Application of Ypt1 gene as a specific target in the detection of Pythium variegata

Abstract

The present invention discloses an application of Ypt1 gene as a specific target in detecting Phytophthora variabilis, and the nucleotide sequence of the specific detection target Ypt1 is shown in SEQ ID NO.1. The new target gene Ypt1 disclosed in the present invention is used to detect Phytophthora variabilis, and the LAMP primer composition designed based on the target gene can be used to quickly and efficiently detect Phytophthora variabilis. 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 The multivariate blight fungus can be identified at any time, which is very practical.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an application of a Ypt1 gene as a specific target in detecting Phytophthora variabilis. Background Art

[0002] Phytopythium mercuriale belongs to the Straminipila kingdom, Oomycota, Oomycetes, Peronosporales, Pythiaceae, Phytopythium. It is an important plant pathogen that can infect a variety of economic crops and fruit trees, such as soybeans, strawberries, tomatoes, beets, sunflowers, citrus, apple trees and peach trees. It is widely distributed in the world and has a wide host range. It causes plant root rot and causes heavy economic losses in the production of infected plants. Phytopythium mercuriale grows faster at room temperature, with the lowest growth temperature of 4°C, the most suitable growth temperature of 25–30°C, and the highest growth temperature of 38°C. The morphological characteristics are that the hyphae are non-septate, up to 6.0μm wide, and the sporangium is nearly spherical with papillae. The oospores are smooth, spherical, thin-walled, and without ootheca. Phytophthora variabilis is easily confused with Phytophthora and Pythium in terms of morphology, which requires extensive and professional experience in pathogen identification. Moreover, it is difficult to distinguish Phytophthora variabilis from similar species based on morphological characteristics.

[0003] At present, there are few detection technologies for this type of oomycetes, and traditional morphological characteristics are usually used for identification, mainly based on morphological characteristics, pathogenicity, physiological and biochemical characteristics, etc. Traditional methods have played an important role in the detection of oomycetes, but they are time-consuming and labor-intensive, and require professional knowledge of oomycete isolation technology and classification and identification. With the development of nucleic acid-related identification methods, PCR technology provides the advantages of rapid, sensitive and accurate diagnosis of plant pathogens, but the detection time is still long and relies on precise temperature cycling devices, which cannot meet the needs of rapid detection and is not suitable for use and promotion in grassroots environments.

[0004] Loop-mediated isothermal amplification (LAMP) is a molecular detection technology based on PCR reaction. This technology uses Bst large fragment polymerase, which has strong chain displacement ability and does not require the template denaturation step usually required by other polymerases, so that the reaction can be carried out under isothermal conditions. The entire LAMP reaction process is carried out at 60-65°C, and the reaction time is within 80 minutes. A simple heating device can meet the reaction, which greatly reduces the complexity and cost of the reaction equipment. This is a prominent advantage of LAMP detection technology. Unlike traditional PCR, LAMP requires at least four pairs of primers that are complementary to six different regions of the target DNA sequence. The final product of the LAMP reaction is a mixture of a series of stem-loop DNA and cauliflower structure DNA containing multiple inverted repeat sequences of the target sequence. The LAMP amplification product can use the color change of hydroxyphenol blue (HNB) to indicate the positive or negative reaction result, which can be determined by observing the color change with the naked eye. At present, LAMP technology can achieve specific, efficient and rapid amplification of target DNA under isothermal conditions. In recent years, it has been widely used in the fields of medical care, food safety, customs quarantine and agricultural production. At present, this technology has been widely used in the rapid detection of fungi, bacteria, viruses and oomycetes, but there are no reports on the detection of Phytopythium mercuriale at home and abroad.

[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] Purpose of the invention: The first purpose of the present invention is to provide an application of a guanosine triphosphate binding protein (Ypt1) gene as a specific target in detecting Pythium variegata. The second purpose of the present invention is to provide a LAMP primer composition and its application for detecting Pythium variegata using the Ypt1 gene as a specific target. The third purpose of the present invention is to provide a LAMP method for detecting Pythium variegata.

[0007] Technical solution: The present invention uses the Ypt1 gene as a specific target in the detection of Pythium solani. The nucleotide sequence of the specific detection target Ypt1 gene is shown in SEQ ID NO.1.

[0008] The present invention discloses a LAMP primer composition for detecting Phytophthora variabilis using the Ypt1 gene as a specific target. The primer composition comprises: a forward inner primer FIP as shown in SEQ ID NO.2, a reverse inner primer BIP as shown in SEQ ID NO.3, a forward outer primer F3 as shown in SEQ ID NO.4, a reverse outer primer B3 as shown in SEQ ID NO.5, and a reverse loop primer LB as shown in SEQ ID NO.6.

[0009] Because the traditional detection method of Phytophthora variabilis is only identified based on morphological characteristics, and the growth of Phytophthora variabilis is unstable due to the influence of temperature, and is affected by similar species at the same time, it is difficult to accurately identify. The present invention is based on the Ypt1 gene sequence of Phytophthora variabilis, and the genome sequence of Phytophthora variabilis is compared with the genome sequence of other Phytophthora variabilis by using Blast software, and a section of Ypt1 gene sequence unique to Phytophthora variabilis is selected and specific LAMP primers are designed. In the LAMP design primer, the target region length of the primer is usually about 200-400bp. The target region is particularly important during the design of LAMP primers, which directly affects the specificity and sensitivity of the primer. The LAMP reaction is through 4 primers (FIP, BIP, F3, B3) to specifically identify 6 independent regions (Table 1) on the target sequence, and its specificity is relatively high. In addition, the reverse loop primer LB can improve the reaction rate, and together with other four primers, when ensuring the accuracy of the reaction, the present invention can quickly detect Phytophthora variabilis. The LAMP reaction uses four primers to specifically recognize six independent regions on the target sequence. Compared with the PCR primers that recognize two independent regions of the target sequence, it has higher specificity and sensitivity.

[0010] The invention relates to the use of the LAMP primer combination in the preparation of a kit.

[0011] The invention discloses a LAMP kit for detecting Pythium multivolume, which comprises the above-mentioned LAMP primer composition.

[0012] Furthermore, the kit also contains 10×ThermoPol Buffer, MgSO4, dNTPs, betaine, BstDNA polymerase, and hydroxynaphthol blue.

[0013] An application of the LAMP primer combination and LAMP kit in detecting Pythium variabilis, wherein the detection is for non-disease diagnosis purposes.

[0014] The invention discloses a LAMP method for detecting Phytophthora variabilis, comprising 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 Phytophthora variabilis exists in the organism to be detected; if the amplified product is purple, it indicates that the detection result is negative, indicating that Phytophthora variabilis does not exist in the organism to be detected.

[0015] 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 LB, 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.

[0016] Furthermore, the procedure of the LAMP reaction is: amplification at 63° C. for 60 min.

[0017] Beneficial effects: The new target gene Ypt1 disclosed in the present invention is used to detect Pythium variegata. The LAMP primer composition designed based on the target gene can be used to quickly and efficiently detect Pythium variegata. 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 100 pg·μL -1 The multivariate blight fungus can be identified at any time, which is very practical.

[0018] Compared with the prior art, the present invention has the following outstanding 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

[0019] Figure 1 is the nucleic acid sequence and primer positions of the Pythium variabilis Ypt1 gene;

[0020] Figure 2 The results of LAMP amplification of DNA samples of Pythium variegata strains and other fungal strains by the LAMP primer combination in Example 1, wherein 1-20 are Phytopythium mercuriale, P. helicoides, P. litorale, P. nanjingense, P. sindhum, P. vexans, Globisporangium irregulare, G. huanghuaiense, G. pengfuense, G. recalcitrans, G. spinosum, G. splendens, G. ultimum var. ultimum, Pythium acanthicum, Py. aphanidermatum, Py. deliense, Phytophthora cactorum, Ph. sojae, Fusarium graminearum and ddH2O (negative control), respectively;

[0021] Figure 3 This is the sensitivity of LAMP in detecting Pythium multiflorum in Example 2. 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 Pythium multiflorum using LAMP

[0024] 1. LAMP primers and primer combinations for detecting Phytopythium mercuriale: Ypt1 nucleic acid sequence as shown in SEQ ID NO.1, forward inner primer FIP as shown in SEQ ID NO.2, reverse inner primer BIP as shown in SEQ ID NO.3, forward outer primer F3 as shown in SEQ ID NO.4, reverse outer primer B3 as shown in SEQ ID NO.5, reverse loop primer LB as shown in SEQ ID NO.6 (primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., detailed information is shown in Table 1, Ypt1 nucleic acid sequence and primer positions are shown in Table 1. 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-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 LB, 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 26 μL; the procedure for the LAMP reaction is: amplify at 63°C for 60 min, and then observe the color change of the amplified product. If it is sky blue, it means the test result is positive and Pythium multivariate is present; if it is purple, it means the test result is negative and Pythium multivariate is not present.

[0026] 3. In order to verify the specificity of the LAMP method, a strain of Pythium multivariate and other 18 oomycetes and fungal strains were used as test materials (Table 2). The LAMP test results showed that only the reaction tube solution with Pythium multivariate as the template showed a sky blue positive reaction, while the other test fungal strains and negative controls showed purple negative reactions ( Figure 2 ).

[0027] Table 1

[0028]

[0029] Table 2

[0030]

[0031] Example 2 Sensitivity of LAMP detection of Pythium variegata

[0032] In order to determine the sensitivity of the LAMP detection method, the concentration of the extracted DNA of Pythium multivariate 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 Pythium multivariate reached 100pg·μL -1 When the solution in the reaction tube turns sky blue ( Figure 3 ).

[0033] Example 3 Validation of primer specificity and sensitivity of LAMP reaction of Pythium variegata

[0034] A total of 20 sets of primers that meet the conditions were designed for the LAMP primer set for Pythium multivariate, 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 reverse loop primer LB). 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:

[0035] FIP1: 5'-ACGTCGTACACGACGATGATGCCTTCCGCACCATCACTAGC-3';

[0036] BIP1: 5'-GACGGACCAGGAGTCGTTCAACCCGAATGCAACGCGAGG-3';

[0037] F31: 5'-CTTTTAGTGGGACACGGCC-3';

[0038] B31: 5'-TGATGACCCGGTACTGAGAT-3';

[0039] LB1: 5'-CGTCAAGCAGTGGCTGCAC-3'.

[0040] The strains used in Example 1 were used as test materials (1 strain of different Pythium polymorphum strains and other 18 oomycete and fungal 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. This shows that the primer combination used in the present invention has high specificity and sensitivity.

Claims

1. A Ypt1 The LAMP primer composition for detecting Pythium variabilis as a specific target gene is characterized in that: The specific detection target Ypt1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the primer composition includes: a forward inner primer FIP as shown in SEQ ID NO.2, a reverse inner primer BIP as shown in SEQ ID NO.3, a forward outer primer F3 as shown in SEQ ID NO.4, a reverse outer primer B3 as shown in SEQ ID NO.5, and a reverse loop primer LB as shown in SEQ ID NO.

6.

2. A method according to claim 1 Ypt1 The invention discloses an application of a LAMP primer composition for detecting Pythium variabilis using a gene as a specific target in the preparation of a kit.

3. A LAMP kit for detecting Pythium variabilis, characterized in that: The kit comprises the LAMP primer composition according to claim 1.

4. The LAMP kit for detecting Pythium variabilis according to claim 3, characterized in that: The kit also contains 10×ThermoPol Buffer, MgSO4, 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 Pythium variabilis, wherein the detection is for non-disease diagnosis purposes.

6. A LAMP method for detecting Pythium variabilis, 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, which means that Phytophthora multiflora is present in the organism to be tested; if the amplified product is purple, it indicates that the test result is negative, which means that Phytophthora multiflora is not present in the organism to be tested.

7. The LAMP method for Phytophthora variabilis according to claim 6, characterized in that: The LAMP reaction system is: 2.5 μL 10×ThermoPol Buffer, 8 mmol·L -1 MgSO4, 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 LB, 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.

8. The LAMP method for Pythium variabilis according to claim 6, characterized in that: The procedure of the LAMP reaction is: amplification at 63° C. for 60 min.

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