CN102108404B - Visible chip-based method for detecting food allergens - Google Patents
Visible chip-based method for detecting food allergens Download PDFInfo
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Abstract
The invention discloses a method for detecting a plurality of food allergens in a sample, which comprises: (1) preparing a visible thin film sensor chip, wherein a plurality of specific probes corresponding to the DNA sequences of the plurality of allergens are fixed on the surface of a chip substrate respectively; (2) performing multiplex polymerase chain reaction (PCR) amplification on the DNA sequence of a test sample by using the plurality of pairs of specific upstream and downstream primers corresponding to the DNA sequences of the plurality of allergens, wherein the 5' terminal of the downstream primer has biotin bonded by a covalent bond; (3) crossing the product of the PCR amplification in the step (2) with the probes on the visible chip, and washing; (4) crossing horseradish peroxidase-marked biotin with the chip, and eluting uncrossed chains; (5) processing and developing the chip by using a horseradish peroxidase substrate; and (6), determining if the sample has the allergens and the types of the allergens according to the generation condition of a visible signal and the position of the probe corresponding to the generated visible signal. The method can quickly and synchronously detect if the sample has various allergens with high throughput.
Description
Technical field
Present invention relates in general to a kind of detection method based on visible die, relate in particular to the detection method of food allergens.
Background technology
Food anaphylaxis is a kind of untoward reaction that people produce food, belongs to a kind of transformation reactions that body produces allogenic material (anaphylactogen).The clinical manifestation of food anaphylaxis comprises the clinical symptom such as headache, asthma, allergic rhinitis, urticaria, enteropathy syndrome, anaphylactic shock or death.Food anaphylaxis also is a food-safety problem that day by day was concerned in recent years, and being defined as eight common large class anaphylactogens (food) by the World Health Organization (WHO) at present is wheat, peanut, soybean, nuts, milk, egg, fish and crustaceans.Treatment there is no effect method for food anaphylaxis now, and strictly avoiding the edible food that contains allergic component is the most effective therapy.Just because of this, the U.S. is in formal enforcement on January 1st, 2006 " food allergens sign and consumer protection rules ".European Union, Japan, Australia and New Zealand etc. have also successively put into effect relevant decree, require clearly to identify the anaphylactogen material composition in the food labelling, therefore set up the anaphylactogen detection method very necessary for the consistence of guaranteeing food labelling and human consumer's safety.
Current two classes that mainly are divided into of the detection method of food allergens.One class is immunological method, allergic protein for food allergy, mainly comprise: enzyme-linked immunosorbent assay, immunochromatographic method, radioimmunology, immunoblotting etc., that wherein the most frequently used, sensitivity is the highest is enzyme-linked immunosorbent assay (ELISA), and operating the easiest is immunochromatographic method.Equations of The Second Kind is nucleic acid amplification method, can for food allergens specific gene sequence, detect the allergen gene in the food.At present, more existing commercial kit of food allergens.As: the food allergen peanut test kit of the R-Biopharm AG company of the Neogen Corporation of the U.S., the Tepnel BioSystems Ltd of Britain and Germany has passed through the approval of AOAC (AOAC), and is used for food test by U.S. U.S. food Drug Administration (FDA) approval.But present food allergens test kit is not contained all food allergenss, and there are the defectives such as undetected in the detection kit of some food allergens; In addition, present test kit detects common only for a kind of anaphylactogen, but in fact, food ingredient is complicated, often has multiple anaphylactogen, therefore is necessary very much to develop high-throughout testing product, realizes the disposable of multiple anaphylactogen detected.
At present, no matter ELISA method or nucleic acid detection method can only detect a kind of anaphylactogen usually simultaneously, and in actual food product detects, need to carry out examination to anaphylactogen to detect, and namely multiple anaphylactogen are detected simultaneously.Demand now developing a kind of high-throughput sensitivity urgently, realize the online detection of exported product, reach save time, low consumption, economy, food allergens new detecting method fast and effectively.
Summary of the invention
For addressing the above problem, the invention provides a kind of can quick, high-throughout detection method based on anaphylactogen in the sample of visual chip.
Anaphylactogen detection method in a kind of test sample provided by the present invention comprises:
(1) preparation visual film sensor chip is wherein fixed respectively a plurality of specific probes corresponding to a plurality of anaphylactogen dna sequence dnas on same chip matrix surface;
(2) utilize corresponding to the many of a plurality of anaphylactogen dna sequence dnas specificity upstream primer and downstream primer are carried out the multiplex PCR amplification to the dna sequence dna of test sample, wherein 5 ' of downstream primer end is with covalently bound vitamin H;
(3) pcr amplification product of step (2) and the probe on the visual chip are hybridized, and washing;
(4) hybridize with biotin antibody and the chip of horseradish peroxidase mark, and the wash-out chain of not hybridizing;
(5) with the substrate of horseradish peroxidase chip is processed and developed the color;
(6) according to whether producing the probe location corresponding to optical signal of optical signal and generation, determine the kind that whether contains anaphylactogen and contain anaphylactogen in the test sample.
Preferably, also be fixed with the positive control sequence on the stromal surface of the visual film sensor chip in the step of the present invention (1), 3 ' end of described positive control sequence is with covalently bound vitamin H, this sequence can be used as the positive control of experiment on the one hand, also can be used as simultaneously the locating point of probe array, be used for assisting to determine the position of probe array hybridization point.Described positive control sequence can for example be SEQ ID NO:25, and namely (ALD refers to aldehyde group modified 5 '-ALD-aaaaaaaaaaaaaaaaaaaa-3 '-Biotin; Biotin is biotin modification).
Preferably, in the described step of the present invention (1), the specific probe corresponding to each anaphylactogen dna sequence dna of being fixed on same chip matrix surface has two at least.Because the specific probe of each anaphylactogen dna sequence dna is fixed with two at least, during test sample, the result can corroborate each other, and further guarantees the reliability that detects thus.
Preferably, described step (1) specifically comprises following steps: with point sample instrument a plurality of specific probes are put on the same chip matrix surface, then be to place under 70% the environment to make probe be fixed on chip surface in 2 hours in humidity, it then is the SDS cleaning chip of 0.1% (w/v) with concentration, clean chip with aqua sterilisa again, final drying, and in 4 ℃ of lower preservations.
Preferably, in the described step (2), the response procedures of multiplex PCR amplification is: after 95 ℃ of denaturation 15min, 94 ℃ of sex change 30s, 58 ℃ of annealing of temperature 30s, 72 ℃ extend 20s, repeat above-mentioned denaturation, sex change, annealing, extension step again, carry out 35 circulations; Carry out 5min at 72 ℃ after circulation is finished and fully extend, at last 4 ℃ of lower preservations.
Preferably, described step (3) specifically comprises following steps: 2 μ L pcr amplification products are diluted to 10 μ L with aqua sterilisa, behind 95 ℃ of sex change 5min, ice bath 3min immediately, the pcr amplification product solution of getting again behind the ice bath of 10 μ L is diluted to 100 μ L with hybridization buffer (5 * SSC and 5mg/ml acid treatment casein), and be added drop-wise on the chip, 45 ℃ of water-bath 10min use 200 μ L, 0.1 * SSC flushing again and dry up.
Preferably, described step (4) specifically comprises following steps: chip is processed 5min with the biotin antibody solution of 100 μ L horseradish peroxidase marks, wash and dry up with 200 μ L, 0.1 * SSC solution.
Preferably, described step (5) specifically comprises following steps: with the substrate solution process chip of 100 μ L horseradish peroxidases, after the room temperature lucifuge is placed 5min, dry up with sterilized water is clean.
In the described step (6), detected result with the naked eye can be analyzed, the preservation of also can taking a picture.
Preferably, the substrate of described horseradish peroxidase is tetramethyl benzidine (TMB).
The present invention also provides a kind of visual film sensor chip for anaphylactogen in the test sample, wherein fixes respectively a plurality of specific probes corresponding to a plurality of anaphylactogen dna sequence dnas on same chip matrix surface.
No matter be in anaphylactogen detection method provided by the present invention, or in visual film sensor chip provided by the present invention, preferably, described a plurality of anaphylactogen is selected from multiple in wheat, peanut, soybean, cashew nut, milk, egg, fish and the crustaceans.
According to a specific embodiment of the present invention, described a plurality of anaphylactogens are soybean, peanut, wheat, cashew nut.At this moment, the employed a plurality of specific probes that correspond respectively to soybean, peanut, wheat, cashew nut allergen dna sequence dna can be respectively SEQ ID NO:3,6,9,12, are respectively SEQ ID NO:1-2,4-5,7-8,10-11 corresponding to specificity upstream primer and the downstream primer of soybean, peanut, wheat, cashew nut allergen dna sequence dna.
According to another embodiment of the present invention, described a plurality of anaphylactogens are egg, milk, fish and shrimp.At this moment, the employed a plurality of specific probes that correspond respectively to egg, milk, fish and shrimp allergen dna sequence dna can be respectively SEQ ID NO:15,18,21,24, are respectively SEQ ID NO:13-14,16-17,19-20,22-23 corresponding to specificity upstream primer and the downstream primer of egg, milk, fish and shrimp allergen dna sequence dna.
According to another embodiment of the present invention, described a plurality of anaphylactogen is soybean, peanut, wheat, cashew nut, egg, milk, fish and shrimp, owing to having fixed most common anaphylactogen on the chip, can satisfy the detection of the common anaphylactogen of all foods.At this moment, 8 species specificity probes corresponding to these 8 kinds of anaphylactogen dna sequence dnas are: SEQ ID NO:3,6,9,12,15,18,21,24; Utilization is corresponding to the 8 pairs of specificity upstream primers and the downstream primer of described 8 kinds of anaphylactogen dna sequence dnas: SEQ ID NO:1-2,4-5,7-8,10-11,13-14,16-17,19-20,22-23 carry out the multiplex PCR amplification to the dna sequence dna of test sample.
Visible die can with specific molecule in conjunction with being transformed into the signal that is observed visually, make the process imagery of interpretation of result as a kind of novel gene chip technology.The matrix of visible die is that the surface is through the silicon chip of special processing.At first probe is fixed on chip surface during use, then uses through biotin labeled primer and carry out pcr amplification, the probe on amplified production and the chip carries out specific cross.Then use the horseradish peroxidase antibody treatment, the chain that flush away is not hybridized, and process with tetramethyl benzidine (TMB).At this moment combine biotin labeled hybrid dna chain and will under the effect of horseradish peroxidase antibody and TMB, produce the thickness that precipitation changes chip, thereby produce the signal that naked eyes can be distinguished.Visible die had both had quick, accurate, efficient, the high-throughout characteristics of gene chip, can break away from again the restriction of expensive gene chip experiment equipment, was conducive to the rapid detection check of food allergen.
At the anaphylactogen detection field, traditional Immunological Method and nucleic acid amplification are because the defective of itself has limited its application at present.And use visual protein chip, then owing to the chip manufacturing system expensive, detecting complicated operation, the defective such as time-consuming, strongly professional, also limited its large-scale application industrially.In the actual life, usually can not only contain a kind of anaphylactogen in a kind of food.Utilize traditional method, need to detect respectively each anaphylactogen, lose time, cost is also high.And method of the present invention is utilized multiple PCR technique, adopts specific primer pair and specific probe for a plurality of anaphylactogens, can detect simultaneously a plurality of anaphylactogens in the same food, and method is easy saves time fast, has reduced cost.Be fixed with the probe for multiple anaphylactogen in the visual film sensor chip provided by the present invention, described multiple anaphylactogen has been included eight kinds of common anaphylactogens, uses this chip can use very easily method of the present invention that test sample is detected.
When utilizing the multiplex PCR amplification technique that the anaphylactogen sample is detected based on the visual film sensor chip, owing to needing to guarantee specificity, usually for the Auele Specific Primer of anaphylactogen to being difficult to select with specific probe, this also is to have no the major reason that it is used at the anaphylactogen detection field in the prior art.Simultaneously, owing in the multi-PRC reaction system, comprising the easily competitive amplification of generation between many to primer pair, different primer pairs, have a strong impact on reaction result; The necessary high degree of specificity of each primer is to avoid occurring non-specific amplification; In addition, different primers is not identical to desired pcr amplification condition yet.Therefore 4 primer pair and probes heavy or that react more than 4 heavy PCR for the particular allergen design are difficult to selection especially.Yet the application has but solved the problems referred to above.The surprised discovery of present inventor adopts selected 8 pairs of primers and probe among the application to be fit to very much the application's anaphylactogen detection technique, primer pair has high degree of specificity each other, reaction result is precise and high efficiency also, utilizes the multiplex PCR amplification technique to carry out the technical barrier of anaphylactogen sample detection thereby overcome based on the visual film sensor chip.
Description of drawings
Below in conjunction with accompanying drawing, further specify the embodiment of technical solution of the present invention.
Figure 1 shows that chip point sample instrument point sample arrangement schematic diagram; Wherein, M represents positive control; 1 expression cashew nut probe; 2 expression peanut probes; 3 expression wheat probes; 4 expression soybean probes; 5 expression egg probes; 6 expression fish probes; 7 expression shrimp probes; 8 expression milk probes.
Figure 2 shows that the as a result figure that utilizes anaphylactogen in the method according to this invention test sample: wherein, 11 is blank detected result figure; 12 is as a result figure of cashew nut sample detection; 13 is peanut sample detected result figure; 14 is wheat samples detected result figure; 15 is soybean sample detected result figure; 16 for changing egg sample detected result figure into; 17 is as a result figure of fish sample detection; 18 is as a result figure of shrimp sample detection; 19 is milk sample detected result figure.
Figure 3 shows that the as a result figure that utilizes the method according to this invention to detect anaphylactogen in the biased sample: wherein, 21 is blank (ddH
2O) detected result figure; 22 is as a result figure of cashew nut DNA detection; 23 is as a result figure of shrimp DNA detection; 24 is milk and shrimp hybrid dna detected result figure; 25 is milk, shrimp and fish hybrid dna detected result figure; 26 is milk, shrimp, fish and egg hybrid dna detected result figure; 27 is soybean and cashew nut hybrid dna detected result figure; 28 is soybean, cashew nut and wheat hybrid dna detected result figure; 29 is soybean, cashew nut, wheat and peanut hybrid dna detected result figure.
Figure 4 shows that the as a result figure that utilizes the method according to this invention to detect actual sample: wherein, 31 is the detected result figure of refined Na white chocolate strawberry oat bar (France) sample; 32 is the detected result figure of chocolate (Korea S) sample of Ao Lien crisp-fried; 33 is the detected result figure of cock vertical bar shaped spaghetti (Spain) sample; 34 is the detected result figure of Switzerland's triangle dark chocolate (Switzerland) sample; 35 is the detected result figure of dace with black bean (China) sample; 36 is blank (ddH
2O) detected result figure.
Embodiment
For 8 common in food class anaphylactogens: wheat, peanut, soybean, cashew nut, milk, egg, fish and crustaceans, according to soybean Lectin gene, wheat Gliadin gene, peanut Arah 3 genes, cashew nut Anao3 gene, fish and shrimp 16S rRNA, ox and chicken Mitochondrial DNA design specific primer sequence and probe.Primer and probe sequence are referring to table 1.
Table 1 utilizes primer and the probe of specific sequence design
* annotate: F is upstream primer; R is downstream primer; P is probe; ALD is aldehyde group modified; Biotin is biotin modification.
False negative occurs in the hybridization system, special synthetic one section sequence that is comprised of 20 VITAMIN B4 is as the positive control of testing, and this positive control also can be used as the locating point of probe array simultaneously, is used for determining the position of probe array hybridization point.If positive control has the signal instruction reaction normal after the hybridization; If no signal then shows and hybridizes unsuccessfully.Positive control is SEQ ID NO:25 (that is: 5 '-ALD-aaaaaaaaaaaaaaaaaaaa-3 '-Biotin).
The matrix of visible die is the silicon chip of surface process special processing, needs at first probe is fixed on the surface of chip matrix during use.8 kinds of probes in positive control and the table 1 are put on the same chip with the chip point sample instrument, and the chip specification is 7mm * 7mm, arranges as shown in Figure 1, and concentration and probe concentration is that 10 μ mol/L and positive control concentration are 1 μ mol/L.After point sample is complete, be to place 2h under 70% the room temperature environment to make probe be fixed on chip surface in humidity, then be that the SDS of 0.1% (W/V) cleans chip 3 times with concentration, aqua sterilisa cleans 3 times again, dry up with drying air stream at last, namely obtain to be fixed with the visual film sensor chip of multiple anaphylactogen probe, and in 4 ℃ of lower preservations.
Then use through biotin labeled primer pair sample DNA and carry out the multiplex PCR amplification.PCR reaction system: dna profiling 5.0 μ L; 10 * Multi Hotstart buffer (contains Mg
2+) 2.5 μ L; DNTPs (2.5mol/L) 1.0 μ L; Primer is divided into two groups, first group: soybean, wheat, peanut and cashew nut, and second group: milk, shrimp, fish and egg, wherein the primer final concentration of soybean, wheat, peanut and cashew nut is respectively: 80nmol/L, 160nmol/L, 160nmol/L and 80nmol/L; The primer final concentration of milk, shrimp, fish and egg is respectively: 80nmol/L, 480nmol/L, 80nmol/L and 64nmol/L; HotMaster Taq DNA polymerase (5U/ μ L) 0.2 μ L; Use ddH
2O mend to cumulative volume be 25 μ L.The amplified reaction program is: 95 ℃ of denaturation 15min, and 94 ℃ of sex change 30s, the equal initial setting of multiplex PCR annealing temperature are 58 ℃ of 30s, 72 ℃ are extended 20s; 35 circulations; After circulation is finished 72 ℃, 5min fully extends.In multiplex PCR when amplification, be divided into two parts with sample, and two duplicate samples take 4 kinds of primers of 4 kinds of primers of first group and second group as primer increases, obtain final multiplex PCR amplified production after amplified production mixes respectively.
Then carry out specific cross with amplified production and according to the probe on the visual film sensor chip of aforesaid method preparation.Then use the horseradish peroxidase antibody treatment, will be less than the chain flush away of hybridization through an elution process, and process with tetramethyl benzidine (TMB).At this time combine biotin labeled hybrid dna chain and will under the effect of horseradish peroxidase antibody and TMB, produce the thickness that precipitation changes chip, thereby produce the signal that naked eyes can be distinguished, and do not hybridize successful probe, can't then not change by binding antibody.Detailed process is as follows: at first, 2 μ L multiplex PCR amplified productions are diluted to 10 μ L with aqua sterilisa, behind 95 ℃ of sex change 5min, ice bath 3min immediately, PCR product solution with 10 μ L ice baths is diluted to 100 μ L with hybridization buffer (5 * SSC and 5mg/ml acid treatment casein) again, and be added drop-wise on the chip, 45 ℃ of water-bath 10min, use again 200 μ L, 0.1 * SSC flushing 3 times and dry up, process 5min with 100 μ L horseradish peroxidase antibody-solutions afterwards, wash 3 times and dry up with 200 μ L, 0.1 * SSC solution.With 100 μ L TMB solution-treated, the room temperature lucifuge is placed 5min, and sterilized water is cleaned and dried up.The result with the naked eye can analyze, with the preservation of taking a picture of Pixera pro 150ES system.
Hereinafter respectively different food is detected wherein whether contain anaphylactogen according to above-mentioned method among the embodiment 1-3.
Choose respectively soybean, wheat, cashew nut, peanut, milk, egg, fish and 8 kinds of food of shrimp as test sample.
According to above-mentioned steps, respectively the multiplex PCR amplification is carried out with the primer shown in the table 1 in the DNA of above-mentioned 8 kinds of food, with the multiplex PCR amplified production with carry out hybridization according to the prepared visual film sensor chip of above-mentioned steps, and carry out blank with aqua sterilisa and test, reaction result is as shown in Figure 2.
With the signaling point suitable with positive control brightness as the specific reaction sign, can find out by the results of hybridization optical signal photo (Fig. 2) to 8 kinds of food allergens, the cross pattern that positive control point all is arranged in according to experimental design, and the purpose probe all presents obvious signal, can distinguish clearly with background, and other non-purpose probe site are then fully consistent with background, no signal.Under the dual assurance of PCR product specificity and probe specificity, all food allergens have preferably specificity, do not have false-positive appearance.
Get 9 of the visual film sensor chips put according to aforesaid method, use respectively the unique DNA of cashew nut and shrimp, soybean and cashew nut hybrid dna, milk and shrimp hybrid dna, soybean, cashew nut and wheat hybrid dna, milk, shrimp and fish hybrid dna, milk, shrimp, fish and egg hybrid dna, soybean, wheat, peanut and cashew nut hybrid dna are template, carry out the multiplex PCR amplification take 8 pairs of primers shown in the table 1 as primer, pcr amplification product is carried out the chip hybridization experiment, and carry out the blank experiment with aqua sterilisa, the result as shown in Figure 3.
By finding out results of hybridization optical signal photo, the cross pattern that positive control point all is arranged in according to experimental design, purpose probe also all present obvious signal, can distinguish clearly with background.Under the dual assurance of PCR product specificity and probe specificity, all food allergens have preferably specificity, do not have false-positive appearance.
The 5 kinds of food (refined Na white chocolate strawberry oat bar, Ao Lien crisp-fried chocolate, cock vertical bar shaped spaghetti, Switzerland's triangle dark chocolate and dace with black bean) that utilize multiple PCR method as described in Example 2 that the supermarket is bought increase, multiplex PCR amplified production and chip are carried out hybridization, and carry out blank with aqua sterilisa and test, the accuracy of checking food labelling mark.
Indicate in the label of 5 kinds of food contain milk constituents have 4 kinds, 4 kinds of the wheat composition, 3 kinds of soybean components, fish and egg ingredients each a kind, contain a kind of micro-nuts and kernels composition.
Multiple pcr amplification product and chip are carried out hybridization, replace template DNA to make blank with aqua sterilisa.Experimental result as shown in Figure 4, the result demonstrates, the purpose probe all presents obvious signal, can distinguish clearly with background, has confirmed high flux property and the specificity of visible die in actual sample detects.
Although this paper describes out preferred embodiment of the present invention, the many changes that clearly can carry out to those skilled in the art and modification can not exceed the scope of broad aspect of the present invention.Therefore, appended what is claimed is in order to attempt to cover all changes and modifications within true spirit of the present invention and scope.
Claims (8)
1. the detection method of a plurality of anaphylactogens in the test sample comprises:
(1) preparation visual film sensor chip is wherein fixed respectively a plurality of specific probes corresponding to a plurality of anaphylactogen dna sequence dnas on same chip matrix surface;
(2) utilize corresponding to the many of a plurality of anaphylactogen dna sequence dnas specificity upstream primer and downstream primer are carried out the multiplex PCR amplification to the dna sequence dna of test sample, wherein 5 ' of downstream primer end is with covalently bound vitamin H;
(3) pcr amplification product of step (2) and the probe on the visual chip are hybridized, and washing;
(4) hybridize with biotin antibody and the chip of horseradish peroxidase mark, and the wash-out chain of not hybridizing;
(5) with the substrate of horseradish peroxidase chip is processed and developed the color;
(6) according to whether producing the probe location corresponding to optical signal of optical signal and generation, determine the kind that whether contains anaphylactogen and contain anaphylactogen in the test sample,
Wherein, described anaphylactogen is selected from soybean, peanut, wheat, cashew nut, egg, milk, fish and shrimp, the 8 species specificity probes that correspond respectively to the dna sequence dna of these 8 kinds of anaphylactogens are: SEQ ID NO:3,6,9,12,15,18,21,24 is respectively corresponding to the 8 pairs of specificity upstream primers and the downstream primer of the dna sequence dna of described 8 kinds of anaphylactogens: SEQ ID NO:1-2,4-5,7-8,10-11,13-14,16-17,19-20,22-23.
2. detection method according to claim 1 is characterized in that, also is fixed with the positive control sequence on the stromal surface of the visual film sensor chip in the described step (1), and 3 ' end of described positive control sequence is with covalently bound vitamin H; Described positive control sequence preference is SEQ ID NO:25.
3. detection method according to claim 1 is characterized in that, in the described step (1), the specific probe corresponding to each anaphylactogen dna sequence dna of being fixed on same chip matrix surface has two at least.
4. arbitrary described detection method according to claim 1-3, it is characterized in that, described a plurality of anaphylactogen is soybean, peanut, wheat, cashew nut, the employed a plurality of specific probes that correspond respectively to soybean, peanut, wheat, cashew nut allergen dna sequence dna are respectively SEQ ID NO:3,6,9,12, are respectively SEQ ID NO:1-2,4-5,7-8,10-11 corresponding to specificity upstream primer and the downstream primer of soybean, peanut, wheat, cashew nut allergen dna sequence dna; Egg, milk, fish and shrimp, the employed a plurality of specific probes that correspond respectively to egg, milk, fish and shrimp allergen dna sequence dna are respectively SEQ ID NO:15,18,21,24, and the specificity upstream primer and the downstream primer that correspond respectively to egg, milk, fish and shrimp allergen dna sequence dna are respectively SEQ ID NO:13-14,16-17,19-20,22-23; Perhaps soybean, peanut, wheat, cashew nut, egg, milk, fish and shrimp, the 8 species specificity probes that correspond respectively to these 8 kinds of anaphylactogen dna sequence dnas are: SEQ ID NO:3,6,9,12,15,18,21,24, utilize the 8 pairs of specificity upstream primers and downstream primer corresponding to described 8 kinds of anaphylactogen dna sequence dnas: SEQ ID NO:1-2,4-5,7-8,10-11,13-14,16-17,19-20,22-23 carry out the multiplex PCR amplification to the dna sequence dna of test sample.
5. visual film sensor chip for detection of anaphylactogen in the sample, wherein on same chip matrix surface, fix respectively a plurality of specific probes corresponding to a plurality of anaphylactogen dna sequence dnas, wherein, described anaphylactogen is selected from soybean, peanut, wheat, cashew nut, egg, milk, fish and shrimp, and the 8 species specificity probes that correspond respectively to the dna sequence dna of these 8 kinds of anaphylactogens are: SEQ ID NO:3,6,9,12,15,18,21,24.
6. visual film sensor chip according to claim 5 is characterized in that, also is fixed with the positive control sequence on the stromal surface of described visual film sensor chip, and 3 ' end of described positive control sequence is with covalently bound vitamin H.
7. visual film sensor chip according to claim 5 is characterized in that, the specific probe corresponding to each anaphylactogen dna sequence dna of being fixed on same chip matrix surface has two at least.
8. arbitrary described visual film sensor chip according to claim 5-7, it is characterized in that, described a plurality of anaphylactogen is soybean, peanut, wheat, cashew nut, and the employed a plurality of specific probes that correspond respectively to soybean, peanut, wheat, cashew nut allergen dna sequence dna are respectively SEQ ID NO:3,6,9,12; Egg, milk, fish and shrimp, the employed a plurality of specific probes that correspond respectively to egg, milk, fish and shrimp allergen dna sequence dna are respectively SEQ ID NO:15,18,21,24; Perhaps soybean, peanut, wheat, cashew nut, egg, milk, fish and shrimp, the 8 species specificity probes that correspond respectively to these 8 kinds of anaphylactogen dna sequence dnas are: SEQ ID NO:3,6,9,12,15,18,21,24.
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| CN103060462A (en) * | 2013-01-18 | 2013-04-24 | 天津生物芯片技术有限责任公司 | Gene chip and detection kit for detecting common food allergens |
| CN103060461A (en) * | 2013-01-18 | 2013-04-24 | 天津生物芯片技术有限责任公司 | Specific primer and kit for detecting common food allergens |
| CN104894278A (en) * | 2015-06-19 | 2015-09-09 | 杭州谱尼检测科技有限公司 | Method for screening detected food allergens rapidly and efficiently |
| CN114606297A (en) * | 2020-12-09 | 2022-06-10 | 成都万众壹芯生物科技有限公司 | Nucleic acid detection chip and application thereof |
| CN113755555A (en) * | 2021-09-03 | 2021-12-07 | 浙江工商大学 | Capture probe set for detecting food allergen, preparation method and application thereof |
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| CN101851669A (en) * | 2009-11-03 | 2010-10-06 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | Real-time fluorescent PCR quantitative detection method of original soybean sensitive components in food |
| CN101899501A (en) * | 2010-02-10 | 2010-12-01 | 江苏出入境检验检疫局动植物与食品检测中心 | Constant temperature amplification detection kit and method for detecting food allergen crustacean gene |
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2010
- 2010-12-08 CN CN 201010588394 patent/CN102108404B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101250582A (en) * | 2008-03-07 | 2008-08-27 | 山东出入境检验检疫局检验检疫技术中心 | Preparation of rapid test kit for fish anaphylactogen in food and detecting method |
| CN101851669A (en) * | 2009-11-03 | 2010-10-06 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | Real-time fluorescent PCR quantitative detection method of original soybean sensitive components in food |
| CN101899501A (en) * | 2010-02-10 | 2010-12-01 | 江苏出入境检验检疫局动植物与食品检测中心 | Constant temperature amplification detection kit and method for detecting food allergen crustacean gene |
Non-Patent Citations (1)
| Title |
|---|
| HIDENORI T..Quantification of genetically modified soybean by quenching probe polymerase chain reaction..《J. Agric. Food Chem.》.2005,第53卷2535-2540. * |
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| CN102108404A (en) | 2011-06-29 |
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