Primers and method for cross primer isothermal amplification detection of acidovorax citrulli

A technology for detection of melon fruit spot bacteria and constant temperature amplification, which is applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve the problems of time-consuming and expensive instrument configuration

Inactive Publication Date: 2012-10-31
CHINESE ACAD OF INSPECTION & QUARANTINE +1
View PDF1 Cites 14 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the above detection methods have time-consuming operating procedures and expe...

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
  • Primers and method for cross primer isothermal amplification detection of acidovorax citrulli
  • Primers and method for cross primer isothermal amplification detection of acidovorax citrulli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Design and synthesis of embodiment 1 primer

[0029] Five primers were designed according to the 16S rDNA sequence of Melon fruit spot pathogen, and the primer sequences are:

[0030] ACLF3: 5'-GGCTAACTACGTGCCAGC-3'

[0031] ACLB3: 5'-ACGCATTTCACTGCTACA-3'

[0032] ACLBIP: 5'-CAGATGTGAAATCCCCGGGCTCTGCCGTACTCCAGCGAT-3'

[0033] ACDF5b1: 5'-GCAAGCGTTAATCGGAATTACT-3'

[0034] ACDF5f2: 5'-CAACCTGGGAACTGCATTTGT-3'

[0035] Among them, ACLF3 and ACLB3 are replacement primers, ACLBIP is a cross primer, ACDF5b1 is a forward detection primer with 5-terminal biotin-labeled, and ACDF5f2 is a reverse detection primer with 5-terminal FITC-labeled.

Embodiment 2

[0036] The establishment of embodiment 2 cross primer amplification method

[0037] Using the total DNA extracted from a commercial plant DNA extraction kit or the isolated bacterial liquid as a template, the 20 μl reaction system contains: 0.8 μmol / L cross primer ACLBIP; 0.1 μmol / L forward detection primer ACDF5b1, 0.3 μmol / L L reverse detection primer ACDF5f2, two displacement primers ACLF3 and ACLB3 at 0.1 μmol / L each, 0.4 mmol / L dNTP, 2 μl 10× reaction buffer; 8 units of Bst DNA polymerase, 2 mmol / L MgSO 4 , and 4 μl of target DNA.

[0038] The amplification reaction was at 63° C. for 60 min, and then the amplification product was detected. The results of the amplification products are interpreted by a commercial disposable fully enclosed rapid detection device for target nucleic acid amplification products. Interpret the results by observing the color development of the detection strip and the quality control strip of the test strip. If both the detection band and the ...

Embodiment 3

[0039] Embodiment 3 cross primer amplification method sensitivity determination

[0040] The melon fruit blight bacteria suspension quantitatively counted by plate culture was diluted tenfold, and 2 μl of the bacterial pure culture of each gradient was used as a reaction template to test the detection sensitivity of the method. After testing, the tested 3.7× 10 2 The detection band and the quality control band can be observed in the bacteria solution with a concentration above cfu / ml, while 3.7×10 2 There are only quality control strips below the cfu / ml concentration, so the sensitivity of the method is determined to be 3.7×10 2 cfu / ml, because 2 μl was added to each reaction, the lowest sensitivity after conversion was 7.4 bacteria.

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 provides primers and a method for cross primer isothermal amplification detection of acidovorax citrulli. Nucleotide sequences of the primers areACLF3:5'-GGCTAACTACGTGCCAGC-3'ACLB3:5'-ACGCATTTCACTGCTACA-3'ACLBIP:5'-CAGATGTGAAATCCCCGGGCTCTGCCGTACTCCAGCGAT-3'ACDF5b1:5'-GCAAGCGTTAATCGGAATTACT-3'ACDF5f2:5'-CAACCTGGGAACTGCATTTGT-3', wherein the end 5 of the primer ACDF5b1 is labeled with biotin, and the end 5 of the primer ACDF5f2 is labeled with FITC (Fluorescein Isothiocyanate). The invention further provides a cross primer isothermal amplification technology for detecting the acidovorax citrulli, wherein with total DNA of a sample or a bacterial liquid as a template, isothermal amplification is carried out by using the primers; and after the reaction is finished, a result is judged directly by using a sealed colloidal gold-labeled DNA detecting device. The primers and the method provided by the invention have the advantages of good specificity, high accuracy, high sensitivity as well as simplicity, convenience and rapidness in operation and provides assurance for import/export safety.

Description

technical field [0001] The invention "primers and method for cross-primer constant temperature amplification detection of melon fruit spot pathogen" is specially used for detecting melon fruit spot pathogen, belongs to the technical field of plant quarantine, and relates to biological detection technology. Background technique [0002] Melon fruit spot disease has been widely occurring in the world since it was discovered in the watermelon growing areas of the United States in 1989, causing serious economic losses to agricultural production. The pathogen of melon fruit spot disease is Acidovorax citrulli, which is a phytosanitary pest imported into my country and can be transmitted long-distance through seeds. Its main hosts include watermelon, muskmelon, cantaloupe, zucchini and other cucurbit crops, and contaminated seeds are the main cause of the outbreak. The best way to control disease occurrence is to plant healthy seeds. The detection methods of the pathogen mainly ...

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): C12Q1/68C12Q1/04C12N15/11
Inventor 赵文军田茜张靓尤其敏商明清朱水芳
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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