Primer group, kit and method for detecting cordate telosma mosaic virus in passion fruit leaves

Abstract

The invention provides a primer group, a kit and a detection method for detecting telosma mosaic virus (DelMV) in passion fruit leaves, and relates to a primer group for detecting the telosma mosaic virus (DelMV) in the passion fruit leaves and a kit for detecting the telosma mosaic virus (DelMV) in the passion fruit leaves. The sequences of the primer group designed based on a TelMV genome coat protein conserved region are shown as sequences 1-5 in a sequence table, a complete LAMP reaction system is established, and visual detection is realized by combining the primer group with a nucleic acid detection test strip. The method does not need complex instruments and professional operation, only needs a simple constant temperature device, and has the characteristics of rapidness, high specificity, high sensitivity and visual and easy result interpretation. By applying the method, virus-carrying seed trees can be removed in time, healthy seedlings can be guided to be cultivated, allocated and transported, technical support is provided for early diagnosis and monitoring of virus diseases, and the method has important significance on prevention and control of the virus diseases of the passion fruits in the field.

Description

Technical Field

[0001] This invention belongs to the field of plant virology technology, specifically relating to a primer set, reagent kit, and method for detecting telosma mosaic virus (TelMV) in passion fruit leaves. Background Technology

[0002] passion fruit( Passiflora edulia Passion fruit, also known as granadilla or egg fruit, is a perennial evergreen vine belonging to the genus Passiflora in the family Passifloraceae. Native to South America, it is now widely cultivated in tropical and subtropical regions worldwide. The passion fruit industry in my country has developed rapidly, forming large-scale planting belts centered on Guangxi, Fujian, Taiwan, Yunnan, Guangdong, Hainan, and Guizhou. It is an important characteristic agricultural industry in my country's rural revitalization strategy in tropical regions. Viral diseases are the most significant threat to passion fruit production, occurring very frequently in orchards during the later stages of growth, with some orchards experiencing infection rates exceeding 90%. This severely impacts the yield and quality of passion fruit. Currently, there is a lack of virus-resistant varieties and effective chemical control measures, making the prevention and control of viral diseases an urgent problem to be solved in production.

[0003] Telosma mosaic virus (TelMV) is a highly pathogenic virus that has become one of the most important pathogens affecting passion fruit production in countries such as China, Thailand, and Vietnam, severely hindering the healthy development of the local passion fruit industry. TelMV belongs to the Potato Virus Y family (…). Potyviridae ) of the genus *Potato* Y ( Potyvirus TelMV virus particles are curved and filamentous, 750–770 nm in length and approximately 14 nm in diameter. The genome is a linear single-stranded positive RNA, approximately 10,000 nt in length. TelMV can cause symptoms in passion fruit plants such as mosaic, yellowing, wrinkling, and stunting, reducing fruit quality and yield; however, under specific environmental conditions, it can manifest as latent infection.

[0004] Planting healthy seedlings and promptly destroying infected plants in the field are effective measures to control virus transmission at the source. Currently, passion fruit production mainly relies on grafted and cutting propagation, making the selection of healthy mother plants crucial. Given that mother plants are prone to carrying viruses, virus testing is necessary during selection; therefore, establishing a rapid detection method suitable for field use is essential. Commonly used virus detection methods include electron microscopy, PCR, and real-time quantitative PCR. These methods are complex, require sophisticated instruments and specialized personnel, and are not suitable for rapid field testing. This invention establishes a visual and rapid detection method for TelMV based on loop-mediated isothermal amplification (LAMP) combined with nucleic acid test strip technology. This method requires only a simple isothermal device (such as a water bath or metal bath) to complete amplification, has a fast reaction speed, high specificity, high sensitivity, and rapid and intuitive result interpretation. It requires minimal operator skill and is particularly suitable for rapid field testing. The application of this invention can promptly remove infected mother trees, guide the cultivation and transportation of healthy seedlings, and provide technical support for the early diagnosis and monitoring of viral diseases, which is of great significance for the prevention and control of viral diseases in passion fruit in the field. Summary of the Invention

[0005] The first aspect of this invention is to provide a primer combination for detecting telosma mosaic virus (TelMV), comprising an outer primer, an inner primer, and a loop primer, the specific sequences of which are as follows: Upstream outer primer TelMV-F3: GCTGTNACACAAATGAARGC (R= A / G, N= A / G / C / T) (SEQ ID NO.1) Downstream outer primer TelMV-B3: CCACAGTTAGAAYTTCGCG (Y = C / T) (SEQ ID NO.2) Upstream inner primer TelMV-FIP: ATCTTCSCCAGTTGTGCTGAGTTGGCACTACDAATAAGATGTT (S=C / G, D=A / G / T) (SEQ ID NO.3) Downstream inner primer TelMV-BIP: RAGGCACACTGCAMGAGATGGTTTACCTAGCCTTTACTGC (R= A / G, M= A / C) (SEQ ID NO.4) Loop primer TelMV-LF: ATGCACTCCTTGCTTGGRGT (R=A / G) (SEQ ID NO.5) The downstream inner primer TelMV-BIP is labeled with 6-carboxyfluorescein (6-FAM) at its 5' end, and the circular primer TelMV-LF is labeled with biotin at its 5' end.

[0006] A second aspect of the present invention is to provide a LAMP amplification reagent for detecting TelMV, comprising the primer set and lysis buffer, binding buffer, washing buffer I, washing buffer II, elution buffer, reaction buffer, BSTMix, nuclease-free water, positive control template, and latex microsphere test strip as described in the first aspect of the present invention.

[0007] The lysis buffer consisted of 4–6 M guanidine isothiocyanate, 30–60 mM Tris-HCl, 0.5%–2% SDS, and 10–20 mM EDTA; the reaction buffer consisted of 150–300 mmol / L Tris-HCl, 300–600 mmol / L KCl, 150–300 mmol / L (NH4)2SO4, 5–10 mmol / L MgSO4, 0.5–1% Tween-20, and 10–50 mmol / L dN(U)TP; the BST Mix contained 6–50 U / μL Bst plus DNA polymerase, 0.1–0.5 U / μL UDGase, 6–50 U / μL Reverse Transcriptase, and 6–50 U / μL MRI. All solutions were prepared using nuclease-free water.

[0008] The LAMP primer set was configured as a 10× primer set mixture. The concentrations of each primer in the 10× primer set mixture were as follows: upstream inner primer TelMV-FIP 16 μmol / L, downstream inner primer TelMV-BIP 16 μmol / L, upstream outer primer TelMV-F3 1~8 μmol / L, downstream outer primer TelMV-B3 1~8 μmol / L, and loop primer TelMV-LB 2~16 μmol / L.

[0009] A third aspect of the present invention is to provide a kit for detecting TelMV, comprising the primer set described in the first aspect of the present invention or the loop-mediated isothermal amplification reagent described in the second aspect of the present invention.

[0010] A fourth aspect of the present invention is to provide a method for detecting TelMV, comprising: performing loop-mediated isothermal amplification detection on passion fruit leaf samples using the primer set described in the first aspect of the present invention, or the loop-mediated isothermal amplification reagent described in the second aspect, or the kit described in the third aspect.

[0011] Specifically, the method for detecting TelMV in passion fruit leaves according to the present invention includes the following steps: (1) Take 100~200 mg of passion fruit leaves, place them in a mortar, add 600 μL of lysis buffer, grind them thoroughly into a homogenate, and let them stand at room temperature for 5 min to obtain the lysis product. (2) Using the lysis product obtained in (1) as a template, prepare a LAMP reaction system. The 25 μL reaction system contains: 6 μL reaction buffer, 1 μL BST Mix, 2.5 μL 10× primer set mixture, 5~10 μL template, and the remainder is made up to 25 μL with Nuclease-Free water. The above reaction system is placed at 63~65℃ for 15~30 min to obtain the amplification product. (3) Add the amplification product from (2) to the sample pad of the nucleic acid test strip and let it stand horizontally for 5-10 minutes to observe the results.

[0012] Positive (+): Both the control line (C line) and the test line (T line) of the test strip show color, indicating that the sample contains TelMV; Negative (-): The C line of the test strip is colored, but the T line is not colored, indicating that the sample does not contain TelMV; Invalid: If the C line of the test strip does not develop color (regardless of whether the T line develops color), it indicates that the test strip used is invalid or the operation is incorrect, and the test needs to be repeated.

[0013] The beneficial effects of this invention are: To address the need for rapid detection of TelMV in passion fruit leaves, this invention provides a detection method with high specificity, high sensitivity, and intuitive result interpretation. This method is simple and fast to operate, requiring no professional background or expensive equipment, making it particularly suitable for direct field use. It is an ideal tool for seedling production enterprises and plant protection monitoring departments for TelMV screening and control. Attached Figure Description

[0014] Figure 1 These are the experimental validation results for the LAMP primer set. Primers 1-10 are: HN-13, HN-15, YN-7, FJ-5, GX-6, GX-9, GZ-5, HN-H1, HN-H2, and HN-H3, respectively.

[0015] Figure 2 The results show the sensitivity detection of LAMP-latex microsphere test strips. Samples 1-4 represent concentrations of 1000 copies / μL, 300 copies / μL, 100 copies / μL, and 10 copies / μL, respectively. NTC serves as the blank control.

[0016] Figure 3 These are the specific detection results for LAMP-latex microsphere test strips. Samples 1-6 contain CMV, EAPV, PWV, PVV, CAbYV, and TYLCV, respectively. PC is the positive control; NC is the negative control; and NTC is the blank control. Detailed Implementation

[0017] To better understand the present invention, the present invention will be further described below with reference to specific embodiments.

[0018] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0019] Example 1: Design and validation of TelMV's LAMP-Latex Microsphere Test Strip Rapid Detection Primer Set (a) Primer design Based on the TelMV genome nucleotide sequence published by NCBI (https: / / www.ncbi.nlm.nih.gov / ), sequence alignment analysis was performed using DNAMAN software to select conserved regions of the genome. Subsequently, a set of LAMP primers was designed using the online LAMP primer design software PrimerExplorer V4 (http: / / primerexplorer.jp / e / ). This primer set targets the conserved region of the TelMV genome's coat protein (CP) and includes: upstream outer primer TelMV-F3, downstream outer primer TelMV-B3, upstream inner primer TelMV-FIP, downstream inner primer TelMV-BIP, and loop primer TelMV-LB (Table 1). The 5' end of the downstream inner primer TelMV-BIP is labeled with 6-carboxyfluorescein (6-FAM), and the 5' end of the loop primer TelMV-LB is labeled with biotin. The primers were manufactured by Sangon Biotech (Shanghai) Co., Ltd.

[0020] Table 1 Primer sequence information used in the LAMP-latex microsphere test strip method for detecting TelMV Primer name sequence Serial Number TelMV-F3 GCTGTNACACAAATGAARGC SEQ ID NO.1 TelMV-B3 CCACAGTTAGAAYTTCGCG SEQ ID NO.2 TelMV-FIP ATCTTCSCCAGTTGTGCTGAGTTGGCACTACDAATAAGATGTT SEQ ID NO.3 TelMV-BIP RAGGCACACTGCAMGAGATGGTTTACCTAGCCTTTACTGC SEQ ID NO.4 TelMV-LB ATGCACTCCTTGCTTGGRGT SEQ ID NO.5 N = A, G, C, or T, R = A or G, Y = C or T, S = C or G, D = A, G, or T, M = A or C (II) Experimental verification of the detection effect of LAMP primer set To verify the detection efficacy of the designed LAMP primer set, we used the LAMP-latex microsphere test strip method to rapidly detect 7 leaf samples of RT-PCR-verified TelMV infected (HN-13, HN-15, YN-7, FJ-5, GX-6, GX-9, GZ-5) and 3 healthy passion fruit samples (HN-H1, HN-H2, and HN-H3). The specific steps are as follows: 1. Take 100 mg of each sample, grind it into a homogenate, add 600 μL of lysis buffer (components: 4 M guanidine isothiocyanate, 30 mM Tris-HCl, 1% SDS and 15 mM EDTA), and let it stand at room temperature for 5 min to obtain the lysis product.

[0021] 2. Using the lysis product obtained in (1) as a template, prepare a reaction system for loop-mediated isothermal amplification. The 25 μL reaction system contains: 6 μL Reaction buffer, 1 μL BST Mix, 2.5 μL 10× primer set, 10 μL template, and 5.5 μL Nuclease-Free water. The reaction buffer consisted of 150 mmol / L Tris-HCl, 300 mmol / L KCl, 150 mmol / L (NH4)2SO4, 5 mmol / L MgSO4, 1% Tween-20, and 10 mmol / L dN(U)TP; the BST Mix contained 10 U / μL Bst plus DNA polymerase, 0.3 U / μL UDGase, 10 U / μL ReverseTranscriptase, and 10 U / μL MRI; the 10× primer set contained 16 μmol / L TelMV-FIP, 16 μmol / L TelMV-BIP, 4 μmol / L TelMV-F3, 4 μmol / L TelMV-B3, and 8 μmol / L TelMV-LB.

[0022] 3. The reaction system was reacted at 63℃ for 30 min to obtain the amplified product; 4. Add the amplification product to the sample pad of the test strip, let it stand for 5 minutes, and then interpret the result.

[0023] Test results as follows Figure 1As shown: In all seven TelMV-infected samples, both the C and T lines on the test strips showed color, indicating a positive result; while in the healthy control samples, only the C line showed color, indicating a negative result. This result confirms that the LAMP primer set provided by this invention can detect TelMV.

[0024] Example 2: Establishment of a TelMV LAMP-Latex Microsphere Test Strip Detection Method This embodiment provides a method for detecting TelMV, and the specific steps are as follows: Using the lysis product of the infected sample HN-3 in Example 1 as a template, the key parameters such as primer concentration, reaction temperature and reaction time were optimized sequentially based on the LAMP reaction system of this example using a single-factor gradient method.

[0025] The primer concentrations in the LAMP reaction system were optimized: the concentration of the inner primer (TELMV-FIP / TELMV-BIP) was kept constant at 1.6 µmol / L, and four concentration combinations of the outer primer (TELMV-F3 / TELMV-B3) and the loop primer (TELMV-LB) were screened (Table 2). The results showed that all four concentration combinations A, B, C, and D could achieve stable detection.

[0026] Table 2 Primer usage settings in LAMP reaction system optimization

[0027] The LAMP reaction temperature was optimized by setting six gradients: 58℃, 61℃, 63℃, 65℃, 67℃, and 70℃. The results showed that both the T and C lines of the test strip developed color within the range of 63–65℃; while at other temperatures, only the C line developed color. Therefore, 63–65℃ can be considered a suitable reaction temperature.

[0028] The LAMP reaction time was optimized: the reaction temperature was set at 63℃, and six reaction time gradients were set: 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min. The results showed that both the T and C lines of the test strip were colored within the range of 15–30 min; while at other reaction times, only the C line was colored. Therefore, 15–30 min can be considered a suitable reaction time.

[0029] Example 3: Preparation of TelMV's LAMP-Latex Microsphere Test Strip Rapid Detection Kit This embodiment provides a kit for detecting TelMV, which includes the lysis buffer, reaction buffer, BST Mix, 10× primer set mixture, Nuclease-Free water, positive control template, latex microsphere test strips, and RNase-Free centrifuge tubes (2.0 mL and 1.5 mL).

[0030] (1) Preparation of lysis buffer required for sample pretreatment The lysis buffer consists of 4–6 M guanidine isothiocyanate, 30–60 mM Tris-HCl, 0.5%–2% SDS, and 10–20 mM EDTA.

[0031] (2) Preparation of solutions required for LAMP reaction system Reaction buffer includes 150~300 mmol / L Tris-HCl, 300~600 mmol / L KCl, 150~300mmol / L (NH4)2SO4, 5~10 mmol / L MgSO4, 0.5~1% Tween-20, 10~50 mmol / L dN(U)TP; BSTMix includes 6~50 U / μL Bst plus DNA polymerase, 0.1~0.5 U / μL UDGase, 6~50 U / μL ReverseTranscriptase and 6~50 U / μL MRI.

[0032] The concentrations of each primer in the 10× primer set mixture are as follows: upstream inner primer TelMV-FIP 16 μmol / L, downstream inner primer TelMV-BIP 16 μmol / L, upstream outer primer TelMV-F3 1~8 μmol / L, downstream outer primer TelMV-B3 1~8 μmol / L, and loop primer TelMV-LB 2~16 μmol / L.

[0033] (3) Preparation of positive control template The partial coding region of the TelMV genome CP gene, targeted by artificially synthesized LAMP primers (see SEQ ID NO. 6 in the sequence listing), was ligated into the pESI-TA / Blunt vector and transformed. E. coli After screening positive clones and verifying them by sequencing, the plasmid is extracted from DH5α CompetentCells and serves as the positive control template for the kit.

[0034] (4) Assembly of latex microsphere test strips The latex microsphere test strip consists of a PVC base plate, a sample pad, a conjugate pad, a nitrocellulose membrane (NC membrane), and an absorbent pad. The conjugate pad is coated with red latex microspheres labeled with FAM monoclonal antibody and blue latex microspheres labeled with chicken IgY antibody. The nitrocellulose membrane has T lines and C lines. The T line is coated with biotin monoclonal antibody, and the C line is coated with goat anti-chicken IgY antibody.

[0035] Example 4: Sensitivity test of TelMV detection using the LAMP-latex microsphere test strip method To determine the sensitivity of the constructed LAMP-latex microsphere test strip for detecting TelMV, the positive control template described in Example 3 was serially diluted to prepare gradient templates with concentrations of 1000, 300, 100, and 10 copies / μL. Subsequently, the LAMP reaction and test strip detection were performed according to the method described in Example 1 (II). The results showed that ( Figure 2 When the template concentration is 1000, 300, and 100 copies / μL, both the T and C lines of the test strip show color; when the template concentration is 10 copies / μL, only the C line shows color. Therefore, the limit of detection for this method is 100 copies / μL.

[0036] Example 5: Specificity verification of the primer combination of the present invention To evaluate the specificity of the primers designed in this invention, common viruses found on passion fruit were selected as test subjects. Passion fruit leaf samples carrying the following viruses were tested according to the steps in Example 4, including cucumber mosaic virus (CMV), East Asian passiflora virus (EAPV), passion fruit woodiness virus (PWV), passion fruit Vietnam virus (PVV), cucumber aphid-borne yellows virus (CABYV), and tomato yellow leaf curl virus (TYLCV). Samples carrying TelMV were used as positive controls, and healthy passion fruit leaf samples were used as negative controls. The test results showed that, except for the positive control, all other test samples were negative. Figure 3 This demonstrates that the detection method established in this invention has high specificity for TelMV and shows no cross-reactivity with other common passion fruit viruses.

Claims

1. The LAMP primer set for detecting telosma mosaic virus (TelMV) in passion fruit leaves consists of outer primers, inner primers, and loop primers; among which, The outer primer consists of an upstream outer primer and a downstream outer primer, with the upstream outer primer being the nucleotide shown in SEQ ID NO.1 of the sequence listing and the downstream outer primer being the nucleotide shown in SEQ ID NO.2 of the sequence listing; the inner primer consists of an upstream inner primer and a downstream inner primer, with the upstream inner primer being the nucleotide shown in SEQ ID NO.3 of the sequence listing and the downstream inner primer being the nucleotide shown in SEQ ID NO.4 of the sequence listing; the loop primer is the nucleotide shown in SEQ ID NO.5 of the sequence listing.

2. The LAMP primer set according to claim 1, characterized in that, The 5' end of the downstream inner primer is labeled with 6-carboxyfluorescein (6-FAM), and the 5' end of the loop primer is labeled with biotin.

3. A kit for detecting TelMV, comprising the LAMP primer set as described in claim 1.

4. The kit according to claim 3, characterized in that: The kit also includes lysis buffer, reaction buffer, BST Mix, nuclease-free water, positive control template, and latex microsphere test strips; The lysis buffer consisted of 4–6 M guanidine isothiocyanate, 30–60 mM Tris-HCl, 0.5%–2% SDS, and 10–20 mM EDTA; the reaction buffer consisted of 150–300 mmol / L Tris-HCl, 300–600 mmol / L KCl, 150–300 mmol / L (NH4)2SO4, 5–10 mmol / L MgSO4, 0.5–1% Tween-20, and 10–50 mmol / L dN(U)TP; the BST Mix contained 6–50 U / μL Bst plus DNA polymerase, 0.1–0.5 U / μL UDGase, 6–50 U / μL ReverseTranscriptase, and 6–50 U / μL MRI; all the above solutions were prepared using nuclease-free water.

5. The kit according to claim 3, characterized in that: The LAMP primer set is configured as a 10× primer set mixture; wherein, the concentration of each primer in the 10× primer set mixture is as follows: upstream inner primer TelMV-FIP 16 μmol / L, downstream inner primer TelMV-BIP 16 μmol / L, upstream outer primer TelMV-F3 1~8 μmol / L, downstream outer primer TelMV-B3 1~8 μmol / L, and loop primer TelMV-LB 2~16 μmol / L.

6. The application of the LAMP primer set according to claim 1 or 2 and the kit according to claim 3 or 4 in the detection of TelMV in passion fruit leaves.

7. A method for detecting TelMV, comprising the following steps, using the kit described in claims 3-5 to detect TelMV in passion fruit leaves: (1) Take 100~200 mg of passion fruit leaves, place them in a mortar, add 600 μL of lysis buffer, grind them thoroughly into a homogenate, and let them stand at room temperature for 5 min to obtain the lysis product. (2) Using the lysis product obtained in (1) as a template, prepare a LAMP reaction system. The 25 μL reaction system contains: 6 μL reaction buffer, 1 μL BST Mix, 2.5 μL 10× primer set mixture, 5~10 μL template, and the remainder is made up to 25 μL with Nuclease-Free water. The above reaction system is placed at 63~65℃ for 15~30 min to obtain the amplification product. (3) Add the amplification product from (2) to the sample pad of the nucleic acid test strip, let it stand horizontally for 5-10 minutes and observe the results; Positive (+): Both the control line (C line) and the test line (T line) of the test strip show color, indicating that the sample contains TelMV; Negative (-): The C line of the test strip is colored, but the T line is not colored, indicating that the sample does not contain TelMV; Invalid: If the C line of the test strip does not develop color (regardless of whether the T line develops color), it indicates that the test strip used is invalid or the operation is incorrect, and the test needs to be repeated.

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