Unlock instant, AI-driven research and patent intelligence for your innovation.

Method for high sensitivity detection of Phytophthora hibernalis Carne

A technology of Phytophthora wintere and high sensitivity is applied in the field of detection of Phytophthora wintere brown rot pathogens of citrus, which can solve the problem that the amplification band of citrus samples with bacteria is not particularly obvious, and achieves high sensitivity, simple operation and repeatability. Good results

Inactive Publication Date: 2013-04-24
HUBEI ENTRY EXIT INSPECTION & QUARANTINE BUREAUOF THE PEOPLES REPUBLIC OF CHINA
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection limit can reach the level of 10 fg genomic DNA per 25 μL reaction system, but the amplified bands of the infected citrus samples are not particularly obvious

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
  • Method for high sensitivity detection of Phytophthora hibernalis Carne
  • Method for high sensitivity detection of Phytophthora hibernalis Carne

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The total volume of the RCA reaction system in this example is 10 μl. The specific detection method is as follows: (1) In a sterile centrifuge tube, add 5.9μl of sterile deionized water, 1μl of 10×phi29 DNA polymerase buffer, and the sense primer PHIB1 with a concentration of 10μM in a volume of 0.5μl and a counter with a concentration of 10μM. The volume of the sense primer PHIB2 is 0.5μl and the volume of the target DNA with a concentration of 200 ng / μl is 1μl, and then they are mixed well and centrifuged, and the reaction solution is centrifuged to the bottom of the tube;

[0028] (2) Heat the centrifuge tube with a PCR machine at 95°C for 3 minutes, and then quickly ice bath for 15 minutes;

[0029] (3) Add 0.5μl of dNTPs mixture at a concentration of 2.5mM each, 0.1μl of BSA at a concentration of 1%, and 0.5μl of phi29 DNA polymerase at a concentration of 10 U / μl in the centrifuge tube, mix well and centrifuge. Centrifuge the reaction solution to the bottom of the tube...

Embodiment 2

[0035] The total volume of the RCA reaction system in this example is 10 μl. The specific detection method is as follows: (1) In a sterile centrifuge tube, add 5.9μl of sterile deionized water, 1μl of 10×phi29 DNA polymerase buffer, and the sense primer PHIB1 with a concentration of 10μM in a volume of 0.5μl and a counter with a concentration of 10μM. The volume of the sense primer PHIB2 is 0.5μl and the volume of the target DNA with a concentration of 110 ng / μl is 1μl, and then they are mixed uniformly and centrifuged, and the reaction solution is centrifuged to the bottom of the tube;

[0036] (2) Heat the centrifuge tube with a PCR machine at 95°C for 5 minutes, and then quickly ice bath for 18 minutes;

[0037] (3) Add 0.5μl of dNTPs mixture at a concentration of 2.5mM each, 0.1μl of BSA at a concentration of 1%, and 0.5μl of phi29 DNA polymerase at a concentration of 10 U / μl in the centrifuge tube, mix well and centrifuge. Centrifuge the reaction solution to the bottom of the...

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 a method for high sensitivity detection of Phytophthora hibernalis Carne. The method comprises the following steps of orderly adding sterile deionized water, a phi29 DNA polymerase buffer solution, a forward primer PHIB1, a reverse primer PHIB2 and an object DNA into a sterile centrifugation tube, uniformly mixing, carrying out centrifugation of the mixed reaction liquid to tube bottom, heating the sterile centrifugation tube, fast cooling in an ice-bath, orderly adding a dNTPs mixture, BSA and a phi29 DNA polymerase into the sterile centrifugation tube to obtain an RCA reaction product, utilizing a novel sterile centrifugation tube, carrying out PCR amplification to obtain a PCR reaction product, and detecting the PCR reaction product. The method can realize fast and sensitive detection of Phytophthora hibernalis Carne. The sensitivity of the method provided by the invention is 10*4<9> times higher than that of the traditional PCR amplification detection method.

Description

Technical field [0001] The invention relates to a method for detecting the brown rot pathogen of citrus Phytophthora winterskin, in particular to a high-sensitivity method for detecting the pathogen of Phytophthora winterskin brown rot in citrus by applying RCA-PCR technology. Background technique [0002] Phytophthora hibernalis Carne, the brown rot pathogen of citrus, has a wide host range, serious damage to citrus production, and a wide distribution. It can carry diseased fruits, tissues or asexual propagation materials for long-distance transmission. my country's citrus planting areas are widely distributed. In recent years, these pathogens have become more and more likely to spread to my country with the introduction of high-quality seed and seedlings. Once the disease is introduced, it will cause great harm to my country's citrus industry. The brown rot disease of Phytophthora spp. in citrus citrus caused by the pathogenic bacteria Phytophthora spp. in citrus was first dis...

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): C12Q1/68C12Q1/04C12R1/645
Inventor 王振华张建坤李凤新曾宪东
Owner HUBEI ENTRY EXIT INSPECTION & QUARANTINE BUREAUOF THE PEOPLES REPUBLIC OF CHINA