A target Rpb1 gene for specific detection of Glomerella intermedius and its application

Abstract

The invention discloses a target Rpb1 gene for specifically detecting intermedium Globisporangium and an application thereof, wherein the nucleotide sequence of the target Rpb1 gene is shown in SEQ ID NO. 1. The target Rpb1 gene for specifically detecting intermedium Globisporangium disclosed by the invention, and a LAMP primer composition designed based on the target gene can be used for rapid and efficient detection of intermedium Globisporangium, 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, and intermedium Globisporangium can be identified when the DNA reaches 10pg·μL‑1, and the practicability is strong.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology detection and relates to a target Rpb1 gene for specifically detecting Intersphaerocephala and an application thereof. Background Art

[0002] Globisporangium intermedium is an important plant pathogen belonging to the phylum Oomycota. It is a broad group of pythium species that can infect many conifers, flowers and crops, such as hydrangea, rose, parsley, parsnip and soybean. It is widely distributed in the world and causes a variety of symptoms such as root rot and damping-off of plants, which cause heavy economic losses in the production of infected plants. Globisporangium intermedium hyphae are septate, septate after old age, irregularly branched, spore sacs are spherical or nearly spherical, and chain-grown. The minimum, optimum and maximum temperatures for mycelial growth are 0℃, 25-28℃ and 35℃, respectively. Globisporangium intermedium is easily confused with species of long spore cysts, narrow spore cysts, phytophthora and dense spore cysts in terms of morphology, and requires rich and professional experience in pathogen identification to distinguish them. In addition, Coccidioides has a high morphological diversity. The morphological characteristics of some species are overlapping, and some species have high intraspecific morphological variation. Some phenotypes are easily affected by environmental conditions and nutrient matrix and undergo significant changes. It is often difficult to determine the interrelationships between these complex and changeable morphological characteristics through naked eye observation.

[0003] At present, there are few detection technologies for Globisporangium intermedium. The traditional detection method mainly uses the plate separation method to identify Globisporangium intermedium based on its morphology. This method is time-consuming and labor-intensive, and requires rich and professional experience in pathogen identification. Moreover, since Globisporangium intermedium is a heterothallic species and does not produce oospores under natural conditions, it is difficult to distinguish Globisporangium intermedium from similar species based on morphological characteristics, and it 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.

[0004] Loop-mediated isothermal amplification (LAMP) is a new nucleic acid amplification technology developed in recent years. This technology has been widely used in the detection of pathogens such as viruses, bacteria, parasites, and fungi. As a constant temperature nucleic acid amplification technology, LAMP technology has the biggest advantages of fast reaction speed, simple equipment, and easy identification of results. It is especially suitable for grassroots inspection and quarantine agencies and medical institutions. At present, there are no reports on the detection of Globisporangium intermedium using LAMP technology at home and abroad.

[0005] In order to prevent and control the spread of Globisporangium intermedium, it is necessary to detect it quickly and accurately. Discovering target genes with good specificity is the core of all current detection technologies. There is a certain gap in the specificity and sensitivity of different target sequence detections. The results will be very different due to different target sequences and fragment sizes. The selection of target genes must ensure that they are highly conserved between different strains within the species, but have high variability between species.

[0006] In summary, discovering high-reliability specific molecular detection targets and establishing a sensitive and accurate detection technology system based on new targets plays an important role in improving the rapid molecular detection research of Globisporangiumintermedium and the early diagnosis of diseases caused by its detection. Summary of the invention

[0007] Purpose of the invention: The first purpose of the present invention is to provide a target RNA polymerase II large subunit sequence (Rpb1) gene for specific detection of Globisporangium intermedium. The second purpose of the present invention is to provide a LAMP primer composition and its application for detecting Globisporangium intermedium with Rpb1 gene as a specific target. The third purpose of the present invention is to provide a LAMP method for detecting Globisporangium intermedium.

[0008] Technical solution: The target Rpb1 gene for specifically detecting Globisporangium intermedium of the present invention, the nucleotide sequence of the target Rpb1 gene is shown in SEQ ID NO.1.

[0009] The invention discloses a LAMP primer composition for detecting Globisporangium intermedium using the Rpb1 gene as a specific target, wherein 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.

[0010] The present invention is based on the Rpb1 gene sequence of Globisporangium intermedium, which is alternately spaced in the genome evolution zone and the conservative zone in Globisporangium intermedium. The genome sequence of Globisporangium intermedium is compared with the genome sequence of other fungi by Blast software, and the unique end sequence of Globisporangium intermedium is selected and specific LAMP primers are designed. The LAMP reaction specifically recognizes 6 independent regions (Table 1) on the target sequence by 4 primers (FIP, BIP, F3, B3), and its specificity is relatively high. In addition, the reverse loop primer LB can improve the reaction rate, and together with the other four primers, the present invention can quickly detect Globisporangium intermedium while ensuring the accuracy of the reaction. The LAMP reaction specifically recognizes 6 independent regions on the target sequence by 4 primers, and the specificity and sensitivity are relatively high relative to the 2 independent regions of the PCR primer recognition target sequence.

[0011] The invention discloses an application of a LAMP primer composition for detecting Globisporangium intermedium by taking the Rpb1 gene as a specific target in preparing a kit.

[0012] The LAMP kit for detecting Globisporangium intermedium of the present invention comprises the above-mentioned LAMP primer combination.

[0013] Furthermore, the kit also contains 10×ThermoPol Buffer, MgSO4, dNTPs, betaine, BstDNA polymerase, and hydroxynaphthol blue.

[0014] An application of the LAMP primer combination and LAMP kit in detecting Globisporangium intermedium, wherein the detection is for non-disease diagnosis purposes.

[0015] The LAMP method for detecting Globisporangium intermedium of the present invention comprises the following steps: extracting DNA of a microorganism to be detected, taking a DNA solution as a reaction template, performing a LAMP reaction with the primer combination, and then observing the color change of an amplified product. If the amplified product is sky blue, it indicates that the detection result is positive, indicating that Globisporangium intermedium exists in the organism to be detected; if the amplified product is purple, it indicates that the detection result is negative, indicating that Globisporangium intermedium does not exist in the organism to be detected.

[0016] 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.

[0017] Furthermore, the procedure of the LAMP reaction is: amplification at 63° C. for 63 min.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: the target Rpb1 gene for specific detection of Globisporangium intermedium disclosed in the present invention, the LAMP primer composition designed based on the target gene can be used for rapid and efficient detection of Globisporangium intermedium, 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 10 pg·μL -1 The inter-type sphaerocystis can be identified immediately, which is very practical. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The nucleic acid sequence and primer positions of Globisporangiumintermedium Rpb1 gene.

[0020] Figure 2 To detect the specificity verification results of Globisporangiumintermedium LAMP primers, 1-9 are different Globisporangiumintermedium strains, 10-31 are G.acanthophoron, G.glomeratum, G.heterothallicum, G.huanghuaiense, G.irregulare, G.middletonii, G.nodosum, G.orthogonon, G.paroecandrum, G.pengfuense, G.recalcitrans, G.spinosum, G.splendens, G.tenuihyphum, G.ultimum var.ultimum, Pythium acanthicum, P.aphanidermatum, P.deliense, P.myriotylum, Phytopythium helicoides, Ph.vexans, Phytophthora cactorum, Ph.sojae, Fusarium graminearum and ddH2O (negative control).

[0021] Figure 3 The results are for sensitivity validation of the LAMP primer combination for detecting Globisporangium intermedium. 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 Globisporangium intermedium using LAMP

[0024] 1. LAMP primer composition for detecting Globisporangium intermedium: 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, Rpb1 gene sequence and primer positions 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 -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, sterile water to 26 μL; the procedure of LAMP reaction is: 63℃ reaction amplification for 63 min, then observe the color change of the amplified product, if it is sky blue, it means the test result is positive and Globisporangium intermedium is present, if it is purple, it means the test result is negative and Globisporangium intermedium is not present.

[0026] 3. In order to verify the specificity of the LAMP method, 9 different strains of Globisporangium intermedium and other 22 oomycete and fungal strains were used as test materials (Table 2). The LAMP test results showed that only the reaction tube solution with Globisporangium intermedium as the template showed a sky blue positive reaction, while the other test microbial strains and negative controls showed purple negative reactions ( Figure 2 ).

[0027] Table 1

[0028]

[0029]

[0030] Table 2

[0031]

[0032]

[0033] Example 2 Sensitivity of Globisporangium intermedium LAMP Detection

[0034] In order to determine the sensitivity of the LAMP detection method, the concentration of the extracted Globisporangium intermedium DNA 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℃, 63min. The HNB color development results showed that when the DNA concentration of Globisporangium intermedium reached 10pg·μL -1 When the solution in the reaction tube turns sky blue ( Figure 3 ).

Claims

1. A method for the specific detection of Glomerella intermedius (Globisporangium intermedium) Target Rpb1 A nucleotide fragment of a gene, characterized in that The target Rpb1 The nucleotide sequence of the nucleotide fragment of the gene is shown in SEQ ID NO.

1.

2. A Rpb1 Gene as a specific target for the detection of Glomerella intermedius (Globisporangium intermedium) The LAMP primer composition is characterized in that 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.

3. A method according to claim 2 Rpb1 Gene as a specific target for the detection of Glomerella intermedius (Globisporangium intermedium) Use of the LAMP primer combination in preparing a kit.

4. A method for detecting intersporicidal cysts (Globisporangium intermedium) The LAMP kit is characterized in that The kit comprises the LAMP primer composition according to claim 2.

5. The method for detecting Metasporum according to claim 4. (Globisporangium intermedium) The LAMP kit is characterized in that The kit further comprises 10×ThermoPol Buffer, MgSO4, dNTPs, betaine, Bst DNA polymerase, and hydroxynaphthol blue.

6. A LAMP primer combination according to claim 2, and a LAMP kit according to any one of claims 4 to 5 for detecting Metasporum (Globisporangium intermedium) The application in the present invention is that the detection is for non-disease diagnosis purposes.

7. A method for detecting the presence of sphaerocystis (Globisporangium intermedium) The LAMP method 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 2, and then observe the color change of the amplified product. If the amplified product is sky blue, it means that the test result is positive, which means that the microorganism to be tested has Intersporangiophora. (Globisporangium intermedium) If the amplification product is purple, it means the test result is negative, which means that there is no interspermocystis in the organism to be tested. (Globisporangium intermedium) .

8. The method for detecting Metasporangium according to claim 7. (Globisporangium intermedium) The LAMP method is 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.

9. The method for detecting Metasporum according to claim 7 (Globisporangium intermedium) The LAMP method is characterized in that The procedure of the LAMP reaction is: amplification at 63° C. for 63 min.

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