Real-time fluorescence quantification PCR primers and probes for identifying three kinds of pratylenchus coffeae on sugarcane and kit thereof

A real-time fluorescent quantitative, short-bodied nematode technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems affecting sugarcane yield and quality, and achieve high sensitivity and good application. Foreground effect

Active Publication Date: 2016-10-26
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the infestation of the roots of sugarcane by Brachybody zea, Brachybody zea pseudomaize and Brachypoda zea, the yield and quality of sugarcane are seriously affected.

Method used

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  • Real-time fluorescence quantification PCR primers and probes for identifying three kinds of pratylenchus coffeae on sugarcane and kit thereof
  • Real-time fluorescence quantification PCR primers and probes for identifying three kinds of pratylenchus coffeae on sugarcane and kit thereof
  • Real-time fluorescence quantification PCR primers and probes for identifying three kinds of pratylenchus coffeae on sugarcane and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Primer probe design and reaction condition optimization

[0043] 1. Primer design

[0044] (1) The primer set qYF2 / qYR2, the specific probe qYP, and the primer set qNF for Brachybody zea zea were designed according to the DNA ITS sequences of Brachybody zea zea, Brachybody zea pseudonym and other plant-parasitic nematodes in NCBI / qNP, specific probe qNP. As shown in Table 1.

[0045] (2) According to the mitochondrial DNA of short-tailed brevis and other plant-parasitic nematodes in NCBI COI Sequence designed the primer set qBF2 and qBR1, and the specific probe qBP. As shown in Table 1.

[0046] Table 1 Primers and probes

[0047]

[0048] 2. Optimization of primer reaction conditions

[0049] (1) Using a single nematode DNA of Brachybody zea zea, Brachybody zea pseudonym, and Brachybody zeatrix as templates, PCR amplification was performed at different annealing temperatures, and the annealing temperature of the above primer sets was optimized.

...

Embodiment 2

[0057] Example 2 specific detection

[0058] 1. According to the primers and probes designed in Example 1, the real-time fluorescent quantitative PCR method for detecting Brachybody zea zea, Brachybody zea pseudomaize and the shortest-tailed Brachybody worm respectively has been established:

[0059] The real-time fluorescent quantitative PCR reaction system is: 2 μL DNA, 0.4 μL (10 μM) of upstream and downstream primers, 0.4 μL (10 μM) of probe, 10 μL real-time fluorescent quantitative PCR premix reagent and the balance of ddH 2 O, a total of 20 μL.

[0060] The real-time fluorescent quantitative PCR reaction conditions of B. maize and B. maize were as follows: pre-denaturation at 95°C for 30 s; 95°C for 5 s, annealing temperature at 60°C for 35 s, a total of 40 cycles;

[0061] The real-time fluorescent quantitative PCR reaction conditions of the short-tailed Brachyphyta nematode were as follows: pre-denaturation at 95°C for 30 s; 95°C for 5 s, annealing at 55°C for 35 s an...

Embodiment 3

[0073] Embodiment 3 detection sensitivity and standard curve

[0074] 1. Under the stereoscope, pick 1 Brachybody zea zea, 1 Brachybody zea pseudonym, and 1 short-tail Brachybody nematode respectively, extract DNA according to the above method, and then carry out gradient dilution on the DNA samples ( as shown in Table 3).

[0075] Table 3 Concentrations of DNA samples used to make the standard curve and the corresponding Ct values

[0076]

[0077] 2. Using the above-mentioned DNA samples of various dilution concentrations as templates, perform real-time fluorescent quantitative PCR amplification respectively, and the PCR reaction system and conditions are the same as those in Example 2.

[0078] 3. The results are attached image 3 As shown, there is a good linear relationship when amplifying 0.01-1 target nematode, indicating that the system can accurately quantify the number of nematodes in the sample.

[0079] In addition, the above results also show that the real-t...

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Abstract

The invention discloses a set of real-time fluorescence quantification PCR primers and probes for identifying three kinds of pratylenchus coffeae on sugarcane and a kit thereof. The set of primers and probes comprises three groups of primers and probes which are the primer group qYF2 / qYR2 and probe qYP, the primer group qNF / qNR and probe qNP and the primer group qBF2 / qBR1and probe qBP, and the nucleotide sequences are shown in SEQ ID NO.1-9. The three kinds of pratylenchus coffeae are corn pratylenchus coffeae, quasi corn pratylenchus coffeae and shortest tail pratylenchus coffeae. By means of the primer groups, the probes and the kit thereof, the corn pratylenchus coffeae, the quasi corn pratylenchus coffeae and the shortest tail pratylenchus coffeae can be specifically identified, quantitative detection can be carried out, the defects that the three kinds of pratylenchus coffeae are difficult to distinct morphologically, the detection workload is large and the cost is high are effectively overcome, the detection sensitivity can reach 0.01 nematode, and the primers, the probes and the kit thereof have very important boosting significance for actual detection work.

Description

technical field [0001] The invention belongs to the technical field of plant pathogen detection. More specifically, it relates to a set of real-time fluorescent quantitative PCR primers and probes for identifying three brevis nematodes on sugarcane and a kit thereof. Background technique [0002] Brachybody nematodes, also known as root-rot nematodes, are an important migratory endoparasitic nematode consisting of more than 70 valid species. The short-bodied nematodes move, puncture and feed in the roots of plants, causing the formation of necrotic spots and cavities in the root tissue, and then necrosis of the root tissue. Plants infested by short-body nematodes, one of the three plant nematodes that cause the greatest economic losses, exhibit symptoms of water and nutrient deficiencies due to root necrosis. [0003] Brachybody maize, Brachybody pseudomaize, and Brachybody shortest tail are three common short-bodied nematodes, all of which can damage sugarcane, and they w...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 卓侃林柏荣刘星彤廖金铃
Owner SOUTH CHINA AGRI UNIV
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