Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

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

A technology of Pseudomonas ayumi and a detection kit, which is applied in the direction of microorganism-based methods, microorganism measurement/inspection, biochemical equipment and methods, etc., can solve problems such as difficult detection and prevention of fish diseases, and achieve wide application sexual effect

Inactive Publication Date: 2013-09-04
NINGBO UNIV
View PDF2 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • 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

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] 1. Fluorescence quantitative PCR detection kit for Pseudomonas ayurus: including SYBR Premix Ex Taq TM II (2×) special reagent 10.0μl, the upstream primer solution and the downstream primer solution with a concentration of 10μM 0.8μl each, the upstream and downstream primers are based on the internal transcription spacer sequence of the rpoD gene of Pseudomonas ayu with the accession number JN688867. The sequence size is 743bp. The above-mentioned pair of Pseudomonas ayus primers with higher specificity were designed with the primer design software Beacon Designer7. The primers were synthesized by Shanghai Shenggong Biological Engineering Co., Ltd.

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

Embodiment 2

[0021] Dilute the DNA template solution to a concentration of 10 3 --10 7 Copies / μl concentration gradient, the same fluorescence quantitative PCR detection as in Example 1 was performed, and the similarity of the amplification curve and melting curve was compared, 10 3 --10 7 The concentration gradients of copies / μl are similar, such as figure 1 Schematic diagram of the amplification curve shown and figure 2 As shown in the melting curve, the sensitivity of the fluorescence quantitative PCR detection kit for Pseudomonas ayuus of the present invention can reach 10 3 copies / μl.

[0022] According to the Ct values ​​of Examples 1 and 2, taking the Ct value as the ordinate, and the log copies of the Pseudomonas ayurus plasmid template solution concentration as the abscissa, we can get as image 3 As shown in the standard curve diagram of the fluorescence quantitative PCR detection of Pseudomonas ayurus, the Ct value of Example 1 is 14.5, and the Ct value of Example 2 is 31.60, 28.42...

Embodiment 3

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Sensitivityaaaaaaaaaa
Login to View More

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 ayu. Background technique [0002] Pseudomonas plecoglossicida (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 croaker. Death has a long-lasting epidemic, strong infectivity, high mortality, and great harm. The average morbidity rate is as high as 30%, and the average mortality rate is over 60%. We have found this bacteria in various sizes of large yellow croaker in marine culture. Because the bacteria reside in the internal organs of the fish (liver, spleen, kidney, etc.), the main symptoms are white nodules in the internal organs, but there are no obvious symptoms on the body surface in the early stage. This brings difficulties to fish disease detection ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/68C12Q1/04C12R1/38
Inventor 王国良张继挺刘顺周涛
Owner NINGBO UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com