Primer set and kit for detecting rice bacterial leaf blight pathogen based on lamp

Abstract

The application discloses a primer group for detecting a rice bacterial blight pathogen based on LAMP, comprising an Xo2967 LAMP primer group, wherein the Xo2967 LAMP primer group is composed of a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3, a forward loop primer LF and a reverse loop primer LB. The application further discloses a kit containing any one of the Xo2967 LAMP primer groups. The method of the application takes the XOO gene of the rice bacterial blight pathogen as a target gene, designs and screens a LAMP primer group capable of specifically detecting the bacterial blight, and has the advantages of high specificity, high sensitivity, low equipment requirement, simple operation, short time consumption, low cost, visual result and the like, and can realize rapid diagnosis of the rice disease in the field.

Description

Technical Field

[0001] This invention belongs to the field of plant pathogenic microorganism detection technology, specifically relating to primer sets and kits for detecting rice bacterial blight pathogens based on LAMP. Background Technology

[0002] Rice (Oryza sativa L.) is one of the world's most important crops and a staple food for more than half the population (Fahad et al., 2014). According to data from the Food and Agriculture Organization of the United Nations (FAO) in 2019, the global annual rice planting area is 160 million hectares, with an annual yield of 750 million tons, of which more than 90% comes from Asian countries (FAOSTAT). In China, rice has a long history of cultivation. As of 2020, rice's planting area and yield were second only to maize, making it China's second largest food crop (China Statistical Yearbook 2020). However, rice diseases (including bacterial, fungal, and viral diseases) are one of the important factors limiting rice yield. Reports of rice disease stress have been made all over the world, especially in Asia, such as China, South Korea, and Japan (SY Cho et al., 2013; Kim et al., 2011; Le et al., 2010; Yang et al., 2013), and these diseases can cause yield losses of 20-70% (Fahad et al., 2014).

[0003] Bacterial leaf blight (BLB) in rice is caused by a Gram-negative bacterium, *Xanthomonas oryzae* pv. oryzae (XOO) (Lee, Chung, Kang, Chung, & Lee, 2016). The pathogen initially enters the plant through hydathodes and wounds. Once inside the vascular system, the bacterium produces irregular, yellowish, wavy-edged lesions that soak and eventually clog the xylem sieve tubes, causing wilting in rice seedlings and later leading to leaf and stem dieback, ultimately resulting in reduced yield (Shen & Ronald, 2002; Zhang et al., 2020). Rapid and effective detection and control of rice diseases are crucial for ensuring rice yield. Currently, methods for detecting rice diseases include conventional PCR diagnostics (Song et al., 2014), fluorescence immunoassay (Awaludin et al., 2020), antigen-antibody assays (Ding, Liu, Wu, Zhou, & Ni, 2014; Wang et al., 2006), microscopic observation (Deng, Li, Jian, Ji, & Zhou, 2013), biological inoculation (Li, Li, Dong, Wang, & Zhou, 2011), and qRT-PCR (Cho et al., 2013; Xu et al., 2017). While these methods can detect rice diseases, they also have limitations. For example, biological inoculation is time-consuming and labor-intensive, microscopic observation is highly dependent on equipment and requires specially trained personnel, and immunological methods typically require the preparation of specific antibodies, which often has a long cycle. Although qRT-PCR has high sensitivity and specificity, the detection equipment is expensive and requires professional operation.

[0004] In recent years, with the development of isothermal amplification technology, loop-mediated isothermal amplification (LAMP) has been widely used in nucleic acid diagnosis due to its advantages such as high sensitivity, high specificity, fast speed, and low equipment requirements (Becherer, Borst, Bakheit, Frischmann, & methods, 2020). LAMP has also been reported in the detection of rice diseases (Le et al., 2010; Zhou, Du, Fan, & Zhou, 2012). Using LAMP to detect rice bacterial blight pathogens can be completed within 1 hour with a sensitivity of 10 aM, but highly specific LAMP detection primers still need to be designed to achieve rapid and accurate diagnosis of rice diseases in the field. Summary of the Invention

[0005] In view of this, the present invention aims to provide a primer set and kit for detecting rice bacterial blight pathogens based on LAMP. It can design and screen LAMP primer sets and detection kits that can specifically detect bacterial blight by using the XOO gene of the rice bacterial blight pathogen as the target gene. The kits are highly sensitive, specific, easy to operate, time-saving, and low-cost, and can realize rapid diagnosis of rice diseases in the field.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows: Primer sets for detecting rice bacterial blight pathogens based on LAMP include the Xo1321 LAMP primer set or the Xo2967 LAMP primer set.

[0007] Furthermore, the Xo1321 LAMP primer set includes: forward inner primer FIP, reverse inner primer BIP, forward outer primer F3, reverse outer primer B3, forward loop primer LF, and reverse loop primer LB, with the following base sequences: FIP:5′-TGATGGACCAGATGCGAAGCAACGGAAAAGGTGGCATCGG-3′; BIP:5′-AGTCATACGCCAGGTTGGTCGTTGCAGACGGCGAATGTG-3′; F3:5′-GGCTGCAGTCACAGTGTT-3′; B3:5′-TCGTCGTTCTCGCATTTGG-3′; LF:5′-GCTTCCAGTCTGCCTGGTATA-3′; LB: 5′-CCTGGGTGCGGTAGTTGGA-3′.

[0008] Furthermore, the Xo2967 LAMP primer set includes: forward inner primer FIP, reverse inner primer BIP, forward outer primer F3, reverse outer primer B3, forward loop primer LF, and reverse loop primer LB, with the following base sequences: FIP:5′-AGCGCGCAGCTGATCTGCCACGAAGCTGTAGCGCAAG-3′; BIP:5′-ATGCGTTCCAGCTCGCGGCGTGCCGTACTGGTCGA-3′; F3:5′-CACGCCATCGGTCTGTG-3′; B3:5′-CACGGCATGCCGTTCTG-3′; LF:5′-TGGGGTGTTTTGTGGTATTGC-3′; LB: 5′-GTGCGGCAAACGAAAGCG-3′.

[0009] Here, this invention uses the gene of *Xanthomonas oryzae* pv. oryzae PXO99A (XOO) as the target gene of rice bacterial blight pathogen, designs and screens a set of LAMP primers capable of detecting bacterial blight, and performs loop-mediated isothermal amplification... Enhanced detection method for the pathogen of rice bacterial blight, Xanthomonas oryzae pv. oryzae PXO99A.

[0010] The present invention also provides a kit for detecting rice bacterial blight pathogens based on LAMP, comprising the Xo1321 LAMP primer set and / or the Xo2967 LAMP primer set.

[0011] Furthermore, the above kit includes a 10 µM FIP / BIP primer, a 10 µM LF / LB primer, and a 10 µM MF3 / B3 primer.

[0012] Here, the 10 µM guide concentration specification is 10 µM / µl.

[0013] Furthermore, the aforementioned kit also includes 10 X Thermol buffer, 100 mM MgSO4, 1mM dNTP, 8000 U Bst larger fragment polymerase.

[0014] Furthermore, the reaction system of the kit is 25 µl, comprising 2.5 µl of 10 X Thermol buffer, 1.5µl 100 mM MgSO4, 3.5µl 1mM dNTP, 2µl 10 µM FIP / BIP primer, 1µl 10 µM LF / LB primer, 0.5µl 10 µM F3 / B3 primer, 1µl 8000 U Bst larger fragment polymerase, 1µl DNA template, and ultrapure water to a final volume of 25µl.

[0015] Here, the concentration of the DNA template should be no less than 20 ng / µl, that is, the initial sample input should be at least 20 ng, requiring a small sample volume and high sensitivity.

[0016] Furthermore, the test kit is used to detect nucleic acid samples extracted from rice seedlings or rice seeds.

[0017] Furthermore, the kit was used to perform a LAMP reaction under the following conditions: incubation at 65°C for 40 min.

[0018] Furthermore, the kit was used to perform a LAMP reaction, and the detection results were determined based on the color change of the reaction solution.

[0019] Here, the LAMP reaction using the above-mentioned kit is simple to operate, requires low equipment, is quick and inexpensive, and provides visualized results, enabling rapid diagnosis of rice diseases in the field.

[0020] The beneficial effects of this invention are as follows: 1) The method of this invention uses the XOO gene of the rice bacterial blight pathogen as the target gene, and designs and screens to obtain a LAMP primer set that can specifically detect bacterial blight, with high specificity; 2) The method of this invention further utilizes this specific LAMP primer set to establish a kit for detecting the rice bacterial blight pathogen, which has high sensitivity, low equipment requirements, and is simple to operate, short in time, low in cost, and visualizes the results, enabling rapid diagnosis of rice diseases in the field. Attached Figure Description

[0021] Figure 1 The images show the XOO LAMP amplification of rice bacterial blight pathogen and the LAMP amplification electrophoresis detection of rice samples in Example 1 of this invention. Figure 2 This is a specific detection diagram of the rice bacterial blight pathogen XOO in Example 2 of the present invention; Figure 3 This is a visual detection image of the rice bacterial blight pathogen XOO in Example 2 of the present invention; Figure 4 This is an electrophoresis detection image of the Xo1321 LAMP amplification sensitivity in Example 3 of the present invention; Figure 5 This is an electrophoresis detection image of the Xo2967 LAMP amplification sensitivity in Example 3 of the present invention; Figure 6 This is a visualization of the Xo1321 LAMP amplification sensitivity in Example 3 of the present invention; Figure 7 This is a visualization of the Xo2967 LAMP amplification sensitivity in Example 3 of the present invention. Detailed Implementation

[0022] To provide a more detailed understanding of the features and technical content of this invention, the implementation of this invention will be described in detail below with reference to the accompanying drawings. The accompanying drawings are for reference only and are not intended to limit this invention.

[0023] Example 1 LAMP Amplification First, DNA was extracted from rice seedlings or seeds infected with the bacterial blight pathogen XOO. The DNA extraction method followed the instructions of the QIAGEN plant DNA Mini kit (#69104). The extracted nucleic acid samples were used as the test samples. The concentration was detected using a Qubit 4.0 fluorescence quantitative PCR instrument, and the samples were diluted to 20 ng / µl with ultrapure water ddH2O.

[0024] The kits mentioned here can be obtained from commercially available channels, depending on the laboratory conditions.

[0025] Two sets of primers were designed against the pathogen XOO of rice bacterial blight: the Xo1321 LAMP primer set or the Xo2967 LAMP primer set, which include a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3, a forward loop primer LF, and a reverse loop primer LB, respectively. The base sequences are as follows: Xo1321 LAMP primer set: FIP:5′-TGATGGACCAGATGCGAAGCAACGGAAAAGGTGGCATCGG-3′; BIP:5′-AGTCATACGCCAGGTTGGTCGTTGCAGACGGCGAATGTG-3′; F3:5′-GGCTGCAGTCACAGTGTT-3′; B3:5′-TCGTCGTTCTCGCATTTGG-3′; LF:5′-GCTTCCAGTCTGCCTGGTATA-3′; LB: 5′-CCTGGGTGCGGTAGTTGGA-3′.

[0026] Xo2967 LAMP primer set: FIP:5′-AGCGCGCAGCTGATCTGCCACGAAGCTGTAGCGCAAG-3′; BIP:5′-ATGCGTTCCAGCTCGCGGCGTGCCGTACTGGTCGA-3′; F3:5′-CACGCCATCGGTCTGTG-3′; B3:5′-CACGGCATGCCGTTCTG-3′; LF:5′-TGGGGTGTTTTGTGGTATTGC-3′; LB: 5′-GTGCGGCAAACGAAAGCG-3′.

[0027] Here, this invention uses the gene of *Xanthomonas oryzae pv. oryzae* PXO99A (XOO), the pathogen of rice bacterial blight, as the target gene. A set of LAMP primers capable of detecting bacterial blight was designed and screened, and the pathogen was specifically detected using loop-mediated isothermal amplification (LAMP) to detect *Xanthomonas oryzae pv. oryzae* PXO99A. The primers were synthesized by Suzhou Hongxun Biotechnology Co., Ltd.

[0028] This embodiment also includes two additional kits: an Xo1321 LAMP kit and an Xo2967 LAMP kit, each comprising the aforementioned Xo1321 LAMP primer set and / or Xo2967 LAMP primer set. The kit components also include 10... X Thermol buffer, 100 mM MgSO4, 1mM dNTP, 8000 U Bst larger fragment polymerase.

[0029] Here, the primer concentrations in the kit are 10 µM / µl, and the primer volume ratio in the kit is FIP / BIP primer : LF / LB primer : F3 / B3 primer = 4 : 2 : 1.

[0030] The usage instructions for the two reagent kits are as follows: Take 200µl of each of the eight reaction tubes and add 2.5µl of 10 to each tube. X Thermol buffer, 1.5 µl 100 mM MgSO4, 3.5 µl 1 mM dNTP, 2 µl 10 µM FIP primer, 2 µl 10 µM BIP primer, 1 µl 10 µM LF primer, 1 µl 10 µM LB primer, 0.5 µl 10 µM F3 primer, 0.5 µl 10 µM B3 primer, 1 µl 8000 U Bst larger fragment polymerase, 1 µl DNA template, and ultrapure water ddH2O to a final volume of 25 µl. Incubate at 65 °C for 40 min.

[0031] Here, the initial sample input is at least 20 ng, and the DNA template volume can exceed 1 µl.

[0032] One sample was prepared using both the Xo1321 LAMP kit and the Xo2967 LAMP kit, and the LAMP amplification results were observed by agarose gel electrophoresis.

[0033] Figure 1 This is an XOO LAMP amplification electrophoresis detection image of the rice bacterial blight pathogen in Example 1 of the present invention. The three samples on the left, from left to right, are: Marker (M), XOO Xo1321 amplification product (Xo1321), and XOO Xo2967 amplification product (Xo2967); the three samples on the right are: Marker (M), rice sample Xo1321 amplification product (Xo1321), and rice sample Xo2967 amplification product (Xo2967). Figure 1As shown, rice samples were successfully amplified using primers Xo1321 and Xo2967, respectively, indicating that the rice samples were infected with the bacterial blight pathogen Xanthomonas oryzae pv. oryzae PXO99A (XOO).

[0034] Example 2: Specificity Detection and Visual Detection XO1321 and XO2967 kits were used to specifically detect the pathogens of rice bacterial blight (Xanthomonas oryzae pv. oryzae PXO99A), banana bacterial wilt (X. campestris pv. musacearum), pepper spot disease (X. campestris pv. vesicatoria), and cowpea wilt (X. axonopodis pv. vignicola). Negative controls were included. The LAMP amplification products were subjected to gel electrophoresis; 2 µl of 1000... X SYBRGreen Ⅰ.

[0035] Here, 1000 X SYBR Green I was purchased from Shanghai Guchen Biotechnology Co., Ltd., product number GCD15143.

[0036] Figure 2 This is a specific detection diagram of the rice bacterial blight pathogen XOO in Example 2 of the present invention, as shown in the figure. Figure 2 As shown, only the rice bacterial blight pathogen X. oryzae pv. oryzae PXO99A (XOO) sample was successfully amplified, indicating that this method can be used for the specific detection of rice bacterial blight pathogen XOO.

[0037] here, Figure 2 In the sample L1, the amplification products of each bacterial species were obtained using the Xo1321 kit; in the sample L2, the amplification products of each bacterial species were obtained using the Xo2967 kit. Add 1000... X After SYBR Green I, if the amplification product is green, the LAMP amplification is successful and the result is positive; if the amplification product is orange, the LAMP amplification fails and the result is negative.

[0038] Figure 3 This is a visual detection image of the rice bacterial blight pathogen XOO in Example 2 of the present invention, as shown below. Figure 3As shown, the two samples on the left are green, while the rest are orange. This means that only the sample of rice bacterial blight pathogen X. oryzae pv. oryzaePXO99A (XOO) was successfully amplified and is positive, while the other samples were negative. Figure 3 The visualized detection results shown are Figure 2 The gel electrophoresis results shown are consistent, indicating that the Xo1321 and Xo2967 primer sets can be used for the visual and specific detection of XOO, the pathogen of rice bacterial blight.

[0039] Example 3: Sensitivity Detection and Visual Detection Sensitivity detection of XOO pathogen of rice bacterial blight: The cloned fragments of primer sets Xo1321 and Xo2967 were constructed into PEASY plasmids, respectively. The construction method was based on the Seamless Assembly Cloning Kit (C5891), which was purchased from Sino-American Taihe Biotechnology (Beijing) Co., Ltd.

[0040] Six copy gradients of plasmid concentration were sequentially set: 10000, 1000, 100, 10, 5, 0. LAMP amplification was performed using Xo1321 and Xo2967 kits, and the amplification products were subjected to gel electrophoresis. Then, 2 µl of 1000... X Visual detection was performed using SYBR Green I. The gel electrophoresis results are as follows: Figure 4 , Figure 5 As shown, the visual detection results are as follows: Figure 6 , Figure 7 As shown.

[0041] Figure 4 This is an electrophoresis detection image of the Xo1321 LAMP amplification sensitivity in Example 3 of the present invention. Figure 5 This is an electrophoresis detection image of the Xo2967 LAMP amplification sensitivity in Example 3 of the present invention. Figure 4 As shown, the detection limit of Xo1321 is 10 copies; Figure 5 As shown, the detection limit for Xo2967 is 5 copies.

[0042] Figure 6 This is a visualization of the Xo1321 LAMP amplification sensitivity in Example 3 of the present invention, as shown below. Figure 6 As shown, amplified samples with 10,000 to 10 copies are green, amplified samples with 5 copies are colorless, and amplified samples with 0 copies are orange. Figure 7 This is a visualization of the Xo2967 LAMP amplification sensitivity in Example 3 of the present invention, as shown below. Figure 7 As shown, amplified samples with 10,000 to 5 copies are green, and amplified samples with 0 copies are orange. Figure 6 , Figure 7 This indicates that the detection limit for Xo1321 is 10 copies, and the detection limit for Xo2967 is 5 copies. Figure 4 , Figure 5 The electrophoresis results were consistent.

[0043] The above examples demonstrate that LAMP reactions using the Xo1321 and Xo2967 kits against rice bacterial blight pathogen XOO are highly specific, sensitive, easy to operate, require minimal equipment, are time-efficient, low-cost, and produce accurate and visualized results, enabling rapid diagnosis of rice diseases in the field.

[0044] The specific steps of the invention methods mentioned above are not limited or described in detail, and the specific components of the reagents mentioned above are not described in detail. As they are common knowledge, those skilled in the art can understand them.

[0045] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A primer set for detecting the pathogen of rice bacterial blight based on LAMP, characterized in that, The Xo2967 LAMP primer set consists of a forward inner primer FIP, a reverse inner primer BIP, a forward outer primer F3, a reverse outer primer B3, a forward loop primer LF, and a reverse loop primer LB. The base sequences are as follows: FIP: 5′-AGCGCGCAGCTGATCTGCCACGAAGCTGTAGCGCAAG-3′; BIP: 5′-ATGCGTGTCCAGCTCGCGGCGTGCCGTACTGGTCGA-3′; F3: 5′-CACGCCATCGGTCTGTG-3′; B3: 5′-CACGGCATGCCGTTCTG-3′; LF: 5′-TGGGGTGTTTTGTGGTATTGC-3′; LB: 5′-GTGCGGCAAACGAAAAGCG-3′.

2. A kit for detecting the pathogen of rice bacterial blight based on LAMP, characterized in that, It contains the Xo2967 LAMP primer set as described in claim 1.

3. The kit for detecting rice bacterial blight pathogens based on LAMP according to claim 2, characterized in that, Includes 10 µM FIP / BIP primers, 10 µM LF / LB primers, and 10 µM F3 / B3 primers.

4. The kit for detecting rice bacterial blight pathogens based on LAMP according to claim 3, characterized in that, Also includes 10 x Thermol buffer, 100 mM MgSO4, 1 mM dNTP, 8000 U Bst large fragment polymerase.

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