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

Primer for performing double-PCR early and rapid detection on streptococcus agalactiae and streptococcus iniae as well as application thereof

A technology of Streptococcus iniae and Streptococcus nisella, applied in the field of microbial detection, can solve the problems of high cost, missed detection or false detection, false positive, etc., and achieve the effect of long solution cycle, low detection cost and good specificity

Active Publication Date: 2019-05-28
JINAN UNIVERSITY
View PDF1 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both Streptococcus iniae and Streptococcus agalactiae can cause fish septicemia, meningitis and other diseases. It is difficult to accurately judge which kind of Streptococcus is caused by the external symptoms and the colony and shape of the two pathogenic bacteria. This will seriously affect the effective management of the disease
[0003] At present, the conventional diagnostic methods for Streptococcus tilapia are difficult to meet the requirements for rapid diagnosis of infected fish samples. At the same time, some physiological reactions may change with changes in the external environment, which can easily lead to misjudgment.
Ordinary PCR can only detect one gene at a time, which may cause missed or false detections
Fluorescent antibody technology and ELISA are expensive and require skilled technical operations; LAMP has false positives; quantitative PCR requires expensive instruments, professional operations, and high costs.

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
  • Primer for performing double-PCR early and rapid detection on streptococcus agalactiae and streptococcus iniae as well as application thereof
  • Primer for performing double-PCR early and rapid detection on streptococcus agalactiae and streptococcus iniae as well as application thereof
  • Primer for performing double-PCR early and rapid detection on streptococcus agalactiae and streptococcus iniae as well as application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 The establishment of a double PCR method for rapid detection of Streptococcus agalactiae and Streptococcus dolphin

[0046] (1) Template preparation: select Streptococcus agalactiae, Streptococcus inus, Aeromonas hydrophila, Edwards tarda, Aeromonas victorini, Vibrio vulnificus, and mild from the strains stored at -80°C Aeromonas, Vibrio alginolyticus, and Escherichia coli were inoculated on the brain heart infusion agar medium under aseptic conditions, and cultured at a constant temperature at 28°C. After 24 hours, the cultured bacteria were collected and cultured respectively. Refer to Gram-positive bacteria DNA extraction method extracts the genomic DNA of various bacteria, and uses the DNA extraction kit (Dalian Bao Biological Engineering Co., Ltd.) to extract the genomic DNA and use it as a reaction template;

[0047] (2) Primers design and synthesis: According to the 16S rDNA partial sequence of Streptococcus agalactiae in GenBank, primers P-1 and P-2 are des...

Embodiment 2

[0056] Example 2 The detection of DNA sensitivity of Streptococcus agalactiae and Streptococcus dolphin by double PCR includes the following steps:

[0057] (1) Take 2-3 mL of activated Streptococcus agalactiae and Streptococcus dolphinus respectively, extract DNA with a commercial DNA extraction kit, and measure its concentration with a spectrophotometer;

[0058] (2) Dilute the genomic DNA of Streptococcus agalactiae to 9.84×10 -2 , 9.84×10 -3 , 9.84×10 -4 , 9.84×10 -5 , 9.84×10 -6 ng / μL, respectively dilute the genomic DNA of Streptococcus inus to 9.30×10 -2 , 9.30×10 -3 , 9.30×10 -4 , 9.30×10 -5 , 9.30×10 -6 ng / μL.

[0059] (3) such as figure 2 As shown, the DNA sensitivity of Streptococcus agalactiae and Streptococcus dolphin are 9.84×10 -5 ng / μL and 9.30×10 -5 ng / μL.

Embodiment 3

[0060] Example 3 Double PCR detection of single bacteria sensitivity of Streptococcus agalactiae and Streptococcus dolphin includes the following steps:

[0061] (1) Take the activated Streptococcus agalactiae and Streptococcus dolphin bacteria respectively, wash twice with 0.9% saline, dilute by ten times gradient, and count on the plate;

[0062] (2) Dilute the concentration of Streptococcus agalactiae to 2.76×10 respectively 6 , 2.76×10 5 , 2.76×10 4 , 2.76×10 3 , 2.76×10 2 , 27.6cfu / mL, dilute the concentration of Streptococcus dolphin to 2.51×10 6 , 2.51×10 5 , 2.51×10 4 , 2.51×10 3 , 2.51×10 2 , 25.1cfu / mL.

[0063] (3) Using the system and procedure of Example 1, boil the bacterial liquid prepared above for 10 minutes, and take the supernatant as a template for the sample to be tested;

[0064] (4) such as image 3 As shown, the sensitivity of pure bacteria of Streptococcus agalactiae and Streptococcus dolphin are 2.76×10 2 cfu / mL and 2.51×10 2 cfu / mL.

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
Sensitivityaaaaaaaaaa
Login to View More

Abstract

The invention discloses a primer for performing double-PCR early and rapid detection on streptococcus agalactiae and streptococcus iniae as well as application thereof. A sample is detected by utilizing the primer disclosed by the invention, and streptococcus agalactiae and streptococcus iniae in the ample can be simultaneously specially detected at one time. A detection method built by the invention can be used for detecting the streptococcus agalactiae of which gene group content is 9.84x10<-5>ng / microliter and the streptococcus iniae of which gene group content is 9.30x10<-5>ng / microliter,and the streptococcus agalactiae with bacteria liquid concentration of 2.76x102cfu / mL and the streptococcus iniae bacteria liquid concentration of 2.51x102cfu / mL. The method disclosed by the invention has the advantages that the method is higher in specificity, sensitivity, repeatability and stability on quickness and correctcness in a detection process of tilapia samples, early molecular warningof tilapia streptococcus diseases can be realized, the application prospect is wide, and an accurate and effective mean is provided for quick diagnosis of the tilapia streptococcus diseases and investigation of epidemiology.

Description

Technical field [0001] The invention belongs to the field of microbial detection, and in particular relates to a primer for early and rapid detection of Streptococcus agalactiae and Streptococcus dolphinus by double PCR and its application. Background technique [0002] Tilapia is one of the aquatic products that are widely cultured worldwide. According to incomplete statistics, in 2014, China's tilapia farming output reached 1,698,500 tons, accounting for about 32% of the global farming output. In recent years, tilapia streptococcal disease has become more and more serious, which has caused serious economic losses to the tilapia breeding industry. Studies have shown that the main pathogens of streptococcus agalactiae are Streptococcus agalactiae and Streptococcus dolphin. In recent years, the most serious damage is Streptococcus agalactiae. Streptococcus agalactiae is a kind of human, animal and fish. The pathogenic bacteria can infect sea bream, tilapia and other freshwater fi...

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): C12Q1/689C12Q1/686C12Q1/14C12N15/11C12R1/46
Inventor 崔淼黎晶晶张辉杰张其中许德麟
Owner JINAN UNIVERSITY
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