CN106480231B

CN106480231B - Multiple RT-PCR method for simultaneously detecting citrus yellow vein clearing virus and tristeza virus and application thereof

Info

Publication number: CN106480231B
Application number: CN201611264705.XA
Authority: CN
Inventors: 刘金香; 赵恒燕; 周常勇; 陈洪明
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2016-12-30
Filing date: 2016-12-30
Publication date: 2020-01-14
Anticipated expiration: 2036-12-30
Also published as: CN106480231A

Abstract

The invention relates to a method for simultaneously detecting citrus yellow vein clearing virus and citrus tristeza virusA multiple RT-PCR method for 2 citrus viruses. The method selects specific primers of a CYCVC and CTV 2 pathogenic coat protein conserved sequence and an internal reference gene to carry out multiplex PCR (polymerase chain reaction) influenced Mg²⁺dNTP concentration, primer concentration and annealing temperature are adjusted and optimized, a one-step method multiple RT-PCR method for simultaneously detecting CYCVC and CTV is established, and the sensitivity is consistent with that of single RT-PCR detection. The results of 33 field samples tested by the method show that the CYVCV and CTV infection rates are 54.5% and 66.7% respectively, and the composite infection rate of the samples is up to 36.4%. The method can be used for simultaneously and rapidly detecting CYVCV and CTV in a large number of field samples, and has important significance for improving the efficient and accurate detection and timely and effective prevention and treatment of citrus yellow vein clearing virus and tristeza virus.

Description

Multiple RT-PCR method for simultaneously detecting citrus yellow vein clearing virus and tristeza virus and application thereof

Technical Field

The invention belongs to the field of plant pathology, and particularly relates to a multiple RT-PCR method for simultaneously detecting citrus yellow vein clearing virus and tristimania virus and application thereof.

Background

Orange yellow vein disease (CYVCV) caused by orange yellow vein clearing virus is a newly-generated disease in China, and poses serious threat to orange industry, especially lemon industry. CYVCV is a positive-sense single-stranded RNA (+ ssRNA) virus consisting of 7529 nucleotides, and belongs to a member of the genus Citrus (Mandarivirus). CYVCV can be transmitted by aphids spiraeolotap (Aphis spiraecolaapatch) and bean aphids (a. cracivora koch) on beans (Phaseolus vulgaris) and cowpeas (vigna anguuiculata) in addition to transmission by grafting, tribological inoculation. Citrus Tristeza Virus (CTV), a Citrus tristeza virus, is a worldwide Citrus viral disease that causes significant economic losses to the Citrus industry worldwide. CTV is a positive-sense single-stranded RNA virus consisting of 19226-19296 nucleotides, belongs to the genus Clostridia, and is the largest virus in the genome of plant viruses known at present. CTV can be transmitted in the field in a non-circulating semi-persistent manner by aphids such as green orange Aphid (toxoplacitrida), cotton Aphid (Aphid gossypii) and binary orange Aphid (t. In order to quickly evaluate the susceptibility conditions of CYVCV and CTV of fruit trees and seedlings in the field, the establishment of a multiplex RT-PCR detection method for simultaneously and quickly detecting various viruses for detecting CYVCV and CTV is urgently needed. There have been many reports on the methods for separately detecting CYVCV and CTV. Adopting an indicator plant identification method; electron microscopy assay; enzyme-linked immunosorbent assay (ELISA), RT-PCR molecular assay, etc. The multiplex RT-PCR technology has the characteristics of sensitivity, rapidness, economy and the like, and is widely applied to plant virus detection. CYVCV and CTV have similar transmission ways, and due to long-term irregular field operation, seedling production and vector insect transmission, the two viruses are widely transmitted and prevalent, so that serious harm is caused to the citrus industry, and the phenomenon of mixed infection of the two diseases in the field often occurs. At present, no report of a CYVCV and CTV simultaneous multiplex PCR detection method exists in China, so that a one-step multiplex RT-PCR detection system for simultaneously detecting the two viruses is established, and a rapid, simple and reliable detection method is provided for evaluating the field infection condition of the two viruses and the virus removal condition in the seedling detoxification process.

Disclosure of Invention

In view of the above, the invention aims to provide a one-step method multiple RT-PCR method for simultaneously detecting CYVCV and CTV in a field, so as to realize simultaneous, rapid and sensitive detection of CYVCV and CTV, and improve the high-efficiency detection efficiency of CYVCV and CTV and timely and effectively prevent and control winning time.

The factors influencing the multiple RT-PCR reaction are many, and the inventor comprehensively considers the concentration of the primer and Mg²⁺The influence of variations in concentration, dNTP concentration, annealing temperature, cycle number and the like on Mg in the system²+ and dNTP amounts were adjusted to increase Mg²⁺Concentration to obtain optimum amplification effect and raise amplification efficiency of PCR reaction, and its main system adopts Mg²⁺The concentration is 0.625mmol/L and the concentration of dNTP is 0.2-0.25 mmol/L. Secondly, the concentration of three pairs of primers influencing the one-step method multiplex RT-PCR amplification is optimized and adjusted, and in order to eliminate the influence of impurities and operation process of total RNA in extraction, a highly conserved Ubiquitin sequence is added as an internal reference gene, so that the result of the multiplex RT-PCR detection system is more real and credible. However, UBQ primers are either specific for citrus or they compete for amplification between primersThe amplification efficiency of UBQ of short segments is lower than that of other two viruses, the amplification segment of UBQ is shorter and is close to primer dimer, so that UBQ amplification results are difficult to distinguish sometimes, so the equilibrium relationship of three pairs of primers in the whole reaction system is coordinated, and finally the concentration of CYVCV primer is determined to be 0.15-0.2 mu mol/L, the concentration of CTV primer is 0.1-0.13 mu mol/L, and the concentration of Ubiquitin primer is 0.5 mu mol/L. And the multiple PCR products are detected by agarose gel electrophoresis, a large-scale detection instrument is not needed, the operation of a conventional experiment is convenient, and the method is suitable for simultaneously and rapidly detecting the infection condition of two viruses in the field.

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

a multiple RT-PCR method for simultaneously detecting citrus yellow vein clearing virus and declining virus comprises the steps of extracting total DNA of a leaf sample to be detected as a template, carrying out multiple RT-PCR amplification by taking sequences shown in SEQ ID NO. 1-SEQ ID NO.6 as primers, judging a result by agarose gel electrophoresis of an amplification product, wherein a specific PCR reaction program of the multiple RT-PCR amplification is as follows: reverse transcription is carried out for 30min at 50 ℃; pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 62-63 ℃ for 50s, extension at 72 ℃ for 1min, and 35 cycles; 10min at 72 ℃; the reaction was terminated at 10 ℃.

Further, the primer pair is as follows:

SEQ ID NO.1 CYVCV F：TACCGCAGCTATCCATTTCC

SEQ ID NO.2 CYVCV R：GCAGAAATCCCGAACCACTA

SEQ ID NO.3 LD F：TCATTGCAAAGTGACGACGAC

SEQ ID NO.4 LD R：TGCCTGACATTGGTAACTACG

SEQ ID NO.5 UBQ F：GATCTTCGCCTTAACGTTGT

SEQ ID NO.6 UBQ R：GTTGATTTTTGCTGGGAAGC

in the multiplex PCR amplification Total reaction System, Mg²⁺The concentration is 0.625mmol/L, the concentration of dNTPs is 0.2-0.25 mmol/L, the concentration of primers SEQ ID NO.1 and SEQ ID NO.2 is 0.15-0.2 mu mol/L respectively, the concentration of primers SEQ ID NO.3 and SEQ ID NO.4 is 0.1-0.13 mu mol/L respectively, and the concentration of primers SEQ ID NO.5 and SEQ ID NO.6 is 0.5 mu mol/L.

The application of the multiplex RT-PCR detection method in detecting citrus yellow vein clearing virus and tristeza virus of plants is provided, wherein the plant is a plant of Citrus in Rutaceae.

Further, the Citrus plant of Rutaceae can be fructus Citri Grandis, fructus Citri sinensis, fructus Citri Limoniae, fructus Citri Sarcodactylis, and fructus Citri Tangerinae.

The invention has the beneficial effects that: the system can simultaneously detect CYVCV and CTV from 4 ng/. mu.L of total nucleic acid of a sample. Compared with single RT-PCR, the method has the advantages of simple and convenient operation, time and cost saving, avoids false positive caused by cross contamination among samples, can achieve the sensitivity with single PCR, and can simultaneously and accurately detect the infection of CYVCV and CTV in the citrus. And a highly conserved Ubiquitin sequence is added as an internal reference gene, so that the result of the multiple RT-PCR detection system is more real and credible.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 shows the results of multiplex RT-PCR amplification with different cycle numbers;

FIG. 2 shows different Mg²⁺Combining the multiple RT-PCR amplification result with the dosage of dNTPs;

FIG. 3 shows the result of multiplex RT-PCR amplification at different annealing temperatures;

FIG. 4 shows the result of multiplex RT-PCR amplification with different primer concentrations for CYVCV and CTV

FIG. 5 shows the results of multiplex RT-PCR amplification of different primer concentrations for reference genes;

FIG. 6 shows the result of multiplex RT-PCR detection of 2 citrus viruses;

FIG. 7 shows the results of comparing the sensitivity of single RT-PCR (A, B) and multiplex RT-PCR (C);

FIG. 8 shows the field sample detection results of multiplex RT-PCR.

Wherein, A in FIG. 1 is the amplification result of 30 cycles; b is the amplification result of 35 cycle times; m: DL 2000 molecular weight standard; 1: healthy sweet oranges; 2: CYVCV + CTV mixed orange; 3: water control;

in FIG. 2, M is DL 2000 molecular weight standard, 1-7 are shown in Table 2;

FIG. 3A is the result of the amplification of the healthy control at different annealing temperatures; b is the amplification result of the infected sample at different annealing temperatures;

m: DL 2000 molecular weight standard, 1-12 annealing temperatures 51.0, 51.3, 52.1, 53.4, 54.8, 56.3, 57.7, 59.1, 60.6, 61.9, 62.7 and 63 ℃ respectively;

in FIG. 4, A is the result of multiplex RT-PCR amplification of CTV with different primer concentrations 1-3: the primer concentrations of CTV are 0.1, 0.13 and 0.15 mu mol/L respectively; m: DL 2000 molecular weight standard; b is multiple RT-PCR amplification results of different primer concentrations of CYVCV; 1-3: the primer concentrations of CYVCV are 0.1, 0.15 and 0.2 mu mol/L respectively; m: DL 2000 molecular weight standard;

m in fig. 5: DL 2000 molecular weight standard 1, 1-3: the concentrations of the primers of the internal reference gene are respectively 0.3 mu mol/L, 0.4 mu mol/L and 0.5 mu mol/L, 4: water control;

m in fig. 6: DL 2000 molecular weight standard 1: CYVCV + CTV mixed orange; 2: CYVCV orange; 3: CTV sweet orange; 4: healthy sweet oranges; 5: water control;

m in fig. 7: DL 2000 molecular weight standard; h: a healthy control;

m in fig. 8: DL 2000 molecular weight standard; 1-11: a field sample; 12: positive control 13: and (4) water control.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

Example 1

1. Test material

The test material infected with CTV and CYVCV 2 viruses is Simmons sweet orange, which is provided by the center for detoxification of citrus seedlings in the institute of Citrus, national academy of agricultural sciences. The leaves of the sweet orange are used as positive materials, and the leaves of healthy sweet orange seedlings are used as negative control materials. The field samples were randomly sampled from 5-10 leaves in Chongqing North at 2016, 9 months.

2. Total nucleic acid extraction

Total nucleic acid of sweet orange leaves (directly collected from a poison source or healthy seedlings or stored in a refrigerator at-80 ℃) is extracted by an RNAasso Plus kit (TaKaRa, Japan) according to an operation instruction, 40 mu L of DEPC water is dissolved, the concentration and the purity are detected by a spectrophotometer, the ratio of A280/A260 ranges from 1.8 to 2.2, and the extracted nucleic acid is used as a template of one-step multiplex RT-PCR.

3. Single RT-PCR in one step

RT-PCR primers for CYVCV, CTV and reference genes are shown in Table 1. CYVCV One-step RT-PCR Using One StepRT-PCR Kit Ver.2 Kit (TaKaRa, Japan) reference Chen et al^[20]The method of (3) can be used for detection, and CTV one-step RT-PCR can be amplified by the same method after being searched. The PCR reaction system was 10. mu.L containing 2X 1Step Buffer 5. mu.L, upstream and downstream primers (Table 1) each 0.2. mu.L, PrimeScript 1Step Enzyme Mix 0.3. mu.L, RNA template 1. mu.L was added, and finally 10. mu.L was made up with sterile water. The PCR reaction program is: reverse transcription is carried out for 30min at 50 ℃; pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 45s for 30 cycles; 5min at 72 ℃; the reaction was terminated at 12 ℃.

TABLE 1 multiplex and Single RT-PCR primers

Example 2

Establishment of one-step method multiple RT-PCR system

Designing specific primers, optimally adjusting the concentrations of CTV and CYVCV primers influencing One-Step multiple RT-PCR amplification by using an One Step RT-PCR Kit Ver.2 Kit (TaKaRa, Japan), and determining the final appropriate primer concentration by comparing the electrophoresis results of 3 pairs of primers with different concentrations of PCR products. For Mg additionally added in a PCR system²⁺And dNTP final concentration were optimized, 7 concentration combinations were set (Table 2), and PCR products were electrophoresed to determine the optimum combination. According to the Tm values of 3 pairs of primers, 12 annealing temperatures (T1-T12) were set by a gradient PCR apparatus (biometraTgradient) with a gradient temperature difference of 12 ℃ for the annealing temperatures: 51.0, 51.3, 52.1, 53.4, 54.8, 56.3, 57.7, 59.1, 60.6, 61.9, 62.7 and 63 ℃, and determining the optimal temperature gradient by the electrophoresis result of the PCR products.

Optimization of PCR cycle number

Taking total nucleic acid extracted from positive orange leaves as a template, wherein 10ul of a total reaction system comprises: 2X 1Step Buffer 5. mu.L, CYVCV primers 0.2. mu.L each, CTV primers 0.13. mu.L each, internal reference primers 0.3. mu.L, PrimeScript 1Step enzymeMix 0.3. mu.L, total nucleic acid template 1. mu.L, Mg²⁺And dNTPs 0.2. mu.L each, sterilized pure water was added to a total volume of 10. mu.L. The PCR reaction conditions are as follows: 30min at 50 ℃; 4min at 94 ℃; 30s at 94 ℃, 50s at 55 ℃, 1min at 72 ℃, 35 cycles or 30 cycles; 10min at 72 ℃; the reaction is terminated at 10 ℃, the amplification conditions of target bands of the multiple RT-PCR system are observed at 30 and 35 cycles, the band of the internal reference gene is unclear at 30 cycles (figure 1, A), and the target bands of the amplified 2 viruses and the internal reference gene are clear at 35 cycles (figure 1, B), so that the PCR cycle is preliminarily determined to be 35.

2.Mg²⁺And optimization of dNTPs concentration

Taking total nucleic acid extracted from positive orange leaves as a template, wherein 10ul of a total reaction system comprises: 2X 1Step Buffer 5. mu.L, CYVCV primers 0.2. mu.L each, CTV primers 0.13. mu.L each, internal reference primers 0.3. mu.L each, PrimeScript 1Step enzymeMix 0.3. mu.L, total nucleic acid template 1. mu.L, Mg²⁺And dNTPs concentrations in Table 2 for 7 combinations, sterile purified water was added to a total volume of 10. mu.L. The PCR reaction conditions are as follows: 30min at 50 ℃; 4min at 94 ℃; 30s at 94 ℃, 50s at 55 ℃, 1min at 72 ℃, 35 cycles or 30 cycles; 10min at 72 ℃; the reaction was terminated at 10 ℃.

By comparing different Mg²⁺Agarose electrophoresis results of one-step multiplex RT-PCR reactions (FIG. 2) combined with 7 combinations of dNTPs concentration, as shown in the figure, the sizes of specific fragments of CYVCV, CTV and UBQ are 614bp, 373bp and 194bp respectively, 7 combinations can amplify target bands, and according to the brightness of the target bands, Mg which needs to be added in a 10 mu L system is determined²⁺And dNTPs at a combined concentration of 0.625mmol/L and 0.2mmol/L or 0.625mmol/L and 0.25mmol/L

TABLE 2 Mg in one-step multiplex PCR System²⁺And dNTPs in combination

3. Optimization of annealing temperature

And performing one-step multiple temperature gradient RT-PCR (figure 3) at the annealing temperature of 51-63 ℃ according to the optimal component dosage and PCR cycle number. Observing and comparing the multiple RT-PCR amplification results of the total nucleic acids of the healthy control and the susceptible sweet orange samples at different temperatures, and showing that 3 clear specific target bands can be simultaneously amplified by the susceptible samples at the annealing temperatures of 51 ℃, 54.8 ℃ and 56.3 ℃ (FIG. 3, A), but the non-specific amplification in the healthy control is obvious (FIG. 3, B); when the annealing temperature is 62.7 ℃, although the target band of the reference gene in the positive sample is weak (fig. 3, a), the healthy control has no non-specific amplification and no tailing phenomenon (fig. 3, B), and the optimal annealing temperature range can be preliminarily determined to be 62-63 ℃.

4. Optimization of primer concentration

(1) CYVCV and CTV primer concentrations

The concentrations of CTV and CYVCV primers affecting the one-step multiplex RT-PCR amplification were initially adjusted optimally, and the PCR reaction conditions were: 30min at 50 ℃; 4min at 94 ℃; 30s at 94 ℃, 50s at 55 ℃, 1min at 72 ℃, 35 cycles, 10min at 72 ℃; the reaction was terminated at 10 ℃. In the multiplex RT-PCR reaction system, the CYVCV primer concentration is 0.1 mu mol/L, the Ubiquitin primer concentration is 0.3 mu mol/L, the CTV primer concentration is increased from 0.1 mu mol/L to 0.15 mu mol/L, the target band of CTV is lightened, the amplification efficiency is increased, the amplification efficiency of CYVCV is reduced, and the target band of CYVCV is weakened (FIG. 4A). In the multiplex RT-PCR reaction system, the concentration of CTV primers is 0.13 mu mol/L, the concentration of Ubiquitin primers is 0.3 mu mol/L, the concentration of CYVCV primers is increased from 0.1 mu mol/L to 0.2 mu mol/L, the target band of CYVCV is gradually lightened, the amplification efficiency is improved, and the brightness change of the target band of CTV is not obvious (FIG. 4B). Preliminarily determining the concentration of CYVCV primer to be 0.15-0.2 mu mol/L, the concentration of CTV primer to be 0.1-0.13 mu mol/L and the concentration of Ubiquitin primer to be 0.3 mu mol/L.

(2) Concentration of internal reference primer

After the annealing temperature is optimized, when the annealing temperature is in the optimal range of 62-63 ℃, the target band of the internal reference gene in the positive sample is weaker, the concentrations of internal reference primers are further adjusted, 0.2 mu L of CYVCV primers and 0.13 mu L of CTV primers in a multiple RT-PCR system respectively, the concentrations of Ubiquitin primers are 0.3, 0.4 and 0.5 mu mol/L respectively, and the PCR reaction is carried out at the annealing temperature of 63 ℃ for 50 s. The bands of the internal reference gene were not clear at the UBQ concentrations of 0.3. mu. mol/L and 0.4. mu. mol/L (FIG. 5), and the bands of interest were clear at the UBQ concentration of 0.5. mu. mol/L for both the 2 amplified viruses and the internal reference gene (FIG. 5), so that the UBQ concentration was finally determined to be 0.5. mu. mol/L.

According to the test results, a one-step multiple RT-PCR reaction system and a one-step multiple RT-PCR reaction program of citrus samples infected with CYVCV and CTV 2 virus diseases are finally determined. The one-step multiplex RT-PCR system (10. mu.L) was: 2X 1Step Buffer 5. mu.L, 25mmol/L Mg²⁺0.25 mu.L, 0.2-0.25 mu.L of 10mmol/L dNTPs, 0.15-0.2 mu.L of CYVCV primer, 0.1-0.13 mu.L of CTV primer, 0.5 mu.L of internal reference primer, 0.3 mu.L of PrimeScript 1Step Enzyme Mix, 1 mu.L of total nucleic acid template, and the volume is supplemented to 10 mu.L by sterilized pure water. The one-step method multiplex RT-PCR reaction program comprises the following steps: 30min at 50 ℃; 4min at 94 ℃; 30s at 94 ℃, 50s at 62-63 ℃, 1min at 72 ℃ and 35 cycles; 10min at 72 ℃; the reaction was terminated at 10 ℃. The established multiplex system is used for carrying out one-step multiplex RT-PCR amplification on the susceptible sample and the healthy sample, and the amplification result shows that the target bands of 2 viruses and internal reference genes are clear, which shows that the established one-step multiplex RT-PCR detection system has better amplification effect (figure 6).

Example 3

1. One-step single and multiple RT-PCR detection sensitivity comparison result

The test result of the detection sensitivity shows that the maximum dilution multiple of the total nucleic acid of the CYVCV can be detected by a single RT-PCR system in one step to the positive sample of the complex infection 2 viruses^-2Fold (FIG. 7, A), maximum dilution of total nucleic acids capable of detecting CTV 10^-2Multiple (fig. 7, B). The maximum dilution multiple of total nucleic acid of CYVCV and CTV detected by a one-step method multiple RT-PCR system is 10^-2And (4) the detection sensitivity of a single RT-PCR system is achieved by times (figure 7, C). The one-step method multiplex RT-PCR method can detect CYVCV and CTV from 4 ng/mu L total nucleic acid at the lowest energy, and the sensitivity of the method is consistent with that of single RT-PCR detection.

2. One-step method for cloning and sequencing multiple RT-PCR products

DNA fragments obtained by multiple RT-PCR amplification are subjected to electrophoresis and gel cutting recovery respectively, cloned to a T vector, sequenced by a company, and subjected to homology search and comparison by using NCBI Blast. The results show that the specific fragment of CYVCV has 99% homology with submitted China Hunan isolate (KT124646) and China Chongqing isolate (KP313240), and has more than 95% homology with other isolates. The CTV specific fragment has homology of up to 99% with Chinese isolate (FJ998200) and two Italian isolates (KJ790175 and KC748392), 97% with American isolate (DQ363901) and more than 96% with other isolates. The amplified fragments of CYVCV and CTV have higher homology with the sequences registered in NCBI, indicating that the DNA fragment obtained by amplification is the desired band.

3. Practical application of one-step method multiple RT-PCR detection system

The optimized one-step multiplex RT-PCR detection system is applied to detect 33 citrus field samples (figure 8), the infection conditions of the 2 viruses in the fields are rapidly evaluated, and the results show that (table 3) in the 33 field samples, the CYVCV infection rate is 54.5%, the CTV infection rate is 66.7%, and the complex infection rate is as high as 36.4%; the CYVCV and CTV complex infested samples were mainly concentrated in sweet oranges and satsuma mandarins. And carrying out one-step RT-PCR detection on the corresponding samples, wherein the result is consistent with the result of the multiple RT-PCR detection.

One-step multiplex RT-PCR detection method for 333 parts of field different citrus samples

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

SEQUENCE LISTING

<110> university of southwest

<120> multiple RT-PCR method for simultaneously detecting citrus yellow vein clearing virus and declining virus and application thereof

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Claims

1. A multiple RT-PCR method for simultaneously detecting orange yellow vein clearing virus and declining virus is characterized in that total nucleic acid of a leaf sample to be detected is extracted as a template, a sequence shown as SEQ ID NO.1 ~ SEQ ID NO.6 is used as a primer to carry out multiple RT-PCR amplification, an amplification product of the multiple RT-PCR amplification is subjected to agarose gel electrophoresis to judge a result, the annealing temperature of the multiple RT-PCR amplification is 62 ℃ ~ ℃, the multiple RT-PCR amplification reaction program is that reverse transcription is carried out at 50 ℃ for 30min, pre-denaturation is carried out at 94 ℃ for 4min, denaturation is carried out at 94 ℃ for 30s, annealing is carried out at 62 ℃ for ~ ℃ for 50s, extension is carried out at 72 ℃ for 1min, 35 cycles is carried out, the temperature is 72 ℃ for 10min, and termination reaction is carried out at 10 ℃, and Mg is added into²⁺The concentration is 0.625mmol/L, the concentration of dNTPs is 0.2 ~ 0.25.25 mmol/L, the concentration of primers SEQ ID NO.1 and SEQ ID NO.2 is 0.15 ~ 0.2.2 μmol/L respectively, the concentration of primers SEQ ID NO.3 and SEQ ID NO.4 is 0.1 ~ 0.13.13 μmol/L respectively, and the concentration of primers SEQ ID NO.5 and SEQ ID NO.6 is 0.5 μmol/L.

2. Use of the method of claim 1 for detecting citrus yellow vein clearing virus and tristeza virus in plants, wherein: the plant species is Rutaceae Citrus plant.

3. Use according to claim 2, characterized in that: the Citrus plant of Rutaceae is selected from fructus Citri Grandis, fructus Citri sinensis, fructus Citri Limoniae, fructus Citri Sarcodactylis, and fructus Citri Tangerinae.