RT-LAMP (reverse transcription loop-mediated isothermal amplification) primer group for detecting rehmannia ignition virus and detection method

Abstract

The invention discloses an RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) detection primer group and a detection method of a rehmannia scorch virus, the RT-LAMP detection primer group comprises a pair of outer primers ReTV-F3 and ReTV-B3, a pair of inner primers ReTV-FIP and ReTV-BIP and a loop primer ReTV-LB, and the RT-LAMP detection primer group is judged according to 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. 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 ReTV.

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 burn virus. Background Art

[0002] Rehmannia glutinosa L. is a perennial herbaceous plant of the genus Rehmannia in the family Scrophulariaceae. It is also known as raw Rehmannia and cooked Rehmannia. It is one of the commonly used Chinese medicinal materials. Commonly used ones include Zhibai Dihuang Pills, Qiju Dihuang Pills, and Liuwei Dihuang Pills. Rehmannia has a long history of cultivation in my country and is mainly distributed in Henan, Shanxi, Shandong, Shaanxi, Hebei and other provinces. Rehmannia has the effects of nourishing yin and kidney, clearing heat and cooling blood, and promoting blood circulation and eliminating blood stasis. Rehmannia is mainly cultivated by asexual reproduction. Viral diseases are very serious, resulting in reduced yield and quality of Rehmannia. Rehmannia Torradovirus virus (ReTV) is a member of the genus Torradovirus. It is a single-stranded positive-sense RNA. The genome consists of RNA1 and RNA2. RNA1 contains a large open reading frame (ORF) encoding a polyprotein and contains a conserved region of a typical domain of a helicase, a protease, and an RNA-dependent RNA polymerase. RNA2 contains two open reading frames (ORF) encoding two proteins. Currently, the detection technology for ReTV mainly includes RT-PCR, which is the most commonly used detection method in laboratories, but 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 can identify 4 or 6 different DNA sequences on the target DNA. It relies on an internal primer containing the sense and antisense sequences of the target DNA to initiate the LAMP amplification reaction, and achieves rapid molecular detection of the target gene under constant temperature conditions. It has been widely used in the detection of various plant viruses. Therefore, applying LAMP technology to ReTV detection and establishing a detection technology that is more advantageous than conventional PCR methods to meet the needs of grassroots and field virus detection are technical issues that need to be solved. Summary of the invention

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

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

[0006] 1. The RT-LAMP primer set for detecting Rehmannia glutinosa burn virus is characterized by comprising a primer set consisting of a pair of outer primers, a pair of inner primers and a loop primer, as follows:

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

[0008] ReTV-F3:5′-TGTCTCATCCACCGGTAGC-3′;

[0009] ReTV-B3:5′-AGACGTAGTCCTGTACGCTG-3′;

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

[0011] ReTV-FIP: 5′-CTGTGCCTGCAGGGAGAAGCACCGAAAAAGCTTCTGGGAA-3′;

[0012] ReTV-BIP: 5′-GCTGGATGGCACTTCTTTGTGGCGCCTGTGAAGGGCTGAAC-3′;

[0013] (3) One loop primer:

[0014] ReTV-LB: 5′-CTTTTCTTCGCCCTTGTTGCTT-3′.

[0015] 2. The method for detecting Rehmannia glutinosa virus using RT-LAMP primer set comprises 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 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 there were no bands, it was negative.

[0020] The reaction system for RT-LAMP amplification was as follows: cDNA template 1 μL, 10× ThermoPol Buffer 2.5 μL, ReTV-FIP and ReTV-BIP primers 1.6 μmol / L each, 4 μL, ReTV-F3 and ReTV-B3 primers 0.2 μmol / L each, 0.5 μL, ReTV-LB primer 0.4 μmol / L, 1 μL, Mg 2+ 6mM, usage 1.5μL, dNTPs 1.4mM, usage 3.5μL, Bst DNApolymerase 8U, usage 1μL, ddH2O is added to 25μL; 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 GreenI nucleic acid dye into the PCR tube cap.

[0021] The reaction conditions of RT-LAMP amplification were as follows: 65°C for 50 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 Rehmannia glutinosa virus detection kit or a detection reagent.

[0023] Beneficial Effects of the Invention

[0024] (1) The present invention obtains an RT-LAMP detection primer set for Rehmannia glutinosa virus by screening RT-LAMP detection primers and optimizing reaction conditions, and establishes a highly sensitive ReTV detection method. The results show that this method can detect 3.39×10 0 The sensitivity of PCR is 100 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 ReTV, ReAV, TMGMV, ReMV, CCYV, and CLVd, in Rehmannia glutinosa, and only ReTV 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 50 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 ReTV 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 ReTV 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 ReTV 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 virus

[0036] The specific steps are as follows:

[0037] Step 1: According to the previous transcriptome sequencing results and the coat protein (CP) nucleotide sequence of ReTV of Rehmannia glutinosa virus registered in GenBank (GenBank accession number: OR453968.1), 3 sets of RT-LAMP detection primers for ReTV were designed online using the NEB LAMP Primer Design Tool website (https: / / lamp.neb.com / ). Each set of detection primers contained 5 primers corresponding to 6 regions of the target gene. After primer screening and removal of false positive related primers, the ReAVRT-LAMP detection primers were determined to be ReTV-F3, ReTV-B3, ReTV-FIP, ReTV-BIP and ReTV-LB; at the same time, the common RT-PCR detection primers ReTV-CPF and ReTV-CPR were designed for comparison experiments using RT-PCR amplification and 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 TM Ⅱ1st 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 reaction conditions: 65°C for 50 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 DNA double strand 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 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.

[0049] Example 2 Identification of RT-LAMP amplification products

[0050] 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.

[0051] The sequencing results were compared with the sequences reported in GenBank using BLAST. The results showed that the sequence was 100% identical to the sequence of the reported ReTV-8 isolate (GenBank accession number: OR453968.1), indicating that the established RT-LAMP detection method can accurately detect ReTV.

[0052] Table 3 Reaction system of recovered products connected to pMD19-T vector

[0053]

[0054] Example 3 Optimization of RT-LAMP amplification reaction temperature

[0055] 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:

[0056] During the RT-LAMP reaction, five temperature gradients were set at 50, 55, 60, 65 and 70°C, and the reaction time was set to 1h. The conditions of the RT-LAMP reaction system were optimized to determine the optimal reaction temperature. The reaction products were detected by agarose gel electrophoresis.

[0057] Results Figure 1 , under the five different reaction temperatures set, there are target bands at 60℃~65℃, among which the band is the clearest at 65℃, so the optimal reaction temperature is 65℃.

[0058] 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.

[0059] Example 4 Optimization of RT-LAMP amplification reaction time

[0060] 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:

[0061] 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.

[0062] The reaction products were detected by agarose gel electrophoresis. Figure 2 , in 6 different reaction times, Figure 2 In A, the target bands were present at 30 min to 70 min, and the target band corresponding to 50 min was the clearest, so 50 min was selected as the optimal reaction time.

[0063] Figure 2 In the figure, A: agarose gel electrophoresis diagram of RT-LAMP product, B: visualization detection diagram of RT-LAMP product; M: 2000 DNA Marker; the times corresponding to 1-6 are 20, 30, 40, 50, 60, and 70 min, respectively.

[0064] Example 5 Specificity verification of RT-LAMP detection

[0065] The method of Example 1 was used to detect the related viruses infecting Rehmannia glutinosa, and the recombinant plasmids of Rehmannia glutinosa burn virus (ReTV), Rehmannia glutinosa onion X virus (ReAV), tobacco mild green mosaic virus (TMGMV), Rehmannia glutinosa mosaic virus (ReMV), cucurbit chlorosis yellows virus (CCYV), and goldfish flower latent viroid (CLVd) were used as templates for RT-LAMP specific detection.

[0066] Results Figure 3 The RT-LAMP amplification products were analyzed by agarose gel electrophoresis, and only the samples infected with ReTV showed waterfall-like bands, while the other samples had no bands; after adding SYBR Green I, only the samples infected with ReTV 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.

[0067] Figure 3 In the figure, M: 2000DNAMarker; the viruses numbered 1-6 correspond to: ReTV, ReAV, TMGMV, ReMV, CCYV, CLVd.

[0068] Example 6 Sensitivity test of RT-LAMP detection

[0069] The initial concentration of ReTV plasmid stored in our laboratory was 339.8 ng / μL, which was converted to 3.39×10 11 copies / μL, and the plasmid was diluted 10-fold to 3.39×10 3 -3.39×10-4 copies / μL, and used this as a template to perform RT-LAMP detection according to the method of Example 1;

[0070] Conventional PCR amplification was carried out at the same time. The PCR amplification system was as follows: 10 μL of 2×Taq Mix, 0.5 μL of ReTV-CPF / ReTV-CPR at a concentration of 10 μmol / L, 1 μL of plasmid DNA, and ddH2O was added to 20 μL. PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 60 s, 35 cycles; extension at 72°C for another 10 min, and storage at 4°C.

[0071] The results showed that conventional PCR could detect 3.39×10 2 copies / μL, RT-LAMP can detect 3.39×10 0 copies / μL, it can be seen that the sensitivity of RT-LAMP is 100 times that of conventional PCR.

[0072] 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: 3.39×10 3 ,3.39×10 2 , 3.39×10 1 ,3.39×10 0 ,3.39×10 -1 ,3.39×10 -2 ,3.39×10 -3 ,3.39×10 -4 copies / μL.

[0073] Example 7 Comparison of the accuracy of RT-LAMP detection and conventional PCR detection results

[0074] 16 Rehmannia root disease samples collected in the field were extracted and amplified by RT-LAMP according to the method of Example 1. After the reaction, SYBR green I was added for visual detection, and numbers 1-16 corresponded to 16 Rehmannia root samples, and number 17 ddH2O was a negative control.

[0075] Conventional PCR detection was performed according to the method of Example 6, and the appearance of a single band after the reaction was completed was considered a positive reaction.

[0076] Results Figure 5A is the conventional RT-PCR detection electrophoresis diagram, and the target bands appear in sample numbers 1-2, 4-9, 11-13, and 15-16; B is the RT-LAMP visualization detection diagram; M: 2000 DNA Marker; No. 1-16 appears green (positive reaction).

[0077] The results showed that 16 samples were positive in the RT-LAMP test results of the present invention; 13 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 Rehmannia glutinosa 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: ReTV-F3:5′-TGTCTCATCCACCGGTAGC-3′; ReTV-B3:5′-AGACGTAGTCCTGTACGCTG-3′; (2) A pair of inner primers: ReTV-FIP: 5′-CTGTGCCTGCAGGGAGAAGCACCGAAAAAGCTTCTGGGAA-3′; ReTV-BIP: 5′-GCTGGATGGCACTTCTTTGTGGCGCCTGTGAAGGGCTGAAC-3′; (3) One loop primer: ReTV-LB: 5′-CTTTTCTTCGCCCTTGTTGCTT-3′.

2. A method for detecting Rehmannia glutinosa virus using the RT-LAMP primer set according to 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 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 there were no bands, it was negative.

3. The method according to claim 2, characterized in that: The reaction system for RT-LAMP amplification is as follows: 1 μL cDNA template, 2.5 μL 10× ThermoPol Buffer, 1.6 μmol / L ReTV-FIP and ReTV-BIP primers, 4 μL each, 0.2 μmol / L ReTV-F3 and ReTV-B3 primers, 0.5 μL each, 0.4 μmol / L ReTV-LB primer, 1 μL Mg 2+ 6mM, usage 1.5μL, dNTPs 1.4mM, usage 3.5μL, Bst DNApolymerase 8U, usage 1μL, ddH2O is added to 25μL; during the reaction, add the reaction system to the PCR tube, gently pipette and mix, then add 30μL of 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 50 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 detection kit or a detection reagent for Rehmannia glutinosa virus.

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