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Recombinase polymerase amplification (RPA) primer, kit and detection method for detecting ditylenchus destructor thorne

A detection method, stem nematode technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of false positives easily, and achieve high sensitivity, accurate detection, and simple operation

Active Publication Date: 2020-05-29
XUZHOU INST OF AGRI SCI IN JIANGSU XUHUAI DISTRICT (JIANGSU XUZHOU SWEETPOTATO CENT)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, this solution requires a constant temperature water bath at 65°C for more than 1 hour, and the product is multiband, which is prone to false positives

Method used

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  • Recombinase polymerase amplification (RPA) primer, kit and detection method for detecting ditylenchus destructor thorne
  • Recombinase polymerase amplification (RPA) primer, kit and detection method for detecting ditylenchus destructor thorne
  • Recombinase polymerase amplification (RPA) primer, kit and detection method for detecting ditylenchus destructor thorne

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Experimental program
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Effect test

Embodiment 1

[0027] Embodiment 1, D. destructor D. destructor RPA amplification primer design and establishment of detection method

[0028] 1.1 Design and screening of D. destructor RPA primers

[0029] According to the comparative analysis of ITS and ribosomal RNA 28s nucleotide sequences of D. destructor and other D. nematodes, three pairs of primers suitable for RPA reactions were designed (Table 1).

[0030] Table 1: Primers used for detection and screening of D. destructor RPA in sweet potato

[0031]

[0032] 1.2 Preparation of D. destructor DNA template

[0033] Collect D. destructor of sweet potato into a 1.5mL centrifuge tube, centrifuge at 5000rpm for 10 minutes, use a pipette to absorb the water in the tube as much as possible, put the centrifuge tube into liquid nitrogen for 30 seconds, and grind it with a grinding pestle. Nematode DNA was extracted from the homogenate after grinding using Animal Genome DNA Rapid Extraction Kit (Shanghai Sangong).

[0034] 1.3 RPA reaction...

Embodiment 2

[0040] Embodiment 2, sweet potato D. destructor RPA specific amplification accuracy and reliability test

[0041] A total of 15 populations of D. destructor, Meloidogyne incognita, Meloidogyne javanica, Root-knot nematode Elephant ear, soybean cyst nematode, cereal cyst nematode, S. bessiei, and Brachypodia coffeee were collected (Table 1) 2) According to Example 1.2, extract DNA from different populations of nematodes as templates, and use ddH 2 O is a negative control, and RPA reaction is carried out and detected according to examples 1.3, 1.4, and 1.5. figure 1 The results showed that only the D. destructor population obtained a 230bp specific amplification product, and no other nematode populations produced bands here.

[0042] Table 2 Nematode populations used in the experiment

[0043]

Embodiment 3

[0044] Embodiment 3, sweet potato D. destructor RPA reaction sensitivity detection

[0045] 3.1 Genomic DNA RPA reaction sensitivity detection of D. destructor population

[0046] Using the extraction method in Example 1.2, extract the DNA of D. destructor D. destructor, use Nanodrop2000 to measure its genomic DNA concentration, and use water to dilute it to 10 ng / μL, 1ng / μL, 10 -1 ng / μL, 10 -2 ng / μL, 10 -3 ng / μL.

[0047] RPA detection: Using the DNA of these five different concentrations of D. destructor D. destructor as templates (1 μL) and water as the control, perform RPA reactions and detection according to 1.3, 1.4, and 1.5.

[0048] PCR detection: DNA template diluted in the above gradient, amplification system 25 μL, DNA template 1 μL, primer pair DdF1 / DdR1 (20 μM) 1 μL, EmeraldAmp® MAX PCR Master Mix (TaKaRa, Dalian) 12.5 μL, ddH 2 O to make up to 25 μL. Negative worm DNA template was used as negative control. Amplification was performed on an Eppendorf PCR cyc...

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Abstract

The invention provides a recombinase polymerase amplification (RPA) primer for detecting ditylenchus destructor thorne by the RPA technology and an RPA kit comprising the RPA primer, and the RPA primer has sequences shown in SEQ ID No: 5 and SEQ ID No: 6. The invention also provides a method for detecting the ditylenchus destructor thorne by using the RPA kit. The method can detect single or mixedditylenchus destructor thorne DNA samples, is accurate in detection, high in sensitivity, simple to operate, short in time and good in repeatability, and provides theoretical guidance for diagnosis detection and scientific pesticide application of the ditylenchus destructor thorne.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a set of primers and methods for detecting D. destructor in sweet potato by using a recombinase polymerase isothermal amplification technique (Recombinase polylymerase amplification, RPA), specifically an RPA primer for detecting D. destructor in sweet potato , kits and detection methods. Background technique [0002] Sweet potato stem nematode disease is also called hollow heart disease, commonly known as "bran heart disease", which is one of the domestic plant quarantine objects. The causative nematode is D. destructor ( Ditylenchus destructor Thorne), the nematode was first found on potatoes, causing potato rot, so it is also called potato rot nematode (Potato rot nematode). In 1980, Zhang Yunmei and others reported for the first time that the pathogen of sweet potato stem nematode disease in Shandong Province was sweet potato stem nematode. In 1982, Ding Zaifu and Lin Maosong i...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
CPCC12Q1/6888C12Q1/6844C12Q2521/507C12Q2522/101
Inventor 马居奎孙厚俊谢逸萍张成玲杨冬静
Owner XUZHOU INST OF AGRI SCI IN JIANGSU XUHUAI DISTRICT (JIANGSU XUZHOU SWEETPOTATO CENT)
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