CN104561271A - Visual DNA (deoxyribonucleic acid) chip kit and method for detecting multiple animal-derived components - Google Patents
Visual DNA (deoxyribonucleic acid) chip kit and method for detecting multiple animal-derived components Download PDFInfo
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Abstract
The invention discloses a visual DNA (deoxyribonucleic acid) chip kit and method for detecting multiple animal-derived components. The visual DNA chip detection method is used for animal derivation of chickens, ducks, geese, pigs, cattle, sheep, goats, tigers, bears and leopards. The method comprises the following steps: designing and synthesizing specific primers and probes according to the sequences of mitochondrial DNAs of the animals above in the GenBank; and carrying out chip sample application, hybridization and color development by using a Dr.chip system, optimizing reaction conditions, carrying out sensitivity and specificity testing and repetitive testing, and carrying out detection on the animal-derived components. The result indicates that the novel visual DNA chip detection method for chickens, ducks, geese, pigs, cattle, sheep, goats, tigers, bears and leopards has the characteristics of high sensitivity, high specificity and high flux.
Description
Technical field
The invention belongs to field of molecular detection, being specifically related to a kind of visual DNA chip test kit for detecting many animals derived component and method.
Background technology
Along with the develop rapidly of international trade, the difficulty of defending international animal epidemic to propagate is continuing to increase.In order to prevent mad cow disease, itch, animal influenza etc. through feed, livestock product propagate, countries in the world government and be organized in specification to animalsderived feedstuffs use while, actively develop the research of animal derived materials detection technique.In addition, at home and in international trade, adulterate in beef or mutton low-cost pork, and shoddy deceptive practices are of common occurrence, while bringing inconvenience to vast friend Moslem, and the grievous injury interests of human consumer.Tiger, bear, leopard are rare wild animals, are hit the illegal effective way of hunting and smuggling to the source of species qualification of wildlife and processed goods thereof.
At present, for above animal derived materials detection method mainly PCR method, the method specificity is good, quick and precisely, is used widely.Simultaneously the deficiency of the detection of PCR method can only detect limited several species, for source background not cleer and peaceful may adulterate multiple meat when, just there is difficulty in Primer selection.
Summary of the invention
For deficiency existing in prior art, the object of the present invention is to provide a kind of visual DNA chip test kit for detecting many animals derived component and detection method.
The technical solution used in the present invention is:
For detecting a visual DNA chip test kit for many animals derived component, it comprises: chip substrate, the specific probe of point sample on chip substrate, Auele Specific Primer; Wherein, chicken, duck, goose, pig, ox, sheep and goat use a set of universal primer to increase, and tiger, bear and leopard use respective Auele Specific Primer respectively, and the sequence of described specific probe and Auele Specific Primer is as shown in the table:
On described chip substrate, probe point sample matrix diagram is as shown in the table:
| PC | Chicken-P1 | Chicken-P2 | Duck-P1 | Duck-P2 |
[0011]
| NC | Goose-P1 | Goose-P2 | Swine-P1 | Swine-P2 |
| Bovine-P1 | Bovine-P2 | PC | Sheep-P1 | Sheep-P2 |
| Goat-P1 | Goat-P2 | Tiger-P1 | Tiger-P2 | NC |
| Bear-P1 | Bear-P2 | Leopard-P1 | Leopard-P1 | PC |
Also containing damping fluid, detection liquid, confining liquid and developer in described test kit.
Utilize mentioned reagent box to detect the method for many animals derived component, comprise the steps:
(1) pcr amplification: extract measuring samples DNA, utilize Auele Specific Primer to carry out pcr amplification;
(2) chip hybridization: get PCR primer and DNA chip is hybridized at 47 ~ 49 DEG C, discards detection liquid, with water rinse to hybridization put clear, camera imaging.Concrete operations be:
1) get PCR primer fully to mix with DR. hybridization buffer, boiling water sex change, be transferred to rapidly on ice;
2) said mixture is transferred to chip indoor, tiling evenly, is avoided producing bubble in bottom, under 47 ~ 49 DEG C of temperature condition, is hybridized 45-60min in vibration incubator; Discard hybridization solution, wash with DR. washings;
3) in hybridization chamber, add the mixed solution of Strep-AP and confining liquid, room temperature reaction, discards confining liquid, with washings washing, absorbs moisture residual on chip;
4) each chip room adds NBT/BCIP and detects the mixed solution of liquid, chip is placed in darkroom and reacts 5-10min, discard detection liquid, rinses to hybridization point is clear, camera imaging with water.
The invention has the beneficial effects as follows:
The visual DNA chip detection method of the chicken that the present invention sets up, duck, goose, pig, ox, sheep, goat, tiger, Xiong Hebao, has highly sensitive, high specificity and high-throughout feature.Carry out disposable amplification employing universal primer to the pcr amplification of chicken, duck, goose, pig, ox, sheep and goat, multiplex PCR is employed to the amplification of tiger, Xiong Hebao, the trouble that decreasing repeatedly increases brings, only need to set up two pcr amplifications to the detection of above 10 kinds of animal derived materials, then carry out the detection of concentrated disposable visual DNA chip and just can show result fast and accurately.
Accompanying drawing explanation
Fig. 1: universal PC R amplification (M.DNA Marker DL2000; 1. chicken; 2. duck; 3. goose; 4. pig; 5. ox; 6. sheep; 7. goat; N. blank).
Fig. 2: tiger, bear, leopard substance pcr amplification result (M.DNA Marker DL2000; 1. northeastern tiger; 2. bangladesh tiger; 3. brave negative control; 4. leopard negative control; 5. leopard positive control; 6. bear negative control; 7. bear's paw; 8. bear gall).
Fig. 3: tiger, bear, leopard multiplexed PCR amplification result (M.DNA Marker DL2000; 1. tiger, leopard, bear multiplex PCR; N. negative control).
Fig. 4: visual DNA chip specific test (A. chicken; B. duck; C. goose; D. pig; E. ox; F. sheep; G. goat; H. tiger; I. bear; J. leopard).
Fig. 5: duck derived component pcr amplification sensitivity test (M.DNA Marker DL2000; 1. duck derived component DNA stoste PCR tests; 2-6. duck derived component DNA stoste dilution 10 to 105 times of PCR tests; N. negative control).
Fig. 6: the visual DNA chip sensitivity test of duck derived component.
Fig. 7: the visual DNA chip replica test of duck derived component (A. first time chip detection result; B. second time chip detection result).
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated, but be not limited thereto.
Embodiment:
1 materials and methods
1.1 material
1.1.1 sample DNA source
Chicken, duck, goose, pork, beef, sheep and goat meat are for preserving sample in this laboratory; Bangladesh tiger (white tiger mutation), each portion of northeastern tiger hair, from Guangzhou Zoo; One, bear's paw, for sample is preserved in this laboratory; Bear gall one, from the immersion bear bile wine of 2 years.The nucleic acid that above sample is obtained by DNA extraction method is for pcr amplification.Leopard positive control is mitochondrial cytochrome b gene, cloned plasmids prepared by this laboratory.
1.1.2 main agents and instrument
DNA extraction kit E.Z.N.A.TM Tissue DNA Kit is U.S. OMEGA Products; 2 × Taq PCR Mastermix is TIANGEN Biotech's product; DNA Marker DL 2000 is purchased from precious biotechnology (Dalian) company limited.
DR.chip DIY Kit (comprising chip substrate, damping fluid, detection liquid, confining liquid, developer etc.), is purchased from Taiwan Jingyu Biological Science and Technology Industry Co Ltd; DR.Fast Spot chip point sample instrument, DR.Mini Oven hybridizes case, biochip reaction apparatus system, is Taiwan Jingyu Biological Science and Technology Industry Co Ltd product; UV-crosslinked instrument, Xin Zhi biotech inc product; Ultraviolet spectrophotometer NanoDrop ND-1000, U.S. Thermo Fisher Scientific product.
1.2 method
1.2.1 primer, probe design synthesis
From GenBank, download chicken, duck, goose, pig, ox, sheep, goat, bear, tiger and leopard Mitochondrial gene sequence respectively, after utilizing Clustal X comparison, find out high conservative region, utilize Oligo7.0 to design Auele Specific Primer and probe.At 5 of primer, end mark Biotin, at 5 of probe, end adds Poly (T) and ensures probe length, and primer and probe are synthesized by Hui Rui bio tech ltd, Shanghai, and after screening, primed probe is in table 1.Wherein, chicken, duck, goose, pig, ox, sheep and goat use a set of universal primer to increase, and tiger, bear and leopard use respective Auele Specific Primer respectively.Often kind of animal derived materials designs two specific hybridization probes, if wherein a positive animal derived materials being these species of probe hybridization result display detects positive.
1.2.2 DNA extraction
Use OMEGA company DNA extraction kit to extract DNA, operation steps is carried out to specifications, and use ultraviolet spectrophotometer analytical concentration and purity, the DNA meeting pcr amplification requirement carries out mark, places-20 DEG C and saves backup.
1.2.3 the preparation of PCR primer
With carried DNA for template, the DNA using 2 × Taq PCR Mastermix test kit to extract with primer pair in table 1 carries out pcr amplification.Wherein the amplification of chicken, duck, goose, pig, ox, sheep and goat DNA uses universal primer.Tiger, bear, leopard use respective Auele Specific Primer, are merged into multiplex PCR after single amplification success.Wherein the positive control of leopard composition detection uses cloned plasmids.All PCR reaction systems are: 2 × Taq PCR Master mix 12.5 μ L, 10 μm of each 1.0 μ L of ol/L upstream and downstream primer, DNA masterplate 2.0 μ L, moisturizing to 25 μ L.Response procedures: 94 DEG C of 5min; 94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s, 35 circulations; 72 DEG C of 5min.After 2% agarose gel electrophoresis, the successful PCR primer that increases delivers to Shanghai Ying Jun Bioisystech Co., Ltd cloning and sequencing, confirms source of species.
1.2.4 the preparation of visual DNA chip
By the probe of synthesis probe dilution liquid adjustment concentration to 20 μm ol/L, by DR.Fast Spot chip point sample instrument system by probe point sample on chip substrate, point sample matrix is in table 2.Hybridization positive control is that test kit carries, and negative control is and the fragment gene of each target gene in this research without any the plant chlorophyll of homology.Drying at room temperature 10min after point sample, fixes (254nm, 1.2J, 10min) at UV-crosslinked instrument, uses Milli-Q water washing successively 3 times, and 95% ethanol infiltrates 20s, puts the dry 10min of 50 DEG C of incubators, for subsequent use.
Table 2 probe point sample matrix diagram
Table 2 Distribution dots of the microarray
| PC | Chicken-P1 | Chicken-P2 | Duck-P1 | Duck-P2 |
| NC | Goose-P1 | Goose-P2 | Swine-P1 | Swine-P2 |
| Bovine-P1 | Bovine-P2 | PC | Sheep-P1 | Sheep-P2 |
[0052]
| Goat-P1 | Goat-P2 | Tiger-P1 | Tiger-P2 | NC |
| Bear-P1 | Bear-P2 | Leopard-P1 | Leopard-P1 | PC |
Note:PC:Positive control；NC:Negative control
1.2.5 visual DNA chip detection method is set up and temperature optimization
Respectively 10 kinds of PCR primer are carried out chip hybridization under 47 DEG C, 48 DEG C, 49 DEG C, 50 DEG C temperature condition, select best hybridization temperature.Concrete operation method: get 10 kinds of each 10 μ L of PCR primer and fully mix with the DR. hybridization buffer of 200 μ L, boiling water sex change 6min, is transferred to rapidly 10min on ice; Said mixture is transferred to chip indoor, tiling evenly, is avoided producing bubble in bottom, under above-mentioned gradient temperature condition, is hybridized 45-60min in vibration incubator; Discard hybridization solution, wash 3 times with DR. washings; In hybridization chamber, add the mixed solution of 0.2 μ L Strep-AP and 200 μ L confining liquids, room temperature reaction 30min, discards confining liquid, washs 3 times with washings, absorbs moisture residual on chip; Each chip room adds the mixed solution that 4 μ L NBT/BCIP and 196 μ L detect liquid, chip is placed in darkroom and reacts 5-10min, discard detection liquid, rinses to hybridization point is clear, camera imaging with water.
1.2.6 visual DNA chip hybrid specificities test
First carry out pcr amplification, then under hybridization temperature condition after optimization, get 10 kinds of PCR primer hybridization respectively, observe between each test item and have no cross reaction.
1.2.7 visual DNA chip hybridization sensitivity and replica test
The DNA of the 10 kinds of animal derived materials extracted, 10 times of gradient dilutions become 6 concentration.With the nucleic acid of 6 concentration for template carries out pcr amplification, with amplified production and chip hybridization, be its sensitivity with finding of naked eye minimum concentration hybridization spot.Carry out agarose gel electrophoresis detection to above-mentioned PCR primer, the minimum concentration band that gel imaging can be seen is the sensitivity of PCR simultaneously, so that compare with visual DNA chip method.
To same animal derived materials in a detection twice in month of being separated by, by visual DNA chip color developing effect, the repeatability of checking detection method.
1.2.8 application is detected
Use visual DNA chip method prepared in this research to 43 parts of animal meats, a animal's paw detects.Wherein animal meat detected result and inspection and quarantine industry standard detected result compare, and animal's paw detected result uses normal PCR cloning and sequencing result verification.
2 results
2.1 PCR primer electrophoresis result
PCR primer in 1.2.3 used 2% agarose gel electrophoresis to detect, amplified band size and object clip size basically identical (see Fig. 1, Fig. 2) can be found out by result.The amplification of tiger, leopard, bear is merged into multiplex PCR, electrophoretic band clear (see Fig. 3).PCR primer sequencing result shows, the source of species of sample is completely the same with expection.
2.2 visual DNA chip specific tests
PCR primer and visual DNA chip are hybridized under condition of different temperatures respectively, result shows, and 49 DEG C time, hybridization signal is the most obvious, the clear and background signal of hybridization spot in tolerance interval, so best hybridization temperature is set as 49 DEG C.
Use universal primer preparation PCR reaction system, pcr amplification is carried out for template respectively with chicken, duck, goose, pig, ox, sheep, goat, use triple PCR reaction system, pcr amplification is carried out for template respectively with tiger, bear and leopard nucleic acid, then hybridize, for verifying the specificity of animal derived materials detection method by PCR primer and visual DNA chip respectively.Find out from results of hybridization, there is hybridization signal spot in often kind of animal component position corresponding in visual DNA chip matrix, without cross influence between different animals derived component results of hybridization, prove visual DNA chip specificity good (see Fig. 4).
2.3 visual DNA chip sensitivity tests
The duck source property DNA stoste of extraction is carried out 10 times of gradient dilutions, then pcr amplification is carried out, the PCR primer of dilution 103 times of DNA can also see band, and the PCR primer electrophoresis diluting 104 times of DNA can't see band (see Fig. 5), and use this PCR primer to carry out chip hybridization can to see obvious hybridization spot (see Fig. 6).
The PCR using other animal derived materials to detect and visual DNA chip detected result relatively can be found out, the sensitivity of visual DNA chip is apparently higher than traditional PCR method 1 to 2 orders of magnitude.Prove that visual DNA chip detection method prepared in this research is highly sensitive in traditional PCR method used.
2.4 visual DNA chip replica tests
In twice detection of one month of being separated by, the detected result of often kind of animal derived materials is all consistent, and illustrate that visual DNA chip detection method is reproducible, result is reliable and stable.Fig. 7 is twice visual DNA chip detected result of duck derived component.
2.5 visual DNA chip detect application test
Visual DNA chip method is used to detect 43 batches of meats, detect chicken derived component 1 part, duck derived component 3 parts, goose derived component 1 part, calf-derived Cyclospora 23 parts, sheep derived component 7 parts, goat derived component 2 parts, pig derived component 5 parts, quarantining with service test, to detect the result obtained completely the same for industry standard.Detect brave derived component 1 part, due to without reference standard, use PCR primer order-checking validate result accurate.
More than experiment shows: the Novel visual DNA chip detection method of the chicken that the present invention sets up, duck, goose, pig, ox, sheep, goat, tiger, Xiong Hebao, has highly sensitive, high specificity and high-throughout feature.
<110> Inspection & Quarantine Technology Center of Zhuhai Entry-Exit Inspection & Quarantine Bureau
<120> mono-kind is for detecting visual DNA chip test kit and the method for many animals derived component
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Claims (5)
1., for detecting a visual DNA chip test kit for many animals derived component, it comprises: chip substrate, the specific probe of point sample on chip substrate, Auele Specific Primer; Wherein, chicken, duck, goose, pig, ox, sheep and goat use a set of universal primer to increase, and tiger, bear and leopard use respective Auele Specific Primer respectively, and the sequence of described specific probe and Auele Specific Primer is as shown in the table:
2. the visual DNA chip test kit for detecting many animals derived component according to claim 1, it is characterized in that, on described chip substrate, probe point sample matrix diagram is as shown in the table:
3. the visual DNA chip test kit for detecting many animals derived component according to claim 1, is characterized in that, also containing damping fluid, detection liquid, confining liquid and developer in described test kit.
4. utilize the test kit described in any one of claim 1 ~ 3 to detect the method for many animals derived component, comprise the steps:
(1) pcr amplification: extract measuring samples DNA, utilize Auele Specific Primer to carry out pcr amplification;
(2) chip hybridization: get PCR primer and DNA chip is hybridized at 47 ~ 49 DEG C, discards detection liquid, with water rinse to hybridization put clear, camera imaging.
5. method according to claim 4, is characterized in that, the concrete operations of step (2) are:
1) get PCR primer fully to mix with DR. hybridization buffer, boiling water sex change, be transferred to rapidly on ice;
2) said mixture is transferred to chip indoor, tiling evenly, is avoided producing bubble in bottom, under 47 ~ 49 DEG C of temperature condition, is hybridized 45-60min in vibration incubator; Discard hybridization solution, wash with DR. washings;
3) in hybridization chamber, add the mixed solution of Strep-AP and confining liquid, room temperature reaction, discards confining liquid, with washings washing, absorbs moisture residual on chip;
4) each chip room adds NBT/BCIP and detects the mixed solution of liquid, chip is placed in darkroom and reacts 5-10min, discard detection liquid, rinses to hybridization point is clear, camera imaging with water.
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| CN108220457A (en) * | 2018-03-22 | 2018-06-29 | 四川华汉三创生物科技有限公司 | A kind of Nucleic acid combinations and its application for animal derived materials detection |
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