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Method for counting archenteric flora of aquatic animals and special primer thereof

An aquatic animal and digestive tract technology, applied in biochemical equipment and methods, microorganism-based methods, microorganisms, etc., can solve the problems of strict aseptic operation requirements, high labor intensity, complicated procedures, etc., and achieve specificity and reliability. Strong, avoid background interference, achieve the effect of multiple reactions

Inactive Publication Date: 2009-12-02
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The plate colony counting method has the characteristics of low cost, simple method, and convenient operation, but the method has the following problems: time-consuming, strict aseptic operation requirements, cumbersome procedures, high labor intensity, and the measurement results are affected by factors such as culture conditions ; When the amount of bacteria in the sample is too small, it can not represent the amount of bacteria in the whole sample (Jiang Zenghui, Wang Xingyan, 2005. Introduce several new methods for detecting the total number of bacteria in water. Urban Public Utilities, 19 (2) 15-16)
[0005] The DAPI staining method can be successfully applied to the counting of bacterial flora in the water environment, but for samples from complex environments, such as fish intestines, because they contain a large amount of digestive content, intestinal epithelial tissue, etc., there will be background interference color, which is serious Affect the total bacterial count (Sabrina N.Hymell, Craig J.plante., 1998. Improved method of bacterial enumeration in sandy and deposit-feeder gut sediments using the fluorescent stain 4, 6-diamidino-2-phenylindole (DAPI). MARINEECOLOGY PROGRESS SERIES 173 299-304.)

Method used

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  • Method for counting archenteric flora of aquatic animals and special primer thereof
  • Method for counting archenteric flora of aquatic animals and special primer thereof
  • Method for counting archenteric flora of aquatic animals and special primer thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1, the acquisition of specific primer pair

[0029] The RNA polymerase β subunit is encoded by the rpoB gene, which has RNA polymerase activity and catalyzes the synthesis of RNA. The rpoB gene of most bacteria is highly conserved (Kapur V, Li LL, Iordanescu S, 1994.Characterization by automated DNA sequencing of mutations in the gene(rpoB) encoding the RNA polymerase beta subunit in rifampin-resistant Mycobacterium tuberculosis strains from New York City and Texas.J ClinMicrobiol 32(4), 1095-1098.), and is a single copy.

[0030] According to the rpoB genes of Escherichia coli, Bacillus subtilis, Serratia liquefaciens MG1, and Helicobacter pylori (NCBI accession numbers: AE000472, 2632267, 677848 and AE000625), their conserved regions are found by comparison, and their conserved regions are not 100% similar, if Designed as degenerate primers, PCR results showed non-monospecific amplification, which violated the special requirements of qtPCR for primers, so ...

Embodiment 2

[0034] Embodiment 2, tilapia intestinal flora count

[0035] Tilapia: local seed hatchery in Jiaxing, Zhejiang.

[0036] 1. Sample source

[0037] 1. Feed preparation

[0038] Prepare three kinds of isonitrogenous and isoenergetic experimental feeds: control group, yeast culture group and antibiotic group. The feed was extruded and granulated by an extruder (Jiangsu Muyang Group, Yangzhou, Jiangsu) at 120°C for 15s. The feed formulation and active ingredients are listed in Table 1. For the determination methods of moisture, crude protein and crude fat content in feed, refer to literature (Xie S, CuiY, Yang Y, Liu J. Energy budget of Nile tilapia (Oreochromis niloticus) inrelation to ration size. Aquaculture 1997; 154: 57-68. ).

[0039] Table 1 Feed formula and chemical composition (%)*

[0040]

[0041] C: control group; A: antibiotic group; Y: yeast culture group;

[0042] 1 Provided by Zhejiang Xinxin Feed Co., Ltd.;

[0043] 2 Provided by Zhejiang Yiwu Huatai ...

Embodiment 3

[0109] Embodiment 3, the verification of detection result

[0110] Verification experiment: use two kinds of bacteria (one G+, one G-) mixed with muscle as a standard curve, and another four kinds of bacteria (two G+, two G-), but the total proportion is not more than 1 / 2, Prepare 5 modes: all G+, all G-, 2G+1G-, 1G+2G-, 2G+2G- to be verified.

[0111] 1. Preparation of standard curve

[0112] (1) Strain culture and counting

[0113] Two strains (Pseudomonas fluorescens CGMCC 1.55 and Bacillus subtilis CGMCC 1.1630) were respectively inoculated in LB liquid medium, cultured overnight at 37°C and 200 rpm, counted on a hemocytometer, and 5 × 10 7 Mix equal amounts of CFU.

[0114] (2) DNA extraction and purification

[0115] Method is with embodiment 2.

[0116] (3) QPCR

[0117] According to the 10-fold gradient dilution DNA, select 1×10 7 -1×10 3 CFUml -1 5 gradients with 3 parallels for each gradient. PCR was carried out using the specific primers obtained in Examp...

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Abstract

The invention discloses a method for counting archenteric flora of aquatic animals and a special primer thereof. The special primer provided by the invention is a primer pair consisting of nucleotide shown in a sequence 1 of a sequence table and nucleotide shown in a sequence 2 of the sequence table. The special primer can be applied to preparation of kit counting the archenteric flora of the aquatic animals or used for carrying out counting of the archenteric flora of the aquatic animals. The special primer and the method can avoid background interference, can make accurate quantification on the number of total flora in a complex micro-ecological environment, and has the characteristics of high sensitivity, stronger specificity and reliability, multiple reaction realization, high automation degree, non pollution, real time, accuracy and the like.

Description

technical field [0001] The invention relates to a method for counting the digestive tract flora of aquatic animals and special primers thereof. Background technique [0002] Environmental bacteria counting methods can be divided into indirect culture counting method and direct counting method (Qiu Dajun, Jiao Nianzhi, Qian Lumin, 2004. The progress of aquatic bacteria count and bacteria size measurement technology. Taiwan Strait, 3(3) 377-385.) . [0003] The indirect culture counting method is to cultivate the bacteria in the sample under suitable culture conditions through a selective medium, so as to count the living bacteria, mainly including the plate colony counting method. The number of bacteria determined by the plate colony count method is the number of cultivable bacteria in the sample, that is, the number of viable bacteria. The plate colony counting method has the characteristics of low cost, simple method, and convenient operation, but the method has the follo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G01N21/64C12R1/39C12R1/19C12R1/125C12R1/01C12Q1/06
Inventor 周志刚何夙旭姚斌曹雅男刘玉春孟昆石鹏君杨培龙
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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