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Molecular identification method for structure of dominant bacteria in digestive canal of aquatic animal and its special primer

A technology for dominant flora and aquatic animals, applied in biochemical equipment and methods, measurement/inspection of microorganisms, material separation, etc., can solve problems such as morphological differences, cumbersome operation processes, and the inability of artificial culture of microorganisms, so as to overcome uncertainty Sexuality and accurate results

Inactive Publication Date: 2006-10-04
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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Problems solved by technology

However, most of these studies focus on the intestinal contents or intestinal tubes containing chyme, and less attention is paid to the intestinal wall flora alone, and the research reports on the gastric flora of aquatic animals are also rare; in addition, these studies All are based on the conventional artificial culture method, which has certain limitations (Feng Yanglian, Beijing: Science Press, 2004, 114-130): ①Many microorganisms in nature cannot be artificially cultivated; ②Most strictly anaerobic microorganisms have not yet been identified and described ; ③ The morphology and characteristics of artificially cultivated microorganisms are significantly different from those of the natural bacteria; ④ The operation process is cumbersome (Ji, N., et al. J. Microbiol. Methods 2004.57: 409-413), for example: it can be cultivated in water environment Bacteria only account for 1% of the total (Amann, R.I., et al.Microbiol.Reviews 1995.59:143-169); fish body surface can cultivate microorganisms less than 0.01% of the total amount (Bernadsky, G., Rosenberg, E., MicrobialEcol .1992.24: 63-74); 50%-89% of rainbow trout intestinal bacteria cannot be cultivated, and 20% of the cultureable bacteria cannot be qualitatively analyzed (Huber, I., et al.J.Applied Microbiol.2004.96: 117 -132)
It can be inferred that the comprehensive information of the digestive tract flora of aquatic animals cannot be obtained by conventional artificial culture methods, which will greatly limit the in-depth research on the digestive tract flora of aquatic animals

Method used

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  • Molecular identification method for structure of dominant bacteria in digestive canal of aquatic animal and its special primer
  • Molecular identification method for structure of dominant bacteria in digestive canal of aquatic animal and its special primer
  • Molecular identification method for structure of dominant bacteria in digestive canal of aquatic animal and its special primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1. Analysis of the structure of the dominant bacterial flora in the digestive tract of seawater fish-Sichuan snapper (Lutjanus sebae)

[0038] 1. Prepare samples

[0039] Healthy Sichuan snapper (weight: 241.9g) was randomly selected from marine culture cages in Lingshui Xincun Harbor, Hainan Province. The water temperature was 30°C and the seawater salinity was 33‰ when the samples were measured. The experimental fish were fed with single-extruded feed ( Containing 8.2% moisture, 45.0% crude protein and 8.5% crude fat), fed 3-4 times a day, and sampled at 15:10 after feeding at 13:00-14:00 on the sampling day. When sampling, use a dipping net to quickly remove the fish from the cage, beat the head with scissors to kill, then put the fish in a sterilized plastic sealed bag, store it in ice, and transfer it to the laboratory within 3 hours, and within 12 hours Process fish samples.

[0040]Fish samples are processed on a sterile operating table. First, steriliz...

Embodiment 2

[0069] Example 2. Analysis of the structure of the dominant bacterial flora in the digestive tract of freshwater fish-Oreochromis niloticus×O.aureus

[0070] The structure analysis of the digestive tract dominant flora of freshwater fish-Oni tilapia (Oreochromisniloticus×O.aureus) is carried out in the same way as in Example 1. The difference in the method is that when the sample is prepared in step 1, the wall of the digestive tract is rinsed The sterile flushing solution used in the process was changed to sterile water; the primer sequence for amplifying the 16s rDNA v6-v8 region fragment in step 3 was V 6 F1 and V 6 R.

[0071] Results The partial structure of the dominant flora in the intestinal wall of the tilapia was as follows: Aeromonas hydrophila, with a relative abundance of 21.3%; Escherichia coli, with a relative abundance of 12.5%; Photobacterium fluorescens, with a relative abundance of 4.0%. The rest of the dominant flora can be obtained by analogy in the same...

Embodiment 3

[0072] Embodiment 3, Crustacean-vannamei white shrimp (Penaeus vannamei) digestive tract dominant flora structure analysis

[0073] The structure analysis of the digestive tract dominant flora of crustacean-Penaeus vannamei (Penaeus vannamei) is basically the same as in Example 1. The difference in the method is that when preparing the sample in step 1, the sterile bacteria used when washing the digestive tract wall The flushing solution is replaced with sterilized and filtered aquaculture water; the primer sequence for amplifying the 16s rDNA v6-v8 region fragment in step 3 is V 6 F1 and V 6 R.

[0074] Results The partial structures of the dominant flora in the gastric wall of Penaeus vannamei were: Lactobacillus sp., with a relative abundance of 10.2%; Closteridium perfringens, with a relative abundance of 3.2%. The rest of the dominant flora can be obtained by analogy in the same way. The flora structure of other parts of the digestive tract can also be obtained by the ...

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Abstract

The disclosed molecular identification method and specific primer with upper / lower primer as SEQ ID No:1 / 2 for dominant flora structure in aquatic animal enteron comprise: 1) obtaining different fragments and contents of enteron as sample; 2)extracting sample total DNA; 3) with former DNA as template, taking PCR amplification; 4) taking denaturized gradient gel electrophoresis to target fragment, obtaining DGGE fingerprint spectrum by Ag dyeing, and setting another sequenching group; 5) taking cluster and semi-quantitative analysis; 6) recovering dominant strip as template for PCR amplification and sequenching; 7) comparing and analyzing the result to determine structure.

Description

technical field [0001] The invention relates to a molecular identification method and special primers for the structure of the dominant bacterial group in the digestive tract of aquatic animals. Background technique [0002] There are a large number of bacterial communities in the digestive tract of aquatic animals. The dominant bacteria in the digestive tract of healthy animals secrete extracellular enzymes (Ghosh, K., et al. The Israeli Journal of Aquaculture 2003.55(1): 13-21); ② synthesize vitamins (Sugita, H., et al.Aquaculture 1991.92: 267-276); ③synthesis of short-chain fatty acids (Li Jing, master's thesis of Huazhong Agricultural University, 2003); ④secretion of acid bacteria [0003] (Acidoline) (Sugita, H., et al. Fish. Sci. 2002.68 (5): 1004-1011) and other pathways play an important role in the normal life activities of the body. Smith began to study fish bacteria as early as the 1930s, and began to study fish gut bacteria in the 1950s (Liston, J., J.Gen.Microb...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N30/02
Inventor 周志刚姚斌苏永全石鹏君王征南孟昆何旭夙
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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