CN102382897B - Real-time fluorescent PCR (Polymerase Chain Reaction) detection method and kit for goose components in food and feedstuff - Google Patents
Real-time fluorescent PCR (Polymerase Chain Reaction) detection method and kit for goose components in food and feedstuff Download PDFInfo
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Abstract
The invention relates to a real-time fluorescent PCR (Polymerase Chain Reaction) detection method and kit for goose components in food and feedstuff. The invention discloses a primer and probe capable of specifically identifying goose components for the first time, and the primer and probe can specifically amplify DNA (deoxyribonucleic acid) containing goose components and can not specifically amplify DNA containing no goose components. Thus, the primer can be well used for identifying goose components, and has favorable repeatability and sensitivity.
Description
Technical field
The present invention relates to molecular biology and nucleic acid detection technique field.Particularly, the present invention relates to real-time fluorescence PCR detection method and the test kit of goose composition in food or the feed.
Background technology
For guaranteeing the verity of food ingredient, quality inspection " 12 " planning outline needs emphasis reinforcement conducting food to mingle authentication technique research during spelling out " 12 ".The Law of the People's Republic of China on Product Quality, " Food Hygiene Law of the People's Republic of China " and " food identity management regulation " laws and regulations such as (State Administration for Quality Supervision and Inspection and Quarantine make No. 102) forbid that the fraud of product mingles behavior and correct sign food labelling.Yet because economic interests drive, some illegal merchants usually with cheap raw material substitution or mix the raw material of high price, perhaps not add the material composition of mark in the course of processing of food.Mingle in the process in the fraud of converted products, use hazardous and noxious substances (as the steamed bun etc. that dyes), harm people's health through regular meeting.
In animal product raw material and converted products, mingling of composition often occur and fake and the unintentional pollution phenomenon.The people mainly is that illegal merchant will reduce cost or increase quality for adulterating, to seek huge economic interests.Thisly mingle fraud, shoddy behavior except influencing China's economy, most importantly another influence HUMAN HEALTH and fowl poultry safety.The worldwide outburst of mad cow disease and bird flu and popular, people more and more pay close attention to food safety and feed safety.Part crowd is because also edible vegetarian diet part only of healthy cause.Mad cow disease be since ruminating animal eaten the feed that contains animal component, so various countries forbid containing the forage feed animal of animal component.The feed that contains the fowl composition may have the risk of propagating bird flu.With these forage feed animals of mingling, can influence the healthy growth of livestock and poultry and our livestock industry safety.
Therefore press for exploitation and effectively identify the method for various bird compositions, with standard market, the protection consumer rights.
Summary of the invention
The object of the present invention is to provide the real-time fluorescence PCR detection method of goose composition in food and the feed.
The test kit that another object of the present invention is to provide the real-time fluorescence PCR of goose composition in food and the feed to detect.
In a first aspect of the present invention, a kind of method of identifying the goose composition is provided, described method comprises:
DNA with testing sample is template, carries out pcr amplification with the primer shown in SEQ ID NO:1 and the SEQ ID NO:2; If specific amplification takes place, then shows to comprise the goose composition in the testing sample.
In a preference, carrying out the real-time fluorescence PCR detection with the primer shown in SEQ ID NO:1 and the SEQ ID NO:2 and the Taqman MGB probe shown in the SEQID NO:3.
In another preference, described testing sample is food or feed.
In another preference, the detection sensitivity of described method is 10
-4Ng/ μ L DNA.
In another aspect of this invention, provide a kind of primer, described primer is that primer is right, and its sequence is shown in SEQ ID NO:1 and SEQ ID NO:2.
In another aspect of this invention, provide a kind of Taqman MGB probe, described probe sequence is shown in SEQ ID NO:3.
In another aspect of this invention, provide the purposes of described primer and/or described Taqman MGB probe, be used for identifying the goose composition from testing sample.
In another aspect of this invention, provide a kind of test kit of identifying the goose composition, comprising described primer; And/or described Taqman MGB probe.
In a preference, also comprise in the described test kit: the examination criteria product (the goose component content is determined in these standard substance) that contain the goose composition.
In another preference, also comprise in the described test kit being selected from following reagent: DNA extraction reagent (or test kit), the Taq enzyme, the PCR damping fluid, archaeal dna polymerase, and/or the working instructions of the method for goose composition are identified in explanation.
Other side of the present invention is because the disclosure of this paper is apparent to those skilled in the art.
Description of drawings
Fig. 1, goose source property primer probe specificity real-time fluorescence PCR detect collection of illustrative plates.Only in goose DNA, produce amplified production, and in other species DNA sample, all do not have amplification.
Fig. 2, goose source property primer probe sensitivity real-time fluorescence PCR detect collection of illustrative plates.
Amplification curve (level and smooth curved line) corresponds respectively to from left to right: 10% goose DNA, 1% goose DNA, 0.1% goose DNA, 0.01% goose DNA, 0.001% goose DNA, 0.0001% goose DNA.
The real-time fluorescence PCR detection sensitivity figure of Fig. 3, goose powder weight ratio.
Amplification curve is the DNA:10% goose powder of (level and smooth curved line) difference (level and smooth curved line) following sample from left to right; 1% goose powder; 0.1% goose powder; 0.01% goose powder; 0.001% goose powder.
Embodiment
The inventor is through extensive and deep research and test, but disclose the primer that a kind of specificity is identified the goose composition first, specific amplification (acquisition positive findings) can take place at the DNA that contains the goose composition in described primer, and specific amplification (acquisition negative findings) is not taken place the DNA that does not have the goose composition.In order to simplify the pcr amplification method, the inventor has also designed and has cooperated Taqman MGB probe described primer, that be used for carrying out real-time fluorescence PCR.Adopt described primer to cooperate Taqman MGB probe, can be applied to identify the goose composition well, and have good reproducibility, sensitivity.
Exploitation at present identifies that effectively the difficult point of the method for various bird compositions is that these compositions are very close on outward appearance, quality usually, causes the difficult branch true and false of people.Even adopt the technology on some genes or the protein level, also because many birds are nearer on sibship, be difficult to find the detection target standard compliant, that detection accuracy is high, practical.For this reason, the inventor has found suitable detection target through deep research and a large amount of screenings, has developed the method for real-time fluorescence PCR detection goose composition based on this.
The inventor passes through the screening to primer, but obtains the primer that a class specificity is identified the goose composition, and specific amplification takes place its DNA for goose, and specific amplification is not taken place the DNA that does not have the goose composition.
Therefore, the invention provides a kind of primer, the nucleotide sequence shown in described primer tool SEQ ID NO:1 and the SEQ ID NO:2.
These primers of the present invention can also carry out mark with radio isotope, vitamin H, enzyme, fluorescein or other chemiluminescent substances.
The present invention also provides a kind of probe, the nucleotide sequence shown in the described probe tool SEQ ID NO:3; Preferably, described probe is Taqman MGB probe, detects thereby be convenient to real-time fluorescence.
Utilize primer of the present invention and probe, only need carry out conventional PCR reaction and/or agarose gel electrophoresis, and by judging having or not of corresponding PCR product, just can judge accurately and rapidly whether testing sample contains the goose composition, and required sample size seldom.
Based on Auele Specific Primer and the probe of identifying the goose composition that be applicable to provided by the present invention, the present invention also provides a kind of method of identifying the goose composition, described method comprises: the DNA with testing sample is template, carries out pcr amplification with the primer shown in SEQ ID NO:1 and the SEQ ID NO:2; If specific amplification takes place, then shows to comprise the goose composition in the testing sample.
Polymerase chain reaction (PCR) technology is technology well known to those skilled in the art, and its ultimate principle is the method for the synthetic specific DNA fragment of external enzymatic.Method of the present invention can adopt conventional round pcr to carry out.
As optimal way of the present invention, utilize described primer, adopt Taqman MGB real time fluorescent PCR method to carry out the evaluation of goose composition.The TaqMan probe method is the quantitative PCR technique of high special, and its core is to utilize 3 of Taq enzyme ' → 5 ' exonuclease activity, cuts off probe, produces fluorescent signal.Because probe is that specificity is combined with template, so the power of fluorescent signal has just represented the quantity of template.The TaqMan probe is divided into two kinds according to the difference of the fluorescent quenching group of its 3 ' end mark: common TaqMan probe and TaqMan MGB probe.The quenching group of TaqMan MGB probe adopts non-fluorescent quenching group (Non-Fluorescent Quencher), and itself does not produce fluorescence, can reduce the intensity of background signal greatly.Also be connected with MGB (Minor Groove Binder) modification group on the probe simultaneously.
The method of obtaining the DNA of testing sample is technology well-known to those skilled in the art, for example can take traditional phenol/chloroform/primary isoamyl alcohol method, the DNA extraction test kit that perhaps can adopt some to be purchased, and this class test kit is well known to those skilled in the art.
The invention still further relates to a kind of test kit for the identification of the goose composition, contain the primer shown in SEQ ID NO:1 and the SEQ ID NO:2 in the described test kit; More preferably, also contain the probe shown in the SEQ ID NO:3 in the described test kit.
In addition, described test kit also can contain other reagent of identifying the goose composition, as (but being not limited to):
(A) various PCR reaction reagent, such as but not limited to: Taq enzyme, PCR damping fluid, dNTP, archaeal dna polymerase etc.; Or
(B) the required reagent of various extraction DNA (namely preparing the PCR reaction template), such as but not limited to: phenol, chloroform, primary isoamyl alcohol, NaCl etc.; Or
(C) test kit of extraction DNA.
In addition, also can contain working instructions and/or the Standard operation procedure SOP of identifying the goose composition in the described test kit.
Test kit of the present invention can be realized the purpose of rapid detection, batch detection goose composition.
Major advantage of the present invention is:
(1) but disclose the primer that a kind of specificity is identified the goose composition first, described primer specificity is good, can realize specific amplification for goose, then can not specific amplification for other material that is typically used as imitated goose composition beyond the goose.And described primer has good reproducibility, the result is reliable and stable.
(2) utilize described primer or contain the detection kit of described primer, can detect the goose composition fast, in large quantity, from testing sample, distinguish true and false goose composition rapidly and accurately, and required sample size is few, simple to operate.
(3) preferably, the present invention uses Taqman MGB real-time fluorescence PCR technology, can realize the accurate evaluation of goose derived component in the food fast.
(4) applying ensureing the quality of product of method of the present invention, protection human consumer's right to know and preference safeguard that normal economic order etc. provides technical support.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example is write according to normal condition such as J. Sa nurse Brooker etc. usually, molecular cloning experiment guide, Science Press, the condition described in 2002, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
I. materials and methods
1.1 sample collection and preparation
Goose, chicken, duck, turkey, pigeon, quail, beef, pork, Goral mutton, meat of a sheep, Cervus nippon meat, Carnis Cameli, horseflesh, donkey meat, rabbit meat, hare meat, Wild boar as food, racoon dog meat, meat of snake, bullfrog, frog meat, grass shrimp, crab, soft-shelled turtle, oyster, mussel, 27 kinds of common animals materials such as snail and soybean, corn, barley, wheat, rice, potato, tomato, pea, cotton, 10 kinds of frequently seen plants materials such as rape are available from the market of farm produce or the supermarket in Shanghai.
6 kinds of chicken and egg product, each 4 kinds of ducks, turkey meat, pigeon, quail meat and egg products, 15 batches of other composition food (jerky, meat pulp, biscuit etc.) are available from the supermarket in Shanghai.The 12 batches of import foie gras, goose dried meat meat, 15 batches of poultry bone powder fodders, 20 batches of other composition feeds (comprising chitling membranin powder, many good fortune albumen, whey powder, feed for pet etc.) are provided by the port sampling of Shanghai City customs.
1.2 reagent
(1) primer and the MGB fluorescent probe sequence of real-time fluorescence PCR detection:
Designed many kinds of primers and probe, through relatively screening repeatedly, obtained to be applicable to specific PCR primer and the probe of amplification goose derived component, sequence is:
Forward primer: 5 '-TTAAAACTCTAAGGACTTGGCGGT-3 ' (SEQ ID NO:1);
Reverse primer: 5 '-CTTGTTAGCGGTGTGCTATTGTGT-3 ' (SEQ ID NO:2);
Probe: 5 '-FAM-CTAGAGGAGCCTGTTCT-MGB-3 ' (SEQ ID NO:3);
(2) liquid nitrogen;
(3) CTAB extracts damping fluid: 20g/L CTAB, and, 1.4mol/L NaCL, 0.1mol/L Tris-HCL, 0.02mol/L Na
2EDTA, pH8.0;
(4) Proteinase K solution (20mg/mL);
(5) CTAB precipitated liquid: 5g/L CTAB, 40mmol/L NaCl;
(6) NaCl solution (1.2mol/L);
(7) Virahol;
(8) 70% ethanol;
(9) TE damping fluid, pH8.0;
(10)ABI?TaqMan?Universal?PCR?Master?Mix(Part?Number?4304437)；
(11) ultrapure water (ddH
2O).
1.3 plant and instrument
ABI 7300 real-time fluorescence PCR instrument; Balance; Liquid-transfering gun; Whizzer; The constant temperature couveuse; Freezing runner milling; DNA lyophilize whizzer; The nucleic acid-protein concentration analyzer; The Eppendorf centrifuge tube; The PCR reaction tubes.
1.4 the DNA extraction of sample
After sample (various animal or plant material) grinding, take by weighing 200mg left and right sides solid sample or 200 μ L liquid samples in the 2mL centrifuge tube, add 1.5mL CTAB and extract damping fluid (20g/L CTAB, 1.4mol/L NaCL, 0.1mol/L Tris-HCL, 0.02mol/L Na
2EDTA is pH8.0) with 10 μ L Proteinase K (20mg/mL) solution.The centrifugal 10min of 13000g after 60 ℃ of shaken overnight shifts supernatant liquor in new centrifuge tube.Use forced oscillation after adding 750 μ L chloroforms, the centrifugal 5min of 13000g transfers to supernatant in the new centrifuge tube.Add the CTAB precipitation solution (5g/L CTAB, 40mmol/L NaCl) of 2 times of volumes, room temperature leaves standstill 60min, the centrifugal 15min of 13000g, supernatant discarded.Adding 350 μ L NaCl solution suspends precipitation.Add 350 μ L chloroforms again, mixing, the centrifugal 10min of 13000g are carried out in the vortex vibration.Shift the Virahol that adds 0.6 times of volume behind the supernatant and be used for precipitate nucleic acids, room temperature is placed 20min, the centrifugal 10min of 13000g, supernatant discarded.Add 500 μ L, 70% ethanolic soln washing precipitation, be dissolved in the 200 μ L TE solution and measure DNA concentration with the nucleic acid-protein content meter.Also the commercialization DNA extraction test kit of available equivalents extracts template DNA.
Wherein, the goose dna solution is diluted to the DNA sample of 10ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L, 0.001ng/ μ L, 0.0001ng/ μ L, 0.00001ng/ μ L respectively.Be used for sensitivity test.
Wherein, mix with the goose powder with chicken powder, be mixed with the biased sample that contains 10%, 1%, 0.1%, 0.01%, 0.001% and 0.0001% goose powder content (W/W) respectively.Aforesaid method extracts DNA.
1.5 goose real-time fluorescence PCR test
1.5.1PCR reaction system
The reaction system of carrying out the real-time fluorescence PCR detection sees Table 1.
The reaction system that table 1, real-time fluorescence PCR detect
1.5.2PCR reaction cycle parameter
Real-time fluorescence PCR reaction cycle parameter: 50 ℃, 2min; 95 ℃, 10min; 95 ℃ of 15s, 60 ℃ of 1min circulate 40 times.
1.618S the amplification of rRNA gene PCR and detection
Use 18S rRNA gene amplification eukaryote native gene, the DNA that is extracted to guarantee is suitable for pcr amplification.Detecting used 18S rRNA gene primer sequence is:
5’-TCTGCCCTATCAACTTTCGATGGTA-3’(SEQ?ID?NO：4)；
5’-AATTTGCGCGCCTGCTGCCTTCCTT-3’(SEQ?ID?NO：5)；
18S rRNA gene primer PCR reaction system: 1 * PCR damping fluid, 2.5mmol/L Mg
2+, 1U Taq enzyme, 200 μ mol/L dNTPs, primer 100nmol/L, template 100ng, reaction volume are 25 μ l.Amplification condition is: 94 ℃, and 3min; 94 ℃, 20s, 54 ℃, 40s, 72 ℃, 40s, 40 circulations; 72 ℃, 5min.
Get 10 μ L PCR products, add 1 μ L10 * sample-loading buffer point sample and carry out electrophoresis.The used gel strength of PCR product electrophoresis detection is 1.5-2.0%.
II. embodiment
The detected result of embodiment 1, eukaryote special primer 18S rRNA primer
With eukaryote 18S rRNA special primer all dna solutions (dna solution that comprises various animal or plant materials) that this experiment extracts that increase, the specific amplified band of 137bp all can appear in all dna solutions.
The above results shows that the dna solution of all extractings is suitable for the PCR test.
The real-time fluorescence PCR specific detection of embodiment 2, goose composition
The real-time fluorescence PCR of setting up with the present invention detects primer and the MGB fluorescent probe of goose derived component, detects to goose with for other 26 kinds of common animals and 10 kinds of frequently seen plants of examination.
Found that, adopt described Auele Specific Primer and probe, only in goose DNA, produce amplified production, and in other species DNA sample, all do not have amplification, as Fig. 1.
The The above results explanation, the detection method that the present invention sets up has species specificity.
The real-time fluorescence PCR detection sensitivity of embodiment 3, goose composition
To 10ng/ μ L (10%), 1ng/ μ L (1%), 0.1ng/ μ L (0.1%), 0.01ng/ μ L (0.01%), 0.001ng/ μ L (0.001%), 0.0001ng/ μ L (0.0001%), 0.00001ng/ μ L (0.00001%) goose DNA detects.Amplification appears in result such as Fig. 2 in greater than 0.0001% goose DNA.
The above results shows that goose source property detection primer has species specificity, and detection sensitivity is 0.0001ng/ μ L (0.0001%) goose DNA, and visible detection primer of the present invention and detection method are highly sensitive.
With the goose powder sample of 10 times of serial mixed preparing different contents of chicken powder, extract and obtain dna solution.Use goose composition primer and probe that the sample DNA corresponding to 10%, 1%, 0.1%, 0.01%, 0.001% and 0.0001% goose powder content (W/W) is detected respectively.
Result such as Fig. 3, amplification curve all appears in 10%, 1%, 0.1%, 0.01% and 0.001% goose powder, and amplification curve appears in 0.0001% goose powder.
The above results shows that on weight percent levels, the detection sensitivity of this method is 0.001% goose powder (W/W), and visible detection primer of the present invention and detection method are highly sensitive, are not subjected to the influence of close composition.
The application of goose composition detection in embodiment 4, the food
Adopt present method that import foie gras, the goose dried meat meat at 12 batches of customs ports are detected, all detect the goose composition.At 6 kinds of chicken and egg product, each 4 kinds of ducks, turkey, pigeon, quail meat and egg products do not detect the goose composition in 15 batches of other composition food.Detect the goose composition at the poultry bone meal of 1 batch of U.S.'s import and the poultry bone powder fodder of 1 batch of Australian import, do not detect the goose composition at other 14 batches of poultry bone powder fodders and 20 batches of other composition feeds.Through comparing and following the tracks of, these analyzing and testing result is accurately.
To sum up, goose composition real-time fluorescence PCR detection method in food of the present invention and the feed, high specificity, highly sensitive, can accurately detect the goose composition in food and the feed, for goose component detection method stdn in food and the feed provides foundation.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. a method of identifying the goose composition is characterized in that, described method comprises:
DNA with testing sample is template, carries out pcr amplification with the primer shown in SEQ ID NO:1 and the SEQ ID NO:2; If specific amplification takes place, then shows to comprise the goose composition in the testing sample.
2. the method for claim 1 is characterized in that, is carrying out the real-time fluorescence PCR detection with the primer shown in SEQ ID NO:1 and the SEQ ID NO:2 and the Taqman MGB probe shown in the SEQ ID NO:3.
3. the method for claim 1 is characterized in that, described testing sample is food or feed.
4. the method for claim 1 is characterized in that, the detection sensitivity of described method is 10
-4Ng/ μ L DNA.
5. a primer is characterized in that, described primer is that primer is right, and its sequence is shown in SEQ ID NO:1 and SEQ ID NO:2.
6. a Taqman MGB probe is characterized in that, described probe sequence is shown in SEQ ID NO:3.
7. the purposes of the described primer of claim 5 or the described Taqman MGB of claim 6 probe is used for identifying the goose composition from testing sample.
8. a test kit of identifying the goose composition is characterized in that, comprising the described primer of claim 5 and the described Taqman MGB of claim 6 probe.
9. test kit as claimed in claim 8 is characterized in that, also comprises in the described test kit: the examination criteria product that contain the goose composition.
10. test kit as claimed in claim 8 is characterized in that, also comprises in the described test kit being selected from following reagent: DNA extraction reagent, and the Taq enzyme, the PCR damping fluid, archaeal dna polymerase, and/or the working instructions of the method for goose composition are identified in explanation.
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| CN105586428B (en) * | 2016-03-10 | 2018-06-29 | 中国热带农业科学院热带生物技术研究所 | A kind of Primer composition, kit and detection method for being used to identify transgenic sugarcane brown sugar |
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