SN/T 5205-2020Quarantine and identification method of Boping melon mosaic virus (English PDF)

5.1 Main equipment and utensils

Micro grinder, microplate reader, plate washer, micro balance (sensitivity. 0.001g), PCR machine, electrophoresis machine, horizontal electrophoresis tank, coagulation Gel imager, high-speed refrigerated benchtop centrifuge, ice maker, vortex shaker, water bath, pH meter, adjustable pipette (1000 µL, 200 µL, 20 µL, 2 µL), 96-well microtiter plate, mortar, etc.

5.2 Main reagents

The main reagents and buffers are shown in Appendix B, C, D.

6.1 Seed sample preparation

Pick out malformed and immature seeds and sown them in sterilized soil in the insect-proof net room. After 3 to 4 true leaves have grown, the plants that will show symptoms Number, the plants that did not show symptoms were grouped (10 plants as a group) and numbered. The collected true leaves were divided into 2 parts, and they were used separately for later use as needed.

Continued trials. Seeds can also be tested directly.

6.2 Seedlings and plant products

Seedlings and plant product numbers with symptoms (such as leaf deformities, mosaics, mottled, etc.) are tested separately. Group and number without symptoms the detection, grouping method and detection method are the same as 6.1.

7.1 Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA)

Set up a negative control and a positive control (healthy plant tissue is used as a negative control, and PepMV infected plant tissue is used as a positive control), the sample extraction buffer is used as a blank control, and the type and material of the negative control (such as seeds or leaves) should be as consistent with the test sample as possible. Do not the same detection reagents or kits are operated according to the instructions of the reagents or kits. See Appendix B for specific operations.

7.2 One-step RT-PCR detection

Extract the total RNA of the sample and the control separately, and perform PCR amplification in one step by RT-PCR. Healthy plant tissue is used as a negative control, Plant tissue infected with PepMV was used as a positive control, and ddH2O was used as a blank control. See Appendix C for specific operations.

7.3 One-step real-time fluorescent RT-PCR detection

The total RNA of the sample and the control were extracted separately, and the one-step real-time fluorescent RT-PCR was performed for detection. Healthy plant tissue is used as a negative control, Plant tissue infected with PepMV was used as a positive control, and ddH2O was used as a blank control. See Appendix D for specific operations.

9.1 Results recording

A complete experiment record includes. the source, type, time of the sample, the time, location, method and results of the experiment, etc., and must have the signature of the handler and the experimenter. The original data of the enzyme-linked plate reaction is required for the enzyme-linked assay; the photo of the electrophoresis result is required for the PCR Fluorescent RT-PCR must have an amplification curve.

9.2 Sample storage

After inspection, it is determined that the samples carrying PepMV should be stored under appropriate conditions. The seeds should be stored at 4°C, and the diseased leaves should be stored at -20°C or Do a good job of marking and registration in the refrigerator at -80°C.

Appendix A

(Informative)

Boping melon mosaic virus related information A.1 Antigen characteristics PepMV has strong antigenicity.

A.2 Chondrial morphology Boping cucurbita mosaic virus particles are linear, with a length of 500 nm and a diameter of 13 nm.

A.3 Genome

Single-stranded linear RNA, the genome length is 6.5kb, the genome is a single component, base ratio. G. A. C. U=25.26.19.30, it accounts for 6% of the particle weight. The genome does not have a Poly(A) tail, but has a tRNA-like structure, encoding 4 proteins, 5' of the viral genome and the 3'ends each contain a non-coding region.

A.4 Host range

Boping melon mosaic virus is mainly found in tomato (Lycopersicon esculentum) and phoenix (Solanum muricatum) in nature.

A.5 Disease symptoms Tomato. Leaf mosaic, twisted, bright yellow mosaic, shrunken, brown spots on old leaves, brown streaks on the stem, and red spots on the fruit.

A.6 Distribution

PepMV was first discovered in Peru in 1974.It has occurred in the Netherlands, many European countries, including Austria, Bulgaria, car Ministers, Italy, Finland, France, Germany, Hungary, Netherlands, Norway, Poland, Slovakia, Spain, Sweden, Switzerland, Turkey Ear it and the United Kingdom.

A.7 Transmission route PepMV can be spread through contact or through seeds.

Appendix B

(Normative)

Double antibody sandwich enzyme-linked immunosorbent assay B.1 Test materials B.1.1 ELISA plate B.1.2 Coated antibody Specific Boping Cucumber Mosaic Virus Antibody B.1.3 Enzyme-labeled antibody Alkaline Phosphatase-labeled Boping Wax Gourd Mosaic Virus Antibody B.1.4 Substrate Disodium p-nitrophenyl phosphate (rhoNPP) B.1.5 PBST buffer (washing buffer pH7.4) B.1.6 Sample extraction buffer (pH7.4) B.1.7 Coating buffer (pH 9.6) B.1.8 Enzyme-labeled antibody dilution buffer (pH7.4) B.1.9 Substrate (rhoNPP) buffer (pH 9.8) B.2 Procedure B.2.1 Coating antibody Dilute the antibody with the coating buffer according to the instructions, add it to the well of the enzyme-linked plate, 100 µL/well, incubate at 37°C for 2 h, and empty the well.

SN/T 5205-2020

PBST wash 3 times.

B.2.2 Sample preparation B.2.3 Sample addition B.2.4 Add enzyme-labeled antibody Use the 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/well, Incubate at 37°C for 4 h, wash with PBST 3 times.

B.2.5 Add substrate

Add the substrate rhoNPP to the substrate buffer to make the final concentration 1 mg/mL (prepared for use now), press 100 µL/well, add to the enzyme in the joint plate, incubate in the dark at room temperature.

B.2.6 Reading

Read the OD value at 405 nm with a microplate reader at 30 min, 1 h and 2 h.

Note. In actual detection, the incubation temperature, PBST washing times and color reading time should be in accordance with the instructions in the detection antibody or kit the regulations are implemented.

B.3 Judgment of results