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

Primer, probe, kit and detection method for detecting cotton root rot bacteria

A detection method and technology of rot fungus, which is applied in the field of kits and detection, primers and probes for cotton root rot fungus detection, can solve the problem that the rapid and accurate detection of ports cannot meet the long time and limitations of inducing spore production and sclerotia production. Problems such as popularization and application of detection methods to achieve reliable detection results, short detection cycle and high sensitivity

Pending Publication Date: 2021-12-07
INSPECTION & QUARANTINE TECH CENT OF NINGBO ENTRY EXIT INSPECTION & QUARANTINE BUREAU
View PDF3 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, in the morphological method, although the morphological characteristics of the cotton root rot fungus are more obvious, for example, the appearance of the conidia is similar to "baasis", can produce hyphae and the lateral hyphae are cross-shaped branches, and can produce brown or black sclerotia, etc., but it takes a long time to induce sporulation and sclerotia production, which is only suitable for experimental research and cannot meet the requirements of rapid and accurate detection at ports
In the molecular biology method, Marek et al. designed a pair of specific primers for the detection of the pathogen, but the sensitivity of the primers is not very high and cannot detect DNA samples with low content
Arif et al. established a multi-gene detection method based on real-time fluorescent PCR and Razorex biological monitoring system, but this method needs to be used in conjunction with the Razor Ex BioDetectionsystem biological monitoring system of Idaho Company in the United States, which limits the popularization and application of this detection method

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
  • Primer, probe, kit and detection method for detecting cotton root rot bacteria
  • Primer, probe, kit and detection method for detecting cotton root rot bacteria
  • Primer, probe, kit and detection method for detecting cotton root rot bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: be used for the primer of cotton root rot pathogen detection, probe design

[0034] The sequence of the conserved fragment (ITS) in the cotton root rot fungus is as described in SEQ ID NO: 1, by amplifying this laboratory preserves the cotton root rot fungus ITS sequence, and uses Bioedit software in conjunction with the pathogenic bacteria in the Genbank gene bank and related The ITS sequences of different species were compared and analyzed, and the primers and probes of cotton root rot were designed by using Primer Express3.0 to determine the conserved fragments of the ITS region. Among them, the synthesis of primers and probes adopts the β-acetonitrile phosphate ammonium chemical synthesis method, and the synthesis of OligoDNA is carried out by using a fully automatic DNA synthesizer. In this embodiment, the primers and probes are synthesized by Bao Biological Engineering (Dalian) Co., Ltd. .

[0035] Wherein, the sequence of the primer used for cotton...

Embodiment 2

[0040] Embodiment 2: the kit that is used for cotton root rot pathogen detection

[0041] The kit in this example includes the primers and probes in Example 1, and also includes CTAB extract, Taq DNA polymerase, positive control solution and negative control solution, as shown in Table 1.

[0042] Table 1 Standardized kit composition (20 μL reaction × 100 times)

[0043] Solution I (CTAB extract) 30mL×2 bottles Solution II (Primer Probe) 1.25mL Solution III (Polymerase) 50μL Solution IV (deionized water) 1.25mL×2 sticks Solution V (positive control) 50μL Solution VI (negative control) 50μL Solution VII (quantitative standard solution) 25 μL

[0044]Wherein, the above solution I contains 0.7mol / L NaCl, 100mmol / L Tris-HC1, pH 8.0 and 20mmol / LEDTA, 10g / L PVP-360, 20g / L CTAB and a final concentration of 2% (V:V) β -Mercaptoethanol. The above solution II is a one-step fluorescence quantitative PCR buffer (purchased from TA...

Embodiment 3

[0045] Embodiment 3: the detection of cotton root rot pathogen

[0046] The detection process of cotton root rot pathogen is as follows:

[0047] Step 1: Take about 0.5g of dried mycelium, put it into a 1.5ml centrifuge tube submerged in liquid nitrogen, crush it with a plastic pestle, then add 600μL of CTAB extract, and bathe in water at 65°C for 30min, during the water bath process from time to time Mix by inversion.

[0048] The second step: centrifuge at 10000r / min for 5min, take the supernatant and add an equal volume of extract, and mix until it becomes milky; in this embodiment, the above extract is a mixture of chloroform and isoamyl alcohol, and the mixture The volume ratio of chloroform and isoamyl alcohol in the solution is 24:1.

[0049] The third step: 4 ℃ 10000rpm centrifugal 5min, take supernatant and add the sodium acetate solution (3mol / L, pH 6.0) of this 1 / 10 volume (being 1 / 10 of this supernatant volume) and 2.5 times of volume ( That is, 2.5 times the vo...

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, a probe, a kit and detection method for detecting cotton root rot bacteria. the sequence of an upstream primer myaF is 5 '-GAGGGCATGCCTGTTCGA-3', the sequence of a downstream primer myaR is 5 '-CCAAGCCAACCCTTTTCAC-3', and the sequence of a probe myaT is 5 '-CGTCAGCATAACAACCTCAAGCACC-3'. A real-time fluorescence PCR detection method is adopted to detect the cotton root rot bacteria, compared with a traditional detection method, the method has the advantages of being short in detection period, high in specificity and sensitivity, free of PCR post-treatment and the like, and the requirements for rapid and accurate port detection are met. In addition, the primer and the probe have the advantages of being high in amplification efficiency, good in specificity, high in sensitivity and the like.

Description

technical field [0001] The invention relates to the field of biological technology, in particular to a primer, a probe, a kit and a detection method for detecting cotton root rot pathogens. Background technique [0002] Cotton root rot, also known as Texas root rot, caused by Phymototrichum omnivorum (Shear) Duggar, was first reported in Texas by Pammel in 1888. This disease is a very destructive disease on cotton. According to the 1980-2008 disease loss assessment price data provided by the National Cotton Council of the United States, it alone causes a loss of US $100 million in cotton production every year. [0003] According to reports, there are more than 2,000 species of cotton root rot hosts, among which cotton, alfalfa, stone fruit trees and shade trees are the most lost. The main root of the infected plant rots, the vascular bundles of the stem change color, the leaves wilt rapidly, and the leaves do not fall off, eventually leading to plant death, and a large numb...

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): C12Q1/6895C12Q1/686C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/686C12Q2563/107C12Q2545/114
Inventor 张慧丽李雪莲段维军赵雷陈先锋
Owner INSPECTION & QUARANTINE TECH CENT OF NINGBO ENTRY EXIT INSPECTION & QUARANTINE BUREAU