CN114703318A - Flue-cured tobacco aphid-borne virus multiplex composite PCR detection method - Google Patents

Abstract

The invention belongs to the technical field of plant virology detection, and particularly relates to a flue-cured tobacco aphid-borne virus multiplex composite PCR detection method, which comprises the following steps: (1) designing specific primers for amplifying Cucumber Mosaic Virus (CMV) and Potato Virus Y (PVY)2 virus gene segments; (2) extracting total RNA of the sample; (3) performing reverse transcription reaction on a tobacco plant total RNA sample containing virus RNA at high temperature by using the sample as a template to synthesize a cDNA first chain; (4) obtaining a PCR amplification product by using a single-tube multiplex PCR amplification reaction; (5) and carrying out agarose gel electrophoresis detection on the obtained PCR amplification product. The detection system can identify various compositely infected viruses through one-time PCR reaction, is suitable for quickly and efficiently identifying a large number of tobacco leaf samples at a basic detection site, and lays a foundation for subsequent virus prevention and control work such as early discovery of infected tobacco plants, antiviral genetic breeding and the like.

Description

Flue-cured tobacco aphid-borne virus multiplex composite PCR detection method

Technical Field

The invention belongs to the technical field of detection, further belongs to the technical field of plant virology detection, and particularly relates to a flue-cured tobacco aphid-borne virus multiplex PCR detection method.

Background

The diseases caused by Potato Virus Y (PVY) are also called tobacco vein belt disease and tobacco vein spot virus disease. Different symptoms are exhibited due to different virus strains. The main types include vein-band floral leaf type, vein spot type and chlorosis spot type. The vein belt type has yellow green flower leaves at the upper part of the tobacco plant, light color between veins, dark green at two sides of the vein, obvious vein belt formation, rolling leaves or burning spots in serious cases, abnormal mature leaves, uneven color, reduced quality and dwarfing of the tobacco plant. The lower leaf of vein spot type is diseased, the leaf is yellowish brown, the main side vein is gray black or reddish brown and necrotic from the base of the leaf, the petiole is crisp, the vascular bundle is browned when the main side vein is picked off, and the stalk is reddish brown or black necrotic. The initial stage of the chlorosis speckle pattern is similar to the pulse zone pattern, but the upper leaves are chlorosis speckles, the middle and lower leaves are brown or white small necrotic spots, the lesions are irregular, and the spots are dense in the whole leaves and form perforations or fall off when the lesions are serious. Clear veins appear at the early stage of the disease of the potato Y virus disease tobacco plant, mosaic mottle is formed soon, then the color between the lobular veins becomes light, and tissues on two sides of the lobular veins are in a dark green belt shape. The veins of diseased plants infected with necrotic strains are brownish to black in color.

Cucumber Mosaic Virus (CMV) is a very serious viral disease and the virus can reach anywhere but the growth point. Cucumber mosaic virus is one of the most host-wide, most widespread, and most economically important plant viruses. All tobacco growing areas around the world have the distribution and harm of the virus. The world is distributed in great part in the United kingdom, Germany, Denmark, Russia, India, Japan, Korea, Greece, Romania, Hungary, Czech, Bulgaria, Brazil, Ireland, Mouduoe, Sweden, Finland, Poland, and so on, as well as in most parts of our country.

The two viruses are both transmitted by vectors such as myzus persicae in a non-continuous manner, the transmission distance is wide, the prevention and control method is limited, and great influence is brought to the quality and the yield of tobacco leaves.

Disclosure of Invention

In order to solve the technical problems of time and labor waste and high cost when detecting the Potato Virus Y (PVY) and the Cucumber Mosaic Virus (CMV), the invention provides a flue-cured tobacco aphid-borne virus multiplex PCR detection method.

The technical scheme is as follows:

A. specific primers for amplifying Cucumber Mosaic Virus (CMV) and Potato Virus Y (PVY)2 virus gene fragments are designed:

CMV: upstream primer (CMV _ CP _ F1) 5'-gcagctggtcgtccaactat-3';

downstream primer (CMV _ CP _ R1) 5'-gactgtcacccacacggtag-3';

PVY: an upstream primer (PVY _ CP _ F1) 5'-ccagccaaacccgaacaaag-3';

downstream primer (PVY _ CP _ R1) 5'-tactgatgccaccgtccaac-3';

B. extracting total RNA of the sample;

C. adding the extracted tobacco plant total RNA sample containing the virus RNA into a PCR tube without RNA enzyme as a template to carry out reverse transcription reaction to synthesize a cDNA first chain;

D. obtaining a PCR amplification product by using a single-tube multiplex PCR amplification reaction;

E. and carrying out agarose gel electrophoresis detection on the obtained PCR amplification product.

Preferably, in step a, Primer design tool (Primer designing tool) of NCBI platform is used to select the Primer pair with largest difference in length of the coverage area for coat proteins in genomes of two Cucumber Mosaic Virus (CMV) and Potato Virus Y (PVY) under the condition of same annealing temperature.

Preferably, the First Strand of cDNA synthesized in step C is the Thermo Scientific RevertAID First Strand cDNA Synthesis Kit, with a reaction total of 25 uL; the method comprises the following specific steps: supplementing 2uL total RNA and 1uL random hexamer primer to 17uL with high-purity water without nuclease, gently mixing, centrifuging for a short time, incubating at 65 ℃ for 5 minutes, cooling on ice, centrifuging, and cooling on ice; then, 5 Xreaction Buffer 4uL, RiboLock RNase Inhibitor enzyme Inhibitor (20U/. mu.L) 1uL, 10mM dNTP Mix 2. mu.L and RevertAId M-MuLV reverse transcriptase (200U/. mu.L) 1. mu.L were added thereto, and the mixture was gently centrifuged, mixed, incubated at 25 ℃ for 5 minutes, then at 42 ℃ for 60 minutes, heated at 70 ℃ for 5 minutes to terminate the Reaction, and stored.

Preferably, in step D the multiplex PCR amplification reaction: the total reaction system for PCR was 25 uL: comprises 1uL of cDNA template, 10mM of each of two groups of upstream and downstream primers (CMV _ CP _ F1, CMV _ CP _ R1, PVY _ CP _ F1 and PVY _ CP _ R1), 13uL of reaction enzyme (Qiagen Multiplex PCR Master Mix) and the balance of sterilized ultrapure water; the reaction procedure is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, with 25 cycles; finally, extension was carried out at 72 ℃ for 7 min.

Preferably, in step D, the multiplex PCR amplifies a 240bp fragment for cucumber mosaic virus and a 660bp fragment for potato virus Y.

Compared with the prior art, the invention has the following two beneficial effects:

(1) the detection system can identify the type of RNA virus according to the length of an amplified strip through one-time PCR reaction, identifies multiple viruses infected in a complex way at one time, has large difference of the sizes of the amplified strips, is suitable for operators with insufficient experience to identify, and reduces the detection time;

(2) the detection method is very suitable for rapidly and efficiently identifying a large quantity of tobacco leaf samples at a basic level detection station, and lays a foundation for subsequent early discovery of infected tobacco plants and virus prevention and control work such as antiviral genetic breeding and the like.

Drawings

FIG. 1 shows the test strip test results of sample D.

FIG. 2 shows the electrophoresis results of two types of virus-borne viruses of flue-cured tobacco aphids.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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.

Biological sample: the sample D was collected from Guizhou tobacco area, and the tobacco was infected with Cucumber Mosaic Virus (CMV) and Potato Virus Y (PVY) at the same time.

Plant Mini Kit (Qiagen); RevertAId First Strand cDNA Synthesis Kit (Thermo Scientific); qiagen Multiplex PCR Master Mix is commercially available.

Example 1

Designing a primer:

firstly, searching and downloading a genome sequence (serial number: NC-001367) of Potato Virus Y (PVY) and a sequence (serial number: NC-001440) of a III-th fragment of Cucumber Mosaic Virus (CMV) genome from GenBank of the National Center for Biotechnology Information (NCBI), determining the range of Coat Proteins (CP) in two genomes, designing specific primers by using Primer design tool (Primer designing tool) of an NCBI platform, and selecting a Primer pair with the largest difference of the length of a coverage area under the condition of annealing temperature of 55 ℃; the primer pairs obtained were as follows:

CMV: upstream primer (CMV _ CP _ F1) 5'-gcagctggtcgtccaactat-3';

downstream primer (CMV _ CP _ R1) 5'-gactgtcacccacacggtag-3';

PVY: an upstream primer (PVY _ CP _ F1) 5'-ccagccaaacccgaacaaag-3';

downstream primer (PVY _ CP _ R1) 5'-tactgatgccaccgtccaac-3';

example 2

Reverse transcription of random primer to synthesize the first strand of cDNA:

the Agdia brand test strip is used for identifying the type of the sample D infecting virus, the CMV test strip (left) and the PVY test strip both have two strips and show positive reaction, and the result is shown in figure 1;

approximately 100mg of leaf tissue (without main veins) was cut from the infected tobacco leaves (sample D), and ground thoroughly in a bowl with liquid nitrogen, using

The procedure of the Plant Mini Kit (Qiagen) extracts total RNA from the milled powder;

the random primer reverse transcription process uses RevertAID First Strand cDNA Synthesis Kit (Thermo Scientific), each reaction establishes 25uL reaction system according to the formula of reaction reagent in the instruction, wherein 2uL total RNA is included as template, the steps are as follows:

(1) sequentially adding reactants in the following table 1 into a sterile nuclease-free PCR tube placed on ice, gently mixing uniformly, centrifuging briefly, incubating at 65 ℃ for 5 minutes, cooling on ice, centrifuging, and then placing on ice for cooling;

TABLE 1

(2) The following Table 2 components were added in order

TABLE 2

5X Reaction Buffer	4μL
		RiboLock RNase Inhibitor (20U/. mu.L)	1μL
10mM dNTP Mix	2μL
		RevertAId M-MuLV reverse transcriptase (200U/. mu.L)	1μL
Total volume	25μL

Slightly centrifuging, mixing, incubating at 25 deg.C for 5min, incubating at 42 deg.C for 60 min, heating at 70 deg.C for 5min to terminate reaction, and storing;

example 3

Multiplex PCR detection:

the experimental system contained cDNA and the blank was a reaction without DNA, and both reactions were repeated twice.

The PCR reaction system was 25 uL: comprises cDNA template 1uL, two groups of upstream and downstream primers (CMV _ CP _ F1, CMV _ CP _ R1, PVY _ CP _ F1 and PVY _ CP _ R1) each of 10mM, reaction enzyme (Qiagen Multiplex PCR Master Mix)13Ul, and the rest volume of sterilized ultrapure water;

the PCR program designed based on similar annealing temperatures (55 ℃) for the two primer pairs was as follows:

example 4

Agarose gel electrophoresis:

1% agarose gel was prepared with 0.5XTAE buffer, cooled and then 5uLPCR products were added and electrophoresed at 5 v/cm. After electrophoresis, the gel was stained in a solution containing ethidium bromide for 5min, rinsed with clear water and observed under an ultraviolet lamp, and the results are shown in FIG. 2 below:

the results show that the primer mixture system amplified two bands from two repeated reactions (D1 and D2) of sample D, the sizes of the bands are 240bp and 660bp respectively, the bands correspond to two RNA viruses of CMV and PVY respectively, no reaction occurs in blank controls (N1 and N2), and the detection result is matched with the result that the sample D is infected with CMV and PVY simultaneously.

Sequence listing

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Claims (5)

1. A detection method of flue-cured tobacco aphid-borne virus multiplex PCR is characterized in that the detection method of tobacco mosaic virus multiplex PCR comprises the following steps:

A. specific primers for amplifying Cucumber Mosaic Virus (CMV) and Potato Virus Y (PVY)2 virus gene fragments are designed:

CMV: upstream primer (CMV _ CP _ F1) 5'-gcagctggtcgtccaactat-3';

downstream primer (CMV _ CP _ R1) 5'-gactgtcacccacacggtag-3';

PVY: an upstream primer (PVY _ CP _ F1) 5'-ccagccaaacccgaacaaag-3';

a downstream primer (PVY _ CP _ R1) 5'-tactgatgccaccgtccaac-3';

B. extracting total RNA of the sample;

C. adding the extracted tobacco plant total RNA sample containing the virus RNA into a PCR tube without RNA enzyme as a template to carry out reverse transcription reaction to synthesize a cDNA first chain;

D. obtaining a PCR amplification product by using a single-tube multiplex PCR amplification reaction;

E. and carrying out agarose gel electrophoresis detection on the obtained PCR amplification product.

2. The detection method according to claim 1, wherein in step A, Primer pairs with the largest difference in length of the coverage area are selected for coat proteins in two genomes of Cucumber Mosaic Virus (CMV) and Potato Virus Y (PVY) under the same annealing temperature conditions by using Primer design tool (Primer designing tool) of NCBI platform.

3. The assay of claim 1, wherein the First Strand of cDNA synthesized in step C is performed using the Thermo Scientific revertID First Strand cDNA Synthesis Kit, with a reaction total of 25 uL; the method comprises the following specific steps: supplementing 2uL total RNA and 1uL random hexamer primer to 17uL with high-purity water without nuclease, gently mixing, centrifuging for a short time, incubating at 65 ℃ for 5 minutes, cooling on ice, centrifuging, and cooling on ice; then, 5 Xreaction Buffer 4uL, RiboLock RNase Inhibitor enzyme Inhibitor (20U/. mu.L) 1uL, 10mM dNTP Mix 2. mu.L and RevertAId M-MuLV reverse transcriptase (200U/. mu.L) 1. mu.L were added thereto, and the mixture was gently centrifuged, mixed, incubated at 25 ℃ for 5 minutes, then at 42 ℃ for 60 minutes, heated at 70 ℃ for 5 minutes to terminate the Reaction, and stored.

4. The detection method according to claim 1, wherein in step D the multiplex PCR amplification reaction: the PCR reaction system was 25 uL: comprises 1uL of cDNA template, 10mM of each of two groups of upstream and downstream primers (CMV _ CP _ F1, CMV _ CP _ R1, PVY _ CP _ F1 and PVY _ CP _ R1), 13uL of reaction enzyme (Qiagen Multiplex PCR Master Mix) and the balance of sterilized ultrapure water; the reaction procedure is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, with 25 cycles; finally, extension was carried out at 72 ℃ for 7 min.

5. The detection method according to claim 1, wherein in step D, the PCR amplification reaction amplifies a 240bp fragment for cucumber mosaic virus and a 660bp fragment for potato virus Y.

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