SN/T 5207-2020Quarantine and identification method of blueberry shock virus (English PDF)

Foreword

This document was drafted in accordance with the rules given in GB/T 1.1-2009.

1 Scope

This document stipulates the principles of blueberry shock virus quarantine and identification, equipment, appliances and reagents, detection and identification, and determination of results and records, as well as the preservation of samples.

This document is applicable to the detection of blueberry shock virus in blueberry seedlings in and out of the country.

2 Normative references

The contents of the following documents constitute the indispensable clauses of this document through normative references in the text. Among them, dated quotations Only the version corresponding to that date is applicable to this document; for undated references, the latest version (including all amendments) is applicable Used in this document.

SN/T 2122 Quarantine sampling method for entry and exit plants and plant products

3 Terms and definitions

There are no terms and definitions that need to be defined in this document.

4 Basic information

The basic information of blueberry shock virus is as follows.

--Scientific name. Blueberry shock virus, abbreviation. BlShV.

--Classification status. member of Bromoviridae and Ilarvirus.

--Classification status, host range, disease symptoms, distribution area, transmission route, morphology of blueberry shock virus, virus See Appendix A for genomes, etc.

5 Principle

The serological and molecular biological characteristics of blueberry shock virus are the main basis for formulating this quarantine identification method.

6 Equipment, utensils and reagents

7 Testing and identification

8 Judgment and record of results

9 Storage of samples

The samples that are judged to be positive by the test result should be properly stored in an ultra-low temperature refrigerator at -20°C~-80°C, and be registered and marked.

Work in preparation for review.

Appendix A

(Informative)

Background information on blueberry shock virus A.1 Classification status Blueberry shock virus is a member of Bromoviridae and Ilarvirus.

A.2 Host range

The reported natural host is blueberry (Vaccinium spp.).

A.3 Disease symptoms the main symptom is the sudden and complete necrosis of flowers and leaves during the flowering period, resulting in no fruit. According to statistics, the virus infects blueberries caused by the loss can reach 34%~90%. BlShV can infect almost all blueberry varieties and show similar symptoms. Since BlShV can communicate Since bees carry infected pollen to spread, it spreads quickly in the field and easily occurs in a large area. Blshv caused by blueberry infection the symptoms are shown in Figure A.1.

A.4 Distribution area Mainly distributed in the United States, Canada and other countries.

A.5 Transmission route it is spread by bees carrying infected pollen, and it can also be spread over long distances through virulent seedlings.

A.6 Chondrial morphology the virions are equiaxially symmetrical and spherical with a diameter of about 27 nm.

A.7 Viral genome

Sense single-stranded RNA virus.

Appendix B

(Normative)

Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) B.1 Reagents B.1.1 Coating antibody Specific blueberry shock virus antibody.

B.1.2 Enzyme-labeled antibody Alkaline phosphatase labeled blueberry shock virus antibody.

B.1.3 Substrate

Disodium p-nitrophenyl phosphate (pNPP).

B.1.4 1×PBST buffer (pH 7.4) B.1.5 Sample extraction buffer (pH 7.4) B.1.6 Coating buffer (pH 9.6) B.1.7 Enzyme-labeled antibody dilution buffer (pH 7.4) B.1.8 Substrate buffer (pH 9.8) B.2 Procedure B.2.1 Sample preparation B.2.1.1 Test sample Weigh 0.5 g~1.0 g of sample tissue, add extraction buffer to the sample to be tested at 1.10 (mass. volume), and grind with a mortar the slurry was centrifuged at 8 000 g for 10 min, and the supernatant was the prepared test sample.

B.2.1.2 Positive control sample the pure virus or tissue extract of blueberry shock virus is provided by the antiserum supplier and prepared with extraction buffer.

B.2.1.3 Negative control sample Provided by the antiserum supplier, prepared with extraction buffer.

B.2.1.4 Blank control sample Use extraction buffer as a blank control.

B.2.2 Coated antibody Dilute the coated antibody with the coating buffer according to the instructions, add it to the well of the enzyme-linked plate, with 100 µL per well, cover, and incubate at 37°C Incubate in a refrigerator for 2 h or 4°C overnight, pour out the solution in the wells of the enzyme-linked plate, and wash with PBST in an amount of.200 µL per well 4-6 times, each time 1 min.

B.2.3 Sample addition Add the prepared test sample, negative control sample, positive control sample, blank control sample, and at least one heavy Repeat, 100 µL per well, cover, incubate at 37°C for 2 h or 4°C in the refrigerator overnight, pour out the solution in the wells of the enzyme-linked plate, press.200 µL per well Wash 4~6 times with PBST for 1 min each time.

B.2.4 Add enzyme-labeled antibody Use enzyme-labeled antibody dilution buffer to dilute the enzyme-labeled antibody to the working concentration according to the instructions, and add it to the enzyme-linked plate, 100 µL per well, Cover, incubate at 37°C for 2 h, pour out the solution in the wells of the enzyme-linked plate, and wash 4-6 times with PBST.

B.2.5 Add substrate

Add the substrate pNPP to the substrate buffer so that the final concentration is 1 mg/mL (prepared for current use), add enzyme per 100 µL per well in the joint plate, incubate in the dark at room temperature.

B.2.6 Reading

Read the optical density (OD) value at 405 nm on the enzyme-linked instrument.

B.3 Judgment of results Detect the OD value of 405 nm in the automatic enzyme-linked immunoassay system. When the OD405 value of the negative control well is less than 0.15, After the OD405 value of the positive control well/the OD405 value of the negative control well is greater than 5 and less than 10, the repeatability of the wells is basically consistent with the quality requirements, the sample OD405 value/negative control OD405 value shows...