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 parallel detection of various vibrio and a detection method thereof. Provides an oligonucleotide probe for the species-specific gene of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi and Vibrio vulnificus, the species-specific gene encoding the heat shock protein hsp60 gene. Needle sequence, the oligonucleotide probe sequence of the virulence gene toxR. The detection method is based on the principle of complementary base pairing, combined with optimized common PCR and fluorescent labeling PCR reaction systems, and achieves the purpose of detection through oligonucleotide probes immobilized on solid-phase aldehyde-based chips. Aiming at the bottleneck of the current detection method for pathogenic microorganisms, the present invention proposes a high-throughput, high-specificity, high-sensitivity, fast and efficient detection method.

Description

A gene chip capable of parallel detection of multiple vibrio species and its detection method

technical field

The invention belongs to the technical field of microorganism detection, and in particular relates to a gene chip capable of parallel detection of various Vibrio and a detection method thereof.

Background technique

There are many methods for microbial detection. At present, traditional physiological and biochemical experimental methods, immune antibody detection technology, PCR detection technology, etc. are widely used. Traditional physiological and biochemical experiments need to isolate and cultivate the strains of the samples to be tested, and identify the strains through a series of tedious characteristics and results of physiological and biochemical experiments. Although this method is a commonly used and classic identification method in microbiology laboratories, However, the process often takes several days, requires a lot of manpower and material resources, and the phenotypic characteristics obtained by the experiment may be affected by the external environment. It is difficult to distinguish some atypical strains and adjacent species according to the phenotype. Especially for some bacteria in the "viable non-culturable state" (VBNC), traditional culture methods are difficult to cultivate and identify, which will leave major hidden dangers in the fields of aquaculture and food safety.

PCR detection technology is to amplify a large number of target gene fragments by designing specific primers, and identify the strains according to the relevant gene information of the strains to be detected. PCR technology is highly sensitive, fast and efficient, especially with the leapfrogging of nucleic acid sequencing technology Development, making the cost of PCR technology lower. However, the sensitivity of its detection is often limited by the sensitivity of agarose gel, and the ability to detect a large number of samples at the same time is limited. In recent years, on the basis of ordinary PCR technology, multiplex PCR technology has been derived to solve this problem to a certain extent, but still Limited by the number of gene species that can be amplified.

The specificity and sensitivity of immune antibody detection technology are high, but often only a single bacterial species can be tested at a time, and antiserum needs to be prepared.

Gene chip technology has developed rapidly since the 1990s, and it has played a huge role in functional genome research. With the continuous development of materials science, mechanical automation, whole genome sequencing and other fields, gene chips have also gained more and more attention in the field of microbial detection. more applications. The basic principle of the gene chip is to artificially synthesize the nucleotide fragments of the target gene and place them on a certain carrier (such as a solid-phase aldehyde-based chip); extract the whole genome DNA of the sample to be tested, and amplify the target gene by PCR or other methods fragments, and label fluorescent substances on these gene fragments; add them to the chip and hybridize under appropriate conditions, fragments with high homology will combine through complementary base pairing, and after cleaning and scanning, they will appear at the corresponding points fluorescent signal.

The detection chip used in the field of microbial detection has also been improved on the basis of the original chip: the artificially synthesized oligonucleotide probe used, combined with other improvements, makes its specificity and sensitivity extremely high; there are various fluorescent labeling methods, most of which are The use of fluorescein Cy3-labeled single bases or random primers and other substances is easy to operate and low in cost; the automation and intelligence of chip spotting and scanning equipment are also gradually improving, which can greatly shorten the detection time and reduce human error; chip The high throughput is reflected in the improvement of chip printing density (currently it can print 10 ⁴ probes per square centimeter), which has obvious advantages in the detection of a large number of complex samples in environmental monitoring and food testing.

The gene chip detection chip of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi and Vibrio vulnificus of the present invention can realize parallel fast, high-throughput, high-specificity and high-sensitivity detection of complex samples, here On the basis of the four pathogenic Vibrio, it also has good scalability, which has practical significance for the monitoring and detection of pathogenic microorganisms in seawater environments.

Contents of the invention

The object of the present invention is to provide a gene chip capable of parallel detection of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi and Vibrio vulnificus and a detection method thereof, so as to make up for the deficiencies in the prior art.

Existing gene chips often use the hsp60 gene encoding heat shock protein as the target gene for strain identification. This gene belongs to the housekeeping gene, which exists widely in bacteria and has species-specific differences. It is usually used as a target for species identification and phylogenetic analysis in current bacterial taxonomy. However, the identification probe established by the housekeeping gene can only identify the type of pathogenic strain. In a complex aquaculture environment, different strains of the same bacteria often have different pathogenicity and strength. Therefore, it is necessary to understand the relevant virulence. The presence or absence of genes has practical implications. However, the applicant has found in long-term research that the toxR gene encoding a transmembrane transcriptional regulatory protein can be used as a molecular target for identifying virulence genes. This gene has a key regulatory function for the production of toxins in pathogenic strains. are also widely present. Therefore, the present invention selects specific fragments to design probes for the two genes of Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, and Vibrio vulnificus, so as to ensure that the probes of each bacterial species are only compatible with the corresponding bacteria. produce a specific signal.

A gene chip capable of parallel detection of multiple Vibrios of the present invention includes a chip carrier on which nuclei for detecting Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi and Vibrio vulnificus are fixed. nucleotide probe;

The probe for detecting Vibrio alginolyticus is designed from the hsp60 gene and/or toxR gene of Vibrio alginolyticus;

The probe for detecting Vibrio parahaemolyticus is designed from the hsp60 gene and/or toxR gene of Vibrio alginolyticus;

The probe for detecting Vibrio harveyi is designed from the toxR gene of Vibrio alginolyticus;

The probe for detecting Vibrio vulnificus is designed from the hsp60 gene of Vibrio alginolyticus.

The sequence of the nucleotide probe for detecting Vibrio alginolyticus is SEQ ID NO: 1 or SEQ ID NO: 2.

The sequence of the nucleotide probe for detecting Vibrio parahaemolyticus is SEQ ID NO: 3 or SEQ ID NO: 4.

The sequence of the nucleotide probe for detecting Vibrio vulnificus is SEQ ID NO:5.

The sequence of the nucleotide probe for detecting Vibrio haeveyi is SEQ ID NO:6.

Table 1: Probe sequence information used in the present invention

The chip of the present invention is also fixed with a positive quality control probe showing the coordinate position of the probe, its nucleotide sequence is SEQ ID NO: 7; and the nucleotide sequence of the positive quality control fragment hybridized with the positive quality control probe is SEQ ID NO: 8 (Table 2).

Table 2: Sequence information of positive quality control probes and positive quality control fragments

The 5' ends of all the above probes were modified with amino groups.

The preparation steps of the gene chip of the present invention are:

After diluting the nucleotide probes to an appropriate concentration, spot the probes on the solid-phase aldehyde-based chip using a spotting instrument under appropriate temperature and humidity conditions; then hydrate and cross-link the probes with the chip; Ligated, loosely ligated probes, blocked to complete the preparation.

The step of diluting the nucleotide probe to an appropriate concentration is to use a buffer to dilute to a concentration of 4-10 μ mol/L, and mix it with 0.3M Na ₂ HPO ₄ solution (pH=8.5) at a ratio of 1:1 Do point system.

Said method of spotting the probes on the solid-phase aldehyde-based chip using a spotting apparatus under appropriate temperature and humidity conditions: Spotting is carried out at room temperature (20° C.) and humidity 60%-70%.

The gene chip of the invention is used to detect Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi and Vibrio vulnificus.

The detection method of the gene chip described in the present invention comprises the following steps in turn: (1) extraction of nucleic acid from the sample to be detected; (2) ordinary or multiplex PCR amplification reaction; (3) recovery and purification of PCR products; (4) fluorescence Mark PCR reaction; (5) Pre-hybridization; (6) Hybridization; (7) Elution; (8) Use a scanner to scan and analyze the results.

The step (1) extraction of nucleic acid of the sample to be tested: extract the whole genome DNA of the sample to be tested according to the method of DNA extraction or use a kit as a template for future use.

The ordinary or multiplex PCR amplification system in the step (2) is: 10×PCR reaction buffer 2.5 μL, 25mM MgCl ₂ 2 μL, 2.5mM dNTPs mixture 4 μL, Taq DNA polymerase 2.5U, 10 μM forward primer and 10 μM reverse 1 μL of each primer, 1-2 μL of DNA of the sample to be tested, and 25 μL of double distilled water. And the conditions of the PCR reaction are: 94°C for 7 minutes; 94°C for 45 seconds, 55°C for 45 seconds, 72°C for 30 seconds, 35 cycles; 72°C for 7 minutes, and storage at 4°C.

The recovery and purification of the PCR product in the step (3): recover according to the operating procedure of the PCR product recovery and purification kit.

The step (4) fluorescent labeling PCR system and steps are: (1) Pretreatment: 5 μL of purified DNA product recovered by PCR, 0.75-1.5 μL of 100 μM Cy3-labeled random primer, 19 μL of double distilled water, and the above mixture was passed through the PCR instrument Denaturation at 95°C for 3 minutes, chilled on ice for 3 minutes; (2) Fluorescence labeling: add 1 μL of Klenow enzyme, 2.5 μL of Klenow buffer, 2.5 μL of 2.5mM dNTPs mixture, and the conditions for fluorescent labeling PCR are: 37°C for 90 minutes , 72°C for 10 minutes.

The steps (5), (6) pre-hybridization and hybridization: the preparation method of the pre-hybridization mixture is deionized formamide 500mL, 50× standard citrate solution 100mL, 50× Denhardt’s solution 100mL, 10% dodecyl Sodium sulfate 50mL, dilute to 1000mL with sterilized double distilled water. Add 100 μg/mL denatured salmon sperm DNA at a ratio of 1:10 before use. The preparation of the hybridization mixture is to add 100 g of dextran sulfate to the formula of the pre-hybridization solution. Add 100 μg/mL denatured salmon sperm DNA at a ratio of 1:10 before use. And the addition ratio of the hybridization solution to the fluorescently labeled product described in step (4) must be approximately 1:1, and 1 μL of 10 μM positive quality control must be added.

The preparation and operation method of the three eluents used in the step (7) elution: the eluent I is a mixture of 1× sodium citrate solution and 0.2% sodium dodecyl sulfate solution; the eluent II is 0.2×sodium citrate solution; eluent III is 0.1×sodium citrate solution. Before elution, each eluent must be preheated at 60°C, and the above three eluents should be shaken and eluted for 15 minutes at 42°C and 150 rpm.

The step (8) uses a scanner to scan and analyze the results: use the scanner to scan the results, and check whether the synthesized points have signals with the naked eye, or use analysis software to quantify the signal values of each point.

The present invention has gone through a series of optimization explorations on the basis of the traditional gene chip, so that the entire sample detection time can be completed within 24 hours, and the detection can be completed within 12 hours if the time for preparing the gene chip is not counted; and the specificity of the probe can reach 100%; the chip can reach the sensitivity level of identifying 3.5fg bacterial whole genome DNA (or 4 CFU/mL bacteria), which is two orders of magnitude higher than PCR detection technology and immune antibody detection technology, and has reached the level of gene chip research at home and abroad. The highest level of sensitivity in other areas of microbial detection; the optimized standard operating procedures avoid the influence of human operations as much as possible, and the stability is better. Generally speaking, the present invention can break through various drawbacks of existing microbial detection technologies, and realize high-throughput, high-specificity, high-sensitivity, efficient and fast detection of pathogenic strains in seawater environments, and is useful for fish farming and aquatic products. Disease monitoring and early warning has practical significance.

Description of drawings

Figure 1: The sensitivity curve of the gene chip of the present invention to detect the gradient concentration of Vibrio alginolyticus whole genome DNA;

Fig. 2: The sensitivity curve of detecting the gradient concentration of Vibrio alginolyticus by the gene chip of the present invention.

Detailed ways

1. Probe Design

The gene chip of the invention is used to detect vibrio alginolyticus, vibrio parahaemolyticus, vibrio harveyi and vibrio vulnificus in fishery breeding environment and aquatic products. The probes used are all designed according to the hypervariable region sequences of the hsp60 and toxR genes of the strain, and their specificity is guaranteed by BLAST comparison. table 3. The primers and probes used were commercially synthesized by specialized companies.

Table 3. Sequence comparison information of the probes applied for in the present invention and similar probes of other published patents

Note: Approximate probe patent publication numbers: 1. CN101475986A; 2. CN101475986A; 3. CN101967510A; 4. CN101967510A; 5. CN101113476A.

2. The construction of the gene chip:

(1) Dilute the commercially synthesized oligonucleotide probe to an appropriate concentration: use 1×TE buffer to dilute to a concentration of 4-10 μmol/L, and mix with 0.3M Na ₂ HPO ₄ solution (pH=8.5) according to 1: A ratio of 1 was mixed and spotted on a solid aldehyde-based chip. At room temperature (20°C) and humidity 60%-70%, use a spotting device to spot probes on the aldehyde-based chip.

Table 4. A kind of sample array of gene chip probe 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 hydration cross-linking: After spotting, it needs to be hydrated and hybridized in a humid chamber at 37°C for 12 hours.

(3) Chip treatment after hydration: wash 3 times with double distilled water, 2 minutes each time, to elute unconnected and loosely connected probes. Seal with 0.2% NaBH ₄ : completely submerge the chip, let it stand for 5 minutes, shake it at 150 rpm for 5 minutes, and let it stand for 5 minutes; finally wash it with double distilled water 3 times, 2 minutes each time. Centrifuge to remove water traces on the surface of the chip, centrifugation parameters: 2000rpm, 2 minutes. Store at 4°C in the dark for later use.

3. The detection method of the gene chip

(1) Extraction of nucleic acid from the sample to be tested: follow the DNA extraction method or use a kit to extract the whole genome DNA of the sample to be tested (such as seawater fish) as a template for later use.

(2) PCR amplification: common or multiplex PCR amplification system: 10×PCR reaction buffer 2.5 μL, 25mM MgCl ₂ 2 μL, 2.5mM dNTPs mixture 4 μL, Taq DNA polymerase 2.5U, 10 μM forward primer and 10 μM reverse 1 μL of each primer, 1-2 μL of DNA of the sample to be tested, and 25 μL of double distilled water. And the conditions of the PCR reaction are: 94°C for 7 minutes; 94°C for 45 seconds, 55°C for 45 seconds, 72°C for 30 seconds, 35 cycles; 72°C for 7 minutes, and storage at 4°C.

(3) Recovery and purification of PCR products: recover according to the operating procedures of commercialized PCR product recovery and purification kits.

(4) Fluorescence-labeled PCR: 5 μL of purified DNA products recovered by PCR, 0.75-1.5 μL of 100 μM Cy3-labeled random primers, and 19 μL of double-distilled water. The above mixture was denatured in a PCR instrument at 95 ° C for 3 minutes, and placed on ice for 3 minutes. Minutes; then add 1 μL of Klenow enzyme, 2.5 μL of Klenow buffer, and 2.5 μL of 2.5mM dNTPs mixture. The conditions for PCR labeling are: 37°C for 90 minutes, 72°C for 10 minutes.

(5) Pre-hybridization: The preparation method of the pre-hybridization solution is 500 mL of deionized formamide, 100 mL of 50× standard citrate solution, 100 mL of 50× Denhardt’s solution, 50 mL of 10% sodium dodecyl sulfate, and sterile double distilled water. Capacity to 1000mL. Add 100 μg/mL denatured salmon sperm DNA at a ratio of 1:10 before use. Add the pre-hybridization mixture to the spotting area of the prepared gene chip according to the ratio of 10 μL/cm ² , cover with a cover slip, and place in a 60°C humid box for 1 hour for pre-hybridization. Then take it out and centrifuge (2000rpm, 2 minutes) for later use.

(6) Hybridization: The preparation of the hybridization solution is to add 100 g of dextran sulfate to the formula of the pre-hybridization solution. Add 100 μg/mL denatured salmon sperm DNA at a ratio of 1:10 before use. Mix the hybridization solution with the fluorescently labeled product described in step (4) at a ratio of approximately 1:1, and add 1 μL of 10 μM positive quality control. According to the ratio of 10 μL/cm ² , add the hybridization mixture to the spotting area of the pre-hybridized gene chip, cover with a cover slip, and place it in a humid chamber at 60°C for 2 hours for hybridization.

(7) Elution: use three eluents for elution: eluent Ⅰ is a mixture of 1× sodium citrate salt solution and 0.2% sodium dodecyl sulfate solution; eluent Ⅱ is 0.2× citric acid Sodium salt solution; eluent III is 0.1× sodium citrate solution. Before elution, each eluent must be preheated at 60°C, and the above three eluents should be shaken and eluted for 15 minutes at 42°C and 150 rpm. Then centrifuge to dry the water (2000rpm, 2 minutes).

(8) Scan the chip and analyze the results: Use a scanner to scan the chip and output the image. Quantify the signal value at each point using analysis software. Positive results have clear hybridization signals.

4. Detection of Vibrio alginolyticus using gene chips

(1). Extraction of nucleic acid from the sample to be tested: follow the DNA extraction method or use a kit to extract the whole genome DNA of Vibrio alginolyticus as a template for future use.

(2).PCR amplification: Common or multiplex PCR amplification system is: 10×PCR reaction buffer 2.5μL, 25mM MgCl ₂ 2μL, 2.5mM dNTPs mixture 4μL, Taq DNA polymerase 2.5U, 10μM forward primer and 10μM reverse primer Add 1 μL of each primer, 1-2 μL of sample DNA to be tested, and make up 25 μL with double distilled water. And the conditions of the PCR reaction are: 94°C for 7 minutes; 35 cycles of 94°C for 45 seconds, 55°C for 45 seconds, 72°C for 30 seconds; 72°C for 7 minutes, and storage at 4°C.

(3). Recovery and purification of PCR products: recover according to the operating procedure of a commercialized PCR product recovery and purification kit.

(4). Fluorescence-labeled PCR: 5 μL of purified DNA product recovered by PCR, 0.75-1.5 μL of 100 μM Cy3-labeled random primers, and 19 μL of double distilled water. The above mixture was denatured at 95 ° C for 3 minutes in a PCR instrument, and placed on ice for cooling 3 minutes; then add Klenow enzyme 1μL, Klenow buffer 2.5μL, 2.5mM dNTPs mixture 2.5μL, PCR labeling conditions: 37°C for 90 minutes, 72°C for 10 minutes.

(5). Pre-hybridization: The preparation method of the pre-hybridization solution is 500 mL of deionized formamide, 100 mL of 50× standard citrate solution, 100 mL of 50× Denhardt’s solution, 50 mL of 10% sodium dodecyl sulfate, and sterilized double distilled water. Dilute to 1000mL. Add 100 μg/mL denatured salmon sperm DNA at a ratio of 1:10 before use. Add the pre-hybridization mixture to the sample area of the prepared gene chip according to the ratio of 10 μL/cm ² , cover with a cover slip, and place it in a 60°C humid box for 1 hour for pre-hybridization. Then take it out and centrifuge (2000rpm, 2 minutes) for later use.

(6). Hybridization: The preparation of the hybridization solution is to add 100 g of dextran sulfate to the formula of the pre-hybridization solution. Add 100 μg/mL denatured salmon sperm DNA at a ratio of 1:10 before use. Mix the hybridization solution with the fluorescently labeled product described in step (4) at a ratio of approximately 1:1, and add 1 μL of 10 μM positive quality control. According to the ratio of 10 μL/cm ² , add the hybridization mixture to the spotting area of the pre-hybridized gene chip, cover with a cover slip, and place it in a humid chamber at 60°C for 2 hours for hybridization.

(7). Elution: Use three eluents for elution: eluent Ⅰ is a mixture of 1× sodium citrate salt solution and 0.2% sodium dodecyl sulfate solution; eluent Ⅱ is 0.2× citric acid Sodium salt solution; eluent III is 0.1× sodium citrate solution. Each eluent must be preheated at 60°C before elution, and the above three eluents should be shaken and eluted for 15 minutes at 42°C and 150 rpm. Then centrifuge to dry the water (2000rpm, 2 minutes).

(8). Scan the chip and analyze the results: use the scanner to scan the chip and output the image. Use the analysis software to quantify the signal value of each point, and also use the naked eye to check whether there is a signal in the printed point. Summarize point array information and scan information to determine detection results.

(9) The detection results of the method of this embodiment show that the gene chip only detects stronger signal values for the hsp60 gene and the toxR gene of Vibrio alginolyticus, and no false negative situation occurs; There was also no non-specific signal at the needle.

5. Detection of Vibrio parahaemolyticus using gene chip

The detection method is the same as in Example 4, and the scanning image of the detection results shows that the gene chip only detects strong signal values for the hsp60 gene and the toxR gene of Vibrio parahaemolyticus, and no false negatives occur; There was also no non-specific signal at the probe.

6. Detection of Vibrio vulnificus using gene chips

The detection method is the same as in Example 4, and the detection results show that the gene chip only detects a strong signal value for the hsp60 gene of Vibrio vulnificus, and no false negative situation occurs; the specificity is good, and it does not appear at the probes of other Vibrio nonspecific signal.

7. Detection of Vibrio harveyi using gene chips

The detection method is the same as in Example 4, and the detection results show that the gene chip only detects a strong signal value to the toxR gene of Vibrio harveyi, and no false negative situation occurs; No nonspecific signal appeared.

8. Sensitivity detection of gene chip

(1). Dilution of bacterial cell concentration: Dilute Vibrio alginolyticus with a cell concentration of 4.0×10 ¹² CFU/mL after overnight pure culture in 1×PBS solution in a 100-fold gradient, and then dilute to 4.0 CFU/mL sequentially. And set a blank control group.

(2). Extraction of nucleic acid from the sample to be tested: Take 1.5 mL of bacterial liquid from each dilution group, and extract the whole genome DNA of Vibrio alginolyticus with various concentration gradients according to the DNA extraction method or using a kit as a template for later use; For the whole genome DNA of Vibrio alginolyticus in Example 4 (1), measure the concentration of the whole genome DNA (3.5×10 ⁸ fg/μl), and dilute it in 1×TE solution in a 100-fold gradient, and successively dilute to 3.5 fg/μl μl, the whole genome DNA of Vibrio alginolyticus in each dilution concentration gradient is also used as a template for future use.

(3).PCR amplification: Common or multiplex PCR amplification system is: 10×PCR reaction buffer 2.5μL, 25mM MgCl ₂ 2μL, 2.5mM dNTPs mixture 4μL, Taq DNA polymerase 2.5U, 10μM forward primer and 10μM reverse primer Add 1 μL of each primer, 1-2 μL of sample DNA to be tested, and make up 25 μL with double distilled water. And the conditions of the PCR reaction are: 94°C for 7 minutes; 35 cycles of 94°C for 45 seconds, 55°C for 45 seconds, 72°C for 30 seconds; 72°C for 7 minutes, and storage at 4°C.

(4). Recovery and purification of PCR products: recover according to the operating procedure of a commercialized PCR product recovery and purification kit.

(5). Fluorescence-labeled PCR: 5 μL of purified DNA product recovered by PCR, 0.75-1.5 μL of 100 μM Cy3-labeled random primer, 19 μL of double-distilled water, and the above mixture was denatured at 95 ° C for 3 minutes in a PCR instrument, and placed on ice for cooling 3 minutes; then add Klenow enzyme 1μL, Klenow buffer 2.5μL, 2.5mM dNTPs mixture 2.5μL, PCR labeling conditions: 37°C for 90 minutes, 72°C for 10 minutes.

(6). Pre-hybridization: The preparation method of the pre-hybridization solution is 500 mL of deionized formamide, 100 mL of 50×standard citrate solution, 100 mL of 50×Denhardt’s solution, 50 mL of 10% sodium dodecyl sulfate, and sterilized double distilled water. Dilute to 1000mL. Add 100 μg/mL denatured salmon sperm DNA at a ratio of 1:10 before use. Add the pre-hybridization mixture to the sample area of the prepared gene chip according to the ratio of 10 μL/cm ² , cover with a cover slip, and place it in a 60°C humid box for 1 hour for pre-hybridization. Then take it out and centrifuge (2000rpm, 2 minutes) for later use.

(7). Hybridization: The preparation of the hybridization solution is to add 100 g of dextran sulfate to the formula of the pre-hybridization solution. Add 100 μg/mL denatured salmon sperm DNA at a ratio of 1:10 before use. Mix the hybridization solution with the fluorescently labeled product described in step (4) at a ratio of approximately 1:1, and add 1 μL of 10 μM positive quality control. According to the ratio of 10 μL/cm ² , add the hybridization mixture to the spotting area of the pre-hybridized gene chip, cover with a cover slip, and place it in a humid chamber at 60°C for 2 hours for hybridization.

(8). Elution: Use three eluents for elution: eluent Ⅰ is a mixture of 1× sodium citrate salt solution and 0.2% sodium dodecyl sulfate solution; eluent Ⅱ is 0.2× citric acid Sodium salt solution; eluent III is 0.1× sodium citrate solution. Each eluent must be preheated at 60°C before elution, and the above three eluents should be shaken and eluted for 15 minutes at 42°C and 150 rpm. Then centrifuge to dry the water (2000rpm, 2 minutes).

(9). Scan the chip and analyze the results: use the scanner to scan the chip and output the image. Quantify the signal value at each point using analysis software. Summarize point array information and scan information to determine detection results.

The detection result of the method of this embodiment shows that the chip can detect 3.5fg of Vibrio alginolyticus whole genome DNA (see accompanying drawing 1); High signal values (see Figure 2). It shows that the gene chip of the present invention has high sensitivity.

9. Detection of Vibrio fischeri and Vibrio resplendents using gene chips

The detection method is the same as that in Example 4, and the scanned images of the detection results show that, except for the hybridization signal at the positive quality control point of the gene chip, no hybridization signal appears at the probes of other Vibrio, which indicates that the gene chip has a hybridization signal. The probe will not produce false-positive results for Vibrio spp.

The above results show that the gene chip of the present invention can detect Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi and Vibrio vulnificus sensitively.

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%.