A LAMP primer composition for detecting citrus brown rot phytophthora and its application

Abstract

The present invention discloses a LAMP primer composition for detecting citrus brown rot Phytophthora and its application. The primer composition comprises: a forward inner primer FIP as shown in SEQ ID NO.1, a reverse inner primer BIP as shown in SEQ ID NO.2, a forward outer primer F3 as shown in SEQ ID NO.3, a reverse outer primer B3 as shown in SEQ ID NO.4, and a forward loop primer LF as shown in SEQ ID NO.5. The present invention designs primers based on the guanosine triphosphate binding protein (Ypt1) gene sequence of citrus brown rot Phytophthora, and the reaction procedure is: amplification at 63°C isothermal conditions for 60 minutes. The LAMP primer composition disclosed in the present invention can be used for rapid and efficient detection of citrus brown rot Phytophthora, and the detection method has the advantages of strong specificity, high accuracy, good sensitivity, simple operation, and the results can be observed with the naked eye. When the DNA reaches 100pg·μL ‑1 Citrus brown rot fungus can be detected at any time, which is very practical.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a LAMP primer composition for detecting citrus brown rot phytophthora and application thereof. Background Art

[0002] Phytophthora citrophthora belongs to the kingdom Stramenopilia, phylum Heterokont, class Oomycetes, order Peronosporales, family Peronosporaceae. Phytophthora citrophthora is widely distributed throughout the world and is an important plant pathogen that can infect a variety of plants such as citrus, orange, grapefruit, lemon, horse chestnut, pear, peach, etc., causing symptoms such as plant foot rot, root rot, leaf blight, leaf spot, and fruit rot, causing heavy economic losses in the production of infected plants. The lesions caused by Phytophthora citrophthora are light brown in the early stage, and the diseased parts are brown and water-soaked in the later stage. Under humid conditions, soft and sparse white hyphae are easy to grow on the diseased parts. The fungus is serious and is of great importance in citrus diseases. The fungus has a short incubation period and develops quickly. It overwinters with hyphae and thick-walled spores on diseased plants or soil residues. The next year, the hyphae grow and produce sporangia to release zoospores, which infect leaves and fruits with water flow or raindrops, causing disease. Humid and rainy weather is conducive to hyphae expansion and sporangium formation, and the number of reinfections is high, resulting in rapid spread. In view of the harmfulness of citrus brown rot Phytophthora, a method that can quickly and accurately identify the pathogen in the early stage of plant disease is needed in production.

[0003] At present, there are few detection technologies for citrus brown rot Phytophthora, and the traditional morphological feature identification method is usually used, that is, the plate separation method and the bait method are used to identify the citrus brown rot Phytophthora based on its morphology. This method is time-consuming and labor-intensive, requires rich and professional experience in pathogen identification, and it is difficult to distinguish citrus brown rot Phytophthora from similar species based on morphological characteristics, which cannot meet the needs of field production. With the development of molecular biology, especially the popularization of PCR technology, more and more molecular biology techniques are applied to the detection of citrus brown rot Phytophthora. However, these detection technologies often need to be carried out in laboratories with professional instruments, reagents and strict environmental conditions. The instruments and reagents are expensive and have high operating requirements. The process is complicated and the detection time is still long. It cannot meet the needs of rapid detection and is not suitable for use and promotion in grassroots environments.

[0004] In recent years, loop-mediated isothermal amplification (LAMP) technology has been applied by more and more researchers to the detection of plant pathogens due to its strong specificity, simple operation, high sensitivity, simple result determination and low cost. This method uses 4-6 primers to achieve rapid amplification of target fragments under isothermal conditions. The LAMP reaction is intense, and a large number of amplification products of varying sizes are generated while a white precipitate visible to the naked eye - magnesium pyrophosphate. If a metal ion indicator such as hydroxyl tea blue HNB is added to the LAMP reaction system, the solution gradually turns sky blue during the LAMP reaction. The visual color reaction greatly simplifies the detection of LAMP amplification products. At present, reports on LAMP technology are mostly seen in health and safety testing, disease diagnosis and environmental safety testing. However, in the detection of plant pathogenic fungi, the application of LAMP technology needs to be further expanded, and no reports on the detection of citrus brown rot Phytophthora by LAMP have been found.

[0005] The selection of target genes is one of the important factors in LAMP detection. Currently, the source of primers designed for LAMP detection is mainly the transcribed spacer (ITS), but there are not enough sites to distinguish similar species. Therefore, developing new detection targets has become a hot spot for detection. Summary of the invention

[0006] Objectives of the invention: The first objective of the invention is to provide a LAMP primer composition and its application for detecting citrus brown rot Phytophthora using Ypt1 gene as a specific target. The second objective of the invention is to provide a LAMP method for detecting citrus brown rot Phytophthora.

[0007] Technical solution: The present invention discloses a LAMP primer composition for detecting citrus brown rot Phytophthora using the Ypt1 gene as a specific target, the primer composition comprising: a forward inner primer FIP as shown in SEQ ID NO.1, a reverse inner primer BIP as shown in SEQ ID NO.2, a forward outer primer F3 as shown in SEQ ID NO.3, a reverse outer primer B3 as shown in SEQ ID NO.4, and a forward loop primer LF as shown in SEQ ID NO.5.

[0008] Since the traditional citrus brown rot Phytophthora detection method is only based on morphological characteristics for identification, and the growth of citrus brown rot Phytophthora is affected by temperature and its morphology is unstable, and it is also affected by similar species, so it is difficult to accurately identify it. According to the Ypt1 gene sequence of citrus brown rot Phytophthora, the present invention uses Blast software to compare the genome sequence of citrus brown rot Phytophthora with the genome sequence of other Phytophthora, selects a Ypt1 gene sequence unique to citrus brown rot Phytophthora and designs specific LAMP primers. In the LAMP design primer, the target region length of the primer is usually about 200-400bp. The Ypt1 gene length of the Phytophthora strain is about 700bp. Due to the excessive length, the full-length sequence of Ypt1 cannot be used as a candidate target. The target region is particularly important when designing LAMP primers, which directly affects the specificity and sensitivity of the primers. The present invention has been tested in different regions, and 20 sets of primer systems have been designed. Through the test of these primer systems, a unique successful primer combination (Table 1) and reaction system have been selected. This primer combination has no reaction to bacterial strains of similar species and similar genera, has strong specificity, and has been proved to have this result through repeated tests. The LAMP reaction of this primer combination specifically recognizes 6 independent regions (Table 1) on the target sequence through 4 primers (FIP, BIP, F3, B3), and its specificity is relatively high. In addition, the forward loop primer LF can improve the reaction rate, and together with the other four primers, the present invention can detect citrus brown rot phytophthora quickly while ensuring the accuracy of the reaction. The LAMP reaction specifically recognizes 6 independent regions on the target sequence through 4 primers, and has relatively high specificity and sensitivity relative to 2 independent regions of the PCR primer recognition target sequence.

[0009] The invention relates to the use of the LAMP primer combination in the preparation of a kit.

[0010] The present invention discloses a LAMP kit for detecting citrus brown rot Phytophthora, wherein the kit comprises the above-mentioned LAMP primer composition.

[0011] Furthermore, the kit also contains 10×ThermoPol Buffer, MgSO4, dNTPs, betaine, BstDNA polymerase, and hydroxynaphthol blue.

[0012] An application of the LAMP primer composition and LAMP kit in detecting citrus brown rot Phytophthora, wherein the detection is for non-disease diagnosis purposes.

[0013] The invention discloses a LAMP method for detecting citrus brown rot Phytophthora, which comprises extracting DNA of a microorganism to be detected, taking a DNA solution as a reaction template, performing a LAMP reaction with the primer composition, and then observing the color change of the amplified product. If the amplified product is sky blue, it indicates that the detection result is positive, indicating that citrus brown rot Phytophthora is present in the organism to be detected; if the amplified product is purple, it indicates that the detection result is negative, indicating that citrus brown rot Phytophthora is not present in the organism to be detected.

[0014] Furthermore, the LAMP reaction system is: 2.5 μL 10×ThermoPol Buffer, 8 mmol·L - 1 MgSO4, 1.2mmol·L -1 dNTPs, inner primers FIP and BIP, 1.6 μmol·L each -1 , outer primers F3 and B3, 0.4 μmol·L -1 , 0.8 μmol·L -1 Loop primer LF, 0.8 μmol·L -1 Betaine, 8U·μL -1 Bst DNA polymerase, 180mmol·L -1 Hydroxynaphthol blue, 2 μL template DNA, and sterile water to make up to 26 μL.

[0015] Furthermore, the procedure of the LAMP reaction is: amplification at 63° C. for 60 min.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: the target gene Ypt1 disclosed in the present invention is used to detect citrus brown rot Phytophthora, and the LAMP primer composition designed based on the target gene can be used to quickly and efficiently detect citrus brown rot Phytophthora, and the detection method has the advantages of strong specificity, high accuracy, good sensitivity, simple operation, and the results can be observed with the naked eye. When the DNA reaches 100pg·μL -1 Citrus brown rot fungus can be identified at any time, which is very practical. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The nucleic acid sequence and primer positions of the Ypt1 gene of Phytophthora citri citri;

[0018] Figure 2 The results of LAMP detection of different citrus brown rot Phytophthora strains and negative controls using the LAMP primer combination in Example 1;

[0019] Figure 3The results of LAMP amplification detection of DNA samples of different citrus brown rot Phytophthora strains and other fungal strains by the LAMP primer combination in Example 1, wherein 1-33 are citrus brown rot Phytophthora, cotton Phytophthora, Phytophthora caerulea, pepper Phytophthora, camphor Phytophthora, taro Phytophthora, cryptophytophthora, digging Phytophthora, infestation Phytophthora, major male Phytophthora, alfalfa Phytophthora, cucurbit Phytophthora, tobacco Phytophthora, palm Phytophthora, microspore Phytophthora, costus Phytophthora, soybean Phytophthora, huanghuai Glomerophyta, ultimate Glomerophyta, irregular Glomerophyta, gorgeous Pythium Glomerophyta, litchi downy mildew, rotten handle Phytophthora, bell-shaped Phytophthora, delhi Pytophthora, group-knotting Pytophthora, alternaria, botrytis, Fusarium graminearum, Fusarium oxysporum, soybean Phomopsis, sheath blight and ddH2O (negative control);

[0020] Figure 4 The sensitivity test results of LAMP for detecting citrus brown rot Phytophthora;

[0021] Figure 5 This is the LAMP detection of pathogens in the soil of citrus diseased fields and pathogens in diseased citrus tissues. 1 is the positive control of citrus brown rot Phytophthora, 2 is the amplification result of healthy soil samples, 3-4 are the LAMP colorimetric results of pathogens in diseased soil around susceptible citrus, 5 is the amplification result of DNA extracted from healthy citrus tissues, 6-7 are the LAMP colorimetric results of pathogens in susceptible citrus, and 8 is the ddH2O negative control. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0023] Example 1 Detection of citrus brown rot Phytophthora using LAMP

[0024] 1. LAMP primer composition for detecting citrus brown rot Phytophthora: forward inner primer FIP as shown in SEQ ID NO.1, reverse inner primer BIP as shown in SEQ ID NO.2, forward outer primer F3 as shown in SEQ ID NO.3, reverse outer primer B3 as shown in SEQ ID NO.4, reverse loop primer LF as shown in SEQ ID NO.5 (primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., detailed information is shown in Table 1, Ypt1 nucleic acid sequence and primer position are shown in Figure 1 shown).

[0025] 2. Detection method: Extract the DNA of the microorganism to be tested, take the DNA solution as the reaction template, add the reaction solution including the above primers to carry out LAMP reaction. The LAMP reaction system is: 2.5μL 10×ThermoPol Buffer, 8mmol·L - 1 MgSO4, 1.2mmol·L -1dNTPs, inner primers FIP and BIP, 1.6 μmol·L each -1 , outer primers F3 and B3, 0.4 μmol·L -1 , 0.8 μmol·L -1 Loop primer LF, 0.8 μmol·L -1 Betaine, 8U·μL -1 Bst DNA polymerase, 180mmol·L -1 Hydroxynaphthol blue, 2 μL template DNA, sterile water to 26 μL; the procedure of LAMP reaction is: 63℃ reaction amplification for 60 min, then observe the color change of the amplified product, if it is sky blue, it means the test result is positive and citrus brown rot Phytophthora is present, if it is purple, it means the test result is negative and citrus brown rot Phytophthora is not present.

[0026] 3. In order to verify the specificity of the LAMP method, four different strains of citrus brown rot Phytophthora and other 31 oomycete and fungal strains were used as test materials (Table 2). The LAMP test results showed that only the reaction tube solution with citrus brown rot Phytophthora as the template showed a sky blue positive reaction ( Figure 2 ), other tested microbial strains and negative control (ddH2O) all showed purple negative reaction ( Figure 3 ).

[0027] Table 1

[0028]

[0029] Table 2

[0030]

[0031]

[0032] Note: NJAU is Nanjing Agricultural University

[0033] Example 2 Sensitivity of LAMP detection of citrus brown rot Phytophthora

[0034] In order to determine the sensitivity of the LAMP detection method, the concentration of the extracted DNA of Phytophthora citri citri was measured by spectrophotometer and diluted in a 10-fold gradient to a mass concentration of 10 ng·μL -1 , 1ng·μL -1 , 100pg·μL -1 , 10pg·μL -1 , 1pg·μL -1 、100fg·μL -1 and 10fg·μL -1, 2 μL was taken as template for LAMP reaction. The reaction procedure was 63℃, 60min. The HNB color development results showed that when the DNA concentration of citrus brown rot phytophthora reached 100pg·μL -1 When the solution in the reaction tube turns sky blue ( Figure 4 ).

[0035] Example 3 Validation of primer specificity and sensitivity in LAMP reaction of Phytophthora citri

[0036] A total of 20 sets of primers that meet the conditions were designed for the LAMP primer set for Phytophthora citrifolia, and finally a set of primers with the most specificity and high sensitivity were screened out, namely the primer sequences used in Example 1 (including forward inner primer FIP, reverse inner primer BIP, forward outer primer F3, reverse outer primer B3 and forward loop primer LF). The remaining designed primers (only one set was randomly selected from the remaining 20 pairs of primers for illustration) were used as controls, and the specific primer sequences are as follows:

[0037] FIP1: 5'-CCAGAGTCTCAGGTGGATGACGAGACCATCAAGCTCCAGATTGT-3';

[0038] BIP1: 5'-TAATGGCTGTGCCAACTCCCTTATCGTGCGGAAACGCTC-3';

[0039] F31: 5'-TACGATCGAGTTGGACGGC-3';

[0040] B31: 5'-CCACGGTAGTAGCTGCTAGT-3';

[0041] LB1: 5'-GTGCTCCTGTAGTGGGACACG-3'.

[0042] The strains used in Example 1 were used as test materials (4 different strains of Phytophthora citrifolia and other 31 oomycetes and fungi strains) and LAMP detection was performed according to the methods of Examples 1 and 2. The results showed that the specificity of the selected control primer set was not high and the sensitivity was also poor, indicating that the primer combination used in the present invention has high specificity and sensitivity.

[0043] Example 4 LAMP detection of citrus brown rot Phytophthora in living tissue

[0044] The NaOH alkaline lysis method was used to extract the DNA of the diseased plants inoculated with citrus brown rot Phytophthora (2 diseased citrus were selected) and used as a template for LAMP amplification. Take 2 μL of DNA solution and perform LAMP reaction according to the method of Example 1. In addition, healthy citrus tissues and the surrounding soil, and the soil around the diseased citrus were tested, and a citrus brown rot Phytophthora in Table 2 was selected as a positive control. The results showed that when LAMP was performed on the diseased plants inoculated with citrus brown rot Phytophthora, the color reaction also showed a positive sky blue; while the healthy plants and the negative control showed purple ( Figure 5 ).

Claims

1. A LAMP primer composition for identifying Phytophthora citrifolia, It is characterized in that The primer composition comprises: a forward inner primer FIP as shown in SEQ ID NO.1, a reverse inner primer BIP as shown in SEQ ID NO.2, a forward outer primer F3 as shown in SEQ ID NO.3, a reverse outer primer B3 as shown in SEQ ID NO.4, and a forward loop primer LF as shown in SEQ ID NO.

5.

2. Use of the LAMP primer combination according to claim 1 in preparing a kit for identifying citrus brown rot Phytophthora.

3. A LAMP kit for detecting citrus brown rot Phytophthora, It is characterized in that The kit comprises the LAMP primer composition according to claim 1.

4. The LAMP kit for detecting Phytophthora citrifolia according to claim 3, It is characterized in that The kit also contains 10×ThermoPol Buffer, MgSO 4 , dNTPs, betaine, Bst DNA polymerase, hydroxynaphthol blue.

5. Use of the LAMP primer composition according to claim 1 and the LAMP kit according to any one of claims 3 to 4 in detecting citrus brown rot Phytophthora, wherein the detection is for non-disease diagnosis purposes.

6. A LAMP method for detecting citrus brown rot Phytophthora, It is characterized in that The following steps are involved: Extract the DNA of the microorganism to be tested, take the DNA solution as a reaction template, perform LAMP reaction with the primer combination described in claim 1, and then observe the color change of the amplified product. If the amplified product is sky blue, it indicates that the test result is positive, indicating the presence of citrus brown rot Phytophthora; if the amplified product is purple, it indicates that the test result is negative, indicating the absence of citrus brown rot Phytophthora.

7. The method according to claim 6, It is characterized in that The LAMP reaction system is: 2.5 μL 10×ThermoPolBuffer, 8 mmol·L -1 MgSO 4 , 1.2 mmol·L -1 dNTPs, inner primers FIP and BIP, 1.6 μmol·L each -1 , outer primers F3 and B3, 0.4 μmol·L -1 , 0.8 μmol·L -1 Loop primer LF, 0.8 μmol·L -1 Betaine, 8U·μL- 1 Bst DNApolymerase, 180mmol·L -1 Hydroxynaphthol blue, 2 μL template DNA, and sterile water to make up to 26 μL.

8. The method according to claim 6, It is characterized in that The procedure of LAMP reaction was as follows: amplification at 63°C for 60 min.

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