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NY/T 2288-2021: Detection and identification method of Cucumber green mottle mosaic virus
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 deioniz...