阅读说明：本技术 一种运用PCR引物检测Diaporthe novem的方法 (Method for detecting Diaporthe novem by using PCR (polymerase chain reaction) primers ) 是由 张莹 胡佳续 王娓辰 刘鹏 张裕君 刘勇 王仲敏 于 2021-08-06 设计创作，主要内容包括：本发明涉及使用PCR技术检测植物及植物产品中Diaporthe novem的方法,属于植物病原真菌检测领域。本发明设计合成了D.novem的特异性引物,用于对D.novem基因组DNA的特异检测。本发明建立了一种快速简便、特异性强、准确可靠的D.novem的分子检测方法,一个工作日内完成整个检测。(The present invention relates to the detection of plants and plant products using PCR technology Diaporthe novem Belonging to the field of plant pathogenic fungi detection. The invention designs and synthesizes D.novem Specific primers of (1), for the pair D.novem Specific detection of genomic DNA. The invention establishes a method which is rapid, simple, convenient, strong in specificity, accurate and reliable D.novem The molecular detection method of (1) completes the whole detection within one working day.)

1. Detection by PCR primerDiaporthe novemIs characterized by being self-designedD. novem PCR specific primer pair with the following sequence:

DnoF1：CTCACACTGGCTGGATTATG

DnoR1：AGCCGCTTATCTCCTATGC

the amplified fragment was 329bp and used forD. novemAnd (3) specific amplification detection of genome DNA.

2. The method of claim 1, wherein the detection is performed using PCR primersD. novemThe method is characterized by comprising the following steps:

(1) extracting plant genome DNA;

(2) establishingD. novemA PCR amplification system of genome DNA, which is used for amplification according to a reaction program;

(3) and detecting the amplification product by agarose gel electrophoresis.

3. The assay as claimed in claim 1D. novemThe specific PCR primer set of (1) or the assay of claim 2D. novemIn the specific detectionD. novemThe application of (1).

Technical Field

The present invention relates to the detection of plants and plant products using PCR technologyDiaporthe novem J.M. Santos, Vrandečić & A.J.L. Phillips, sp. nov.Belonging to the field of plant pathogenic fungi detection.

Background

Diaporthe novemBelongs to the kingdom of Fungi (Fungi), phylum Ascomycota (Ascomycota), class Ascomycetes (Ascomycetes), order Sitophyllales (Diaporthales), family Sitophyllaceae (Diaporthaceae), genus Sitophyllum (A)Diaporthe) In asexual stages thereofPhomopsissp.9. It is associated with the pathogenic bacteria of northern tuber ulcer of soybean which seriously harms soybeanD. caulivoraSouthern Stem ulcer pathogen of soybeanD. aspalathiPhomopsis thalianaD. longicollaThe species are similar to each other, wherein the soybean northern stem canker pathogen and the soybean southern stem canker pathogen are listed in the entry plant quarantine pest directory of China.

D. novemOriginally officially named by Joge Manual Santos et al in 2011, the existing data show that the pathogen is distributed in areas such as Chilean, Crohn's disease, Italy, Portugal, south Africa and Australia. The method is characterized in that the method harms important economic crops such as soybean, sunflower, kiwi fruit, grape, kumquat, lime and the like, seeds and fruits are rotted, so that serious economic loss is caused, and the method also has separation reports on hydrangea, south African black tea and rubiaceae Raglania, and China has not reported yet.

The soybean is used as an important grain and oil crop and feed raw material, has great demand in China, is one of the most important imported agricultural products in China, and the imported total amount of the soybean in China in 2019 reaches 8851 ten thousand tons. Repeated detection of port in ChinaD. novemSince the pathogen is not in the quarantine directory, the harm is easy to be overlooked. The pathogen causes stalk ulcers and seed rot in soybeans, resulting in reduced yield and quality of soybeans. Soybean oil produced from the damaged seeds and other products such as feed, fertilizer and soybean cake obtained after oil extraction from soybean are low in quality and not suitable for use as feed. In addition, the host of the pathogen is wide, and once the pathogen is introduced into China along with the imported soybeans, the pathogen causes inestimable loss on the production and agricultural ecology of the soybeans and other crops in China.

In view ofD. novemTransmitted into ChinaRisk and severity of pathogen harm, and establishes a set of rapid, accurate and complete methods for protecting the production safety of agriculture and forestry in China, maintaining the benefits of China, further standardizing the quarantine of the important disease, improving the accuracy and the verification speed of port epidemic situation detection, preventing the pathogen from transmitting into ChinaD. novemThe detection method of (2) is imminent.

Disclosure of Invention

The invention collects the diaporthe 8 fungi (see table 1), establishes a rapid, simple, convenient, strong-specificity, accurate and reliable molecular detection method for the diaporthe fungi, and can detect the diaportheD. novemAccurately identified. The method is rapid in detection and reliable, the whole process is completed within one working day, and the method can be effectively popularized and applied to port detection.

The specific technical scheme is as follows:

the invention aims to provide detectionD. novemA method for PCR of genomic DNA comprising the steps of:

(1) extracting plant genome DNA;

（2）D. novemthe specific primer has the following sequence:

DnoF1：CTCACACTGGCTGGATTATG

DnoR1：AGCCGCTTATCTCCTATGC

the primer is used forD. novemThe genome DNA is specifically detected, and the amplified fragment is about 329 bp.

Compared with the prior art, the invention has the beneficial effects that: the invention establishes the characteristics of rapidness, simplicity, convenience, strong specificity, accuracy and reliabilityD. novemA molecular detection method ofD. novemIs distinguished from other species of the saddle shell. The method is rapid in detection and reliable, the whole process is completed within one working day, and the method can be effectively popularized and applied to port detection.

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

Drawings

FIG. 1 shows a schematic view of aD. novemAmplification of genomic DNA specific primers

In the figure, lanes 1 to 9 areD. novem, D. longicolla , D. caulivora, D. aspalathi, Diaporthe sp., D. eres , D.phaseolorum, D. vacciniiAnd a negative control, lane M is 2000 bp DNA marker.

Detailed Description

Example 1: extraction of genomic DNA from plant material

This experimentD. novemThe strain is from plant inspection laboratory of Tianjin customs animal food planting center. The total number of the strains is 8, and the related information is shown in the table 1.

TABLE 1 code of test materials, Latin name, Chinese name and year of Collection

Serial number	Latin name	Name of Chinese	Year of collection
				1	Diaporthe novem	-	2019
2	Diaporthe longicolla	Phomopsis longissima (L.) F.sp.	2019
				3	Diaporthe caulivora	Bacterial canker of northern tuber of soybean	2019
4	Diaporthe aspalathi	Bacterial canker of south Stem of Soybean	2019
				5	Diaporthesp.	Sunflower seat shell	2020
6	Diaporthe eres	Cherry house seat shell	2020
				7	Diaporthephaseolorum	Kidney bean room seat shell	2020
8	Diaporthe vaccinii	Blueberry fruit rot fungi	2020

Picking crimp bandSterilizing the diseased bean with lesion on the surface with 1% sodium hypochlorite for 5 min, washing with sterilized water for 3 times, maintaining moisture at 25 deg.C, culturing for 24 hr, freezing at-20 deg.C for 24 hr, culturing on PDA culture medium at 22 deg.C under 12 hr in dark. After 5d, the culture results were observed. The suspicious colony is separated and purified by PDA, a proper amount of mycelium blocks are taken, liquid nitrogen is fully ground, DNA is extracted by a Dneasy plant mini kit plant gene extraction kit of Germany QIAGEN, and the DNA is dissolved in 100μThe cells were stored at-20 ℃ in L1 XTE buffer.

Example 2: design of specific primers

Artificially synthesizedD. novemThe specific primer of (1) has the following primer sequence:

DnoF1：CTCACACTGGCTGGATTATG

DnoR1：AGCCGCTTATCTCCTATGC

example 3:D. novemPCR amplification of specific primers

1. Preparation of reaction mixture

The reaction system configuration is shown in Table 2.

TABLE 2D. novemSpecific amplification system

Reagent	Sample addition amount/. mu.l
		2× Premix	12.5
ddH2O	10.0
		DnoF1	0.5
DnoR1	0.5
		Form panel	1.5
Total	25.0

2. PCR reaction procedure

Pre-denaturation: 94 ℃ for 3min

Denaturation: 94 ℃ for 30s

Annealing: 60 ℃ for 20s

Extension: 72 ℃ and 20s

The number of cycles: 35 are provided with

Extension: 72 ℃ for 6min

2.3 analysis of results

Genomic DNA from 8 experimental materials was amplified with specific primers DnoF1/DnoR1, and sterile water was added as a template for negative control. The amplified product is electrophoresed through 1.5% agarose gel, and EB staining can observe a band with a specific target size, and the amplified fragment of the universal primer is about 329bp, and only the amplified fragment isD. novemShowing a band of interest of a specific size (see lane 1 in FIG),D. longicolla , D. caulivora, D. aspalathi, Diaporthe sp., D. eres , D.phaseolorum, D. vaccinii and negative control has no corresponding product (shown in lanes 2-9, and lanes 1-9 in the figure areD. novem, D. longicolla, D. caulivora, D. aspalathi, Diaporthe sp., D. eres, D.phaseolorum, D. vacciniiAnd negative control), lane M is 2000 bp DNA marker.

Sequence listing

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