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

Method for rapidly detecting bacillus velezensis and application thereof

A Bacillus, specific primer pair technology, applied in the biological field, can solve the problems of long detection cycle, time-consuming and labor-intensive, unable to quickly detect beneficial microorganisms, etc.

Inactive Publication Date: 2021-07-13
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
View PDF1 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional detection of bio-control bacteria is mainly through the plate detection method and selective medium, which is not only time-consuming and labor-intensive, the detection cycle is long, and it is impossible to quickly detect beneficial microorganisms in the natural environment

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 rapidly detecting bacillus velezensis and application thereof
  • Method for rapidly detecting bacillus velezensis and application thereof
  • Method for rapidly detecting bacillus velezensis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1, be used for the screening of the specific primer pair of amplification Veles bacillus (Bacillus velezensis)

[0064] 1. Design and synthesis of specific primer pairs for amplifying Bacillus Velez

[0065] According to the nucleotide alignment results of the conserved gene sequence and flagellin synthesis gene sequence of Bacillus veilesi ZF2 and Bacillus veileii FZB42 (type strain), Bacillus amyloliquefaciens DSM7, and Bacillus subtilis 168, and based on the The nucleotide sequence of the ZF2 specific gene of Bacillus lacei is designed and artificially synthesized to amplify the specific primer pair of Bacillus velei. Specific steps are as follows:

[0066] 1. Download Bacillus Velez ZF2 (CP032154.1), Bacillus Velez FZB42 (CP000560.1), and Amyloliquefaciens from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) Nucleotide sequences of 16S rRNA, atpD, galE, gap, gyrB, metC, pdhA, pgk, rho, rpoD of Bacillus DSM7 (FN597644.1) and Bacillus subtilis 168 (AL00...

Embodiment 2

[0104] Embodiment 2, establishment of Bacillus velezensis ZF2 PMA-qPCR detection system

[0105] 1. Screening of the optimum PMA concentration

[0106] Propidium azide bromide (PMA) can selectively pass through the cell membrane of dead bacteria and bind to the DNA of dead bacteria under light conditions, thereby preventing the PCR amplification of dead bacteria. Screening the optimal concentration of PMA can distinguish live bacteria from dead bacteria and detect active Bacillus velei.

[0107] 1. Inoculate a single colony of Bacillus Velez ZF2 in LB liquid medium, culture at a constant temperature at 28°C for 24 hours, and adjust the OD 600nm is 0.8. Divide the bacterial solution into two parts on average, group A and group B. Group A is placed in an ice bath for standby use, and group B is incubated in a water bath at 99°C for 30 minutes to inactivate the cells. Spread 100 μL of the treated bacterial suspension on the LB solid plate and incubate at 28°C for 48 hours to v...

Embodiment 3

[0127] Embodiment 3, the colonization dynamic monitoring of Bacillus velezensis (Bacillus velezensis) ZF2 in soil

[0128] 1. Rifampicin domestication of Bacillus veleisi ZF2

[0129] Rifampicin is a broad-spectrum bacterial antibiotic. After being domesticated by rifampicin, Bacillus Velez ZF2 can grow on LB plates or medium containing rifampicin, while other bacteria cannot grow, thus distinguishing Strain ZF2 and other bacteria, convenient and accurate detection of colonization ability of strain ZF2. Rifampicin itself is red and has a strong identity. It is currently the most commonly used antibiotic for strain domestication.

[0130] 1. A single colony of Bacillus Velez ZF2 was inoculated in 5 mL of LB liquid medium containing 0.5 ng / μL rifampicin, and shaken at 28°C and 180 rpm for 24 hours to obtain ZF2 bacterial liquid.

[0131]2. Subculture the ZF2 bacterial liquid from low concentration to high concentration of rifampicin resistance, and the rifampicin concentration...

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 discloses a method for rapidly detecting bacillus velezensis and application thereof. According to the method, genome DNA of a bacterium to be detected is taken as a template, a specific primer pair is adopted for PCR amplification, and then the following judgment is carried out: if a PCR amplification product contains a DNA fragment with the size of 193bp, the bacterium to be detected is bacillus velezensis or is suspected to be bacillus velezensis; the specific primer pair is composed of a primer F as shown in SEQ ID NO: 2 and a primer R as shown in SEQ ID NO: 3. Experiments prove that the specific primer pair can be used for detecting bacillus velezensis, and is high in sensitivity and good in specificity; the method can also be used for evaluating the colonization ability of bacillus velezensis in soil to be detected, namely, monitoring the colonization dynamic state of bacillus velezensis in the soil. The method has an important application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for rapidly detecting Bacillus Velez and its application. Background technique [0002] The use of beneficial microorganisms to prevent and control plant diseases has the advantages of being green and pollution-free, and it is also a hot spot in current research. Biocontrol Bacillus exists widely in soil, and has become the most widely used beneficial microorganism because of its strong stress resistance and easy isolation. Biocontrol bacillus has also been widely reported in disease control, but there are few studies on the detection of bacillus. The traditional detection of bio-control bacteria is mainly through the plate detection method and selective medium, which is not only time-consuming and labor-intensive, but also has a long detection cycle, and it is impossible to quickly detect beneficial microorganisms in the natural environment. [0003] There are...

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): C12Q1/689C12Q1/04C12N15/11C12R1/07
CPCC12Q1/689
Inventor 李宝聚李磊许帅石延霞谢学文柴阿丽
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI