CN102559919B - Real-time PCR (Polymerase Chain Reaction) detection method of buffalo components in food and feed - Google Patents
Real-time PCR (Polymerase Chain Reaction) detection method of buffalo components in food and feed Download PDFInfo
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Abstract
The invention relates to a real-time PCR (Polymerase Chain Reaction) detection method of buffalo components in food and feed. The invention firstly discloses a primer for specific identification of buffalo components, and the primer can generate specific amplification of DNA (Deoxyribonucleic Acid) containing buffalo components, and can not generate specific amplification of DNA containing no buffalo components. The invention also provides a simplified PCR detection method, which can be well applied to the identification of the buffalo components, and has good reproducibility 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 buffalo composition in food or feed.
Background technology
As a class high-grade nutrient food, " new lover " that buffalo milk goods day by day become people to consume, is described as " king in milk ".Buffalo milk, buffalo cheese, buffalo milk healthcare product and infant food series product are milk preparations of high added value, and value of the product and nutritive value are all higher than ordinary milk goods several times.As commercial extremely valuable composition, sugared content, the milk-protein of buffalo milk are higher than ox milk with casein content, and rich in mineral substances and trace element, be of high nutritive value, buffalo's milk frankincense is dense thick, with rich flavor, mouthfeel is full, is all-ages nutrient excellent product.Buffalo milk is local characteristic product.Italy's buffalo milk industry production level is high, and product category is many, and market is large, and Mozzarella cheese is world-famous, is deeply subject to the welcome of European & American Market.Buffalo cheese is found a good sale in the countries and regions such as English, method, U.S., mainly in high-grade restaurant and delicatessen, sells, and is worth extremely expensive.In China, maximum with a Guangxi buffalo number, be thereafter Yunnan, Guangdong, Guizhou, Hubei, Sichuan, Hunan, Jiangxi and Anhui successively.The product of buffalo milk processing is ginger milk curd, double-skin milk etc.Because buffalo milk output is not high, and price is more expensive, and so the phenomenon of mixing ordinary milk current in buffalo milk and goods is very general.
At present about the adulterated detection method of milk-product, there are electrophoresis, chromatogram, immunochemistry, DNA technique etc., take DNA as the basic methods such as PCR are by DNA cloning being carried out to the analysis of dairy products kind, sensitive, special, easy, quick, good stability.But, current reagent and the method that does not also detect respond well specific detection buffalo composition.
Summary of the invention
The object of the present invention is to provide real-time fluorescence PCR detection method and the test kit of buffalo composition in food or feed.
In a first aspect of the present invention, a kind of method of identifying buffalo composition is provided, described method comprises:
The DNA of testing sample of take is template, with the primer shown in SEQ ID NO:1 and SEQ ID NO:2, carries out pcr amplification; If generation specific amplification, shows to comprise in testing sample buffalo composition.
In a preference, with the primer shown in SEQ ID NO:1 and SEQ ID NO:2 and the Taqman probe shown in SEQ ID NO:3, carry out real-time fluorescence PCR detection.
In another preference, described testing sample is food or feed.
In another preference, the detection sensitivity of described method is 10
-3ng/ μ L DNA.
In another aspect of this invention, provide a kind of primer, described primer is primer pair, and its sequence is as shown in SEQ ID NO:1 and SEQ ID NO:2.
In another aspect of this invention, provide a kind of Taqman probe, it is characterized in that, described probe sequence is as shown in SEQ ID NO:3.
In another aspect of this invention, the purposes of the primer described in providing and/or described Taqman probe, for identifying buffalo composition from testing sample.
In another aspect of this invention, provide a kind of test kit of identifying buffalo composition, comprising described primer and/or described Taqman probe.
In a preference, in described test kit, also comprise: the examination criteria product that contain buffalo composition.
In another preference, in described test kit, also comprise and be selected from following reagent: DNA extraction reagent, Taq enzyme, PCR damping fluid, archaeal dna polymerase, and/or the working instructions of the method for buffalo composition are identified in explanation.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
The species specificity real-time fluorescence PCR of Fig. 1, buffalo composition detects figure.Amplification curve is from left to right respectively: in Murrah, Buddhist nun-and the DNA of La Fei buffalo, Murrah and Guangxi buffalo first cross buffalo, Murrah and Guangxi buffalo hybridization buffalo of low generation, Murrah and the high milk sample for hybridization buffalo of Guangxi buffalo.
The real-time fluorescence PCR detection sensitivity figure of Fig. 2, buffalo DNA proportioning.Amplification curve is from left to right respectively: the DNA of 10ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L, 0.001ng/ μ L buffalo milk sample.
The real-time fluorescence PCR detection sensitivity figure of Fig. 3, buffalo milk weight ratio.Amplification curve is respectively from left to right (W/W): 10% buffalo milk; 1% buffalo milk; 0.1% buffalo milk; The DNA that 0.01% buffalo milk extracts.
Embodiment
The inventor is through extensive and deep research and test, disclose first a kind of primer that can specificity identification buffalo composition, for the DNA that contains buffalo composition, can there is specific amplification (acquisition positive findings) in described primer, and to not having the DNA of buffalo composition that specific amplification (acquisition negative findings) does not occur.In order to simplify pcr amplification method, the inventor also designed coordinate described primer, for carrying out the Taqman probe of real-time fluorescence PCR.Adopt described primer to coordinate Taqman probe, can be applied to well identify buffalo composition, and there is good reproducibility, sensitivity.
Exploitation at present effectively identifies that the difficult point of the method for various animals milk product compositions is that these compositions are conventionally very close in outward appearance, quality, causes the difficult minute true and false of people.Even if adopt the technology on some genes or protein level, also because some animal varietiess are nearer in sibship, be difficult to find the detection target standard compliant, detection accuracy is high, practical.For this reason, the inventor, through deep research and a large amount of screenings, has found suitable detection target, based on this, has developed the method for real-time fluorescence PCR detection buffalo composition.
As used herein, described " buffalo composition " refers to that specificity comes from the composition of buffalo, such as the milk of buffalo, buffalo meat etc.
As used herein, described " food " has comprised beverage.
The inventor is by the screening to primer, obtains the primer that a class can specificity identification buffalo composition, and specific amplification occurs its DNA for buffalo, and to not having the DNA of buffalo composition that specific amplification does not occur.
Therefore, the invention provides a kind of primer, described primer tool SEQ ID NO:1 and the nucleotide sequence shown in 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 described probe tool SEQ ID NO:3; Preferably, described probe is Taqman MGB probe, thereby be convenient to real-time fluorescence, detects.
Utilize primer of the present invention and probe, only need carry out real-time fluorescence PCR reaction, and by judging having or not of corresponding PCR product, just can judge accurately and rapidly whether testing sample contains buffalo composition, and required sample size seldom.
Based on Auele Specific Primer and the probe that is applicable to identify buffalo composition provided by the present invention, the present invention also provides a kind of method of identifying buffalo composition, described method comprises: the DNA of testing sample of take is template, with the primer shown in SEQ ID NO:1 and SEQ ID NO:2, carries out pcr amplification; If generation specific amplification, shows to comprise in testing sample buffalo composition.
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 buffalo composition.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 and template are specific bindings, so the power of fluorescent signal has just represented the quantity of template.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 greatly reduce the intensity of background signal.On probe, be also connected with MGB (Minor Groove Binder) modification group 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, or the DNA extraction test kit that 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 buffalo composition, in described test kit, contain the primer shown in SEQ ID NO:1 and SEQ ID NO:2; More preferably, in described test kit, also contain the probe shown in SEQ ID NO:3.
In addition, described test kit also can contain other reagent of identifying buffalo 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 (preparing PCR reaction template), such as but not limited to: phenol, chloroform, primary isoamyl alcohol, NaCl etc.; Or
(C) extract the test kit of DNA.
In addition, in described test kit, also can contain working instructions and/or the Standard operation procedure SOP of identifying buffalo composition.
Test kit of the present invention can be realized the object of rapid detection, batch detection buffalo composition.
Major advantage of the present invention is:
(1) disclose first a kind of primer that can specificity identification buffalo composition, described primer specificity is good, composition (as buffalo milk) for buffalo source can be realized specific amplification, can not specific amplification for other material that is typically used as imitated buffalo composition beyond buffalo.And described primer has good reproducibility, result is reliable and stable.
(2) utilize described primer or the detection kit that contains described primer, can detect fast, in large quantity buffalo composition, from testing sample, distinguish rapidly and accurately true and false buffalo composition, and required sample size is few, simple to operate.
(3) preferably, the present invention's application Taqman MGB real-time fluorescence PCR technology, can realize the precise Identification of buffalo derived component in food fast.
(4) applying ensureing the quality of product of method of the present invention, Protection of consumer right to know and preference, safeguard that normal economic order etc. provides technical support.For market surveillance department and the inspection and quarantine department of food provides technical support.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, writes molecular cloning experiment guide, Science Press, the condition described in 2002, or the condition of advising according to manufacturer conventionally as J. Pehanorm Brooker etc. according to normal condition.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Unless otherwise defined, the same meaning that all specialties of using in literary composition and scientific words and one skilled in the art are familiar.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
I. materials and methods
1.1 sample collections, test materials, reagent and instrument
From in Murrah, Buddhist nun-the high aqua pura milk for hybridization buffalo etc. of first cross buffalo, Murrah and Guangxi buffalo hybridization buffalo of low generation, Murrah and Guangxi buffalo of La Fei buffalo, Murrah and Guangxi buffalo provides by Guangxi buffalo institute.He Sitanniu produces milk, beef (Carnis Bovis seu Bubali), Henan product beef (Carnis Bovis seu Bubali), Qingdao product beef (Carnis Bovis seu Bubali), market, Shanghai beef (Carnis Bovis seu Bubali), Qinghai Yak meat etc. are produced purchased from the market of farm produce, Shanghai City or supermarket in Jilin.Australia ox (ox) meat meal tankage is provided by Shanghai customs port.Buffalo meat is bought by market, Jiangxi.
The animal components such as horseflesh, donkey meat, Goral mutton, meat of a sheep, Carnis Cameli, cat meat, pork, chicken, duck, goose, quail meat, bream meat, butterfish meat, mackerel meat, turbot meat, mouse meat are purchased from the market of farm produce, Shanghai City or supermarket.
The plant constituents such as rice, corn, soya bean, glutinous millet, mung bean, buckwheat, Ipomoea batatas, potato, cashew nut, Semen Phaseoli Vulgaris, almond, honey peach, mango, pomegranate, sea-tangle, celery are purchased from the market of farm produce, Shanghai City or supermarket.
Import buffalo product: 6 crowdes of Ai Baisi board water logging Mo Zeruila cheese (Ambrosi Mozzarella Cheese, Italy produces, lot number L1194, L1179, L0911, L1319, L1320, L11333), 1 batch of horse Soviet Union lira buffalo cheese (Italy produces), 1 batch of outstanding thunder Sa board buffalo horse Soviet Union's lira cheese (Italian CIRESA SNC DI CIRESA V.E.A. produces), (Italy produces 2 batches of buffalo milk horse Soviet Union lira cheese, lot number LF1200B, LF1341B), very swing license support horse Soviet Union lira buffalo cheese (Italy's product for 2 parts, lot number L299, L327), (brand is respectively the Raphael's buffalo milk that rubs to 3 parts of domestic buffalo milks, Raphael buffalo papaya milk rubs, original flavor buffalo milk) purchased from the market of farm produce, Shanghai City or supermarket.Be used for the buffalo meat sample of example detection purchased from Guangxi, Jiangxi and market, Zhejiang.
Ordinary milk for detection of sensitivity preparation is the how U.S. fresh whole milk of German import, purchased from supermarket, Shanghai.
1.2 method
1.2.1 sample preparation
Use refrigeration grinding machine (SPEX 6850) by the solid sample grinds powder in above-mentioned sample.For species specificity, detect.
1.2.2 DNA extraction
Use a day root Animal genome to extract test kit (purchased from Tian Gen biochemical technology company limited; Catalog number (Cat.No.): DP323) extract animal specimen DNA, day root Plant Genome extraction test kit (purchased from Tian Gen biochemical technology company limited; Catalog number (Cat.No.): DP305) extract plants sample DNA, extracting method refers to test kit process specifications.With nucleic acid-protein concentration determination instrument (Eppendorf company), measure the concentration of DNA solution.DNA solution is placed in-20 ℃ and saves backup.
1.2.3 real-time fluorescence PCR detection, primer and probe
Through testing widely and comparing, the inventor finally determines the target sequence that utilizes ATPase 8 gene conserved sequences to differentiate as buffalo composition, design primer and probe, and primer and probe sequence are:
Upstream primer: 5 '-TTCATTGAYCTCCCTGCTCC-3 ' (SEQ ID NO:1);
Downstream primer: 5 '-GGAATAGGCCGGTGAGGATT-3 ' (SEQ ID NO:2);
Probe: 5 ' FAM-ACTTTGGCTCTCTCC-MGB 3 ' (SEQ ID NO:3).
Real-time fluorescence PCR detects and adopts ABITaqMan Universal PCR Master Mix enzyme premixed liquid (Part Number4304437).Cumulative volume is 25 μ L, containing 2 * Real Time PCR Buffer, and 100nmol/L primer, 100nmol/L probe, 50～100ng DNA profiling amount.
Pcr amplification condition: 50 ℃, 2min; 95 ℃, 10min; 95 ℃, 15s; 60 ℃, 60s, totally 40 circulations.Use ABI7300 real-time fluorescence PCR instrument to carry out qualitative detection.
1.2.4 the amplification of 18S rRNA gene PCR and detection
Use 18S rRNA gene amplification eukaryote native gene, the DNA being extracted to guarantee is suitable for pcr amplification.Detecting 18S rRNA gene primer sequence used is:
5 '-TCTGCCCTATCAACTTTCGATGGTA-3 ' (SEQ ID NO:4); With
5’-AATTTGCGCGCCTGCTGCCTTCCTT-3’(SEQ?ID?NO：5)。
PCR reaction system is: 1 * PCR damping fluid, 2.5mmol/L Mg
2+, 1U Taq enzyme, 200 μ mol/LdNTPs, primer 100nmol/L, template 50-100ng, reaction volume is 25 μ L.
Pcr amplification condition is: 94 ℃, and 3min; 94 ℃, 20s, 54 ℃, 40s, 72 ℃, 40s, 40 circulations.
II. embodiment
The detected result of embodiment 1, eukaryote special primer 18S rRNA primer
With the eukaryote 18S rRNA special primer all DNA solutions that this experiment extracts that increase, all can there is the specific amplified band of 137bp in all DNA solutions.Result shows, the DNA solution of all extractings is all suitable for PCR test.
The real-time fluorescence PCR specific detection of embodiment 2, buffalo composition
Use buffalo composition detection primer and probe to detect 45 kinds of animals and plants material DNA samples for examination.There is obvious amplification curve in the detected result of the milk sample of wherein in Murrah, Buddhist nun-La Fei buffalo, Murrah and Guangxi buffalo first cross buffalo, Murrah and Guangxi buffalo hybridization buffalo of low generation, Murrah and Guangxi buffalo assorted buffalo of high generation, and all without obvious, increase in other 39 species DNA samples, as Fig. 1.The summary of detected result is in Table 1.
Table 1, buffalo composition specific detection result
The above results explanation, detection method of the present invention has species specificity.
The real-time fluorescence PCR detection sensitivity of embodiment 3, buffalo composition
With 10 times of serial dilution buffalo DNA solutions of ordinary milk (the fresh whole milk of many U.S.s of German import, purchased from supermarket, Shanghai) DNA solution.Use real-time fluorescent PCR testing primer and probe to carry out real-time fluorescence PCR test to 10ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L, 0.001ng/ μ L and 0.0001ng/ μ L buffalo milk sample DNA solution.Experiment repeats 6 times.
In 6 times are detected, in 10ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L, 0.001ng/ μ L buffalo DNA solution, all occur amplification curve, and there is not amplification curve in 0.0001ng/ μ L buffalo milk sample DNA, as Fig. 2.
Experiment shows, in the level of DNA concentration, the detection sensitivity of method of the present invention is 0.001ng/ μ L buffalo milk sample DNA.
With ordinary milk (the fresh whole milk of many U.S.s of German import, purchased from supermarket, Shanghai) the buffalo milk sample of 10 times of serial mixed preparing different contents, obtain respectively the sample of 10%, 1%, 0.1%, 0.01%, 0.001% and 0.0001% buffalo milk content (W/W), extract the DNA of each sample.Use real-time fluorescent PCR testing primer and probe to detect DNA.In 12 times are detected, all there is amplification curve in the DNA of 10%, 1%, 0.1%, 0.01% buffalo milk sample extraction, and amplification curve does not appear in the DNA of 0.001% and 0.0001% buffalo milk sample extraction, as Fig. 3.
The above results shows, in weight percent levels, the detection sensitivity of the method is 0.01% buffalo milk (W/W).
The application of buffalo composition detection in embodiment 4, food
Adopt above-mentioned real time fluorescent PCR method, on market, the sample such as the foreign trade approach buffalo of collecting and Cattle, yak detects.
Result, at 12 parts of import buffalo milk samples (the outstanding thunder Sa board buffalo horse of the 6 batches of Ai Baisi board water logging Mo Zeruila cheese, the 1 batch of horse Soviet Union lira buffalo cheese, a 1 batch Soviet Union lira cheese, 2 parts very swing a license support horse Soviet Union lira buffalo cheese, 2 batches of buffalo milk horses Soviet Union lira cheese) and 3 parts of domestic buffalo milk samples, pcr amplification is positive, contains buffalo composition.In 3 parts of buffalo meat samples, detect buffalo composition.At 50 parts of other food (food labelling does not mark buffalo composition) such as 5 parts of ordinary milks, 5 parts, ordinary milk junket, 2 parts, butter, 6 parts of fresh beef (Carnis Bovis seu Bubali), 2 parts of dried beef, 2 parts of dried yak beefs, biscuit, jam, beverage etc., all do not present the pcr amplification positive, do not detect buffalo composition.In 10 parts of beef bone meal, 20 parts of other feeds, do not detect buffalo composition.The result of further checking and verify according to the source of above-mentioned sample segment conforms to the detected result of method of the present invention.
The above results is visible, and the present invention has found the specific primer of buffalo, can distinguish well buffalo composition and become to grade with Cattle composition, yak, also can distinguish well buffalo composition and other various food, forage component.And, primer of the present invention for be equal conservative region in the buffalo genome in various sources, it can detect the buffalo of various different sourcess.
Conclusion
The present invention utilizes specific primer and probe to carry out buffalo composition real-time fluorescence PCR in food and feed and detects, and method has specificity.The method DNA concentration sensitivity is 0.001ng/ μ L, and weight sensitivity is 0.01%.The experiment proved that, the method is simple to operate fast, can accurately detect the buffalo composition in food and feed, and provides technology platform for other components in qualitative and quantitative detection milk-product.
All documents of mentioning in the present invention are all quoted as a reference in this application, just as each piece of document, are quoted as a reference separately.In addition should be understood that 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 limited range equally.
Claims (8)
1. a method of identifying buffalo composition, is characterized in that, described method comprises:
The DNA of testing sample of take is template, with the primer shown in SEQ ID NO:1 and SEQ ID NO:2, carries out pcr amplification; If generation specific amplification, shows to comprise in testing sample buffalo composition.
2. the method for claim 1, is characterized in that, with the primer shown in SEQ ID NO:1 and SEQ ID NO:2 and the Taqman probe shown in SEQ ID NO:3, carries out real-time fluorescence PCR detection.
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
-3ng/ μ L DNA.
5. a test kit of identifying buffalo composition, is characterized in that, comprising:
Primer pair, its sequence is as shown in SEQ ID NO:1 and SEQ ID NO:2; With
Taqman probe, described probe sequence is as shown in SEQ ID NO:3.
6. test kit as claimed in claim 5, is characterized in that, in described test kit, also comprises: the examination criteria product that contain buffalo composition.
7. test kit as claimed in claim 5, is characterized in that, also comprises and be selected from following reagent in described test kit: DNA extraction reagent, and Taq enzyme, PCR damping fluid, archaeal dna polymerase, and/or the working instructions of the method for buffalo composition are identified in explanation.
8. the purposes of test kit claimed in claim 5, for identifying buffalo composition from testing sample.
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| CN105296476B (en) * | 2015-11-20 | 2018-11-02 | 华中农业大学 | Buffalo specific primer, kit and its application in the identification of buffalo derived component |
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