CN105177188A - Quadruple RT-PCR (reverse transcription-polymerase chain reaction) detection primers and method capable of simultaneously detecting multiple chili viruses - Google Patents

Abstract

The invention discloses quadruple RT-PCR (reverse transcription-polymerase chain reaction) detection primers and a method capable of simultaneously detecting multiple chili viruses. The method adopts four pairs of primers disclosed as SEQ ID NO:1-SEQ ID NO:8; and one RT-PCR process can simultaneously detect four chili viruses: Broad bean wilt virus 2 (BBWV 2), Pepper vein yellows virus (PeVYV), Chilli veinal mottle virus (ChiVMV) and Pepper mildmottle virus (PMMoV). Compared with the existing single RT-PCR and duplex RT-PCR detection methods, the method disclosed by the invention can simultaneously detect the four chili viruses by one RT-PCR process, and is capable of detecting more virus varieties, greatly enhancing the detection efficiency and obviously lowering the detection cost.

Description

Quadruple RT-PCR detection primers and method for simultaneous detection of multiple pepper viruses

technical field

The invention belongs to the technical field of crop virus detection, and in particular relates to a quadruple RT-PCR detection primer and a detection method for simultaneous detection of multiple pepper viruses.

Background technique

Capsicum (Capsicumannuum L.) is a plant of the genus Capsicum in the family Solanaceae. It is an annual or limited perennial herbaceous plant. The fruit is usually conical or oblong, green when immature, and bright red, green or purple when mature. Most common, its original range was from Mexico to Colombia; it is now cultivated all over the world. Capsicum has been cultivated in my country for hundreds of years and is an important vegetable and condiment. It is currently one of the main types of vegetables grown in large areas in my country. In addition to being an important vegetable crop, pepper has high nutritional value. The content of vitamin C occupies the first place in vegetables, which is 7-15 times that of tomato. In addition, capsaicin and dihydrocapsaicin in pepper are the main spicy flavors. The source of seasoning, the red pigment in chili is one of the most widely used natural food coloring agents at present, and chili also has the effect of sweating and repelling insects.

Since the 1970s, due to the frequent exchanges among various varieties, the scope of exchanges has continued to expand, and the farming system and other reasons, the occurrence of pepper virus diseases in my country has become wider and the degree of damage has increased, which has seriously affected the yield and quality of peppers. Yang Yonglin and others collected 3618 samples of pepper (sweet) pepper virus in six regions of Beijing, Tianjin, Liao, Jiangsu, Jilin and Xin, and identified tobacco mosaic virus (TMV ), Cucumbermosaicvirus (CMV), Potatovirus X (PotatovirusX, PVX), Potatovirus Y (PotatovirusY, PVY), Tobacco etchvirus (Tobaccoetchvirus, TEV), Alfalfa MosaicVirus (AMV), etc. Various viruses. According to current reports, there are at least 40 kinds of viruses that can infect pepper (sweet) peppers, and tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) are the most common in most countries. There are 17 RNA viruses identified on hot (sweet) peppers in my country, including CMV, TMV, Tobaccoetchvirus (TEV), Potatovirus Y (PotatovirusY, PVY), Potatovirus X virus (PotatovirusX, PVX), Peppermildmottle virus (PMMoV), alfalfa mosaic virus (AMV), broadbean wilt virus 2 (Broadbeanwiltvirus2, BBWV2), tobacco crisp virus (Tobaccorattlevirus, TRV), pepper ringspot virus (Chilliringspotvirus, ChiRSV) , tomato spotted wilt virus (Tomatospottedwiltvirus, TSWV), peanut yellow spot virus (Groundnutyellowspotvirus, GYSV), pepper chlorosis virus (Capsicumchlorosisvirus, CaCV), pepper vein mottle virus (Chilliveinalmottlevirus, ChiVMV), tobacco light green mosaic virus (Tobaccomildgreenmosaicvirus, TMGMV) ), the tomato chlorosis virus (Tomatochlorosisvirus, ToCV) and the pepper vein yellowing virus (Pepperveinyellowsvirus, PeVYV) detected in Hunan for the first time this year, these viral diseases generally cause 10% to 30% of the damage, serious It can reduce production by 50% to 80%.

At present, the commonly used methods for detecting plant viruses include biological methods (indicator plant method), serological methods (enzyme-linked immunosorbent assay), electron microscopy, and reverse transcription polymerase chain reaction (Reverse transcription polymerase chain reaction, RT-PCR). Among them, the biological method is time-consuming, heavy workload, and poor sensitivity. Different viruses often show similar symptoms after infection, and the symptomatic response will also be affected by combined infection. Serological detection methods may have false negatives and false positives within a certain range, and for tests with small test samples, the cost of the kit is relatively high. Each detection of biological methods and serological methods can only target a single virus, and the detection efficiency is low. Electron microscopy cannot determine the classification of Viridae or below the genus, and the equipment is expensive, so the number of samples should not be too large. RT-PCR has become the preferred method for pathogen detection because of its strongest specificity and highest sensitivity, and is widely used in plant virus detection. For complex infection of multiple viruses, multiple RT-PCR methods can be used to detect multiple viruses at the same time, which can detect a large number of samples in a short time, improve the detection efficiency, reduce the detection cost, and the detection cost is low, which is better than other detection methods. Incomparable. However, there are many studies on single-plex RT-PCR detection of pepper virus, but few studies on multiple RT-PCR. KumarS et al established a dual RT-PCR that can simultaneously detect TMV and ToMV in pepper or tomato. A variety of multiple detection methods have been developed for common viruses such as TMV, CMV, PVY, and ToMV at home and abroad. However, no corresponding multiple detection methods have been found for the domestically reported and newly discovered pepper viruses.

According to the conserved sequence of the whole genome or partial sequence of the virus released by Genbank, the present invention designs universal primers, aiming to establish a simple, efficient and sensitive quadruple RT-PCR detection technology, and simultaneously detect the relatively common viruses and the latest discovery of pepper found in China Viruses that may have an outbreak trend: Broadbean wilt virus 2 (BBWV2), pepper mild mottle virus (PMMoV), pepper vein mottle virus (ChiVMV), pepper vein yellow virus (Pepperveinyellowsvirus), for the research of domestic The epidemic situation of pepper virus disease and the virus detection of virus-free pepper species provide technical support.

Contents of the invention

The technical problem to be solved by the present invention is: aiming at the above-mentioned deficiencies in the prior art, provide a kind of high-efficiency detection primer and method for quadruple RT-PCR detection of multiple capsicum viruses at the same time, for four kinds of capsicum viruses found in China: broad bean wilting Virus No. 2 (BBWV2), Pepper Vein Yellowing Virus (PeVYV), Pepper Vein Mottle Virus (ChiVMV) and Pepper Mild Mottle Virus (PMMoV), according to the conserved sequences of the whole genome or partial sequences of the four viruses released by Genbank, design primers, and provide the quadruple RT-PCR detection method of the present invention to detect the four viruses simultaneously.

In order to solve the above-mentioned technical problems, the technical scheme adopted in the present invention is: for 4 kinds of capsicum viruses in my country (Broad Bean Wilt Virus No. Mottle virus (PMMoV)) genome conserved sequence designed 4 pairs of primers, and established a quadruple RT-PCR reaction system and procedures to simultaneously detect the 4 pepper viruses.

The detection method of the present invention is specifically as follows:

1. Primer design:

According to the conserved sequences of the above-mentioned 4 kinds of capsicum virus genomes or partial sequences released by Genbank, and according to the requirements of the different sizes of the quadruple RT-PCR products, four pairs of primers (Table 1) were designed, wherein the primer pairs BBWV2-F and BBWV2- R is the primer for detecting broad bean wilt virus No. 2 (BBWV2), the fragment size is 693bp; the primer pair PevYV-F and PevYV-R are the primers for detecting pepper vein yellowing virus (PeVYV), the fragment size is 383bp; F and ChiVMV-R are primers for detecting pepper vein mottle virus (ChiVMV), and the fragment size is 513bp; the primer pair PMMoV-F and PMMoV-R are primers for detecting pepper vein mottle virus (PMMoV), and the fragment size is 236bp.

Table 1 Quadruple RT-PCR detects the primers of capsicum virus and the virus it detects

2. RT-PCR process:

1) Acquisition of RNA from pepper plant leaves: take young pepper leaves with mosaic symptoms in the field, store them on ice, and extract total RNA by Trizol method;

2) Using the RNA obtained in step 1) as a template, using the 6-base random primer RandomPrimers as a reverse primer, and obtaining cDNA by reverse transcription;

3) Using the cDNA obtained in step 2) as a template, perform PCR amplification with four pairs of primers in Table 1.

The above-mentioned quadruple PCR reaction system, based on 50 μl: 10xPCRBUFFER 5 μl, dNTPs 5 μl, PCRTaq 2 μl, template (cDNA) 2.5 μl, broad bean wilt virus No. 2 forward primer and reverse primer 0.5 μl each, pepper vein yellowing virus forward primer and reverse primer Add 1.25 μl each of the primers, 1 μl each of the forward primer and reverse primer of pepper vein mottle virus, 1.25 μl each of the forward primer and reverse primer of pepper vein mottle virus, and make up to the required volume with ultrapure water.

PCR program: pre-denaturation at 94°C for 10 minutes; 40 cycle parameters: 94°C for 30s, 54°C for 30s, 72°C for 1min; finally 72°C for 10min.

4) Gel electrophoresis of the PCR product, taking pictures with the gel imaging system, and judging the virus infection according to the size of the target fragment and the positive control.

The present invention has the following beneficial effects: four common pepper viruses are detected by quadruple RT-PCR, compared with the current single RT-PCR and double RT-PCR detection methods, four viruses can be detected by one RT-PCR, and the The types of viruses increase, the detection efficiency is improved, and the cost of detection is significantly reduced.

Description of drawings

Figure 1 is the electrophoresis of broad bean wilt virus 2 (BBWV2), pepper vein yellowing virus (PeVYV), pepper vein mottle virus (ChiVMV) and pepper mild mottle virus (PMMoV) detected by single and quadruple RT-PCR.

Among them, M: DNAmarker; 1 is the quadruple PCR product of broad bean wilting virus 2 (BBWV2), pepper vein yellowing virus (PeVYV), pepper vein mottle virus (ChiVMV) and pepper mild mottle virus (PMMoV); 2, 3 , 4, and 5 are the PCR products of broad bean wilt virus 2 (BBWV2), pepper vein mottle virus (ChiVMV), pepper mild mottle virus (PMMoV) and pepper vein yellowing virus (PeVYV), respectively.

Figure 2 is the electrophoresis of broad bean wilt virus 2 (BBWV2), pepper vein yellowing virus (PeVYV), pepper vein mottle virus (ChiVMV) and pepper mild mottle virus (PMMoV) detected by quadruple RT-PCR at different annealing temperatures.

Among them, M: DNAmarker; 1-6 represent the temperatures of 60°C, 58°C, 56°C, 54°C, 52°C and 50°C in sequence.

Figure 3 is the electrophoresis of broad bean wilt virus 2 (BBWV2), pepper vein yellowing virus (PeVYV), pepper vein mottle virus (ChiVMV) and pepper mild mottle virus (PMMoV) detected by quadruple RT-PCR with different dilution multiple templates.

Among them, M: DNAmarker; the initial template concentration is 125ng/μl, and the RNA concentrations represented by 1-6 are 10 ⁰ , 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 and 10 ^-5 times the initial concentration.

Figure 4 is the electrophoresis of broad bean wilting virus 2 (BBWV2), pepper vein yellowing virus (PeVYV), pepper vein mottle virus (ChiVMV) and pepper mild mottle virus (PMMoV) detected by quadruple RT-PCR in Hunan pepper plant samples .

Among them, M: DNAladder; positive indicates positive control; CK indicates negative control; lanes 1-10 are samples from Liuyang City, Hunan Province, and lanes 11-15 are pepper samples from Furong District, Changsha City, Hunan Province.

Detailed ways

The present invention will be further explained below in conjunction with specific examples, but the specific examples do not limit the present invention in any way.

Example 1

1. Primer design

Due to the need to detect 4 kinds of pepper viruses, in order to establish a multiplex RT-PCR detection method with the characteristics of many types of viruses, simple, accurate and economical detection steps, Primer6. 0 Design primers Design primer pair (forward primer BBWV2-F and reverse primer BBWV2-R), the target fragment size of amplification is 693bp; According to the conservative sequence of pepper vein yellowing virus (PeVYV), design primer pair (forward Primer PevYV-F and reverse primer PevYV-R), the amplified target fragment size is 383bp; also design primer pairs (forward primer ChiVMV-F and reverse primer ChiVMV-R, and forward primer PMMoV-F and reverse primer PMMoV-R), the amplified target fragment sizes are 513bp and 236bp respectively; the primer sequences are shown in Table 1. The sizes of the fragments amplified by each pair of primers are 693, 383, 513 and 236bp respectively. The size of the fragments is quite different, there will be no overlap, and they can be clearly separated. When designing the primers, the differences between each pair of primers and several other viruses are excluded Mismatches and complementarity between primers. The target fragments were cloned and sequenced, and all of them were specific fragments of each virus.

The above 4 pairs of primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

2. Extraction of capsicum total RNA

Take pepper leaf samples that show symptoms such as bright veins, mosaic leaves, dwarfing, shrinkage, curling, yellowing, dead leaves, and arbuscular branches in the field. Take 2.5g of each sample, add liquid nitrogen to grind, and use RNA kit Total RNA was extracted, and the quality and concentration of RNA were detected by electrophoresis and ultraviolet spectrophotometer.

3. Use the above primers to perform single-plex RT-PCR and quadruple RT-PCR amplification on the extracted total RNA, respectively.

The reverse transcription process is as follows:

RNA denaturation: Take 2.5 μl of RNA and incubate at 65°C for 8 minutes, then cool on ice;

Prepare reverse transcription reaction solution (7.5 μl): 2 μl of 5× reaction buffer, 1 μl of 100 mM DTT, 1 μl of 10 mM dNTPs, 0.125 μl of RNase inhibitor, 0.5 μl of reverse transcriptase, 0.5 μl of 10 μM random primer, and make up to 7.5 μl with ultrapure water.

Add 7.5 μl of reaction solution to 2.5 μl of denatured RNA, mix well, extend at 42°C for 1 hour, and denature at 95°C for 2 minutes.

PCR process:

Single-plex PCR reaction system (50 μl): 5 μl of 10xPCRBUFFER, 5 μl of dNTPs, 2 μl of PCRTaq, 2.5 μl of template (cDNA), 2 μl of each forward primer and reverse primer in Table 1, and make up to the required volume with ultrapure water.

Quadruple PCR reaction system (50 μl): 10xPCRBUFFER 5 μl, dNTPs 5 μl, PCRTaq 2 μl, template (cDNA) 2.5 μl, broad bean wilt virus No. 2 forward primer and reverse primer 0.5 μl each, pepper vein yellowing virus forward primer and reverse primer 1.25 μl each, 1 μl each of the forward primer and reverse primer of pepper vein mottle virus, 1.25 μl each of the forward primer and reverse primer of pepper vein mottle virus, and make up to the required volume with ultrapure water.

PCR program: pre-denaturation at 94°C for 2min; 40 cycle parameters: 94°C for 30s, 54°C for 30s, 72°C for 1min; finally 72°C for 10min.

4. Gel electrophoresis

Take 10 μl of the PCR product and use 2.0% agarose gel electrophoresis, and take pictures with a gel imaging system. The results are shown in Figure 1. The results showed that quadruple RT-PCR could detect broad bean wilt virus 2 (BBWV2), pepper vein yellowing virus (PeVYV), pepper vein mottle virus (ChiVMV) and pepper mild mottle virus (PMMoV) simultaneously.

Example 2 Effects of different annealing temperatures and different template dilution factors on the detection effect of quadruple RT-PCR

Since the Tm values of each pair of primers are not the same, it is necessary to select a suitable annealing temperature to obtain four target fragments by quadruple RT-PCR and improve detection sensitivity. The annealing temperature was set to 6 temperature gradients of 60°C, 58°C, 56°C, 54°C, 52°C and 50°C. The electrophoresis results of PCR products are shown in Figure 2. The results showed that when the annealing temperature was 58-54°C, four clear target fragments could be obtained by quadruple RT-PCR.

In order to test the sensitivity of quadruple RT-PCR detection, the initial RNA concentration was 125ng/μl, diluted 10, 100, 1000 and 10000 times and 100000 respectively, that is, the RNA concentration was 10 ^-1 , 10 ^-2 , 10 ^-3 of the initial concentration and 10 ^-4 and 10 ^-5 times. The electrophoresis results of the PCR products are shown in Figure 3. It can be seen that when the initial RNA is diluted 10-100 times, 4 target fragments can be amplified, which shows that the sensitivity of this RT-PCR detection technology is high.

Embodiment 3 quadruple RT-PCR detection field capsicum plant sample

Pepper plants with yellowing and mosaic symptoms were investigated in the field in Furong District, Changsha City, Hunan Province and the main pepper producing areas of Liuyang City. The plants were brought back to the laboratory with soil, and the young leaves were selected, ground with liquid nitrogen, and RNA was extracted. A good quadruple RT-PCR detects its virus infection, and the test results are shown in Figure 4. The results showed that the quadruple RT-PCR can detect the viruses in the pepper samples from the main production areas of Hunan very well, and the pepper plant samples from the two main production areas of Hunan, Changsha and Liuyang, generally have 3-4 kinds of virus co-infection.

Claims (5)

1. a kind of quadruple RT-PCR detection primer that detects multiple capsicum viruses simultaneously, this capsicum virus is: broad bean wilt virus No. 2, capsicum vein yellowing virus, capsicum vein mottle virus and capsicum light mottle virus, it is characterized in that, The primers include the following 4 pairs of primers: The primer pair for detecting broad bean wilt virus No. 2 is: BBWV2-F: CTCCTGTGGTGGTTATTCA, BBWV2-R: TTCCTATGCGTGTTATCGT; The primer pair for detecting pepper vein yellowing virus is: PevYV-F:GAAGAAGCCGAGATAGAGTT, PevYV-R: ATAGAGCAGCTGAATTGAT; The primer pair for detecting pepper vein mottle virus is: ChiVMV-F: AACCTTGAACACCTTCTTGA, ChiVMV-R: GTCCAGTCCGAACATCCT; The primer pair for detection of pepper light mottle virus is: PMMoV-F:GTCCAACAGCAGTTCTCT, PMMoV-R: CTAATAGCCACCGTAGCAT. 2. A quadruple RT-PCR detection method for simultaneously detecting multiple capsicum viruses is characterized in that the method is to extract capsicum total RNA; with the obtained RNA as a template, by reverse transcription, obtain cDNA; with the obtained cDNA As a template, carry out PCR amplification to it with 4 pairs of primers described in claim 1, and identify by electrophoresis at last. 3. the quadruple RT-PCR detection method of simultaneously detecting multiple capsicum viruses as claimed in claim 2, is characterized in that: described PCR reaction system is: 10xPCRBUFFER5 μ l, dNTPs5 μ l, PCRTaq2 μ l, cDNA template 2.5 μ l, broad bean wilt virus 2 No. forward primer and reverse primer 0.5 μl each, pepper vein yellowing virus forward primer and reverse primer 1.25 μl, pepper vein mottle virus forward primer and reverse primer each 1 μl, pepper light mottle virus forward primer and 1.25 μl of each reverse primer, supplemented with ultrapure water to 50 μl. 4. The quadruple RT-PCR detection method for simultaneously detecting multiple pepper viruses as claimed in claim 2, characterized in that: the PCR reaction program is: 92°C for 10min; 94°C for 30s, 54-58°C for 30s, 72°C 1min at ℃, 40 cycles; 10min at 72℃. 5. the quadruple RT-PCR detection kit that contains the primer pair described in claim 1 to detect multiple capsicum viruses simultaneously.