Fluorescent quantitative PCR detection kit of Pseudomonas plecoglossicida, and detection method thereof

A kind of Pseudomonas ayu, detection kit technology, applied in the directions of microorganism-based method, microbial determination/inspection, biochemical equipment and method, etc. sexual effect

Inactive Publication Date: 2015-02-04
NINGBO UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

We have found the bacteria in various sizes of large yellow croakers cultured in seawater. Since the bacteria live in the viscera (liver, spleen, kidney, etc.) of the fish, the main symptom is white nodules appearing in the viscera, but there are no obvious symptoms on the body surface in the early stage. Bringing difficulties to fish disease detection and control

Method used

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  • Fluorescent quantitative PCR detection kit of Pseudomonas plecoglossicida, and detection method thereof
  • Fluorescent quantitative PCR detection kit of Pseudomonas plecoglossicida, and detection method thereof
  • Fluorescent quantitative PCR detection kit of Pseudomonas plecoglossicida, and detection method thereof

Examples

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Embodiment 1

[0017] 1. Fluorescent quantitative PCR detection kit for Pseudomonas ayumi: including SYBR Premix Ex Taq TM 10.0 μl of II (2×) special reagent, 0.8 μl of upstream primer solution and downstream primer solution with a concentration of 10 μM, the upstream and downstream primers are based on the transcriptional spacer sequence in the rpoD gene of Pseudomonas ayutthus with accession number JN688867 as the template, the The sequence size is 743bp, and the above-mentioned pair of Pseudomonas ayutii primers with high specificity were designed with the primer design software Beacon Designer7, and the primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0018]2. Preparation of total DNA template of Pseudomonas ayumi: put 1.5ml of the cultured Pseudomonas ayuba suspension in a microcentrifuge tube and centrifuge at 6000rpm for 5min, discard the supernatant, add 450μl of TE Buffer solution and the concentration 20 μl of 50 mg / ml lysozyme, placed in a 37°C water bath for...

Embodiment 2

[0021] The DNA template solution was diluted to a concentration of 10 3 --10 7 copies / μl concentration gradient, carry out the same fluorescent quantitative PCR detection of embodiment 1, through amplification curve and melting curve similarity comparison, 10 3 --10 7 copies / μl concentration gradients are similar as figure 1 Schematic representation of the amplification curve shown and figure 2 Shown melting curve, the sensitivity of Pseudomonas ayumi fluorescent quantitative PCR detection kit of the present invention can reach 10 3 copies / μl.

[0022] According to the Ct value of Examples 1 and 2, with the Ct value as the ordinate, and the Pseudomonas ayumi plasmid template solution concentration copy number logarithmic value (Log copies) as the abscissa, it can be drawn as image 3 The standard curve of Pseudomonas ayumi fluorescent quantitative PCR detection shown, wherein the Ct value of Example 1 is 14.5, and the Ct value of Example 2 is respectively 31.60, 28.42, 2...

Embodiment 3

[0024] Grind the viscera of large yellow croaker into tissue fluid, take 1.5ml of tissue fluid and put it in a microcentrifuge tube, centrifuge at 6000rpm at room temperature for 2min, add sterile water to the sediment to half the tube, centrifuge at 6000rpm at room temperature for 5min, add sterile water to the sediment Water to half tube, centrifuge at 6000rpm at room temperature for 5min, then add TE Buffer solution to the pellet, centrifuge at room temperature at 6000rpm for 5min, discard supernatant to obtain bacterial extract; then follow the same method as in Example 1 Carry out total DNA template preparation and fluorescent quantitative PCR detection, with such as figure 1 and figure 2 The amplification curve and melting curve are basically the same, and the Ct value is 31.7, indicating that the large yellow croaker has Pseudomonas ayutii, and the detected Ct value is substituted into the established standard curve of Pseudomonas ayutii DNA , resulting in a DNA conte...

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Abstract

The invention discloses a fluorescent quantitative PCR detection kit of Pseudomonas plecoglossicida, and a detection method thereof. The kit comprises 10.0mul of an SYBR Premix Ex TaqTMII reagent, 0.8mul of an upstream primer solution having a concentration f 10muM, and 0.8mul of a downstream primer solution having a concentration of 1.0muM. The detection method comprises the steps of bacterium extraction, bacterium enzymatic hydrolysis, DNA crude extract preparation, DNA crude extract purification and detection. The detection kit can sensitively, rapidly, quantitatively and specifically detect early-stage Pseudomonas plecoglossicida, and the qualitative and quantitative sensitivities can reach 10<3>copies; and the detection kit can detect whether aquatic farming animals are infected with the Pseudomonas plecoglossicida nor not, the content of the Pseudomonas plecoglossicida in the farming water environment, whether a farming feed and a fresh bait are polluted by the Pseudomonas plecoglossicida or not, and the like.

Description

technical field [0001] The invention relates to a PCR detection technology for bacteria, in particular to a fluorescent quantitative PCR detection kit and a detection method for Pseudomonas ayumi. Background technique [0002] Pseudomonas plecoglossicida was first reported as a bacterium of sweetfish in the 1990s. In recent years, it has been found that the bacterium is also highly pathogenic to large yellow croakers. The continuation of the death epidemic is long, the infection is strong, the mortality rate is high, and the hazard is large. The average incidence rate is as high as 30%, and the average mortality rate reaches more than 60%. We have found the bacteria in large yellow croakers of various sizes cultured in seawater. Since the bacteria live in the viscera (liver, spleen, kidney, etc.) It has brought difficulties to fish disease detection and prevention. Therefore, it is particularly important and urgent to establish the detection technology of Pseudomonas ayumi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/38
Inventor 王国良张继挺刘顺周涛
Owner NINGBO UNIV
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