CN111690777B

CN111690777B - Specific primer, kit and method for RT-RPA detection of citrus leaf mottle virus

Info

Publication number: CN111690777B
Application number: CN202010680187.XA
Authority: CN
Inventors: 宋震; 周常勇; 段玉; 许建建; 马志敏; 宾羽
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2020-07-15
Filing date: 2020-07-15
Publication date: 2023-06-02
Anticipated expiration: 2040-07-15
Also published as: CN111690777A

Abstract

The invention discloses a specific primer for RT-RPA detection of citrus leaf mottle virus, which comprises an upstream primer: 5'-ATGAACACTCACGGCGATGAAATTCCCACA-3', downstream primer: 5'-GGATCCCCCATTAAATTCCATAAGCCAGTC-3'. Also discloses a kit for detecting RT-RPA of the citrus leaf mottle virus, which contains the specific primer. Also disclosed is a method for detecting RT-RPA of the citrus leaf mottle virus: 1) Extracting RNA of a sample to be detected; 2) Carrying out RT-RPA reaction by using the extracted sample RNA, wherein the specific primer is the specific primer; 3) And detecting a reaction product after the RT-RPA reaction is finished, wherein the detected target amplified object is a positive sample. The method has the advantages of strong specificity, equivalent sensitivity to RT-PCR, simple and convenient operation and short detection time.

Description

Specific primer, kit and method for RT-RPA detection of citrus leaf mottle virus

Technical Field

The invention relates to the technical field of virus detection, in particular to a specific primer, a kit and a method for RT-RPA detection of citrus leaf mottle virus.

Background

The citrus leaf mottle virus (Citrus leaf blotch virus, CLBV) is a positive-sense single-stranded RNA virus of the family betalinear virus (betaflexiviidae) citrus (Citrivirus), which is a representative species of the genus citrus. CLBV was first detected and reported in kumquat in 1984, and later in several citrus varieties in australia, japan, the united states and spain. CLBV was first discovered in the investigation of the fowlery kiwi fruits in 1995 in China, and subsequent detection shows that the virus has a tendency of wide distribution and increasingly serious hazard on the kiwi fruits. The virus infects almost all citrus plants and related species, and most of the citrus plants are cryptogam, but the virus can cause symptoms such as disregulation of bud joints of kumquats and fructus aurantii, mottle of orange leaves, and citrus fruit stem acnes. Under natural conditions, the virus is mainly transmitted through asexual propagation paths such as grafting and the like, and can also be transmitted through citrus seeds, but the transmission rate is very low.

So far, early detection and application of detoxified seedlings are important means for effectively preventing the virus from being harmful, so that the establishment of a simple, convenient and quick CLBV detection method has important significance for effectively preventing and controlling the virus. With the intensive research of CLBV, various methods for identifying CLBV are established, from long and expensive indicator plants to spot hybridization and tissue blot hybridization, to molecular detection, such as RT-PCR detection, multiplex RT-PCR detection techniques, real-time fluorescent quantitative PCR, and the like. However, most of the existing molecular detection methods are time-consuming and labor-consuming and not simple enough. The RT-LAMP detection method breaks through the limitation of thermal cycle, but the complex primer design and longer reaction time make the method not simple and quick enough.

In recent years, a new isothermal nucleic acid amplification technique, recombinase polymerase amplification (Recombinase polymerase amplification, RPA), has received attention because of its rapidity, sensitivity, and ease of operation. RPA is amplified at constant temperature using an enzymatic mixture comprising a recombinase, a single-stranded DNA binding protein, and a DNA polymerase with substitution activity, and the target product can be presented in gel electrophoresis. As early as 2006, the technology is applied to rapid and sensitive detection of DNA, and is widely applied to disease detection of animals and plants, such as rapid detection of transgenic crops and detection of Potato virus (PMTV) virus; in the aspect of fruit tree disease detection, an RPA detection method of banana bunchy top virus (Banana bunchy top virus, BBTV) has been reported, the whole detection reaction can be completed within 15min, the operation is simple and convenient, and the method is suitable for field rapid detection of the disease. RT-RPA is used to detect viruses with RNA genomes.

Although the detection method of the CLBV is not few, the detection method is time-consuming and labor-consuming in operation implementation, and has high requirements on operation skills and test instruments. RT-LAMP can realize constant temperature detection of CLBV, and is simple to operate, but requires complex probe design and long reaction time.

Disclosure of Invention

The invention aims at solving the problems and provides a specific primer for RT-RPA detection of citrus leaf mottle virus, which has the nucleotide sequence as follows:

an upstream primer: 5'-ATGAACACTCACGGCGATGAAATTCCCACA-3' the number of the individual pieces of the plastic,

a downstream primer: 5'-GGATCCCCCATTAAATTCCATAAGCCAGTC-3'.

The invention also provides a kit for detecting RT-RPA of the citrus leaf mottle virus, which contains the specific primer.

The kit also comprises a plant total RNA extraction kit, an RT-RPA amplification reaction reagent, a negative control and a positive control.

In yet another aspect, the invention provides a method for RT-RPA detection of citrus leaf mottle virus, comprising the steps of:

1) Extracting RNA of a sample to be detected;

2) Carrying out RT-RPA reaction by using the extracted sample RNA, wherein the specific primer is the specific primer;

3) And detecting a reaction product after the RT-RPA reaction is finished, wherein the detected target amplified object is a positive sample.

In the technical scheme, the reaction system of the RT-RPA reaction is as follows: 10. Mu.M/. Mu.L of each of the upstream/downstream primers, 2. Mu.L of sample RNA, and RT-RPA amplification reaction reagent.

The RT-RPA reaction conditions are as follows: the reaction temperature is 34-44 ℃ and the reaction time is 26-34 minutes.

Preferably, the RT-RPA reaction conditions are: the reaction temperature was 40℃and the reaction time was 30 minutes.

In the above technical scheme, after the RT-RPA reaction in the step 3) is completed, the reaction product is detected by agarose gel electrophoresis analysis or nucleic acid sequencing of the amplified product.

When agarose gel electrophoresis analysis is carried out, the detection of a 112bp band indicates that the sample to be detected is a positive sample.

The beneficial effects of the invention are as follows: the RT-RPA rapid detection system of the CLBV is established for the first time at home and abroad; the system is simple and convenient to operate, quick and sensitive in reaction and strong in specificity, can rapidly present a detection result, breaks through the temperature limit of conventional RT-PCR, does not need a separate reverse transcription step, does not need a complex instrument, does not need light-proof operation and a precise instrument to perform fluorescence acquisition like RT-qPCR, establishes a standard curve and analyzes complex fluorescence data, consumes a great deal of time and energy, has lower technical requirements on operators, is convenient for basic staff to rapidly detect CLBV, and has wide application prospect. The detection method has the advantages of strong specificity, high sensitivity, equivalent sensitivity to RT-PCR, simple and convenient operation, short detection time, complete detection within 35min, suitability for rapid detection of the CLBV of the citrus sample, particular suitability for detection application in places with insufficient conditions such as a base layer and the like, and high practicability.

Drawings

FIG. 1 shows the result of screening CLBV RT-RPA detection specific primers, wherein M is DNA molecular mass marker; 1,2,5,6,9, 10: CLBV positive nucleic acid; 3,7, 11: healthy controls; 4,8, 12: ddH ₂ O; 1. the primer RPACF1/RPACR1 is used as 2, the primer RCLBV-F/RCLBV-R1 is used as 5 and 6, the primer RCLBV-F/RCLBV-R2 is used as 9 and 10, and the primer RCLBV-F/RCLBV-R2 is used as the rest of lanes.

FIG. 2 is a screen of CLBV RT-RPA detection of different reaction temperatures, wherein M is DNA molecular mass marker.

FIG. 3 is a screen of CLBV RT-RPA assay for different reaction durations, wherein M is a DNA molecular mass marker.

FIG. 4 is a specific detection result of an RT-RPA detection system of CLBV, wherein M is DNA molecular mass marker; CTV-CLBV: different types of citrus disease positive samples; CK: healthy controls; h ₂ O: water control.

FIG. 5 is the sensitivity test results of the RT-RPA test system of CLBV, wherein A: RT-PCR detection sensitivity; b: RT-RPA detection sensitivity; m, DNA molecular mass marking; 10 ^-1 —10 ^-6 The concentration is 1.17 multiplied by 10 ^-1 μg.μL ^-1 —1.17×10 ^-6 μg.μL ^-1 CLBV positive nucleic acid.

FIG. 6 is an RT-PCR, RT-RPA and RT-qPCR assay for a portion of a citrus sample, wherein A: RT-PCR detection; b: RT-RPA detection; c, RT-qPCR detection; m, DNA molecular mass marking; 1-7: citrus sample nucleic acid; -CK: negative control.

Detailed Description

The invention is further illustrated, but is not limited, by the following examples.

The experimental methods in the following examples are conventional methods unless otherwise specified.

Example 1

1 materials and methods

1.1 test materials and Primary reagents

Test materials:

the citrus disease positive materials to which the present application relates include CLBV, citrus tristeza virus (Citrus tristeza virus, CTV), citrus yellow vein ming virus (Citrus yellow vein clearing virus, CYVCV), citrus split virus (Citrus exocortis viroid, CEVd), citrus split virus (Citrus tatter leaf virus, CTLV), citrus scale virus (Citrus psorosis virus, CPsV), citrus unshiu atrophy virus (Satsuma dwarf virus, SDV), citrus canker pathogen (Xanthomonas citri subsp. Citr., xcc), and citrus yellow dragon germ asian species (Candidatus Liberibacter asiaticus, CLas) are all provided by the national center for citrus nursery stock detoxification at the university of west south.

The main reagent comprises:

biospin totipotent plant genome DNA extraction kit, polysaccharide polyphenol plant total RNA extraction kit and Prime Script ^TM The One Step RT-PCR Kit Ver.2 Kit was purchased from Bao Ri doctor materials technology (Beijing) Co., ltd,

qPCR Master Mix kit was purchased from Promega, inc.;

the RT-RPA nucleic acid amplification kit adopts an RT-basic nucleic acid amplification reagent (RAA method) product of Hangzhou public detection biotechnology Co Ltd, and the product is the product number S003ZC, wherein the kit comprises an A buffer solution and a B buffer solution, namely an A buffer and a B buffer in the kit.

The remaining reagents were all analytically pure and commercially available.

1.2 nucleic acid preparation

Taking 50mg of citrus leaf tissue sample, extracting sample nucleic acid according to the instruction of an RNA extraction kit, carrying out CLBV detection by using a CLBV1F/CLBV5R primer and adopting RT-PCR, and storing positive sample nucleic acid at the temperature of-20 ℃ for later use. The other citrus virus positive nucleic acids are also extracted according to the instruction of the RNA extraction kit, and the nucleic acids are detected to be virus positive and then stored at the temperature of-20 ℃ for standby. The citrus canker pathogen and the citrus yellow dragon disease are used for extracting nucleic acid according to the instruction of the DNA kit, and are stored at-20 ℃ for standby after being detected positive.

1.3 primer design and primer specific screening

3 pairs of primers were designed by the Premier 5.0 software based on the Coat Protein (CP) gene conserved sequence of the citrus She Banbo virus gene (accession No. MG 572236.1) (table 1). The primer design is carried out by avoiding mismatch, dimer and hairpin structure, the optimal primer length is between 30 and 35bp, and the product is between 100 and 150 bp. RT-RPA screening System for primers: 2 mu L (10 mu M/mu L) of each of the upstream/downstream primers, 41.5 mu L of A buffer, 2.5 mu L of B buffer and 2 mu L of RNA are covered by a unit cover, the mixture is fully and uniformly mixed by shaking, subjected to instantaneous centrifugation for 10s and 30min at 37 ℃, the chloroform with the same volume as the reaction system is added, the mixture is fully and uniformly mixed, subjected to centrifugation at 12000rpm for 5min, 10 mu L of supernatant products are sucked, and 2.5% agarose gel electrophoresis analysis is carried out. The RT-RPA reaction product detected as positive was sequenced and was completed by Huada gene company. The RT-PCR detection primer CLBV1F/CLBV5R of the CLBV refers to sequence synthesis reported by Chavan and the like, and the amplified fragment size is 425bp; sequence synthesis reported by RT-qPCR primer CLBVF/CLBVR with reference to Ruiz-Ruiz, et al. The sequences of the primers are shown in Table 1:

TABLE 1 primer sequences

1.4 optimization of reaction temperature and optimization of reaction time duration

Setting a temperature difference from 34 ℃ to 44 ℃, taking 2 ℃ as a temperature gradient, detecting system conditions according to the conditions of primer design and primer specificity screening of 1.3, wherein the primer adopts RCLBV-F/RCLBV-R2, the reaction time is 30min, the temperature of an RT-RPA reaction system is optimized, the reaction product is subjected to agarose gel electrophoresis analysis, and the reaction temperature with optimal amplification efficiency is screened.

Setting a reaction time difference from 26min to 34min, taking 2min as a time gradient, detecting system conditions according to the conditions of the primer design and the primer specificity screening in the previous description, wherein the primer adopts RCLBV-F/RCLBV-R2, the reaction temperature is 40 ℃, the reaction time of an RT-RPA reaction system is optimized, the reaction product is subjected to agarose gel electrophoresis analysis, and the reaction time with the optimal amplification efficiency is screened.

1.5 Specificity evaluation of RT-RPA detection System

CLBV, CTV, CYVCV, CEVd, CTLV, CPsV, SDV, xcc and CLas positive sample nucleic acid prepared in the previous step 1.2 are provided with CLBV positive control, healthy control and water control, and detection is carried out according to the RT-RPA method established in the step 1.4, so that the specificity of the established RT-RPA detection system is evaluated.

1.6 Sensitivity comparison of RT-RPA detection System

The CLBV positive nucleic acid is selected as a template, diluted by 10 times gradient, and parallel detection is carried out by respectively adopting the established RT-RPA and the RT-PCR reported in the literature, and the detection sensitivity is compared.

RT-PCR detection system: CLBV1F/CLBV5R 0.2 μl (10 μΜ/μl) each, prime Script 1Step Enzyme Mix 0.4 μl,2×1Step Buffer 5 μl, nuclease water 2.2 μl, RNA2 μl. Detection procedure: reverse transcription at 50 ℃ for 30min, pre-denaturation at 94 ℃ for 3min, denaturation at 94 ℃ for 30s, melting at 58 ℃ for 30s, extension at 72 ℃ for 30s, circulation for 35 times, extension at 72 ℃ for 5min again, termination of reaction at 12 ℃ and preservation at 4 ℃.

1.7 CLBV detection of citrus samples

And (3) extracting nucleic acid from 72 suspected CLBV citrus samples according to an RNA extraction kit, respectively adopting three methods of RT-PCR, RT-RPA and RT-qPCR to detect according to the reaction system and the procedure of the step (1.6), and respectively counting the CLBV positive detection rate of each detection method.

RT-qPCR detection system: CLBVF/CLBVR of 0.4 μl (10 μm/μl) each,

qPCR Master Mix 10. Mu.L, nuclease water 7.2. Mu.L, cDNA 2. Mu.L. Detection procedure: pre-denaturation at 95℃for 5min, denaturation at 95℃for 15s, melting at 58℃for 30s, extension at 72℃for 30s, fluorescence collection for 15s,35 cycles.

2 results

2.1 Establishment and optimization of RT-RPA detection system

2.1.1 screening of specific primers

Nucleic acid of a CLBV positive sample is selected as a template, healthy control and water control are set, and 3 pairs of designed RT-RPA primers are used for amplification. The results show (FIG. 1) that the primer RPACF1/RPACR1 can only amplify a relatively weak band with low amplification efficiency, while the primer RCLBV-F/RCLBV-R1 cannot amplify a target band.

The primer RCLBV-F/RCLBV-R2 can specifically amplify the orange sample infected with CLBV, the amplified band is single and bright, the amplified product size is 112bp, the size of the amplified product meets the size of the target band, and no specific band appears in the healthy control and the water control. The products amplified by the RT-RPA detection system are sent to Hua big gene company for sequencing, and the sequencing result is subjected to sequence comparison on NCBI, so that the sequence consistency of the sequencing result and the target gene sequence is 100%, and the primer RCLBV-F/RCLBV-R2 has the specificity of amplifying CLBV.

2.1.2 screening of reaction temperature and reaction time period conditions

RCLBV-F/RCLBV-R2 showed good amplification efficiency at 6 different temperature conditions: amplification efficiency increased with increasing temperature before 40 ℃; after 40 ℃, the amplification efficiency did not change significantly with increasing reaction temperature. Thus, 40℃is the optimal temperature for the reaction of the RT-RPA reaction system (FIG. 2).

Under the condition of 5 different reaction time periods, the RT-RPA detection system can carry out specific amplification: amplification yield increased with the extension of the reaction time before 30min of reaction; after 30min of reaction, the amplification yield gradually decreased with the extension of the reaction time. Thus, 30min is the optimal reaction time for the RT-RPA reaction system (FIG. 3).

2.2 Specificity of RT-RPA detection System

RT-RPA detection is carried out on the citrus positive samples infected with CTV, CYVCV, CEVd, CTLV, CPsV, SDV, xcc, CLas and CLBV by using an optimized RT-RPA detection system, the electrophoresis result is shown in figure 4, the established RT-RPA system only detects the specific strip of the CLBV on the citrus positive samples of the CLBV, and the detection on other citrus pathogens (CTV, CYVCV, CEVd, CTLV, CPsV, SDV, xcc and CLas) infection samples, healthy control and water control is negative. The established RT-RPA detection system has good specificity.

2.3 Sensitivity of RT-RPA detection system

Nucleic acid of CLBV positive sample is extracted, and detection shows that its concentration is 1.17 mug. Mu.L ^-1 . Serial concentration gradient templates are obtained through 10-time gradient dilution, RT-PCR and RT-RPA detection are carried out in parallel, and sensitivity comparison is carried out. As shown in FIG. 5, the minimum concentration of CLBV nucleic acid detectable by RT-RPA and RT-PCR was 1.17X10 ^-3 μg.μL ^-1 . From this, it was shown that the sensitivity of RT-RPA detection was comparable to that of RT-PCR.

2.4 citrus sample detection

Meanwhile, 72 citrus sample nucleic acid is detected by using three detection methods of RT-PCR, RT-RPA and RT-qPCR, the statistical result is shown in table 2, 11 citrus samples with CLBV are detected by all three detection methods, and the detection rate is 15.28% (figure 6, table 2). The detection results of the three detection methods are consistent. Therefore, the RT-RPA detection method established by the application is stable and reliable, and can be used for detecting the CLBV of the citrus sample. The detection rate of the CLBV is 15.2%, and the detection rate does not represent the probability that the field citrus plants commonly carry the CLBV, but only represents the number of plants carrying the CLBV in the citrus samples related to the application.

TABLE 2 CLBV detection of citrus samples

The application designs a specific primer on the CP gene conservation sequence of the CLBV, optimizes the reaction time, the reaction temperature and the like, and establishes an RT-RPA rapid detection system of the CLBV for the first time at home and abroad; the system is simple and convenient to operate, quick and sensitive in reaction, strong in specificity, capable of rapidly presenting detection results, free of temperature limitation of conventional RT-PCR, low in technical requirements on operators, convenient for basic-level personnel to rapidly detect CLBV, and wide in application prospect.

At present, an RT-PCR method and an RT-qPCR method are commonly adopted for detecting the CLBV, wherein the RT-qPCR method needs to be operated in a dark place and a precise instrument is required to carry out fluorescence acquisition, a standard curve is established and complex fluorescence data is analyzed, a great deal of time and energy are consumed, and the method is not simple and convenient; the common RT-PCR detection system is long in time consumption and has strong dependence on instruments. Compared with the two detection methods, the RT-RPA detection has the advantages of no need of light shielding, no dependence on precise instruments, no need of a separate reverse transcription step and capability of carrying out detection reaction under the constant temperature condition. Compared with the reverse transcription loop-mediated isothermal amplification (RT-LAMP) which is another constant temperature detection method, the RT-RPA method does not need to design complex probes and 4-6 primers, the reaction temperature is lower, the reaction time is shortened by half, all detection reactions can be completed only by 35min, the products are single, the result is clear, and the components of the reaction products of the RT-LAMP are complex, so that the subsequent sequencing analysis of the products is not facilitated. Therefore, the RT-RPA method has certain advantages over the RT-PCR method, the RT-qPCR method and the RT-LAMP method.

RNA is easy to degrade, and the reverse transcription efficiency problem exists in the reverse transcription process, so that in order to more accurately compare the detection sensitivity of the RT-RPA detection method and the common RT-PCR detection method, RNA directly extracted from citrus sample seeds is adopted as a template, thus reflecting the reverse transcription efficiency and the amplification efficiency, and having higher authenticity. In this study, the minimum CLBV nucleic acid concentration of RT-RPA and normal RT-PCR was 1.17X10 ^-3 μg.μL ^-1 The detection sensitivity of RT-RPA and common RT-PCR is shown to be in an order of magnitude.

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Claims

1. A method for detecting the RT-RPA of the citrus leaf mottle virus, which is characterized by comprising the following steps:

1) Extracting RNA of a sample to be detected;

2) Carrying out RT-RPA reaction by using the extracted sample RNA, wherein the nucleotide sequence of the specific primer is as follows:

an upstream primer: 5'-ATGAACACTCACGGCGATGAAATTCCCACA-3', downstream primer: 5'-GGATCCCCCATTAAATTCCATAAGCCAGTC-3';

the RT-RPA reaction conditions are as follows: the reaction temperature is 40 ℃ and the reaction time is 30 minutes;

3) And detecting a reaction product after the RT-RPA reaction is finished, and detecting a 112bp band indicates that the sample to be detected is a positive sample.

2. The method of claim 1, wherein the RT-RPA reaction is performed in a reaction system comprising: 10 mu.M of each of the upstream/downstream primers was 2. Mu.L, and 2. Mu.L of sample RNA was used as the RT-RPA amplification reagent.

3. The method of claim 1, wherein the reaction product is detected after completion of the RT-RPA reaction in step 3) by subjecting the amplified product to agarose gel electrophoresis or nucleic acid sequencing.