Specific PCR (Polymerase Chain Reaction) detection primer IKF5 / IKR4 for Karst colletotrichum gloeosporioides and application thereof

Abstract

The invention discloses a specific PCR (Polymerase Chain Reaction) detection primer IKF5 / IKR4 of Karst colletotrichum gloeosporioides and application of the specific PCR detection primer IKF5 / IKR4, the specific detection primers IKF5: 5 '-GTAGGCCGTCCCCTGAAAAG-3' and IKR4: 5 '-CCAAGCAGCTTGAGGGTT-3' on the basis of ITS (International Transcription Sequence) sequences, and the sensitivity of the specific detection primers IKF5: 5 '-GTAGGCCGTCCCTGAAAAG-3' and IKR4: 5 '-CCAAGCAGCTTGAGGGTT-3' is 100 pg. Mu L <-1 >. Research results show that the pair of primers can efficiently and specifically detect the Karst colletotrichum in the mango leaves, and a reliable technical tool is provided for early diagnosis of mango anthracnose, analysis of a disease occurrence rule and construction of a precise prevention and control strategy.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of molecular biology, and particularly relates to a group of specific PCR detection primers IKF5 / IKR4 for mango Colletotrichum karstii and application thereof. BACKGROUND

[0002] Mangifera indica L. is known as the "King of Fruits", and China is the second largest mango producer in the world after India, mainly concentrated in Guangxi, Hainan, Yunnan, Sichuan, Guangdong, Fujian, Guizhou and other seven provinces, among which the planting area of Guangxi ranks first. Mango anthracnose is the most important disease before and after mango harvesting, which seriously reduces the yield, commodity value and shortens the shelf life of mango, causing 30-60% loss per year, and the highest can reach 100%, which is an important limiting factor for the increase of mango yield and the improvement of quality. It has been confirmed that the pathogen composition of mango anthracnose in the main producing areas of China is C. asianum, C. fructicola, C. siamense, C. karstii, C. gloeosporioides, C. musae, C. endophytica, C. tropicale, C. cordylinicola, C. scovillei, C. cliviicola or C. cliviae, C. gigasporum and C. liaoningense.

[0003] Colletotrichum has the characteristics of latent infection, which can complete the infection process in tender tissues such as tender leaves, tender shoots and young fruits, but does not show symptoms, and only shows symptoms when the environmental conditions are suitable. This hidden infection mode makes it difficult to achieve rapid and early identification of diseases through conventional visual inspection and pathogen isolation and culture diagnosis. At present, specific detection primers for C. asianum, C. fructicola and C. siamense have been developed, but there is a lack of detection primers for C. karstii. Therefore, it is of great significance to establish a rapid and accurate molecular detection system for C. karstii for early identification, prevention and control of mango anthracnose pathogen species and sustainable development of the industry. SUMMARY

[0004] In view of the above-mentioned needs, the purpose of the present application is to provide a group of specific PCR detection primers for mango Colletotrichum karstii, which have the characteristics of strong specificity, good stability and high sensitivity.

[0005] The application also provides application of the specific PCR detection primer.

[0006] The application also provides the specific PCR detection kit.

[0007] To achieve the above-mentioned purposes, the application adopts the following measures:

[0008] A set of specific PCR detection primers for mango Guizhou Gnomoniella, the nucleotide sequences of which are as follows:

[0009] IKF5: 5'-GTAGGCCGTCCCCTGAAAAG-3',

[0010] IKR4: 5'-CCAAGCAGAGCTTGAGGGTT-3'.

[0011] The PCR rapid detection method for mango Guizhou Gnomoniella extracts DNA of a sample to be detected, and adopts the specific PCR detection primer to perform PCR detection, and the amplification product is 313 bp, so that the sample contains Guizhou Gnomoniella.

[0012] The mango Guizhou Gnomoniella is mango Guizhou Gnomoniella in a pathogenic bacterial population of mango anthracnose.

[0013] The DNA of the sample to be detected is DNA of a mango leaf.

[0014] In the PCR detection, the PCR amplification system is as follows: Premix Taq 12.5 μL, 1 μL of IKF5 and IKR4 primers each with a concentration of 10 μmol / L, 1 μL of DNA template, and finally adding sterilized double-distilled water to 25 μL.

[0015] In the PCR detection, the PCR amplification program of the primer IKF5 / IKR4 is as follows: 94 ℃ for 5 min, 94 ℃ for 30 s, 58 ℃ for 30 s, 72 ℃ for 1 min, 30 cycles, 72 ℃ for 10 min, and 4 ℃ for preservation.

[0016] A kit for detecting Guizhou Gnomoniella, comprising the primer.

[0017] The kit further comprises Premix Taq.

[0018] To accurately detect the pathogen of mango anthracnose, Colletotrichum karstii, the present application carries out molecular marker screening and primer design based on multiple gene loci. Using the MAFFT Alignment function in Geneious Prime software, the ITS region and ACT, CHS, and GAPDH gene sequences of 51 strains of 15 kinds of Colletotrichum reference strains are subjected to multiple sequence alignment, and specific sites of C. karstii are analyzed and screened. By comparing the ITS regions of different species of Colletotrichum, it is found that this region has high sequence polymorphism, and specific nucleotide variation sites of C. karstii are identified. Based on these specific variation sites, multiple pairs of primers are designed for specific amplification of C. karstii. Using the Geneious Prime system, the sequences of 51 strains of 15 kinds of Colletotrichum reference strains in the ITS region and four genes ACT, CHS, and GAPDH are analyzed, and molecular marker sites with significant specificity are screened. Ten upstream primers and ten downstream primers are designed in the ITS site, ten upstream primers and ten downstream primers are designed according to ACT, and five upstream primers and six downstream primers are designed according to GADPH. Since there is no obvious specific site in the CHS region, no primers are designed in this region. Through the combination and pairing of different primers, a total of 38 pairs of candidate primers are formed. Through screening, one pair of primers based on the ITS site with high sensitivity and high specificity is finally determined. The species-specific PCR detection system established in this study can realize the rapid and accurate identification of the pathogen of mango anthracnose, C. karstii. The application of this technology will significantly improve the early diagnosis efficiency of the pathogen of mango anthracnose, C. karstii, and provide important technical support for the development of precise prevention and control strategies, which has important significance for the sustainable development of the mango industry.

[0019] The specificity, stability, and sensitivity of the mango C. karstii specific primer were verified:

[0020] To verify the specificity and stability of the primers, 19 strains of 15 different species of Colletotrichum (including mango C. karstii strain YN34-1) and 12 strains of 12 different species of fungi other than Colletotrichum were subjected to PCR amplification. The results showed that primer pair IKF5 / IKR4 only stably amplified a clear specific band in mango C. karstii YN34-1, and no amplification product was obtained in all other tested strains (including closely related Colletotrichum species and other fungi). This result fully demonstrates that primer pair IKF5 / IKR4 has high species specificity for the target strain and good detection stability. The DNA of mango C. karstii (YN34-1) was diluted by 10 times to obtain the following eight concentration gradients: 10 ng·μL -1 , 1 ng·μL -1 , 100 pg·μL -1 , 10 pg·μL -11 pg·μL -1 100 fg·μL -1 10 fg·μL -1 and 1 fg·μL -1 Amplification was performed using primers IKF5 / IKR4, and the results showed that the lowest detectable concentration using IKF5 / IKR4 was 100 pg·μL. -1 .

[0021] Application of primers for PCR detection of *Anthrax karstii* in mangoes:

[0022] To verify the detection capability of this primer pair against *Anthracis mangosteen* in mixed DNA samples, this study conducted an artificial inoculation experiment using detached mango leaves. Specifically, healthy mango leaves grown in our laboratory greenhouse for over two years were inoculated with 6 mm diameter *Anthracis mangosteen* mycelial blocks. Leaves inoculated with blank PDA were used as a negative control to verify the reliability of the primers' specific amplification of the target band in a mixed DNA background. Results showed that 24 hours after inoculation, no disease symptoms were observed on the leaves, and clear bands were visible using the IKF5 / IKR4 primers. 48 hours after inoculation, mild symptoms began to appear, and the band signal gradually increased. By 72 hours, the symptoms were significant, and clear bands were observed in all samples detected by the primers. No amplification bands were observed in the negative control throughout the entire process. These results indicate that the target bacterium could be detected in the DNA of all detached mango leaves inoculated with *Anthracis mangosteen*, confirming that the detection primers can directly detect *Anthracis mangosteen* DNA from mixed DNA samples.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] This method allows for the direct and specific detection of *Anthracis mangosteen* from the mixed DNA of mango leaves infected with *Anthracis mangosteen*, eliminating the need for traditional, cumbersome steps such as pathogen isolation and culture, multi-gene phylogenetic analysis, and pathogenicity determination. Furthermore, the primers used in this method exhibit high specificity, high stability, and high sensitivity, enabling early detection and prevention. Attached Figure Description

[0025] Figure 1 Detection results based on 11 primer pairs designed for the ACT site of Bacillus karstii;

[0026] Figure 2 Detection results based on 8 primer pairs designed for the GAPDH site of Bacillus karstus;

[0027] Figure 3 Detection results based on 19 primer pairs designed for the ITS site of Bacillus karstus;

[0028] in, Figures 1-3 The specificity test results of 15 tested strains (corresponding to numbers 1-15 in Table 3) are shown. In each figure, lane M: DL 2000 molecular weight standard, and "negative" is the lane with ddH2O as the negative control.

[0029] Figure 4 Design site for the karst anthrax-specific primers IKF5 / IKR4.

[0030] Figure 5 The specificity of primers IKF5 / IKR4 for detecting Bacillus anthracis.

[0031] Lane M: DL 2000 molecular weight standard; Lanes 1-31: correspond to strains numbered 1-31 in Table 3 respectively; Lane negative: ddH2O.

[0032] Figure 6 Gel image showing the sensitivity of primers IKF5 / IKR4 to different concentrations of DNA from *A. mangostomata* (YN34-1); 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 DNA template; swim lane negative: ddH2O.

[0033] Figure 7 Gel image of mango leaves artificially inoculated with *A. mangoescarponema karstiensis* (YN34-1) using primers IKF5 / IKR4; Lane M: DL 2000 molecular weight standard; Lane PON: DNA of *A. mangoescarponema karstiensis* (YN34-1); Lanes 1-3: DNA of mango leaf tissues after 24 h, 48 h, and 72 h of inoculation, respectively; Lanes 4-6: DNA of mango leaf tissues after 24 h, 48 h, and 72 h of ddH2O inoculation; Lane PON: ddH2O. Detailed Implementation Plan

[0034] The present invention will now be clearly and completely described in conjunction with specific embodiments and accompanying drawings.

[0035] All biological materials used in this invention are preserved in the applicant's laboratory and are available for public distribution.

[0036] Example 1: Screening of specific sites for karst anthrax bacteria and primer design

[0037] This experiment utilized the MAFFT Alignment function in Geneious Prime software to perform multiple sequence alignment (Table 1) on the ITS region and ACT, CHS, and GAPDH gene sequences of 51 anthrax reference strains from 18 species. The specific site IKF5 / IKR4 of *Anthrax karstii* was analyzed and screened. Figure 4 ), and designed karst anthrax-specific primers using NCBI's Primer-BLAST tool. The primer sequences are shown in Table 2.

[0038] Table 1. Information on 51 reference strains from 15 different strains used for multiple sequence alignment.

[0039]

[0040]

[0041] Note: C. cliviicola and C. cliviae are synonyms of the same species.

[0042] Table 2 Primer sequences designed based on specific sites in the ITS region and ACT and GAPDH genes of Bacillus karstiensis.

[0043]

[0044]

[0045] Example 2: Verification of primer specificity among 15 anthrax species

[0046] The primers designed in Table 2 were used to amplify the DNA of 15 anthrax bacteria (including *Anthrax moniliforme* strain YN34-1) by PCR. The PCR program was: 94 ℃ for 5 min; 94 ℃ for 30 s, 55 ℃ for 30 s, 72 ℃ for 1 min, 30 cycles; extension at 72 ℃ for 5 min. The results showed that primer IKF5 / IKR4 could only stably amplify a clear, specific band from the DNA of *Anthrax moniliforme* strain YN34-1, while no amplification products were observed from any of the other tested strains. The remaining primers showed non-specific amplification in the DNA of other anthrax bacteria and closely related species; therefore, these primers were discarded. Figures 1-3 ).

[0047] Example 3: Screening of the specificity, stability, and sensitivity of primers IKF5 / IKR4 for detecting *Anthrax karstii* in mangoes.

[0048] The primers IKF5 / IKR4 selected from the initial screening were used to perform PCR amplification on DNA from 19 different strains of 15 species of anthracnose (including *Anthracnose mangifera* strain YN34-1) and 12 different fungal species other than *Anthracnose*. The results showed that primers IKF5 / IKR4 could only stably amplify a clear and specific band from the DNA of *Anthracnose mangifera* strain YN34-1. Figure 5 The DNA of *Anthracis mangostomata* (YN34-1) was serially diluted 10-fold to obtain the following eight concentration gradients: 10 ng / μL. No amplification products were observed in any of the other tested strains (including closely related anthracnose species and non-anthrax fungi), demonstrating its high species specificity and detection stability. Finally, the sensitivity of primers IKF5 / IKR4 was determined by serially diluting the DNA of *Anthracis mangostomata* (YN34-1) 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 IKF5 / IKR4, and the results showed that the lowest detectable concentration using primers IKF5 / IKR4 was 100 pg·μL. -1 ( Figure 6 The PCR amplification program for primer IKF5 / IKR4 PCR detection was as follows: 94 ℃ for 5 min, 94 ℃ for 30 s, 58 ℃ for 30 s, 72 ℃ for 1 min, 30 cycles, 72 ℃ for 5 min. Information on all tested strains is shown in Table 3. All tested strains in this invention were isolated, identified, and preserved in our laboratory.

[0049] Table 3 Information on 31 tested strains

[0050]

[0051] a: ITS accession number, ITS = Internal Transcriptional Spacer.

[0052] N: Fungi were identified based on morphological and ITS sequence analysis; accession number not uploaded.

[0053] Example 4: Application of the primers IKF5 / IKR4 for the specific detection of *Anthrax karstii* in mangoes

[0054] To verify that the primer pair could accurately detect the specific band of *Anthracis mangosteen* from mixed DNA of mango leaves inoculated with *Anthracis mangosteen*, artificial wound inoculation was performed on detached leaves of healthy mango plants grown in our laboratory greenhouse for more than two years using *Anthracis mangosteen* mycelial blocks (YN34-1). *Anthracis mangosteen* mycelial blocks with a diameter of 6 mm were used to inoculate the wound sites on the leaves. Leaves inoculated with blank PDA were used as a negative control. After inoculation, leaf samples were collected every 24 hours to extract genomic DNA. One sample each from the *Anthracis mangosteen* (YN34-1) inoculated and the blank control leaves were randomly collected each time, with each sample containing 5 leaves. A total of 3 samplings were performed, and PCR detection was performed using primers IKF5 / IKR4. The detection results are as follows: Figure 7 As shown, the target band of *Anthracis mangosteen* was detected in the DNA of all mango leaves inoculated with *Anthracis mangosteen*, while the target band was not amplified in the control group. This indicates that the detection primers can directly detect *Anthracis mangosteen* DNA from mixed DNA samples.

Claims

1. The specific PCR detection primer pair for *Colletotrichum karstii* in mangoes, with the following nucleotide sequence: IKF5:5'-GTAGGCCGTCCCCTGAAAAG-3', IKR4:5'-CCAAGCAGAGCTTGAGGGTT-3'.

2. A rapid PCR detection method for *Anthrax karstii* in mangoes: 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 313 bp, then the sample contains *Anthrax karstii*.

3. The method according to claim 2, wherein the mango karst anthracnose fungus is mango karst anthracnose fungus from the mango anthracnose pathogenic fungus group.

4. The method according to claim 3, wherein the DNA sample to be tested is DNA from mango leaves.

5. The method according to claim 4, wherein the PCR amplification system in the PCR detection is: 12.5 μL of Premix Taq, 1 μL each of IKF5 and IKR4 primers at a concentration of 10 μmol / L, 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, 58 ℃ for 30 s, 72 ℃ for 1 min, 30 cycles; extension at 72 ℃ for 5 min; storage at 4 ℃.

7. A kit for detecting *Anthrax karstii* in mangoes, 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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