Reagent and detection method for detecting proportion of vibrio in total bacteria in prawn seeds or prawn culture water

Abstract

The invention provides a reagent for detecting the proportion of vibrio in prawn seeds or prawn culture water in total bacteria. The reagent comprises a vibrio detection primer and probe, a bacteria detection primer and probe, Taq DNA polymerase, a dNTP mixture, UNG enzyme, a freeze-drying protective additive and a PCR buffer solution. According to the method, the specific DNA of the vibrio and the bacteria in the sample is quantitatively detected by utilizing the fluorescent PCR technology, the proportion of the vibrio in the total bacteria is obtained through the obtained quantitative detection data, the proportion of the vibrio in the total bacteria in shrimp seeds and shrimp culture water can be obtained in a short time, and guidance is provided for shrimp breeding and culture.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and particularly relates to a reagent and a detection method for detecting the proportion of Vibrio in total bacteria in shrimp fry or shrimp breeding water. Background Art

[0002] my country introduced whiteleg shrimp from Hawaii in the 1980s. Artificial breeding was initially successful in the 1990s. After 2000, whiteleg shrimp was widely cultivated in my country. During its growth and development, whiteleg shrimp go through different stages, such as nauplii, zoea, mysid larvae, and shrimplets. At different developmental stages, the external morphology, internal structure, feeding habits, and movement patterns of shrimp will change. Its developmental characteristics make shrimp farming difficult. The cultivation of whiteleg shrimp is affected by a variety of environmental factors, among which Vibrio is one of the important factors.

[0003] Vibrio is a type of conditional pathogen. When environmental conditions deteriorate and the resistance of shrimp decreases, Vibrio will take advantage of the situation and enter. The symptoms of shrimp such as death, enteritis, slow growth, and white stool are closely related to Vibrio. In addition, the symptoms of shrimp such as black gills, brown spots, red legs and red tails, yellow gills, shell ulcers, and white muscle turbidity are also closely related to Vibrio. Vibrio is one of the common bacterial groups in the marine environment. This type of bacteria has strong adaptability and resistance, so it has become a dominant population in the seawater environment and often causes outbreaks of diseases as a pathogen. Common Vibrio in shrimp farming include Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio vulnificus, and Vibrio anguillarum; Vibrio bacteria can cause vibrio disease in shrimp, and it is still the most common and most harmful bacterial disease in shrimp farming. As an important part of the shrimp farming ecology, Vibrio has a great competitive relationship with other beneficial bacteria. The higher the proportion of Vibrio in the total number of bacteria, the greater the competitive advantage of Vibrio compared with other bacteria, and the greater the harm to shrimp.

[0004] RDNA is a gene encoding rRNA on bacterial chromosomes. According to different sedimentation coefficients, bacterial rRNA can be divided into three types: 5S, 16S and 23S. Among them, 16S rDNA corresponding to 16S rRNA is the most commonly used research object in the systematic classification research of bacteria. 16S rDNA is of moderate size, about 1.5Kb, and its gene sequence includes conservative regions and variable regions distributed at intervals. Bacteria generally contain ten conservative regions and 9 variable regions, and different types of bacteria have the same conservative region sequence and different variable region sequences. The bacterial detection primer probe designed using the bacterial 16S rDNA conservative region sequence in the present invention, and the Vibrio detection primer probe designed using the bacterial 16S rDNA variable region sequence, can be respectively quantitatively detected for all bacteria and Vibrio in shrimp and shrimp breeding water, and then according to the quantitative detection data of the two, the proportion data of Vibrio in total bacteria are obtained. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a reagent and a detection method for detecting the proportion of Vibrio in total bacteria in shrimp fry or shrimp breeding water.

[0006] The present invention is achieved through the following technical solutions:

[0007] The present invention provides a first solution, a reagent for detecting the proportion of Vibrio in total bacteria in shrimp fry or shrimp breeding water, the reagent comprising: Vibrio detection primers and probes, bacteria detection primers and probes, Taq DNA polymerase, dNTP mixture, UNG enzyme, lyophilization protectant and PCR buffer;

[0008] The nucleotide sequence of the Vibrio detection primer is:

[0009] 5′-GGGGCTCAACCTCGGAATWG-3′;

[0010] 5′-CTTCGCCACCGGTATTCCTT-3′;

[0011] The nucleotide sequence of the Vibrio detection probe is:

[0012] 5′-ATTTGAAACTGGCAGACTAG-3′;

[0013] The nucleotide sequence of the bacterial detection primer is:

[0014] 5′-GCGAAAGCGTGGGGAGC-3′;

[0015] 5′-CATCGTTTACGGCGTGGAC-3′;

[0016] 5′-CGCCCATTGTGCAATATTCC-3′;

[0017] 5'-TGAGACACGGTCCAGACTCCTA-3';

[0018] The nucleotide sequence of the bacteria detection probe is:

[0019] 5'-AACAGGATTAGATACCCTGGTA-3';

[0020] 5′-ACTGCTGCCTCCC-3′.

[0021] Preferably, the Vibrio detection probe has a FAM fluorescent reporter group at its 5' end and a MGB fluorescent quencher group at its 3' end, and the bacteria detection probe has a VIC fluorescent reporter group at its 5' end and a MGB fluorescent quencher group at its 3' end.

[0022] Preferably, the lyoprotectant comprises at least one of trehalose, sucrose, glucose, fucose, sorbitol, raffinose, mannitol, dextran, and polyethylene glycol.

[0023] The present invention also provides a second scheme, a method for preparing a reagent for detecting the proportion of Vibrio in total bacteria in shrimp fry or shrimp breeding water, comprising the following process: mixing 1-5 units of Taq DNA polymerase, 5%-13% (W / V) freeze-drying protectant, 0.1-0.5mmol / L dNTP mixture, 0.05-0.4umol / L of each primer and probe, 0.1-0.5 units of UNG enzyme, and PCR buffer in a PCR reaction tube, placing the PCR tube containing the fluorescent PCR reagent in a freeze dryer for freeze drying, and the freeze drying program is: -45°C, 1h; -45°C, 20Pa, 2h; -40°C, 10Pa, 10h; -20°C, 10Pa, 1h; 0°C, 10Pa, 30m; 25°C, 10Pa, 2h.

[0024] The present invention also provides a third solution, a method for detecting the proportion of Vibrio in total bacteria in shrimp seedlings or shrimp breeding water, using the above reagent for detection, comprising the following steps:

[0025] S1. DNA was extracted from the overnight cultured Vibrio solution, and the Vibrio DNA was diluted 10 times with sterile water. The DNA solutions of four consecutive dilution gradients were simultaneously tested by PCR with reagents to prepare a fluorescent PCR amplification standard curve;

[0026] S2. Extract DNA from shrimp fry or shrimp farming water samples to be tested;

[0027] S3. The extracted DNA is subjected to fluorescent PCR detection using reagents;

[0028] S4. Substitute the fluorescent PCR Ct value obtained in step S3 into the standard curve in step S1 to obtain the concentration values ​​of Vibrio and total bacteria, respectively, and calculate the proportion of Vibrio in the total bacteria.

[0029] The beneficial effects of the present invention are:

[0030] The fluorescent PCR technology is used to quantitatively detect the specific DNA of Vibrio and bacteria in the sample, and the proportion of Vibrio in the total bacteria is obtained through the obtained quantitative detection data. The present invention can obtain the proportion of Vibrio in the total bacteria in shrimp fry and shrimp breeding water in a short time, providing guidance for shrimp breeding and farming. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 , a fluorescent PCR reagent for detecting the proportion of Vibrio in total bacteria in shrimp fry and shrimp breeding water prepared by the method in Example 1.

[0032] Figure 2 , the fluorescence PCR standard curve prepared by the method in Example 2.

[0033] Figure 3 , Example 3: Fluorescence PCR curve for detecting the proportion of Vibrio in total bacteria in shrimp fry samples.

[0034] Figure 4 , Example 4 Fluorescence PCR curve for detecting the proportion of Vibrio in total bacteria in shrimp farming water samples.

[0035] Figure 5 , the concentration values ​​of Vibrio and bacteria in the samples after the plate count colony counts in Example 3 and Example 4 were converted, and the concentration values ​​of Vibrio and bacteria quantified by fluorescent PCR. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0037] Embodiment 1:

[0038] Preparation of fluorescent PCR reagents for detecting the proportion of Vibrio in total bacteria in shrimp fry and shrimp farming water:

[0039] Mix 1.5uL 10×PCR buffer, 5uL 20% trehalose (W / V), 0.3uL dNTP / dUTP mixture, Vibrio detection primers and probes (100umol / L) SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, 0.015uL each, bacteria detection primers and probes (100umol / L) SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9, 0.015uL each, UNG enzyme (1U / uL) 0.15uL, Taq 0.3uL of DNA polymerase was supplemented with pure water to 15uL and placed in a freeze dryer. The freeze drying program was as follows: -45°C, 1h; -45°C, 20Pa, 2h; -40°C, 10Pa, 10h; -20°C, 10Pa, 1h; 0°C, 10Pa, 30m; 25°C, 10Pa, 2h to prepare a freeze-dried fluorescent PCR reagent.

[0040] in,

[0041] SEQ ID NO.1: 5'-GGGGCTCAACCTCGGAATWG-3';

[0042] SEQ ID NO.2: 5'-CTTCGCCACCGGTATTCCTT-3';

[0043] SEQ ID NO.3: 5'-ATTTGAAACTGGCAGACTAG-3';

[0044] SEQ ID NO.4: 5'-GCGAAAGCGTGGGGAGC-3';

[0045] SEQ ID NO.5: 5'-CATCGTTTACGGCGTGGAC-3';

[0046] SEQ ID NO.6: 5'-AACAGGATTAGATACCCTGGTA-3';

[0047] SEQ ID NO.7: 5'-CGCCATTGTGCAATATTCC-3';

[0048] SEQ ID NO.8: 5'-TGAGACACGGTCCAGACTCCTA-3';

[0049] SEQ ID NO.9: 5'-ACTGCTGCCTCCC-3'.

[0050] Embodiment 2:

[0051] Preparation of fluorescent PCR standard curve:

[0052] Take out the Vibrio alginolyticus seed liquid from -80℃, inoculate it into 3ml 3% sodium chloride alkaline peptone water culture medium, and culture it at 36℃±1℃ for 24 hours. Dilute the cultured bacterial liquid 10 times with sterile physiological saline, dilute it continuously for 9 gradients, take 1mL of the eighth and ninth dilution gradient bacterial liquid, spread it on the nutrient agar plate, and count the number of colonies on the plate after culturing at 36℃±1℃ for 24 hours. Take 1mL of the first to fourth dilution gradient bacterial liquid, extract DNA respectively, and detect DNA with the fluorescent PCR reagent prepared in Example 1. The PCR reaction program is: 50℃, 2min; 95℃, 2min; 95℃, 5sec, 60℃, 30sec, 45 cycles, FAM and VIC channels detect fluorescence, set the sample type to "standard", and enter the corresponding concentration value according to the plate count result. The experiment is completed to obtain the fluorescent PCR standard curve.

[0053] Embodiment 3:

[0054] The proportion of Vibrio in the total bacteria in the shrimp fry samples tested:

[0055] Take a shrimp fry sample, grind it thoroughly, take 10 mg of grinding liquid to extract DNA, and use the PCR reagent in Example 1 to detect DNA. The PCR reaction program is: 50°C, 2 min; 95°C, 2 min; 95°C, 5 sec, 60°C, 30 sec, 45 cycles, FAM and VIC channels to detect fluorescence, set the sample type to "to be tested", and after the experiment, import the standard curve of Example 2, convert the CT value into a concentration value, and calculate the ratio of the concentration values ​​of the FAM and VIC fluorescence channels. Take another 10 mg of grinding liquid and dilute it 100 and 1000 times with sterile physiological saline, take 1 mL of the dilution and apply it on a TCBS plate and a nutrient agar plate, respectively, and culture at 32°C for 24-48 hours, and calculate the number of Vibrio colonies on the TCBS plate or the number of bacterial colonies on the nutrient agar plate, respectively.

[0056] Embodiment 4:

[0057] Detection of the proportion of Vibrio in total bacteria in shrimp farming water samples:

[0058] Take 1 mL of shrimp culture water, centrifuge at 12000 rpm for 2 min, discard the supernatant, and precipitate for DNA extraction. The DNA is detected using the PCR reagent in Example 1. The PCR reaction program is: 50°C, 2 min; 95°C, 2 min; 95°C, 5 sec, 60°C, 30 sec, 45 cycles, FAM and VIC channels detect fluorescence, and the sample type is set to "to be tested". After the experiment, the standard curve of Example 2 is imported, the CT value is converted into a concentration value, and the concentration value ratio of the FAM and VIC fluorescence channels is calculated. Take another 1 mL of shrimp culture water and dilute it with physiological saline 100 and 1000 times, take 1 mL of the dilution and apply it on a TCBS plate and a nutrient agar plate, respectively, and culture at 32°C for 24-48 hours, and calculate the number of Vibrio colonies on the TCBS plate or the number of bacterial colonies on the nutrient agar plate, respectively.

[0059] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A reagent for detecting the proportion of Vibrio in total bacteria in shrimp seedlings or shrimp breeding water, characterized in that: The reagents include: primers and probes for detecting Vibrio, primers and probes for detecting bacteria, Taq DNA polymerase, dNTP mixture, UNG enzyme, lyophilization protectant and PCR buffer; The nucleotide sequence of the Vibrio detection primer is: 5′-GGGGCTCAACCTCGGAATWG-3′; 5′-CTTCGCCACCGGTATTCCTT-3′; The nucleotide sequence of the Vibrio detection probe is: 5′-ATTTGAAACTGGCAGACTAG-3′; The nucleotide sequence of the bacterial detection primer is: 5′-GCGAAAGCGTGGGGAGC-3′; 5′-CATCGTTTACGGCGTGGAC-3′; 5′-CGCCCATTGTGCAATATTCC-3′; 5'-TGAGACACGGTCCAGACTCCTA-3'; The nucleotide sequence of the bacteria detection probe is: 5'-AACAGGATTAGATACCCTGGTA-3'; 5′-ACTGCTGCCTCCC-3′.

2. A reagent for detecting the proportion of Vibrio in total bacteria in shrimp seedlings or shrimp breeding water according to claim 1, characterized in that: The Vibrio detection probe has a FAM fluorescent reporter group at its 5' end and a MGB fluorescent quencher group at its 3' end. The bacteria detection probe has a VIC fluorescent reporter group at its 5' end and a MGB fluorescent quencher group at its 3' end.

3. A reagent for detecting the proportion of Vibrio in total bacteria in shrimp seedlings or shrimp breeding water according to claim 1, characterized in that: The lyophilization protective agent includes at least one of trehalose, sucrose, glucose, fucose, sorbitol, raffinose, mannitol, dextran, and polyethylene glycol.

4. A method for preparing a reagent for detecting the proportion of Vibrio in total bacteria in shrimp fry or shrimp breeding water as claimed in claim 1, characterized in that: The method comprises the following steps: mixing 1-5 units of Taq DNA polymerase, 5%-13% (W / V) freeze-drying protective agent, 0.1-0.5 mmol / L dNTP mixture, 0.05-0.4 umol / L of each primer and probe, 0.1-0.5 units of UNG enzyme and PCR buffer in a PCR reaction tube, and placing the PCR tube containing the fluorescent PCR reagent in a freeze dryer for freeze drying, wherein the freeze drying procedure is: -45°C, 1h; -45°C, 20Pa, 2h; -40°C, 10Pa, 10h; -20°C, 10Pa, 1h; 0°C, 10Pa, 30m; 25°C, 10Pa, 2h.

5. A method for detecting the proportion of Vibrio in total bacteria in shrimp fry or shrimp breeding water, characterized in that: The detection is carried out using the reagent for detecting the proportion of Vibrio in total bacteria in shrimp fry or shrimp breeding water as described in any one of claims 1 to 3.

6. A method for detecting the proportion of Vibrio in total bacteria in shrimp seed or shrimp breeding water according to claim 5, characterized in that: The steps include: S1. DNA was extracted from the overnight cultured Vibrio solution, and the Vibrio DNA was diluted 10 times with sterile water. The DNA solutions of four consecutive dilution gradients were simultaneously tested by PCR with reagents to prepare a fluorescent PCR amplification standard curve; S2. Extract DNA from shrimp fry or shrimp farming water samples to be tested; S3. The extracted DNA is subjected to fluorescent PCR detection using reagents; S4. Substitute the fluorescent PCR Ct value obtained in step S3 into the standard curve in step S1 to obtain the concentration values ​​of Vibrio and total bacteria, respectively, and calculate the proportion of Vibrio in the total bacteria.

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