Specific primer set, kit and application for analyzing the diversity of Fusarium soybean root rot fungus

A technology of specificity and primer set, applied in the direction of microorganism-based methods, microorganisms, recombinant DNA technology, etc., can solve the problem of inability to determine whether there are pathogenic bacteria, unable to separate pathogenic Fusarium, unsuitable for Fusarium identification, etc. problems, to achieve high sensitivity, low cost, and strong specificity

Active Publication Date: 2018-10-30
四川农科源检测技术有限公司
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Multiple DNA molecular markers represented by rDNA-ITS sequences have been used in the detection of Fusarium, but rDNA-ITS is relatively conserved between species or compound species of Fusarium, and there are few mutation sites, so it cannot be well detected. Separate various pathogenic Fusarium species. For example, O'Donnell et al. (2015) also found that rDNA-ITS was in the genus Relatively conserved among species, not suitable for population identification of Fusarium
Moreover, most of the current methods involve the identification or detection of a single Fusarium species. Considering the diversity of Fusarium species that cause soybean root rot, it is urgent to develop a series of molecular techniques for the detection of Fusarium diversity.
[0004] The application number is 201310353895.2, which discloses a specific primer for analyzing the diversity of Fusarium soybean root rot fungus. This application only uses one specific primer to realize the analysis and detection of various pathogenic bacteria. The principle is based on the yeast genome A pair of specific primers were designed for the 18sRNA gene in China to distinguish various fungi according to the size of the amplified fragments. However, due to the relatively conservative sequence of the gene, the differences between the sequences are small. Although the application detected 11 related fungi, the It is not ruled out that other fungi in environmental samples may also be amplified with the same bands, so that it is impossible to determine whether the corresponding pathogenic bacteria exist. Moreover, since the system requires two steps of ordinary PCR and DGGE stratification, its detection process It takes about 12 hours

Method used

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  • Specific primer set, kit and application for analyzing the diversity of Fusarium soybean root rot fungus
  • Specific primer set, kit and application for analyzing the diversity of Fusarium soybean root rot fungus
  • Specific primer set, kit and application for analyzing the diversity of Fusarium soybean root rot fungus

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Embodiment 1

[0047] Example 1 specific primer

[0048] 1. Acquisition of specific primers

[0049] Using the universal primer EF1 / EF2 of the elongation factor EF-1α gene sequence to perform PCR amplification on different soybean root rot pathogenic Fusarium, and obtain the EF-1α gene sequence of various soybean root rot pathogenic Fusarium And submitted to GenBank, gene accession numbers are KX966232, KX966220, KX966207 and KX966200. According to the EF-1α gene sequence of Fusarium, use primer design software such as Vector NTI 11.0software (Invitrogen, USA) or NCBI online platform (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to sequence Compare and find the difference sites, and design the specific primers with obvious differences in the size of the amplified fragments of the four pathogenic bacteria according to the difference sites, and obtain the specific primer sequences shown in SEQ ID NO.1-4. The reverse primers are designed in the conservative segment, the common reverse primer sho...

Embodiment 2

[0068] The specific detection of embodiment 2 specific primers

[0069] 1. Acquisition and cultivation of samples to be tested

[0070] Fusarium oxysporum (F.oxysporum), Fusarium solani (F.solani) and Fusarium equiseti (F.eqμiseti) obtained from the roots of soybean root rot diseased plants were selected and identified in our laboratory. and Fusarium graminearum (F. graminearum) four purified strains of pathogenic bacteria as samples to be tested.

[0071] Inoculate the above-mentioned 4 kinds of monospore purified strains of Fusarium spp. into potato dextrose agar medium (200g potato, 10g glucose, 20g agar powder, PDA) PDA medium respectively, and place them in an incubator at 25-28°C. Cultivate for 3 to 5 days and save for later use.

[0072] 2. Extraction of DNA

[0073] Fusarium DNA was extracted by CTAB method. The specific steps are as follows: Scrape the mycelium on the PDA medium into a mortar, grind it in liquid nitrogen until it is powdery, put it into a 2mL cent...

Embodiment 3

[0085] Sensitivity detection of embodiment 3 specific primers

[0086] The DNA (100ng / μL) of a single Fusarium was detected with a Nanodrop 2000 ultra-micro spectrophotometer, and the concentration of the mixed DNA was adjusted to 100ng / μL, and then 10 gradient dilutions were made to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 7 、10 -8 、10 -9 、10 -10 、10 -11 、10 -12 、10 -13 With different concentration gradients, the forward primers shown in SEQ ID NO.1-4 provided by the present invention are mixed at a volume ratio of 1:1:1:1, and the reverse primer shown in SEQ ID NO.5 is used as a common reverse primer. To the primers, and the established multiplex PCR system for amplification and electrophoresis detection, as shown in image 3 The electrophoresis results shown, according to image 3 The electropherogram shown shows that when the DNA is diluted to 10 -8 Fusarium can still be seen when folded. It can be seen that compared with the prior art, the sensitivity o...

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Abstract

The invention provides a specific primer group for analyzing the soybean fusarium root rot fungus diversity, a kit and application thereof, and relates to the field of biological detection. The specific primer group consists of a first forward primer shown as SEQ ID NO. 1, a second forward primer shown as SEQ ID NO. 2, a third forward primer shown as SEQ ID NO. 3, one or a plurality of fourth forward primers shown as SEQ ID NO. 4 and a shared reverse primer shown as SEQ ID NO. 5. When the specific primer group is used for the detection of soybean root rot caused by fusaria, the fusaria of the soybean root rot can be fast recognized; the types of the fusaria are determined through the strip belt size displayed according to the detecting result, so that a basis is provided for comprehensive prevention and control of the soybean root rot.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a detection method for analyzing the diversity of Fusarium soybean root rot fungi. Background technique [0002] Soybean root rot (Soybean root rot) is a fungal soil-borne disease with wide distribution, serious damage and difficult control in soybean production. The pathogenic group of soybean root rot is complex and diverse, and Fusarium is one of its important pathogenic groups. The main ones that have been reported at home and abroad are Fusarium solani (Fusarium solani), Fusarium oxysporum (F.oxysporum), Fusarium graminearum (F. graminearum), Fusarium equiseti (F.equiseti), F. proliferatum, F. pseudoproliferatum, F. avenaceum, F. semimitectum, F. tricinctum and F. armeniacum Wait. In Southwest my country, Fusarium solani (F.solani), Fusarium oxysporum (F.oxysporum), Fusarium graminearum (F.graminearum) and Fusarium equiseti (F.equiseti) are mainly used as soybean Root ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04C12N15/11C12R1/77
CPCC12Q1/686C12Q1/6895C12Q2600/16C12Q2537/143C12Q2565/125
Inventor 常小丽杨文钰付瑶何宛芹杨继芝武晓玲雍太文尚静
Owner 四川农科源检测技术有限公司
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