CN104328167B - Can the genetic chip of ten kinds of Main Pathogenic Bacterias of parallel detection mastitis for milk cows and detection method - Google Patents

Abstract

The invention discloses a gene chip and a detection method capable of parallel detection of ten main pathogenic bacteria of dairy cow mastitis. Nucleotide probes for Streptococcus lactis, Staphylococcus epidermidis, Streptococcus uberis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Salmonella typhi, also immobilized positive controls, hybridization positive controls and Hybridization negative control. The gene chip designed multiple probes for different sites of gyrB and fliC, effectively avoiding the difference in the nucleic acid sequence of different subtypes of bacteria and the missed detection caused by a few base mutations in the genome of the bacteria, and realizing the detection of dairy cow udder The high-throughput, high-specificity, high-sensitivity, efficient and rapid detection of ten major pathogenic bacteria in mastitis has practical significance for the monitoring and early warning of pathogenic bacteria in dairy cow breeding and dairy cow mastitis.

Description

Gene chip and detection method for parallel detection of ten main pathogenic bacteria of dairy cow mastitis

Technical field:

The invention belongs to the technical field of microbial detection, and in particular relates to a gene chip capable of parallel detection of ten main pathogenic bacteria of cow mastitis and a detection method.

Background technique:

Gene chip technology (Gene chip) is a high-tech biotechnology for gene analysis developed in the early 1990s. It is to place the artificially synthesized nucleotide fragment (probe) of the target gene on a certain carrier, and then Extract the whole genome DNA of the microorganism to be tested, amplify the target gene fragments by PCR, and mark the fluorescent substances on these gene fragments, and hybridize them with the probes on the chip under appropriate conditions, and the fragments with high homology After hybridization and complementary pairing, after cleaning and scanning, fluorescent signals will be displayed at the corresponding matrix probe sites. The target fragments detected by the gene chip can be PCR products, genomic DNA, total RNA, cDNA, plasmid DNA or oligonucleotides, etc. (Schena et al, 2000). Currently reported detection methods are mainly based on multiplex PCR combined with gene chip method and universal primer combined with gene chip method. The application of gene chip technology in microbial identification has the characteristics of strong specificity and high sensitivity, especially when dealing with a large number of complex samples, the advantages are more obvious, convenient and efficient, low cost, and small error. Its outstanding features are integration, miniaturization, automation, high-throughput, etc. It can accurately identify a variety of target genes in high-throughput and parallelization, and can be used for identification of modern microbial strains, determination of virulence factors, and drug resistance. Provide a complete technical platform for detection (Call et al, 2001). Therefore, it is necessary to establish a high-throughput, rapid and accurate monitoring system for a variety of infectious pathogenic bacteria using high-tech molecular biology techniques in a short period of time to ensure correct diagnosis such as cow mastitis in a short period of time. Infectious diseases caused by various pathogenic bacteria in order to improve the prevention, diagnosis and control capabilities of multi-infectious pathogenic bacteria in my country.

Cow mastitis pathogenic bacteria have wide infection ability and transmission routes. At the same time, due to the large-scale use of antibiotics and the problem of drug residues, people are often helpless in the face of high incidence and mass outbreaks of dairy cow mastitis. A variety of pathogenic bacteria pass through sporadic, Mass disease has caused great losses to the dairy industry, which in turn has a great impact on people's consumption of dairy products, and has brought immeasurable losses on the normal quality of life of consumers and the sustainable development of the social economy. It is imminent to realize rapid parallel detection of the main pathogenic bacteria of pneumonia. There are many types of pathogenic bacteria that cause mastitis in dairy cows. Currently, there are more than 220 known pathogenic bacteria, of which more than 20 are common, and 90% of the pathogenic bacteria of cow mastitis are mainly Escherichia coli and Staphylococcus aureus. , Staphylococcus epidermidis, Klebsiella pneumoniae, Proteus mirabilis, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus agalactiae, Salmonella typhi, Pseudomonas aeruginosa, Shigella, Bacillus cereus, etc. In the prior art, there are researches on the detection gene chip and detection method of the main pathogenic bacteria of dairy cow mastitis, such as the invention patent CN 101492739A discloses a detection gene chip and detection method of the main pathogenic bacteria of dairy cow mastitis, which is for the Research on gene chip detection methods for six main pathogenic bacteria of cow mastitis (Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and Staphylococcus aureus). Including a solid phase carrier and specific detection probes immobilized on the solid phase carrier, the specific detection probes include 6 main pathogenic factors of cow mastitis among the universal primer sequences designed based on the Escherichia coli 16S rDNA conservative region The specific oligonucleotide hybridization probe designed in the variable region of the bacteria, and the degradation temperature of the PCR reaction is limited, and the conditions for hybridization of the nucleic acid fragment amplified by PCR and the gene chip are optimized, and a good hybridization effect is obtained. Accurate detection results are finally obtained. This method uses the 16S rDNA conserved region as the base sequence to design universal primers and use it as the target molecule identified by the bacterial detection box. And a small number of base mutations in the strain genome will cause missed detection.

Invention content:

The purpose of the present invention is to overcome the deficiencies of the prior art, to provide a method for parallel detection of ten main pathogenic bacteria Proteus mirabilis, Streptococcus dysgalactiae, Streptococcus agalactiae, Staphylococcus epidermidis, Streptococcus uberis, pneumonia Gene chips and detection methods for Klebsiella, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Salmonella typhi realize parallel, rapid, high-throughput, high-specificity, and high-sensitivity detection of complex samples.

The existing gene chip of the main pathogenic bacteria of dairy cow mastitis uses the conserved region of 16S rRNA as the base sequence to design universal primers and use it as the target molecule identified by the bacterial detection box. However, the resolution of 16S rRNA is not enough for bacteria with close relatives After long-term research, the applicant found that gyrB was used as the target gene for bacterial identification and classification. The gyrB gene is a gene encoding the B subunit of DNA helicase. The 16srRNA gene can better distinguish similar species, and can meet the requirements of Proteus mirabilis, Streptococcus dysgalactiae, Streptococcus agalactiae, Staphylococcus epidermidis, Streptococcus uberis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli Identification requirements for nine species of bacteria. In addition, due to the confusion of the current nomenclature of Salmonella, through the conservative analysis of 16s rRNA and gyrB sequences, the design of probes based on the two gene sequences may only identify Salmonella, but it cannot be guaranteed that the specificity of the designed probes is only for Salmonella typhimurium. After extensive literature research, the applicant adopted fliC as the target gene for identifying Salmonella typhi. Therefore, the present invention selects specific probes respectively according to the two genes of gyrB and fliC for the above-mentioned ten kinds of common cow mastitis pathogenic bacteria, so as to ensure that the bacterial species corresponding to the sum of the probes of each bacterial species produces a specific signal, and at the same time targets Multiple probes were designed for the gyrB and fliC genes of the ten kinds of bacteria, which effectively avoided the missed detection phenomenon caused by the difference in nucleic acid sequences of different subtypes of bacteria and the mutation of a few bases in the genome of the bacteria .

The present invention is realized by taking the following technical solutions:

A gene chip that can detect ten main pathogenic bacteria of cow mastitis in parallel, including a chip carrier, on which are immobilized a gene chip for detecting Proteus mirabilis, Streptococcus dysgalactiae, Streptococcus agalactiae, Staphylococcus epidermidis, breast Nucleotide probes for Streptococcus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Salmonella typhi.

The probes for detecting Proteus mirabilis, Streptococcus dysgalactiae, Streptococcus agalactiae, Staphylococcus epidermidis, Streptococcus uberis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli are obtained from designed in the gyrB gene.

The said probe for detecting Salmonella typhi is designed from gyrB and fliC genes.

The sequence of the nucleotide probe for detecting Proteus mirabilis (pro) is SEQ ID NO:1.

The sequence of the nucleotide probe for detecting Streptococcus dysgalactiae (sdy) is SEQ ID NO:2.

The sequence of the nucleotide probe for detecting Streptococcus agalactiae (sag) is SEQ ID NO:3.

The sequence of the nucleotide probe for detecting Staphylococcus epidermidis (sep) is SEQ ID NO:4.

The sequence of the nucleotide probe for detecting Streptococcus uberis (supi) is SEQ ID NO:5.

The sequence of the nucleotide probe for detecting Klebsiella pneumoniae (kpn) is SEQ ID NO:6.

The sequence of the nucleotide probe for detecting Pseudomonas aeruginosa (pae) is SEQ ID NO:7.

The sequence of the nucleotide probe for detecting Staphylococcus aureus (sau) is SEQ ID NO:8.

The sequence of the nucleotide probe for detecting Escherichia coli (eco) is SEQ ID NO:9.

The sequence of the nucleotide probe for detecting Salmonella typhi (sen) is SEQ ID NO:10.

Table 1: Probe sequence information table used in the present invention

Also fixed on the chip of the present invention are fixed positive control (HEX), hybridization positive control (PC) and hybridization negative control (NC), and its nucleotide sequence is SEQ ID NO:11, SEQ ID NO:12 and SEQID NO :13.

The positive quality control fragment hybridized with the positive quality control probe is hybrid PC-TAMRA, and its nucleotide sequence is SEQ ID NO:14. (The sequence information of quality control probes and positive quality control fragments is shown in Table 2).

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

The gene chip preparation steps of the present invention are:

After diluting the nucleotide probe to an appropriate concentration, on the Crystal Core PersonalArrayer 16 Personal Spotting Instrument (CapitalBio), the 384-well spotting plate, glass slide, needle rack, dot matrix, sample and cleaning parameters were respectively carried out according to the actual situation. Set up, run the crystal core PersonalArrayer 16 personal spotter to spot the probe on the solid-phase amino chip; then use Boao crystal core LuxScan10K scanner to detect the chip spotting situation.

Dilute the nucleotide probes in the above steps to an appropriate concentration, using sterile water to dissolve each probe to 40 μM, and mix it with the crystal core ^@ gene chip sample solution at a ratio of 1:1, so that the final concentration of each probe is 20 μM; Then, the probes were sequentially added to the 384-well sample plate according to the designed probe arrangement table.

The detection method of the gene chip that can parallelly detect ten kinds of main pathogenic bacteria of dairy cow mastitis described in the present invention, comprises the following steps successively:

(1) Extraction of the nucleic acid of the sample to be tested: the bacteria were isolated from the collected cow mastitis milk sample; then the genomic DNA of the bacteria was extracted by the CTAB/NaCl method as a template for subsequent use;

(2) Common or multiplex PCR amplification reaction: Common or multiplex PCR amplification system is: 2 μL 10×PCR reaction buffer, 0.4 μL 10 mM dNTPs, 1 U Taq DNA polymerase, 0.5 μL 10 μM upstream primer and 0.5 μL 10 μM downstream primer , 1-2 μL (about 100ng) of the DNA of the sample to be tested was made up to a total volume of 20 μL with double distilled water. And the conditions of the PCR reaction are: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30s, annealing at 55°C (adjusted according to the primer annealing temperature) for 30s, extension at 72°C for 1min and 30s, 35 cycles; extension at 72°C for 10 minutes, and storage at 4°C;

(3) Fluorescence-labeled PCR reaction: ① Pretreatment: Take 5 μL of the PCR product directly as a template into a sterile and clean 0.2ml centrifuge tube, then add 3 μL of random primers to each sample tube, replenish water to 19 μL, shake and mix well , and centrifuged instantaneously; then put the centrifuged sample tube into the PCR instrument, denature at 95°C for 3 minutes, immediately bathe in ice for 3 minutes, and centrifuge instantaneously; ②Fluorescence labeling: take a new 0.2ml centrifuge tube on ice, and place Add 2.5μL 5×Klenow Buffer, 1μL Klenow Enzyme, 2.5μL 2.5mM dNTPs; then add 6μL of fluorescent labeling reaction system Mix into the pretreated sample tube, shake and mix, and centrifuge briefly; finally put the sample tube into PCR In the instrument, first react at 37°C for 90 minutes, then react at 72°C for 10 minutes, and store at 4°C;

(4) Hybridization: ① Prepare the hybridization system: Melt the hybridization buffer at 42°C, take a sterile and clean 0.2ml centrifuge tube, add 5 μL of hybridization buffer and 15 μL of fluorescent markers in sequence, and put it on ice for later use; ② Hybridization: Open the chip hybridization box, put the hybridization box flat on the table, add about 80 μL sterile water to the hybridization and the bottom groove to prevent a large amount of volatilization of the hybridization solution during the hybridization process; put the chip face up (the label faces the operator) Put it between the two positioning pins in the hybridization box; put the cover slip on the chip (the side with the boss facing the chip), the upper end first touches the chip, and then slowly cover it; use a pipette to slowly add the sample hole through the cover glass Inject 20 μL of denatured hybridization solution, the hybridization solution will form a liquid film between the boss under the cover glass and the surface of the chip by virtue of the surface tension of the liquid, do not shake the cover slip or chip to avoid damage to the liquid film; put the cover plate of the hybridization box Fasten it, and make sure that the positioning pins at both ends of the slot plate are respectively inserted into the positioning pin holes at both ends of the cover plate; vibration to prevent the hybridization solution from splashing out of the array area; in addition, the metal clip needs to be fully snapped in to ensure the airtightness of the hybridization box; put the hybridization box into the BioMixerTMⅡ hybridization instrument at 42°C for 2 hours or overnight.

(5) Elution: use The SlibeWasherTM8 chip cleaning instrument cleans the chip: ①The configuration of two eluents: the eluent Ⅰ is a mixture of 2× sodium citrate salt solution and 0.2% sodium dodecyl sulfate solution, and the eluent Ⅱ is 0.2× Sodium citrate solution: ② Elution operation: at 42°C, first wash twice with eluent I, 120s each time; then wash three times with eluent II, 80s each time, and centrifuge at 1500rpm for 1min to dry;

(6) Use a scanner to detect and analyze the results: Use a scanner to scan out the results, and use analysis software to quantify the signal value of each point.

The present invention has gone through a series of optimization studies on the basis of the existing gene chip, and the whole sample detection time can be completed within 6 hours without counting the time for preparing the gene chip, and because multiple Probes, so the gene chip can effectively avoid the missed detection of a single probe in the identification of different subtypes of microorganisms on the one hand, and on the other hand can effectively reduce the missed detection of a single probe in the identification of microorganisms caused by gene mutations; optimized The standard operating procedure avoids the influence of human operation as far as possible, and the stability is better. In general, the present invention can break through various drawbacks of existing microbial detection technologies, and realize high-throughput, high-specificity, high-sensitivity, efficient and rapid detection of ten main pathogenic bacteria in dairy cow mastitis, and is useful for dairy cow breeding, dairy cow udder The monitoring and early warning of pathogenic bacteria in inflammation has practical significance.

Description of drawings:

Fig. 1 is the result figure of gene chip specific detection of the present invention-unrelated bacteria test;

Fig. 2 is the result figure of gene chip specific detection-standard bacteria test of the present invention;

Fig. 3 is the result figure of gene chip specific detection of the present invention-two strains of standard bacteria test;

Fig. 4 is a graph showing the results of sensitivity detection of the gene chip of the present invention-Streptococcus agalactiae.

detailed description:

The present invention will be described in further detail below in conjunction with specific embodiments and accompanying drawings.

1. Probe Design

The gene chip of the present invention is used to detect ten kinds of common pathogenic bacteria in milk samples of cow mastitis disease, including Proteus mirabilis, Streptococcus dysgalactiae, Streptococcus agalactiae, Staphylococcus epidermidis, Streptococcus uberis, Klebsiella pneumoniae bacteria, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Salmonella typhi. The probes used were designed according to the gyrB and fliC gene sequences of the strains, and their specificity was guaranteed by BLAST comparison. See Table 3 for the sequence information of the probes and primers used. The probes and primers used are synthesized by specialized companies.

Table 3: Sequence information of probes and primers used in the present invention

2. The construction of the gene chip:

(1) Dilute the commercially synthesized oligonucleotide probes to an appropriate concentration: use sterile water to dissolve each probe to 40 μM, and mix it with the crystal core ^@ gene chip sample solution at a ratio of 1:1 to make each probe final The concentration is 20 μM. Then, according to the actual situation, set the 384-well spotting plate, glass slide, needle rack, dot matrix, sample and cleaning parameters respectively, and spot the probe on the solid-phase amino chip by running the crystal core PersonalArrayer 16 personal spotting instrument. A spotting array of the gene chip probe constructed above is shown in Table 4.

Table 4: A spotting array of the above gene chip probes

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 twenty one eco_1 eco_2 eco_3 eco_4 eco_5 eco_6 twenty two HEX NC NC PC PC PC NC HEX

Note: HEX is spot quality control probe; NC is hybridization negative quality control probe; PC is hybridization positive quality control probe.

(2) Hydration and cross-linking of probes and chips: After spotting, water and hybridization in a humid chamber at 37°C are required for 12 hours.

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

3. The detection method of the gene chip

(1) Extraction of the nucleic acid of the sample to be tested: first, isolate the bacteria from the collected cow mastitis milk sample; then use the CTAB/NaCl method to extract the genomic DNA of the gained bacteria as a template for subsequent use;

(2) PCR amplification: ①Common or multiplex PCR amplification system: 2μL 10×PCR reaction buffer, 0.4μL 10mM dNTPs, 1U Taq DNA Polymerase, 0.5μL 10μM upstream primer and 0.5μL 10μM downstream primer, 1-2μL (about 100ng ) DNA of the sample to be tested, made up to a total volume of 20 μL with double distilled water. ②PCR reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C (adjusted according to the primer annealing temperature) for 30 s, extension at 72°C for 1 min 30 s, 35 cycles; extension at 72°C for 10 min, and storage at 4°C;

(3) Fluorescence-labeled PCR product: ① Pretreatment: Take 5 μL of the PCR product directly as a template into a sterile and clean 0.2ml centrifuge tube, then add 3 μL of random primers to each sample tube, replenish water to 19 μL, shake and mix well , and centrifuged instantaneously; then put the centrifuged sample tube into the PCR instrument, denature at 95°C for 3 minutes, immediately bathe in ice for 3 minutes, and centrifuge instantaneously; ②Fluorescence labeling: take a new 0.2ml centrifuge tube on ice, and place Add 2.5μL 5×Klenow Buffer, 1μL Klenow Enzyme, 2.5μL 2.5mM dNTPs; then add 6μL of fluorescent labeling reaction system Mix into the pretreated sample tube, shake and mix, and centrifuge briefly; finally put the sample tube into PCR In the instrument, first react at 37°C for 90 minutes, then react at 72°C for 10 minutes, and store at 4°C;

(4) Hybridization: ① Preparation of hybridization system: Melt the hybridization buffer at 42°C, take a sterile and clean 0.2ml centrifuge tube, add 5 μL of hybridization buffer and 15 μL of fluorescent markers in sequence, and put it on ice for later use; ② Hybridization: Open the chip hybridization box, put the hybridization box flat on the table, add about 80 μL sterile water to the hybridization and the bottom groove to prevent a large amount of volatilization of the hybridization solution during the hybridization process; put the chip face up (label facing the operator) into the Between the two positioning pins in the hybridization box; put the chip cover (the side with the boss facing the chip), the upper end first touches the chip, and then slowly cover it; use a pipette to slowly inject 20 μL through the sample hole of the cover glass After denaturing the hybridization solution, the hybridization solution will form a liquid film between the boss under the cover glass and the surface of the chip by virtue of the surface tension of the liquid. Do not shake the cover slip or chip to avoid damaging the liquid film; buckle the cover of the hybridization box , make sure that the positioning pins at both ends of the slot plate are respectively inserted into the positioning pin holes at both ends of the cover plate; after buckling the cover plate, snap the two metal clips into both sides respectively. Note: the action should be gentle, and do not let the hybrid box have a large vibration , to prevent the hybridization solution from splashing out of the dot matrix area; in addition, the metal clip needs to be completely stuck in to ensure the airtightness of the hybridization box; put the hybridization box into the BioMixerTMⅡ hybridization instrument at 42°C, and react for 2 hours or overnight;

(5) Elution: use SlibeWasherTM8 chip cleaning instrument cleans the chip. ① Configuration of two eluents: eluent Ⅰ is a mixture of 2× sodium citrate solution and 0.2% sodium dodecyl sulfate solution, and eluent Ⅱ is 0.2× sodium citrate solution: ② Elution Operation: at 42°C, wash twice with eluent I, each time for 120s; then wash three times with eluent II, each time for 80s, centrifuge at 1500rpm for 1min to dry;

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

4. Detection of Proteus mirabilis using gene chips

(1) Extraction of nucleic acid from the sample to be tested: extract the genomic DNA of Proteus mirabilis according to the CTAB/NaCl method as a template for subsequent use;

(2) PCR amplification: ①Common or multiplex PCR amplification system: 2μL 10×PCR reaction buffer, 0.4μL 10mM dNTPs, 1U Taq DNA Polymerase, 0.5μL 10μM upstream primer and 0.5μL 10μM downstream primer, 1-2μL (about 100ng ) DNA of the sample to be tested, made up to a total volume of 20 μL with double distilled water. ②PCR reaction conditions: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C (adjusted according to the primer annealing temperature) for 30 seconds, extension at 72°C for 1 minute and 30 seconds, 35 cycles; extension at 72°C for 10 minutes, and storage at 4°C;

(3) Fluorescence-labeled PCR product: ① Pretreatment: Take 5 μL of the PCR product directly as a template into a sterile and clean 0.2ml centrifuge tube, then add 3 μL of random primers to each sample tube, replenish water to 19 μL, shake and mix well , and centrifuged instantaneously; then put the centrifuged sample tube into the PCR instrument, denature at 95°C for 3 minutes, immediately bathe in ice for 3 minutes, and centrifuge instantaneously; ②Fluorescence labeling: take a new 0.2ml centrifuge tube on ice, and place Add 2.5μL 5×Klenow Buffer, 1μL Klenow Enzyme, 2.5μL 2.5mM dNTPs; then add 6μL of fluorescent labeling reaction system Mix into the pretreated sample tube, shake and mix, and centrifuge briefly; finally put the sample tube into PCR In the instrument, first react at 37°C for 90 minutes, then react at 72°C for 10 minutes, and store at 4°C;

(4) Hybridization: ① Preparation of hybridization system: Melt the hybridization buffer at 42°C, take a sterile and clean 0.2ml centrifuge tube, add 5 μL of hybridization buffer and 15 μL of fluorescent markers in sequence, and put it on ice for later use; ② Hybridization: Open the chip hybridization box, put the hybridization box flat on the table, add about 80 μL sterile water to the hybridization and the bottom groove to prevent a large amount of volatilization of the hybridization solution during the hybridization process; put the chip face up (label facing the operator) into the Between the two positioning pins in the hybridization box; put the chip cover (the side with the boss facing the chip), the upper end first touches the chip, and then slowly cover it; use a pipette to slowly inject 20 μL through the sample hole of the cover glass After denaturing the hybridization solution, the hybridization solution will form a liquid film between the boss under the cover glass and the surface of the chip by virtue of the surface tension of the liquid. Do not shake the cover slip or chip to avoid damaging the liquid film; buckle the cover of the hybridization box , make sure that the positioning pins at both ends of the slot plate are respectively inserted into the positioning pin holes at both ends of the cover plate. After fastening the cover, snap the two metal clips into the two sides respectively. Note: the action should be gentle and do not let the hybridization box have a large vibration, so as to prevent the hybridization liquid from splashing out of the dot matrix area; in addition, the metal clips need to be completely snapped in , to ensure the tightness of the hybridization box; put the hybridization box into the BioMixerTMⅡ hybridization instrument at 42°C, and react for 2 hours or overnight;

(5) Elution: use SlibeWasherTM8 chip cleaning instrument cleans the chip. ① Configuration of two eluents: eluent Ⅰ is a mixture of 2× sodium citrate solution and 0.2% sodium dodecyl sulfate solution, and eluent Ⅱ is 0.2× sodium citrate solution: ② Elution Operation: at 42°C, wash twice with eluent I, each time for 120s; then wash three times with eluent II, each time for 80s, centrifuge at 1500rpm for 1min to dry;

(6) Scan the chip and analyze the result: use a scanner to scan the chip and output an image. Quantify the signal value at each point using analysis software. Summarize the point array information and scan information to clarify the detection results;

(7) The detection results of the method in this embodiment show that the gene chip only detected a strong signal value for the gyrB gene of Proteus mirabilis, no false negatives occurred, and the specificity was good.

5. Detection of Streptococcus dysgalactiae using gene chip

The detection method is the same as that in Example 4. The scanned images of the detection results show that the gene chip only detects a strong signal value for the gyrB gene of Streptococcus dysgalactiae, and no false negatives occur, and the specificity is good.

6. Detection of Streptococcus agalactiae using gene chip

The detection method is the same as that in Example 4, and the scanned images of the detection results show that the gene chip only detects a strong signal value for the gyrB gene of Streptococcus agalactiae, and no false negative occurs; and the specificity is good.

7. Detection of Staphylococcus epidermidis using gene chips

The detection method is the same as that in Example 4. The scanned images of the detection results show that the gene chip only detects a strong signal value for the gyrB gene of Staphylococcus epidermidis, no false negatives occur, and the specificity is good.

8. Detection of Streptococcus uberis using gene chip

The detection method is the same as that in Example 4, and the scanned images of the detection results show that the gene chip only detects a strong signal value for the gyrB gene of Streptococcus uberis, no false negatives occur, and the specificity is good.

9. Detection of Klebsiella pneumoniae using gene chip

The detection method is the same as that in Example 4, and the scanned image of the detection results shows that the gene chip only detects a strong signal value for the gyrB gene of Klebsiella pneumoniae, no false negatives occur, and the specificity is good.

10. Detection of Pseudomonas aeruginosa using gene chip

The detection method is the same as that in Example 4. The scanned images of the detection results show that the gene chip only detects a strong signal value for the gyrB gene of Pseudomonas aeruginosa, no false negatives occur, and the specificity is good.

11. Detection of Staphylococcus aureus using gene chip

The detection method is the same as that in Example 4, and the scanned images of the detection results show that the gene chip only detects a strong signal value for the gyrB gene of Staphylococcus aureus, no false negatives occur, and the specificity is good.

12. Detection of Escherichia coli using gene chips

The detection method is the same as that in Example 4. The scanned images of the detection results show that the gene chip only detects a strong signal value for the gyrB gene of Escherichia coli, no false negatives occur, and the specificity is good.

13. Detection of Salmonella typhi using gene chip

The detection method is the same as that in Example 4. The scanned images of the detection results show that the gene chip only detects a strong signal value for the gyrB gene of Salmonella typhi, no false negatives occur, and the specificity is good.

The specific detection of the gene chip in this embodiment includes the test of unrelated bacteria, the test of standard bacteria and the test of two strains of standard bacteria, as shown in Figures 1 to 3, and the detection results are as follows:

(1) gene chip specific detection - unrelated bacteria test: unrelated bacteria are A-Streptococcus pneumoniae 5-D, B-proteus vulgaris 1527, C-hemolytic staphylococcus CN4, as shown in Figure 1: 1 is PCR amplification As a result, 2 is the hybridization result of the gene chip. When PCR amplification amplifies the three kinds of bacteria A, B and C, the primer specificity is good, and there is no hybridization signal between the PCR product and the gene chip. The detection result shows that the specificity of the chip good.

(2) Specific detection of gene chip - standard bacteria test: for ten kinds of pathogenic bacteria: Proteus mirabilis, Streptococcus dysgalactiae, Streptococcus agalactiae, Staphylococcus epidermidis, Streptococcus uberis, Klebsiella pneumoniae, Pseudomonas aeruginosa Bacillus, Staphylococcus aureus, Escherichia coli and Salmonella typhi were individually detected by the gene chip, as shown in Figure 2, A is the PCR amplification result, B is the gene chip hybridization result, the results show: PCR products and gene There is no hybridization signal between the chips, and the test results show that there are no false negatives, and the specificity is good.

(3) gene chip specific detection - two strains of standard bacteria test: carry out the detection of gene chip specificity to Proteus mirabilis standard strain and Staphylococcus epidermidis standard strain hybridization, as shown in Figure 3, 2, 3 rows: Proteus mirabilis standard strain Hybridization signals, lines 8 and 9: hybridization signals of Staphylococcus epidermidis, lines 1 and 22: quality control probes, the results show: positive results have clear hybridization signals, good specificity, and high throughput.

14. Sensitivity detection of gene chip

(1) Extraction of nucleic acid from the sample to be tested: extract the genomic DNA of Streptococcus agalactiae as a template according to the CTAB/NaCl method, then accurately measure the nucleic acid concentration of Streptococcus agalactiae with Qubit2. Genome copy number, sample dilution by copy number gradient is 1×10 ⁵ , 1×10 ⁴ , 5×10 ³ , 1×10 ³ , 5×10 ² , 1×10 ² , 5×10 ¹ and 1×10 ¹ spare.

(2) PCR amplification product: ①General or multiplex PCR amplification system: 2μL 10×PCR reactionbuffer, 0.4μL 10mM dNTPs, 1U Taq DNA Polymerase, 0.5μL 10μM upstream primer and 0.5μL 10μM downstream primer, 1-2μL (about 100ng ) each gradient sample DNA, make up to a total volume of 20 μL with double distilled water. ②PCR reaction conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C (adjusted according to the primer annealing temperature) for 30 s, extension at 72°C for 1 min and 30 s, 35 cycles; extension at 72°C for 10 min, and storage at 4°C.

(3) Fluorescence-labeled PCR product: ① Pretreatment: Take 5 μL of the PCR product directly as a template into a sterile and clean 0.2ml centrifuge tube, then add 3 μL of random primers to each sample tube, replenish water to 19 μL, shake and mix well , and centrifuged instantaneously; then put the centrifuged sample tube into the PCR instrument, denature at 95°C for 3 minutes, immediately bathe in ice for 3 minutes, and centrifuge instantaneously; ②Fluorescence labeling: take a new 0.2ml centrifuge tube on ice, and place Add 2.5μL 5×Klenow Buffer, 1μL Klenow enzyme, 2.5μL 2.5mM dNTPs; then add 6μL fluorescent labeling reaction system Mix into the pretreated sample tube, shake and mix, and centrifuge briefly; finally put the sample tube into PCR In the instrument, react at 37°C for 90 minutes, then react at 72°C for 10 minutes, and store at 4°C.

(4) Hybridization: ① Preparation of hybridization system: Melt the hybridization buffer at 42°C, take a sterile and clean 0.2ml centrifuge tube, add 5 μL of hybridization buffer and 15 μL of fluorescent markers in sequence, and put it on ice for later use; ② Hybridization: Open the chip hybridization box, put the hybridization box flat on the table, add about 80 μL sterile water to the hybridization and the bottom groove to prevent a large amount of volatilization of the hybridization solution during the hybridization process; put the chip face up (label facing the operator) into the Between the two positioning pins in the hybridization box; put the chip cover (the side with the boss facing the chip), the upper end first touches the chip, and then slowly cover it; use a pipette to slowly inject 20 μL through the sample hole of the cover glass After denaturing the hybridization solution, the hybridization solution will form a liquid film between the boss under the cover glass and the surface of the chip by virtue of the surface tension of the liquid. Do not shake the cover slip or chip to avoid damaging the liquid film; buckle the cover of the hybridization box , make sure that the positioning pins at both ends of the slot plate are respectively inserted into the positioning pin holes at both ends of the cover plate. After fastening the cover, snap the two metal clips into the two sides respectively. Note: the action should be gentle and do not let the hybridization box have a large vibration, so as to prevent the hybridization liquid from splashing out of the dot matrix area; in addition, the metal clips need to be completely snapped in , to ensure the airtightness of the hybridization box; put the hybridization box into the BioMixerTM II hybridization instrument at 42°C, and react for 2 hours or overnight.

(5) Elution: use SlibeWasherTM8 chip cleaning instrument cleans the chip. ① Configuration of two eluents: eluent Ⅰ is a mixture of 2× sodium citrate solution and 0.2% sodium dodecyl sulfate solution, and eluent Ⅱ is 0.2× sodium citrate solution: ② Elution Operation: at 42°C, wash twice with eluent I, each time for 120s; then wash three times with eluent II, each time for 80s, and centrifuge at 1500rpm for 1min to dry.

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

The method in this example uses Streptococcus agalactiae as a representative strain to detect and analyze the sensitivity of the gene chip. The gradient concentration of the whole genome DNA of Streptococcus agalactiae in the test: 1-1×10 ⁵ , 2-1×10 ⁴ , 3-5×10 ³ , 4-1×10 ³ , 5-5×10 ² , 6-1×10 ² , 7-5×10 ¹ , 8-1×10 ¹ , 9-NC, 1×10 ⁵ ~1×10 ¹ represents the genome copy of 1ul template amplified by PCR, as shown in Figure 4, the results show that the gene chip can detect as low as 5×10 ² copies of pathogenic nucleic acid, so the results show that the gene chip has a higher sensitivity high. Similarly, the implementation method is adopted for other nine pathogenic bacteria Proteus mirabilis, Streptococcus dysgalactiae, Staphylococcus epidermidis, Streptococcus uberis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Salmonella typhi Example method, the sensitivity test of the gene chip was carried out, and the results all showed that the sensitivity of the gene chip was high.

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.