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NY/T 2288-2021 (NYT2288-2021)

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BASIC DATA
Standard ID NY/T 2288-2021 (NY/T2288-2021)
Description (Translated English) (Cucumber green mottle mosaic virus quarantine, detection and identification method)
Sector / Industry Agriculture Industry Standard (Recommended)
Classification of Chinese Standard B16
Date of Issue 2021-05-07
Date of Implementation 2021-11-01
Older Standard (superseded by this standard) NY/T 2288-2012
Drafting Organization National Agricultural Technology Extension Service Center
Administrative Organization National Agricultural Technology Extension Service Center
Regulation (derived from) Ministry of Agriculture and Rural Affairs Announcement No. 424

Standards related to: NY/T 2288-2021

NY/T 2288-2021
AGRICULTURAL INDUSTRY STANDARD
ICS 65.020
CCS B 16
Replacing NY/T 2288-2012
Detection and identification method of Cucumber green
mottle mosaic virus
ISSUED ON. MAY 07, 2021
IMPLEMENTED ON. NOVEMBER 01, 2021
Issued by. Ministry of Agriculture and Rural Affairs of PRC
Table of Contents
Foreword... 3 
1 Scope... 5 
2 Normative references... 5 
3 Terms and definitions... 5 
4 Principles... 5 
5 Reagents and materials... 6 
6 Instruments and utensils... 6 
7 Sampling... 6 
8 Testing... 6 
9 Results judgement and reporting... 7 
10 Sample processing... 8 
11 Archive preservation... 8 
Appendix A (Informative) Reagent preparation method... 9 
Appendix B (Informative) Basic information of cucumber green mottle mosaic virus
... 10 
Appendix C (Informative) Rapid diagnostic test strip or test kit for cucumber green
mottle mosaic virus... 13 
Appendix D (Informative) Double antibody sandwich enzyme-linked immunosorbent
assay for cucumber green mottle mosaic virus... 14 
Appendix E (Informative) Reverse transcription polymerase chain reaction (RT-PCR)
assay of cucumber green mottle mosaic virus... 16 
Appendix F (Informative) Identification report of plant pest sample... 19 
Detection and identification method of Cucumber green
mottle mosaic virus
1 Scope
This document specifies the sampling methods, quarantine detection, result
determination and reporting, sample processing, file preservation of cucumber mottle
mosaic virus.
This document is applicable to the quarantine detection and identification of cucumber
mottle mosaic virus, in agricultural plant quarantine.
2 Normative references
The provisions in following documents become the provisions of this Standard through
reference in this Standard. For the dated documents, only the versions with the dates
indicated are applicable to this document; for the undated documents, only the latest
version (including all the amendments) is applicable to this standard.
GB 15569 Quarantine protocol for the movement of agricultural plants and plant
products
GB/T 28071 Detection and identification of cucumber green mottle mosaic virus
3 Terms and definitions
There are no terms and definitions, that need to be defined in this document.
4 Principles
Cucumber green mottle mosaic virus (CGMMV) belongs to the genus Tobacco mosaic
virus, which spreads with seeds, scions, rootstocks. The important basis for the virus
quarantine detection and identification is typical symptoms and other rapid diagnosis.
It is combined with the detection results of virus double antibody sandwich enzyme-
linked immunosorbent (ELISA) or the results of reverse transcription polymerase chain
reaction (RT-PCR), for comprehensive judgement.
5 Reagents and materials
Sodium carbonate (Na2CO3), sodium bicarbonate (NaHCO3), anhydrous disodium
hydrogen phosphate (Na2HPO4), anhydrous potassium dihydrogen phosphate
(KH2PO4), sodium chloride (NaCl), potassium chloride (KCl), magnesium chloride
(MgCl2), anhydrous sodium sulfite (Na2SO3), sodium hydroxide (NaOH), sodium azide
(NaN3), ethanol (CH3CH2OH), disodium p-nitrophenylphosphonate (PNPP), diethyl
pyrocarbonate (DEPC), deionized water, commercially available antibodies,
commercially available enzyme-labeled antibodies, commercially available PCR
reagents, commercially available Trizol reagents, etc. The ratio of reagents, which are
required in the testing, is as shown in Appendix A.
All reagents are of analytical pure.
6 Instruments and utensils
PCR instrument, desktop high-speed refrigerated centrifuge, spectrophotometer,
electronic balance, enzyme-linked plate, enzyme-linked reader, enzyme-free plate
machine, micropipette, water bath, electrophoresis instrument and horizontal
electrophoresis tank, gel imaging system, incubators, refrigerators, etc.
7 Sampling
7.1 Seeds
The sampling quantity and method shall be carried out, according to the provisions of
GB 15569.The sampling shall be sufficient and representative.
7.2 Leaves and fruits
Randomly select the diseased or suspected diseased leaves or fruits, mainly young
leaves and fruits. Each fruit sample shall be not less than 200 g; the leaves shall be not
less than 10 g. They shall be sealed in a Ziplock bag and stored at 4 °C, indicating the
information, such as the name of the plant, collection time, place, collector.
8 Testing
8.1 Symptom inspection
Check the leaves, stems, fruits, for comparison with typical symptoms of cucumber
green mottle mosaic virus disease (see Appendix B).
8.2 Rapid diagnosis
See Appendix C, for rapid diagnostic test strips or diagnostic kits. If using commercially
available test strips or kits, follow the instructions to operate.
8.3 Enzyme linked immunosorbent assay (ELISA)
See Appendix D, for enzyme linked immunosorbent assay of cucumber green mottle
mosaic virus. If a commercially available kit is used, follow the instructions to operate.
8.4 Reverse transcription polymerase chain reaction detection (RT-PCR)
Refer to Appendix E, for the reverse transcription polymerase chain reaction detection
(RT-PCR) of cucumber green mottle mosaic virus. If using a commercially available
kit, follow the instructions to operate.
9 Results judgement and reporting
9.1 Result judgment
9.1.1 Typical symptoms
If the fruit is diseased and has typical symptoms, it can be initially judged.
9.1.2 Rapid diagnostic results
If the test result of rapid diagnostic test strip or diagnostic kit is positive, it can be judged
as highly suspected.
9.1.3 Enzyme linked immunosorbent assay (ELISA)
Under the premise that the OD value of the negative control optical density value is ≤
0.1, AND positive control's OD value is greater than 2 times the negative control's OD
value, if the sample hole's OD value is greater than 2 times the negative control's OD
value, it is determined as a positive reaction, that is, the sample has cucumber green
mottle mosaic virus.
9.1.4 Reverse transcription polymerase chain reaction detection (RT-PCR)
Observe the electrophoresis results. On the premise that the positive control has a band
at 654 bp, whilst the blank and negative controls have no band, if the sample to be tested
has a band at 654 bp, THEN, it is judged as a positive reaction, that is, the sample has
cucumber green mottle mosaic virus.
9.1.5 Result report
Fill in the identification results, in the plant pest sample identification report, in
Appendix F.
Appendix A
(Informative)
Reagent preparation method
A.1 Coating buffer
Take 1.59 g of Na2CO3, 2.93 g of NaHCO3, 0.2 g of NaN3.Use deionized water to dilute
it to 1000 mL. Adjust the pH to 9.6.Store it at 4 °C.
A.2 Washing buffer
Take 1.15 g of Na2HPO4, 0.2 g of KH2PO4, 8.0 g of NaCl, 0.2 g of KCl, 0.5 g of Tween-
20.Use deionized water to dilute it to 1000 mL. Adjust pH to 7.4.Store it at room
temperature.
A.3 Extraction buffer
Take 2.0 g of egg white powder, 20.0 g of polyvinylpyrrolidone (PVP, K30) (molecular
weight 44000 ~ 54000), 1.3 g of Na2SO3, 0.2 g of NaN3, 20.0 g of Tween-20.Dissolve
it in 1000 mL of 1XPBST. Adjust pH to 7.4.Store it at 4 °C.
A.4 Enzyme-labeled antibody buffer
Take 2.0 g of bovine serum albumin, 10.0 g of polyvinylpyrrolidone (PVP, K30), 0.2 g
of NaN3.Dissolve it in 1000 mL of 1XPBST. Adjust to pH 7.4.Store it at 4 °C.
A.5 Substrate buffer
Take 97.0 mL of diethanolamine, 0.1 g of MgCl2, 0.2 g of NaN3.Use deionized water
to dilute it to 800 mL. Adjust pH to 9.8.Store it at 4 °C.
Appendix B
(Informative)
Basic information of cucumber green mottle mosaic virus
B.1 Host range
Watermelon, gourd, cucumber, pumpkin, gourd and other melon crops.
B.2 Biological characteristics
B.2.1 Lethal temperature
90 °C ~ 100 °C.
B.2.2 Dilution limit
10-7 mL/L ~ 10-6 mL/L.
B.2.3 In vitro survival period
Up to 119 d or longer. It can be kept for several years at 0 °C. The virus can survive in
the seeds for 240 d ~ 540 d. After the diseased residue is buried in the soil for 420 d,
the virus still has infection activity.
B.2.4 Morphology of virions
The virions are rod-shaped by electron microscope, about 300 nm long and 15 nm wide.
B.3 Typical symptoms
B.3.1 Symptoms of watermelon infection
The leaves have irregular chlorotic, in mottled mosaic; the green part is uplifted; the
leaf margins are rolled up; the leaves are slightly narrower and thinner. Fruiting pedicels
sometimes show brown necrotic streaks. There are filamentous yellow fibers in the pulp;
the pulp around the seeds becomes dark red and water-stained. In severe cases, the pulp
becomes dark red, with a lot of cavities in it; it becomes softening, rotten, smelly, which
is inedible (see Figure B.1 and Figure B.2).
Appendix D
(Informative)
Double antibody sandwich enzyme-linked immunosorbent assay for cucumber
green mottle mosaic virus
D.1 Preparation of sample
D.1.1 Seeds
Take a few seeds. Peel them. Add sample extraction buffer (W.V = 1.8). Grind and
homogenize it. Centrifuge it, at 5000 r/min at 4 °C for 5 min. Store it for later use.
D.1.2 Leaves
Take leaves. Add extraction buffer (W.V = 1.8). Grind and homogenize it. Centrifuge
at 5000 r/min for 5 min at 4 °C. Store for later use.
D.2 Coating
Use coating antibody buffer solution, to dilute the coating antibody of CGMMV,
according to the dilution ratio specified by the kit. Mix well. Add it to the enzyme-
linked plate. Add 100 µL to each hole. Put it in a humidor. Incubate at 37 °C, for 4 h.
D.3 Washing
After the incubation, pour out the reagents in the reaction holes. Use PBST washing
solution, to rinse it 3 ~ 4 times, for 15 s each time. Then invert the microplate on
absorbent paper, to naturally dry the residual liquid in the holes.
D.4 Add antigen (sample to be tested)
Add the antigen (sample to be tested) to the enzyme-linked plate. At the same time, add
100 µL of positive control, negative control, blank control (sample extraction buffer).
Repeat 3 times for the sample to be tested. Incubate for 2 h, in an incubator or at room
temperature (25 °C) h. Store the remaining samples, in a -20 °C refrigerator, for future
reference.
D.5 Washing
It is carried out, according to D.3.
D.6 Add enzyme-labeled antibody
Use enzyme-labeled antibody buffer, to dilute the enzyme-labeled antibody of CGMMV,
Appendix E
(Informative)
Reverse transcription polymerase chain reaction (RT-PCR) assay of cucumber
green mottle mosaic virus
E.1 Pretreatment of utensils
To prevent RNA degradation by RNA enzyme, the vessels are treated by DEPC water
(V.V=1.1000) before the test. Place the mortar, gun tip, centrifuge tube, etc. in DEPC
water at 37 °C. Soak it for 24 h. Take out. Sterilize it at 121 °C for 30 min. Bake it dry
for later use.
E.2 Extraction of total RNA
Take symptomatic leaves. Grind them fully in liquid nitrogen. Transfer them into a 1.5
mL centrifuge tube. Add 1 mL of Trizol reagent. Let it stand for 5 min, at room
temperature. Then add 200 µL of chloroform. Vortex-shake it for 15 s. Place at room
temperature for 5 min. Centrifuge it at 12000 r/min, at 4 °C for 15 min. Transfer the
supernatant to a new centrifuge tube. Add 600 µL of isopropanol. Mix well. Let it stand
at room temperature for 10 min. Centrifuge it at 12000 r/min, at 4 °C for 15 min. Discard
the supernatant. Add 1 mL of 75% ethanol into the precipitate, to wash it once. Absorb
the supernatant. Dry it at room temperature for 3 min ~ 5 min. Add 30 µL of DEPC
ddH2O, to dissolve the total RNA.
E.3 RT-PCR reaction system
E.3.1 Primer sequence (CGMMV-654F/R)
Upstream primer (Primer F). 5'-CGTGGTAAGCGGCATTCTAAACCTC-3';
Downstream primer (Primer R). 5'-CCGCAAACCAATGAGCAAACCG-3'.
E.3.2 RT-PCR experimental steps
E.3.2.1 cDNA synthesis
Prepare the RT reaction system (20 µL), according to the following components. The
reaction is performed, by adding ddH2O without RNA enzyme as a negative control.
The reaction conditions are 42 °C for 1 h, to synthesize cDNA, inactivate reverse
transcriptase at 70 °C for 10 min. The reaction system is as shown in Table E.1.
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