CN102305727B - Sweet potato viral disease serum detection sampling method - Google Patents
Sweet potato viral disease serum detection sampling method Download PDFInfo
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- CN102305727B CN102305727B CN 201110210962 CN201110210962A CN102305727B CN 102305727 B CN102305727 B CN 102305727B CN 201110210962 CN201110210962 CN 201110210962 CN 201110210962 A CN201110210962 A CN 201110210962A CN 102305727 B CN102305727 B CN 102305727B
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Abstract
The invention relates to crop viral disease serum detection, in particular to a sweet potato viral disease serum detection sampling method which belongs to the technical field of agricultural plant protection. The method comprises the following steps of: obtaining ten sweet potato viral detection (NCM-ELISA) enzyme linked bags from CIP (International Potato Center) by a sweet potato viral detecting rate comparing test on leaves, petioles and the combining part of the leaves and the petioles of a sweet potato; acquiring detoxified original seedling Xu potato No. 18 and Beijing 553 which are bloomed for two years from the field; respectively extracting leaf extract liquid, petiole extract liquid and the combining part extract liquid of the leaves and the petioles for carrying out corresponding detection; and recording a developing result, wherein the masculine gender represents that the sweet potato contains viruses. A detecting result indicates that the combining part extract liquid of the leaves and the petioles is used as a sample for carrying out viral serology detection, and the viral masculine gender detecting rate is the highest.
Description
Technical field
The present invention relates to a kind of crop disease viral disease serum and detect, relate to specifically a kind of sweet potato viral disease serum detection sampling method, belong to the agricultural plant protection technical field.
Background technology
Virus Diseases of Sweet Potato is one of Major Diseases during sweet potato produces, and extensively is present in the potato seed seedling, has had a strong impact on the yield and quality of sweet potato.There is data to show, the field kind incidence of disease 100% of Virus Diseases of Sweet Potato, East Africa, South America are because virosis causes the significant underproduction of sweet potato even total crop failure.The U.S., Japan have reported in succession that also Virus Diseases of Sweet Potato causes yield of sweet potato reduction, quality to descend, and Virus Diseases of Sweet Potato is the important disease in Chinese sweet potato producing region, is the major reason that causes the sweet potato degeneration underproduction.The infection of virosis can make yield reducation 30%～50%, the method of preventing and treating of virosis mainly is kind to be carried out detoxification cultivate, sweet potato behind the Virusfree can increase substantially output and quality, volume increase is more than 20% after the general kind detoxification, high person reaches 200% more than, and between kind larger difference is arranged.The Sweetpotato Viruses Elimination potato seed can prevent and treat the generation of virosis effectively producing utilization, but the quality of virus-free seed potato is the important step that guarantees that prophylactico-therapeutic measures is implemented, and therefore, the band of identification of species poison rate is the key that guarantees the virus-free seed potato quality effectively and quickly.It is the important means that Virus Diseases of Sweet Potato detects that serology detects, and the sample that in the past detected is that blade take sweet potato is as main, thanked to the ease duckweed carried out this partly improving in 2002, take the sample of petiole as detecting, for this reason, viral recall rate has improved 20%, 2010, the position that the people such as Xie Yiping exist in the sweet potato body according to different sweet potato viruses has again been carried out again improving to SD sampling method, thereby has been improved the detection efficiency of Sweetpotato Viruses Elimination potato seed.
Summary of the invention
The objective of the invention is provides a kind of sweet potato viral disease serum detection sampling method for overcoming above-mentioned the deficiencies in the prior art part.Research report according to international Virus Diseases of Sweet Potato, the position that different sweet potato viruses exist in sweet potato is different, the virus that has content in sweet potato mesophyll is higher, the virus that has content in the petiole of sweet potato is higher, the method is by the sweet potato viruses recall rate comparison test to Sweet Potato Leaf, petiole and blade and petiole joint portion, carry out correspondence from CI section extract and detect, record colour developing result, positive expression contains virus.
The present invention realizes with following technical scheme: a kind of sweet potato viral disease serum detection sampling method is characterized in that: carry out as follows:
(1) preparation of samples of participating in the experiment: fetch Virus-free Sweetpotato sample to be detected (get blade and add petiole) from the field;
(2) sampling: polybag is numbered, and the joint portion with diameter 15mm test tube is got blade and petiole needs every getting outside polybag, only stays the joint portion of blade and petiole, and all the other are removed;
(3) preparation of test sample: add the 1ml Extraction buffer, mill outside polybag with glass test tube, room temperature was placed 20-30 minute, and water is fully extracted;
(4) point sample: a, the filter paper of a drying is placed on the experiment table, above film is placed on, avoids Bubble formation; B, with extract supernatant part (15-20 microlitre) in the aseptic rifle head plastic uptake pocket, film is not run into the rifle head in some grid center on film, and sample is with a rifle head, wear gloves in the point sample process always and use the tweezers contact membranes, finger-marks may cause false positive results;
(5) establish positive control: every film positive control is set at last, point sample is finished film is forwarded on the filter paper of a drying, dry 15-20 minute, can clip the film preservation with two filter paper before continuing experiment;
(6) serology detects: serology detects according to the step in the enzyme connection bag carries out: confining liquid hatch 60 minutes → TBS wash fast film → addings antiviral antibody liquid 50rpm overnight incubation → outwell antiviral antibody liquid add T-TBS100rpm wash film 3 times → the anti-liquid 50rpm of adding enzyme mark hatch 1 hour → remove the anti-liquid of enzyme mark, wash film 4 times → film is put into the double dish that dyeing liquor is housed with 30mlT-TBS, 50rpm hatch 30 minutes → wash film 3 times with distilled water 100rpm, color development stopping → filter paper suck dry moisture, record colour developing result, positive expression contains virus.
Advantage of the present invention is: carry out serum virus with blade and petiole joint portion extract as sample and learn detection, improved the virus-positive recall rate, utilize virus concentration height in the petiole juice, impurity is few, and petiole mesophyll is taken into account, effectively eliminated the limitation of blade and petiole sampling, because different sweet potato viruses are different at the position that blade exists, virus is mainly propagated by vascular bundle, most of virus can be bred in vascular bundle, contain a large amount of vascular bundles in the petiole, mainly be breeding and exist in mesophyll and the minority sweet potato viruses is arranged, alone petiole detects then can't detect all viruses, can eliminate error in the sampling with blade and petiole joint portion extract as test sample.Blade and petiole joint portion extract are lighter simultaneously, have avoided chlorophyllous deposition affects.Improve virus-free seed potato virosis recall rate, the band poison rate of virus-free seed potato in producing for overall understanding, instruct the significant and realistic function of development and use of virus-free potato.
Embodiment
The invention will be further described below in conjunction with embodiment:
Embodiment,
Sweet potato viral disease serum detects the sampling new method, and we carry out the recall rate comparison test to leaf, petiole, blade and petiole joint portion, carry out as follows:
(1) preparation of samples of participating in the experiment: fetch the sample of Xu's potato 18 and Beijing 553 from the field, every kind is got 15;
(2) preparation of test sample: polybag is numbered, each sample gets respectively blade, petiole, blade and petiole and the correspondence detection is carried out in the joint portion, add the 1ml Extraction buffer in each polybag, mill outside polybag with glass test tube, room temperature was placed 20-30 minute, and water is fully extracted;
(3) point sample: a. is placed on the filter paper of a drying on the experiment table, above film is placed on, avoids Bubble formation; B. use extract supernatant part (15-20 microlitre) in the aseptic rifle head plastic uptake pocket, point is the grid center on film, do not run into film with the rifle head, sample is with a rifle head, if positive control, wear gloves in the point sample process and use the tweezers contact membranes, finger-marks may cause false positive results; C. film is forwarded on the filter paper of a drying, dry 15-20 minute, adopts two filter paper to clip film and preserve:
(4) serology detects:
A. all hatch and wash at room temperature carry out, hatch vibration shaking speed 50rpm, washing 100rpm;
B. add the 30ml confining liquid in double dish, the film miter angle that tilts is immersed, avoid Bubble formation, hatched 50rpm 60 minutes;
Join simultaneously primary antibodie (antiviral antibody): 0.1ml9# bag and add 30ml enzyme mark damping fluid, 10 kinds of monoclonal antibodies each one;
C. outwell confining liquid, wash fast film with TBS, add antibody liquid (primary antibodie) in double dish, cover lid seals the double dish edge with the parafilm film, prevents volatilization, overnight incubation, 50rpm;
D. outwell primary antibodie, wash film with 30mlT-TBS, 4 times, each 3 minutes, 100rpm; Remove not binding antibody;
Prepare simultaneously to be equipped with solution (Conjugate solution, two anti-solution): the 0.1ml10# bottle two anti-30ml that add
TwoAnti-damping fluid, the Conjugatebuffer:6# bag adds 300mlTBS;
E. clamp film with thieving paper and remove unnecessary T-TBS, film is put into the double dish that two anti-liquid are housed, hatched 50rpm 1 hour;
Substrate buffer solution (substrate buffer): pH9.5,250ml, the 8# bag adds 250ml distilled water, adds 0.5mlHCl (2# bag), transfers pH;
F. remove two anti-solution, wash film with 30mlT-TBS, 4 times, each 3 minutes, 100rpm.Remove not binding antibody;
When washing film at last, join dyeing liquor (NBT/BCIP): mark in each bottle of NBT and BCIP inhaling in the 12# bottle in the 1ml adding 11# bottle, fully after the dissolving, after each inhaled the 0.1ml mixing, adding 25ml substrate buffer solution, 4 degree were preserved 6-8 month;
G. clamp film with thieving paper and remove unnecessary T-TBS, film is put into the double dish that dyeing liquor is housed, hatched 50rpm 30 minutes;
H. with distillation washing film, color development stopping, 3 times, each 10 minutes, 100rpm;
I. film filter paper suck dry moisture records the colour developing result, is clipped in the filter paper and preserves;
(5) investigation result: the record positive rate, leaf, petiole and blade and petiole joint portion sweet potato viruses testing result see Table:
Conclusion:
Can find out from same sample different parts testing result: the positive rate of different parts is variant, and the viral recall rate of blade and petiole joint portion is the highest, and petiole takes second place, and blade is minimum.Therefore, for the virus detection of virus-free seed potato in producing, adopt blade and petiole joint portion to carry out virus and detect the most effective.
Claims (1)
1. sweet potato viral disease serum detection sampling method is characterized in that: carry out as follows:
(1) preparation of samples of participating in the experiment: fetch Virus-free Sweetpotato sample to be detected from the field and get blade and add petiole;
(2) sampling: polybag is numbered, and the joint portion with diameter 15mm test tube is got blade and petiole needs every getting outside polybag, only stays the joint portion of blade and petiole, and all the other are removed;
(3) preparation of test sample: add the 1ml Extraction buffer, mill outside polybag with glass test tube, room temperature was placed 20-30 minute, and water is fully extracted;
(4) point sample: a, the filter paper of a drying is placed on the experiment table, above film is placed on, avoids Bubble formation; B, with extract supernatant part 15-20 microlitre in the aseptic rifle head plastic uptake pocket, film is not run into the rifle head in some grid center on film, and sample is with a rifle head, wear gloves in the point sample process always and use the tweezers contact membranes, finger-marks may cause false positive results;
(5) establish positive control: every film positive control is set at last, point sample is finished film is forwarded on the filter paper of a drying, dry 15-20 minute, clips the film preservation with two filter paper before continuing experiment;
(6) serology detects: serology detects according to the step in the enzyme connection bag carries out: confining liquid hatches 60 minutes → TBS and washes fast film → addings antiviral antibody liquid 50rpm overnight incubation → outwell antiviral antibody liquid and add T-TBS100rpm and wash film 3 times → adding enzyme labelled antibody liquid 50rpm hatch 1 hour → remove enzyme labelled antibody liquid, wash film 4 times → film is put into the double dish that dyeing liquor is housed with 30mlT-TBS, 50rpm hatch 30 minutes → wash film 3 times with distilled water 100rpm, color development stopping → filter paper suck dry moisture, record colour developing result, positive expression contains virus.
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| CN101230346A (en) * | 2008-01-15 | 2008-07-30 | 河南省农业科学院植物保护研究所 | Method for preparing and detecting specific primer, explorer of sweet potato latent virus |
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