CN108330110B

CN108330110B - Primer and method for detecting pathogen of chrysanthemum dwarfing disease

Info

Publication number: CN108330110B
Application number: CN201810106337.9A
Authority: CN
Inventors: 王蓉; 李勇; 丁万隆
Original assignee: Institute of Medicinal Plant Development of CAMS and PUMC
Current assignee: Institute of Medicinal Plant Development of CAMS and PUMC
Priority date: 2018-02-02
Filing date: 2018-02-02
Publication date: 2021-02-26
Anticipated expiration: 2038-02-02
Also published as: CN108330110A

Abstract

The application relates to a primer and a method for detecting pathogens of chrysanthemum dwarfing disease. Specifically, the inventor identifies a new pathogen of carnation latent virus on chrysanthemum dwarf plants, and the pathogen is reported for the first time. Because a detection method aiming at the pathogen does not exist, the application designs a specific detection primer according to the whole genome sequence of the pathogen and establishes a method for detecting the pathogen. The sensitivity of the method can reach 2.1 multiplied by 10³The copy is effective on various chrysanthemum varieties, and the detection range of chrysanthemum virus diseases is expanded.

Description

Primer and method for detecting pathogen of chrysanthemum dwarfing disease

Technical Field

The application belongs to the field of plant disease diagnosis, and relates to a method for detecting chrysanthemum pathogens.

Background

Chrysanthemum morifolium Ramat is a plant of Compositae, is not only an ornamental flower, but also can be used for medicine and drinking, and is one of the traditional Chinese medicinal materials in China.

The production of chrysanthemum is seriously harmed by virus. To date, more than 20 viruses are reported in the world to harm chrysanthemum. At present, 7 kinds of chrysanthemum-infecting viruses are reported in China: chrysanthemum Virus B (CVB), Tomato Aspermy Virus (TAV), Cucumber Mosaic Virus (CMV), Tobacco Mosaic Virus (TMV), Potato Virus Y (PVY), Potato Virus X (PVX), and Zucchini Yellow Mosaic Virus (ZYMV); viroid 2 species: chrysanthemum dwarfing viroid (CSVd) and Chrysanthemum withered yellow spot viroid (CChMVd) (Wu hong Zhi et al. investigation and identification of Chrysanthemum virosis in Kunming region. Yunnan university of agriculture proceedings 2002,17(1): 24-27; Niu EB 2015First Report of Zucchi yellow mosaic virus in Chrysanthemum Dis 99: 1289; ZHao X2015. Amultiplex RT-PCR for sizing and materials detection and identification of five viruses and wo viruses in Chrysanthemum section virol 160: 1145: 1152).

In 2017, when disease investigation is carried out on chrysanthemum in North China, a large number of chrysanthemum dwarf plants are found. The pathogen of the chrysanthemum dwarfing disease is determined to be a new virus of carnation latent virus (Carlavus) by a small RNA high-throughput sequencing method. In the context of the present application, the virus is referred to as Chrysanthemum Virus R (CVR).

The chrysanthemum is mainly subjected to vegetative propagation, and virus-free seedlings are mostly used in production in order to prevent the virus from being spread to the next generation through root retaining or cutting branches. The establishment of a comprehensive, efficient and rapid virus detection system is an important step for screening the virus-free seedlings. Aiming at common chrysanthemum virus diseases in China, a multiple Reverse Transcription (RT) PCR detection system of TAV, CVB, CMV, TMV, PVY, CSVd and CChMVd (ZHao X, 2015.A multiple RT-PCR for multiple gene detection and identification of five viruses. Arch virus.160: 1145-. However, no effective detection method for new viral CVR is currently available.

RT-PCR is one of the most commonly used virus detection methods at present, and is a nucleic acid level detection method. The detection methods for biological detection, electron microscopy detection, serology detection and the like comprise the following steps: (1) the sensitivity is high, the trace nucleic acid can be rapidly amplified to a detectable level, and the sensitivity can reach pg level; (2) the specificity is strong: unaffected by in vivo heteroproteins); (3) fast (sample detection can be completed within 24 h) and the like.

The application designs a specific detection primer aiming at a new virus CVR on chrysanthemum, and provides an effective tool for the detection of the CVR.

Disclosure of Invention

According to some embodiments, an isolated organism is provided. In some embodiments, the isolated organism is new, a new species not currently reported. In the context of the present application, the isolated organism is isolated and not in a state that exists in nature.

In particular embodiments, an isolated organism is provided. The organism is a microorganism. In particular embodiments, an isolated organism is provided, the organism being a virus; preferably an RNA virus. In a specific embodiment, there is provided an isolated organism, according to the ICTV specifications for the new species identification of the carnation latent virus genus, said organism being carnation latent virus caravivirus. In a specific embodiment, an isolated organism is provided, said organism being a Chrysanthemum R virus (CVR).

In particular embodiments, there is provided an isolated organism comprising a nucleotide selected from (any one or combination of) the group consisting of:

-the nucleotide shown as SEQ ID No.7 or a fragment thereof;

-a nucleotide complementary to the nucleotide represented by SEQ ID No. 7; or

-a nucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to the nucleotide shown in SEQ ID No. 7.

Wherein the nucleotide represented by SEQ ID No.7 is the genetic material of said organism; such as genomic nucleotides (e.g., RNA).

According to some embodiments, there is provided an isolated nucleotide selected from the group consisting of:

the nucleotide shown as SEQ ID No.1 or a fragment thereof;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 1;

a nucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to the nucleotide set forth in SEQ ID No. 1;

the nucleotide shown as SEQ ID No.2 or a fragment thereof;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 2;

a nucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to the nucleotide set forth in SEQ ID No. 2;

the nucleotide shown as SEQ ID No.3 or a fragment thereof;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 3;

a nucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to the nucleotide set forth in SEQ ID No. 3;

the nucleotide shown as SEQ ID No.4 or a fragment thereof;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 4;

a nucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to the nucleotide set forth in SEQ ID No. 4;

the nucleotide shown as SEQ ID No.5 or a fragment thereof;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 5;

a nucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to the nucleotide set forth in SEQ ID No. 5;

the nucleotide shown as SEQ ID No.6 or a fragment thereof;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 6;

a nucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to the nucleotide set forth in SEQ ID No. 6;

the nucleotide shown as SEQ ID No.7 or a fragment thereof;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 7;

a nucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to the nucleotide set forth in SEQ ID No. 7.

In a particular embodiment, the nucleotides shown in SEQ ID No.1 to SEQ ID No.6 represent the 6 open reading frames (ORF1 to ORF6) of the new virus, respectively: ORF1 encodes an egg associated with viral replication; ORFs 2 to 4 encode TGB1, TGB2 and TGB3, respectively, constituting a three-gene cassette, associated with the movement of the virus; ORF5 encodes a Coat Protein (CP); ORF6 encodes a cysteine-rich protein (CRP).

According to some embodiments, a primer or probe is provided that is specific for a new organism in the present application. In particular embodiments, a primer or probe is provided that is specific for a nucleotide selected from the group consisting of:

the nucleotide shown as SEQ ID No.1 or a fragment thereof;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 1;

the nucleotide shown as SEQ ID No.2 or a fragment thereof;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 2;

the nucleotide shown as SEQ ID No.3 or a fragment thereof;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 3;

the nucleotide shown as SEQ ID No.4 or a fragment thereof;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 4;

the nucleotide shown as SEQ ID No.5 or a fragment thereof;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 5;

the nucleotide shown as SEQ ID No.6 or a fragment thereof;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 6;

the nucleotide shown as SEQ ID No.7 or a fragment thereof;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 7;

In a specific embodiment, the primer or probe is a nucleotide selected from the group consisting of the nucleotides shown in the following sequences: SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.19, SEQ ID NO. 28.

In a particular embodiment, the primer may be in the form of a primer pair selected from the group consisting of:

SEQ ID NO.22 and SEQ ID NO. 23;

SEQ ID No.24 and SEQ ID No. 25;

SEQ ID NO.26 and SEQ ID NO. 27;

SEQ ID NO.19 and SEQ ID NO. 28.

The skilled artisan will appreciate that although specific examples are provided for the detection of new organisms with a particular pair of primers, the pair of primers is merely exemplary and should not be construed as limiting the scope of the present application. Indeed, when the skilled person is aware of the genomic sequence of the new organisms in the present application, and functional fragments thereof (e.g. ORFs), specific primers or probes can be designed based on the sequence to achieve specific recognition, binding, identification, detection, quantification or characterization.

As an example, in a preferred embodiment of the present application, specific primers for new organisms can be designed by the following principle:

1) the length of the primer is 15bp to 30bp (including end points, the same below);

2) the content of G + C in the primer is 40-60%;

3) the primers cannot have continuous complementarity of 4 bases in and between the primers;

4) the Tm value of the primer is 55 +/-5 ℃;

5) the length of an amplification product obtained by the primer is 300bp to 1200 bp;

6) the primer is complementary to a nucleotide selected from the group consisting of:

-the nucleotide shown as SEQ ID No.1 or a fragment thereof;

-a nucleotide complementary to the nucleotide represented by SEQ ID No. 1;

-a nucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to the nucleotide as represented in SEQ ID No. 1;

-the nucleotide shown as SEQ ID No.2 or a fragment thereof;

-a nucleotide complementary to the nucleotide represented by SEQ ID No. 2;

-a nucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to the nucleotide represented by SEQ ID No. 2;

-the nucleotide shown as SEQ ID No.3 or a fragment thereof;

-a nucleotide complementary to the nucleotide represented by SEQ ID No. 3;

-a nucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to the nucleotide represented by SEQ ID No. 3;

-the nucleotide shown as SEQ ID No.4 or a fragment thereof;

-a nucleotide complementary to the nucleotide represented by SEQ ID No. 4;

-a nucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to the nucleotide represented by SEQ ID No. 4;

-the nucleotide shown as SEQ ID No.5 or a fragment thereof;

-a nucleotide complementary to the nucleotide represented by SEQ ID No. 5;

-a nucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to the nucleotide represented by SEQ ID No. 5;

-the nucleotide shown as SEQ ID No.6 or a fragment thereof;

-a nucleotide complementary to the nucleotide represented by SEQ ID No. 6;

-a nucleotide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity to the nucleotide represented by SEQ ID No. 6;

-the nucleotide shown as SEQ ID No.7 or a fragment thereof;

-a nucleotide complementary to the nucleotide represented by SEQ ID No. 7;

According to some embodiments, there is also provided the use of said primer or probe. In some embodiments, there is provided the use of a primer or probe of the present application in the diagnosis of stunting disease in plants of the asteraceae family. It has not been found that this new organism infects plants of other families. However, it is not excluded that it is capable of infecting plants of other families. In some embodiments, the compositae plants include, but are not limited to: dulisneria, vernonia, Alternanthera, elephantopus, aster, ageratum, eupatorium, pseudoeupatorium, solidago, euglenopsis, cupflower, hypericum, leptospermum, centella, celosia, orostachys, inula, echinacea, marjoram, aster, emerald, dolomia, aster, asterium, galbanaba, galbanum, erigerontium, salviae, asterias, latifolius, latifolia, asterias, latifolius, asterias, callianthus, asteria, asterias, echinacea, meadowrue, meadowfoam, meadow, The plant may be selected from the group consisting of plants of the genera chamomilla, Inula, Phragmites, Philippine, Hedonia, Echinacea, Chrysanthemum, Lespedeza, Cruciferae, Xanthium, ragweed, Echinacea, Bordetella, Echinacea, Helianthus, Heliothis, Aronia, Chrysanthemum, Helianthus, Chrysanthemum, Calophyllum, Bidens, Matricaria, Tagetes, Chrysanthemum, Artemisia, Chamomilla, Echinacea, Achillea, Artemisia, Chamomilla, Echinacea, Ec, Callistephus, Chamomilla, Echinacea, karst, Artemisia, Hibiscus, Coriandrum, Gynura, Callicarpa, Doiscorea, Eriocheir, Echinacea, Pseudobulbus crepidii, Costus, Petasites, Potentilla, Echinacea, Potentilla, Costus, Potentilla, Chrysanthemum, Achillea, Calophyllum, Chrysanthemum, Calophyllum, Aleurites, Everonica, ligularia, Calophyllum, Echinacea, Sophora, Gynura, Tremella, Echinacea, Echin, Cardigan, Oligochaeta, Silybum, semiaquilegia, Serenoa, Echinacea, Filifolium, Phragmites, Carthamus, Echinacea, Cnaphalium, Centaurium, Echinacea, Chrysanthemum, Calliopsis, Cyprinus, Peucedanum, Costus, Cyprinus, Costus, Lactuca, Lacca, Crassulaceae, Scorpion, Brucella, Cochloes, Cochloa, Borrelia, Lactuca, Lacca, Crataegus, Echinacea, Cochloa, Echinopsis, Echinacea, Ec, Lactuca, Nerygium, Ixeris, Oldenlandia, Cucumis, Ecliptae, Lactuca, Ecliptae, Filipendula, Isorhynchosia, Echinacea, and Taraxacum.

In some embodiments, the Asteraceae plants are in particular those of the Asteraceae family which have a medicinal, ornamental, and edible economic value. For example, but not limited to, chrysanthemum parietans, chrysanthemum early-maturing, chrysanthemum late-maturing, chrysanthemum tribute, chrysanthemum morifolium, chrysanthemum keemun, chrysanthemum morifolium, chrysanthemum clitoris, chrysanthemum camomile, and chrysanthemum indicum.

According to further embodiments, there is also provided use of the primer or probe for identifying an organism according to further embodiments, there is also provided use of the primer or probe for identifying a pathogen. In some embodiments, the organism/pathogen refers to an isolated organism as described herein. In the context of the present application, the identification comprises a meaning selected from: determining whether the organism to be tested is an isolated organism as described herein; determining the presence or absence of an isolated organism as described herein; alternatively, the isolated organisms described herein are quantified.

According to other embodiments, there is also provided the use of the primer or probe in the preparation of a biological detection device. In some embodiments, the organism refers to an isolated organism described herein. The skilled person will appreciate that the detection means may be embodied in any known or future form, such as in the form of reagents and/or chips. When the detection device is in the form of a reagent (or kit), it comprises a primer or probe of the present application. When the detection device is in the form of a chip, the primers or probes of the present application are bound thereto; optionally, it may also carry a detectable label.

According to further embodiments, there is also provided a detection device comprising a primer or probe of the present application.

According to further embodiments, there is also provided a method of detecting an organism or pathogen, comprising the steps of:

1) providing a sample to be tested, wherein the sample to be tested is derived from a plant of the Compositae family; 2) obtaining the nucleotide of the sample to be detected; 3) contacting the nucleotide or amplification product or transcription product thereof of the test sample with a primer or probe of the present application; 4) determining the presence or absence of an isolated nucleotide as described herein.

Drawings

FIGS. 1A to 1D are genomic amplification electropherograms of CVR.

FIG. 1A: m represents a molecular weight marker; lane 1 shows the amplified fragments with primers CV1F and CV 807R.

FIG. 1B: lane 2 shows the amplified fragments for primers CV518F and CV 2353R; lane 3 shows the amplified fragments of primers CV1406F and CV 4047R; lane 4 shows the amplified fragments with primers CV3968F and CV 6177R; lane 5 shows the amplified fragments of primers CV5579F and CV 7213R; lane 6 shows the amplified fragments with primers CV7162F and CV 8802R.

FIG. 1C: 3 'RACE represents the 3' RACE fragment.

FIG. 1D: 5 'RACE represents the 5' RACE fragment.

FIG. 2 is a diagram of the CVR genome.

Fig. 3 shows the establishment of the CVR detection method. 1 represents a healthy chrysanthemum sample as a negative control; 2 represents the fragment amplified by the primer pair CV7162F/CV8217R in the sample No.2 chrysanthemum; m represents a molecular weight marker.

FIG. 4 shows the sensitivity analysis of RT-PCR detection of CVR. M represents a molecular weight marker; 1 to 8 each represents a template copy number of 2.1X 10⁸、2.1×10⁷、2.1×10⁶、2.1×10⁵、2.1×10⁴、2.1×10³、2.1×10²And 21.

FIG. 5 shows RT-PCR detection of CVR in chrysanthemum samples. 1 to 4 represent 4 samples of the chrysanthemum; 5-6 represent 2 samples of early-maturing chrysanthemum respectively; 7-10 represent 4 samples of late-maturing chrysanthemums, respectively; 11-12 represent 2 samples of florists chrysanthemum respectively; 13-14 represent 2 samples of Huanggong chrysanthemum, respectively; -representing a healthy chrysanthemum sample as a negative control; + represents chrysanthemum sample No.2 as positive control; m represents a molecular weight marker.

Detailed Description

Example 1 discovery of New pathogen for Chrysanthemum dwarfing disease

In the investigation of chrysanthemum virus diseases in Beijing area, chrysanthemum dwarfing disease is found to be an important disease which endangers chrysanthemum production. In Yanqing district of Beijing, 4 Chrysanthemum dwarf virus samples are taken, and RT-PCR method is used for detecting common Chrysanthemum Virus B (CVB), Tomato sterility virus (TAV) and Chrysanthemum dwarf virus (CSVd). However, in sample No.2, the virus was not detected, and therefore, it was presumed that the strain of chrysanthemum was dwarfed by infection with a new pathogen (for example, a virus with a high probability).

Example 2 acquisition, sequencing and database search of genomic RNA of New pathogens

The present application identifies pathogens in sample No.2 using Illumina small RNA high throughput sequencing methods. The specific method comprises the following steps:

1. weighing 0.1g of chrysanthemum leaf blades, fully grinding in liquid nitrogen, and extracting total RNA of the chrysanthemum leaf blades by a TRNzol method (the application refers to the TRNzol method for extracting the total plant RNA, but is not limited to the TRNzol method).

2. The method comprises the steps of constructing a small RNA library by taking a total RNA sample as a template, and carrying out Illumina small RNA high-throughput sequencing on the library, wherein the sequencing reading number is not less than 20M.

3. The analysis of the raw data generated by sequencing mainly comprises: using a Velet algorithm to remove low-quality sequencing reads and linkers in the reads, and splicing the processed reads into a contig (contig); the resulting contigs were aligned to the virus database in GenBank (ftp:// ftp. ncbi. nlm. nih. gov/refseq/release/viral /) using the BLAST program.

BLAST results show that: the 26 contigs with length 46bp to 183bp have higher nucleotide similarity (74.04% to 88.89% identity) with the carnation latent virus (Carlavus) members, and are preliminarily presumed to be a new pathogen (most likely a virus).

5. Primers were designed based on contig sequences and the whole genome sequence of the new pathogen was amplified by RT-PCR and RACE PCR.

The information of the new pathogen amplified fragments is shown in FIGS. 1A to 1D. The amplification primer information is shown in Table 1.

Table 1: novel pathogen genome amplification primer for chrysanthemum dwarf disease

6. Results

(1) The genome structure is as follows:

the amplified new pathogen fragment is connected into a pMD18-T vector, and the whole genome information of the pathogen is obtained through sequencing (the genome structure diagram is shown in figure 2, SEQ ID No. 7). The pathogen has a full length of 8874bp (no polyA tail), and is predicted to encode 6 Open Reading Frames (ORFs) from ORF1 to ORF6(SEQ ID No.1 to SEQ ID No. 6):

ORF1 encodes proteins involved in viral replication;

ORF 2-ORF 4 encode TGB1, TGB2 and TGB3, respectively, constituting a three-gene cassette, associated with movement of the virus;

ORF5 encodes a Coat Protein (CP);

ORF6 encodes a cysteine-rich protein (CRP).

(2) The new species is identified:

according to the definition reported in the ninth classification of ICTV (International Committee on Taxomy of Virus) for the identification of new species of carnation latent virus, a virus is identified as a new species when the nucleotide similarity of the Polymerase protein (Polymerase) or Coat Protein (CP) of the virus is less than 72% (or the amino acid similarity is less than 80%).

BLAST analysis showed that the polymerase protein of the new pathogen has the highest similarity to the CVB-Uttarakhand isolate (GenBank accession No. AM765838), with 64% nucleotide and 67% amino acid; the coat protein has the highest similarity to common latent Narcissus virus (NCLV, AM158439), with a nucleotide level of 58% and an amino acid level of 51%.

The Chrysanthemum new virus is a new virus of the carnation latent virus genus, and is temporarily named as Chrysanthemum R virus (CVR).

Example 3 establishment of a method for detecting Chrysanthemum R Virus (CVR)

1. Designing a primer:

allows the design of detection primers for CVR based on any fragment of CVR at any position in the nucleotide sequence encoding replicase, triple-gene cassette and coat protein (i.e., ORF1 to 5). The principle of primer design is as follows:

1) the primer specifically binds to any fragment of the CVR genome;

2) the length of the primer is 15bp to 30 bp;

3) the G + C content is between 40% and 60%;

4) the complementarity of continuous 4 bases can not exist in the primers and between the primers;

5) the Tm value of the primer is 55 +/-5 ℃;

6) the length of the amplification product is 300bp to 1200 bp.

The detection primers designed according to the above design principles are, for example:

TABLE 2 designed detection primers

2. The detection method comprises the following steps:

taking the chrysanthemum No.2 sample for high-throughput sequencing as a material, and taking CV7162F (SEQ ID NO.19) and CV8217R (SEQ ID NO.28) of a pair of amplified coat protein partial sequences as examples, a CVR detection method is established.

(1) Weighing 0.1g of leaves of the No.2 sample of the chrysanthemum, fully grinding the leaves in liquid nitrogen, and extracting total RNA of the leaves of the chrysanthemum by a TRNzol method, wherein the specific method refers to the operation steps of extracting total RNA of the radix tianae.

(2) Total RNA concentration was determined using a Nano drop2000, and 500 to 2000ng of total RNA was used to generate complementary cDNA strands of the RNA by reverse transcriptase. The reaction system adds 0.5 to 2 mu g of total RNA into the PE tube without RNase in turn according to the instruction of the M-MLV reverse transcriptase; 1. mu.l dNTPs (2.5 mM each, without RNase); random primer Hexamer 1. mu.l; incubating at 70 deg.C for 5 min; rapidly standing on ice for 1 min; sequentially adding 5 mul of 5 XM-MLV reverse transcriptase reaction buffer solution into a PE tube; RNase inhibitor (50U/. mu.l) 0.5. mu.l; M-MLV reverse transcriptase (200U/. mu.l) 1. mu.l; DEPC ddH₂O make up system to 25 μ l; water bath at 37 ℃ for 1 h. Meanwhile, healthy chrysanthemum samples are used as controls. The reverse transcription product was stored at-20 ℃.

(3) The PCR detection reaction was carried out using the synthesized cDNA strand as a template, and the 25. mu.l system was as follows: cDNA template 1 to 2. mu.l, dNTP (2.5 mM each) 1. mu.l, primers CV7162F and CV8217R 0.2. mu.M to 1. mu.M, 10 XDNA polymerase reaction buffer 2.5. mu.l, DNA polymerase 0.25. mu.l; ddH₂O make up system to 25. mu.l.

The PCR reaction program is: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 50-60 ℃ for 30s, extension at 72 ℃ for 1min for 10s, 32 cycles; final extension at 72 ℃ for 10 min.

(4) Mu.l of RT-PCR product was analyzed on 1% agarose gel, and the results are shown in FIG. 3, and a target fragment of about 1100bp in size was amplified in CVR-infected chrysanthemum samples by RT-PCR using CV7162F/CV8217R primer set. On the contrary, the target band could not be amplified in the healthy chrysanthemum sample. Sequencing experiments further confirmed that the amplified band was a CVR fragment.

Example 4 sensitivity analysis of Chrysanthemum R Virus RT-PCR detection

1. This example was carried out using the recombinant pMD19-T plasmid with the amplified fragment as a template. The recombinant plasmid is obtained by connecting a CVR fragment (1641bp) obtained by amplifying CV7162F/CV8802R primer into a pMD19-T (TaKaRa) vector, and is a double-stranded circular DNA molecule of 4333bp (1641bp +2692 bp).

2. The concentration of the recombinant plasmid was determined using a Nano drop2000 microspectrophotometer, and diluted to 1 ng/. mu.l, according to which 1ng of the recombinant plasmid contained 2.1X 10⁸And (6) copying.

The calculation formula is as follows:

1ng of plasmid contained (1X 10) copies^-9g×6.02×10²³) Average molecular mass of plasmid

Average molecular mass of double-stranded DNA × 660 base numbers

3. Diluting the recombinant plasmid DNA with 1 ng/mul by 10 times gradient step by step to respectively obtain stock solution and 10^-1、10^-2、10^-3、10^-4、10^-5、10^-6、10^-7The number of copies contained in 1. mu.l of each dilution was 2.1X 10⁸、2.1×10⁷、2.1×10⁶、2.1×10⁵、2.1×10⁴、2.1×10³、2.1×10²And 21.

Mu.l of each of the dilutions was used as a template, and 1. mu.l of dNTP (2.5 mM each), CV7162F and CV8217R primers 0.2. mu.M to 1. mu.M, 10 XDNA polymerase reaction buffer 2.5. mu.l, DNA polymerase 0.25. mu.l, ddH were added in this order₂O make up system to 25. mu.l.

4. 5 mul RT-PCR product is analyzed by 1% agarose gel, the sensitivity result of the RT-PCR detection of the chrysanthemum virus R is shown in figure 4, and the sensitivity of the CVR RT-PCR detection can reach 2.1 multiplied by 10³。

EXAMPLE 5 Chrysanthemum sample CVR RT-PCR detection

To verify that the pair of detection primers can detect CVR viruses in different chrysanthemum varieties:

1. in a chrysanthemum planting base in Yanqing district of Beijing, 14 medicinal chrysanthemum samples are collected, including 4 chrysanthemum counter-chrysanthemum, 2 chrysanthemum early maturing, 4 chrysanthemum late maturing, 2 chrysanthemum floribunda and 2 chrysanthemum floribunda.

2. 0.1g of chrysanthemum leaf blade is weighed for each sample, and total RNA of the chrysanthemum leaf blade is extracted by using a TRNzol method, wherein the specific method refers to the operation step of total RNA extraction of radix asparagi.

3. Total RNA concentration was determined using a Nano drop2000, and 500 to 2000ng of total RNA was used to generate complementary cDNA strands of the RNA by reverse transcriptase. Sequentially adding 0.5-2 mu g of total RNA into the PE tube without RNase; 1. mu.l dNTPs (2.5 mM each, without RNase); random primer hexamer 1. mu.l; incubating at 70 deg.C for 5 min; rapidly standing on ice for 1 min; sequentially adding 5 mul of 5 XM-MLV reverse transcriptase reaction buffer solution into a PE tube; RNase inhibitor (50U/. mu.l) 0.5. mu.l; M-MLV reverse transcriptase (200U/. mu.l) 1. mu.l; DEPC ddH₂O make up system to 25 μ l; water bath at 37 ℃ for 1 h. Meanwhile, the No.2 sample of chrysanthemum is used as a positive control, and the healthy sample of chrysanthemum is used as a negative control. The reverse transcription product was stored at-20 ℃.

4. The PCR detection reaction was carried out using the synthesized cDNA strand as a template, and the 25. mu.l system was as follows: cDNA template 1 to 2. mu.l, dNTP (2.5 mM each) 1. mu.l, primers CV7162F and CV8217R 0.2. mu.M to 1. mu.M, 10 XDNA polymerase reaction buffer 2.5. mu.l, DNA polymerase 0.25. mu.l; ddH₂O make up system to 25. mu.l.

5. Mu.l of RT-PCR product was analyzed on a 1% agarose gel, and the results are shown in FIG. 5, where 4 samples of the contra-chrysanthemum were all infected with CVR, 2 samples of the early-maturing chrysanthemum were not infected with CVR, 1 sample of the late-maturing chrysanthemum was infected with CVR, 1 sample of the silk tribute chrysanthemum was infected with CVR, and 1 sample of the yellow tribute chrysanthemum was infected with CVR. 4 positive samples are randomly taken for a sequencing experiment, and an amplified band is further determined to be a CVR fragment.

Sequence listing

<110> institute of medicinal plants of academy of Chinese medical science

<120> primer and method for detecting pathogen of chrysanthemum dwarf disease

<130> 380017CG

<160> 28

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6228

<212> RNA

<213> CVR Virus (Chrysanthemum virus R)

<220>

<221> misc_feature

<222> (1)..(6228)

<223> ORF1

<400> 1

auggcgcuca cuuuccgcag cccuauugca gauguuauua caaauuucga uccauccauc 60

aacuccauua uagccacuac agcugcugaa cacuacaaga ggcuugagga agaaaauuuc 120

gcgcucuuca acuaugcaau gucgccacag acgacgcagc auuugauaga cgccggaauu 180

uacuuaagcc ccuucucugg gguaccucau ucucacccag cuuguaagac ucuggagaau 240

uacauauugu auauaguacu uccccaauau auagauaaua guuuuuauuu ugucgguauu 300

aaagauuuua aauuaaaugc acucaagcuc cggcauaaac auacuaacau ggugcaaugu 360

auuaacaggu acgucacaag ccaagauaaa augcgcuacc ccucagauuu cgucauuagg 420

uacucuaaac cacaugaggg guugucacgg caccgcaugu cauuggaaag ccacacacuc 480

cgugaucugg uaccugagcu aauguccagg agcugcaaga agaucuuccu acaugaugag 540

cuacauuauu ggaagauaga ugagcuuugc acguuuuugg aaguugugca gccuaaagua 600

gucauaggga caauuguuua cccgccagaa uuacugaagg gcucuaagaa aagcuuaaac 660

aauugguguu auacuuacga ggucaaaggg cgcgcgcuua gauucuaccc agauggggug 720

cguggcgaag guuaugagca gccacugaau gguggcuuuu ugcuagagug ugggaagaua 780

acacucuacg acgggacggu guacaugguc gauauccucu gcagcaaauu ugcgcaccac 840

uuaguugcac ucacccgugg ggucgccgca gggccaacaa ugagaucauu uggcccguuu 900

gaagccacug cgugcaaugg ucuugauccc cuuacuaggg auguuaccug caguuucccg 960

aucccguaug aggugguguc uaaaguuuau agguaccuga agacucucag gaagccagau 1020

gagcaaucau cuauggccaa auugacacaa cuuuugccau gccccacagg cgaagagauu 1080

gucuuugugc aggaguuuuc cagucuaguu aucuccacga acaccaucaa gaccaugauc 1140

agugcugaca ggcucaaagu auucuugggc aaguaccuga guaaguugcc caaaauacug 1200

gcugagcguu uugaaacugu gaaagaacug agucugcaug aauuuaucaa aaaucuugag 1260

ccauucaccg uggacgugca acucaaagaa cucaaaugga acaacgucug gacgcucgaa 1320

uugagggaug acgggauggc ugaugaacug cuggauccau cacggcuaau ggaggagaag 1380

uacacgcaug gcguuuacaa cgcugugccu gaucggagga gugccggcua uuauguuagg 1440

aaagaaaugc uugagcgcau gagggggccu cuaaucgaaa uugagcgcaa uguuuugaaa 1500

aacacccuag cagcaauaug uuacaaaagc uugagcaaua gcgauggaac ucuugcuucc 1560

auaaacgaaa uuggugcuuu uuuuaggaug auuugugcga ggccuuuccu gagaccagcg 1620

aaguacaaag ugcaugaucu guauggaagc aauggagcua ucgggcucca gaucuacaug 1680

agaagucguu gggcgagacu agucaaguuu uauugggcua auauugguau uuugugguuc 1740

aggagcaauc acaaauucua ucaaaaauac cuuuguucug ugccugaugg cauucaaacu 1800

uaccggacuu ucacgcaacc guggaaaacu guaguccacg aggucguuug cgugaagcau 1860

acacucaaga aaacuggcaa uuacucaugg uugaggugcg cuaggguagc gcaaaagcau 1920

ggagguacua augaagagcc uauccgggca gauagaagca cugagaccgc aggaggugga 1980

ucacaguuca augaggcuag aacagaucag gacaacaaaa caaauggggg ggacgcgccu 2040

aaugaagggg aguggacacc uccucaaggc agcaaguaug aggaaaugcc accgccaccg 2100

caacggcaag ggagccagag agaggaacag cgugcgauca aaugcaccug uaauuuagua 2160

uugccaauuu cuacugauga agccacacgc caucacaagu uccuaacucc ggaccgacuu 2220

aagaauaggu gcgccgguuu uuauucaaaa aauaaagagg gguacacuua caauggcggu 2280

ucccaucaaa guuuggggug gccuggcugg aucaaugcuu ggaugcgccu gaucggcaua 2340

ccagaauucu acaauugcug ccuaguacaa cgcuacgacg agggggcuaa gcucggcuug 2400

cauagugaug augaagagug uuuuguccag gguagcccug uguuuacugu aaauauugaa 2460

ggucaugcaa cuuuccucau ugcaugcaag aacucgccug aaaggagggu gaccagaucc 2520

auaguggggc cgggugauuu guuugagaug ccuggugguu uucaagaaac ccacaaacau 2580

ggagucugcg aaacugagaa aggcaggcuc ucgguuacau uucgagugau gaagaaaaac 2640

ccucauagug gugaagaaaa agaccacgga gcaagcgagg gccaccagaa uggaacagag 2700

aaugugaaug accaaaguga aucagaaacc caugaggaaa aaccccagca agaaggagug 2760

aaggaaagag aaaguguaca ggaaaguggu gagggcauuu cugaagaaca caaacaagaa 2820

cagagugacu gccccaauaa cgguacagcu ggagcuggga cagaggcauu cucaagacac 2880

uuuaaugguu gcaaagugag cgugcgcaaa gaggaacuug cgcacauaua caguacaacu 2940

gauuguggcg gggguggcaa cuguuuuugg cuuugcuugg ccacugagau gggcugcgac 3000

ccuuuuagag gaaagaaauu ggcacuagag uaugaucugg guuccgcagg ggcaaaugaa 3060

ucugcacgca guugugcugg cgagggugug uaugcuguag augaagccau ugcaugugcu 3120

gcaagugugu ucagggucgu cauuaaaaua uaccaacccg aguugaaugu caugacuacc 3180

uuugagccag caaaagggga gcggguccuu cacuuggaau uagaggggga acacuucaga 3240

cucaugagaa uaaucaacgg gugcguaauu agagccauaa gcucugcacu caaucgcaga 3300

gagggugaag ugaugcacgu gauugaagag aauugugacc ccacaaugau cacugaccuu 3360

uggaaagggg cggguguuga uuugagugua uuucacuccc uccuugagcu uuucucuauu 3420

aaagcuuuga uuuuugaagg aucuagggaa guguuguaca acagugaagg gcgcuucgaa 3480

gcauccuucg aaauaaaagg ugaucacaua gaacauguau ugaggaagaa gggugcgugc 3540

aauguccuuu uugaggaaug ugggaagacu uucgagguga aggccgaauc ucuagaacua 3600

cugaacagag caggcacauu auuaaaguac aagagcacuc uugcuagggc aaaaagacuc 3660

gcagacagcu uguguuccgg aacgacugga guuguuagcu caucuuuauu caauaagaag 3720

ccuaaccuug caggccaauu uagggaaaag acagagucuc uugauaggga uguucuugcu 3780

gugauuggaa cuuucggauc aggcaagagu acuauccuca agaacuuuuu caaaaucaau 3840

uugggucgaa aagugcuaua ugugucccca cgcagagcac ucuuaaauga auuccagaga 3900

agugcuugcg gugacuuuaa agacaagaag gaacgcaaga augcaagaaa gaaaggccaa 3960

gagaacuggg auuucaugac uuucgaaacc uucauacuua aaugcucuag uuuaccuuca 4020

gguauggcug uaguccuuga ugagauacaa uuguacccac caggguaucu ggauaugcuc 4080

uguuaccuag uuaaggaagg ugugcauuug auagucgcag gagauccagc ucaaagugau 4140

uacgauagcg agaaagaucg agcuugguug cauggcgucg aaccggauca cucgaaauua 4200

cugacuggga augaguauaa guacgucacu uugaguaaga gaaugcaaaa cuccauguuu 4260

gaaggcagau ugccgugcac auuaaaagua gccaaaaaug cggacaccaa ugaagauuuc 4320

gaaauuagag guggugugga gauuauacac aaacucgaua gcaaguggag agaaugcuuc 4380

cucgugucuu ccuucgaaga aaaacgauug auugaguacu accagacuga gagcucaaaa 4440

gauaaagagu gcauccuuac uuuuggagaa ucuacgggcc uuacuuucga uuacgguagc 4500

auccuaauca cucaaaguuc agagaagacg aacgagcagc gguggauaac ugccuugagc 4560

cgauuucgga agagcgucug cuucauuaau gccacaggca ugacgcuaca ugaacuauua 4620

cuugucuaua aagaucgggc ucucggcagg uucuugggua agcgagcgag ugucgaggau 4680

cuaaagaagu uccugccagg aagcccucaa uucgcagaag aauacguacc cucuauggga 4740

gcugagugug gcucuaggga ggagaagcug gcaggggacc ccugguuaaa gacgaugauc 4800

gaucuuuugc agauagagga uguggaagag gaagucggcg ugguugaaga aauagcccgu 4860

gaggaguggu ucaagacgca uuuaccucag gaagaacugg agagcgucag agcgaggugg 4920

gugcacaaga uuuuggccaa ggaagcuaga gaggucagaa ugggugacau uguuucugaa 4980

caguucacug augagcacag uaaggaaaaa gguaagcaac ucaccaaugc agcggagcgu 5040

uuugaaacaa uauacccuag gcauaggucg agugacacgg ugacuuucau aauggcggua 5100

cgaaagaggc ugagguucuc caagcccaca guggagaugc ccaaauugag ggccgcaaaa 5160

cccuauggac caauguugcu caagaaguuc uugaagcaug ugccacucaa accgaauaaa 5220

gacccgauac ugauggccag ggcaaagaug gaauuugagg aaaagaaggu cagcaaaucc 5280

gcggcaacua uugagaauca caguguacgg ucaaccagag acuggcuagu ggacguuggg 5340

cagauuuuuu cuaagaguca aauaugcacc aaauuugaaa aacguuucag ugcugcgaaa 5400

gcggcacaau ccauugugug cuuccaacau ucgguacucg uuagguuugc ucccuauaug 5460

cgcuacauug agcuuaaauu gagggaaucc cugcccgaua gauucuauau acauucgggc 5520

aagggcuugg augagcucga uucuugggug cgcucgcaug gcuuuagcgg gauguguaca 5580

gaaucugauu acgaagccuu cgaugcaucc caagaccaau auauugucgc cuuugagauu 5640

gcucucauga aguaucuggg ucugccgcaa aguuuaauag cagacuauga guucaucaag 5700

acgcaccugg gaucaaaacu aggaucauuc gcuaucauga gauucucagg ggaagcaagu 5760

accuuccuuu ucaacacuau ggccaauaug cuauucacgu ucaugcgcua ugauauuaac 5820

aacaaggauu gcaucugcuu ugcuggggau gauaugugug cuucaagacg uuugccauua 5880

agcaaagagu acgaaagcuu uuuauccaaa uugaaacuga aagccaaagu gcaguucacg 5940

gagaagccua cauucugugg uuggaaucuc uccccugaug guaucuauaa gaaaccucaa 6000

uugguucucg agagaaugug uauagcgaag gagaccaaua accugcacaa uugcauagac 6060

aauuacgcua ucgaaguuuc cuacgcguac aagaugggcg aaaaagcagu caauaggaug 6120

gacgaagagg aauuggccgc cuacuacaac ugugugcgua ucaucauuaa gaauaaacau 6180

uugcuacguu cgaauauuuc ugagguguac aguaauaggg cuugguag 6228

<210> 2

<211> 690

<212> RNA

<213> CVR Virus (Chrysanthemum virus R)

<220>

<221> misc_feature

<222> (1)..(690)

<223> ORF2

<400> 2

auggaugugu uaguuaauuu agcuaagaau uuugguuuuc auagggugug uacuacauug 60

aauaaaccca uuguuuucca cuguguuccg ggugcaggca aguccaccuu gauucgugau 120

ugcuguaggg cagauaguag auucguugcg gucacuuugg gcaaagagga cgagagggau 180

uugaccgggg uaggcauuac aaaauauucu gguaccaucc cagauugcga guacccgcuc 240

uuagacgaau auacccuugc ggaaagcuug ucagaaggcu gguuugcaau cuuuggugau 300

ccaauccaag cagcuaaucg cgugguacug cgugcacauu ucacuugcaa ucuaagccgc 360

agauucggua agugcacugc ccaauuccuu agggaguugg gauuugacgu aguugcugag 420

ggugaggaua ccguucagau cucugggauu uacguagagg acccgcggga uaaaauaaua 480

uauuacgagc cugagguggg uugcuuacua aggagucacu caguugaagc uagccacauc 540

acagaaguaa uuggucaaac uuuugauucc gugacuuuug uuacugcuag ugaauaucca 600

guagaucgcu cgagugcuua ucaguguuug acgagacauc gcaagagcuu acucauucua 660

gcacccaaug ccucuuacuc cuccgccuga 690

<210> 3

<211> 324

<212> RNA

<213> CVR Virus (Chrysanthemum virus R)

<220>

<221> misc_feature

<222> (1)..(324)

<223> ORF3

<400> 3

augccucuua cuccuccgcc ugaucauaca aaggcauuuu uagcuauagc uguggguuuu 60

ucuugugcau uaaucauaag ccuauuuacu cguaguaccu uaccucaagu cggagaucaa 120

auccauucgc ugccgcacgg cgguugguac aaagacggaa caaagcaaau cuacuacgga 180

cgaccgaaua aauugaacuc aguagagaaa caaggcuucc ucauugguca gcccugggcc 240

aucguaauca uacuuauagc ucugaucauc cuuucaaguu uccguggucc uggcaggugu 300

auggugugcg gccagaggca cuaa 324

<210> 4

<211> 192

<212> RNA

<213> CVR Virus (Chrysanthemum virus R)

<220>

<221> misc_feature

<222> (1)..(192)

<223> ORF4

<400> 4

auguauguuu auaucuuguu gagugugcuu gugugcuugu uuucauuaua cuuucugcgu 60

aauuauaguc auccuaguga guguguggua guaguuacug gcgagucuuu uagagugauu 120

aauugugcaa uaaauaggga uuugauugag uugucuaagg caaucaaacc uaggguuugg 180

ggcugccuuu ag 192

<210> 5

<211> 924

<212> RNA

<213> CVR Virus (Chrysanthemum virus R)

<220>

<221> misc_feature

<222> (1)..(924)

<223> ORF5

<400> 5

augccgcaga aaacagaagc agaauuggcu gcagaagcag cauuugcgaa aacugcagaa 60

gaggcgaaaa gacgggcagc agaaauggaa gccaaaaaga gagaagagag gggcaagucu 120

ccagagaccg gccagggcag cggugaagag ucauucgagg aagaagacaa auuagaggcu 180

augcuggaua agcucguuga guaccgccgu aaggaguaca ggaccucacg gaucaccaau 240

gccggguaug aaacugguag accccuuaug cguauccccg gcagaaugcg uggugauuca 300

gccaacaugu acaauaugcu guccaucgau gaucucuaug cgaucaaacc aaaagcuguc 360

ucgaacaaua uggcaaccac agaagauaug gcaaaaguga agguuucucu ugaggcucuu 420

ggagugccca ccgaagaggu guccaggcuc guugcgcaag cagccauuua uugcaaagau 480

acgagcagcu cagaauauau ggacccuagg ggcaccuucg aaacuucugc uggugccauu 540

ucaauggacg cuguauuugc agcgcugaag acccagggag ggaccuugcg ucgugugugc 600

cgacuuuaug cggcaaucac uuggaacuac auguuauugc augaggcgcc uccuucagac 660

uggucagcua ugggcuuuaa aagaaaugaa cgcuucgcug cuuuugacug cuuagacuau 720

guggagaacu cugcagccau uccucccaaa gaaggcuuga uucgcaaacc uacaaguaau 780

gaguacaucg cucauaauac uuacaagcga auagcauuag aucguucuaa ucgcaaugag 840

guauuuggca auuugcaaac agaaauaacu ggcggccguu uuggugauga gauugagcgc 900

aaucacuuaa gaucgaauaa auaa 924

<210> 6

<211> 324

<212> RNA

<213> ORF6(Chrysanthemum virus R)

<220>

<221> misc_feature

<222> (1)..(324)

<223> ORF6

<400> 6

auggaaagca gaaggcuugc ggaagccuug uccgauugcu uucuaggguu guguggugug 60

ucuaauuuag augugugcua cuuaauaauu caaaaggcua ggggucauac ccccggccuu 120

ggcaaaagcu ccuacgccag acguaggcgg gcugcaaagg ugagcaggug ucaccgaugu 180

uaccggguaa cuccuggcuu uuauuauacu acuagaugug auggcaaaac gugugugcca 240

ggcauuuccu acaaugagaa aauagagacc uacguacgua ccggaguaac ugaggugaua 300

ccggacguuc uaggucguuu guga 324

<210> 7

<211> 8890

<212> RNA

<213> CVR Virus (Chrysanthemum virus R)

<220>

<221> misc_feature

<222> (1)..(8890)

<223> genomic sequence

<400> 7

ggauaaacca aacauaacau aaacauacuu uaaauaaauc acuauucgau ucauuuugaa 60

guacguuagc auggcgcuca cuuuccgcag cccuauugca gauguuauua caaauuucga 120

uccauccauc aacuccauua uagccacuac agcugcugaa cacuacaaga ggcuugagga 180

agaaaauuuc gcgcucuuca acuaugcaau gucgccacag acgacgcagc auuugauaga 240

cgccggaauu uacuuaagcc ccuucucugg gguaccucau ucucacccag cuuguaagac 300

ucuggagaau uacauauugu auauaguacu uccccaauau auagauaaua guuuuuauuu 360

ugucgguauu aaagauuuua aauuaaaugc acucaagcuc cggcauaaac auacuaacau 420

ggugcaaugu auuaacaggu acgucacaag ccaagauaaa augcgcuacc ccucagauuu 480

cgucauuagg uacucuaaac cacaugaggg guugucacgg caccgcaugu cauuggaaag 540

ccacacacuc cgugaucugg uaccugagcu aauguccagg agcugcaaga agaucuuccu 600

acaugaugag cuacauuauu ggaagauaga ugagcuuugc acguuuuugg aaguugugca 660

gccuaaagua gucauaggga caauuguuua cccgccagaa uuacugaagg gcucuaagaa 720

aagcuuaaac aauugguguu auacuuacga ggucaaaggg cgcgcgcuua gauucuaccc 780

agauggggug cguggcgaag guuaugagca gccacugaau gguggcuuuu ugcuagagug 840

ugggaagaua acacucuacg acgggacggu guacaugguc gauauccucu gcagcaaauu 900

ugcgcaccac uuaguugcac ucacccgugg ggucgccgca gggccaacaa ugagaucauu 960

uggcccguuu gaagccacug cgugcaaugg ucuugauccc cuuacuaggg auguuaccug 1020

caguuucccg aucccguaug aggugguguc uaaaguuuau agguaccuga agacucucag 1080

gaagccagau gagcaaucau cuauggccaa auugacacaa cuuuugccau gccccacagg 1140

cgaagagauu gucuuugugc aggaguuuuc cagucuaguu aucuccacga acaccaucaa 1200

gaccaugauc agugcugaca ggcucaaagu auucuugggc aaguaccuga guaaguugcc 1260

caaaauacug gcugagcguu uugaaacugu gaaagaacug agucugcaug aauuuaucaa 1320

aaaucuugag ccauucaccg uggacgugca acucaaagaa cucaaaugga acaacgucug 1380

gacgcucgaa uugagggaug acgggauggc ugaugaacug cuggauccau cacggcuaau 1440

ggaggagaag uacacgcaug gcguuuacaa cgcugugccu gaucggagga gugccggcua 1500

uuauguuagg aaagaaaugc uugagcgcau gagggggccu cuaaucgaaa uugagcgcaa 1560

uguuuugaaa aacacccuag cagcaauaug uuacaaaagc uugagcaaua gcgauggaac 1620

ucuugcuucc auaaacgaaa uuggugcuuu uuuuaggaug auuugugcga ggccuuuccu 1680

gagaccagcg aaguacaaag ugcaugaucu guauggaagc aauggagcua ucgggcucca 1740

gaucuacaug agaagucguu gggcgagacu agucaaguuu uauugggcua auauugguau 1800

uuugugguuc aggagcaauc acaaauucua ucaaaaauac cuuuguucug ugccugaugg 1860

cauucaaacu uaccggacuu ucacgcaacc guggaaaacu guaguccacg aggucguuug 1920

cgugaagcau acacucaaga aaacuggcaa uuacucaugg uugaggugcg cuaggguagc 1980

gcaaaagcau ggagguacua augaagagcc uauccgggca gauagaagca cugagaccgc 2040

aggaggugga ucacaguuca augaggcuag aacagaucag gacaacaaaa caaauggggg 2100

ggacgcgccu aaugaagggg aguggacacc uccucaaggc agcaaguaug aggaaaugcc 2160

accgccaccg caacggcaag ggagccagag agaggaacag cgugcgauca aaugcaccug 2220

uaauuuagua uugccaauuu cuacugauga agccacacgc caucacaagu uccuaacucc 2280

ggaccgacuu aagaauaggu gcgccgguuu uuauucaaaa aauaaagagg gguacacuua 2340

caauggcggu ucccaucaaa guuuggggug gccuggcugg aucaaugcuu ggaugcgccu 2400

gaucggcaua ccagaauucu acaauugcug ccuaguacaa cgcuacgacg agggggcuaa 2460

gcucggcuug cauagugaug augaagagug uuuuguccag gguagcccug uguuuacugu 2520

aaauauugaa ggucaugcaa cuuuccucau ugcaugcaag aacucgccug aaaggagggu 2580

gaccagaucc auaguggggc cgggugauuu guuugagaug ccuggugguu uucaagaaac 2640

ccacaaacau ggagucugcg aaacugagaa aggcaggcuc ucgguuacau uucgagugau 2700

gaagaaaaac ccucauagug gugaagaaaa agaccacgga gcaagcgagg gccaccagaa 2760

uggaacagag aaugugaaug accaaaguga aucagaaacc caugaggaaa aaccccagca 2820

agaaggagug aaggaaagag aaaguguaca ggaaaguggu gagggcauuu cugaagaaca 2880

caaacaagaa cagagugacu gccccaauaa cgguacagcu ggagcuggga cagaggcauu 2940

cucaagacac uuuaaugguu gcaaagugag cgugcgcaaa gaggaacuug cgcacauaua 3000

caguacaacu gauuguggcg gggguggcaa cuguuuuugg cuuugcuugg ccacugagau 3060

gggcugcgac ccuuuuagag gaaagaaauu ggcacuagag uaugaucugg guuccgcagg 3120

ggcaaaugaa ucugcacgca guugugcugg cgagggugug uaugcuguag augaagccau 3180

ugcaugugcu gcaagugugu ucagggucgu cauuaaaaua uaccaacccg aguugaaugu 3240

caugacuacc uuugagccag caaaagggga gcggguccuu cacuuggaau uagaggggga 3300

acacuucaga cucaugagaa uaaucaacgg gugcguaauu agagccauaa gcucugcacu 3360

caaucgcaga gagggugaag ugaugcacgu gauugaagag aauugugacc ccacaaugau 3420

cacugaccuu uggaaagggg cggguguuga uuugagugua uuucacuccc uccuugagcu 3480

uuucucuauu aaagcuuuga uuuuugaagg aucuagggaa guguuguaca acagugaagg 3540

gcgcuucgaa gcauccuucg aaauaaaagg ugaucacaua gaacauguau ugaggaagaa 3600

gggugcgugc aauguccuuu uugaggaaug ugggaagacu uucgagguga aggccgaauc 3660

ucuagaacua cugaacagag caggcacauu auuaaaguac aagagcacuc uugcuagggc 3720

aaaaagacuc gcagacagcu uguguuccgg aacgacugga guuguuagcu caucuuuauu 3780

caauaagaag ccuaaccuug caggccaauu uagggaaaag acagagucuc uugauaggga 3840

uguucuugcu gugauuggaa cuuucggauc aggcaagagu acuauccuca agaacuuuuu 3900

caaaaucaau uugggucgaa aagugcuaua ugugucccca cgcagagcac ucuuaaauga 3960

auuccagaga agugcuugcg gugacuuuaa agacaagaag gaacgcaaga augcaagaaa 4020

gaaaggccaa gagaacuggg auuucaugac uuucgaaacc uucauacuua aaugcucuag 4080

uuuaccuuca gguauggcug uaguccuuga ugagauacaa uuguacccac caggguaucu 4140

ggauaugcuc uguuaccuag uuaaggaagg ugugcauuug auagucgcag gagauccagc 4200

ucaaagugau uacgauagcg agaaagaucg agcuugguug cauggcgucg aaccggauca 4260

cucgaaauua cugacuggga augaguauaa guacgucacu uugaguaaga gaaugcaaaa 4320

cuccauguuu gaaggcagau ugccgugcac auuaaaagua gccaaaaaug cggacaccaa 4380

ugaagauuuc gaaauuagag guggugugga gauuauacac aaacucgaua gcaaguggag 4440

agaaugcuuc cucgugucuu ccuucgaaga aaaacgauug auugaguacu accagacuga 4500

gagcucaaaa gauaaagagu gcauccuuac uuuuggagaa ucuacgggcc uuacuuucga 4560

uuacgguagc auccuaauca cucaaaguuc agagaagacg aacgagcagc gguggauaac 4620

ugccuugagc cgauuucgga agagcgucug cuucauuaau gccacaggca ugacgcuaca 4680

ugaacuauua cuugucuaua aagaucgggc ucucggcagg uucuugggua agcgagcgag 4740

ugucgaggau cuaaagaagu uccugccagg aagcccucaa uucgcagaag aauacguacc 4800

cucuauggga gcugagugug gcucuaggga ggagaagcug gcaggggacc ccugguuaaa 4860

gacgaugauc gaucuuuugc agauagagga uguggaagag gaagucggcg ugguugaaga 4920

aauagcccgu gaggaguggu ucaagacgca uuuaccucag gaagaacugg agagcgucag 4980

agcgaggugg gugcacaaga uuuuggccaa ggaagcuaga gaggucagaa ugggugacau 5040

uguuucugaa caguucacug augagcacag uaaggaaaaa gguaagcaac ucaccaaugc 5100

agcggagcgu uuugaaacaa uauacccuag gcauaggucg agugacacgg ugacuuucau 5160

aauggcggua cgaaagaggc ugagguucuc caagcccaca guggagaugc ccaaauugag 5220

ggccgcaaaa cccuauggac caauguugcu caagaaguuc uugaagcaug ugccacucaa 5280

accgaauaaa gacccgauac ugauggccag ggcaaagaug gaauuugagg aaaagaaggu 5340

cagcaaaucc gcggcaacua uugagaauca caguguacgg ucaaccagag acuggcuagu 5400

ggacguuggg cagauuuuuu cuaagaguca aauaugcacc aaauuugaaa aacguuucag 5460

ugcugcgaaa gcggcacaau ccauugugug cuuccaacau ucgguacucg uuagguuugc 5520

ucccuauaug cgcuacauug agcuuaaauu gagggaaucc cugcccgaua gauucuauau 5580

acauucgggc aagggcuugg augagcucga uucuugggug cgcucgcaug gcuuuagcgg 5640

gauguguaca gaaucugauu acgaagccuu cgaugcaucc caagaccaau auauugucgc 5700

cuuugagauu gcucucauga aguaucuggg ucugccgcaa aguuuaauag cagacuauga 5760

guucaucaag acgcaccugg gaucaaaacu aggaucauuc gcuaucauga gauucucagg 5820

ggaagcaagu accuuccuuu ucaacacuau ggccaauaug cuauucacgu ucaugcgcua 5880

ugauauuaac aacaaggauu gcaucugcuu ugcuggggau gauaugugug cuucaagacg 5940

uuugccauua agcaaagagu acgaaagcuu uuuauccaaa uugaaacuga aagccaaagu 6000

gcaguucacg gagaagccua cauucugugg uuggaaucuc uccccugaug guaucuauaa 6060

gaaaccucaa uugguucucg agagaaugug uauagcgaag gagaccaaua accugcacaa 6120

uugcauagac aauuacgcua ucgaaguuuc cuacgcguac aagaugggcg aaaaagcagu 6180

caauaggaug gacgaagagg aauuggccgc cuacuacaac ugugugcgua ucaucauuaa 6240

gaauaaacau uugcuacguu cgaauauuuc ugagguguac aguaauaggg cuugguagcu 6300

uagguuguug cuaguuuagu ugacguaugg auguguuagu uaauuuagcu aagaauuuug 6360

guuuucauag gguguguacu acauugaaua aacccauugu uuuccacugu guuccgggug 6420

caggcaaguc caccuugauu cgugauugcu guagggcaga uaguagauuc guugcgguca 6480

cuuugggcaa agaggacgag agggauuuga ccgggguagg cauuacaaaa uauucuggua 6540

ccaucccaga uugcgaguac ccgcucuuag acgaauauac ccuugcggaa agcuugucag 6600

aaggcugguu ugcaaucuuu ggugauccaa uccaagcagc uaaucgcgug guacugcgug 6660

cacauuucac uugcaaucua agccgcagau ucgguaagug cacugcccaa uuccuuaggg 6720

aguugggauu ugacguaguu gcugagggug aggauaccgu ucagaucucu gggauuuacg 6780

uagaggaccc gcgggauaaa auaauauauu acgagccuga gguggguugc uuacuaagga 6840

gucacucagu ugaagcuagc cacaucacag aaguaauugg ucaaacuuuu gauuccguga 6900

cuuuuguuac ugcuagugaa uauccaguag aucgcucgag ugcuuaucag uguuugacga 6960

gacaucgcaa gagcuuacuc auucuagcac ccaaugccuc uuacuccucc gccugaucau 7020

acaaaggcau uuuuagcuau agcugugggu uuuucuugug cauuaaucau aagccuauuu 7080

acucguagua ccuuaccuca agucggagau caaauccauu cgcugccgca cggcgguugg 7140

uacaaagacg gaacaaagca aaucuacuac ggacgaccga auaaauugaa cucaguagag 7200

aaacaaggcu uccucauugg ucagcccugg gccaucguaa ucauacuuau agcucugauc 7260

auccuuucaa guuuccgugg uccuggcagg uguauggugu gcggccagag gcacuaaugu 7320

auguuuauau cuuguugagu gugcuugugu gcuuguuuuc auuauacuuu cugcguaauu 7380

auagucaucc uagugagugu gugguaguag uuacuggcga gucuuuuaga gugauuaauu 7440

gugcaauaaa uagggauuug auugaguugu cuaaggcaau caaaccuagg guuuggggcu 7500

gccuuuaggu uuacaggcua gagauuguua acgaaaucuc acacagaaaa ugccgcagaa 7560

aacagaagca gaauuggcug cagaagcagc auuugcgaaa acugcagaag aggcgaaaag 7620

acgggcagca gaaauggaag ccaaaaagag agaagagagg ggcaagucuc cagagaccgg 7680

ccagggcagc ggugaagagu cauucgagga agaagacaaa uuagaggcua ugcuggauaa 7740

gcucguugag uaccgccgua aggaguacag gaccucacgg aucaccaaug ccggguauga 7800

aacugguaga ccccuuaugc guauccccgg cagaaugcgu ggugauucag ccaacaugua 7860

caauaugcug uccaucgaug aucucuaugc gaucaaacca aaagcugucu cgaacaauau 7920

ggcaaccaca gaagauaugg caaaagugaa gguuucucuu gaggcucuug gagugcccac 7980

cgaagaggug uccaggcucg uugcgcaagc agccauuuau ugcaaagaua cgagcagcuc 8040

agaauauaug gacccuaggg gcaccuucga aacuucugcu ggugccauuu caauggacgc 8100

uguauuugca gcgcugaaga cccagggagg gaccuugcgu cgugugugcc gacuuuaugc 8160

ggcaaucacu uggaacuaca uguuauugca ugaggcgccu ccuucagacu ggucagcuau 8220

gggcuuuaaa agaaaugaac gcuucgcugc uuuugacugc uuagacuaug uggagaacuc 8280

ugcagccauu ccucccaaag aaggcuugau ucgcaaaccu acaaguaaug aguacaucgc 8340

ucauaauacu uacaagcgaa uagcauuaga ucguucuaau cgcaaugagg uauuuggcaa 8400

uuugcaaaca gaaauaacug gcggccguuu uggugaugag auugagcgca aucacuuaag 8460

aucgaauaaa uaauggaaag cagaaggcuu gcggaagccu uguccgauug cuuucuaggg 8520

uuguguggug ugucuaauuu agaugugugc uacuuaauaa uucaaaaggc uaggggucau 8580

acccccggcc uuggcaaaag cuccuacgcc agacguaggc gggcugcaaa ggugagcagg 8640

ugucaccgau guuaccgggu aacuccuggc uuuuauuaua cuacuagaug ugauggcaaa 8700

acgugugugc caggcauuuc cuacaaugag aaaauagaga ccuacguacg uaccggagua 8760

acugagguga uaccggacgu ucuaggucgu uugugauguu gcauauaaag ccuaaauaau 8820

uuauaagugu guaacuauaa aaagagugaa guuuuaaagu auuuuuccuu cuuuaaaaaa 8880

aaaaaaaaaa 8890

<210> 8

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(18)

<223> primer

<400> 8

aatgctgcgt cgtctgtg 18

<210> 9

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(22)

<223> primer

<400> 9

atggcactca catacagaag tc 22

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(21)

<223> primer

<400> 10

cagtggctgc tcataacctt c 21

<210> 11

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(19)

<223> primer

<400> 11

gcaccgcatg tcattggaa 19

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(21)

<223> primer

<400> 12

ggaaccgcca ttgtaagtgt a 21

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(20)

<223> primer

<400> 13

tggctgatga actgctggat 20

<210> 14

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(22)

<223> primer

<400> 14

atgaaatccc agttctcttg gc 22

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(20)

<223> primer

<400> 15

agaagtgctt gcggtgactt 20

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(20)

<223> primer

<400> 16

gctttttcgc ccatcttgta 20

<210> 17

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(19)

<223> primer

<400> 17

atacattcgg gcaagggct 19

<210> 18

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(22)

<223> primer

<400> 18

ggaagccttg tttctctact ga 22

<210> 19

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(22)

<223> primer

<400> 19

atctactacg gacgaccgaa ta 22

<210> 20

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(22)

<223> primer

<400> 20

gcaacatcac aaacgaccta ga 22

<210> 21

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(21)

<223> primer

<400> 21

ggagtaactg aggtgatacc g 21

<210> 22

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(21)

<223> primer

<400> 22

ctccacgaac accatcaaga c 21

<210> 23

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(22)

<223> primer

<400> 23

gctccattgc ttccatacag at 22

<210> 24

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(22)

<223> primer

<400> 24

ggtatggctg tagtccttga tg 22

<210> 25

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(20)

<223> primer

<400> 25

tgacgctctc cagttcttcc 20

<210> 26

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(22)

<223> primer

<400> 26

cttgattcgt gattgctgta gg 22

<210> 27

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(20)

<223> primer

<400> 27

atcaggcgga ggagtaagag 20

<210> 28

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_feature

<222> (1)..(20)

<223> primer

<400> 28

ggctgaccag tctgaaggag 20

Claims

1. An isolated organism comprising a nucleotide selected from the group consisting of:

the nucleotide shown as SEQ ID No. 7;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 7.

2. The isolated organism of claim 1, wherein:

the organism is a microorganism.

3. The isolated organism of claim 1, wherein said organism is a virus.

4. The isolated organism of claim 3, wherein the organism is Carlavus of the genus Dianthus caryophyllus.

5. The isolated organism of claim 1, wherein said nucleotide is RNA.

6. An isolated nucleotide, which is a nucleotide selected from the group consisting of:

the nucleotide shown as SEQ ID No. 1;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 1;

the nucleotide shown as SEQ ID No. 2;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 2;

the nucleotide shown as SEQ ID No. 3;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 3;

the nucleotide shown as SEQ ID No. 4;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 4;

the nucleotide shown as SEQ ID No. 5;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 5;

the nucleotide shown as SEQ ID No. 6;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 6;

the nucleotide shown as SEQ ID No. 7;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 7.

7. A primer or probe specific for a nucleotide selected from the group consisting of:

the nucleotide shown as SEQ ID No. 1;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 1;

the nucleotide shown as SEQ ID No. 2;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 2;

the nucleotide shown as SEQ ID No. 3;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 3;

the nucleotide shown as SEQ ID No. 4;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 4;

the nucleotide shown as SEQ ID No. 5;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 5;

the nucleotide shown as SEQ ID No. 6;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 6;

the nucleotide shown as SEQ ID No. 7;

a nucleotide complementary to the nucleotide shown in SEQ ID No. 7.

8. The primer or probe of claim 7, wherein the primer is a primer pair selected from any one of the group consisting of:

a primer shown as SEQ ID NO.22 and a primer shown as SEQ ID NO. 23;

a primer shown as SEQ ID NO.24 and a primer shown as SEQ ID NO. 25;

a primer shown as SEQ ID NO.26 and a primer shown as SEQ ID NO. 27;

a primer shown as SEQ ID NO.19 and a primer shown as SEQ ID NO. 28.

9. Use of the primer or probe of claim 7 for the diagnosis of stunting disease in plants of the Asteraceae family.

10. Use according to claim 9, wherein the Asteraceae plant is selected from the group consisting of: flos Chrysanthemi, early-maturing flos Chrysanthemi, late-maturing flos Chrysanthemi, HUANGGONGJU, SILICANGJU, flos Chrysanthemi, HUAJU, Bobo flos Chrysanthemi, QIJU, Chuzhou flos Chrysanthemi, multiheaded flos Chrysanthemi, solely flos Chrysanthemi, herba Chrysanthemum morifolium Ramat, flos Chrysanthemi Indici, flos Matricariae Chamomillae, flos Matrica.

11. Use of the primer or probe of claim 7 for identifying an organism, wherein:

the organism is the isolated organism of any one of claims 1 to 5;

the identification refers to that: determining whether the organism to be tested is an isolated organism according to any one of claims 1 to 5; determining the presence or absence of the isolated organism of any one of claims 1 to 5; alternatively, quantifying the isolated organism of any one of claims 1 to 5.

12. Use of the primer or probe of claim 7 in the preparation of a biological detection device.

13. The use of claim 12, the organism being the isolated organism of claim 1 or 2.

14. The use according to claim 12, the detection device being in the form of a reagent or a chip.

15.A detection device comprising the primer or probe of claim 7 or 8.

16. The test device of claim 15, which is in the form of a reagent or a chip.

17. A method of detecting a pathogen comprising the steps of:

1) providing a sample to be detected;

2) obtaining the nucleotide of the sample to be detected;

3) contacting the nucleotide or amplification product or reverse transcription product thereof of the test sample with the primer or probe of claim 7 or 8;

4) determining the presence or absence of the isolated nucleotide of claim 6.

18. The method of claim 17, wherein the test sample is isolated from a plant of the family Compositae.

19. The method of claim 18, wherein the Asteraceae plant is selected from the group consisting of: flos Chrysanthemi, early-maturing flos Chrysanthemi, late-maturing flos Chrysanthemi, HUANGGONGJU, SILICANGJU, flos Chrysanthemi, HUAJU, Bobo flos Chrysanthemi, QIJU, Chuzhou flos Chrysanthemi, multiheaded flos Chrysanthemi, solely flos Chrysanthemi, herba Chrysanthemum morifolium Ramat, flos Chrysanthemi Indici, flos Matricariae Chamomillae, flos Matrica.

20. The method of claim 17, wherein the nucleotide is RNA.