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

Reagent kit and and detection method capable of detecting three fish-derived streptococcus simultaneously

A technology of Streptococcus lactis and Streptococcus iniae, applied in the biological field, can solve the problems of high equipment requirements, expensive reagent consumables, and low accuracy, and achieve the effects of high sensitivity, low cost of reagent consumables, and strong specificity

Active Publication Date: 2020-07-10
ZHEJIANG INST OF FRESH WATER FISHERIES
View PDF6 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The present invention aims to solve the problems of long time-consuming, low efficiency, low accuracy, high requirements for instruments and equipment, expensive reagent consumables and the like in the prior art to detect the three fish-derived streptococci, and thus provides a simultaneous detection device of the present invention. Describe the kits and detection methods of three kinds of Streptococcus, which can realize the rapid detection and accurate identification of Streptococcus agalactiae, Streptococcus iniae and Streptococcus dysgalactiae

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
  • Reagent kit and and detection method capable of detecting three fish-derived streptococcus simultaneously
  • Reagent kit and and detection method capable of detecting three fish-derived streptococcus simultaneously
  • Reagent kit and and detection method capable of detecting three fish-derived streptococcus simultaneously

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 multiplex PCR detects

[0041] 1. Establishment of single-gene PCR reaction system and reaction conditions

[0042] The genomic DNAs of the three streptococci and the control strain were respectively extracted, used as templates for PCR amplification, the specificity of each primer was detected, and the PCR amplification conditions were optimized.

[0043] The 16 control strains verified for specificity are all common aquatic animal pathogens, including: Aeromonas sobria ATCC43979; Aeromonas caviae ATCC15468; Aeromonas hydrophila TPS; Aeromonas velonia MRM0908; Vibrio alginolyticus VAN100117; Vibrio parahaemolyticus VPZJ; Vibrio harveii VHHA; Vibrio mimeticum VM18531K; Bacillus hsy18920S; Elisabeth meningosepticum HBW190701B; Lactococcus gasseri ET060821; Lactococcus lactis sg-5; Enterococcus faecalis M18523-5.

[0044] With three pairs of primers described in the present invention, Sa.tkt-F and Sa.tkt-R, Si.RecA-F and Si.RecA-R, Sd.AtoB-F and Sd.AtoB-R, ...

Embodiment 2

[0069] The formation of embodiment 2 kit

[0070] The kit for simultaneous detection of three fish-derived streptococci of the invention consists of DNA extraction reagents and PCR reaction reagents.

[0071] 1. DNA extraction reagent components are 5-15mM Tris, 0.5-1.5mM EDTA pH 8.0 TE buffer; proteinase K, concentration 15-20mg / mL; 100% chloroform; 100% isopropanol; ethanol, concentration 70%; double distilled water;

[0072] 2. The PCR reaction reagents are divided into several PCR reaction tubes, and each reaction tube contains 2.5 μL of 10×PCR reaction buffer, 0.5 μL of dNTPs with a concentration of 10 μM, and 0.3 μL of Taq DNA polymerase with a concentration of 5 U / μL. 10 μM specific oligonucleotide primer Sa.tkt-F 0.25 μL, 10 μM specific oligonucleotide primer Sa.tkt-R 0.25 μL, 10 μM specific oligonucleotide primer Sd.AtoB-F 0.25 μL, 10 μM specific oligonucleotide primer Sd.AtoB-R 0.25 μL, 10 μM specific oligonucleotide primer Si.RecA-F 0.75 μL, 10 μM specific oligonu...

Embodiment 3

[0073] The sensitivity assessment of embodiment 3 multiplex PCR detection kit

[0074] Adjust the concentration of Streptococcus agalactiae, Streptococcus iniae and Streptococcus dysgalactiae to 3×109 cfu / mL, and extract DNA. Each genomic DNA was serially diluted 10 times, and the bacterial concentration corresponding to the DNA template in each test sample was 3×109 cfu / mL, 3×108 cfu / mL, 3×107 cfu / mL, 3×106 cfu / mL, 3 ×105cfu / mL, 3×104cfu / mL, 3×103cfu / mL. Using the kit of the present invention and the multiplex PCR detection method established in Example 1, the sensitivity detection of the templates with different dilutions of the three kinds of Streptococci was carried out. The result is as Figure 5 Shown, show that multiplex PCR method of the present invention detects three kinds of streptococci strong sensitivity, is 3 * 105cfu / 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

No PUM Login to View More

Abstract

The invention relates to a primer combination capable of detecting three kinds of streptococcus simultaneously. The primer combination consists of upstream and downstream primers capable of detectingstreptococcus agalactiae, as shown in SEQ ID MO:1, 2, upstream and downstream primers capable of detecting streptococcus dysgalactiae, as shown in SEQ ID MO:3, 4, and upstsream and downstream primerscapable of detecting streptococcus iniae, as shown in SEQ ID MO:5, 6. A reagent kit consists of a DNA extraction reagent and a PCR reaction reagent, wherein the DNA extraction reagent consists of a TEbuffer solution, proteinase k, chloroform, isopropyl alcohol, ethanol and redistilled water, and the PCR reaction reagent contains a reaction buffer solution, dNTPs, DNA polymerase, the 6 primers anddeionized water. A method for detecting the three kinds of streptococcus simultaneously comprises the steps of extracting DNA in samples through the extraction reagent, performing PCR amplification on the DNA through the reaction reagent, and performing gel electrophoresis on amplification products. The three kinds of streptococcus can be detected simultaneously, detection is simple, quick, highin sensitivity and high in specificity, and the primer combination and the method can meet requirements for large-scale molecular epidemiology investigation and analysis of aquatic animals and qualitysafety detection of aquatic products.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a detection technology of target DNA fragments, in particular to a rapid detection kit and a detection method for simultaneously detecting Streptococcus agalactiae, Streptococcus iniae and Streptococcus dysgalactiae. [0002] technical background [0003] Fish streptococcosis is an infectious disease caused by bacteria of the genus Streptococcus, including Streptococcus iniae, Streptococcus agalactiae and Streptococcus dysgalactiae. Streptococcus infection has a wide range of hosts, a high mortality rate, and a long duration of disease, which has caused huge economic losses to the aquaculture industry. Streptococcus agalactiae is one of the most important pathogenic bacteria in tilapia, and it can also cause sepsis in various aquatic animals such as rainbow trout, channel catfish, flounder, turbot, red sea bream and Schizothorax and meningitis. Streptococcus iniae, originally isolated ...

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
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/46
CPCC12Q1/689C12Q1/686C12Q2600/16C12Q2537/143Y02A50/30
Inventor 蔺凌云潘晓艺沈锦玉姚嘉赟尹文林曹铮刘忆瀚夏焱春
Owner ZHEJIANG INST OF FRESH WATER FISHERIES
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