Kit for diagnosing hypoxia stress of hexagrammos otakii by using intestinal bacteria Achromobacter as marker

Abstract

The invention discloses a kit for diagnosing hypoxia stress of hexagrammos otakii by taking an intestinal bacterium Achromobacter as a marker. The kit contains a PCR (Polymerase Chain Reaction) specific primer for detecting the intestinal bacterium Achromobacter of the hexagrammos otakii. According to the present invention, the abundance of Achromobacter is significantly reduced after the Hexagrammos otakii is subjected to the hypoxia stress, such that the kit for diagnosing the hypoxia stress of the Hexagrammos otakii by using the Achromobacter as the marker is invented; according to the method, the relative expression quantity of the intestinal bacteria Achromobacter of the Hexagrammos otakii can be detected only by noninvasively collecting intestinal secretions of the Hexagrammos otakii, so that the hypoxia stress in Hexagrammos otakii culture is accurately diagnosed, and basic data is provided for preventing hypoxia in high-density Hexagrammos otakii culture.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, in particular to a method for Achromobacter A kit for diagnosing hypoxia stress in Hexagram ottaki using markers. Background Art

[0002] With the Otaki six-line fish ( Hexagrammos otakii ) The scale of aquaculture and increasing its aquaculture density have become research goals. At the same time, due to the low oxygen tolerance of the bighead carp, the dissolved oxygen content in the aquaculture water has become one of the important environmental factors that cannot be ignored. At present, the main method for monitoring the dissolved oxygen content in aquaculture water is to set up dissolved oxygen monitoring equipment around the aquaculture pond, which has the following problems: 1. It cannot accurately detect the dissolved oxygen status of the entire water body, especially the dissolved oxygen status of the lower water in the pool where the bigeye fish likes to live, so that it cannot timely discover the hypoxia stress caused by local hypoxia in the water body to the bigeye fish, which has a negative impact on the growth, reproduction, immunity and other aspects of the fish, and in severe cases will bring huge losses to the aquaculture industry; 2. The water quality dissolved oxygen monitoring equipment works in a high humidity and high salt environment for a long time, has a short service life, and needs to be replaced regularly, which increases the investment cost of aquaculture equipment.

[0003] Achromobacter Achromobacter ) is a probiotic colonizing in the intestines of fish. Under normal circumstances, the abundance of Achromobacter in Hexagram ottakii is very high and fluctuates within a certain range. So far, there has been no report on a significant decrease in the abundance of Achromobacter in Hexagram ottakii after hypoxia stress, nor has there been any report on a significant decrease in the abundance of Achromobacter in Hexagram ottakii after hypoxia stress. Achromobacter A kit for diagnosing hypoxia stress in Hexagram ottaki using markers. Summary of the invention

[0004] The present invention is to solve the above technical problems existing in the prior art and provide a Achromobacter A kit for diagnosing hypoxia stress in Hexagram ottaki using markers.

[0005] The technical solution of the present invention is: a method based on intestinal bacteria Achromobacter A kit for diagnosing hypoxia stress in Hexagram otaki, containing a marker for detecting intestinal bacteria in Hexagram otaki Achromobacter Specific PCR primers.

[0006] The preferred DNA sequences of PCR specific primers are as follows: Upstream primer: F 5′-GGTAGGCGGAATGTGACAAGTAGC-3′; Downstream primer: R5′-TGCCTCAGCGTCAATAACACCATAG-3′.

[0007] The present invention is to find that after hypoxia stress, Achromobacterium ( Achromobacter ) decreased significantly, thus inventing Achromobacter A kit for diagnosing hypoxia stress in Hexagram otaki. The intestinal bacteria of Hexagram otaki can be detected by non-invasively collecting intestinal secretions of Hexagram otaki. Achromobacter The relative expression levels of the genes involved in the expression of these genes can be used to accurately diagnose hypoxia stress in the culture of Hexagram ottaki, and provide basic data for preventing hypoxia in high-density culture of Hexagram ottaki. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 The intestinal bacteria of each group of Hexapoda taki in the embodiments of the present invention are Achromobacter Electrophoresis diagram of .

[0009] Figure 2 The intestinal bacteria of each group of Hexapoda taki in the embodiments of the present invention are Achromobacter Schematic diagram of relative expression levels. DETAILED DESCRIPTION

[0010] The present invention is based on intestinal bacteria Achromobacter A kit for diagnosing hypoxia stress in Hexagram otaki, containing a marker for detecting intestinal bacteria in Hexagram otaki Achromobacter The PCR specific primers have the following DNA sequences: Upstream primer: F 5′-GGTAGGCGGAATGTGACAAGTAGC-3′; Downstream primer: R5′-TGCCTCAGCGTCAATAACACCATAG-3′.

[0011] experiment: 1. Experimental animals: Healthy Hexalocista otakii, weighing 117.35 ± 2.11 g, were provided by the Key Laboratory of Fish Biology and Aquaculture, Dalian Ocean University.

[0012] 2. Experimental materials: Fluorescence quantitative PCR reagent (TIANGEN, FP207-01), use YSI (USA, 6600) to detect dissolved oxygen concentration.

[0013] 3. Temporary management of Otaki six-line fish Healthy Otaki hexapods were kept in three 100 L plastic tanks, 30 in each tank, and fed twice a day at 8:00 and 16:00. One-third of the water was changed every day. The water temperature was 12 ± 0.6℃, and the dissolved oxygen concentration was 7.3 mg / L. The fish were kept for 7 days. 4. Sample Collection After the temporary rearing, the abdomen of the bigeye hexapod was gently squeezed, and the intestinal contents were collected with a sterile pipette as the control group; nitrogen was then filled into each tank to maintain the dissolved oxygen concentration at 3.5±0.2 mg / L. A fish was randomly taken from each breeding tank on the 3rd, 9th, and 15th days, and the abdomen was gently squeezed, and the intestinal contents were collected with a sterile pipette as the hypoxia 1-3 groups.

[0014] 5. Use the intestinal contents obtained from each group as templates and perform PCR tests according to the following steps: (1) Extraction of total DNA from the intestinal contents of Hexacanthus otakii; (2) Then, using total DNA as a sample, PCR reaction was performed using universal primers for intestinal bacteria to obtain 16rDNA; (3) Amplifying 16S rDNA using the PCR primers of the embodiment of the present invention; the PCR reaction system: 12.5 μl 2×Taq PCR MasterMix, 3 μl BSA (2 ng / μl), 2 Primer (5 μM), 2 μl template DNA, and 5.5 μl ddH2O; reaction parameters: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 45 s, annealing at 55°C for 50 s, extension at 72°C for 45 s, 32 cycles; extension at 72°C for 10 min.

[0015] (4) Agarose gel electrophoresis a. Take 0.25g agarose and add 1×TAE, then heat in a microwave to dissolve and prepare 1% agarose gel. Let stand at room temperature for 3 min before adding nucleic acid dye. Seal both ends of the electrophoresis template, pour in the agarose gel solution, insert the comb, pull out the comb after condensation, and place the electrophoresis gel in the electrophoresis tank; b. Take 10 μL of the above PCR products from the control group and hypoxia groups 1-3 respectively, add 1 / 5 volume of loading buffer, mix well and add the solution to the sample wells, and add Maker to the first lane at the same time; maintain the power at 2-4 V / cm, and stop electrophoresis when bromophenol blue reaches 2 / 3 of the bottom of the gel.

[0016] c. After electrophoresis, take out the agarose gel and then put it into a gel imager for observation and photography.

[0017] The electrophoresis results are as follows Figure 1 shown. Figure 1 M in the figure is Marker, 1 is 0h, 2 is 3d, 3 is 9d, 4 is 15d, i.e. 1 is control group, 2 is hypoxia group 1, 3 is hypoxia group 2, 4 is hypoxia group 3. Figure 1 It can be seen that specific bands were detected in the intestine of the fish and the brightness of the bands decreased after hypoxia stress.

[0018] rightFigure 1 Image J software was used to quantify the gray value, and the obtained data were then normalized with the control group to obtain the following Figure 2 Different hypoxia groups 1-3 shown Achromobacter The relative expression level. Figure 2 The results showed that after 3, 9 and 15 days of hypoxia stress Achromobacter The relative expression levels of the control group were 0.72, 0.38 and 0.13 times, which means that the Achromobacterium ( Achromobacter ) begins to decrease significantly, so it can be used as a hypoxic stress marker to prepare a detection kit.

Claims

1. A kind of intestinal bacteria Achromobacter A kit for diagnosing hypoxia stress in Hesperus otakii using markers, characterized in that Contains intestinal bacteria detected in Otaki six-line fish Achromobacter Specific PCR primers.

2. The method according to claim 1 Achromobacter A kit for diagnosing hypoxia stress in Hesperus otakii using markers, characterized in that The DNA sequence of the PCR specific primer is as follows: Upstream primer: F 5′-GGTAGGCGGAATGTGACAAGTAGC-3′; Downstream primer: R5′-TGCCTCAGCGTCAATAACACCATAG-3′.

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