CN112779356B - Method for detecting GRSPaV by using double nested RT-PCR - Google Patents

Abstract

The invention discloses a method for detecting GRSPaV by using double nested RT-PCR, belonging to the technical field of molecular biology. The invention discloses a method for detecting GRSPaV by using double nested RT-PCR, which comprises two rounds of PCR amplification; the method can simultaneously carry out nested amplification on two genes of a virus, and the detection rate of the method is the same as the sum of the detection rates of two fragment single nested PCR; the method can effectively reduce the detection cost, improve the detection speed, has simple operation and good stability, and provides powerful support for the rapid inspection and detoxification research of GRSPaV.

Description

Method for detecting GRSPaV by using double nested RT-PCR

Technical Field

The invention relates to the technical field of molecular biology, in particular to a method for detecting GRSPaV by using double nested RT-PCR.

Background

The sand grape stem pox accompanying virus (grapevine rupestris stem pitting-associated virus, GRSPaV) occurs in the grape producing area worldwide, is the most widely distributed grape virus, and is related to various diseases such as grape vein necrosis, sand grape stem pox, sira grape decay and the like. GRSPaV is phloem-restricted virus and is mainly transmitted through grafting; the virus belongs to the genus of the sunk virus, and the genome has higher variability.

Therefore, it is a need for a solution to the problem of the art to provide a method for detecting GRSPaV using dual nested RT-PCR.

Disclosure of Invention

In view of the above, the invention provides a method for detecting GRSPaV by using double nested RT-PCR, which has simple operation and good stability.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

a method for detecting GRSPaV by using double nested RT-PCR comprises the following specific steps:

(1) Extracting total RNA of a sample;

(2) cDNA synthesis;

(3) Round 1 PCR system: cDNA2 mu L, 10 XPCR buffer2.5 mu L, 2.5mmol/L each dNTPs0.7 mu L, 10 mu mol/L primer combination P1/P2-P3/P4 0.4-0.4/0.4 mu L, 5U/mu L Taq DNA polymerase 0.05 mu L, and constant volume to 25 mu L;

primer combination P1/P2-P3/P4 amplification procedure: pre-denaturation at 94℃for 3min;94 ℃ for 30s,55 ℃ for 45s and 72 ℃ for 50s, and 30 cycles are total; 72 ℃ for 10min;

(4) Round 2 PCR system: taking the PCR amplification product of round 1 as a template, the dosage is 1 mu L, the rest components are 10 XPCR Buffer2.5 mu L, 2.5mmol/L each dNTPs1 mu L, the dosage of the primer combination P5/P6-P7/P8 of 10 mu mol/L is 0.3/0.3-0.3/0.3 mu L, 5U/mu L Taq DNA polymerase of 0.25 mu L, and the volume is fixed to 25 mu L;

primer combination P5/P6-P7/P8 amplification procedure: pre-denaturation at 94℃for 3min;94 ℃ for 30s,55 ℃ for 45s and 72 ℃ for 50s, and 35 cycles are total; 72 ℃ for 10min;

(5) And (3) after the amplification of the 2 nd round, taking the PCR product to separate in agarose gel, and observing the amplification result in a BIO-RAD gel imaging system.

Further, the primer sequences of the primer combinations P1/P2-P3/P4 in the step (3) are as follows:

P1：5’-CTCCAGAGGTGCTGGTTGGTTCTC-3’；SEQ ID NO.1；

P2：5’-CGGCAAAAGAACGATATGACCAACT-3’；SEQ ID NO.2；

P3：5’-AGGGCTACAGGGGAGTCAAT-3’；SEQ ID NO.5；

P4：5’-TACGGTATTCCAGCGAACAGGCTTA-3’；SEQ ID NO.6；

the primer sequence of the primer combination P5/P6-P7/P8 in the step (4) is as follows:

P5：5’-CCAGATGGCAATTGGAATGAGATG-3’；SEQ ID NO.3；

P6：5’-CATAAGCAGAGAGCCACTCCT-3’；SEQ ID NO.4；

P7：5’-TGAAGGCTTTAGGGATTAGCC-3’；SEQ ID NO.7；

P8：5’-AACCTAGCCTTGAAATCAGG-3’；SEQ ID NO.8。

compared with the prior art, the invention discloses a method for detecting GRSPaV by using double nested RT-PCR, which can simultaneously carry out nested amplification on two genes of a virus, and the detection rate is the same as the sum of the detection rates of two fragment single nested PCR; the method can effectively reduce the detection cost (2 PCR reactions and corresponding agarose gel electrophoresis are reduced, and the reagent consumption is reduced), improve the detection speed, has simple operation and good stability, and provides powerful support for rapid inspection and detoxification research of GRSPaV.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 is a diagram showing the results of dual nested RT-PCR detection according to the present invention;

wherein M is DNA marker II; ck-: negative control (template water); ck+: positive control (GRSPaV infected jingxiu grape); 1-84: detecting a sample;

note that: the amplified band of sample No. 18 is far from the two target bands, and is judged as negative.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Taq DNA polymerase and dNTPs, available from Takara Bio-engineering (Dalian) Inc.; moloney murine leukemia Virus (maloney-murine leukemia virus, M-MLV), 6 base random primers, purchased from Bio-engineering (Shanghai) Inc.; purchased from pluroge (beijing) biotechnology limited; DNA marker II, tiangen Biochemical technology (Beijing); other reagents are all of domestic analytical purity. Primers were synthesized by the division of biological engineering (Shanghai).

The GRSPaV infected grape samples were collected in 2019 from the national center for detoxification of deciduous fruit trees at the national institute of agricultural sciences. The samples to be tested were 84 parts total of post-detoxification regenerated grape plants from laboratory stored heat treatment (13 parts) and chemical treatment (71 parts), respectively.

EXAMPLE 1 Total RNA extraction and cDNA Synthesis

(1) Total RNA extraction

For 84 samples to be tested, respectively taking 0.05-0.1g of regenerated grape plant leaves after detoxification into a plastic bag, adding 1mL of lysate for grinding, transferring homogenate into a 1.5mL centrifuge tube which is added with 150 mu L of 10% N-lauroylsarcosine (NLS) in advance, replacing each gun head, wiping the gun heads, and if more samples are added, putting on ice, and cooling to room temperature. The mixture was incubated at 72℃for 10min, mixed several times, and centrifuged at 13000rpm for 10min on ice for 5 min. 600. Mu.L of the supernatant was taken, 300. Mu.L of absolute ethanol was added, the mixture was stirred for 45s by reversing, 750. Mu.L of the mixture was added to the column, each time with a cap, and the mixture was centrifuged at 12000rpm for 1min, and the waste liquid was discarded. 700. Mu.L deproteinized solution was added thereto, left at room temperature for 2min, centrifuged at 12000rpm for 1min, and the waste liquid was discarded. 700. Mu.L of the rinse solution was added, centrifuged at 12000rpm for 1min, and the waste solution was discarded. Adding 500 mu L of rinsing liquid, centrifuging at 12000rpm for 1min, and discarding the waste liquid; the column was returned to the empty collection tube, centrifuged at 12000rpm for 2min, and the rinse solution was removed. Taking out the adsorption column, placing into a centrifuge tube without RNase pollution, adding 40 μl RNase free water at the middle position of the adsorption membrane according to the expected RNA yield, standing at room temperature for 1min, centrifuging at 12000rpm for 1min, and immediately using the RNA solution obtained by centrifuging for reverse transcription or preserving in a low-temperature refrigerator of-70deg.C (or-20deg.C).

(2) cDNA Synthesis

The total RNA 3. Mu.L and 6. Mu. L, DEPC treated deionized water 6. Mu.L were mixed in a centrifuge tube, denatured in boiling water for 6min, and then placed on ice for 3min. Sequentially adding to the solution: 5 xRT buffer2. Mu. L, dNTPs (10 mmol/L) 1. Mu. L, M-MLV (200U/. Mu.l) 0.5. Mu.L, the total system was made up to 20. Mu.L with water. Mixing the above solutions, inactivating at 37deg.C for 5min, at 37deg.C for 50min, and at 70deg.C for 3min on ice, and standing at-20deg.C.

Example 2 double nested RT-PCR detection of GRSPaV

(1) Primer design

Referring to the GRSPaV whole genome sequence (105) registered on NCBI, a set of nested amplification primers were designed at the viral replicase gene and the coat protein gene, respectively: repF1R 1- & gt repF2R2 and cpF1R 1- & gt cpF R2 (Table 1), the final amplified fragment sizes were 438bp and 902bp, respectively.

TABLE 1 nested PCR amplification primers for GRSPaV

(2) Double nest type RT-PCR amplification and reaction conditions

Round 1 PCR system: cDNA 2. Mu.L, 10 XPCR Buffer (Mg) ²⁺ plus) 2.5 mu L, dNTPs (2.5 mmol/L each) 0.7 mu L, primer (10 mu mol/L) combined P1/P2-P3/P4 in an amount of 0.4/0.4-0.4/0.4 mu L, taq DNA polymerase (5U/. Mu.L) 0.05 mu.L, and constant volume to 25. Mu.L.

Primer combination P1/P2-P3/P4 amplification procedure: pre-denaturation at 94℃for 3min;94 ℃ for 30s,55 ℃ for 45s and 72 ℃ for 50s, and 30 cycles are total; and at 72℃for 10min.

2 ndRound PCR system: the 1 st round PCR amplification product was used as a template in an amount of 1. Mu.L, and the remaining components were 10 XPCRBuffer (Mg ²⁺ plus) 2.5 mu L, dNTPs (2.5 mmol/L each) 1. Mu.L, primer combination (10. Mu. Mol/L) P5/P6-P7/P8 in an amount of 0.3/0.3-0.3/0.3. Mu.L, taq DNA polymerase (5U/. Mu.L) 0.25. Mu.L, and constant volume to 25. Mu.L.

Primer combination P5/P6-P7/P8 amplification procedure: pre-denaturation at 94℃for 3min;94 ℃ for 30s,55 ℃ for 45s and 72 ℃ for 50s, and 35 cycles are total; and at 72℃for 10min.

After the amplification of round 2, 6. Mu.L of the PCR product and 1. Mu.L of a 6×Loading buffer (containing gel red dye) were separated in 1.2% agarose gel, and the amplification result was observed in a BIO-RAD gel imaging system. The results are shown in FIG. 1, and a total of 78 samples were detected.

Comparative example 1 conventional RT-PCR amplification

The conventional RT-PCR amplification primers for GRSPaV are shown in Table 2.

TABLE 2 conventional RT-PCR amplification primers for GRSPaV

(1) 2. Mu.L of the cDNA template synthesized in example 1 was added to the following PCR amplification system: 10 XPCR buffer 2.5. Mu.L, dNTPs (2.5 mmol/L each) 0.5. Mu.L, RSP 52/RSP 53 (10. Mu. Mol/L) each 0.5. Mu.L, taq DNA polymerase (5U/. Mu.L) 0.125. Mu.L, and finally water was added to fix the volume to 25. Mu.L.

The primer RSP52/53 amplification procedure was: pre-denaturation at 94℃for 3min;94 ℃ for 30s,53 ℃ for 40s and 72 ℃ for 50s, and 35 cycles are total; and at 72℃for 10min.

After the amplification was completed, 6. Mu.L of the PCR product and 1. Mu.L of a 6×Loading buffer (containing gel red dye) were separated in 1.2% agarose gel, and the amplification result was observed in a BIO-RAD gel imaging system. As a result, it was found that 46 samples out of 84 samples had amplification bands, i.e., 46 samples were detected as positive in total.

(2) 2. Mu.L of the cDNA template synthesized in example 1 was added to the following PCR amplification system: 10 XPCR buffer 2.5. Mu.L, dNTPs (2.5 mmol/L each) 0.5. Mu.L, RSP 9F/RSP 9R (10. Mu. Mol/L) 0.5. Mu.L each, taq DNA polymerase (5U/. Mu.L) 0.125. Mu.L, and finally water was added to a volume of 25. Mu.L.

The primer RSP9P/9R amplification procedure was: pre-denaturation at 94℃for 3min;94 ℃ for 30s,54 ℃ for 30s and 72 ℃ for 40s, and 35 cycles are total; and at 72℃for 10min.

After the amplification was completed, 6. Mu.L of the PCR product and 1. Mu.L of a 6×Loading buffer (containing gel red dye) were separated in 1.2% agarose gel, and the amplification result was observed in a BIO-RAD gel imaging system. As a result, it was found that 46 samples out of 84 samples had amplification bands, i.e., 46 samples were detected as positive in total.

Comparative example 2 Single nested RT-PCR amplification

(1) Round 1 PCR amplification System

mu.L of the cDNA synthesized in example 1 was taken, and 10 XPCR buffer 2.5. Mu. L, dNTPs (2.5 mmol/L each) 0.5. Mu.L, each of the homologous primer/complementary primer (10. Mu. Mol/L) (P1/P2 or P3/P4) 0.5. Mu.L, and Taq DNA polymerase (5U/. Mu.L) 0.125. Mu.L were sequentially added thereto, and water was added thereto to fix the volume to 25. Mu.L.

Primer P1/P2 amplification procedure: pre-denaturation at 95℃for 3min, denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 40s, 30 cycles total, extension at 72℃for 7min.

Primer P3/P4 amplification procedure: pre-denaturation at 95℃for 3min, denaturation at 94℃for 30s, annealing at 55℃for 40s, extension at 72℃for 50s, 30 cycles total, extension at 72℃for 7min.

(2) Round 2 PCR amplification system: using the 1 st round PCR amplified product as a template, the amount was 1. Mu.L, 10 XPCR buffer 2.5. Mu. L, dNTPs (2.5 mmol/L each) 0.5. Mu.L, homologous and complementary primers (10. Mu. Mol/L) (P5/P6 or P7/P8) 0.5. Mu.L each, and Taq DNA polymerase (5U/. Mu.L) 0.125. Mu.L were added in sequence, and the volume was fixed to 25. Mu.L.

Primer P5/P6 amplification procedure: pre-denaturation at 95℃for 3min, denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 40s, total of 35 cycles, extension at 72℃for 7min.

Primer P7/P8 amplification procedure: pre-denaturation at 95℃for 3min, denaturation at 94℃for 30s, annealing at 57℃for 40s, extension at 72℃for 50s, total of 35 cycles, extension at 72℃for 7min.

After the amplification of round 2, 6. Mu.L of the PCR product and 1. Mu.L of a 6×Loading buffer (containing gel red dye) were separated in 1.2% agarose gel, and the amplification result was observed in a BIO-RAD gel imaging system.

Wherein, repF1R 1- & gtrepF 2R2 is used as a primer, 73 samples of 84 samples have amplified bands, namely 73 samples are detected as positive. Using cpF R1- > cpF2R2 as primer, 68 samples of 84 samples have amplified bands, i.e. 68 samples were detected as positive.

The results of example 2 and comparative examples 1 to 2 were counted and are shown in Table 3.

TABLE 3 detection effects of different methods

* The calculation basis of the "aggregate" in the table is:

A. routine RT-PCR: if only one pair of primers detects the target band, the sample is a positive sample, and the detection result is the sum of the results of the two pairs of primers RSP52/53 and RSP 9F/R;

B. single nested RT-PCR: if only one primer group detects the target band, the sample is a positive sample, and the detection result is the sum of the results of the two primer groups repF1R 1- & gt repF2R2 and cpF1R 1- & gt cpF R2;

C. double nested RT-PCR: as long as one primer detects the target band, the sample is a positive sample, and the detection result is the sum of the results of the primer combination repF1R1/cpF1R 1- & gtrepF 2R2/cpF R2.

The results in Table 3 show that the detection rate of the regenerated 84 grape test tube seedlings is respectively detected by using the conventional RT-PCR, the single nested RT-PCR and the double nested RT-PCR, and the detection rate of the double nested RT-PCR is identical to the sum of the detection rates of the two single nested RT-PCR and is 92.9%, which is improved by 25% compared with the sum of the two conventional RT-PCR.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

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Claims (1)

1. A method for detecting GRSPaV by using double nested RT-PCR is characterized by comprising the following specific steps:

(1) Extracting total RNA of a sample;

(2) cDNA synthesis;

(3) Round 1 PCR system: cDNA 2. Mu.L, 10 XPCRBufer 2.5. Mu.L, 2.5mmol/LeachdNTPs 0.7. Mu.L, 10. Mu. Mol/L primer combination P1/P2-P3/P4 in an amount of 0.4/0.4-0.4/0.4. Mu.L, 5U/. Mu.LTaqDNA polymerase 0.05. Mu.L, and constant volume to 25. Mu.L;

primer combination P1/P2-P3/P4 amplification procedure: pre-denaturation at 94℃for 3min;94 ℃ for 30s,55 ℃ for 45s and 72 ℃ for 50s, and 30 cycles are total; 72 ℃ for 10min;

(4) Round 2 PCR system: taking the PCR amplification product of round 1 as a template, the dosage is 1 mu L, the rest components are 10 XPCRBuferr 2.5 mu L, 2.5mmol/LeachdNTPs1 mu L, the dosage of the primer combination P5/P6-P7/P8 of 10 mu mol/L is 0.3/0.3-0.3/0.3 mu L, 5U/mu LTaqDNA polymerase of 0.25 mu L, and the volume is fixed to 25 mu L;

primer combination P5/P6-P7/P8 amplification procedure: pre-denaturation at 94℃for 3min;94 ℃ for 30s,55 ℃ for 45s and 72 ℃ for 50s, and 35 cycles are total; 72 ℃ for 10min;

(5) Separating the PCR product in agarose gel after the amplification of the 2 nd round is finished, and observing the amplification result in a BIO-RAD gel imaging system;

the primer sequence of the primer combination P1/P2-P3/P4 in the step (3) is as follows:

P1：5’-CTCCAGAGGTGCTGGTTGGTTCTC-3’；SEQ ID NO.1；

P2：5’-CGGCAAAAGAACGATATGACCAACT-3’；SEQ ID NO.2；

P3：5’-AGGGCTACAGGGGAGTCAAT-3’；SEQ ID NO.5；

P4：5’-TACGGTATTCCAGCGAACAGGCTTA-3’；SEQ ID NO.6；

the primer sequence of the primer combination P5/P6-P7/P8 in the step (4) is as follows:

P5：5’-CCAGATGGCAATTGGAATGAGATG-3’；SEQ ID NO.3；

P6：5’-CATAAGCAGAGAGCCACTCCT-3’；SEQ ID NO.4；

P7：5’-TGAAGGCTTTAGGGATTAGCC-3’；SEQ ID NO.7；

P8：5’-AACCTAGCCTTGAAATCAGG-3’；SEQ ID NO.8。

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