CN102128832A - Colloidal gold nucleic acid test strip for watermelon bacterial fruit blotches as well as preparation and application thereof - Google Patents
Colloidal gold nucleic acid test strip for watermelon bacterial fruit blotches as well as preparation and application thereof Download PDFInfo
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- CN102128832A CN102128832A CN 201010606747 CN201010606747A CN102128832A CN 102128832 A CN102128832 A CN 102128832A CN 201010606747 CN201010606747 CN 201010606747 CN 201010606747 A CN201010606747 A CN 201010606747A CN 102128832 A CN102128832 A CN 102128832A
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- aptamer
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- spot virus
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- 235000013399 edible fruits Nutrition 0.000 title claims abstract description 45
- 241000219109 Citrullus Species 0.000 title claims abstract description 42
- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 title claims abstract description 42
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 22
- 238000011330 nucleic acid test Methods 0.000 title claims abstract description 11
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 230000001580 bacterial effect Effects 0.000 title description 3
- 241000700605 Viruses Species 0.000 claims abstract description 32
- 108091023037 Aptamer Proteins 0.000 claims abstract description 28
- 238000012360 testing method Methods 0.000 claims abstract description 25
- 238000001514 detection method Methods 0.000 claims abstract description 24
- 239000000020 Nitrocellulose Substances 0.000 claims description 18
- 229920001220 nitrocellulos Polymers 0.000 claims description 18
- 230000008878 coupling Effects 0.000 claims description 13
- 238000010168 coupling process Methods 0.000 claims description 13
- 238000005859 coupling reaction Methods 0.000 claims description 13
- 108010090804 Streptavidin Proteins 0.000 claims description 9
- 239000000463 material Substances 0.000 claims description 5
- 238000000034 method Methods 0.000 claims description 5
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 239000000853 adhesive Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 229920001817 Agar Polymers 0.000 claims description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 2
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 2
- 239000008272 agar Substances 0.000 claims description 2
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 claims description 2
- 229960005091 chloramphenicol Drugs 0.000 claims description 2
- 239000008103 glucose Substances 0.000 claims description 2
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 2
- 230000002745 absorbent Effects 0.000 claims 11
- 239000002250 absorbent Substances 0.000 claims 11
- 239000012528 membrane Substances 0.000 claims 7
- 230000003902 lesion Effects 0.000 claims 2
- 238000012258 culturing Methods 0.000 claims 1
- 238000011081 inoculation Methods 0.000 claims 1
- 238000013508 migration Methods 0.000 claims 1
- 230000005012 migration Effects 0.000 claims 1
- 239000000843 powder Substances 0.000 claims 1
- 238000002791 soaking Methods 0.000 claims 1
- 239000008223 sterile water Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 4
- 230000027455 binding Effects 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract 3
- 230000002860 competitive effect Effects 0.000 abstract 1
- 235000013305 food Nutrition 0.000 abstract 1
- 238000003307 slaughter Methods 0.000 abstract 1
- 230000000274 adsorptive effect Effects 0.000 description 22
- 244000241257 Cucumis melo Species 0.000 description 11
- 239000010931 gold Substances 0.000 description 10
- 229910052737 gold Inorganic materials 0.000 description 10
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 8
- 235000015510 Cucumis melo subsp melo Nutrition 0.000 description 7
- FJJCIZWZNKZHII-UHFFFAOYSA-N [4,6-bis(cyanoamino)-1,3,5-triazin-2-yl]cyanamide Chemical compound N#CNC1=NC(NC#N)=NC(NC#N)=N1 FJJCIZWZNKZHII-UHFFFAOYSA-N 0.000 description 7
- 206010039509 Scab Diseases 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 239000008363 phosphate buffer Substances 0.000 description 5
- 230000035484 reaction time Effects 0.000 description 5
- 235000009847 Cucumis melo var cantalupensis Nutrition 0.000 description 4
- 229960002685 biotin Drugs 0.000 description 4
- 235000020958 biotin Nutrition 0.000 description 4
- 239000011616 biotin Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
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- 238000009533 lab test Methods 0.000 description 3
- 239000000084 colloidal system Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000012925 reference material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000709721 Hepatovirus A Species 0.000 description 1
- 101000905241 Mus musculus Heart- and neural crest derivatives-expressed protein 1 Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 235000021393 food security Nutrition 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
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Abstract
一种西瓜果斑病毒金标核酸试纸条及其制备和应用，属于胶体金检测技术领域。本发明利用吸水垫形成的毛细管虹吸效应，使被检测物质首先与胶体金偶联Aptamer①发生竞争形式的结合，当偶联Aptamer①过量时，多余的适配体漂移到检测带，与streptavidin-Aptamer②结合并显色；而与检测物结合的胶体金偶联Aptamer①，其V区结合位点被检测物质占据，只能跨越检测带漂移到参照带以C区位点与streptavidin-Aptamer③非特异性结合，同检测带进行比色半定量检测样品中西瓜果斑病毒残留量，能够满足食品安全对西瓜果斑病毒残留量检测需求，适用于饲料、屠宰企业及政府检测机构，具有使用方便、经济快捷、制作容易、成本低廉的特点。
A watermelon fruit spot virus gold-labeled nucleic acid test strip and its preparation and application belong to the technical field of colloidal gold detection. The present invention uses the capillary siphon effect formed by the water-absorbing pad to make the detected substance firstly combine with the colloidal gold-coupled Aptamer① in a competitive form. When the coupled Aptamer① is excessive, the excess aptamer drifts to the detection zone and combines with the streptavidin-Aptamer② and develop color; while the colloidal gold-coupled Aptamer ① combined with the detection substance, its V-region binding site is occupied by the detection substance, and can only drift across the detection zone to the reference zone. The colorimetric semi-quantitative detection of watermelon fruit spot virus residues in samples can meet the food safety requirements for watermelon fruit spot virus residue detection. It is suitable for feed, slaughter enterprises and government testing institutions. Easy and low cost features.
Description
Technical field
A kind of gold test strip bar of aptamers of colloid gold label detects the method for watermelon fruit spot virus, belongs to collaurum detection technique field.
Background technology
Watermelon, muskmelon disease take place general, and some is controlled effectively.But the melon bacterial fruit rot is also referred to as fruit blotch (Bacterial Fruit Blotch is called for short BFB) and as a kind of disease in rising trend in recent years, melon production has been constituted very big threat.This control of medication after being ill takes place fruit does not have effect basically, and fruit rot loses commodity value, and the melon grower suffers heavy losses.Because the main circulation way of this disease is a seed-borne fungi, so grow industry also for watermelon, the muskmelon kind of China and seed export has brought serious impact.
The generation of bacillary fruit rot has become one of urgent problem in watermelon, the muskmelon production, and how preventing, find early and carrying out effectively preventing also becomes the top priority that watermelon, muskmelon are produced.At this disease, the research more than contrasting in the aspects such as processing of domestic and international evaluation with regard to pathogen, detection technique, infected seed still waits fundamental research still very weak about infecting circulation at present.
Summary of the invention
The gold mark nucleic acid test strips that the purpose of this invention is to provide the aptamers of colloid gold label is used to detect watermelon fruit spot virus, and this method has quick, highly sensitive, simple operation and other advantages.
Technical scheme of the present invention:
Aptamer①:3-HS?–AAAAAAAAAAAACGGTAGGGCGAAGAAACCAACACC，
Aptamer②:5-Biotin-TTT?TTT?TTT?TTT?TTT?TTT，
Aptamer③:5-Biotin-?GCCATCCCGCTTCTTTGGTTGTGG，
2. streptavidin and biotin-Aptamer react and generate streptavidin-Aptamer 2., 3. streptavidin and Aptamer react and generate streptavidin-Aptamer 3., Aptamer 1., Aptamer 2., Aptamer 3., streptavidin-Aptamer 2., streptavidin-Aptamer 3., all give birth to worker's bioengineering company limited available from Shanghai.
Streptavidin: Aladdin reagent company buys
A kind of watermelon fruit spot virus gold mark nucleic acid test strips, by adsorptive pads 1 down, collaurum coupling Aptamer 1. 2, and streptavidin-Aptamer 2. 3, and streptavidin-Aptamer 3. 4, last adsorptive pads 5, nitrocellulose filter 6 and base plate 7 are formed; Nitrocellulose filter 6 is fixed on the base plate 7, and nitrocellulose filter 6 one ends and last adsorptive pads 5 link; And the other end or link with following adsorptive pads 1, perhaps 1. the collaurum coupling Aptamer by being fixed in nitrocellulose filter 6 one ends 2 links to each other with following adsorptive pads 1;
Collaurum coupling Aptamer 1. 2, streptavidin-Aptamer 2. 3,3. streptavidin-Aptamer be compounded on the nitrocellulose filter 64 compartment of terrains in proper order;
Wherein, with streptavidin-Aptamer 2. 3 as detecting band, with streptavidin-Aptamer 3. 4 as with reference to being with;
The test paper width is not less than 3.5 mm, and flat appearance, the material adhesion-tight, and the liquid speed of dividing a word with a hyphen at the end of a line is not less than 5 mm/min.
The preparation method of described watermelon fruit spot virus gold mark nucleic acid test strips:
(1) 2. 2. the aptamers Aptamer of biotin modification generate streptavidin-Aptamer with streptavidin 1:4 reaction in molar ratio;
(2) Aptamer that modifies of biotin 3. with streptavidin in molar ratio 1:4 generate streptavidin-Aptamer 3. with reaction;
(3) last adsorptive pads 5 and following adsorptive pads 1 are affixed on respectively on the base plate 7 and connect with nitrocellulose filter 6; Following adsorptive pads 1 or with the collaurum coupling Aptamer that is fixed in nitrocellulose filter 6 one ends 1. 2 parts overlap;
(4) following adsorptive pads 1, collaurum coupling Aptamer 1. 2 and go up adsorptive pads 5 bottom surfaces and have self-adhesive paper to pull on 7 end of for fixing to.
Detect the method for watermelon fruit spot virus with described test strips:
(1) specimen preparation
After sterilizing with 5% liquor natrii hypochloritis in scab place, watermelon surface, cut around the scab respectively and the inner tissue that the scab expansion is not arranged as yet, after 30min is left standstill in grinding in sterilized water, dip in the oese of sterilization and to get soak solution in the surface line of the YDC of drying culture medium flat plate, place 28 ℃ of constant temperature ovens to cultivate, obtain watermelon fruit spot virus after cultivating a week, scrape and get double dish surface watermelon fruit spot virus and be dissolved in the ultrapure water, promptly obtain sample to be tested;
Described YDC nutrient culture media is a dusty yeast glucose chloramphenicol agar nutrient culture media;
(2) operation
Test paper arrow end is immersed in the sample to be tested, does not surpass adsorptive pads down, immerse 30s, taking-up is set level, and 10min reads the result;
(3) testing result
Negative:
Detection band, reference are with two bands all to develop the color, and detect and be with color to be deeper than reference band color, do not contain watermelon fruit spot virus in the interpret sample;
Detection band, reference are with two bands all to develop the color, and it is identical or approaching with reference band color to detect the band color, and the watermelon fruit spot viral level of interpret sample is lower than 10ng/mL;
Positive:
Detection band, reference are with two bands all to develop the color, and are shallower than reference band color but detect the band color, illustrate that melon and fruit pinta malicious content in test sample Chinese and Western surpasses 10ng/mL;
Detect band and do not develop the color, with reference to the band colour developing, the watermelon fruit spot viral level in the interpret sample is higher than 15 μ mol/L.
It is the capillary siphoning effect of utilizing adsorptive pads to form that the present invention detects principle, make detected material at first 1. compete combining of form with collaurum coupling Aptamer, its consequence is, when coupling Aptamer is 1. excessive, unnecessary aptamers floats to and detects band, 2. combines with streptavidin-Aptamer and colour developing; And the collaurum coupling Aptamer that combines with the detection thing 1., its V district detected material of binding site occupies, can only cross over and detect band and float to reference to band, carry out colorimetric and obtain testing result with detecting to be with 3. non-specific binding of C position point and streptavidin-Aptamer.
Beneficial effect of the present invention: the present invention uses one step of chromatography type competition law principle, the colorimetric of band up and down of test strips is come half-quantitative detection sample Chinese and Western melon and fruit pinta poison residual quantity, in 15min, detect sample rapidly and accurately and whether contain watermelon fruit spot virus, to determine whether watermelon fruit spot virus exceeds standard, can satisfy food security watermelon fruit spot virus residual quantity is detected demand, be applicable to feed, meat producing plant and testing agency of government.
The present invention compared with prior art, its effect is self-evident, promptly have easy to use, economical quick, make characteristics easy, with low cost.
Description of drawings
The side view of Fig. 1 watermelon fruit spot virus of the present invention gold mark nucleic acid test strips.1, following adsorptive pads; 2, collaurum coupling Aptamer 1.; 3, streptavidin-Aptamer 2.; 4, streptavidin-Aptamer 3.; 5, go up adsorptive pads; 6, nitrocellulose filter; 7, base plate.
Embodiment
Be described further below in conjunction with accompanying drawing.
1, watermelon fruit spot virus gold mark nucleic acid test strips
Wherein,, 3. be with 2. as detecting band with streptavidin-Aptamer with streptavidin-Aptamer as reference.
Film bar width is not less than 3.5 mm, and flat appearance, the material adhesion-tight, and the liquid speed of dividing a word with a hyphen at the end of a line is not less than 5 mm/min.
2, the preparation of watermelon fruit spot virus gold mark nucleic acid test strips
2. 2. the aptamers Aptamer that biotin modifies generate streptavidin-Aptamer with streptavidin 1:4 reaction in molar ratio,
3. 3. the Aptamer that biotin modifies generate streptavidin-Aptamer with streptavidin 1:4 reaction in molar ratio
Last adsorptive pads 5 and following adsorptive pads 1 are affixed on respectively on the base plate 7 connects with nitrocellulose filter 6; Following adsorptive pads 1 or with the collaurum coupling Aptamer that is fixed in nitrocellulose filter 6 one ends 1. 2 parts overlap;
Following adsorptive pads 1, collaurum coupling Aptamer 1. 2 and go up adsorptive pads 5 bottom surfaces and have self-adhesive paper to pull on 7 the end of for fixing to.
3, test paper requirement
(1) negative reference material coincidence rate
With phosphate buffer (PBS:0.01mol/L, pH 7.4) compound concentration is that the hepatitis B standard solution of 100ng/mL carries out 10 parallel detections, and with normal or correct the visual observation result, the reaction time is observations when 10min, should be negative.
With phosphate buffer (PBS:0.01mol/L, pH 7.4) compound concentration is that the hepatitis A virus standard solution of 100ng/mL carries out 10 parallel detections, and with normal or correct the visual observation result, the reaction time is observations when 10min, should be negative.
(2) positive reference material coincidence rate
With phosphate buffer (PBS:0.01mol/L, pH 7.4) compound concentration is 1,5,10, the watermelon fruit spot of 15ng/mL virus standard items carry out parallel detection, each concentration is carried out 10 parallel laboratory tests, and the reaction time is observations when 10min, feminine gender must not occur.
(3) limit of identification
With phosphate buffer (PBS:0.01mol/L, pH 7.4) respectively compound concentration be 0,1,5,10,15,20, the watermelon fruit spot of 100ng/mL virus standard solution; each concentration is carried out 10 parallel laboratory tests; reaction time is when 10min; with normal or rectification visual observation result, limit of identification is not higher than 10ng/mL.
(4) repeatability
With phosphate buffer (PBS:0.01mol/L, pH 7.4) respectively compound concentration be the watermelon fruit spot virus standard solution of 50ng/mL; carry out 10 parallel laboratory tests, the reaction time is when 10min, with normal or rectification visual observation result; unanimity as a result, colour developing degree homogeneous.
(5) stability test
Place after 10 days for 37 ℃, every index should meet above requirement.
4, test paper using method
(1) specimen preparation
After sterilizing with 5% sodium hypochlorite in sick melon surface, cut around the scab respectively and the inner tissue that the scab expansion is not arranged as yet, in sterilized water, grind leave standstill 30min after, dip in the oese of sterilization and to get soak solution and rule on the YDC of drying culture medium flat plate surface, place 28 ℃ of constant temperature ovens to cultivate.Obtain watermelon fruit spot virus after cultivating a week.Scrape and get double dish surface virus and be dissolved in the ultrapure water, promptly obtain sample to be tested.
(2) operation
Test paper arrow end is immersed in the sample to be tested, does not surpass adsorptive pads 1 down.Immerse the 30sec taking-up and set level, 10min reads the result.
(3) testing result
Negative:
Detection band, reference are with two bands all to develop the color, and detect and be with color to be deeper than reference band color, do not contain watermelon fruit spot virus in the interpret sample.
Detection band, reference are with two bands all to develop the color, and it is identical or approaching with reference band color to detect the band color, and the watermelon fruit spot viral level of interpret sample is lower than 10ppb (10ng/mL).
Positive:
Detection band, reference are with two bands all to develop the color, and are shallower than reference band color but detect the band color, illustrate that melon and fruit pinta malicious content in test sample Chinese and Western surpasses 10ng/mL.
Detect band and do not develop the color, with reference to the band colour developing, the watermelon fruit spot viral level in the interpret sample is higher than 15 μ M (15 μ mol/L).
Claims (3)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN 201010606747 CN102128832A (en) | 2010-12-27 | 2010-12-27 | Colloidal gold nucleic acid test strip for watermelon bacterial fruit blotches as well as preparation and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 201010606747 CN102128832A (en) | 2010-12-27 | 2010-12-27 | Colloidal gold nucleic acid test strip for watermelon bacterial fruit blotches as well as preparation and application thereof |
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| CN102128832A true CN102128832A (en) | 2011-07-20 |
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Cited By (5)
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| CN103013906A (en) * | 2012-09-14 | 2013-04-03 | 博生吉医药科技(苏州)有限公司 | Biological membrane and preparation method and application thereof |
| CN105154565A (en) * | 2015-10-12 | 2015-12-16 | 武汉中帜生物科技股份有限公司 | A method and kit for multiple nucleic acid detection using colloidal gold chromatography |
| CN105572393A (en) * | 2016-01-21 | 2016-05-11 | 武汉慧禹信息科技有限公司 | Nucleic acid aptamer based on estradiol in saliva and gold mark test strip for detection |
| CN108333348A (en) * | 2018-01-11 | 2018-07-27 | 北京化工大学 | Test strips and preparation method thereof for detecting chloramphenicol |
| CN111381037A (en) * | 2020-03-03 | 2020-07-07 | 苏州阿科索生物科技有限公司 | 2019-nCoV aptamer test strip and preparation method thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103013906A (en) * | 2012-09-14 | 2013-04-03 | 博生吉医药科技(苏州)有限公司 | Biological membrane and preparation method and application thereof |
| CN103013906B (en) * | 2012-09-14 | 2015-04-01 | 博生吉医药科技(苏州)有限公司 | Biological membrane and preparation method and application thereof |
| CN105154565A (en) * | 2015-10-12 | 2015-12-16 | 武汉中帜生物科技股份有限公司 | A method and kit for multiple nucleic acid detection using colloidal gold chromatography |
| CN105572393A (en) * | 2016-01-21 | 2016-05-11 | 武汉慧禹信息科技有限公司 | Nucleic acid aptamer based on estradiol in saliva and gold mark test strip for detection |
| CN105572393B (en) * | 2016-01-21 | 2017-11-17 | 武汉慧禹信息科技有限公司 | The aptamer and detection gold label test strip of a kind of estradiol based in saliva |
| CN108333348A (en) * | 2018-01-11 | 2018-07-27 | 北京化工大学 | Test strips and preparation method thereof for detecting chloramphenicol |
| CN108333348B (en) * | 2018-01-11 | 2019-10-15 | 北京化工大学 | Test strip for detecting chloramphenicol and preparation method thereof |
| CN111381037A (en) * | 2020-03-03 | 2020-07-07 | 苏州阿科索生物科技有限公司 | 2019-nCoV aptamer test strip and preparation method thereof |
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Application publication date: 20110720 |