Multiplex RT-PCR primer set, kit and application for simultaneous detection of four CTV genotypes

Abstract

The present invention provides a multiplex RT-PCR primer set, a kit and an application for simultaneously detecting four CTV genotypes, belonging to the technical field of plant virus detection, wherein the four citrus tristeza virus genotypes are T30, T68, JY and HA; the multiplex RT-PCR primer set is composed of a primer for detecting T30, a primer for detecting T68, a primer for detecting JY and a primer for detecting HA. The multiple pairs of specific primers and the kit designed by the present invention have the characteristics of strong specificity and high sensitivity. The kit of the present invention makes the CTV genotype detection process simpler and more efficient, greatly saves the genotype detection cycle, and plays a positive role in improving the detection and prevention and control of CTV genotypes.

Description

Technical Field

[0001] The invention belongs to the technical field of plant virus detection, and in particular relates to a multiplex RT-PCR primer set, a kit and an application thereof for simultaneously detecting four CTV genotypes. Background Art

[0002] Citrus tristeza virus (CTV) is an economic disease that is extremely harmful to citrus and occurs widely around the world. CTV belongs to the genus Closterovirus. The virus particles are 11×2000nm long linear. The genome consists of positive single-stranded RNA (+ssRNA) composed of about 19,300 nucleotides, which is the largest virus in the known plant virus genome. Citrus tristeza virus can be divided into three symptom types according to the phenotypes of indicator plants: stem pitting type, rapid decay type and seedling yellowing type. CTV has complex strain differentiation phenomenon. At present, 11 CTV genotypes have been detected: VT, T30, T36, T3, A18, T68 (B165), RB, HA16-5, S1, L1, M1; in addition, a new CTV genotype has been detected in wild citrus species: JY. Furthermore, the same citrus plant is often infected with a variety of CTV genotypes, making it difficult to clearly define the relationship between genotype and phenotype.

[0003] At present, there are three main commonly used CTV genotype detection systems. The first is to use the restriction endonuclease HinfⅠ to digest the amplified product of the coat protein gene of CTV at 37℃, and divide it into 7 groups according to the patterns of different fragment lengths after digestion. Combined with the different biological symptoms of CTV on indicator plants, the 7 groups are divided into potential weak strains and strong strains; but this method cannot correspond the 7 RFLP groups to the CTV genotypes, and gradually more and more unclassifiable groups are found, resulting in less and less application of this method. The second is to design 10 pairs of primers for detecting VT, T30, T3 and T36 genotypes by comparing the sequence differences of the 5′URT, K17 and POL regions of the CTV 5′ end genome; this method is more cumbersome, and there are also CTV isolates that cannot be classified. The third specific detection primers designed by Roy et al. for detecting B165, VT, T30, T3 and T36 genotypes are currently the more commonly used CTV genotype detection system. In addition, the discoverers of the RB, HA16-5, S1, L1, and M1 genotypes all designed specific primers to detect their respective new genotypes. Due to the large number of CTV genotypes, the numerous detection primers, and the diverse genotype detection systems, the CTV genotype detection procedure is complicated and time-consuming, and the detection results between different systems are often inconsistent, which brings troubles to CTV genotype detection and CTV pathogenic mechanism research. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a multiplex RT-PCR primer set, a kit and an application for simultaneously detecting four CTV genotypes. The primer set and the kit of the present invention can quickly and simultaneously detect four citrus tristeza virus genotypes, T30, T68, JY and HA. Not only can it be quickly and accurately determined whether the sample to be tested is infected with citrus tristeza virus, but it can also determine whether the infected citrus tristeza virus genotype is one or more of T30, T68, JY and HA, which plays a positive role in improving the detection and prevention and control of CTV genotypes.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a multiplex RT-PCR primer set for simultaneously detecting four genotypes of citrus tristeza virus, wherein the four genotypes of citrus tristeza virus are T30, T68, JY, and HA; the multiplex RT-PCR primer set is composed of a primer for detecting T30, a primer for detecting T68, a primer for detecting JY, and a primer for detecting HA;

[0007] The nucleotide sequence of the T30 upstream primer is shown in SEQ ID No. 1, and the nucleotide sequence of the T30 downstream primer is shown in SEQ ID No. 2;

[0008] The nucleotide sequence of the T68 upstream primer is shown in SEQ ID No. 3, and the nucleotide sequence of the T68 downstream primer is shown in SEQ ID No. 4;

[0009] The nucleotide sequence of the JY upstream primer is shown in SEQ ID No. 5, and the nucleotide sequence of the JY downstream primer is shown in SEQ ID No. 6;

[0010] The nucleotide sequence of the HA upstream primer is shown in SEQ ID No.7, and the nucleotide sequence of the HA downstream primer is shown in SEQ ID No.8.

[0011] The present invention also provides the use of the multiplex RT-PCR primer set in preparing a reagent for simultaneously detecting T30, T68, JY and HA citrus tristeza virus genotypes.

[0012] The present invention also provides a kit for simultaneously detecting T30, T68, JY and HA citrus tristeza virus genotypes, comprising the above-mentioned multiple RT-PCR primer set.

[0013] Preferably, the kit comprises a PCR reaction system, an RT reaction system, a positive quality control product and a negative quality control product.

[0014] Preferably, the RT reaction system includes 5 μL of denaturing solution, 4 μL of 5×RT-buffer, 0.5 μL of 30 U / L RNase inhibitor RNasin, 1 μL of 100 U / μL reverse transcriptase RTase, 4.5 μL of RNase-free ultrapure water, and 1 μL of 10 μmol / L reverse transcription primer.

[0015] Preferably, the melting solution consists of 1.0 μL of 10 μmol / L random primers, 1.0 μL of 2.5 mM / L dNTPs, and 3.0 μL of RNA template.

[0016] Preferably, the PCR reaction system includes 13.1 μL of RNase-free ultrapure water, 4.5 μL of 10×PCR-buffer, 2 μL of 2.5 mM / L dNTPs, 0.5 μL of 10 μmol / L upstream and downstream primers of the above-mentioned multiple RT-PCR primer set, 0.4 μL of r-Taq enzyme, and 1 μL of reverse transcription product cDNA template.

[0017] The present invention also provides a method for simultaneously detecting four citrus tristeza virus genotypes using the above-mentioned kit, comprising the following steps: using the multiple RT-PCR primer set to perform PCR amplification to obtain PCR amplification products, performing fragment analysis on the PCR amplification products to determine whether the sample to be tested is infected by one or more CTV genotypes of T30, T68, JY, and HA.

[0018] Preferably, the PCR amplification program is: 95°C pre-denaturation for 3 minutes, 95°C denaturation for 30 seconds, 54°C annealing for 30 seconds, 72°C extension for 50 seconds, and 35 cycles.

[0019] Preferably, the fragment analysis is to perform agarose gel electrophoresis analysis on the PCR amplification products, and determine whether the sample to be tested is infected by one or more CTV genotypes among T30, T68, JY and HA based on the electrophoresis results.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides a multiplex RT-PCR primer set, a kit and an application for simultaneously detecting four CTV genotypes. The present invention designs multiple pairs of specific primers according to the differences in the full gene sequences of the currently common CTV genotypes. The multiplex RT-PCR primer set or the kit of the present invention has the characteristics of strong specificity and high sensitivity. When the T30, T68, JY and HA CTV genotypes exist at the same time, the multiplex RT-PCR primer set can specifically detect the four CTV genotypes of T30, T68, JY and HA, and the size of the DNA amplified fragment is appropriate, and the multiplex PCR results are easy to identify. When the cDNA template is diluted to 10 -2The four genotypes T30, T68, JY, and HA could still be detected when the template was diluted to 10 -3 The kit of the present invention makes the CTV genotype detection process simpler and more efficient, greatly saves the genotype detection cycle, and plays a positive role in improving the detection and prevention of CTV genotypes. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Specific detection results of multiple RT-PCR primer sets for CTV genotypes, where +: positive samples of corresponding genotypes; -: healthy plants as negative controls; 1: mixed infection of T30 and T68; 2: mixed infection of T30 and JY; 3: mixed infection of T30 and HA; 4: mixed infection of T68 and JY; 5: mixed infection of T68 and HA; 6: mixed infection of JY and HA; 7: mixed infection of T30, T68 and JY; 8: mixed infection of T30, T68 and HA; 9: mixed infection of T68, JY and HA; 10: mixed infection of T30, JY and HA; 11: mixed infection of T30, T68, JY and HA;

[0023] Figure 2 This is the sensitivity test result of the multiplex RT-PCR primer set for CTV genotype. DETAILED DESCRIPTION

[0024] The present invention provides a multiplex RT-PCR primer set for simultaneously detecting four genotypes of citrus tristeza virus, wherein the four genotypes of citrus tristeza virus are T30, T68, JY, and HA; the multiplex RT-PCR primer set is composed of a primer for detecting T30, a primer for detecting T68, a primer for detecting JY, and a primer for detecting HA;

[0025] The nucleotide sequence of the T30 upstream primer is shown in SEQ ID No. 1, and the nucleotide sequence of the T30 downstream primer is shown in SEQ ID No. 2;

[0026] The nucleotide sequence of the T68 upstream primer is shown in SEQ ID No. 3, and the nucleotide sequence of the T68 downstream primer is shown in SEQ ID No. 4;

[0027] The nucleotide sequence of the JY upstream primer is shown in SEQ ID No. 5, and the nucleotide sequence of the JY downstream primer is shown in SEQ ID No. 6;

[0028] The nucleotide sequence of the HA upstream primer is shown in SEQ ID No.7, and the nucleotide sequence of the HA downstream primer is shown in SEQ ID No.8.

[0029] The present invention designs multiple pairs of specific primers based on the differences in the full gene sequences of the currently common CTV genotypes. The above-mentioned multiple RT-PCR primer set can quickly and accurately distinguish the four CTV genotypes of T30, T68, JY and HA at the same time, greatly improving the rate and efficiency of CTV genotype detection.

[0030] The present invention also provides the use of the multiplex RT-PCR primer set in preparing a reagent for simultaneously detecting T30, T68, JY and HA citrus tristeza virus genotypes.

[0031] The present invention also provides a kit for simultaneously detecting T30, T68, JY and HA citrus tristeza virus genotypes, comprising the above-mentioned multiple RT-PCR primer set.

[0032] In the present invention, the kit preferably includes a PCR reaction system, an RT reaction system, a positive quality control product and a negative quality control product. The RT reaction system preferably includes 5 μL of a denaturing solution, 4 μL of 5×RT-buffer, 0.5 μL of 30 U / L of an RNase inhibitor RNasin, 1 μL of 100 U / μL of a reverse transcriptase RTase, 4.5 μL of RNase-free ultrapure water, and 1 μL of a 10 μmol / L reverse transcription primer. The denaturing solution preferably consists of 1.0 μL of 10 μmol / L random primers (6N), 1.0 μL of 2.5 mM / L dNTPs, and 3.0 μL of an RNA template. The PCR reaction system preferably includes 13.1 μL of RNase-free ultrapure water, 4.5 μL of 10×PCR-buffer, 2 μL of 2.5 mM / L dNTPs, 0.5 μL of the upstream and downstream primers of the above-mentioned multiple RT-PCR primer set of 10 μmol / L, 0.4 μL of r-Taq enzyme, and 1 μL of cDNA template of the reverse transcription product. In the present invention, the positive quality control product is a citrus tristeza virus sample containing the T30 genotype, a citrus tristeza virus sample containing the T68 genotype, a citrus tristeza virus sample containing the JY genotype, and a citrus tristeza virus sample containing the HA genotype. The negative quality control product is a sample of a healthy plant. The present invention does not specifically limit the sources of RT-buffer, RNase inhibitor RNasin, reverse transcriptase RTase, RNase-free ultrapure water, PCR-buffer, dNTPs, and r-Taq enzyme, and products known in the art can be used.

[0033] The present invention also provides a method for simultaneously detecting four citrus tristeza virus genotypes using the above-mentioned kit, comprising the following steps: using the multiple RT-PCR primer set to perform PCR amplification to obtain PCR amplification products, performing fragment analysis on the PCR amplification products to determine whether the sample to be tested is infected by one or more CTV genotypes of T30, T68, JY and HA.

[0034] In the present invention, the method for simultaneously detecting four genotypes of citrus tristeza virus using the above kit preferably comprises the following steps:

[0035] 1) Extracting RNA from the sample to be tested;

[0036] 2) Reversely transcribing the RNA in step 1) to synthesize cDNA;

[0037] 3) using the cDNA in step 2) as a template, performing PCR amplification using the multiplex RT-PCR primer set to obtain a PCR amplification product;

[0038] 4) Performing fragment analysis on the PCR amplification product in step 3) to determine whether the sample to be tested is infected by one or more CTV genotypes among T30, T68, JY and HA.

[0039] In the present invention, the transcription reaction procedure is preferably: 30°C reaction for 10 minutes, 42°C reverse transcription for 50 minutes, and 70°C heating for 15 minutes. The PCR amplification procedure is preferably: 95°C pre-denaturation for 3 minutes, 95°C denaturation for 30 seconds, 54°C annealing for 30 seconds, 72°C extension for 50 seconds, and 35 cycles. The fragment analysis is preferably performed on the PCR amplification product by agarose gel electrophoresis analysis, and whether the sample to be tested is infected by one or more CTV genotypes among T30, T68, JY and HA is determined according to the electrophoresis results.

[0040] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0041] Example 1

[0042] A multiplex RT-PCR primer set for simultaneously detecting four citrus tristeza virus genotypes, wherein the four citrus tristeza virus genotypes are T30, T68, JY, and HA; the multiplex RT-PCR primer set consists of a primer for detecting T30, a primer for detecting T68, a primer for detecting JY, and a primer for detecting HA, and the specific primer information is shown in Table 1.

[0043] Table 1 Multiplex RT-PCR primer sets for four genotypes of Citrus tristeza virus

[0044]

[0045] Example 2

[0046] A method for simultaneously detecting the above four citrus tristeza virus genotypes based on RT-PCR technology, the steps are as follows:

[0047] (1) Extract RNA from the sample to be tested:

[0048] S1. Take 0.1 g of citrus leaves, grind them thoroughly with liquid nitrogen, and add them to a 1.5 mL centrifuge tube pre-filled with 1 mL of Trizol solution (Invitrogen, USA). After vigorous shaking to mix, place them at room temperature for 10 minutes. Centrifuge the centrifuge tube at 12900 rpm and 4°C for 10 minutes.

[0049] S2. Take 1 mL of the supernatant and place it in a new 1.5 mL centrifuge tube. Add 200 μL of chloroform:isoamyl alcohol (24:1), shake and mix well. Place at 4°C for 5 minutes and then centrifuge at 12,900 rpm at 4°C for 5 minutes.

[0050] S3. Take the supernatant after centrifugation in step S2 (only take the top layer) and place it in a new 1.5 mL centrifuge tube. Add an equal volume of isopropanol, gently invert to mix, and place at -20°C to settle for more than 2 hours.

[0051] S4. Centrifuge the centrifuge tube in step S3 at 12000 rpm and 4°C for 10 minutes, discard the supernatant, add 1 mL of 75% ethanol and mix by inversion, centrifuge at 8000 rpm and 4°C for 3 minutes, discard the supernatant; centrifuge again for 15 seconds, discard the supernatant with a pipette tip, and repeat twice.

[0052] S5. Place the centrifuge tube containing RNA precipitate in step S4 in a fume hood and dry at room temperature for 2 to 3 minutes, then add 30 μL DEPC water to dissolve the RNA.

[0053] (2) Using the RNA extracted in step (1) as a template, a reverse transcription reaction is performed using random primers to obtain a reverse transcription product cDNA.

[0054] Before the reverse transcription reaction, a melting reaction is required: 5 μL of melting solution is composed of 3 μL RNA reaction template, 1 μL dNTPs with a concentration of 2.5 mM / L, and 1 μL random primer (6N) with a concentration of 10 μmol / L. After melting at 65°C for 5 minutes, the reverse transcription system mixture is added for reverse transcription reaction. The reaction system is 15 μL, including 5 μL of melting solution, 4 μL 5×RT-buffer, 0.5 μL of RNase inhibitor RNasin with a concentration of 30 U / L, 1 μL of reverse transcriptase RTase with a concentration of 100 U / μL, and 4.5 μL of RNase-free ultrapure water; the reaction procedure is: 30°C reaction for 10 minutes, 42°C reverse transcription for 50 minutes, and 70°C heating for 15 minutes.

[0055] (3) Using the reverse transcription product cDNA obtained in step (2) as a template, RT-PCR reaction was performed using the above-mentioned primers for each genotype, and the reaction system was 25 μL, wherein the reaction system included 13.1 μL of RNase-free ultrapure water, 4.5 μL of 10×PCR-buffer, 2 μL of dNTPs with a concentration of 2.5 mM / L, 0.5 μL of each of the upstream and downstream primers of each genotype described in Example 1 with a concentration of 10 μmol / L in the reaction system, 0.4 μL of r-Taq enzyme, and 1 μL of the reverse transcription product cDNA template; the PCR amplification program was: pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 30 seconds, annealing at 54°C for 30 seconds, extension at 72°C for 50 seconds, and 35 cycles.

[0056] Example 3

[0057] In this example, one or more of the multiplex RT-PCR primer sets in Example 1 were mixed to perform specificity evaluation according to the method for detecting genotypes of citrus tristeza virus in Example 2. The results are shown in FIG. Figure 1 .

[0058] The results are as follows Figure 1 As shown, when only one of the genotypes T30, T68, JY, and HA is present, the multiplex RT-PCR primer set can specifically amplify the fragment corresponding to the genotype; when two genotypes are combined arbitrarily, the multiplex RT-PCR primer set can specifically amplify the fragments corresponding to the two genotypes; when three genotypes are combined arbitrarily, the multiplex RT-PCR primer set can specifically amplify the fragments corresponding to the three genotypes; when four genotypes exist at the same time, the multiplex RT-PCR primer set can specifically amplify the fragments corresponding to the four genotypes. Therefore, the above multiplex RT-PCR primer set can specifically detect the four CTV genotypes T30, T68, JY, and HA.

[0059] Example 4

[0060] The sensitivity evaluation of the multiplex RT-PCR primer set of Example 1 was performed according to the following steps:

[0061] The reverse transcribed cDNA template obtained in Example 1 was diluted 10-fold in a total of 6 gradients (10 0 ~10 -6 ) was used as a cDNA template, and PCR amplification was performed according to the detection method of Example 2, and the primer sensitivity was evaluated.

[0062] Depend on Figure 2 It can be seen that when the cDNA template is diluted to 10 -2 The four genotypes T30, T68, JY, and HA could still be detected when the template was diluted to 10 -3Three genotypes, T68, JY and HA, can be detected.

[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A multiplex RT-PCR primer set for simultaneous detection of four genotypes of citrus tristeza virus, characterized in that: The four genotypes of citrus tristeza virus are T30, T68, JY, and HA; the multiplex RT-PCR primer set is composed of a primer for detecting T30, a primer for detecting T68, a primer for detecting JY, and a primer for detecting HA; The nucleotide sequence of the T30 upstream primer is shown in SEQ ID No. 1, and the nucleotide sequence of the T30 downstream primer is shown in SEQ ID No. 2; The nucleotide sequence of the T68 upstream primer is shown in SEQ ID No. 3, and the nucleotide sequence of the T68 downstream primer is shown in SEQ ID No. 4; The nucleotide sequence of the JY upstream primer is shown in SEQ ID No. 5, and the nucleotide sequence of the JY downstream primer is shown in SEQ ID No. 6; The nucleotide sequence of the HA upstream primer is shown in SEQ ID No.7, and the nucleotide sequence of the HA downstream primer is shown in SEQ ID No.

8.

2. Use of the multiplex RT-PCR primer set according to claim 1 in the preparation of a reagent for simultaneously detecting T30, T68, JY, and HA citrus tristeza virus genotypes.

3. A kit for simultaneously detecting T30, T68, JY, and HA citrus tristeza virus genotypes, characterized in that: The method comprises the multiplex RT-PCR primer set according to claim 1.

4. The kit according to claim 3, characterized in that The kit comprises a PCR reaction system, an RT reaction system, a positive quality control product and a negative quality control product.

5. The kit according to claim 4, characterized in that The RT reaction system includes 5 μL of denaturing solution, 4 μL of 5×RT-buffer, 0.5 μL of 30 U / L RNase inhibitor RNasin, 1 μL of 100 U / μL reverse transcriptase RTase, 4.5 μL of RNase-free ultrapure water, and 1 μL of 10 μmol / L reverse transcription primer.

6. The kit according to claim 4, characterized in that The PCR reaction system includes 13.1 μL of RNase-free ultrapure water, 4.5 μL of 10×PCR-buffer, 2 μL of 2.5 mM / L dNTPs, 0.5 μL of 10 μmol / L upstream and downstream primers of the multiple RT-PCR primer set of claim 1, 0.4 μL of r-Taq enzyme, and 1 μL of reverse transcription product cDNA template.

7. The kit according to claim 5, characterized in that The denaturing solution consists of 1.0 µL of 10 µmol / L random primers, 1.0 µL of 2.5 mM / L dNTPs, and 3.0 µL of RNA template.

8. A method for simultaneously detecting four genotypes of citrus tristeza virus using the kit according to any one of claims 3 to 7, characterized in that: The following steps are involved: The multiplex RT-PCR primer set is used to perform PCR amplification to obtain PCR amplification products, and the PCR amplification products are subjected to fragment analysis to determine whether the sample to be tested is infected by one or more CTV genotypes among T30, T68, JY and HA.

9. The method according to claim 8, characterized in that The PCR amplification program was: 95°C pre-denaturation for 3 minutes, 95°C denaturation for 30 seconds, 54°C annealing for 30 seconds, 72°C extension for 50 seconds, and 35 cycles.

10. The method according to claim 8, characterized in that The fragment analysis is to perform agarose gel electrophoresis analysis on the PCR amplification products, and determine whether the sample to be tested is infected by one or more CTV genotypes among T30, T68, JY and HA according to the electrophoresis results.

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