Reverse transcription loop-mediated isothermal amplification (RT-LAMP) visual detection kit for sweet potato pinniform mottle virus

Abstract

The invention discloses a sweet potato pinniform mottle virus RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) visual detection kit. Comprising LAMP primers, freeze-dried microspheres and water. The freeze-dried microspheres are prepared from the following components: HotStart Bst4. 2DNA / RNA (Ribonucleic Acid) polymerase, Helicaser, dNTP (Deoxyribose Nucleic Acid), Mg < 2 + >, a reaction buffer, an L-HNB color change indicator Leuco-HNB, a leuco HNB dye and a stabilizer. The RT-LAMP visual rapid detection method provided by the invention is high in sensitivity and good in specificity, and is suitable for rapid field detection of SPFMV. Dextran sulfate and polyethylene glycol 8000 are used as compound stabilizers in the freeze-dried microspheres, so that the detection sensitivity is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of sweet potato feathery mottle virus detection, and particularly relates to a sweet potato feathery mottle virus RT-LAMP visualization detection kit. Background Art

[0002] Sweet potato (Ipomoea batatas) is the world's seventh largest food crop after wheat, rice, corn, potato, barley and cassava. It is also an important feed and light industrial raw material. Viral diseases are important diseases that threaten sweet potato production. Sweet potato losses caused by viral diseases are as high as 30%-50% per year worldwide, and the economic losses in China reach 4 billion yuan per year. Sweet potato feathery mottle virus (SPFMV) is an important virus on sweet potato. It belongs to the genus Potyvirus. It is transmitted in a non-persistent manner through sap or aphids. It is found in almost all major sweet potato producing areas in the world. SPFMV can infect 8 species of Convolvulaceae, including sweet potato. The symptoms shown in different growth stages of the plant are also different, which seriously damages the yield and quality of sweet potato. In recent years, SPFMV has occurred in many provinces (cities) in China, and the distribution area has been expanding year by year. Therefore, it is of great significance to establish a simple and rapid detection method for early warning of SPFMV and understand the law of disease occurrence. In 2010, Zhang Yehui et al. established a multiplex PCR detection method for three sweet potato viruses including SPFMV. Subsequently, ELISA, RT-PCR, fluorescence quantitative RT-PCR, reverse dot hybridization and other detection methods for SPFMV were gradually established. In the early stage, the author's laboratory has detected SPFMV and other sweet potato viruses in Shandong Province through biological identification, electron microscopy observation and RT-PCR. The above detection methods provide technical guarantees for understanding the field incidence of SPFMV. Reverse transcription loop-mediated isothermal amplification technology (RT-LAMP) is a new nucleic acid amplification method developed by Notomi in 2000. This technology designs 4 primers for the target sequence, specifically recognizes 6 sequence regions, and uses DNA strand displacement polymerase (BstDNApolymerase) for efficient amplification at a constant temperature of 60-65℃. RT-LAMP has been widely used in the detection of human and plant pathogens, and has played an important role in the detection of viral diseases of grains, vegetables, fruit trees and cash crops. So far, there has been no report on the one-step visual detection of SPFMV using this technology at home and abroad.

[0003] Although existing molecular detection technologies such as RT-PCR and fluorescent quantitative PCR have the characteristics of high specificity and sensitivity, they require specific instruments and professional laboratory personnel to complete and are not suitable for grassroots testing. Serological testing requires specific antiserum, the production process is complicated, the cost is expensive, and false positives are prone to occur. Biological identification and electron microscopy observation are time-consuming and labor-intensive, and require high virus content in the test sample. As a new detection technology, RT-LAMP has the characteristics of simplicity, rapidity, and specificity. It does not require thermal denaturation of the template and can be carried out under isothermal conditions. The test results can be obtained in a short time. However, high-concentration DNA products are prone to produce aerosols, causing laboratory environmental pollution. Summary of the invention

[0004] The purpose of the present invention is to establish a RT-LAMP lid-free visual rapid detection technology for SPFMV, and to facilitate the operation of operators and avoid cross-contamination of reagents during the operation. The present invention innovatively prepares the components of the RT-LAMP detection system together with primers into freeze-dried microspheres. Only one freeze-dried microsphere is needed for one reaction, and a detection template and water are added.

[0005] The present invention screens the most suitable RT-LAMP detection primers, verifies the specificity and sensitivity of RT-LAMP amplification, establishes a novel, lid-free, rapid, convenient and sensitive visual detection kit for SPFMV, and provides technical support for the monitoring and prevention of sweet potato virus diseases.

[0006] A sweet potato feathery mottle virus RT-LAMP visualization detection kit comprises LAMP primers, freeze-dried microspheres and water.

[0007] The LAMP primers include F3-1, B3-1, FIP-1, BIP-1, LoopF-1, and LoopB-1, and their nucleotide sequences are shown in the sequence table SEQ ID NO: 1-6 respectively.

[0008] The usage of the LAMP primers is as follows: 0.2 μmol of the primer F3-1, 0.2 μmol of the primer B3-1, 1.5 μmol of the primer FIP-1, 1.5 μmol of the primer BIP-1, 0.8 μmol of the primer LoopF-1, and 0.8 μmol of the primer LoopB-1.

[0009] The freeze-dried microspheres are made of the following components: HotStart Bst4.2 DNA / RNA polymerase 6-10U, Helicaser 2-6ng, dNTP 0.2-0.6μmol, Mg 2+10-15mmol, reaction buffer 0.2-0.6μg, L-HNB color indicator Leuco-HNB 0.2-0.6μL, leuco HNB dye 0.1-0.3μL, stabilizer 0.1-0.3μg, water 6-10uL.

[0010] The reaction buffer is: ethylenediaminetetraacetic acid.

[0011] The stabilizer is: dextran sulfate and polyethylene glycol 8000.

[0012] The application of the kit includes:

[0013] 1) Detecting or assisting in detecting whether the sample to be tested contains or is suspected of containing sweet potato feathery mottle virus;

[0014] 2) Identify or assist in identifying whether the virus to be tested is a candidate sweet potato feathery mottle virus.

[0015] A sweet potato feathery mottle virus RT-LAMP visualization detection method comprises the following steps: preparing a reaction system with one freeze-dried microsphere, 0.3-1 μL of template RNA, and ddH2O to a total volume of 25 μL; after the reaction system is prepared, placing the reaction system at 70°C for 30 minutes, and observing the color after the reaction is completed, with purple being negative and green being positive.

[0016] The template RNA is total RNA from sweet potato leaves.

[0017] Beneficial effects of the present invention: The RT-LAMP visual rapid detection method provided by the present invention has high sensitivity and good specificity, and is suitable for rapid field detection of SPFMV. Dextran sulfate and polyethylene glycol 8000 are used as composite stabilizers in the freeze-dried microspheres to improve the sensitivity of the detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Primer screening results for RT-LAMP visualization assay.

[0019] Figure 2 It is the specific detection result; In the figure, M: marker; C: control; B: blank.

[0020] Figure 3 It is the sensitivity test result; In the figure, M: marker; C: control; B: blank.

[0021] Figure 4 Comparison of detection sensitivity of different freeze-dried microspheres; in the figure, M: marker; C: control; B: blank.

[0022] Figure 5This is the field test result of RT-LAMP lid-free visual detection technology; in the figure, M: marker; C: control; B: blank. DETAILED DESCRIPTION

[0023] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0024] Example 1

[0025] The nucleotide sequence of SPFMV coat protein (CP) gene was obtained from GenBank, and three groups of 18 RT-LAMP specific primers were designed using Primer Explorer V5:

[0026] Group 1:

[0027] F3-1 5′-GGACTATGATGGATGGTGA-3′ (SEQ ID NO: 1);

[0028] B3-1 5′-GTTGAATGCAGCTCGTAG-3′ (SEQ ID NO: 2);

[0029] FIP-1 5′-AGCTTCAGCAACGTCACTGCCATTGTTGGACCATGC-3′ (SEQ ID NO: 3);

[0030] BIP-1 5′-CGAAACCGTACAAAGGCGTACGCAAGACTCATATCAGT C-3′ (SEQ ID NO: 4);

[0031] LoopF-1 5′-AAGTGCGTCATAATCTGCCT-3′ (SEQ ID NO: 5);

[0032] LoopB-1 5'-TGCCGAGGTATGGTCTACAA-3' (SEQ ID NO: 6).

[0033] Group 2:

[0034] F3-2 5′-CCATTGTTGGACCATGC-3′ (SEQ ID NO: 7);

[0035] B3-2 5′-GGCTGCCTTCATCTGTAA-3′(SEQ ID NO: 8);

[0036] FIP-2 5′-TACGCCTTTGTACGGTTTCGAGGCAGATTATGACG CA-3′(SEQ ID NO: 9);

[0037] BIP-2 5′-TGCCGAGGTATGGTCTACAATGAATGCAGCTCGTAG AA-3′(SEQ ID NO: 10);

[0038] LoopF-2 5′-AGCTTCAGCAACGTCACTG-3′(SEQ ID NO: 11);

[0039] LoopB-2 5′-TGAGTCTTGCGCGATATGC-3′(SEQ ID NO: 12).

[0040] Group 3:

[0041] F3-3 TGGATTAATGGTTTGGTGC-3′(SEQ ID NO: 13);

[0042] B3-3 CGCAAGACTCATATCAGTC-3′(SEQ ID NO: 14);

[0043] FIP-3GCACTGCATGGTCCAACAAATGGCGTGTGGACTAT-3′(SEQ ID NO: 15);

[0044] BIP-3ATGACGCACTTCAGTGACGATACCTCGGCATGTACG-3′(SEQ ID NO: 16);

[0045] LoopF-3CACTTGCTCATCACCATCCA-3′(SEQ ID NO: 17);

[0046] LoopB-3AATGCGAAACCGTACAAAGG-3′(SEQ ID NO: 18).

[0047] At the same time, two specific primers for reverse transcription polymerase chain reaction (RT-PCR) were designed: SPFMV-F (5′-AAGCATGGTATGAGGGAGTGAAAG-3′ SEQ ID NO: 19) and SPFMV-R (5′-GTATCTTCTTCTTGCGTGGAGACG-3′ SEQ ID NO: 20).

[0048] The sweet potato feathery mottle virus RT-LAMP visualization detection kit comprises LAMP primers, freeze-dried microspheres and water; the dosage of the LAMP primers is as follows: 0.2 μmol of the primer F3, 0.2 μmol of the primer B3, 1.5 μmol of the primer FIP, 1.5 μmol of the primer BIP, 0.8 μmol of the primer LoopF, and 0.8 μmol of the primer LoopB.

[0049] The freeze-dried microspheres were made of the following components: HotStart Bst4.2 DNA / RNA polymerase 8U (purchased from Harbin Xinhai Gene Testing Co., Ltd.), Helicaser 4ng (purchased from NEB), dNTP 0.4μmol, Mg 2+ 12mmol, reaction buffer 0.4μg, L-HNB color indicator Leuco-HNB 0.4μL (purchased from Harbin Xinhai Gene Testing Co., Ltd.), hidden HNB dye 0.2μL (purchased from Harbin Xinhai Gene Testing Co., Ltd.), stabilizer 0.2μg, water 8uL.

[0050] The above components are fully mixed and freeze-dried to prepare freeze-dried microspheres.

[0051] The reaction buffer is: ethylenediaminetetraacetic acid.

[0052] The stabilizer is: dextran sulfate and polyethylene glycol 8000.

[0053] RT-LAMP specific detection was performed using total RNA from leaves carrying SPFMV, sweet potato virus C (SPVC), sweet potato chlorotic stunt virus (SPCSV), sweet potato virus 2 (SPV2), sweet potato latent virus (SPLV), sweet potato virus G (SPVG), sweet potato chlorotic fleck virus (SPCFV) and healthy sweet potato leaves as templates; SPFMV positive plasmid (100 ng / μL) was diluted in 10-fold concentration gradient, 10 -2 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 and 10 -9 The dilution was used as a template for LAMP sensitivity assay.

[0054] Detection reaction system: 1 freeze-dried microsphere, 0.5μL template RNA, ddH2O to a total volume of 25μL. After the reaction system is prepared, place it at 70℃ for 30 minutes. After the reaction is completed, observe the color. Purple means negative and green means positive amplification.

[0055] The results showed that the first set of primers could effectively develop color in the reaction system, with strong specificity and high effectiveness. Therefore, the first set of primers was selected as the specific primers of the SPFMV RT-LAMP visualization detection system kit ( Figure 1 ).

[0056] Specificity test:

[0057] Using RNA from sweet potato leaves containing SPVC, SPCSV, SPV2, SPLV, SPVG, SPCFV and SPFMV viruses as templates, amplification was performed using conventional RT-PCR and RT-LAMP methods.

[0058] The results showed that the amplification results of RT-LAMP and RT-PCR were consistent, and only the bands could be amplified in the sweet potato samples carrying SPFMV, while no bands were amplified in the sweet potato samples carrying other viruses, indicating that the established SPFMV RT-LAMP visual detection system has good specificity ( Figure 2 ).

[0059] Sensitivity test:

[0060] The SPFMV positive plasmid was diluted in 10-fold concentration gradient, 10 -2 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 and 10 -9 The dilution was used as a template for LAMP sensitivity assay.

[0061] The results showed that the RT-LAMP method established in this study has a detection sensitivity 1000 times higher than that of the conventional RT-PCR method ( Figure 3 ).

[0062] Comparison of freeze-dried microspheres:

[0063] Group 1: The lyophilized microspheres were made of the following components: HotStart Bst4.2 DNA / RNA polymerase 8U, Helicaser 4ng, dNTP 0.4μmol, Mg 2+ 12mmol, ethylenediaminetetraacetic acid 0.4μg, L-HNB color indicator Leuco-HNB 0.4μL, hidden color HNB dye 0.2μL, dextran sulfate 0.1μg, polyethylene glycol 8000 0.1μg, water 8uL; the above components are fully mixed and lyophilized to prepare lyophilized microspheres.

[0064] Group 2: The lyophilized microspheres were made of the following components: HotStart Bst4.2 DNA / RNA polymerase 8U, Helicaser 4ng, dNTP 0.4μmol, Mg 2+ 12mmol, ethylenediaminetetraacetic acid 0.4μg, L-HNB color indicator Leuco-HNB 0.4μL, hidden color HNB dye 0.2μL, dextran sulfate 0.2μg, water 8uL; the above components are fully mixed and lyophilized to prepare lyophilized microspheres.

[0065] Group 3: The lyophilized microspheres were made of the following components: HotStart Bst4.2 DNA / RNA polymerase 8U, Helicaser 4ng, dNTP 0.4μmol, Mg 2+ 12mmol, ethylenediaminetetraacetic acid 0.4μg, L-HNB color indicator Leuco-HNB 0.4μL, hidden color HNB dye 0.2μL, polyethylene glycol 8000 0.2μg, water 8uL; the above components are fully mixed and lyophilized to prepare lyophilized microspheres.

[0066] The SPFMV positive plasmid was diluted in 10-fold concentration gradient, 10 -2 , 10-4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 and 10 -9 The dilution was used as a template for LAMP sensitivity assay.

[0067] The results showed that the sensitivity of the freeze-dried microspheres in group 1 was 100 times higher than that in groups 2 and 3 ( Figure 4 ).

[0068] Field detection application of RT-LAMP non-opening visual detection technology:

[0069] RT-LAMP and RT-PCR tests were performed on suspected diseased sweet potato plants collected from Yanjin, Henan Province. The results showed that 5 SPFMV-positive samples were detected by RT-LAMP detection method among 7 samples, which was consistent with the results of RT-PCR detection ( Figure 5 ), indicating that the RT-LAMP lid-free visualization detection technology established in this study can be used for the rapid visualization detection of SPFMV in sweet potato samples in the field.

[0070] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A sweet potato feathery mottle virus RT-LAMP visualization detection kit, characterized in that: Includes LAMP primers, lyophilized microspheres, and water.

2. The sweet potato feathery mottle virus RT-LAMP visualization detection kit according to claim 1, characterized in that: The LAMP primers include F3-1, B3-1, FIP-1, BIP-1, LoopF-1, and LoopB-1, and their nucleotide sequences are shown in the sequence table SEQ ID NO: 1-6 respectively.

3. The sweet potato feathery mottle virus RT-LAMP visualization detection kit according to claim 2, characterized in that: The dosage of the LAMP primers is as follows: 0.2 μmol of the primer F3-1, 0.2 μmol of the primer B3-1, 1.5 μmol of the primer FIP-1, 1.5 μmol of the primer BIP-1, 0.8 μmol of the primer LoopF-1, and 0.8 μmol of the primer LoopB-1.

4. The sweet potato feathery mottle virus RT-LAMP visualization detection kit according to claim 1, characterized in that: The freeze-dried microspheres are made of the following components: HotStart Bst4.2 DNA / RNA polymerase 6-10U, Helicaser 2-6ng, dNTP 0.2-0.6μmol, Mg 2+ 10-15mmol, reaction buffer 0.2-0.6μg, L-HNB color indicator Leuco-HNB 0.2-0.6μL, leuco HNB dye 0.1-0.3μL, stabilizer 0.1-0.3μg, water 6-10uL.

5. The sweet potato feathery mottle virus RT-LAMP visualization detection kit according to claim 4, characterized in that: The reaction buffer is: ethylenediaminetetraacetic acid.

6. The sweet potato feathery mottle virus RT-LAMP visualization detection kit according to claim 4, characterized in that: The stabilizer is: dextran sulfate and polyethylene glycol 8000.

7. Use of the kit according to claim 1, characterized in that: include: 1) Detecting or assisting in detecting whether the sample to be tested contains or is suspected of containing sweet potato feathery mottle virus; 2) Identify or assist in identifying whether the virus to be tested is a candidate sweet potato feathery mottle virus.

8. A RT-LAMP visualization detection method for sweet potato feathery mottle virus, characterized in that: Prepare a reaction system by taking one freeze-dried microsphere, 0.3-1 μL template RNA, and ddH2O to a total volume of 25 μL. After the reaction system is prepared, place it at 70°C for 30 minutes. After the reaction is completed, observe the color: purple is negative and green is positive.

9. The RT-LAMP visualization detection method for sweet potato feathery mottle virus according to claim 8, characterized in that: The template RNA is total RNA from sweet potato leaves.

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