Specific PCR (Polymerase Chain Reaction) detection primer THF1 / THR2 of colletotrichum gloeosporioides and application of specific PCR detection primer THF1 / THR2

Abstract

The invention discloses a specific PCR (Polymerase Chain Reaction) detection primer THF1 / THR2 of colletotrichum gloeosporioides and an application of the specific PCR detection primer THF1 / THR2. And the specific detection primers THF1: 5 '-GGGGCTAACCAGAGTTCTTTCA-3', and THR2: 5 '-TTAGCGGTCAACCACCAATGT-3' are designed on the basis of a TUB2 (beta-tubulin) sequence, and the specific detection primers THF1: 5 '-GGGGCTAACCAGAGTTCTTTCA-3' and THR2: 5 '-TTAGCGGTCAACCACCAATGT-3' are designed on the basis of a The pair of primers has very strong specificity, the sensitivity of the primers THF1 / THR2 is 100 pg.mu L <-1 >, and the primers THF1 / THR2 can be used for detecting the condition that persimmon leaf samples in the field carry the colletotrichum harerae, and provide technical support for early diagnosis of persimmon anthracnose, research of disease occurrence rules and formulation of prevention and control strategies.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of molecular biology, and particularly relates to a specific PCR detection primer THF1 / THR2 for Colletotrichum horii and application thereof. BACKGROUND

[0002] Persimmon (Diospyros kaki) is a deciduous tree fruit of the Ebenaceae family, which is originally from China and has a cultivation history of more than 2,000 years, especially in Fuping County, Shaanxi Province, Gongcheng County and Pingle County, Guangxi Zhuang Autonomous Region, etc. According to the monitoring data of the Food and Agriculture Organization of the United Nations (FAOSTAT), the persimmon yield in China showed a continuous growth trend from 1994 to 2020, with an average annual growth rate of 4.8%. Persimmon anthracnose is an important fungal disease in persimmon production, and the pathogen is mainly Colletotrichum spp. fungi, which mainly infects leaves and fruits, causing leaves to be unable to carry out normal photosynthesis, fruits to turn brown and black, and seriously affecting the yield and quality of persimmons. After systematic identification, there are mainly five pathogens of persimmon anthracnose in China, which are Colletotrichum fructicola, C. horii, C. karsti, C. cliviae and C. siamense, among which the first three are dominant species.

[0003] The latest research progress reveals that Colletotrichum spp. exhibits typical latent infection biological characteristics during the process of infecting persimmon trees. During the latent stage of the pathogen, the pathogen has completed the infection process, but the host has not shown obvious symptoms, which makes it difficult to achieve early and accurate detection by conventional morphological diagnosis methods. At present, pathogen identification still mainly relies on traditional isolation and culture technology, which has the disadvantages of time-consuming, labor-intensive and high cost. At present, there are specific detection primers for C. fructicola, but specific detection primers for C. horii have not been developed yet. Therefore, establishing a specific detection system for C. horii based on molecular biology technology can provide key technical support for early warning and precise prevention and control of persimmon anthracnose. SUMMARY

[0004] In view of the problems in the prior art that it is difficult to quickly and accurately detect and identify C. horii in persimmons, the application provides a group of PCR detection primers for C. horii with strong specificity, good stability and high sensitivity, so as to realize efficient molecular detection of the pathogen.

[0005] The application also provides the application of the specific PCR detection primer in early diagnosis of persimmon anthracnose.

[0006] In addition, the application also provides a specific PCR detection kit.

[0007] To achieve the above object, the present application adopts the following technical measures:

[0008] 1. Specific site screening: using the MAFFT Alignment function in Geneious Prime software, the TUB2 gene sequences of 15 kinds of 51 reference strains are subjected to multiple sequence alignment, and the specific site of Colletotrichum harzianum is analyzed and screened.

[0009] 2. Based on the above specific site, the primer for targeting the reverse complementary sequence of the TUB2 gene of Colletotrichum harzianum is designed using DNAMAN to ensure its specific targeting. The designed primer is verified for specificity, sensitivity and stability, and the PCR reaction system is optimized.

[0010] The specific PCR detection primer pair of C. harzianum of persimmon has the following nucleotide sequences:

[0011] THF1: 5'-GGGGCTAACCAGAGTTCTTTCA-3',

[0012] THR2: 5'-TTAGCGGTCAACCACCAATGT-3'.

[0013] The PCR rapid detection method of C. harzianum of persimmon extracts DNA of the sample to be tested, and uses the above specific PCR detection primer pair for PCR detection, and the amplification product is 211 bp, so the sample contains C. harzianum.

[0014] The C. harzianum of persimmon is C. harzianum in the pathogenic bacterial flora of persimmon anthracnose.

[0015] The DNA of the sample to be tested is persimmon leaf DNA.

[0016] The PCR amplification system in the PCR detection is: Premix Taq 12.5 μL, 1 μL of THF1 and THR2 primers with a concentration of 10 μmol / L each, 1 μL of DNA template, and finally adding sterilized double distilled water to 25 μL.

[0017] The PCR amplification procedure in the PCR detection is: 94 ℃ for 5 min; 94 ℃ for 30 s, 62 ℃ for 30 s, 72 ℃ for 45 s, 30 cycles; 72 ℃ extension for 5 min; 4 ℃ storage.

[0018] A kit for detecting C. harzianum of persimmon includes the above specific PCR detection primer pair.

[0019] The kit further comprises Premix Taq.

[0020] TUB2 has both conservation and variability, which is a reliable molecular marker for fungal classification, evolution and drug resistance research, especially suitable for fine distinction of closely related species. Since no obvious specific site was found in the ITS region, primers were not designed for this region. For persimmon anthracnose caused by G. harae, the differences in TUB2 gene sequences of 15 species and 51 strains of Colletotrichum were analyzed using Geneious Prime system, and molecular marker sites with significant specificity were screened through multiple sequence alignment. According to the TUB2 gene site, two upstream primers and one downstream primer were designed. Through the combination of different primers, two pairs of candidate primers were formed: THF1 / THR2, THF3 / THR2. Through screening, one pair of primers with high sensitivity and high specificity was finally determined to realize the early and rapid diagnosis of the pathogen, and provide technical support for disease control.

[0021] The specificity, stability and sensitivity of the G. harae specific primer were verified:

[0022] To verify the specificity and stability of the primers, the DNA of 15 species and 19 strains of different types of Colletotrichum (including persimmon G. harae strain PLG3-1), 12 species and 12 strains of different species of fungi other than Colletotrichum, and 5 species and 5 strains of different species of bacteria were subjected to PCR amplification. The results showed that primer THF1 / THR2 could only stably amplify a clear specific band from the DNA of persimmon G. harae strain PLG3-1, while all other test strains (including closely related Colletotrichum species, non-Colletotrichum fungi and bacteria) did not produce any amplification products, proving its high species specificity and detection stability. The DNA of G. harae (PLG3-1) was diluted by 10 times to obtain the following 8 concentration gradients: 10 ng·μL -1 , 1 ng·μL -1 , 100 pg·μL -1 , 10 pg·μL -1 , 1 pg·μL -1 , 100 fg·μL -1 , 10 fg·μL -1 and 1 fg·μL -1 , and amplified with primers THF1 / THR2, respectively. The results showed that the minimum concentration that could be detected by primers THF1 / THR2 was 100 pg·μL -1 .

[0023] Application of G. harae PCR detection primer:

[0024] To verify the ability of the primers to detect G. harveyan in complex background, the spore suspension was used to inoculate the leaves of Diospyros kaki in vitro in Gongcheng County, Guilin, and the leaves inoculated with ddH2O were used as negative control. The leaves were sampled every 24 hours after inoculation, and the genomic DNA of the leaves was extracted as template for PCR detection using the primers THF1 / THR2. The results showed that: 24 hours after inoculation, no disease symptoms were observed in the leaves, and no target band was detected; 48 hours after inoculation, the leaves began to show mild disease symptoms, and weak bands appeared; 72 hours after inoculation, the disease symptoms were aggravated, and the band signal was significantly enhanced. The negative control had no amplification in the whole process. The results show that the primers can specifically detect G. harveyan from the mixed DNA of host plants, and the detection sensitivity increases with the extension of the infection time, which is suitable for early and latent pathogen detection.

[0025] Compared with the prior art, the present application has the following remarkable advantages:

[0026] The detection method established by the present application can directly use the total DNA of persimmon leaf samples for PCR detection, without relying on the traditional complex processes such as pathogenic fungus isolation and purification, multi-gene phylogenetic identification and pathogenicity determination, and can specifically detect G. harveyan. In addition, the primers used in the present application have high specificity, good stability and high sensitivity, which provides reliable technical support for early monitoring and precise prevention and control of persimmon anthracnose. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Design site of G. harveyan specific primers THF1 / THR2.

[0028] Figure 2 The specificity detection results of the primers designed based on the TUB2 specific site of G. harveyan on 15 test strains (corresponding to serial numbers 1~15 in Table 3) are shown. In the figure, lane M: DL 2000 molecular weight standard.

[0029] Figure 3 Specificity of G. harveyan detection primers THF1 / THR2. Lane M: DL 2000 molecular weight standard; lanes 1~36: corresponding to serial numbers 1~36 in Table 3, lane negative: ddH2O.

[0030] Figure 4 Sensitivity detection gel of primers THF1 / THR2 on different concentrations of DNA of G. harveyan (PLG3-1); lane M: DL 2000 molecular weight standard; lanes 1~8: corresponding to concentrations of 10 ng·μL -1 , 1 ng·μL -1 , 100 pg·μL -1 , 10 pg·μL-1 , 1 pg·μL -1 , 100 fg·μL -1 , 10 fg·μL -1 and 1 fg·μL -1 of DNA template; Lane N: ddH2O.

[0031] Figure 5 Gel map of primer THF1 / THR2 detection on persimmon leaf artificially inoculated with G. harveyan (PLG3-1); Lane M: DL 2000 molecular weight marker; Lanes 1~3: DNA of persimmon leaf tissue inoculated for 24 h, 48 h and 72 h, respectively; Lanes 4~6: DNA of persimmon leaf tissue inoculated with ddH2O for 24 h, 48 h and 72 h; Lane N: ddH2O. Specific embodiments

[0032] The application will be clearly and completely explained below in combination with specific examples and drawings.

[0033] All biological materials in the application are preserved in the laboratory of the applicant and can be distributed to the public.

[0034] Example 1 Screening of G. harveyan specific sites and primer design

[0035] In this experiment, the MAFFT Alignment function in Geneious Prime software was used to perform multiple sequence alignment on the ITS region and TUB2 gene sequences of 15 kinds of 51 reference strains (Table 1). Based on the alignment results, G. harveyan specific sites were analyzed and screened out (Table 2). Figure 1 Finally, according to the obtained specific sites, the primer targeting the reverse complementary sequence downstream of the TUB2 gene of G. harveyan was designed using the DNAMAN software, and the primer sequence is shown in Table 2.

[0036] Table 1 Information of 15 kinds of 51 reference strains used for multiple sequence alignment

[0037]

[0038]

[0039] Note: C. cliviicola and C. cliviae are synonymous.

[0040] Table 2 Primer sequence designed based on G. harveyan specific sites on TUB2

[0041]

[0042] Example 2 Verification of specificity of primers in 15 species of Colletotrichum gloeosporioides complex

[0043] The primers designed in Table 2 were used to amplify DNA from 15 species of Colletotrichum, including C. gloeosporioides strain PLG3-1, using the following PCR program: 5 min at 94 °C; 30 s at 94 °C, 30 s at 55 °C, 1 min at 72 °C, 30 cycles; 5 min at 72 °C. The results showed that only primers THF1 / THR2 could stably amplify a clear specific band from DNA of C. gloeosporioides strain PLG3-1 ( Figure 2 ), while no amplification product was observed for any of the other tested strains. Primers THF3 / THR2 showed non-specific amplification or no specific amplification in the amplification of DNA from other Colletotrichum species and related species, so this pair of primers was discarded ( Figure 2 ).

[0044] Example 3 Screening of specificity, stability and sensitivity of detection primers THF1 / THR2 for C. gloeosporioides

[0045] The primers THF1 / THR2, which were initially screened, were used to amplify DNA from 19 strains of 15 different species of Colletotrichum (including C. gloeosporioides strain PLG3-1), 12 strains of 12 different species of fungi other than Colletotrichum, and 5 strains of different species of bacteria. The results showed that primers THF1 / THR2 could only stably amplify a clear specific band from DNA of C. gloeosporioides PLG3-1 ( Figure 3 ), while no amplification product was observed for any of the other tested strains (including related Colletotrichum species, non-Colletotrichum fungi, and bacteria), proving that they have high species specificity and detection stability. Finally, the sensitivity of primers THF1 / THR2 was determined by performing gradient dilutions of DNA from C. gloeosporioides (PLG3-1) at 10-fold concentrations, resulting in the following eight concentration gradients: 10 ng·μL -1 , 1 ng·μL -1 , 100 pg·μL -1 , 10 pg·μL -1 , 1 pg·μL -1 , 100 fg·μL -1 , 10 fg·μL -1 , and 1 fg·μL -1 . Amplification was performed using primers THF1 / THR2. The results showed that the lowest concentration that could be detected by primers THF1 / THR2 was 100 pg·μL -1 Figure 4 ​The PCR amplification program for the THF1 / THR2 primer detection was as follows: 94 ℃ for 5 min, 94 ℃ for 30 s, 62 ℃ for 30 s, 72 ℃ for 45 s, 30 cycles, 72 ℃ for 5 min. The PCR amplification system used in the above experiments consisted of: 12.5 μL Premix Taq (TaKaRa Taq Version 2.0 plus dye), 1 μL each of 10 μmol / L forward and reverse primers, 1 μL DNA template, and finally, sterile double-distilled water to a final volume of 25 μL. Information on all tested strains is shown in Table 3. All tested strains in this invention were isolated, identified, and preserved in our laboratory.

[0046] Table 3 Information on 36 tested strains

[0047]

[0048] a: ITS accession number, ITS = Internal Transcriptional Spacer.

[0049] b: Accession number of 16S rRNA, 16S rRNA = 16S ribosomal RNA.

[0050] N: Fungi were identified based on morphological and ITS sequence analysis, and bacteria were identified based on morphological and 16S rRNA sequence analysis. No accession numbers were uploaded.

[0051] Example 4: Application of the THF1 / THR2 primers for the specific detection of *Haryella anthracis*

[0052] To verify that the primer pair could accurately detect the specific band of *Anthracis harei* from mixed DNA inoculated with *Anthracis harei*, artificial wound inoculation was performed on detached leaves of healthy persimmon plants collected from Gongcheng, Guilin, using a suspension of *Anthracis harei* spores (PLG3-1). 10 μL of a spore suspension of *Anthracis harei* spores (concentration 10 μL) was taken. 6 Inoculate the wound with 10 μL of a 10 μg / mL solution. 6 A spore suspension of *P. persimmon* anthracnose (PLG3-1) at a concentration of spores / mL was inoculated onto leaf wounds, with leaves inoculated with an equal amount of ddH2O serving as a negative control. All inoculation sites were covered with sterile absorbent cotton to maintain humidity. Leaf samples were collected every 24 hours from inoculation, with each sample containing 5 leaves, for a total of 3 samplings. PCR detection was performed using primers THF1 / THR2. The results are as follows: Figure 5As shown, no disease symptom was observed on the leaves inoculated for 24 hours, and no target band was detected; the leaves started to show slight disease symptom and weak band appeared at 48 hours post inoculation; at 72 hours, the disease symptom was aggravated and the band signal was significantly enhanced, while the negative control had no amplification at all. The results showed that the primers could specifically detect G. persiana from the mixed DNA of host plants, and the detection sensitivity was improved with the extension of infection time, which was suitable for early and latent stage pathogen detection.

Claims

1. The specific PCR detection primer pair for *Colletotrichum horii* is shown below, with the following nucleotide sequence: THF1:5'-GGGGCTAACCAGAGTTCTTTCA-3', THR2:5'-TTAGCGGTCAACCACCAATGT-3'.

2. A rapid PCR detection method for *Haryonium halys* in persimmons: DNA is extracted from the sample to be tested, and PCR detection is performed using the specific PCR detection primer pair described in claim 1. If the amplification product is 211 bp, then the sample contains *Haryonium halys*.

3. The method according to claim 2, wherein the *Haryella persimmonis* is *Haryella persimmonis* from the *Haryella persimmonis* anthracnose pathogen flora.

4. The method according to claim 2, wherein the DNA sample to be tested is persimmon leaf DNA.

5. The method according to claim 2, wherein the PCR amplification system in the PCR detection is: 12.5 μL of Premix Taq, 1 μL each of 10 μmol / L THF1 and THR2 primers, 1 μL of DNA template, and finally, sterile double-distilled water to a final volume of 25 μL.

6. The method according to claim 5, wherein the PCR amplification program in the PCR detection is: 94 ℃ for 5 min; 94 ℃ for 30 s, 62 ℃ for 30 s, 72 ℃ for 45 s, 30 cycles; extension at 72 ℃ for 5 min; storage at 4 ℃.

7. A kit for detecting *Haryonius persimmonis* anthrax, comprising the specific PCR detection primer pair as described in claim 1.

8. The kit according to claim 7, further comprising Premix Taq.

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