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People's Republic of China Entry-Exit Inspection and Quarantine Industry Standard SN/T2055—2008
Identification of cowpea severe mosaic virus Quarantine and identification method
Identification of cowpea severe mosaic virus Issued on April 29, 2008
People's Republic of China
General Administration of Quality Supervision, Inspection and Quarantine
Implementation on November 1, 2008
Appendix A of this standard is an informative appendix.
This standard is proposed and managed by the Certification and Accreditation Administration of the People's Republic of China. SN/T2055—2008
Drafting units of this standard: China Inspection and Quarantine Scientific Research Institute, Beijing Entry-Exit Inspection and Quarantine Bureau of the People's Republic of China Main drafters of this standard: Li Guifen, Li Mingfu, Wei Meisheng, Zhang Yongjiang, Xiang Ning, Wang Jinhua This standard is the first issued entry-exit inspection and quarantine industry standard I
1 Scope
Quarantine and identification methods for cowpea severe mosaic virus
This standard specifies the quarantine and identification methods for cowpea severe mosaic virus. This standard is applicable to the quarantine and identification of imported seedlings or propagation materials that may carry rainbow bean severe mosaic virus. 2 Principle
SN/T2055—2008
Scientific name of cowpea severe mosaic virus: cowpeas everemosaic virus, abbreviation: CPSMV. The biological and serological characteristics of Cowpea Mosaic Virus are the main basis for the formulation of this standard; the taxonomic status, host range, disease symptoms, geographical distribution, transmission routes, particle morphology, etc. of the virus are shown in Appendix A.
Instruments and appliances
3.1 Instruments
3.1.1 ELISA detector
3.1.2 Balance (0.001g).
3.1.3 Water bath.
3.1.4 Isolation greenhouse.
3.2 Appliances
3.2.1 Adjustable pipette, adjustable pipette tip3.2.2 ELISA plate.
3.2.3 Eppendorf tube.
3.2.4 Mortar.
4 Quarantine and Identification Methods
Pre-testing of Bean Seeds
The seeds should be sown in soil that has been sterilized at high temperature, cultured in an isolated greenhouse and observed for growth. When 6 to 8 true leaves have grown, the test should be conducted. First, plants with suspected virus symptoms should be selected for testing, and plant samples should also be randomly selected for testing. 4.2 Double Antibody Sandwich ELISA 4.2.1 Test Materials
4.2.1.1 ELISA Plate
Use ELISA plates produced by quality-assured manufacturers. 4.2.1.2 Coated Antibodies
Anti-bean Heavy Mosaic Virus Antibodies with Good Specificity 4.2.1.3 Enzyme-labeled Antibodies
Anti-rainbow bean heavy mosaic virus antibodies labeled with alkaline phosphatase. 4.2.1.4 Coating buffer (pH 9.6)
Sodium carbonate (NazCO3)
Sodium bicarbonate (NaHCOs)www.bzxz.net
Sodium azide (NaN3)
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Make up to 1L with distilled water, store at 4℃
4.2.1.5 Washing buffer (PBST, pH 7.4) Sodium chloride (NaCI)
Potassium dihydrogen phosphate (KH2PO)
Sodium dihydrogen phosphate (Na2HPO)
Potassium chloride (KCI)
Tween-20 (Tween-20)
Sodium azide (NaN)
Make up to 1L with distilled water.
4.2.1.6 Sample extraction buffer
Sodium sulfite (Na2SO3)
Polyvinyl pyrrolidone (PVP, MV24000~40000) Dissolve to 1L with washing solution (PBST), store at 4℃. 4.2.1.7 Enzyme-labeled antibody dilution buffer
Polyvinyl pyrrolidone (PVP.MV24000~40000) Bovine serum albumin (BSA)
Dissolve to 1L with washing solution (PBST), store at 4℃. 4.2.1.8 Substrate buffer
Diethanolamine (C4HiNO2)
Distilled water (H2O)
Adjust pH to 9.8 with hydrochloric acid (HCl). Then dilute to 1L with distilled water, store at 4℃. 4.2.2 Experimental steps
4.2.2.1 Dilute the coating antibody with coating buffer according to the required concentration and volume, and add 100μL to each well. Cover the ELISA plate or wrap it with plastic wrap and incubate it at 37℃ for 2h. 4.2.2.2 Empty the solution in the wells of the ELISA plate, fill each well with PBST, and pour out the washing solution in the wells after 3min. Pat dry on absorbent paper. Repeat the above washing process twice.
4.2.2.3 Add the sample extraction buffer to the sample to be tested at 1: (2~10) (mass concentration), grind it in a mortar, centrifuge it at low speed, and draw the supernatant and add it to the wells of the ELISA plate (100uL/well). Set up 2 replicates for each sample to be tested, and set up positive control wells, negative control wells, and buffer control wells. Cover or wrap with plastic wrap, incubate overnight in a refrigerator at 4℃4.2.2.4 Empty the solution in the wells, rinse thoroughly 3 times under tap water; then fill each well with PBST, pour it out after 3min, and pat dry on absorbent paper. Repeat the above PBST washing process twice. 4.2.2.5 Dilute the enzyme-labeled antibody with enzyme-labeled antibody dilution buffer according to the required concentration, add 100μL to each well, cover or wrap with plastic wrap, incubate at 37℃ for 4h,
4.2.2.6 Empty the solution in the wells, rinse thoroughly 3 times under tap water; then fill each well with PBST, pour it out after 3min, and pat dry on absorbent paper. Repeat the above PBST washing process twice. 4.2.2.7 Dissolve disodium p-nitrophenyl phosphate in substrate solution at a concentration of 1 mg/mL (prepare before use and keep away from light to prevent discoloration). Add 100L of substrate solution to each well. Cover or wrap with plastic wrap, incubate at room temperature away from light until the color is appropriate and read.
4.2.2.8 Measure the absorbance of each well at a wavelength of 405nm using an ELISA reader. 4.2.3 Result determination
4.2.3.1 The OD405 value of the control wells (buffer wells, negative control wells and positive control wells) should be within the quality control range, that is, the OD405 value of the buffer 2
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well and the negative control well is less than 0.15. When the OD4 When the OD405 value is less than 0.05, it is calculated as 0.05; the positive control OD405 value/negative control OD405 value is greater than 5; the repeatability of the wells is consistent. 4.2.3.2 After meeting the quality requirements of 4.2.3.1, the results can be judged as follows: the sample OD405 value/negative control OD405 value is greater than or equal to 2, which is judged as positive. If necessary, the enzyme-linked test needs to be repeated to exclude false positive reactions and obtain accurate results. The sample OD405 value/negative control OD405 value is less than 2 and is judged as negative. 4.2.3.3 If the quality requirements of 4.2.3.1 are not met, the result judgment cannot be carried out. 4.3 Biological assay
4.3.1 Test materials
4.3.1.1 Identification of hosts||t t||Chenopodium amaranticolor, Phaseolus vulgaris variety Pinto, Vigna unguiculata variety Blackeye Early Ramshorn. 4.3.1.2 Grinding buffer
0.01mol/L sodium phosphate buffer (pH7.2)4.3.1.3 Diatomaceous earth.
4.3.2 Method
4.3.2.1 Grinding
Add grinding buffer to the sample at 1: (2-10) (mass concentration) and grind it in a mortar. After grinding, add diatomaceous earth (concentration of 0.5%) and mix it with the diseased juice. ||tt ||4.3.2.2 Inoculation
Age of inoculation of Amaranthaceae: 4 to 6 true leaves. Age of inoculation of Phaseolus vulgaris cultivar Pinto: cotyledon stage or the first true leaf appears. Age of inoculation of Vigna unguiculata cultivar Blackeye Early Ramshorn: cotyledon stage or the first true leaf appears. First, pierce the leaves of the plant to be inoculated with a toothpick or other tools as a mark of the inoculated leaves, and then gently apply the sample juice to the upper surface of the host leaf with your hands.
4.3.2.3 Rinse
Rinse the leaf surface with distilled water.
4.3.2.4 Label
Make a label and place it in an isolated greenhouse.
4.3.2.5 Control of greenhouse conditions
Under natural light, the temperature is controlled at 20℃～30℃. 4.3.2.6 Observation
Observe and record the host reaction in time.
4.3.3 Identification of host symptoms
Broad-colored quinoa: Necrotic spot symptoms appear on the inoculated leaves, and there are no systemic symptoms. Kidney beans (variety Pinto): Ring dead spot symptoms appear on the inoculated leaves, and there are no systemic symptoms. Beans (variety Blackeye Early Ramshorn): Chlorotic spot symptoms appear on the inoculated leaves, and usually there are red necrotic spots. Some main veins turn red and necrotic. Systemic symptoms are mosaic, yellow veins, deformity, and necrosis. 5 Determination of quarantine identification results
The result of the sample enzyme-linked test is positive, and the biological determination of the host reaction is consistent with the description. It can be determined that the sample to be tested carries bean mosaic virus.
SN/T 2055—2008
Sample preservation
Seed samples should be preserved for 6 months. If the samples are found to be infected, they should be preserved for 1 year. If the virus is found in bean plants, the diseased leaves should be collected, dried and stored at -20℃. The original test records, photos and other documents should be registered, marked and archived in accordance with relevant regulations so that they can be reviewed when necessary. A.1 Classification status
Appendix A
(Informative Appendix)
Background information on cowpea mosaic virus
Comoviridae, Comovirus. A.2 Host range
A.2.1 Natural hosts
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Calopogoniummucunoides, Canavaliaensiformis, Centrosemapubescens, Crotalariajuncea, Desmodiumcanescens, Glycinemar, Macroptiliumlathyroides, Phaseolussuulgaris, Psophocarpustetragonolobus, Vignaradiata, Vigna unguiculata, Crotalaria paulineaA.2.2 Test host
The test host includes plants from 3 to 9 families,
Leguminosae: Cajanuscajan, Pisumsativum, Crotalariajuncea, Desmodiumcanescens, Sesbaniaeraltata, Trifoliumincarnatum, Cassiaoccidentalis. Apocynaceae: Catharanthusroseus. Monaceae: Chenopodiumalbum, Chenopodiumamaranticolor, Chenopodiumcapitatum, Chenopodiumquinoa.
Solanaceae: Datura Daturamonium, Nicotiana clevelandii, Petuniahybrida, Nicotiana tabacum, Nicotiana glutinosa. Amaranthaceae: Gomphren aglobososa. A.3 Disease symptoms
Almost all hosts develop chlorotic or necrotic spots on inoculated leaves, systemic symptoms are mottled or mosaic, and young leaves usually develop obvious blister-like protrusions and deformities. Some hosts show systemic necrosis. In Brazil, infected dwarf beans show plant stunting, bud blight, reduced pod number, and reduced yield. Hairy beans show severe mosaic and blister-like protrusions; spur beans show chlorotic and mosaic symptoms; Tibetan hemp leaves show chlorotic mottles and deformities or chlorotic spots; mung bean leaves show mild mosaic and necrotic spots; cowpea leaves show chlorotic mosaic and severe deformities. A.4 Geographical distribution
United States, Trinidad (Republic of Trinidad and Tobago), Puerto Rico, El Salvador, Costa Rica, Venezuela, Suriname, Brazil, Peru, Mexico.
A.5 Transmission route
Mechanical transmission; pollen and seed transmission; beetle transmission, mainly Chrysomelidae beetles, about 10 species, of which Cerotomaruficornis and C.trifurcata are the most important transmission vectors. 5
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Particle morphology
The Cowpea Heavy Mosaic Virus particle is an equiaxially symmetrical icosahedron with a diameter of 25nm and no envelope63 Disease symptoms
Almost all hosts develop chlorotic or necrotic spots on the inoculated leaves, systemic symptoms are mottled or mosaic, and young leaves usually develop obvious blister-like protrusions and deformities. Some hosts show systemic necrosis. In Brazil, infected dwarf beans show plant stunting, bud blight, reduced pod number, and reduced yield. Hairy beans show severe mosaic and blister-like protrusions; spur beans show chlorotic and mosaic symptoms; Tibetan hemp leaves show chlorotic mottles and deformities or chlorotic spots; mung bean leaves show mild mosaic and necrotic spots; cowpea leaves show chlorotic mosaic and severe deformities. A.4 Geographical distribution
United States, Trinidad (Republic of Trinidad and Tobago), Puerto Rico, El Salvador, Costa Rica, Venezuela, Suriname, Brazil, Peru, Mexico.
A.5 Transmission route
Mechanical transmission; pollen and seed transmission; beetle transmission, mainly Chrysomelidae beetles, about 10 species, of which Cerotomaruficornis and C.trifurcata are the most important transmission vectors. 5
SN/T2055—2008
Particle morphology
The Cowpea Heavy Mosaic Virus particle is an equiaxially symmetrical icosahedron with a diameter of 25nm and no envelope63 Disease symptoms
Almost all hosts develop chlorotic or necrotic spots on the inoculated leaves, systemic symptoms are mottled or mosaic, and young leaves usually develop obvious blister-like protrusions and deformities. Some hosts show systemic necrosis. In Brazil, infected dwarf beans show plant stunting, bud blight, reduced pod number, and reduced yield. Hairy beans show severe mosaic and blister-like protrusions; spur beans show chlorotic and mosaic symptoms; Tibetan hemp leaves show chlorotic mottles and deformities or chlorotic spots; mung bean leaves show mild mosaic and necrotic spots; cowpea leaves show chlorotic mosaic and severe deformities. A.4 Geographical distribution
United States, Trinidad (Republic of Trinidad and Tobago), Puerto Rico, El Salvador, Costa Rica, Venezuela, Suriname, Brazil, Peru, Mexico.
A.5 Transmission route
Mechanical transmission; pollen and seed transmission; beetle transmission, mainly Chrysomelidae beetles, about 10 species, of which Cerotomaruficornis and C.trifurcata are the most important transmission vectors. 5
SN/T2055—2008
Particle morphology
The Cowpea Heavy Mosaic Virus particle is an equiaxially symmetrical icosahedron with a diameter of 25nm and no envelope6
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