CN112779356A - Method for detecting GRSPaV by using dual nested RT-PCR - Google Patents

Abstract

The invention discloses a method for detecting GRSPaV by using dual nested RT-PCR, belonging to the technical field of molecular biology. The invention discloses a method for detecting GRSPaV by using dual nested RT-PCR, which comprises two rounds of PCR amplification; the method can simultaneously perform 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 single nested PCR of two fragments; the method can effectively reduce the detection cost and improve the detection speed, is simple to operate and good in stability, and provides powerful support for the rapid detection and detoxification research of GRSPaV.

Description

Method for detecting GRSPaV by using dual 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 dual nested RT-PCR.

Background

Sandland grapevine stalk pox associated virus (GRSPaV) occurs in grape producing areas worldwide, is the most widely distributed grape virus, and is associated with a variety of diseases such as grape vein necrosis, sandland grapevine stalk pox, and grapevine recession. GRSPaV is a phloem-restricted virus, which is mainly transmitted by grafting; the virus belongs to the genus of the dimorphic virus, and the genome has high variability.

Therefore, it is an urgent problem to be solved by those skilled in 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 is simple to operate and has good stability.

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

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

(1) extracting total RNA of the sample;

(2) synthesizing cDNA;

(3) round 1 PCR system: 2 mu L of cDNA, 10 XPCR buffer2.5 mu L, 0.7 mu L of 2.5mmol/L each dNTPs, 0.4/0.4-0.4/0.4 mu L of 10 mu mol/L primer combination P1/P2-P3/P4 and 0.05 mu L of 5U/mu L Taq DNA polymerase, and the volume is up to 25 mu L;

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

(4) round 2 PCR system: 1 muL of the 1 st round PCR amplification product is taken as a template, the other components are 10 XPCR Buffer2.5 muL, 2.5mmol/L each dNTPs 1 muL, 10 mumol/L primer combination P5/P6-P7/P8, the dosage is 0.3/0.3-0.3/0.3 muL, and 5U/muL Taq DNA polymerase is 0.25 muL, and the constant volume is 25 muL;

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

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

Further, the primer sequences of the primer combination 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。

according to the technical scheme, compared with the prior art, the invention discloses and provides the method for detecting GRSPaV by using the dual nested RT-PCR, the method can simultaneously perform 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 single nested PCRs of fragments; the method can effectively reduce the detection cost (2 PCR reactions and corresponding agarose gel electrophoresis are reduced, the reagent dosage is reduced), improve the detection speed, has simple operation and good stability, and provides powerful support for the 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 used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a diagram showing the result of the dual nested RT-PCR assay of the present invention;

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

note: the band amplified from sample No. 18 was far from both target bands, and was judged to be negative.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Taq DNA polymerase and dNTPs, available from Bao bioengineering (Dalian) Inc.; moloney-murin leukemia virus (M-MLV), 6-base random primers, purchased from Biotechnology engineering (Shanghai) Inc.; purchased from promo-mark (beijing) biotechnology limited; DNA marker II, Tiangen Biochemical technology (Beijing) Ltd; other reagents are all domestic analytical purifiers. The primers were synthesized by Biotechnology engineering (Shanghai) Inc.

Samples of GRSPaV-infected grapes were harvested in 2019 from the detoxification center of deciduous fruit trees, national institute of fruit tree, academy of agricultural sciences. The samples to be tested were 84 parts of laboratory-stored regenerated grape plants detoxified by heat treatment (13 parts) and chemical treatment (71 parts), respectively.

Example 1 Total RNA extraction and cDNA Synthesis

(1) Total RNA extraction

Aiming at 84 samples to be detected, respectively taking 0.05-0.1g of regenerated grape plant leaves after detoxification, putting the grape plant leaves into a plastic bag, adding 1mL of lysate for grinding, transferring the homogenate into a 1.5mL centrifuge tube added with 150 mu L of 10% N-lauroylsarcosine (NLS) in advance, changing the gun head one by one, wiping the gun head, and if more samples are added, putting the samples on ice, and less samples can be put at room temperature. Keeping the temperature at 72 ℃ for 10min, mixing the mixture for several times, standing the mixture on ice for 5min, and centrifuging the mixture at 13000rpm for 10 min. Collecting supernatant 600 μ L, adding 300 μ L anhydrous ethanol, reversing for 45s, mixing, adding 750 μ L mixture into adsorption column, covering one cover each time, centrifuging at 12000rpm for 1min, and discarding waste liquid. Adding 700 μ L deproteinized solution, standing at room temperature for 2min, centrifuging at 12000rpm for 1min, and discarding waste liquid. Adding 700 μ L of rinsing solution, centrifuging at 12000rpm for 1min, and discarding the waste liquid. Adding 500 μ L of rinsing solution, centrifuging at 12000rpm for 1min, and removing waste liquid; the column was returned to the empty collection tube and centrifuged at 12000rpm for 2min to remove the rinse. Taking out the adsorption column, placing into a centrifuge tube without RNase contamination, adding 40 μ L 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 subjecting the centrifuged RNA solution to reverse transcription or storing in a low temperature refrigerator at-70 deg.C (or-20 deg.C).

(2) cDNA Synthesis

Taking 3 mu L of the total RNA and 6 mu L of deionized water treated by 6-base random primer 1 mu L, DEPC, placing the mixture in a centrifuge tube, uniformly mixing, performing denaturation in boiling water for 6min, and then placing the mixture on ice for 3 min. Adding the following components into the solution in sequence: 5 × RT buffer2 μ L, dNTPs (10mmol/L)1 μ L, M-MLV (200U/. mu.l) 0.5 μ L, made up to 20 μ L total with water. Mixing the above solutions, inactivating at 42 deg.C for 10min, 37 deg.C for 50min, 70 deg.C for 5min, freezing for 3min, and standing at-20 deg.C.

Example 2 Dual nested RT-PCR detection of GRSPaV

(1) Primer design

With reference to GRSPaV whole genome sequence (105) registered at NCBI, a set of nested amplification primers were designed at the viral replicase and coat protein genes, respectively: repF1R1 → repF2R2 and cpF1R1 → cpF2R2 (Table 1), the sizes of the final amplified fragments are 438bp and 902bp, respectively.

TABLE 1 nested PCR amplification primers for GRSPaV

(2) Dual nested RT-PCR amplification and reaction conditions

Round 1 PCR system: cDNA 2. mu.L, 10 XPCR Buffer (Mg)²⁺plus)2.5μL、dNTPs(2.5mmol/L each)0.7μL、The primer (10 mu mol/L) combination P1/P2-P3/P4 is used in an amount of 0.4/0.4-0.4/0.4 mu L, Taq DNA polymerase (5U/mu L) is used in an amount of 0.05 mu L, and the volume is up to 25 mu L.

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

Round 2 PCR system: using the 1 st round PCR amplification product as a template, the dosage is 1 mu L, and the rest is 10 XPCRBbuffer (Mg)²⁺plus)2.5 μ L, dNTPs (2.5mmol/L each)1 μ L, primer combination (10 μmol/L) P5/P6-P7/P8 in an amount of 0.3/0.3-0.3/0.3 μ L, Taq DNA polymerase (5U/μ L) in an amount of 0.25 μ L, and a volume of 25 μ L.

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

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

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) 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 (2.5mmol/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 make up to 25. mu.L.

The primer RSP52/53 amplification program is as follows: pre-denaturation at 94 ℃ for 3 min; 35 cycles of 94 ℃ for 30s, 53 ℃ for 40s and 72 ℃ for 50 s; 10min at 72 ℃.

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

(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 (2.5mmol/L each) 0.5. mu.L, RSP 9F/RSP 9R (10. mu. mol/L) each 0.5. mu.L, Taq DNA polymerase (5U/. mu.L) 0.125. mu.L, and finally water is added to the mixture to make the volume to 25. mu.L.

The primer RSP9P/9R amplification program is as follows: pre-denaturation at 94 ℃ for 3 min; 35 cycles of 94 ℃ for 30s, 54 ℃ for 30s and 72 ℃ for 40 s; 10min at 72 ℃.

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

Comparative example 2 Single nested RT-PCR amplification

(1) 1 st round PCR amplification system

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

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

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

(2) Round 2 PCR amplification system: using the product of 1 st round of PCR amplification as a template and 1. mu.L, 0.5. mu.L of 10 XPCR buffer 2.5. mu. L, dNTPs (2.5mmol/L each), 0.5. mu.L of each of the homologous and complementary primers (10. mu. mol/L) (P5/P6 or P7/P8), and 0.125. mu.L of Taq DNA polymerase (5U/. mu.L) were sequentially added to the mixture to make a volume of 25. mu.L.

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

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

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

Wherein, the primer is repF1R1 → repF2R2, 73 samples of 84 samples have amplification bands, namely 73 samples are detected as positive. cpF1R1 → cpF2R2 is taken as a primer, 68 samples of 84 samples have amplification bands, and 68 samples are detected to be positive.

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

TABLE 3 examination of the effectiveness of the different methods

The calculation of "total" in the table is based on:

A. conventional RT-PCR: if only one pair of primers detects a 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 group 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 groups of primers, namely repF1R1 → repF2R2 and cpF1R1 → cpF2R 2;

C. double nested RT-PCR: if only 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/cpF1R1 → repF2R2/cpF2R 2.

The results in Table 3 show that the detection rates of the double nested RT-PCR and the two single nested RT-PCR are consistent with the sum of the detection rates of the two single nested RT-PCR, both are 92.9 percent, and are improved by 25 percent compared with the sum of the two conventional RT-PCR when the conventional RT-PCR, the single nested RT-PCR and the double nested RT-PCR are used for respectively detecting the regenerated 84 test-tube plantlets of the grapes.

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.

Sequence listing

<110> research institute of fruit trees of Chinese academy of agricultural sciences

<120> method for detecting GRSPaV by using dual nested RT-PCR

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 1

ctccagaggt gctggttggt tctc 24

<210> 2

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 2

cggcaaaaga acgatatgac caact 25

<210> 3

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 3

ccagatggca attggaatga gatg 24

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 4

cataagcaga gagccactcc t 21

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 5

agggctacag gggagtcaat 20

<210> 6

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 6

tacggtattc cagcgaacag gctta 25

<210> 7

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 7

tgaaggcttt agggattagc c 21

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 8

aacctagcct tgaaatcagg 20

<210> 9

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 9

tgaaggcttt aggggttag 19

<210> 10

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 10

cttaacccag ccttgaaat 19

<210> 11

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 11

ggccaaggtt cagtttg 17

<210> 12

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 12

acacctgctg tgaaagc 17

Claims (2)

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

(1) extracting total RNA of the sample;

(2) synthesizing cDNA;

(3) round 1 PCR system: 2 mu L of cDNA, 10 XPCR buffer2.5 mu L, 0.7 mu L of 2.5mmol/L each dNTPs, 0.4/0.4-0.4/0.4 mu L of 10 mu mol/L primer combination P1/P2-P3/P4 and 0.05 mu L of 5U/mu L Taq DNA polymerase, and the volume is up to 25 mu L;

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

(4) round 2 PCR system: 1 muL of the 1 st round PCR amplification product is taken as a template, the other components are 10 XPCR Buffer2.5 muL, 2.5mmol/L each dNTPs 1 muL, 10 mumol/L primer combination P5/P6-P7/P8, the dosage is 0.3/0.3-0.3/0.3 muL, and 5U/muL Taq DNA polymerase is 0.25 muL, and the constant volume is 25 muL;

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

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

2. The method for detecting GRSPaV by using dual nested RT-PCR as claimed in claim 1, wherein the primer combination P1/P2-P3/P4 in step (3) has the following primer sequences:

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。

Images