A LAMP primer composition for detecting Pythium sindei and its application

Abstract

The present invention discloses a LAMP primer composition for detecting Phytopythium sindhum and its application. The LAMP 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, a forward loop primer LF as shown in SEQ ID No.5, and a reverse loop primer LB as shown in SEQ ID No.6. The LAMP primer composition disclosed in the present invention can be used for rapid and efficient detection of Phytopythium sindhum. 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 virus can detect Pythium spp. at any time, which is very practical.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology detection and relates to a LAMP primer composition for detecting Pythium sindei and an application thereof. Background Art

[0002] Phytopythium is a new genus separated from the genus Pythium in the broad sense. It belongs to the kingdom Straminipila, phylum Oomycota, class Oomycetes, order Peronosporales, family Pythiaceae. Phytopythium is an important plant pathogen that can infect a variety of plants. It is widely distributed in the world and has a wide host range. It causes plant root rot, damping-off and soft rot, causing heavy economic losses in the production of infected plants. Phytopythium sindhum is the model species of the genus Phytopythium. It has been reported in China and Pakistan and can infect soybean, banana, guava, wheat and rice. It is a potential important plant pathogen. Pythium sindei is rose-shaped on CMA medium and can grow 14 mm per day at 25°C. The minimum growth temperature is 5°C, the most suitable growth temperature is 30°C, and the maximum growth temperature is 38°C. The sporangium is spherical to nearly spherical with papillae. Pythium sindei is easily confused with Phytophthora and Pythium in morphology, requiring rich and professional experience in pathogen identification, and it is difficult to distinguish Pythium sindei from similar species based on morphological characteristics.

[0003] At present, there are few detection technologies for Pythium sindei. The traditional detection method mainly uses the plate separation method to identify Pythium sindei 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 Pythium sindei from similar species based on morphological characteristics, which cannot meet the needs of field production. Ordinary PCR technology requires precision variable temperature equipment and advanced and complex analytical instruments, or requires high proficiency and professional level of operators, and has a long reaction time, which is not conducive to grassroots promotion. With the development of molecular biology, loop-mediated isothermal amplification (LAMP) technology provides rapid, sensitive and accurate advantages for plant pathogen diagnosis, and its position in the identification and detection of pathogens is becoming increasingly important. Since the establishment of LAMP detection technology 23 years ago, this technology has been widely used in the detection and research of pathogens such as viruses, bacteria, parasites, and fungi, but there are few reports on the detection of plant pathogenic oomycetes, and no reports on the detection of Pythium sindei have been seen at home and abroad.

[0004] In recent years, the source of primers designed in the oomycete LAMP detection method is mainly the intertranscribed spacer (ITS). However, more and more studies have shown that the ITS sequences of some oomycetes are very different, and primers designed with this as the target are difficult to distinguish different species. There is a certain gap in the specificity and sensitivity of different target sequence detections. Due to the different target sequences and fragment sizes selected, the results will be very different. Therefore, the discovery of target genes with good specificity is the core of the current detection technology of Pythium sindei. In summary, discovering high-reliability specific molecular detection targets and establishing a sensitive and accurate detection technology system based on new targets is important for improving the rapid molecular detection research of Pythium sindei and the early diagnosis of diseases caused by its detection. Summary of the invention

[0005] Objectives of the invention: The first objective of the present invention is to provide a set of LAMP primer compositions and applications thereof that can be used to quickly identify Pythium sindeyi; the second objective of the present invention is to provide a LAMP method for identifying Pythium sindeyi.

[0006] Technical solution: The LAMP primer composition of the present invention for detecting Pythium sindeyi with RPL10 gene as a specific target 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, a forward loop primer LF as shown in SEQ ID NO.5, and a reverse loop primer LB as shown in SEQ ID NO.6.

[0007] The present invention is based on the 60S ribosomal protein L10 (RPL10) gene of Pythium sindei (Genbank accession number: OK533438), and the sequence is alternately spaced in the genome evolution region and the conservative region in Pythium sindei. The genome sequence of Pythium sindei is compared with the genome sequences of other fungi using Blast software, a sequence unique to Pythium sindei 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 RPL10 gene length of the Pythium sindei strain is about 460bp. Due to the excessive length, the full-length sequence of RPL10 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 tested different regions, designed 20 sets of primer systems, and selected a unique successful primer combination (Table 1) and reaction system through the test of these primer systems. This primer combination is all unresponsive to bacterial strains of similar species and similar genera, has strong specificity, and proves this result through repeated tests for many times. And 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, forward loop primer LF and reverse loop primer LB can improve the reaction rate, and together with other four primers, enable the present invention to quickly carry out the detection of Pythium sindei under the situation of ensuring the accuracy of 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.

[0008] The invention discloses an application of the LAMP primer composition for detecting Pythium sindei using the RPL10 gene as a specific target in the preparation of a kit.

[0009] The LAMP kit for detecting Pythium sindei of the present invention comprises the above-mentioned LAMP primer composition.

[0010] Furthermore, the kit also contains 10×ThermoPol Buffer, MgSO4, dNTPs, betaine, BstDNA polymerase, and hydroxynaphthol blue.

[0011] The use of the LAMP primer composition and LAMP kit of the present invention in detecting Pythium sindei, wherein the detection is for non-disease diagnosis purposes.

[0012] The LAMP method for detecting Pythium sindeyi of the present invention extracts DNA of the microorganism to be detected, takes the DNA solution as a reaction template, performs LAMP reaction with the above-mentioned primer combination, and then observes the color change of the amplified product. If the amplified product is sky blue, it indicates that the test result is positive, indicating that Pythium sindeyi is present in the organism to be detected; if the amplified product is purple, it indicates that the test result is negative, indicating that Pythium sindeyi is not present in the organism to be detected.

[0013] 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 , loop primers LF and LB, 0.8 μmol·L -1 , 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.

[0014] Furthermore, the procedure of the LAMP reaction is: amplification at 63° C. for 60 min.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: the target RPL10 gene for specific detection of Pythium sindei disclosed in the present invention, the LAMP primer composition designed based on the target gene can be used for rapid and efficient detection of Pythium sindei, 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 virus can be used to identify Pythium spp. in China, which is very practical. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The nucleic acid sequence and primer positions of the RPL10 gene of Pythium sindei;

[0017] Figure 2 The specificity validation results of the LAMP primer combination for detecting Pythium sindei;

[0018] Figure 3 The sensitivity validation results of the LAMP primer combination for detecting Pythium sindei.

[0019] Figure 4The specificity verification result of the LAMP primer combination for detecting Pythium sindei in Example 3 is shown;

[0020] Figure 5 This is the sensitivity verification result of the LAMP primer combination for detecting Pythium sinensis in Example 3. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0022] Example 1 Detection of Pythium sindei using LAMP

[0023] 1. A LAMP primer composition for detecting Pythium sindei: 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, a forward loop primer LF as shown in SEQ ID NO.5, and a reverse loop primer LB as shown in SEQ ID NO.6 (the primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the detailed information is shown in Table 1. The RPL10 gene sequence and primer positions are shown in Table 1. Figure 1 shown).

[0024] 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 MgS04, 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 , loop primers LF and LB, 0.8 μmol·L -1 , 0.8 μmol·L -1 Betaine, 8U·μL -1 Bst DNA polymerase, 180mmol·L -1 Hydroxynaphthol blue, 2 μL template DNA, the total volume of the system is 26 μL; the procedure of the LAMP reaction is: 63°C reaction amplification for 60 minutes, and then observe the color change of the amplified product. If it is sky blue, it means the test result is positive and Pythium sindei is present. If it is purple, it means the test result is negative and Pythium sindei is not present.

[0025] 3. In order to verify the specificity of the LAMP method, a strain of Pythium sindei and 20 other oomycetes and fungal strains were used as test materials (Table 2). The LAMP test results showed that only the reaction tube solution with Pythium sindei as the template showed a sky blue positive reaction, while the other test fungal strains and negative controls showed purple negative reactions ( Figure 2 ).

[0026] Table 1

[0027]

[0028] Table 2

[0029]

[0030]

[0031] Example 2 Sensitivity of LAMP Detection

[0032] In order to determine the sensitivity of the LAMP detection method, the concentration of the extracted DNA of Pythium sindei 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 sindei 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 sinensis

[0034] A total of 20 sets of primers that meet the conditions were designed for the LAMP primer set of Pythium sindei, 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:

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

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

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

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

[0039] LF1: 5'-AGCGGATGCGCATGTGG-3'.

[0040] The strains used in Example 1 were used as test materials (one strain of Pythium sindei and other 20 oomycete and fungal strains were used as test materials) 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 ( Figure 4 ), and the sensitivity is also poor ( Figure 5 ). This indicates that the primer combination used in the present invention has high specificity and sensitivity.

Claims

1. A method for identifying Pythium sindei Phytopythium sindhum ) is a LAMP primer composition, characterized in that The primer composition includes: 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, a forward loop primer LF as shown in SEQ ID NO.5, and a reverse loop primer LB as shown in SEQ ID NO.

6.

2. Use of the LAMP primer combination according to claim 1 in preparing a kit for identifying Pythium sindei.

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

4. The LAMP kit for detecting Pythium sindei 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 sindei, wherein the detection is for non-disease diagnosis purposes.

6. A LAMP method for detecting Pythium sindei, 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 composition 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 Pythium sindei; if the amplified product is purple, it indicates that the test result is negative, indicating the absence of Pythium sindei.

7. The LAMP method for detecting Pythium sindei 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 , loop primers LF and LB, 0.8 μmol·L -1 , 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 detecting Pythium sindei 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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