Molecular detection method for Edrus deodara (Roxb) Lobd blight and primers therefor

A technology of cedar and Phytophthora, applied in the field of primers for detection of Phytophthora cedarii, can solve the problems that affect the accurate isolation of pathogenic bacteria, difficult pathogens, and complex morphological characteristics of Phytophthora

Inactive Publication Date: 2012-04-25
中华人民共和国昆山出入境检验检疫局
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]The disease is difficult to diagnose, and many bacteria will be produced when isolated according to conventional methods, which will affect the accurate isolation of pathogenic bacteria
At the same time, Phytophthora has complex morphological characteristics and rapid variation, and it is difficult to separate P. lateralis from other pathogenic bacteria such as P. cinnamomi and P. gonapodyides ( Torgeson D C, Young R A, Milbrath J A. Phytophthora root rot diseases of Lawson cypress and other ornamentals[M]. Agricultural Experiment Station, Oregon State College, 1954)
Therefore, conventional disease diagnosis techniques are difficult to quickly and accurately identify the pathogen

Method used

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  • Molecular detection method for Edrus deodara (Roxb) Lobd blight and primers therefor
  • Molecular detection method for Edrus deodara (Roxb) Lobd blight and primers therefor
  • Molecular detection method for Edrus deodara (Roxb) Lobd blight and primers therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1: design, synthesize primer and set up the PCR reaction system of cedar Phytophthora detection kit

[0029] 1. Primer design and synthesis

[0030] Design of specific primer T1 / T2: for GeneBank P. lateralis Comparative analysis with tRNA sequences in other Phytophthora ( figure 1 ), designed a pair of specific primers T1 / T2, the sequence is as follows:

[0031] T1: 5'-GCTTTATATATTTATTAGAT-3' (SEQ ID NO. 1)

[0032] and T2: 5'-CTCCTTTTTTGATATTAT-3' (SEQ ID NO. 2). The designed primers were re-analyzed by BLAST in GeneBank to verify their specificity.

[0033] At the same time, a pair of nested PCR outer primers T3 / T4 were designed, according to P. lateralis tRNA sequence, so that this primer is outside the specific primer, design a pair of outer primers T3 / T4, the sequence is as follows:

[0034] T3: 5'-GTTCGAATCCCTTTTTTTGC-3' (SEQ ID NO. 3)

[0035] T4: 5'-CAACAAATTTACAGTCTGCCGC-3' (SEQ ID NO. 4).

[0036] All primers were synthesized by Shangha...

Embodiment 2

[0043] Embodiment 2: Preparation of DNA template

[0044] Extract DNA from various samples as templates for PCR reactions. The specific process is as follows:

[0045] 1. Mycelium cultivation, collection and DNA extraction

[0046] (1) Preparation of medium

[0047] 10% V8 medium: 100 mL V8 juice, CaCO 3 0.2 g, add water to make up to 1 L.

[0048] PDA medium (Potato Dextrose Agar): 20 g of agar powder, 200 g of potatoes, 20 g of glucose, and dilute to 1 L with water.

[0049] (2) Mycelium cultivation and collection

[0050] Transfer the tested Phytophthora to V8 solid medium plate, culture in the dark at 20°C for 3 days, cut 10 pieces of 2 mm × 2 mm colony pieces from the edge of the colony, transfer to V8 liquid medium, and culture with shaking at 25°C for 5- After 7 days, the mycelium was collected by filtration, dried and ground into powder by freezing, and stored at -20°C for future use.

[0051] (2) Extraction of hyphal DNA

[0052] Take a small amount of myce...

Embodiment 3

[0058] Example 3: Detection of specificity and sensitivity of primers

[0059] 1. Specific detection

[0060] The bacterial strains used in the present invention and related information are shown in Table 1. Adopt specific primer T1 / T2 designed by the present invention to carry out PCR amplification to all test bacterial strain genomic DNAs in table 1, can only be tested from 2 P. lateralis A 192 bp band was specifically amplified in the strain, while the other tested strains and the blank control had no amplified band (Table 1, figure 2 ). Indicates that the primer has species specificity and can be P. lateralis Distinguished from other closely related species and other related species.

[0061] Table 1 The bacterial strains used for screening primer specificity in this example

[0062]

[0063] In the above table: + indicates the specific amplification band with primer T1 / T2, the length is 192 bp; - indicates no amplification product.

[0064] 2. Sensitivity dete...

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Abstract

The invention discloses a molecular detection method for Edrus deodara (Roxb) Lobd blight and primers therefor, and establishes a set of quick, high-sensitivity, stable and reliable molecular detection technology for detecting the Edrus deodara (Roxb) Lobd blight. The method has the advantages of accuracy, quickness, simplicity in operation and the like, can identify pathogens in an early stage of contagious disease, can be referentially applied to inspection and quarantine and field investigation of imported and exported plants and plant products, and has an important significance for controlling the Edrus deodara (Roxb) Lobd blight to spread in China. Meanwhile, the establishment of the system provides technical guidance and theoretical basis for detecting other pathogenic bacteria.

Description

technical field [0001] The invention relates to a primer for detecting Phytophthora cedarii and a molecular detection method using the primer to detect Phytophthora cedarii. Background technique [0002] Phytophthora cedarum root rot ( Phytophthora lateralis Tucker et Mibrath) cause cedar root rot. The disease was first discovered in Seattle, Washington, USA in 1920. At present, the pathogen is widely distributed all over the world (Smith I M. Phytophthora lateralis[ J]. EPPO Bulletin, 2009. 39(1):43-47.). The disease was first reported in Taiwan Province of my country in 2010 (Brasier C M, Vettraino A M, Chang T T, et al . Phytophthora lateralis discovered in an old growth Chamaecyparis forest in Taiwan[J]. Plant Pathology, 2010, 59(4): 595-603), which has not been found in mainland China, and is a dangerous plant quarantine disease. Phytophthora cedarum root rot not only harms cedar, but also can infect American cypress, yew yew, American pointed cypress, kiwi fr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/645
Inventor 纪睿张正光廖太林李百胜王健生
Owner 中华人民共和国昆山出入境检验检疫局
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