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RPA primer, kit, method and system for fast detecting potato black leg diseases (pectobacterium spp.)

A technology for potato and black shank, which is applied in the field of RPA detection primers for potato black shank, can solve the problems of long detection period of potato black shank, high detection requirements, and difficulty in detecting potato black shank in time, and achieves a system and a device. Fast and efficient detection, high sensitivity and high application value

Active Publication Date: 2020-05-15
CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to solve the problems in the prior art that the detection period of potato black shank is long, the detection requirements are high, and it is difficult to detect potato black shank in time, the present invention provides an RPA primer that can quickly and accurately detect potato black shank within 20 minutes, The kit, method and system are capable of timely on-site detection of potato pathogenic bacteria under harsh conditions (such as in the field), early detection and timely prevention and control, thereby ensuring healthy potato production

Method used

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  • RPA primer, kit, method and system for fast detecting potato black leg diseases (pectobacterium spp.)
  • RPA primer, kit, method and system for fast detecting potato black leg diseases (pectobacterium spp.)
  • RPA primer, kit, method and system for fast detecting potato black leg diseases (pectobacterium spp.)

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Experimental program
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Effect test

Embodiment 1

[0062] Example 1 RPA primers

[0063] Recombinase polymerase amplification (RPA) is a nucleic acid isothermal amplification technology, its amplification mechanism is completely different from the reaction mechanism of PCR amplification and LAMP amplification, and the requirements for primers are also different from those of PCR primers and LAMP. Therefore, in order to obtain RPA primers that are longer than PCR primers and can amplify template DNA with high specificity and sensitivity within 20 minutes at a temperature of 37-42°C, the inventors sequenced the causative bacteria of potato black shank, and then After NCBI blsat comparison, specific and conserved segments were found, and primers were designed according to the principle of primer design. After a large number of repeated primer designs, the inventors used recombinase polymerase (Recombinase Polymerase) to quickly target gene under constant temperature conditions. RPA amplification detection was carried out, and aft...

Embodiment 2

[0066] Example 2 Kit

[0067] The kit for rapid detection of early potato black shank provided by the present invention includes the RPA primers shown in Example 1, recombinase polymerase and reaction solution.

[0068] Further, the reaction solution is: a total volume of 50 μL, containing 2 μL of template DNA, 29.5 μL of Rehydration Buffer, 2.4 μL of primers F and R, ddH 2 O 11.2 μL, and finally 2.5 μL of magnesium acetate solution (MgOAc, 280 mmol / L) was added to prepare a 50 μL system.

[0069] Further, the recombinase, single-chain binding protein and polymerase.

[0070] The conditions for using the kit are: constant temperature amplification at 39°C for 20 minutes, and stop the reaction on ice.

Embodiment 3

[0071] Embodiment 3 detection method

[0072] A kind of method of rapid detection potato black shank of the present invention can utilize the RPA primer that embodiment 1 provides or the test kit that embodiment 2 provides, concrete steps are as follows:

[0073] 1. Isolation and purification of pathogenic bacteria

[0074] Pathogens were isolated by conventional plate streaking method. Select a potato stem or tuber diseased tissue with typical black shank symptoms, cut a small piece and soak it in 75% ethanol for 15 seconds for surface disinfection, and wash it with sterile water for 3 times. After microscopic examination, use a sterile inoculation loop to dip a ring of leachate from the diseased tissue where bacteria spraying was observed, and draw a line on the NA medium plate. Inverted culture at 28°C for 24-48 hours, pick a single colony and transfer it to a new NA plate for 1-2 times to obtain a purified strain. The purified strain was expanded and cultured, and final...

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Abstract

The invention discloses an RPA (Recombinase Polymerase Amplification) primer, kit and fast detecting method for fast detecting potato black leg diseases (pectobacterium spp.), and relates to the fieldof plant disease detection. The RPA primer has nucleotide sequences shown as SEQ ID NO.1-2. The potato black leg diseases (pectobacterium spp.) can be detected by only performing isothermal amplification on the extracted DNA of potato black leg disease (pectobacterium spp.) bacteria for 20 min by using the RPA primer. The RPA detection primer of the potato black leg diseases (pectobacterium spp.)has the advantages that the specificity is high; the sensitivity is high; the detection is efficient and fast; the operation is simple and convenient; a special instrument is not needed; and the technical basis can be provided for early diagnosis of the potato black leg diseases (pectobacterium spp.).

Description

technical field [0001] The invention relates to the technical field of detection, identification and prevention of crop diseases, in particular to an RPA detection primer, a kit, a rapid detection method and a system for potato blackleg (Pectobacterium spp.). Background technique [0002] Potatoes are asexually propagated crops mainly propagated by tubers. During production, they are often infected by pathogens, and seed potatoes with bacteria will infect the next generation of seed potatoes, thereby affecting the normal growth and development of potatoes, reducing potato yields, and especially the seed quality. degradation. Potato blackleg is a bacterial disease caused by Pectobacterium spp. and Dickeyaspp., and is a soil-borne and storage disease. Potato blackleg, also known as blackfoot, is named after the symptoms of the blackening of the stem base of the potato plant. The disease occurs to varying degrees in the potato-producing areas of Northeast, North China, and Nor...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12M1/38C12M1/00C12R1/01
CPCC12Q1/689C12Q1/6844C12Q1/6806C12Q2565/125C12Q2521/507C12Q2522/101
Inventor 汤浩李华伟邱思鑫罗文彬许国春许泳清纪荣昌刘中华李国良林赵淼张鸿邱永祥
Owner CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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