also for subsequent use as template.

(3) .PCR amplification: common or multiplexed PCR amplification system is: 10 × PCR reaction buffer 2.5 μ L, 25mM MgCl ₂2 μ L, 2.5mM dNTPs mixture 4 μ L, Taq archaeal dna polymerase 2.5U, 10 μMs of forward primers and 10 μMs of reverse primer each 1 μ L, measuring samples DNA 1-2 μ L, distilled water supplies 25 μ L.And PCR reaction condition be: 94 DEG C 7 minutes; 94 DEG C 45 seconds, 55 DEG C 45 seconds, 72 DEG C 30 seconds, 35 circulations; 72 DEG C 7 minutes, 4 DEG C of preservations.

(4) the recovery purifying of .PCR product: the operating process reclaiming purification kit according to business-like PCR primer reclaims.

(5). fluorescent mark PCR:PCR reclaims purify DNA product 5 μ L, the random primer 0.75-1.5 μ L of 100 μMs of Cy3 marks, and distilled water supplies 19 μ L, and said mixture through 95 DEG C of sex change 3 minutes, is placed in cold shock on ice 3 minutes in PCR instrument; Add Klenow enzyme 1 μ L again, Klenow buffer 2.5 μ L, 2.5mM dNTPs mixture 2.5 μ L, PCR mark condition be: 37 DEG C 90 minutes, 72 DEG C 10 minutes.

(6). prehybridization: the compound method of prehybridization solution is deionized formamide 500mL, 50 × standard saline citrate 100mL, 50 × Denhardt ' s solution 100mL, 10% sodium lauryl sulphate 50mL, sterilizing distilled water is settled to 1000mL.100 μ g/mL sex change salmon sperm DNAs are added according to 1:10 ratio before using.According to 10 μ L/cm ²ratio add prehybridization mixture in the point sample district of the gene chip prepared, cover cover plate, be placed in 60 DEG C of wet box prehybridizations 1 hour.Then take out centrifugal (2000rpm, 2 minutes) for subsequent use.

(7). hybridization: the preparation of hybridization solution adds T 500 100g in the formula of prehybridization solution.100 μ g/mL sex change salmon sperm DNAs are added according to 1:10 ratio before using.Fluorescent mark product described in same for hybridization solution step (4) is mixed according to the ratio of approximate 1:1, separately need add 10 μMs of positive quality control 1 μ L.According to 10 μ L/cm ²ratio add hybrid mixed liquid in the point sample district of the good gene chip of prehybridization, cover cover plate, be placed in 60 DEG C of wet boxes hybridization 2 hours.

(8). wash-out: use three kinds of elutriants to carry out wash-out: elutriant I is 1 × Trisodium Citrate salts solution and 0.2% sodium dodecyl sulfate solution mixture; Elutriant II is 0.2 × Trisodium Citrate salts solution; Elutriant III is 0.1 × Trisodium Citrate salts solution.And each elutriant must 60 DEG C of preheatings before wash-out, 42 DEG C, use above-mentioned three kinds of elutriants concussion wash-out 15 minutes successively under the condition of 150rpm.Centrifuge dripping moisture (2000rpm, 2 minutes) subsequently.

(9). scanning chip, analytical results: use scanner scanning chip, output image.Analysis software is used to quantize the signal value of each point.Aggregation point array information and scanning information thus determine detected result.

The detected result of the present embodiment method shows, described chip can detect the vibrio alginolyticus complete genome DNA (see accompanying drawing 1) of 3.5fg; In addition, when cell concentration is 4 CFU/mL, gene chip can detect higher signal value (see accompanying drawing 2).Illustrate that gene chip sensitivity of the present invention is higher.

9. use genechip detection Fermi operator and Vibrio splindidus

Detection method is with embodiment 4, and detected result scan image shows, gene chip is except hybridization signal appears in positive quality control point, and all do not occur hybridization signal at the probe place of other vibrios, this shows this gene chip.Probe can not produce false positive results to nearly edge vibrios.

Above-mentioned result shows, the detection vibrio alginolyticus that gene chip of the present invention can be sensitive, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio vulnificus.

Claims (3)

1. can the gene chip of parallel detection Vibrio, include chip carrier, it is characterized in that, chip carrier is fixed with the nucleotide probe for detecting vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi and Vibrio vulnificus; Described is design from the hsp60 gene of vibrio alginolyticus for detecting the probe of vibrio alginolyticus, and its sequence is SEQ ID NO:1;

Described is design from the hsp60 gene of vibrio alginolyticus for detecting the probe of Vibrio parahaemolyticus, and its sequence is SEQ ID NO:3;

Described is design from the toxR gene of vibrio alginolyticus for detecting the probe of Vibrio harveyi, and its sequence is SEQ ID NO:5;

Described is design from the hsp60 gene of vibrio alginolyticus for detecting the probe of Vibrio vulnificus, and its sequence is SEQ ID NO:6.

2. gene chip according to claim 1 is also fixed with the positive quality control probe of display probe coordinate position, and its nucleotides sequence is classified as SEQ ID NO:7; And be classified as SEQ ID NO:8 with the nucleotides sequence of the positive quality control fragment of positive quality control probe hybridization.

3. the preparation of gene chip according to claim 1, comprises the steps:

After nucleotide probe is diluted to proper concn, under suitable temperature, humidity condition, use some instrument point manufacturing probe processed on solid phase aldehyde radical chip; Then by probe and chip hydrated cross-linked; That wash-out goes not connect, connect untight probe, closed preparation;

Described step nucleotide probe is diluted to proper concn, is to use damping fluid to be diluted to concentration for 4-10 μm of ol/L, with 0.3M Na ₂hPO ₄solution; A system is carried out according to after the ratio mixing of 1:1;

The described some instrument point manufacturing probe processed that uses under suitable temperature, humidity condition is on solid phase aldehyde radical chip: point sample carries out under the condition of room temperature (20 DEG C), humidity 60%-70%.