CN103602732B - Primers and probes used for detecting ingredients of racoon dog origin, and detection method of ingredients of racoon dog origin - Google Patents
Primers and probes used for detecting ingredients of racoon dog origin, and detection method of ingredients of racoon dog origin Download PDFInfo
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Abstract
The invention relates to qualitative detection methods of ingredients of animal origin in food, forage, fur, and products of food, forage and fur, and more specifically relates to primers and probes used for detecting ingredients of racoon dog origin, and a detection method of the ingredients of racoon dog origin. According to the detection method, gene sequences of mitochondrion cytochrome oxidase b are selected, a set of specific primers and probes is designed, and the specific primers and probes are used for real-time fluorescent PCR, so that simple, convenient and fast detection of the ingredients of racoon dog origin in food, forage, fur, and the products of food, forage and fur is realized.
Description
Technical field
The present invention relates to the detection of animal derived materials, be specifically related to a kind of racoon dog derived component detection primer and probe and detection method thereof.
Background technology
The cultivation scale of racoon dog constantly expands, the fur goods of racoon dog has market very much, simultaneously, the cultivation amount of racoon dog is very large, the single individuality only of racoon dog is large, and as the by product of racoon dog fur industry, racoon dog meat is often added in food or feed, therefore, provide a kind of method that quick and precisely can detect racoon dog derived component very necessary.Heritage official method qualification racoon dog derived component, judge according to color and luster, smell, tissue characteristics etc., easily affect by subjective factor, and need the accumulation of experience, especially with the addition of pigment, perfume compound and be subject to the goods of deep processing, further increase the risk of qualification difficulty and monitoring and supervising.At present, still do not carry out to racoon dog derived component the detection method differentiating qualification from gene level, in fur goods, food and feeds, racoon dog derived component exists blank on detecting and applying.Based on genetic material DNA real-time fluorescence PCR technology, successful Application is in some common domestic animals and fowls species, and as ox, horse, chicken etc., technical qualification are ripe.Species special gene sequence, for one section is only had genetic information specific to target detect living species, by finding the genetic sequence of comparison racoon dog and other species, search the specific gene of racoon dog, obtain genomic DNA sequence information, design primer, probe on this basis, the sample in different plant species source can be distinguished, make the sample containing racoon dog derived component produce specific amplified fluorescent signal, thus racoon dog derived component is identified.
Summary of the invention
The object of the present invention is to provide a kind of racoon dog derived component detection primer and probe and detection method thereof.
Object of the present invention is achieved through the following technical solutions:
A kind of racoon dog derived component detection primer and probe:
Upstream primer sequence is: 5 '-TACAGCTGATCTCCTCACGTTAACA-3 ';
Downstream primer sequence is: 5 '-TTGGCCGATGATGATGAAAG-3 ';
Probe sequence is: 5 '-AATTGCAGGCCAACCGGTCGAAC-3 ', its 5 ' end flag F AM reporter fluorescence group, 3 ' end mark TAMRA quenching fluorescence group.
Apply the method that above-mentioned racoon dog derived component detection primer and probe carry out the detection of racoon dog derived component, comprise the steps:
(1) sample DNA is extracted: get liposuction and to dry and the sample 100mg grinding to form fine powder puts into 2mL centrifuge tube, add CTAB extracting solution 600 μ L and proteolytic enzyme 10 μ L, 65 DEG C of heating in water bath 1 hour, add the saturated phenol of Tris respectively, the each 400 μ L of chloroform isoamyl alcohol shake up respectively, centrifuging and taking supernatant adds in 1.5mL centrifuge tube, add the CTAB precipitated liquid of this supernatant amount 2 times of volumes, room temperature leaves standstill 1 hour, centrifugally abandon supernatant, first add sodium chloride aqueous solution 400 μ L and shake up dissolution precipitation, add 400 μ L chloroforms again to shake up, centrifuging and taking supernatant, add in new 1.5mL centrifuge tube, the Virahol adding supernatant amount 0.8 times of volume in this centrifuge tube shakes up standing 1 hour, centrifugally abandon supernatant, add 70% ethanol wash of 1000 μ L, centrifugal, leave standstill and dry, be dissolved in 100 μ L water,
(2) real-time fluorescence PCR detection is carried out: the reaction system that real-time fluorescence PCR detects is 25 μ L: comprise 12.5 μ L and contain dNTP, Mg
2+and 2 × real time PCR Buffer of Taq enzyme; Described upstream primer and each 1 μ L of downstream primer, final concentration is 0.2 μM; Described probe 1 μ L, final concentration is 0.2 μM; The sample DNA template 2 μ L that step (1) is extracted; Pure water 7.5 μ L; The response procedures that real-time fluorescence PCR detects is: denaturation: temperature 95 DEG C, 10 minutes time, then carries out pcr amplification: temperature 95 DEG C, 15 seconds time, 1 minute temperature 60 C time, circulates 45 times.
Beneficial effect of the present invention is: the primer and the probe that are designed for real-time fluorescence PCR detection according to the species special gene sequence of racoon dog, sample pre-treatments, PCR amplification system condition etc. are optimized simultaneously, provide a kind of real-time fluorescence PCR detection method of racoon dog derived component, can accurately, Rapid identification racoon dog derived component, effectively can resist personation animal derived materials product adulterated, the qualification of food, feed and fur series products has great practical value.Simultaneously also by the method, its composition is identified for some disease propagated through animal racoon dog, and control it and pass in and out, prevent the propagation of these disease pathogens and spread.
The present invention improves sample pre-treatments DNA extraction method by optimization, and effectively can reduce the impurity interference in extracted DNA, DNA purity is higher, improves the sensitivity of detection, more more economical than business-like test kit.The PCR amplification system taked for primer and probe sequence also ensure that specificity and the sensitivity of detection.
The present invention is directed to the PCR Molecular Identification technology of the special genetic material DNA of species of racoon dog, have the advantages that specificity is high, with strong points, highly sensitive, easy fast, low to detection sample requirement, accurately objectively can provide experimental evidence.
Accompanying drawing explanation
Fig. 1 is racoon dog derived component real-time fluorescence PCR detection method specificity experiments figure;
Fig. 2 is racoon dog derived component real-time fluorescence PCR detection method sensitivity experiments figure.
Embodiment
The present invention will be further described in conjunction with the embodiments.
Embodiment:
(1), the design of real-time fluorescence PCR detection method Auele Specific Primer
Download the various animal mitochondria gene orders announced in GenBank, by DNAMAN software compare of analysis, the mitochondrial cytochrome oxydase b gene sequences Design choosing racoon dog goes out to differentiate that the fluorescent PCR Auele Specific Primer of detection racoon dog derived component and probe (can only amplify racoon dog DNA restriction fragment, and other animal DNA restriction fragment that can not increase), primer and probe are distinguished as follows:
Upstream primer sequence: 5 '-TACAGCTGATCTCCTCACGTTAACA-3 ';
Downstream primer sequence: 5 '-TTGGCCGATGATGATGAAAG-3 ';
Probe sequence: 5 ' (FAM)-AATTGCAGGCCAACCGGTCGAAC-3 ' (TAMRA).
(2), the extraction of DNA
(2.1) sample thief grinds to form rotten shape or shape in small, broken bits through shredder, is placed in thermostatic drying chamber and dries, and the sample that the lipid contents such as meat are high can wrap up aseptic filter paper and absorb excess oil;
(2.2) dry sample and grind to form even finely powdered through shredder;
(2.3) take above-mentioned finely powdered sample 100mg, add CTAB extracting solution 600 μ L and proteolytic enzyme 10 μ L, 65 DEG C of heating in water bath 1 hour;
(2.4) add the saturated phenol of Tris respectively, each 400 μ L of chloroform isoamyl alcohol shake up respectively, centrifuging and taking supernatant adds in 1.5ml centrifuge tube;
(2.5) add the CTAB precipitated liquid of 2 times of volumes, room temperature leaves standstill 1 hour, centrifugally abandons supernatant;
(2.6) first add sodium chloride aqueous solution 400 μ L and shake up dissolution precipitation, then add 400 μ L chloroforms and shake up, centrifuging and taking supernatant, adds in new 1.5mL centrifuge tube;
(2.7) Virahol adding 0.8 times of volume shakes up standing 1 hour, centrifugally abandons supernatant;
(2.8) add 70% ethanol wash of 1000 μ L, centrifugal leaving standstill is dried, and is dissolved in 100 μ L water.
Extract genomic dna and all measure its purity and concentration through ultraviolet spectrophotometer.Measure OD260/OD280 value and be that about 1.8, DNA purity is higher meets PCR amplification requirement.Above DNA is extracted as optimization improved method of CTAB, and DNA purity is high, and amount is large, and DNA fragmentation is more complete, and high-quality DNA extraction method is very important for the follow-up PCR sensitivity of raising.
(3), racoon dog derived component real-time fluorescence PCR detects
Utilize Auele Specific Primer and the probe of above-mentioned design, with the DNA extracted for template carries out PCR amplification.
(3.1) real-time fluorescence PCR reaction system is 25 μ L, comprises 12.5 μ L 2 × real time PCR Buffer and (includes dNTP, Mg
2+and Taq enzyme), the upstream primer of design and each 1 μ L of downstream primer (final concentration is 0.2 μM), probe 1 μ L (final concentration is 0.2 μM), the product dna template 2 μ L of extraction, pure water 7.5 μ L;
(3.2) PCR response procedures is: denaturation: temperature 95 DEG C, 10 minutes time, then carries out pcr amplification: temperature 95 DEG C, 15 seconds time, 1 minute temperature 60 C time, circulates 45 times;
(3.3) apply quantitative fluorescent PCR instrument accompanying software, setting reaction parameter, reaction terminates rear preservation file, opens analysis software, analysis design mothod result.
There is positive amplification curve in sample, between general Ct value 14-20, negative control (other species DNA) and blank (water) without amplification or CT value and positive difference obvious, Ct value is greater than 30, positive control (racoon dog DNA) produces typical positive amplification curve, can judge that this sample detects racoon dog composition accordingly.
Explanation is verified in the specificity of present method and sensitivity.
(1) specificity verification of primer and probe
Utilize Auele Specific Primer and the probe of above-mentioned design, respectively with to the DNA of racoon dog, ermine, dog, chicken, duck, cat, pig, quail, fox, ox, sheep, donkey, rabbit, goose, horse animal for template, carry out real-time fluorescence PCR, the specificity of checking primer.Detected result as shown in Fig. 1, in Fig. 1: 1 is racoon dog; 2 is ermine; 3 is dog; 4 is chicken; 5 is duck; 6 is cat; 7 is pig; 8 is quail; Fox, ox, sheep, donkey, rabbit, goose, horse do not detect signal in addition.The positive amplification curve that indivedual species occur, but its signal CT value (>15) mutually far short of what is expected with racoon dog, show above-mentioned primer and probe has good specificity.
(2) the sensitivity checking of real-time fluorescence PCR
With ultrapure water 10 times of gradient dilution pure racoon dog meat sample DNA, until 10
-11, carry out real-time PCR detection, result is as Fig. 2.In Fig. 2: 1 is pure racoon dog meat DNA sample; 2-8 is respectively the sample with ultrapure water 10 times of gradient dilutions.Therefore detect bottom line and at least can reach 10
-7pure racoon dog meat sample DNA concentration, namely the sensitivity of this primer is 10
-7pure racoon dog meat sample.
Entry-Exit Inspection and Quarantine Bureau's inspection and quarantine technique center, <110> Qinhuangdao
<120> racoon dog derived component detection primer and probe and detection method thereof
<160>3
<210>1
<211>25
<212>DNA
<213> synthetic
<400>1
tacagctaatctcctcacgttaaca 25
<210>2
<211>20
<212>DNA
<213> synthetic
<400>2
ttggccgatgatgatgaaag 20
<210>3
<211>23
<212>DNA
<213> synthetic
<400>3
aattgcaggccaaccggtcgaac 23
Claims (2)
1. racoon dog derived component detection primer and a probe, is characterized in that:
Upstream primer sequence is: 5 '-TACAGCTGATCTCCTCACGTTAACA-3 ';
Downstream primer sequence is: 5 '-TTGGCCGATGATGATGAAAG-3 ';
Probe sequence is: 5 '-AATTGCAGGCCAACCGGTCGAAC-3 ', its 5 ' end flag F AM reporter fluorescence group, 3 ' end mark TAMRA quenching fluorescence group.
2. application rights requires that the racoon dog derived component detection primer described in 1 and probe carry out a method for racoon dog derived component detection, it is characterized in that comprising the steps:
(1) sample DNA is extracted: get liposuction and to dry and the sample 100mg grinding to form fine powder puts into 2mL centrifuge tube, add the Proteinase K 10 μ L of CTAB extracting solution 600 μ L and 20mg/mL, 65 DEG C of heating in water bath 1 hour, add the saturated phenol of Tris respectively, the each 400 μ L of chloroform isoamyl alcohol shake up respectively, centrifuging and taking supernatant adds in 1.5mL centrifuge tube, add the CTAB precipitated liquid of this supernatant amount 2 times of volumes, room temperature leaves standstill 1 hour, centrifugally abandon supernatant, first add sodium chloride aqueous solution 400 μ L and shake up dissolution precipitation, add 400 μ L chloroforms again to shake up, centrifuging and taking supernatant, add in new 1.5mL centrifuge tube, the Virahol adding supernatant amount 0.8 times of volume in this centrifuge tube shakes up standing 1 hour, centrifugally abandon supernatant, add 70% ethanol wash of 1000 μ L, centrifugal, leave standstill and dry, be dissolved in 100 μ L water,
(2) real-time fluorescence PCR detection is carried out: the reaction system that real-time fluorescence PCR detects is 25 μ L: comprise 12.5 μ L and contain dNTP, Mg
2+and 2 × real time PCR Buffer of Taq enzyme; Described upstream primer and each 1 μ L of downstream primer, final concentration is respectively 0.2 μM; Described probe 1 μ L, final concentration is 0.2 μM; The sample DNA template 2 μ L that step (1) is extracted; Pure water 7.5 μ L; The response procedures that real-time fluorescence PCR detects is: denaturation: temperature 95 DEG C, 10 minutes time, then carries out pcr amplification: temperature 95 DEG C, 15 seconds time, 1 minute temperature 60 C time, circulates 45 times.
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| CN104032012B (en) * | 2014-06-12 | 2016-03-16 | 中国动物疫病预防控制中心 | The PCR primer pair of qualification or assistant identification domesticated dog tissue and/or organ and application thereof |
| CN104611455A (en) * | 2015-03-02 | 2015-05-13 | 中国农业科学院特产研究所 | Specific primers for detecting raccoon dog-sourced species components and application thereof |
| CN107488707A (en) * | 2016-06-12 | 2017-12-19 | 中国检验检疫科学研究院 | The primed probe and method and kit precisely quantitatively detected for racoon dog derived component digital pcr |
| CN110117643A (en) * | 2018-02-07 | 2019-08-13 | 东北林业大学 | A kind of fluorescence PCR detecting method of the racoon dog fur products true and false |
| CN114657256B (en) * | 2020-12-23 | 2024-07-30 | 广东一方制药有限公司 | Primer combination for PCR identification of Bungarus Parvus medicinal material, standard decoction and traditional Chinese medicine formula particles, application thereof and identification method |
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