阅读说明：本技术 一种仿刺参肠道中芽孢杆菌cqn-2相对定量检测方法 (Relative quantitative detection method for bacillus CQN-2 in stichopus japonicus intestinal tract ) 是由 杨求华 周宸 陆振 黄瑞芳 林琪 杨福元 叶坤 李忠琴 于 2019-12-09 设计创作，主要内容包括：本发明提供了一种仿刺参肠道中芽孢杆菌CQN-2相对定量检测方法,过程包括芽孢杆菌CQN-2特异性检测引物筛选；荧光定量PCR体系构建及标准曲线制作；仿刺参肠道中芽孢杆菌CQN-2相对带菌量计算,利用芽孢杆菌CQN-2的gyrA-4基因与仿刺参的单拷贝基因β-actin分别设计特异性引物,建立基于SYBR GreenⅠ荧光定量反应体系和PCR技术的仿刺参肠道中芽孢杆菌CQN-2含量的相对定量检测方法。(The invention provides a relative quantitative detection method of bacillus CQN-2 in an apostichopus japonicus intestinal tract, which comprises the steps of screening bacillus CQN-2 specific detection primers; constructing a fluorescent quantitative PCR system and making a standard curve; calculating the relative bacterial carrying quantity of bacillus CQN-2 in the stichopus japonicus intestinal tract, respectively designing specific primers by utilizing gyrA-4 gene of bacillus CQN-2 and single copy gene beta-actin of the stichopus japonicus, and establishing a relative quantitative detection method for the bacillus CQN-2 content in the stichopus japonicus intestinal tract based on SYBR Green I fluorescent quantitative reaction system and PCR technology.)

1. A relative quantitative detection method for bacillus CQN-2 in stichopus japonicus intestinal tracts is characterized by comprising the following steps: the detection method comprises the following steps:

s1, screening a bacillus CQN-2 specific detection primer;

s2, constructing a fluorescent quantitative PCR system and making a standard curve;

s3, calculating the relative bacterial carrying quantity of bacillus CQN-2 in the stichopus japonicus intestinal tract.

2. The method for the relative quantitative determination of bacillus CQN-2 in the intestinal tract of apostichopus japonicus according to claim 1, wherein the method comprises the following steps: the step S1 specifically includes

S11, selecting a strain used in an experiment and extracting DNA in bacteria;

s12, designing specific primers for the detected whole gene sequence of the bacillus CQN-2, and comparing the specific primers with the gene sequence of the strain for analysis;

s13, screening out a primer sequence which has specific amplification to the bacillus CQN-2 through PCR amplification.

3. The method for the relative quantitative determination of bacillus CQN-2 in the intestinal tract of apostichopus japonicus according to claim 1, wherein the method comprises the following steps: the step S2 specifically includes

S21, purifying PCR amplification primers;

s22, constructing a fluorescent quantitative PCR system of the purified PCR amplification primer and calculating the corresponding DNA molecule copy number according to a formula 1, wherein the formula 1 is shown in the specification;

wherein 6.022 is multiplied by 10²³(molecules/mole) is the Avogastron constant, 660daltons is the average molecular weight of a single base pair;

s23, finally, taking the logarithm value of the copy number of the DNA molecule after dilution by multiple times as an abscissa, taking the CT value of the fluorescence quantitative PCR as an ordinate, and constructing a standard curve between the copy number of the DNA and the CT value, wherein the equation of the standard curve is that the CT is K multiplied by log, the copy number of the DNA is plus C

Wherein

4. The method for the relative quantitative determination of bacillus CQN-2 in the intestinal tract of apostichopus japonicus according to claim 1, wherein the method comprises the following steps: the step S3 specifically comprises the steps of taking total DNA extracted from the intestinal tissues of apostichopus japonicus as a template, taking beta-actin of the apostichopus japonicus as an internal reference gene, taking gyrA of bacillus CQN-2 as a target gene, wherein,

copy number of gyrA gene:

copy number of β -actin gene:

relative amount of bacteria N_P/N_L

According to the prepared standard curve and the fluorescent quantitative PCR detection result, the copy numbers of the bacillus CQN-2gyrA gene and the apostichopus japonicus beta-actin gene in each sample are calculated, the copy number of the CQN-2gyrA gene measured in the same sample is divided by the corresponding copy number of the apostichopus japonicus beta-actin gene, and the obtained ratio is the relative bacterial carrying capacity which is equivalent to the relative quantity of bacillus CQN-2 detected in the intestinal tissue cells of the apostichopus japonicus with the same quantity.

5. The method for the relative quantitative determination of bacillus CQN-2 in the intestinal tract of apostichopus japonicus according to claim 1, wherein the method comprises the following steps: the fluorescent system PCR system construction comprises a reaction system and a reaction program,

the reaction system is as follows: 2 XChamQ Universal SYBR qPCR Master Mix 10.0. mu.l, gyrA F4/R4 or beta-actin F/R upstream and downstream primers (10. mu.M) each 0.5. mu.l, DNA template 4.0. mu.l, ddH₂O 5.0μl；

The reaction procedure is as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 10s, annealing at 61 ℃ for 30s, for 45 cycles; denaturation at 95 ℃ for 10s, annealing at 65 ℃ for 60s, and annealing at 95 ℃ for 1 s; cooling at 37 deg.C for 30 s.

6. The method for the relative quantitative determination of bacillus CQN-2 in the intestinal tract of apostichopus japonicus according to claim 2, wherein: the PCR amplification in the step S13 comprises a reaction system, an initial reaction program and an optimized reaction program,

the reaction system is as follows: DNA template: 2 mu L of the solution; PCR mix: 25 mu L of the solution; and (3) primer F: 2 mu L of the solution; and (3) primer R: 2 mu L of the solution; ddH₂O19 mu L, total 50 mu L;

the initial reaction procedure was: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, renaturation at 53 deg.C for 30s, extension at 72 deg.C for 90s, 35 cycles, final incubation at 72 deg.C for 7min, and storage at 4 deg.C;

the optimization reaction program comprises the following steps: after selecting appropriate primer pairs, PCR amplification was performed at renaturation annealing temperatures of 51 ℃, 53 ℃, 57 ℃, 59 ℃, 61 ℃ and 63 ℃ respectively, under the same procedure as the other primers.

Technical Field

The invention relates to the technical field of bacillus detection, in particular to a relative quantitative detection method for bacillus CQN-2 in an apostichopus japonicus intestinal tract.

Background

The bacillus CQN-2 is bacillus amyloliquefaciens which is one of probiotics separated from intestinal tracts of healthy apostichopus japonicus, and after the apostichopus japonicus is fed by the bacillus CQN-2 added into feed, the weight gain rate and the specific growth rate of the apostichopus japonicus can be obviously improved, and meanwhile, the disease resistance of the apostichopus japonicus against pathogenic bacteria infection can be effectively improved. However, no effective detection method has been found for the situation of the colonization of bacillus CQN-2 in the intestinal tract of apostichopus japonicus after feeding.

Based on the above series of problems, the following technical solutions have been developed.

Disclosure of Invention

The invention aims to provide a relative quantitative detection method for bacillus CQN-2 in an apostichopus japonicus intestinal tract, which is characterized in that specific primers are respectively designed by utilizing gyrA-4 gene of bacillus CQN-2 and single copy gene beta-actin of the apostichopus japonicus, and a SYBR Green I fluorescent quantitative reaction system and a PCR technology based relative quantitative detection method for bacillus CQN-2 content in the apostichopus japonicus intestinal tract is established.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention provides a relative quantitative detection method for bacillus CQN-2 in an apostichopus japonicus intestinal tract, which comprises the following steps:

s1, screening a bacillus CQN-2 specific detection primer;

s2, constructing a fluorescent quantitative PCR system and making a standard curve;

s3, calculating the relative bacterial carrying quantity of bacillus CQN-2 in the stichopus japonicus intestinal tract.

Further, the step S1 specifically includes

S11, selecting a strain used in an experiment and extracting DNA in bacteria;

s12, designing specific primers for the detected whole gene sequence of the bacillus CQN-2, and comparing the specific primers with the gene sequence of the strain for analysis;

s13, screening out a primer sequence which has specific amplification to the bacillus CQN-2 through PCR amplification.

Further, the step S2 specifically includes

S21, purifying PCR amplification primers;

s22, constructing a fluorescent quantitative PCR system of the purified PCR amplification primer and calculating the corresponding DNA molecule copy number according to a formula 1, wherein the formula 1 is shown in the specification;

wherein 6.022 is multiplied by 10²³(molecules/mole) is the Avogastron constant, 660daltons is the average molecular weight of a single base pair;

s23, finally, taking the logarithm value of the copy number of the DNA molecule after dilution by multiple times as an abscissa, taking the CT value of the fluorescence quantitative PCR as an ordinate, and constructing a standard curve between the copy number of the DNA and the CT value, wherein the equation of the standard curve is that the CT is K multiplied by log, the copy number of the DNA is plus C

WhereinE is the amplification efficiency of the primer.

Further, the step S3 specifically includes using total DNA extracted from intestinal tissues of apostichopus japonicus as a template, apostichopus japonicus beta-actin as an internal reference gene, and gyrA of bacillus CQN-2 as a target gene, wherein,

copy number of gyrA gene:

copy number of β -actin gene:

relative amount of bacteria N_P/N_L

According to the prepared standard curve and the fluorescent quantitative PCR detection result, the copy numbers of the bacillus CQN-2gyrA gene and the apostichopus japonicus beta-actin gene in each sample are calculated, the copy number of the CQN-2gyrA gene measured in the same sample is divided by the corresponding copy number of the apostichopus japonicus beta-actin gene, and the obtained ratio is the relative bacterial carrying capacity which is equivalent to the relative quantity of bacillus CQN-2 detected in the intestinal tissue cells of the apostichopus japonicus with the same quantity.

Further, the fluorescent system PCR system construction comprises a reaction system and a reaction program,

the reaction system is as follows: 2 XChamQ Universal SYBR qPCR Master Mix 10.0. mu.l, gyrAF4/R4 or beta-actin F/R upstream and downstream primers (10. mu.M) each 0.5. mu.l, DNA template 4.0. mu.l, ddH₂O 5.0 μl；

The reaction procedure is as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 10s, annealing at 61 ℃ for 30s, for 45 cycles; denaturation at 95 ℃ for 10s, annealing at 65 ℃ for 60s, and annealing at 95 ℃ for 1 s; cooling at 37 deg.C for 30 s.

Further, the PCR amplification in the step S13 includes a reaction system, an initial reaction program and an optimized reaction program,

the reaction system is as follows: DNA template: 2 mu L of the solution; PCR mix: 25 mu L of the solution; and (3) primer F: 2 mu L of the solution; and (3) primer R: 2 mu L of the solution; ddH₂O19 mu L, total 50 mu L;

the initial reaction procedure was: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 30s, renaturation at 53 deg.C for 30s, extension at 72 deg.C for 90s, 35 cycles, final incubation at 72 deg.C for 7min, and storage at 4 deg.C;

the optimization reaction program comprises the following steps: after selecting appropriate primer pairs, PCR amplification was performed at annealing temperatures of 51 ℃, 53 ℃, 57 ℃, 59 ℃, 61 ℃ and 63 ℃ respectively, under the same procedure.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention utilizes gyrA-4 gene of bacillus CQN-2 and single copy gene beta-actin of apostichopus japonicus to respectively design specific primers, and establishes a relative quantitative detection method for the content of bacillus CQN-2 in the intestinal tract of the apostichopus japonicus based on SYBR Green I fluorescent quantitative reaction system and PCR technology;

2. by using the measurement method provided by the invention, the colonization condition of the bacillus CQN-2 in the stichopus japonicus intestinal tract can be effectively known.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is basic information on strains involved in a preferred embodiment of the present invention;

FIG. 2 shows gene information and sequences of primers according to a preferred embodiment of the present invention;

FIG. 3 is the result of PCR detection of 12 strains of bacteria of different species amplified by different primers in experimental verification according to a preferred embodiment of the present invention;

FIG. 4 shows the result of PCR amplification detection at different annealing temperatures using gyrA F4/R4 as amplification primers in experimental verification of a preferred embodiment of the present invention;

FIG. 5 is the copy number of the corresponding gene detected by quantitative PCR after different multiple dilutions of DNA after recovery and purification of gel in the experimental verification of the preferred embodiment of the present invention;

FIG. 6 is the detection of the real-time fluorescent quantitative amplification effect of the primers gyrA F4/R4 and beta-actin F/R in the preferred embodiment of the invention;

FIG. 7 is a standard curve for the amplification of primer gyrA F4/R4;

FIG. 8 is a standard curve for beta-actin F/R amplification;

FIG. 9 shows CQN-2 relative bacterial load in intestinal tracts of stichopus japonicus of different experimental groups in experimental verification of a preferred embodiment of the invention;

figure 10 is a flow chart of the detection method of the present invention,

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.

The invention provides a relative quantitative detection method for bacillus CQN-2 in an apostichopus japonicus intestinal tract. The technical solution provided by the present invention will be explained in more detail with reference to fig. 1 to 10.

As shown in figures 1-10, in order to solve the problem of the research on the field planting condition of bacillus CQN-2 in the stichopus japonicus intestinal tract after feeding, the invention provides a relative quantitative detection method for bacillus CQN-2 in the stichopus japonicus intestinal tract, and the detection principle of the method is that specific primers are respectively designed by utilizing the gyrA-4 gene of bacillus CQN-2 and the single copy gene beta-actin of stichopus japonicus, and a relative quantitative detection method for the content of bacillus CQN-2 in the stichopus japonicus intestinal tract based on a SYBR Green I fluorescent quantitative reaction system and a PCR technology is established.

The process of the invention comprises:

s1, screening a bacillus CQN-2 specific detection primer;

s2, constructing a fluorescent quantitative PCR system and making a standard curve;

s3, calculating the relative bacterial carrying quantity of bacillus CQN-2 in the stichopus japonicus intestinal tract.

The detection process of the invention preferably adopts a method of experimental comparison and experimental verification, and specifically comprises the following steps:

1. bacillus CQN-2 specific detection primer screening

1) Strains used for experiments

The basic information of the strains used in the method is shown in figure 1, and the total 12 strains comprise 9 strains of bacillus and 3 strains of vibrio, wherein 5 strains of bacillus are separated from intestinal tracts of healthy apostichopus japonicus, 4 strains of bacillus are separated from normal seawater, 2 strains of vibrio are separated from focal tissues of diseased apostichopus japonicus, and 1 strain of vibrio is separated from focal tissues of diseased hippocampus.

2) Extraction of DNA

The method is carried out according to the instruction of a bacterial genome DNA extraction kit (centrifugal column type) of Shanghai Czeri bioengineering company Limited, and comprises the following specific steps:

firstly, a proper amount of bacteria in exponential growth phase (5 multiplied by 10 at most) is added⁸) Centrifuge at 8,000rpm for 1 minute and discard the medium thoroughly.

② adding 200 mul TE to suspend bacteria, adding 1 mul Lysozyme with 400 mul g/ml to dissolve for 3-5 minutes, and mixing evenly by a suction head. Then adding 4. mu.l RNase A and mixing well, adding 4. mu.l Proteinase K, adding 200. mu.l lysine Solution70 ℃ and keeping the temperature for 10 minutes.

③ cooling to room temperature, adding 200 mul of absolute ethyl alcohol, and mixing evenly. If the precipitate appears, the mixture is fully and evenly stirred by a 1ml gun head, and the extraction is not influenced.

Transferring all the liquid into a GenClean column, centrifuging at 8,000rpm at room temperature for 1 minute, removing the GenClean column, and discarding the waste liquid in the collecting pipe.

Fifthly, the GenClean column is put back into the collecting tube, 500 mul AW1 Solution is added, and the mixture is centrifuged for 1 minute at the room temperature of 8,000 rpm. The GenClean column was removed and the waste stream from the collection tube was discarded.

Sixthly, the GenClean column is put back into the collecting tube, 500. mu.l AW2Solution is added, and the mixture is centrifuged for 1 minute at the room temperature of 8,000 rpm. The GenClean column was removed and the waste stream from the collection tube was discarded. And repeated once again.

Seventhly, put the GenClean column back into the collection tube and centrifuge at 12,000rpm for 1 minute at room temperature to remove the residual AW2 Solution.

Eighthly, putting the GenClean column into a new clean 1.5ml centrifuge tube, adding 50-100 ul AE Beffer into the center of the GenClean column, and standing for 2 minutes at room temperature or 37 ℃. Centrifuging at 12,000rpm for 1 minute at room temperature, and obtaining the liquid in the centrifuge tube as the genome DNA. Storing at 4 deg.C or-20 deg.C.

3) Primer design and validation

Specific primers are designed according to the measured whole gene sequence of the bacillus CQN-2, and are compared with gene sequences of other strains (table 1) for analysis, sequence fragments of four genes of 16S, 23S, gyrA and gyrB are selected to design 21 pairs of primers for PCR reaction, so as to screen and verify the specificity of the primers. A single copy gene beta-actin in the apostichopus japonicus genome is selected as an internal reference gene, and 22 primers required by the method are synthesized by Yinxie fundi (Shanghai) trade company Limited, and the primer sequence is shown in FIG. 2.

The primers synthesized were first tested for amplification and specificity by PCR using Bacillus CQN-2 as templates, respectively, and the obtained PCR product was sent to Yinxie Weiji (Shanghai) trade Co., Ltd for sequencing verification.

4) PCR amplification system and amplification condition design

Reaction system: DNA template: 2 mu L of the solution; PCR mix: 25 mu L of the solution; and (3) primer F: 2 mu L of the solution; and (3) primer R: 2 mu L of the solution; ddH₂O19. mu.L, 50. mu.L total.

Initial reaction procedure: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, renaturation at 53 ℃ for 30s, extension at 72 ℃ for 90s, 35 cycles, final incubation at 72 ℃ for 7min, and storage at 4 ℃.

Optimizing a reaction program: after selecting appropriate primer pairs, PCR amplification was performed at renaturation annealing temperatures of 51 ℃, 53 ℃, 57 ℃, 59 ℃, 61 ℃ and 63 ℃ respectively, under the same procedure as the other primers.

The DNA of 12 bacterial strains of different species (Table 1) selected by the method is taken as a template, PCR amplification is carried out, 1.5% agarose gel electrophoresis is used for detection, and a primer sequence with specific amplification to bacillus CQN-2 is screened out.

2. Fluorescent quantitative PCR system construction and standard curve making

1) Purification of PCR amplification products

The bacillus CQN-2DNA and the apostichopus japonicus body wall tissue DNA are used as templates, optimal specific primers obtained by screening are respectively used for PCR amplification, and the recovery and purification of amplification products are carried out according to a thin agarose gel DNA recovery kit of Shanghai Jieli bioengineering Co. The method comprises the following specific steps:

balancing treatment of a DNA adsorption column: 200 μ l buffer CBS was added to the column, centrifuged at 12,000rpm for 1min, the collector tube was decanted, and the column was replaced in the collector tube.

And secondly, cutting off the gel containing the target fragment from the agarose gel, and estimating the weight or accurately weighing the weight.

③ adding 100 mul of Binding Solution into every 100mg of agarose gel, and carrying out water bath at 50-60 ℃ for 3-5min, wherein the mixture is mixed by slightly reversing every 2-3min until the gel block is completely melted.

Transferring the mixed solution to an adsorption column GC-2u sleeved with a 2mL collecting pipe, standing for 2min at room temperature, centrifuging for 1min at 6,000rpm at room temperature, taking out the adsorption column GC-2u, and pouring out waste liquid in the collecting pipe.

Fifthly, the adsorption column GC-2u is placed back into the collecting pipe again, 500 mul WA Solution is added, the mixture is centrifuged for 1min at the room temperature of 12,000rpm, and waste liquid in the collecting pipe is poured out.

Sixthly, the adsorption column GC-2u is placed back into the collecting pipe again, 500 mu l of Wash Solution is added, the mixture is centrifuged for 1min at the room temperature of 12,000rpm, and waste liquid in the collecting pipe is poured out.

Seventhly, repeating the step (c) once.

Eighthly, putting the adsorption column GC-2u back into the collecting pipe again, centrifuging at 12,000rpm at room temperature for 1min, then opening the cover of the adsorption column GC-2u, and standing at room temperature for 5-10min or 50 ℃ for 3-5min to completely remove the Wash Solution.

Ninthly, putting the adsorption column GC-2u into a clean 1.5mL collecting tube (self-contained in the kit), suspending and adding 15-20 μ l of precipitation Buffer into the center of the membrane, covering the cover, placing for 2min at 37 ℃, centrifuging for 1min at 12,000rpm, and obtaining the solution containing the target DNA fragment from the liquid in the centrifuge tube.

2) Fluorescent quantitative PCR system construction

Purifying PCR amplification products of bacillus CQN-2gyrA gene and apostichopus japonicus beta-actin gene by using a colloidal back kit, measuring the concentration by using an M200 Pro enzyme-linked immunosorbent assay (TECAN), and measuring the concentration according to the ratio of 10³、10⁴、 10⁵、10⁶、10⁷、10⁸、10⁹And (3) performing 10-fold gradient dilution, and calculating the corresponding DNA molecule copy number according to formula 1.

Wherein 6.022 is multiplied by 10²³(molecules/mole) is the Avogastron constant, 660daltons is the average molecular weight of a single base pair.

DNA diluted in gradient double ratios was used as a template, and Roche was used

96 quantitative PCR instrument, according to ChamQ^TMUniversal

Instructions for the qPCR Master Mix kit were used for the procedure.

The reaction system and reaction conditions of the real-time fluorescent quantitative PCR are as follows:

reaction system: 2 XChamQ Universal SYBR qPCR Master Mix 10.0. mu.l, gyrA F4/R4 or beta-actin F/R upstream and downstream primers (10. mu.M) each 0.5. mu.l, DNA template 4.0. mu.l, ddH₂O 5.0μl。

Reaction procedure: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 10s, annealing at 61 ℃ for 30s, for 45 cycles; denaturation at 95 ℃ for 10s, annealing at 65 ℃ for 60s, and annealing at 95 ℃ for 1 s; cooling at 37 deg.C for 30 s.

3) Standard Curve preparation

And (3) constructing a standard curve between the DNA copy number and the CT value by taking the DNA molecule copy number logarithmic value (Log) after dilution by multiple times as an abscissa and the CT value of the fluorescence quantitative PCR as an ordinate.

The equation of the standard curve is shown in formula 2:

k × log DNA copy number + C (formula 2)

Wherein

E is the amplification efficiency of the primer.

3. Calculating relative bacteria carrying amount of bacillus CQN-2 in intestinal tract of apostichopus japonicus

Taking total DNA (with the concentration of 20 ng/. mu.L) extracted from the apostichopus japonicus intestinal tissue as a template, the apostichopus japonicus beta-actin as an internal reference gene and the gyrA of the bacillus CQN-2 as a target gene, carrying out fluorescent quantitative PCR detection, carrying out 3 times of repetition on each sample, and calculating the relative bacterium carrying amount by taking an average value.

As can be seen from equation 1:

copies of the gyrA GeneNumber:

copy number of β -actin gene:

relative amount of bacteria N_P/N_L(formula 5)

According to the prepared standard curve and the fluorescent quantitative PCR detection result, the copy numbers of the bacillus CQN-2gyrA gene and the apostichopus japonicus beta-actin gene in each sample are calculated, the copy number of the CQN-2gyrA gene measured in the same sample is divided by the corresponding copy number of the apostichopus japonicus beta-actin gene, and the obtained ratio is the relative bacterial carrying capacity which is equivalent to the relative quantity of bacillus CQN-2 detected in the intestinal tissue cells of the apostichopus japonicus with the same quantity.

The invention carries out the following experimental verification on the experimental result:

1. bacillus CQN-2 specific primer screening

The primer gyrA F4/R4, the primer gyrB F3/R3 and the primer gyrB F5/R5 have better primer specificity to the bacillus CQN-2 (figure 1).

Three pairs of primers, namely, primer gyrA F4/R4, primer gyrB F3/R3 and primer gyrB F5/R5, are used as candidate primers, and PCR amplification is carried out at the designed annealing temperatures of 51 ℃, 53 ℃, 57 ℃ and 59 ℃, and the result shows that the PCR amplification result is not particularly ideal at the annealing temperature, and a single targeted band cannot be amplified against bacillus CQN-2. However, gyrA F4/R4 was the most effective of the three primer pairs, so this primer was used for further annealing temperature optimization. The primers gyrA F4/R4 are designed to be annealing temperatures of 61 ℃ and 63 ℃ for PCR amplification, and the results show that a single specific band aiming at the bacillus CQN-2 can be obtained by carrying out PCR amplification by using gyrA F4/R4 as amplification primers and using 61 ℃ or 63 ℃ as an annealing temperature, as shown in a figure 3-4.

Different columns in each primer set respectively correspond to bacteria numbered 1-12 in Table 1, wherein the 1 st column is Bacillus CQN-2.

2. Fluorescent quantitative PCR system construction and standard curve making

The gel recovery electrophoresis band result shows that the size of the bacillus CQN-2gyrA gene amplification band is about 260 bp, the size of the apostichopus japonicus beta-actin gene amplification band is about 270bp, the expected size is met, and the recovery and purification of the PCR amplification product gel are qualified.

The results of the enzyme-linked immunosorbent assay show that the concentration of the DNA recovered from the bacillus CQN-2gyrA gene glue is 52.8 ng/mu L, the concentration of the DNA recovered from the apostichopus japonicus beta-actin gene glue is 15.2 ng/mu L, and the corresponding DNA copy numbers are respectively 1.82 multiplied by 10¹¹copies/. mu.L and 5.23X 10¹⁰copies/μL。

The DNA stock solutions obtained by the above recovery were each 10 th³、10⁴、10⁵、10⁶、10⁷、10⁸、10⁹And performing 10-fold gradient dilution, and performing fluorescent quantitative PCR detection by using the diluted solution as a template. The corresponding copy numbers of the bacillus CQN-2gyrA gene and the apostichopus japonicus beta-actin gene after dilution with different fold ratios calculated according to the formula 1 are shown in figure 5.

As shown in FIGS. 6 to 8, a standard curve was constructed with the logarithm of DNA copy number as the abscissa and the CT value of the results of fluorescent quantitative PCR as the ordinate, and FIG. 3 includes standard curves for the amplification of primers gyrA F4/R4 and β -actin F/R, respectively; amplification curves for primers gyrA F4/R4 and beta-actin F/R. The standard curve equations of the primers gyrA F4/R4 and beta-actin F/R obtained according to formula 1 and formula 2 are respectively-2.4398 x +26.902 (R)²0.9962) and y-2.9916 x +30.822 (R)²＝0.9977)。

3. Relative bacteria carrying amount of bacillus CQN-2 in intestinal tract of apostichopus japonicus

The experimental group used in this example was a simulated stichopus japonicus intestinal tissue that had been fed with bacillus CQN-2 for 8 weeks; the control group was intestinal tissue of apostichopus japonicus fed with normal feed without CQN-2. Extracting total DNA (the concentration is 20 ng/. mu.L) of the tissue as a template, carrying out fluorescent quantitative PCR detection by taking apostichopus japonicus beta-actin as an internal reference gene and gyrA of bacillus CQN-2 as a target gene, carrying out 3 times of repeated detection on each sample, and calculating the relative bacterial load by taking an average value.

The calculation results of the relative amount of bacteria are shown in fig. 9. Where the ordinate is-Log relative bacterial load, lower ratios indicate higher relative bacterial loads. As can be seen from the figure, after 8 weeks of continuous 8-week mixed feeding of the bacillus CQN-2, the relative bacterial load of CQN-2 in the intestinal tract of the apostichopus japonicus is significantly higher than that of the control group, which indicates that the bacillus CQN-2 can be well planted in the intestinal tract of the apostichopus japonicus.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.