Fluorescent probe PCR (polymerase chain reaction) method for simultaneously detecting Edwardsiella tarda and Edwardsiella ictaluri

A slow-Edward, detection method technology, applied in the field of microbial detection, can solve the problems of cumbersome and time-consuming detection methods, and achieve the effects of shortening detection time, reducing cross-contamination, and improving detection efficiency

Inactive Publication Date: 2014-11-26
INSPECTION & QUARANTINE TECH CENT OF XIAMEN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
View PDF3 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the Edwardsiella gene detection technology established at home and abroad includes the common PCR detection technology for detecting Edwardsiella tarda, and the common PCR detection technology for detecting Edwardsiella spp., these methods need many times to detect these two bacteria, and G...

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 probe PCR (polymerase chain reaction) method for simultaneously detecting Edwardsiella tarda and Edwardsiella ictaluri
  • Fluorescent probe PCR (polymerase chain reaction) method for simultaneously detecting Edwardsiella tarda and Edwardsiella ictaluri
  • Fluorescent probe PCR (polymerase chain reaction) method for simultaneously detecting Edwardsiella tarda and Edwardsiella ictaluri

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] 1. Main instruments and reagents

[0043] Fluorescence quantitative PCR instrument: Model 7300 of ABI Company of the United States. Nucleic acid purification system: KingFisher ml type, Thermo Company, USA. Nucleic acid magnetic bead method extraction kit: Mag-Si Tissue DNA kit from Omega Biotechnology Company, USA. Real-time fluorescent PCR reaction solution: RealMasterMix (Probe) kit from Beijing Tiangen Bioengineering Co., Ltd.

[0044] 2 Primers and probes

[0045] The Edwardsiella gyrb gene sequence recorded in GenBank was selected, and Primer express 3.0 software was used to design primers in the conserved region of the gene sequence, and a TaqMan probe was designed in the specific region of Edwardsiella species, and its specificity was tested by NCBI Blast. Two sets of specific primers and TaqMan-MGB probes were designed and screened. The 5' end of the detection probe of Edwardsiella tarda was labeled with the FAM fluorescent reporter group, and the 5' end of...

Embodiment 2

[0066] A detection kit is used to detect the real-time fluorescent PCR detection of Edwardsiella tarda and Edwardsiella spp. simultaneously, and said kit contains the following primers and probes:

[0067] Table 1 Sequences of primers and probes for detection of Edwardsiella tarda and Edwardsiella chrysalis

[0068]

[0069] Among them, E.t-F1 represents the upstream primer of Edwardsiella tarda, E.t-R1 represents the downstream primer of Edwardsiella tarda, E.i-F2 represents the upstream primer of Edwardsiella chrysalis, and E.i-R2 represents Seq ID No.3 as Edwardsi spp. Downstream primers for bacteria.

[0070] Among them, the FAM labeled at the 5' end of the E.t probe is a fluorescent reporter group, the VIC labeled at the 5' end of the E.i probe is a fluorescent reporter group, and the TAMRA labeled at the 3' end of the E.t probe and the E.i probe is a fluorescent quencher group. group.

[0071] The kit also includes instructions for use.

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 microbe detection method and detection kit, particularly a fluorescent probe PCR (polymerase chain reaction) method for simultaneously detecting Edwardsiella tarda and Edwardsiella ictaluri. The method comprises the following step: carrying out real-time fluorescence PCR reaction by using bacteria DNA (deoxyribonucleic acid) as a template by utilizing specific probes and primer pairs. The method is characterized in that the primer primers are respectively sequences disclosed as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4; the SEQ ID NO.1 is SEQ ID NO.1: 5'-CTGCCAAGCAGGGACGTAA-3', SEQ ID NO.2: 5'-CGAGGAGAGCATCTTGTCGAA-3', SEQ ID NO.3: 5'-TCCATTCGTCTGTGCGACAA-3', and SEQ ID NO.4: 5'-AAAACACGTTCGGATGGATTG-3'; the probes are nucleotide sequences which is identical to or complementary with the sequences between the primer pair on the Edwardsiella tarda or Edwardsiella ictaluri genome DNA; the 5' end and 3' end of the probes are respectively labeled with a fluorescence report group and a fluorescent quenching group; the probes are sequences disclosed as SEQ ID NO.5 and SEQ ID NO.6; and the SEQ ID NO.5 is 5'-ATCCTCAACGTCGAGAAGGCGCG-3', and the SEQ ID NO.6 is 5'-CACTATGAGGGTGGGATCAAGGCGTTT-3'.

Description

technical field [0001] The invention relates to a detection method and a detection kit for microorganisms, in particular to primers and probe sequences and a kit for simultaneous detection of Edwardsiella tarda and Edwardsiella spp. for real-time fluorescent PCR detection. Background technique [0002] Edwardsiella tarda ( Edward Siella sp.) is widely distributed in seawater and freshwater, infects various farmed aquatic animals, and is extremely harmful to aquaculture. It is pathogenic to eels, catfish, squid and other animals, and can cause serious economic losses. The pathogenic bacteria that cause fish disease are mainly Edwardsiella tarda ( E. tarda ) and Edwardsiella ( E. ictaluri ). It is now known that Edwardsiella tarda is an important pathogenic bacterium of a variety of freshwater and seawater cultured fish. It causes diseases of fish kidney and liver abscess lesions, and the loss is serious. It is a pathogenic bacterium that is more harmful in aquaculture a...

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): C12Q1/68C12Q1/04
CPCC12Q1/686C12Q2563/107C12Q2561/113
Inventor 郭书林陈信忠贾鹏龚艳清杨俊萍陈炳颖孔繁德
Owner INSPECTION & QUARANTINE TECH CENT OF XIAMEN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap