RT-LAMP primer group and detection method for detecting shallot X virus of rehmannia

Abstract

The invention relates to an RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) detection primer group and a detection method of a rehmannia onion X virus, the RT-LAMP detection primer group comprises a pair of outer primers ReAV-F3 and ReAV-B3, a pair of inner primers ReAV-FIP and ReAV-BIP and a loop primer ReAV-LB, and the color presented by a nucleic acid dye solution after an RT-LAMP amplification reaction or the agarose gel electrophoresis result of an RT-LAMP product is judged. The detection method disclosed by the invention is high in speed, high in sensitivity, strong in specificity, simple to operate and visual in detection result, and a convenient, rapid and reliable method is provided for field detection of the shallot X virus of rehmannia glutinosa.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to an RT-LAMP detection primer set and a detection method for Rehmannia glutinosa X virus. Background Art

[0002] Rehmannia glutinosa L. is a perennial herbaceous plant of the genus Rehmannia in the family Scrophulariaceae. Its tubers are used as medicine and it is one of the major Chinese medicinal materials in my country. The main chemical components of Rehmannia include iridoids, ionones, phenylethanoid glycosides, flavonoids, etc. It has the effects of clearing heat and cooling blood, nourishing yin and promoting fluid production. It also has a positive effect on improving the body's immunity, anti-cancer, and cancer prevention. Rehmannia glutinosa is asexually propagated with tubers. Long-term asexual reproduction is prone to cause viral diseases, resulting in a decrease in Rehmannia yield and quality. Rehmannia Allexivirusvirus (ReAV) is a member of the genus Allexivirus. It is a single-stranded positive RNA with a genome length of 7249nt and contains 5 open reading frames (ORFs), encoding a total of 5 proteins including replicase, TGB1, TGB2, TGB3, and CP. Currently, the main detection technology for ReAV is RT-PCR. Conventional RT-PCR amplification requires a PCR instrument, and result observation requires an electrophoresis instrument and an ultraviolet imaging instrument. It has the disadvantages of expensive instruments, long time consumption and complicated operation.

[0003] The loop mediated isothermal amplification (LAMP) technology established by Notomi et al. in 2000 designs 4 to 6 specific primers for 6 regions of the target gene, and uses DNA strand displacement polymerase to quickly detect the target gene under constant temperature conditions. It has been widely used in the detection of various plant viruses. Therefore, how to apply LAMP technology to ReAV detection and establish a detection technology that is more advantageous than conventional PCR methods to meet the needs of grassroots and field virus detection is a technical problem that needs to be solved. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides an RT-LAMP detection primer set and a detection method for Allium foetidum X virus.

[0005] The technical solution adopted by the present invention is:

[0006] 1. The RT-LAMP primer set for detecting the Allium radix virus X includes a pair of outer primers, a pair of inner primers and a loop primer, as follows:

[0007] (1) A pair of external primers:

[0008] ReAV-F3:5′-AGGAGCTGGGCGACG-3′;

[0009] ReAV-B3:5′-AGAACGGTACTCCCGCG-3′;

[0010] (2) A pair of inner primers:

[0011] ReAV-FIP:5′-GGTATTGCGCCTGCGCCATAGTCAAACTCCATCGCCACACA-3′;

[0012] ReAV-BIP:5′-TCTCATCACGCTCGCCATGAGGCCCTCCAGCGTTACG-3′;

[0013] (3) One loop primer:

[0014] ReAV-LB: 5′-CTACCACAATGGGGCGTCC-3′.

[0015] 2. A method for detecting Allium chinense X virus using an RT-LAMP primer set, comprising the following steps:

[0016] (1) Extract the total viral RNA and then reverse transcribe it into cDNA using RNA as a template;

[0017] (2) using the cDNA as a template and using an RT-LAMP primer set to perform an RT-LAMP amplification reaction;

[0018] (3) After the RT-LAMP amplification reaction is completed, centrifuge briefly to remove the nucleic acid dye in the lid. If the RT-LAMP reaction solution turns green, it is considered positive; if the reaction solution turns yellow, it is considered negative.

[0019] Alternatively, the RT-LAMP amplification product was analyzed by agarose gel electrophoresis. If waterfall-like bands appeared, it was judged as positive, and if no bands were present, it was negative.

[0020] The reaction system for RT-LAMP amplification was as follows: template cDNA 1 μL, 10×ThermoPol Buffer 2.5 μL, ReAV-FIP and ReAV-BIP primers 1.6 μmol / L each, 4 μL, ReAV-F3 and ReAV-B3 primers 0.2 μmol / L each, 0.5 μL, ReAV-LB primer 0.4 μmol / L, 1 μL, Mg 2+6mM, dosage 1.5μL, dNTPs 1.4mM, dosage 3.5μL, BstDNApolymerase 8U, dosage 1μL, ddH 2 During the reaction, add the reaction system to the PCR tube, gently pipette and mix, then add 30 μL of paraffin oil to seal; at the same time, add 0.5 μL of SYBR Green I nucleic acid dye into the PCR tube cap.

[0021] The reaction conditions of RT-LAMP amplification were as follows: 65°C for 40 min, 85°C for 5 min, and termination of the reaction on ice.

[0022] 3. The RT-LAMP primer set is used in the preparation of a detection kit or reagent for Allium radix X virus.

[0023] Beneficial Effects of the Invention

[0024] (1) The present invention obtains an RT-LAMP detection primer set for Rehmannia glutinosa virus X by screening RT-LAMP detection primers and optimizing reaction conditions, and establishes a highly sensitive ReAV detection method. The results show that this method can detect 6.99×10 0 The sensitivity of PCR is 10 times that of conventional PCR, which is higher.

[0025] (2) Specificity tests showed that the method of the present invention detected six RNA viruses, namely ReAV, TMGMV, CCYV, ReMV, YoMV, and CLVd, of Rehmannia glutinosa, and only ReAV was detected positive, indicating that the method of the present invention has good specificity.

[0026] (3) The present invention optimizes the RT-LAMP amplification reaction temperature and time. The results show that the optimal reaction temperature of the RT-LAMP amplification reaction is 65°C, and the optimal reaction time is 40 min.

[0027] (4) Field detection application tests show that the accuracy of the RT-LAMP detection of the present invention is higher than that of ordinary PCR, and can be applied to the detection of Rehmannia glutinosa field samples.

[0028] (5) The ReAV loop-mediated constant temperature rapid amplification method of the present invention realizes visual detection by adding SYBR green I nucleic acid dye, directly observes the detection results, is simple to operate, and provides a convenient and quick method for ReAV field detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Agarose gel electrophoresis diagram of the products of RT-LAMP under different temperature conditions.

[0030] Figure 2 Agarose gel electrophoresis diagram of the products of RT-LAMP under different reaction time conditions.

[0031] Figure 3 This is a diagram for validating the specificity of RT-LAMP of the present invention.

[0032] Figure 4 This is the detection electrophoresis diagram of the RT-LAMP sensitivity of the present invention.

[0033] Figure 5 This is a comparative experiment of detecting ReAV using the RT-LAMP of the present invention and conventional RT-PCR. DETAILED DESCRIPTION

[0034] The specific implementation modes of the present invention are further described in detail below in conjunction with the embodiments and drawings.

[0035] Example 1 RT-LAMP detection primer set and detection method for Rehmannia glutinosa X virus

[0036] The specific steps are as follows:

[0037] Step 1: According to the previous transcriptome sequencing results and the ReAV coat protein (CP) nucleotide sequence registered in GenBank (GenBank accession number: PP097219.1), three sets of ReAV RT-LAMP detection primers were designed online using the NEB LAMP Primer Design Tool website (https: / / lamp.neb.com / ). Each set of detection primers contained five primers corresponding to six regions of the target gene. After screening and removing false positive related primers, the ReAV RT-LAMP detection primers were determined to be ReAV-F3, ReAV-B3, ReAV-FIP, ReAV-BIP and ReAV-LB; at the same time, common RT-PCR primers ReAV-CPF and ReAV-CPR were designed, and RT-PCR amplification was used for comparative experiments with RT-LAMP. All primers were synthesized by Shanghai Shenggong Biotechnology Co., Ltd., and the sequences of each primer are shown in Table 1.

[0038] Table 1 Detection primers for RT-LAMP and conventional RT-PCR

[0039]

[0040] Step 2: Extraction of total viral RNA and synthesis of cDNA: Take about 0.1 g of Rehmannia glutinosa leaves, freeze them in liquid nitrogen and quickly grind them. According to the instructions of the column-type plant total RNA extraction kit, extract the total RNA of the leaves. Use RNA as a template according to the reverse transcription kit PrimeScript TMⅡ1st Strand cDNA Synthesis Kit instructions to synthesize cDNA;

[0041] Step 3: Using the synthesized cDNA as a template, perform RT-LAMP amplification according to the reaction system in Table 2;

[0042] Add 25 μL of the reaction system to a PCR tube, gently pipette and mix, then add 30 μL of paraffin oil to seal the tube. At the same time, add 0.5 μL of SYBR Green I nucleic acid dye to the PCR tube cap and perform amplification under the following conditions.

[0043] RT-LAMP amplification reaction conditions: 65°C for 40 min, 85°C for 5 min, and terminate the reaction on ice.

[0044] Table 2 RT-LAMP reaction system

[0045]

[0046] Step 4: Result judgment

[0047] After the reaction is finished, centrifuge briefly to remove the SYBR Green I nucleic acid dye in the PCR tube cap. The dye will penetrate into the minor groove of the double-stranded DNA and bind to the double-stranded DNA to release fluorescence. If the sample solution turns green, it is judged as positive, and if the sample solution turns orange, it is judged as negative.

[0048] At the same time, gel electrophoresis analysis was performed, and 5 μL of RT-LAMP product was subjected to agarose gel electrophoresis for 35 min at 1×TBE and 120 V. If the sample showed waterfall-like bands, it was judged as positive, and if the sample had no bands, it was judged as negative. Example 2 Identification of RT-LAMP amplification products

[0049] After the RT-LAMP product of Example 1 was separated by agarose electrophoresis, a band of about 200 bp was cut out, and the product was recovered using a gel recovery kit, and connected to the pMD19-T vector reaction system according to the configuration of Table 3, and the reaction system was placed in a constant temperature water bath at 16°C for 3 hours. 10 μL of the ligation product was transformed into 100 μL of E. coli TG1 competent cells, and positive clones were selected for sequencing.

[0050] The sequencing results were compared with the sequences reported in GenBank using BLAST. The results showed that the sequence was 96.15% identical to the sequence of the reported ReAV-59 isolate (GenBank accession number: PP097219.1), indicating that the established RT-LAMP detection method can accurately detect ReAV.

[0051] Table 3 Reaction system of recovered products connected to pMD19-T vector

[0052]

[0053] Example 3 Optimization of RT-LAMP amplification reaction temperature

[0054] In order to study the RT-LAMP reaction conditions of the present invention, the reaction temperature was optimized in the same manner as in Example 1, as follows:

[0055] During the RT-LAMP amplification reaction, five temperature gradients were set at 50, 55, 60, 65 and 70°C, and the reaction time was set to 1 h. The RT-LAMP reaction conditions were optimized to determine the optimal reaction temperature; the reaction products were detected by agarose gel electrophoresis.

[0056] Results Figure 1 , among the five set reaction temperatures, there are target bands at 55℃~65℃, among which the band is the clearest at 65℃, so 65℃ is selected as the optimal reaction temperature.

[0057] Figure 1 In the figure, A: agarose gel electrophoresis of RT-LAMP product, B: visualization detection of RT-LAMP product; M: 2000 DNA Marker; 1-5 correspond to the temperatures of 50, 55, 60, 65 and 70°C, respectively.

[0058] Example 4 Optimization of RT-LAMP amplification reaction time

[0059] In order to study the RT-LAMP reaction conditions of the present invention, the reaction time was optimized in the same manner as in Example 1, as follows:

[0060] During the RT-LAMP amplification reaction, 6 time periods of 20, 30, 40, 50, 60 and 70 min were set, and the reaction temperature was set to 65°C; the RT-LAMP reaction time was optimized to determine the optimal reaction time.

[0061] The reaction products were detected by agarose gel electrophoresis. Figure 2 Among the six different reaction times, there were target bands at 40 min to 70 min, and the target band corresponding to 40 min was the clearest, so 40 min was selected as the best reaction time.

[0062] Figure 2 In the figure, A: agarose gel electrophoresis of RT-LAMP product, B: visualization detection of RT-LAMP product; M: 2000 DNA Marker; the times corresponding to 1-6 are 20, 30, 40, 50, 60, and 70 min, respectively.

[0063] Example 5 Specificity verification of RT-LAMP detection

[0064] The method of Example 1 was used to detect the related viruses infecting Rehmannia glutinosa, and the recombinant plasmids of Rehmannia glutinosa mosaic virus (ReAV), tobacco mild green mosaic virus (TMGMV), cucurbit chlorosis yellows virus (CCYV), Rehmannia glutinosa mosaic virus (ReMV), rapeseed mosaic virus (YoMV), broad bean wilt virus (BBWV-2), and goldfish flower latent viroid (CLVd) were used as templates for RT-LAMP specific detection.

[0065] Results Figure 3 The RT-LAMP amplification products were analyzed by agarose gel electrophoresis, and only the samples infected with ReAV showed waterfall-like bands, while the other samples had no bands; after adding SYBR Green I, only the samples infected with ReAV were green, while the other samples were orange; the above results showed that the RT-LAMP method established in the present invention has good specificity.

[0066] Figure 3 In the figure, M: 2000DNAMarker; the viruses numbered 1-7 correspond to: ReAV, TMGMV, CCYV, ReMV, BBWV-2, YoMV, CLVd.

[0067] Example 6 Sensitivity test of RT-LAMP detection

[0068] The initial concentration of ReAV plasmid stored in our laboratory was 134.4 ng / μL, which was converted to 6.99×10 10 copies / μL, and the plasmid was diluted 10-fold to 6.99×10 3 -6.99×10 -4 copies / μL, and used this as a template to perform RT-LAMP detection according to the method of Example 1;

[0069] At the same time, conventional PCR amplification was performed. The PCR amplification system was: 2×Taq Mix 10 μL, ReAV-CPF / ReAV-CPR at a concentration of 10 μmol / L, 0.5 μL each, plasmid DNA 1 μL, ddH 2 O to 20 μL; PCR reaction conditions: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 60 s, 35 cycles; 72℃ extension for another 10 min, 4℃ storage.

[0070] The results showed that conventional PCR could detect 6.99×10 1 copies / μL, RT-LAMP can detect 6.99×100 copies / μL, the sensitivity of RT-LAMP is 10 times that of conventional PCR.

[0071] Results Figure 4 , where A is the electrophoresis diagram of common RT-PCR detection, and B is the visualization detection diagram of RT-LAMP products; M in the figure: 2000DNAMarker; the plasmid concentrations corresponding to numbers 1-8 are: 6.99×10 3 , 6.99×10 2 , 6.99×10 1 , 6.99×10 0 , 6.99×10 -1 , 6.99×10 -2 , 6.99×10 -3 , 6.99×10 -4 copies / μL.

[0072] Example 7 Comparison of the accuracy of RT-LAMP detection and conventional PCR detection

[0073] 21 Rehmannia glutinosa disease samples collected in the field were extracted and amplified by RT-LAMP according to the method of Example 1. After the reaction was completed, SYBR green I was added for visual detection. Numbers 1-21 corresponded to 21 Rehmannia glutinosa samples, and number 22ddH 2 O is the negative control.

[0074] Conventional PCR detection was performed by referring to the method of Example 6, and a single band after the reaction was completed was considered a positive reaction.

[0075] Results Figure 5 , where A is the conventional RT-PCR detection electrophoresis diagram, sample numbers 1, 4, 6-8, 12-21 show target bands; B is the RT-LAMP visualization detection diagram; M: 2000 DNA Marker; number 1, 4, 6-21 shows green (positive reaction).

[0076] The results showed that 18 samples were positive in the RT-LAMP test results; 15 samples were positive in the conventional PCR test results, proving that the accuracy of the RT-LAMP visualization detection method established by the present invention is higher than that of conventional PCR.

Claims

1. The RT-LAMP primer set for detecting Allium chinense X virus is characterized by: The primer set includes a pair of outer primers, a pair of inner primers and a loop primer, as follows: (1) A pair of external primers: ReAV-F3:5′-AGGAGCTGGGCGACG-3′; ReAV-B3:5′-AGAACGGTACTCCCGCG-3′; (2) A pair of inner primers: ReAV-FIP:5′-GGTATTGCGCCTGCGCCATAGTCAAACTCCATCGCCACACA-3′; ReAV-BIP:5′-TCTCATCACGCTCGCCATGAGGCCCTCCAGCGTTACG-3′; (3) One loop primer: ReAV-LB: 5′-CTACCACAATGGGGCGTCC-3′.

2. A method for detecting Allium chinense X virus using the RT-LAMP primer set of claim 1, characterized in that: The following steps are involved: (1) Extract the total viral RNA and then reverse transcribe it into cDNA using RNA as a template; (2) using the cDNA as a template and using an RT-LAMP primer set to perform an RT-LAMP amplification reaction; (3) After the RT-LAMP amplification reaction is completed, centrifuge briefly to remove the nucleic acid dye in the lid. If the RT-LAMP reaction solution turns green, it is considered positive; if the reaction solution turns yellow, it is considered negative. Alternatively, the RT-LAMP amplification product was analyzed by agarose gel electrophoresis. If waterfall-like bands appeared, it was judged as positive, and if no bands were present, it was negative.

3. The method according to claim 2, characterized in that: The reaction system for RT-LAMP amplification is as follows: template cDNA 1 μL, 10×ThermoPol Buffer 2.5 μL, ReAV-FIP, ReAV-BIP primers 1.6 μmol / L each, usage 4 μL, ReAV-F3, ReAV-B3 primers 0.2 μmol / L each, usage 0.5 μL, ReAV-LB primer 0.4 μmol / L, usage 1 μL, Mg 2+ 6mM, usage 1.5μL, dNTPs 1.4mM, usage 3.5μL, Bst DNApolymerase 8U, usage 1μL, make up to 25μL with ddH2O; during the reaction, add the reaction system to the PCR tube, gently pipette and mix, then add 30μL paraffin oil to seal; at the same time, add 0.5μL SYBR Green I nucleic acid dye into the PCR tube cap.

4. The method according to claim 3, characterized in that: The reaction conditions of the RT-LAMP amplification are: 65° C. for 40 min, 85° C. for 5 min, and terminating the reaction on ice.

5. Use of the RT-LAMP primer set according to claim 1 in preparing a kit or reagent for detecting Allium radix X virus.

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