CN116397049A - Multiplex PCR detection method for multiple fusarium species of wheat stem-based rot and application - Google Patents

Abstract

The invention discloses a multiplex PCR detection method and application of various Fusarium species of wheat stem base rot and belongs to the technical field of PCR molecular detection application in molecular biotechnology. The primer combination designed by the present invention can quickly detect Fusarium graminearum, Fusarium graminearum, Fusarium exfoliate and Fusarium verticillium in a single PCR reaction through a multiplex PCR amplification system, and the multiplex PCR has good specificity , only 4 target strains can be amplified from 23 test strains, and the genomic DNA of the pathogen as low as 10pg can be detected; it makes up for the long time required for the single-plex PCR to identify the pathogenic bacteria of wheat stem rot in the past Insufficient, the amplification and electrophoresis time of four kinds of Fusarium bacteria is only 2h. Therefore, the designed primers and detection method of the present invention can rapidly, specifically and sensitively identify four different Fusarium fungi that cause wheat stalk rot.

Description

Multiplex PCR detection method and application of multiple Fusarium species of wheat stem rot

technical field

The invention belongs to the technical field of PCR molecular detection application in molecular biology technology, and in particular relates to a multiplex PCR detection method and application of multiple Fusarium species of wheat stem rot disease.

Background technique

Wheat is a grass plant widely grown all over the world. Wheat caryopsis is one of the most important food crops, providing 19% of human daily calories and 21% of protein. Wheat stalk rot is a soil-borne disease and one of the most serious wheat diseases. It has been observed to affect the whole growth period of wheat in wheat cultivation in many arid and semi-arid regions of the world. The production and quality of the region caused great losses. In recent years, the harm of wheat stalk rot in the Huanghuai wheat region of China has gradually deteriorated. For example, in many wheat growing areas in Henan Province, the yield loss caused by wheat stalk rot is as high as 30-50%. In some high-incidence areas, wheat stalk rot has caused more than 70% of wheat yield losses, with an average annual decrease of 9-35% from 2008 to 2019.

Wheat stalk rot is often caused by a variety of Fusarium fungi, such as Fusarium pseudograminearum, F. graminearum, F. culmorum, F. avenaceum, F. verticillioides, and F. proliferatum. Previous studies have shown that F. pseudogminaceum is the dominant pathogen of FCR, which often combines with other Fusarium species to cause wheat stalk rot infecting wheat. And Zhang et al. (2015) pointed out that in Anhui, Jiangsu, Henan, Shandong and Hebei provinces, F. graminiferum is the dominant pathogen of wheat stem rot. The composition of the pathogenic bacteria of wheat stalk rot is complex, and the dominant pathogenic bacteria are different in different regions, which may be related to the location of sample collection and field ecological environment. Our previous investigations showed that wheat stalk rot in Hubei area was mainly caused by combined infection of F. pseudograminearum, F. graminearum, F. proliferatum and F. verticillioides. As a typical soil-borne disease, wheat stalk rot is often caused by a variety of Fusarium species, making it difficult to distinguish and diagnose the pathogenic species. Therefore, it is of great significance to quickly and accurately identify the dominant pathogens of wheat stalk rot and the species of wheat stalk rot pathogens in each growth stage of wheat for timely and targeted control of wheat stalk rot.

With the rapid development of molecular biology techniques, many molecular biology-based molecular detection methods for pathogenic bacteria have been developed. Compared with the traditional detection method based on isolation culture and morphological observation combined with biochemical characteristic detection and analysis, the molecular identification method of pathogenic bacteria is accurate and efficient. Currently, the molecular detection methods used to identify wheat stalk rot are based on the detection of a single pathogen. However, the identification of a single pathogen cannot meet the needs of identification of multiple pathogens of wheat stem rot. Compared with single-plex PCR, multiplex PCR has higher detection efficiency and can detect multiple pathogens at the same time, thereby reducing costs and saving time. The multiplex PCR molecular detection method can simultaneously amplify multiple target sequences, and has been applied to the detection of pathogens in related fields such as medicine, environment, and agricultural science. However, the current multiplex PCR molecular detection technology designs different primer pairs for the target genes of different pathogenic bacteria, resulting in too many primer combinations in one PCR reaction. In a multiplex PCR reaction system, too many primers for simultaneous PCR reactions may lead to cross-combination of primers to form primer dimers, thereby reducing amplification efficiency. By reducing the number of primers in multiplex PCR, the detection efficiency can be improved. With the rapid development of genome sequencing technology and bioinformatics, comparative genomics can be used to screen new molecular detection targets for pathogenic bacteria, providing a simpler and more efficient option for establishing a multiplex PCR molecular detection system. Therefore, based on comparative genomics, the common upstream primers of F. pseudograminearum, F. graminearum, F. proliferatum and F. verticillioides were screened, and then pathogen-specific downstream primers with different fragment sizes were designed sequentially, and then PCR reaction conditions were optimized and specificity detection was performed. and other experiments to establish a rapid diagnosis and detection of Fusarium species identification technology that causes wheat stalk rot.

Contents of the invention

One of the objects of the present invention is to provide a primer set for multiplex PCR detection of multiple Fusarium, said primer set includes an upstream primer and a downstream primer; said upstream primer sequence is shown in SEQ ID NO.1; said The downstream primer sequences are shown in SEQ ID NO.2-5.

Preferably, the plurality of Fusarium species are Fusarium pseudoglycerinus, Fusarium graminearum, Fusarium laminarum and Fusarium verticillium.

The second object of the present invention is to provide a reagent for detecting multiple Fusarium species by multiplex PCR, the reagent comprising the above primer set.

The third object of the present invention is to provide a kit for detecting multiple Fusarium species by multiplex PCR, which contains the above primer set or the above reagent.

The fourth object of the present invention is to provide a multiplex PCR detection method for a variety of Fusarium. The multiplex PCR detection method uses the total DNA of the sample as a template, and utilizes the above primer set to perform PCR amplification. After the reaction is completed, according to the agarose gel Electrophoresis judgment results.

Preferably, the amplification program of PCR is as follows: pre-denaturation at 94°C for 5 min; entering cycle, denaturation at 94°C for 30 s, annealing at 53°C for 30 s, extension at 72°C for 1 min, 32 cycles; final extension at 72°C for 10 min.

More preferably, the total volume of the PCR reaction system is 25 μL, including 2.5 μL of 10×PCR buffer, 1.5 μL of 25 mM Mg ²⁺ , 2 μL of 10 mM dNTP, 0.125ul 5U/μL Taq DNA polymerase, and a concentration of 10 μM upstream 4 μL of primer SEQ ID NO.1, 1 μL of each of downstream primers SEQ ID NO.2, 3, 4, and 5 at a concentration of 10 μM, 1 μL of DNA template, and 9.875 μL of ddH ₂ O.

More preferably, the DNA template is extracted from wheat.

The fifth object of the present invention is to provide the application of the above primer set in identifying various Fusarium species, such as Fusarium pseudograminee, Fusarium graminearum, Fusarium laminarum and Fusarium verticillium.

The sixth object of the present invention is to provide the application of the above-mentioned reagents or kits in the identification of various Fusarium species, the various Fusarium species being Fusarium graminearum, Fusarium graminearum, Fusarium laminarum and Fusarium verticillium .

Compared with the prior art, the present invention has the following beneficial effects:

The advantage of the present invention is that the designed primer combination can quickly detect Fusarium pseudograminee, Fusarium graminearum, Fusarium laminarum and Fusarium verticillium in a single PCR reaction through a multiplex PCR amplification system. Multiplex PCR has very good specificity, only 4 target strains can be amplified from 23 test strains, and the genomic DNA of pathogenic bacteria as low as 10pg can be detected; it makes up for the previous single-plex PCR identification of wheat stem rot The time required for the pathogenic bacteria is too long, and the amplification and electrophoresis time of the four kinds of Fusarium bacteria is only 2 hours. Therefore, the designed primers and detection method of the present invention can quickly, specifically and sensitively identify four different Fusarium fungi that cause wheat stalk rot.

Description of drawings

Fig. 1 is the agarose gel electrophoresis figure of multiplex PCR primer combination in embodiment 2 to four kinds of Fusarium specificity detection, wherein M: 2000bp DNA Marker; Mixed DNA of Fusarium and Fusarium verticillium; Lane 2: DNA of Fusarium verticillium; Lane 3: DNA of Fusarium exfoliates; Lane 4: DNA of Fusarium graminearum; Lane 5: DNA of Fusarium pseudogamine; Lane 6: Fusarium solani DNA; Lane 7: Fusarium rubrum DNA; Lane 8: Fusarium equiseti DNA; Lane 9: Fusarium oxysporum DNA; Lane 10: Fusarium asiatica DNA; Lane 11: Fusarium brevisporum DNA; Lane 12: DNA of Fusarium fujikura; Lane 13: DNA of Fusarium aculoides; Lane 14: DNA of Alternaria alternata; Lane 15: DNA of Sclerotinia sclerotiorum; Lane 16: DNA of Sclerotinia sclerotiorum; Rhizoctonia DNA; Lane 18: Phomopsis peach DNA; Lane 19: Polychaetosporum tea; Lane 20: Verticillium dahliae DNA; Lane 21: Botrytis cinerea DNA; Lane 22: Botrytis cinerea DNA; Lane 23: DNA of P. melon blight; Lane 24: DNA of P. melon anthracnose.

Fig. 2 is the agarose gel electrophoresis figure of multiplex PCR primer combination in embodiment 3 to the sensitivity detection of four kinds of Fusarium, wherein M: 2000bp DNA Marker; Swimming lane 1: 10ng/μL; Swimming lane 2: 1ng/μL; Swimming lane 3 Lane 4: 10 pg/μL; Lane 5: 1 pg/μL; Lane 6: 100 fg/μL; Lane 7: 10 fg/μL.

Fig. 3 is the agarose gel electrophoresis picture of multiplex PCR detection field wheat sample in embodiment 4, wherein M: 2000bpDNA Marker; PC: Fusarium graminearum, Fusarium graminearum, Fusarium laminarum and Fusarium verticillium mixed DNA, positive control; NC: sterile water, negative control; lanes 1-22: field wheat samples.

Detailed ways

Example 1

Establishment of Molecular Detection Methods for Fusarium pseudograminee, Fusarium graminearum, Fusarium laminarum and Fusarium verticillium

1. Primer design

Download F. pseudograminearum Class2-1C (GenBank Accession No. CP064755.1), F. graminearum PH-1 (GenBank Accession No. HG970332.2), F. proliferatum FP-A8 from NCBI (National Center for Biotechnology Information) database (GenBank Accession No. MRDB01000001.1), F. verticillioides 7600 (GenBank Accession No. CM000579.1), F. equiseti D25-1(GenBank Accession No.QOHM01000001.1), F. oxysporum f. sp. lycopersici 4287 (GenBank Accession No. bank Accession No.NC_030986.1), F. solani JS-169 (GenBank Accession No. NGZQ01000001.1), F.incarnatum MOD1-FUNGI18 (GenBank Accession No. RBBZ01000100.1) and F.asiaticum KCTC 16664 (GenBank Accession No. CP088257.1) of the whole genome sequence, conduct multi-genome comparison analysis on all the whole genome sequences, obtain the homologous sequence of the genome, find a 20bp base sequence in the homologous sequence, and use this sequence as the multiplex of four Fusarium species The upstream universal primer (Fu-4F) of the PCR reaction, and the specific downstream primers (Fgram-R, Fpseu-R, Fprol-R and Fvert-R) with different sequence fragment sizes were designed sequentially after the universal upstream primer sequence. Compare the designed specific downstream primers in the NCBI database, initially identify the specificity of the primer sequences, and finally obtain the specificity detection of multiplex PCR of Fusarium pseudograminee, Fusarium graminearum, Fusarium laminarum and Fusarium verticillium Primers, primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.:

Upstream primers:

Fu-4F (SEQ ID NO.1): CTTGAACCTGAGACCTTCGC;

Downstream primers:

Fpseu-R (SEQ ID NO. 2): CGCACATTGCTTATTGCTTA;

Fgram-R (SEQ ID NO.3): CTCATAGCGATATTCTCGTATAC;

Fprol-R (SEQ ID NO.4): ATTCACGGATGAGAATCAAG;

Fvert-R (SEQ ID NO. 5): TCAAAGGAATGTCCGGTAGA.

2. Extraction of Genomic DNA of Pathogenic Bacteria

Extraction was carried out according to the instruction of TIANGEN's plant genome extraction kit.

3. Multiplex PCR molecular detection of four kinds of Fusarium

PCR amplification was performed using the sample DNA as a template. The total volume of the PCR reaction system is 25ul, including: 2.5μL 10×PCR buffer, 1.5μL Mg ²⁺ (concentration 25mM), 2μL dNTP (concentration 10mM), 0.125ul Taq DNA polymerase (5U/μL), upstream primer Fu- 4F 4 μL (concentration 10 μM), downstream primers Fpseu-R, Fgram-R, Fprol-R, Fvert-R 1 μL each (concentration 10 μM), four Fusarium DNA templates 1 μL each, ddH ₂ O 6.875 μL. The PCR reaction program is: pre-denaturation at 94°C for 5 minutes; entering cycle, denaturation at 94°C for 30 seconds, annealing at 53°C for 30 seconds, extension at 72°C for 1 minute, 32 cycles; final extension at 72°C for 10 minutes; complete amplification, and store the amplified product at 4°C . Take 5 μL of the PCR product and electrophoresis in 1.5% m/v agarose gel. After staining with ethidium bromide, observe the size of the amplified product under ultraviolet light. The amplified 206bp DNA band is Fusarium graminearum, and the amplified 482bp DNA The band is Fusarium pseudograminee, the amplified 680bp DNA band is Fusarium laminarum, and the amplified 963bp DNA band is Verticillium fusarium.

Example 2

Specific Validation of Primers for Four Fusarium Multiplex PCR Detection

1. DNA extraction from samples to be tested

Using the above method to extract Fusarium pseudocereal, Fusarium graminearum, Fusarium laminarum, Fusarium verticillium, Fusarium solani, Fusarium reddening, Fusarium equisetum, Fusarium oxysporum, Fusarium asiatica, Fusarium sclerotium, Fusarium fujikura, Fusarium acuminate, Alternaria alternata, Sclerotinia sclerotiorum, Sclerotinia sclerotiorum, Rhizoctonia graminearum, Phomopsis acrescens, Phomopsis amoides, Trichospermia tea, Dahlia DNA from Cladosporium, Botrytis cinerea, Botrytis cinerea, Melonella spp., Melon anthracnose;

2. Specific detection

PCR amplification was carried out using the genomic DNA of the above-mentioned tested fungi as templates. The total volume of the PCR reaction system is 25ul, including: 2.5μL 10×PCR buffer, 1.5μL Mg ²⁺ (concentration 25mM), 2μL dNTP (concentration 10mM), 0.125ul TaqDNA polymerase (5U/μL), upstream primer Fu-4F 4 μL (concentration 10 μM) and downstream primers Fpseu-R, Fgram-R, Fprol-R, Fvert-R each 1 μL (concentration 10 μM), DNA template 1 μL, ddH ₂ O 9.875 μL. The PCR reaction program is: pre-denaturation at 94°C for 5 minutes; entering cycle, denaturation at 94°C for 30 seconds, annealing at 53°C for 30 seconds, extension at 72°C for 1 minute, 32 cycles; final extension at 72°C for 10 minutes; complete amplification, and store the amplified product at 4°C . 5 μL of the PCR product was electrophoresed on a 1.5% m/v agarose gel, and the size of the amplification product was observed under ultraviolet light after staining with ethidium bromide. As shown in Figure 1, multiplex PCR can only specifically amplify the 206bp DNA band of Fusarium graminearum, the 482bp DNA band of Pseudomonas graminearum, the 680bp DNA band of Fusarium exfoliate, and the 963bp DNA band of Verticillium Fusarium, while the other strains had no amplified bands.

Example 3

Sensitivity Validation of Four Fusarium Multiplex PCR Detection Primers

1. DNA extraction from samples to be tested

Using the above method to extract Fusarium pseudograminee, Fusarium graminearum, Fusarium laminarum and Fusarium verticillium.

2. Sensitivity testing

Use a NanoDrop ultra-micro spectrophotometer to measure the concentration of the above-mentioned genomic DNA, and sequentially dilute to 10ng/μL, 1ng/μL, 100pg/μL, 10pg/μL, 1pg/μL, 100fg/μL and 10fg/μL with ddH ₂ O, respectively. . PCR amplification was carried out using the above-mentioned different concentrations of DNA as templates. The total volume of the PCR reaction system is 25ul, including: 2.5μL 10×PCR buffer, 1.5μL Mg ²⁺ (concentration 25mM), 2μL dNTP (concentration 10mM), 0.125ul Taq DNA polymerase (5U/μL), upstream primer Fu- 4F 4 μL (concentration 10 μM), downstream primers Fpseu-R, Fgram-R, Fprol-R, Fvert-R each 1 μL (concentration 10 μM), four Fusarium DNA templates each 1 μL, ddH ₂ O 6.875 μL. The PCR reaction program is: pre-denaturation at 94°C for 5 minutes; entering cycle, denaturation at 94°C for 30 seconds, annealing at 53°C for 30 seconds, extension at 72°C for 1 minute, 32 cycles; final extension at 72°C for 10 minutes; complete amplification, and store the amplified product at 4°C . Take 5 μL of PCR products and electrophoresis in 1.5% m/v agarose gel. After staining with ethidium bromide, observe the size of the amplified products under ultraviolet light. Multiplex PCR can only specifically amplify the 206bp DNA band of cereal Fusarium, 482bp DNA band of Fusarium pseudocereal, 680bp DNA band of Lamellar Fusarium, 963bp DNA band of Fusarium verticillium. As shown in Figure 2, the sensitivity verification results show that the minimum limit of multiplex PCR detection of DNA concentration of Fusarium graminearum, Fusarium laminarum and Verticillium verticillium is 10 pg/μL, while the detection minimum concentration limit of Fusarium pseudogaminergi 100pg/μL.

Example 4

Practical testing of wheat samples in the field

1. Extraction of total DNA from the plant material to be tested

Take about 0.5g of wheat root system, fully grind it into powder in liquid nitrogen, and then extract it according to the instructions of TIANGEN's plant genome extraction kit.

2. Pathogen detection of wheat samples in the field

The above-mentioned wheat sample DNA was used as a template for PCR amplification. The total volume of the PCR reaction system is 25ul, including: 2.5μL 10×PCR buffer, 1.5μL Mg ²⁺ (concentration 25mM), 2μL dNTP (concentration 10mM), 0.125ul Taq DNA polymerase (5U/μL), upstream primer Fu-4F 4 μL (concentration 10 μM) and downstream primers Fpseu-R, Fgram-R, Fprol-R, Fvert-R each 1 μL (concentration 10 μM), wheat sample DNA template 1 μL, ddH ₂ O 9.875 μL. The PCR reaction program is: pre-denaturation at 94°C for 5 minutes; entering cycle, denaturation at 94°C for 30 seconds, annealing at 53°C for 30 seconds, extension at 72°C for 1 minute, 32 cycles; final extension at 72°C for 10 minutes; complete amplification, and store the amplified product at 4°C . 5 μL of the PCR product was electrophoresed on a 1.5% m/v agarose gel, and the size of the amplification product was observed under ultraviolet light after staining with ethidium bromide. As shown in Figure 3, the amplified 206bp DNA band was Fusarium graminearum, the amplified 482bp DNA band was Fusarium graminearum, and the amplified 963bp DNA band was Verticillium verticillium, respectively. Fifteen, 10, and 3 samples were identified as Fusarium graminearum, Fusarium graminearum and Fusarium verticillium, among which 10 wheat samples were identified with Fusarium gramineae and Fusarium gramineae at the same time, 2 F. graminearum and F. graminearum were identified simultaneously in two samples, and F. graminearum, F. graminearum and F. verticillium were simultaneously identified in one sample.

The above-mentioned embodiments are only to describe the preferred mode of the present invention, and are not intended to limit the scope of the present invention. Variations and improvements should fall within the scope of protection defined by the claims of the present invention.

Claims (10)

1. A primer set for multiplex PCR detection of a plurality of fusarium species, wherein the primer set comprises an upstream primer and a downstream primer; the upstream primer sequence is shown as SEQ ID NO. 1; the sequence of the downstream primer is shown as SEQ ID NO. 2-5.

2. The primer set of claim 1, wherein the plurality of fusarium species are fusarium pseudograminearum, fusarium graminearum, fusarium layering, and fusarium verticillatum.

3. A reagent for multiplex PCR detection of a plurality of fusarium species, comprising the primer set of claim 1.

4. A kit for multiplex PCR detection of a plurality of fusarium species, comprising the primer set of claim 1 or the reagent of claim 3.

5. A multiplex PCR detection method for a plurality of fusarium is characterized in that the multiplex PCR detection method uses total DNA of a sample as a template, uses the primer group of claim 1 to carry out PCR amplification, and judges the result according to agarose gel electrophoresis after the reaction is finished.

6. The multiplex PCR detection method according to claim 5, wherein the PCR amplification procedure is as follows: pre-denaturation at 94℃for 5min; the mixture is subjected to cycle, denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 30s and extension at 72 ℃ for 1min for 32 cycles; finally, the temperature is 72 ℃ for 10min.

7. The multiplex PCR detection method according to claim 6, wherein the total volume of the PCR reaction system is 25. Mu.L, and the multiplex PCR detection method comprises 2.5. Mu.L of 10 XPCR buffer and 1.5. Mu.L of Mg at a concentration of 25mM ²⁺ 2. Mu.L of 10mM dNTP,0.125ul 5U/. Mu.L of Taq DNA polymerase, 10. Mu.M upstream primer SEQ ID NO.1 4. Mu.L, 10. Mu.M downstream primers SEQ ID NO.2, 3, 4, 5 each 1. Mu.L, DNA template 1. Mu.L, ddH ₂ O 9.875μL。

8. The multiplex PCR detection method as claimed in claim 7 wherein the DNA template is extracted from wheat.

9. The use of the primer set of claim 1 for identifying a plurality of fusarium species, wherein the plurality of fusarium species is fusarium pseudograminearum, fusarium graminearum, fusarium layering, and fusarium verticillatum.

10. Use of the reagent of claim 3 or the kit of claim 4 for identifying a plurality of fusarium species, wherein the plurality of fusarium species is fusarium pseudograminearum, fusarium graminearum, fusarium layering, and fusarium verticillatum.

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