RT-PCR Primers and Methods for Detecting ParV2, a New Virus from Paris polyphylla

Abstract

The present invention discloses RT-PCR detection primers and methods for ParV2, a new virus of Paris polyphylla. The present invention identifies a new pathogen of the genus Potyvirus Y on plants of the family Solanaceae, which is reported for the first time. Since there is no detection method for the pathogen, the present invention designs specific detection primers / probes based on the full genome sequence of the pathogen, establishes a method for detecting the pathogen, and expands the detection range of diseases related to virus infection of precious Chinese medicinal materials (such as Paris polyphylla, a plant of the family Solanaceae).

Description

Technical Field

[0001] The invention belongs to the technical field of plant disease diagnosis, and specifically relates to an RT-PCR detection primer and method for a new Paris polyphylla virus ParV2. Background Art

[0002] Paris polyphylla is a perennial herbaceous plant of the genus Paris in the family Melanthiaceae. It is mainly distributed in Yunnan, Guizhou, Fujian, Hubei and other places. It is a traditional precious Chinese medicinal material. The steroidal saponins rich in its rhizomes are its main active substances, which have anti-viral, anti-tumor, anti-myocardial ischemia and other effects. The main ones with higher medicinal value are Paris polyphylla var. yunnanensis and Paris polyphylla var. chinensis.

[0003] Paris polyphylla is very susceptible to virus infection and damage during its growth. So far, there are 9 viruses that harm Paris polyphylla, including Paris virus 1 (ParV1), Turnip mosaic virus (TuMV), Paris mosaic necrosis virus (PMNV), and Chilli veinal mottle virus (ChiVMV) of the genus Potyvirus, Paris polyphylla virus X (PVX) of the genus Potexvirus, Pepper Mild Mottle Virus (PMMoV) and Tobacco Mosaic Virus (TMV) of the genus Tobamovirus, Yunnan paris negative-stranded virus (YPNSV) of the genus Coguvirus, and Paris alphapartitivirus 1 (ParAPV1) of the genus Alphapartitivirus.

[0004] In 2020, during a disease survey of Paris polyphylla in Fujian Province and North China, some Paris polyphylla plants were found to have mosaic, mottled and curled leaves. Through high-throughput sequencing, the pathogen was identified as a new virus of the genus Potyvirus. In the context of this application, the virus is referred to as Parispotyvirus 2 (ParV2).

[0005] Paris polyphylla is a perennial herb, and its roots can only be harvested for medicinal purposes after 5-7 years of planting. Therefore, it is particularly important to promptly detect and identify pathogens and to prevent and control diseases during the growth cycle. Establishing a comprehensive, efficient, and rapid virus detection system is an important step in identifying emerging viruses. For common Paris polyphylla virus diseases in my country, detection technologies for ParV1, TuMV, PMNV, PVX, PMMoV, and TMV have been established, but there is no effective detection method for the new virus ParV2. Summary of the invention

[0006] The purpose of the present invention is to provide a RT-PCR detection primer and method for a new Paris polyphylla virus ParV2.

[0007] An isolated organism comprises a nucleotide selected from the following: a nucleotide shown in SEQ ID No: 2 in the sequence list or a fragment thereof, or a nucleotide complementary to the nucleotide shown in SEQ ID No: 2 in the sequence list.

[0008] The organism is Potyvirus; and the nucleotide is RNA.

[0009] An isolated nucleotide comprises a nucleotide selected from the following: a nucleotide shown in SEQ ID No: 1 in the sequence listing or a fragment thereof; a nucleotide complementary to the nucleotide shown in SEQ ID No: 1 in the sequence listing; a nucleotide shown in SEQ ID No: 2 in the sequence listing or a fragment thereof; a nucleotide complementary to the nucleotide shown in SEQ ID No: 2 in the sequence listing.

[0010] A primer or probe, which is specific to or complementary to the following nucleotides: the nucleotide shown in SEQ ID No: 1 in the sequence listing or a fragment thereof; the nucleotide complementary to the nucleotide shown in SEQ ID No: 1 in the sequence listing; the nucleotide shown in SEQ ID No: 2 in the sequence listing or a fragment thereof; the nucleotide complementary to the nucleotide shown in SEQ ID No: 2 in the sequence listing.

[0011] The primer or probe is selected from the nucleotides shown in SEQ ID No: 15-24 in the sequence listing.

[0012] The use of the primers or probes in diagnosing plant virus infection, the plant is one or more of Polygonum multiflorum, Polygonum aviculare, Polygonum multiflorum, Polygonum hainanensis, Polygonum lingyun, Polygonum xichou, Polygonum nanense, Polygonum burma, Polygonum jinxian, Polygonum ovale, Polygonum dali, Polygonum multiflorum, Polygonum dwarfum, Polygonum whitii, Polygonum narrow-leaved, Polygonum wide-leaved, Polygonum long-seed, Polygonum large-calyx, Polygonum rolled, Polygonum hairy, Polygonum variegated, Polygonum luxuriant, Polygonum spherical, Polygonum spherical, Polygonum spherical with wide petals, Polygonum spherical with short petals, Polygonum spherical, Polygonum black-seeded, Polygonum no-seed, Polygonum roxburghii, Polygonum chinense with red fruits, Polygonum flat-cut, Polygonum long-column, Polygonum wrinkled, Polygonum dlong, Polygonum bashanensis, Polygonum niger, Polygonum niger, Polygonum japanensis, Polygonum no-seed, Polygonum niger, Polygonum spherical, Polygonum niger,

[0013] The technician understands that although a pair of specific primer pairs is used for the detection of new organisms in the specific embodiments, this pair of primer pairs is only exemplary and cannot be interpreted as limiting the scope of protection of this application. The technician knows the principle of primer amplification of the target sequence and the principle of probe binding to the target sequence according to textbooks and the principle of nucleotide sequence complementarity (for example, "Molecular Cloning Laboratory Guide" J. Sambrook, MR Green, 2017; P450 "Design of PCR primers using Primer3 Plus"; Chapter 13 "Preparation of labeled DNA probes, RNA probes and oligonucleotide probes"), and is also clear about the design principles of primers and probes. There are a variety of primer / probe design software in the prior art, such as Primer Premier, Oligo7, BeaconDesigner, etc. When the technician knows the genome sequence of the organism or its functional fragment (such as ORF), the sequence information and structural information of the specific primer or probe can be obtained.

[0014] As an example, in a preferred embodiment of the present invention, specific primers for organisms can be designed by the following principles:

[0015] 1) The length of the primer is 15 bp to 30 bp (including the end point, the same below);

[0016] 2) the G+C content of the primer is 40% to 60%;

[0017] 3) There should not be four consecutive bases of complementarity within or between the primers;

[0018] 4) The Tm value of the primer is 55±5°C;

[0019] 5) The length of the amplification product obtained by the primers is 300 bp to 3000 bp (preferably 2000, preferably 1000 bp);

[0020] 6) The primer is complementary to a nucleotide selected from the following:

[0021] - the nucleotide sequence shown in SEQ ID No: 1 or a fragment thereof;

[0022] - a nucleotide complementary to the nucleotide shown in SEQ ID No: 1;

[0023] - the nucleotide sequence shown in SEQ ID No: 2 or a fragment thereof;

[0024] - a nucleotide complementary to the nucleotide shown in SEQ ID No: 2;

[0025] According to some embodiments, the use of the primer or probe is also provided. In some embodiments, the use of the primer or probe of the present application in diagnosing virus infection-related diseases (such as mosaic disease and leaf roll disease) of plants of the family Polygonum (such as Paris polyphylla) is provided.

[0026] So far, this new organism has not been found to infect plants of other families. However, it cannot be ruled out that it can infect plants of other families.

[0027] The primers or probes are used in identifying pathogens, wherein the identification refers to: determining whether the pathogen to be detected is the isolated organism; or determining whether the isolated organism exists in the sample.

[0028] The primer or probe is used in preparing a pathogen detection device, wherein the pathogen is the isolated organism; and the detection device is in the form of a reagent and / or a chip.

[0029] A detection device comprises the primer or probe; the detection device is in the form of a reagent and / or a chip.

[0030] A method for detecting pathogens, comprising the steps of:

[0031] 1) providing a sample to be tested; the sample to be tested is derived from a plant of the genus Paris;

[0032] 2) obtaining nucleotides of the sample to be tested; the nucleotides are RNA;

[0033] 3) contacting the nucleotides of the sample to be tested or its amplified product or reverse transcription product with the primers or probes described in claims 4-5;

[0034] 4) Determining whether the isolated nucleotide according to claim 3 is present.

[0035] Beneficial effects of the present invention: The present inventors have identified a new pathogen of the genus Potyvirus Y on plants of the family Melanophoraceae, and the pathogen is reported for the first time. Since there is no detection method for the pathogen, the present invention designs specific detection primers / probes based on the full genome sequence of the pathogen, establishes a method for detecting the pathogen, and expands the detection range of diseases related to virus infection of precious Chinese medicinal materials (such as Polygonum multiflorum of the family Melanophoraceae). BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a picture of the symptoms of Paris polyphylla mosaic disease.

[0037] Figure 2 This is the genome structure of the new Paris polyphylla virus ParV2; it encodes a polyprotein, which is cut into 10 mature proteins by 9 hydrolysis cleavage sites such as VQHY / S, namely: P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa, NIb, and CP.

[0038] Figure 3 Phylogenetic tree based on the nucleotide sequences of ParV2 and 21 viral polyproteins most similar to ParV2; phylogenetic analysis was performed using the maximum likelihood statistical method with MEGA X software; phylogenetic analysis was performed using the bootstrap method (with 1000 replicates). The phylogenetic tree is shown below the branches.

[0039] Figure 4 The detection method of ParV2 was established. Lanes 1-7 are the fragments amplified by primer pairs CLpoty589F / CLpoty1522R, CLpoty3128F / CLpoty3828R, CLpoty3128F / CLpoty3701R, CLpoty2924F / CLpoty3701R, CLpoty2924F / CLpoty3828R, CLpoty4948F / CLpoty5736R, and CLpoty6861F / CLpoty7471R in Paris polyphylla samples; M: DNA molecular weight marker. DETAILED DESCRIPTION

[0040] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0041] Example 1. Identification and sequencing of pathogens

[0042] In September 2020, the inventors found Paris polyphylla (Paris polyphylla) with mosaic symptoms in Fujian and Hebei provinces, China ( Figure 1 ).

[0043] Using mirVana TM Total RNA was extracted from symptomatic leaves using the microRNA (miRNA) Isolation kit (Ambion, Thermo Fisher Scientific, Waltham, MA, United States). Total RNA was purified using the RNA Clean XP Kit (Beckman Coulter, Brea, CA, United States) and the RNase-Free DNase Set (QIAGEN GmbH, Hilden, Germany). Ribosomal RNA was removed using the RiboZero Magnetic Kit (Epicentre, Lucigen, Middleton, WI, United States), and RNA libraries were constructed using the TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA, United States) and paired-end sequencing was performed on the Illumina HiSeq X platform.

[0044] After sequencing, a total of 80,165,252 reads were obtained. After removing low-quality reads, the first assembly was performed using CLC Genomics Workbench 6.0.4 (Qiagen, Valencia, CA, United States), and the second assembly was performed using CAP3 software, resulting in 384,495 contigs. These contigs were aligned to the NCBI non-redundant (Nr) database using BLASTx (E-value < 1e -5 ), the results showed that 48 overlapping groups ranging from 249bp to 10418bp had high similarity with multiple members of the Potyvirus genus. This indicates that the Paris polyphylla plants showing mosaic symptoms carry a Potyvirus Y virus.

[0045] In order to identify the characteristics of Potyvirus in Paris polyphylla, the inventors used viral amplification primers designed according to the contig sequences of HTS (Table 1) and Ex Taq (TaKaRa, Dalian, China) to amplify 5 overlapping fragments and obtain the 1,235nt to 10,246nt region of the viral genome. The 5' and 3' ends of the viral genome were obtained using the SMARTer RACE 5' / 3' kit (Takara, CA, USA) and the classic RACE PCR, respectively. All the obtained PCR fragments were cloned into the pMD19 vector (Takara) for bidirectional sequencing. Three positive clones were sequenced for each PCR fragment.

[0046] Table 1. Primers used for ParV2 genome cloning

[0047]

[0048]

[0049] The genome sequence of the target virus is 10,418 nt (SEQ ID No: 2), including 5'-UTR (1-911 nt) and 3'-UTR (10164-10418 nt), and the 3' end contains a poly (A) tail. BLASTn analysis shows that the complete sequence of the virus has the highest similarity with several Potyvirus viruses, with a nucleotide similarity of 74.82%-76.20%. The inventors speculate that the identified virus is a new Potyvirus virus, tentatively named Paris virus 2 (ParV2) (GenBank accession number OR767666, not published before the present application).

[0050] Genome analysis showed that ParV2 encoded a large open reading frame (ORF) with typical potato Y virus genome structure characteristics, encoding a polyprotein composed of 3083 amino acids with a molecular mass of approximately 342kDa. According to the criteria proposed by Adams et al., the proteolytic cleavage sites in the ParV2 polyprotein were predicted. The polyprotein was cleaved into 10 mature proteins, namely P1 (nt: 912-1856, 315 aa), HC-Pro (nt: 1857-3230, 458 aa), P3 (nt: 3231-4271, 347 aa), 6K1 (nt: 4272-4427, 52 aa), CI (nt: 4428-6329, 634 aa), 6K2 (nt: 6330-6488, 53 aa), VPg (nt: 6489-7058, 190 aa), NIa (nt: 7059-7787, 243 aa), NIb (nt: 7788-9338, 517 aa), CP (nt: 9339-10163, 274 aa) ( Figure 2 ).

[0051] Phylogenetic analysis was performed using the maximum likelihood statistical method (1000 bootstrap replicates) in MEGA 5 software. Sequence alignment was performed using the ClustalW program. Phylogenetic trees were generated based on the nucleotide and amino acid sequences of the polyproteins of ParV2 and 26 potyviruses that were most similar to ParV2. Cardamom mosaic virus (CdMV) of the genus Macluravirus was used as a distant control. The phylogenetic tree constructed based on the amino acid sequence of the polyprotein showed that ParV2 was closely related to soybean mosaic virus (SMV) and watermelon mosaic virus (WMV). Figure 3 ). Parisvirus 1 (ParV1), Turnip mosaic virus (TuMV), Parisosaic necrosis virus (PMNV), and Chilli veinal mottle virus (ChiVMV) are four other Potyvirus species previously identified to infect Parisia plants. The amino acid phylogenetic tree of the polyprotein showed that ParV2 was most closely related to Parisosaic necrosis virus ( Figure 3 ).

[0052] The genome sequence of ParV2 was preliminarily aligned by BLASTx on NCBI, and DNAMAN (version 6) software was used to compare ParV2 with nine viruses most similar to ParV2. The results showed that the polyprotein of ParV2 had the highest identity with kudzu chlorotic ring blotch virus (KCRBV), with a nucleotide identity of 71.56% and an amino acid identity of 75.64%; the P1 protein of ParV2 had the highest identity with soybean virus A (SVA) and wisteria vein mosaic virus (WVMV), with a nucleotide identity of 57.68% and an amino acid identity of 47.34%; the CP protein of ParV2 had the highest identity with soybean mosaic virus (SMV), with a nucleotide identity of 72.61% and an amino acid identity of 78.10%. The identities of these sequences were all lower than the threshold for species classification of the genus Potyvirus (nucleotide identity of polyprotein <76%, amino acid identity <82%; nucleotide identity of P1 <58%, nucleotide identity of other coding regions <74-78%; nucleotide identity of CP <76-77%, amino acid identity <80%). Based on the comparison results and phylogenetic tree analysis, ParV2 can be considered a new virus of the genus Potyvirus.

[0053] Example 2. Establishment of a detection method for Parv2

[0054] 1. Primer design:

[0055] Based on the complete genome sequence of ParV2 virus, Primer Premier 6 software was used to design ParV2 detection primers, and the primer design sites included any fragment at any position in the nucleotide sequence encoding ParV2 polyprotein (ie, ORF1).

[0056] The principles of primer design are:

[0057] 1) Primers specifically bind to any fragment of the ParV2 genome;

[0058] 2) Primer length is 15 bp to 30 bp;

[0059] 3) G+C content between 40% and 60%;

[0060] 4) There should not be four consecutive bases of complementarity within or between primers;

[0061] 5) The Tm value of the primer is 55±5℃;

[0062] 6) The length of the amplified product is 300 bp to 3000 bp.

[0063] Exemplary detection primers designed according to the above design principles, for example:

[0064] Table 2. Exemplary detection primers designed

[0065]

[0066] 2. The detection method is as follows:

[0067] Using the Paris polyphylla samples used for high-throughput sequencing as materials, and taking a pair of CLpoty6861F and CLpoty7471R, which amplified partial coat protein sequences, as examples, a detection method for ParV2 was established.

[0068] 0.1 g of Paris polyphylla leaves were thoroughly ground in liquid nitrogen, and total RNA from the leaves was extracted using TRNzol reagent (Tiangen, Beijing). The specific steps were referred to the instruction manual. The extracted total RNA was dissolved in 30 μL DEPC ddH 2 O and stored at -80℃ for future use.

[0069] The concentration of total RNA was determined using Nanodrop2000, and 500 to 2000 ng of total RNA was used as template, with random hexamer primers and Oligo d(T) 18 As primers, reverse transcription was performed using M-MLV reverse transcriptase (Promega, USA) to synthesize the first strand of cDNA. The reaction system was based on the instructions for use of M-MLV reverse transcriptase: 500 to 2000 ng of total RNA, 1 μL of dNTP (2.5 mmol / L each, RNase-free), random hexamer primers and Oligo d(T) 18 0.5 μL each, DEPC ddHO 2 O supplement system to 18.5μL, incubate at 70℃ metal bath for 5min, place on ice for 1min, centrifuge briefly, add 5μL 5×M-MLV reverse transcriptase buffer, 1μL M-MLV reverse transcriptase (200U / μl), 0.5μL RNase inhibitor (50U / μl), and incubate at 37℃ water bath for 1h. The obtained reverse transcription product was stored at -20℃.

[0070] The synthesized cDNA chain was used as a template for PCR detection reaction. The PCR reaction system was as follows: 1 to 2 μL of cDNA template, 1 μL of dNTPs (2.5 mmol / L each), 0.2 μM to 1 μM of FZ-Car-7600F2 and FZ-Car-8100R primers, 0.25 μL of DNA polymerase (5 U / μL), 2.5 μL of 10× DNA polymerase buffer, and ddH 2HO to make up to 25 μL.

[0071] PCR reaction program: 94 to 95°C pre-denaturation for 3 min; 94 to 95°C denaturation for 30 s, 50 to 60°C annealing for 30 s; 72°C extension for 45 s to 3 min, 32 cycles; 72°C final extension for 10 min. Take 5 μL of PCR product and run it on 1% agarose gel electrophoresis.

[0072] Take 5 μl of RT-PCR product and analyze it on 1% agarose gel. Figure 4 , using primer pairs CLpoty589F / CLpoty1522R, CLpoty3128F / CLpoty3828R, CLpoty3128F / CLpoty3701R, CLpoty2924F / CLpoty3701R, CLpoty2924F / CLpoty3828R, CLpoty4948F / CLpoty5736R, CLpoty6861F / CLpoty7471R, the target fragments of about 1000bp, 700bp, 600bp, 800bp, 900bp, 800bp, and 600bp were amplified in ParV2-infected Paris polyphylla samples by RT-PCR. Sequencing experiments further confirmed that the amplified bands were ParV2 CP fragments.

[0073] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. An isolated organism, characterized in that Contains a nucleotide selected from any of the following: The nucleotides shown in SEQ ID No: 2 in the sequence listing; A nucleotide that is completely complementary to the nucleotide shown in SEQ ID No:

2.

2. The isolated organism according to claim 1, characterized in that The organism is a potato virus Potyvirus virus; said nucleotide is RNA.

3. An isolated nucleotide, characterized in that It comprises a nucleotide selected from any one of the following: The nucleotides shown in SEQ ID No: 2 in the sequence listing; A nucleotide that is completely complementary to the nucleotide shown in SEQ ID No:

2.

4. A primer pair, characterized in that: The primer pair is selected from the nucleotides specific for or complementary to the nucleotides shown in SEQ ID No: 2 in the sequence table; the primer pair is selected from any of the following primer pairs: The primer pair shown in SEQ ID No: 15 and 16; The primer pair shown in SEQ ID No: 17 and 18; The primer pair shown in SEQ ID No: 17 and 20; The primer pair shown in SEQ ID No: 19 and 20; The primer pair shown in SEQ ID No: 19 and 18; The primer pair shown in SEQ ID No: 21 and 22; The primer pair shown in SEQ ID Nos: 23 and 24.

5. Use of the primer pair according to claim 4 in diagnosing plant virus infection, characterized in that: The plant is any one of the following: Polygonum multiflorum, Polygonum aviculare, Polygonum multiflorum, Polygonum hainanensis, Polygonum lingyun, Polygonum xichou, Polygonum nanense, Polygonum burma, Polygonum jinxian, Polygonum ovate, Polygonum dali, Polygonum dwarfum, Polygonum whitii, Polygonum narrow-leaved, Polygonum broad-leaved, Polygonum longiflorum, Polygonum large-calyx, Polygonum hairy, Polygonum variegated, Polygonum aviculare, Polygonum luquanum variegated, Polygonum spherical, Polygonum short-petaled, Polygonum black-seeded, Polygonum aviculare, Polygonum chinense, Polygonum aviculare with five fingers, Polygonum chinense with red fruits, Polygonum flat-cut, Polygonum long-column, Polygonum wrinkled-leaf, Polygonum dulong, Polygonum bashanense, Polygonum norvegicus, Polygonum japanense, Polygonum truncatum with four leaves, Polygonum truncatum with four leaves, and Polygonum japanense, or a combination thereof.

6. Use of the primer pair according to claim 4 in identifying pathogens, characterized in that: The identification refers to: determining whether the pathogen to be tested is the isolated organism described in claim 1 or 2; or determining whether the isolated organism described in claim 1 or 2 exists in the sample.

7. Use of the primer pair according to claim 4 in preparing a pathogen detection device, characterized in that: The pathogen is the isolated organism according to claim 1 or 2; the detection device is in the form of a reagent or a chip.

8. A detection device, characterized in that: It comprises the primer pair described in claim 4; the detection device is in the form of a reagent or a chip.

9. A method for detecting pathogens, comprising the steps of: 1) Providing a sample to be tested; the sample to be tested is derived from the genus Paris polyphylla; 2) obtaining nucleotides of the sample to be tested; the nucleotides are RNA; 3) contacting the nucleotides of the sample to be tested or its amplified product or reverse transcription product with the primer pair of claim 4; 4) Determining whether the isolated nucleotide of claim 3 is present.

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