CN1210266A

CN1210266A - Inspection of hidden virus in apple

Info

Publication number: CN1210266A
Application number: CN 97106075
Authority: CN
Inventors: 石荫萍; 王强生; 杨建明; 孙清荣; 隋从义
Original assignee: Shandong Institute of Pomology
Current assignee: Shandong Institute of Pomology
Priority date: 1997-09-03
Filing date: 1997-09-03
Publication date: 1999-03-10

Abstract

A method for testing latent virus of apple includes such steps as grinding apple leaf in 0.02 M phosphorate buffering liquid, adding equal chloroform, centrifugal extratraction of antigenic liquid, diluting it with rabbit's serum resisting against leaf-spot virus and micropteres virus to obtain antiserum liquid, enzyme labelling protein A with horse-radish peroxide, dissolving with 0.02 M phosphorate, diluting with antigenic buffering liquid before using it, making a circular trace on nitrocellulose membrane, dropping 2 microlitres of antigenic liquid on it, adding antiserum enzyme labelled protein A liquid and the target solution, and judging if there is latent virus according to the colour of spot.

Description

Inspection of hidden virus in apple

The present invention relates to the inspection of hidden virus in apple technical field.

With Enzyme-multiplied immune technique test apple latent virus is the detection technique of current widespread use, because it is longer that this technology for detection process takes, sensitivity, accuracy and stability are high not enough, and data poor repeatability, antigen, antibody and reagent chemicals consumption are big, expensive high, detect sample and can not preserve.

The objective of the invention is to invent a kind of inspection of hidden virus in apple, make shortening detection time, sensitivity, accuracy and stability high, good reproducibility, it is low to detect cost, and sample can be preserved.

The present invention is achieved in that apple blade to be checked is put into the 0.02M phosphate solution to be ground, and adding equal amounts of chloroform then, to get supernatant in centrifugal 10 minutes be antigen liquid; Get anti-apple chlorotic leaf spot virus serum of rabbit and the anti-apple stem grooving virus serum of rabbit and make antiserum liquid with the dilution of antibody damping fluid, the antibody damping fluid is by the 0.02M phosphate solution, 0.05% polysorbas20,2% polyvinylpyrrolidone and 0.2% bovine serum albumin(BSA) are made; The A albumen of horseradish peroxidase-labeled is made enzyme mark A protein liquid with the dilution of antibody damping fluid with the dissolving of 0.02M phosphate during use; Nitrocellulose filter is beaten the circular hole trace with card punch, splash into 2 μ l antigen liquids on the circular hole trace, immersed then in the sealing damping fluid 30 minutes, the sealing damping fluid is by 0.02M phosphate, 0.05% polysorbas20,2% polyvinylpyrrolidone and 2% bovine serum albumin(BSA) are made; Take out nitrocellulose filter then and blot, add the washing of 0.05 polysorbas20 liquid, blot with 0.02M phosphate, add antiserum liquid then, add 25 μ on every circular hole trace and preserve moisture for 1,28 ℃ and hatch 1 hour, and then add 0.05% polysorbas20 liquid washing with the 0.02M phosphate solution, blot; Immerse antibody damping fluid sealing 15 minutes then, wash, blot; Add enzyme mark A protein liquid again, add 25 μ l on every circular hole trace, preserve moisture for 28 ℃ and hatch 1 hour, wash, blot; Add 25 μ l substrate solutions on every circular hole trace, the lucifuge of preserving moisture was hatched 10-30 minute, malicious in spite of illness sampling point colour developing; Substrate solution adds imidazoles by diaminobenzidine four hydrochlorides to be made, and the time spent adds hydrogen peroxide; With the nitrocellulose filter water flushing of colour developing, with the distilled water washing, blot again, be all no apple latent virus as the spot on the circular hole trace mutually with the nitrocellulose filter color, be that tawny or brown are for there being apple latent virus as spot colors.

Advantage of the present invention is: testing process takes shorter, and sensitivity, accuracy and stability are high, and good reproducibility, antigen, antibody and reagent chemicals consumption are little, and cost is low, detects sample and can preserve.

Below in conjunction with embodiment, the present invention will be further described:

Adopt the apple blade time and be advisable with two seasons of spring and autumn, each sample to be checked is adopted 1-2 sheet leaf, and the sample sack of packing into reinstalls polybag, and preserving moisture, it is to be checked to deposit; Get the 0.02M phosphate buffer that apple blade to be checked 0.5 gram adds 2 times of volumes and grind, adding equal amounts of chloroform, to get supernatant in centrifugal 10 minutes be antigen liquid; Get anti-apple chlorotic leaf spot virus serum of rabbit and the anti-apple stem grooving virus serum of rabbit and make antiserum liquid with the dilution of antibody damping fluid, the antibody damping fluid is by the 0.02M phosphate solution, 0.05% polysorbas20,2% polyvinylpyrrolidone and 0.2% bovine serum albumin(BSA) are made; The A albumen of horseradish peroxidase-labeled is made enzyme mark A protein liquid with the dilution of antibody damping fluid with the dissolving of 0.02M phosphate during use; Every kind of viral antiserum should be separately with 1 nitrocellulose filter, and each repeats also can be singly with 1, and the size of nitrocellulose filter accounts for 1cm according to waiting inspection sample number to determine by each spot ²Cut out, use diameter 0.5cm ²Card punch on this film, beat the circular hole trace, draw Pareto diagram by designing requirement, on the circular hole trace, splash into 2 μ l antigen liquids, immersed then in the sealing damping fluid 30 minutes, the sealing damping fluid is by 0.02M phosphate, 0.05% polysorbas20,2% polyvinylpyrrolidone and 2% bovine serum albumin(BSA) are made; Take out nitrocellulose filter then and blot, add 0.05% polysorbas20 liquid washing 3 times, each 5 minutes, blot with 0.02M phosphate; Add antiserum liquid then, add 25 μ l on every circular hole trace, preserve moisture for 28 ℃ and hatch 1 hour, and then add 0.05% polysorbas20 washing 3 times, each 5 minutes, blot with the 0.02M phosphate solution; Immerse antibody damping fluid sealing 15 minutes then, wash 3 times, each 5 minutes, blot; Add enzyme mark A protein liquid again, add 25 μ l on every circular hole trace, preserve moisture for 28 ℃ and hatch 1 hour, wash 3 times, each 5 minutes, blot; Add 25 μ l substrate solutions on every circular hole trace, the lucifuge of preserving moisture was hatched 10-30 minute, malicious in spite of illness sampling point colour developing, and substrate solution adds imidazoles by diaminobenzidine four hydrochlorides to be made, and the time spent adds 0.1% hydrogen peroxide; With the nitrocellulose filter water flushing of colour developing, with the distilled water washing, blot again, the stopping of reaction, spot colour developing degree is judged as follows per sample:

Negative :-sample point is identical with white nitrocellulose filter color

+ sample point khaki the trace that mays be seen indistinctly

The positive+sample point is khaki trace or the only peripheral tawny that shows

Do not develop the color in annulus, semicircular ring, centre

++ the sample point tawny

++ deep yellow brown of+sample point or brown

Testing result is charged to Pareto diagram, write out examining report, connect and file preservation in the lump with nitrocellulose filter.

Claims

1, inspection of hidden virus in apple is characterized in that: apple blade to be checked is put into the 0.02M phosphate solution grind, adding equal amounts of chloroform then, to get supernatant in centrifugal 10 minutes be antigen liquid; Get anti-apple chlorotic leaf spot virus serum of rabbit and the anti-apple stem grooving virus serum of rabbit and make antiserum liquid with the dilution of antibody damping fluid, the antibody damping fluid is by the 0.02M phosphate solution, 0.05% polysorbas20,2% polyvinylpyrrolidone and 0.2% bovine serum albumin(BSA) are made; The A albumen of horseradish peroxidase-labeled is made enzyme mark A protein liquid with the dilution of antibody damping fluid with the dissolving of 0.02M phosphate during use; Nitrocellulose filter is beaten the circular hole trace with card punch, splash into 2 μ l antigen liquids on the circular hole trace, immersed then in the sealing damping fluid 30 minutes, the sealing damping fluid is by 0.02M phosphate, 0.05% polysorbas20,2% polyvinylpyrrolidone and 2% bovine serum albumin(BSA) are made; Take out nitrocellulose filter then and blot, add the washing of 0.05% polysorbas20 liquid, blot with 0.02M phosphate; Add antiserum liquid then, add 25 μ l on every circular hole trace, preserve moisture for 28 ℃ and hatch 1 hour, and then add the washing of 0.05% polysorbas20 liquid, blot with the 0.02M phosphate solution; Immerse antibody damping fluid sealing 15 minutes then, wash, blot; Add enzyme mark A protein liquid again, add 25 μ l on every circular hole trace, preserve moisture for 28 ℃ and hatch 1 hour, wash, blot; Add 25 μ l substrate solutions on every circular hole trace, the lucifuge of preserving moisture was hatched 10-30 minute, malicious in spite of illness sampling point colour developing; Substrate solution adds imidazoles by diaminobenzidine four hydrochlorides to be made, and the time spent adds hydrogen peroxide; With the nitrocellulose filter water flushing of colour developing, with the distilled water washing, blot again, be all no apple latent virus as the spot on the circular hole trace mutually with the nitrocellulose filter color, be that tawny or brown are for there being apple latent virus as spot colors.