CN107475365A - Fluorescent PCR differentiates composition, kit and the method for sheep Niu Chengfen in sheep breast - Google Patents
Fluorescent PCR differentiates composition, kit and the method for sheep Niu Chengfen in sheep breast Download PDFInfo
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- CN107475365A CN107475365A CN201710531875.8A CN201710531875A CN107475365A CN 107475365 A CN107475365 A CN 107475365A CN 201710531875 A CN201710531875 A CN 201710531875A CN 107475365 A CN107475365 A CN 107475365A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The present invention relates to sheep in sheep breast（Sheep and goat）, goat and common mix pseudo- ox（Ox, yak, buffalo）The real time fluorescent PCR method of composition.The invention further relates to the Oligonucleolide primers probe compositions for methods described.The invention further relates to the real-time fluorescent PCR reagent case for including the composition.Real-time PCR detection is carried out using the composition of the present invention, simple, quick, special and delicately mammal sheep in the goat milk product such as detection liquid sheep breast, sheep milk powder can be passed through（Sheep and goat）, goat and common mix pseudo- ox（Ox, yak, buffalo）Derived component.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to qualitative detection sheep（Sheep and goat）、
Goat, ox（Ox, yak, buffalo）Composition Taqman real time fluorescent PCR methods, the antisense oligonucleotide primer physical prospecting for methods described
Injection composition, and include the kit of the composition.
Background technology
The nutritive value of goat milk product is high, and the correlative study of modern nutriology shows, sheep breast contains more than 200 kinds of nutriment
And bioactie agent, its protein, the total content of minerals and vitamins are above cow's milk.In sheep breast protein curd it is thin and
Soft, lipochondrion is smaller, absorption easy to digest, and human body is up to more than 94% to the absorptivity of sheep breast, is particularly suitable for infant, old age
People and person in poor health drink.Especially because in sheep breast dry matter content it is higher than cow's milk by about 10%, it is higher than breast milk by about 5%, from nutrition into
It is grouped into and foundation structure, each nutrient proportioning is all closest with breast milk to nutritive peculiarity, therefore in infant food
It is with the obvious advantage.Allergenic protein is free of in sheep breast simultaneously, many advantages, such as not being easily caused allergy, cow's milk is recommended as by nutritionist
The optimal replacement dairy products of allergic human population.Therefore, in recent years, the prevalence of sheep breast Related product is gradually into trend, in the market product kind
Class is more and more, and goat milk powder product, liquid goat milk product, fruity goat milk, goat cheese, goat milk piece are developed into by single fresh goat milk
And cosmetics goat milk fat etc..The production and processing of sheep breast is also increasingly fashionable in the international market with selling, goat milk product it is American-European with
The country such as Australia has turned into food essential in people's life, and its occupation rate of market is up to 80%.
However, milk goat is typical short-day seasonal breeding animal, 3 ~ October gives milk phase for it, and milk goat
Daily milk yield is far below cow's milk, only 5 kilograms or so, limits throughput, therefore sheep breast valency lattice are far above cow's milk.In economic interests
Under driving, some illegal retailers and enterprise mix cow's milk to reduce cost in sheep breast, seek exorbitant profit.Because cattle and sheep milk composition connects
Closely, property is similar, and it is difficult to be detected from sense organ and conventional index that a certain amount of cow's milk is mixed in sheep breast.This is not only related to
And the problems such as economy, nutritive value and food security, the health of consumer is more directly affected, while also upset the order in market
Sequence, constrain the normal development of goat milk industry.In order to ensure sheep breast and products thereof quality, it is ensured that manufacturer and consumer
Interests, it is more and more important to establish technical system that is easy, fast and accurately detecting newborn source in sheep breast.Real-time fluorescence PCR technology
Source of species is analyzed from gene level, sensitivity is higher, specificity is stronger, and its result provides very for the true and false of proof dairy products
Real, reliable foundation, the technology are convenient, accurate, rapid, it has also become the focus of recent domestic scholar's research.
The content of the invention
It is an object of the present invention to simply, quickly, specifically and delicately detect the sheep in goat milk product, goat and easily mix
Pseudo- Niu Chengfen, can effectively prevent from adulterating adulterated cow's milk in sheep milk product, or even the fraud mode of ox source property foreign protein.
For the present inventor according to lactation growth hormone gene GH (growth hormone gene), devising can
It is common to the real-time fluorescence PCR detection method of mammal internal reference；Simultaneously respectively using GH genes as target, devising can be special
Property detection sheep（Containing sheep, goat）, goat, ox（Containing ox, yak, buffalo）The primer and probe of composition, wherein mammal
Internal reference target sequence is 66 bp or so, and sheep target sequence is 79 bp, and the bp of goat target sequence 119, ox target sequence is 81 bp, is ensured
Target animal composition can be detected from the dairy products of different processing stages.
In one aspect of the invention, there is provided be common to the Oligonucleolide primers upstream of mammal composition internal reference
MGH-F: 5’- CTCAGCAGGGTCTTCACCAACA -3’（SEQ ID No.1）, and downstream MGH-R: 5’-
TGCCTTCCTCTAGGTCCTTCAGC -3’（SEQ ID No.2）.Probe is MGH-P: 5’-
TGGTGTTTGGCACCTCGGACCGT -3’（SEQ ID No.3）, a fluorescent quenching group is connected with 3 ' ends of probe
TAMRA, 5 ' ends are connected with a fluorescent reporter group FAM.The primer can specific recognition mammal composition MGH sequences,
The bp of expanding fragment length 82.Specificity extension self-increasing reaction condition is：50℃ 2 min；95 DEG C of min of pre-degeneration 10；95℃ 15
S, 60 DEG C of 1 min, 45 circulations.
In one aspect of the invention, specific detection sheep is additionally provided（Goat, sheep）The specific oligonucleotides of composition
Sour primer upstream SGH-F：5’- TGCCAGCCATCTGTTGTTACC -3’（SEQ ID No.4）, and downstream SGH-R：5’-
AAAGGACAGTGGGCACTGGAG -3’（SEQ ID No.5）.Probe is SGH-P：5’-
CCCGTGCCTTCCTAGACCCTGGAAG -3’（SEQ ID No.6）, a fluorescent quenching group is connected with 3 ' ends of probe
TAMRA, 5 ' ends are connected with a fluorescent reporter group FAM.The primer can specific recognition sheep（Goat, sheep）GH sequences,
The bp of expanding fragment length 79.Specificity extension self-increasing reaction condition is：50℃ 2 min；95 DEG C of min of pre-degeneration 10；95℃ 15
S, 60 DEG C of 1 min, 45 circulations.
In one aspect of the invention, the specific oligonucleotide primer upstream of specific detection goat composition is additionally provided
GGH-F：5’- GGAGGGAACTAAGGACCTCAGTG -3’（SEQ ID No.7）, and downstream GGH-R：5’-
GGTGTGTGGTTCCCCTCACTG -3’（SEQ ID No.8）.Probe is GGH-P：5’-
CCTTATTCGGAACCCTCCCCACCCCA -3’（SEQ ID No.9）, a fluorescent quenching base is connected with 3 ' ends of probe
Group TAMRA, 5 ' ends are connected with a fluorescent reporter group FAM.The primer can specific recognition goat GH sequences, amplified fragments
The bp of length 119.Specificity extension self-increasing reaction condition is：50℃ 2 min；95 DEG C of min of pre-degeneration 10；95 DEG C of 15 s, 60 DEG C 1
Min, 45 circulations.
In one aspect of the invention, specific detection ox is additionally provided（Ox, buffalo, yak）The specificity of composition is few
Nucleotide primer upstream BGH-F：5’- GTTGCCAGCCATCTGTTGTTTG -3’（SEQ ID No.10）, and downstream BGH-R：
5’- ATTAGGAAAGGACAGTGGGAGTGG -3’（SEQ ID No.11）.Probe is BGH-P：5’-
TCCCGTGCCTTCCTTGACCCTGG -3’（SEQ ID No.12）, a fluorescent quenching group is connected with 3 ' ends of probe
TAMRA, 5 ' ends are connected with a fluorescent reporter group FAM.The primer can specific recognition goat GH sequences, amplified fragments length
Spend 81 bp.Specificity extension self-increasing reaction condition is：50℃ 2 min；95 DEG C of min of pre-degeneration 10；95 DEG C of 15 s, 60 DEG C 1
Min, 40 circulations.
In another aspect of the present invention, there is provided include the composition of above-mentioned oligonucleotide sequence., the composition bag
Containing selected from following（1）Extremely（4）In any one or more groups of primer and probe sequences：
（1）Mammal internal reference gene Oligonucleolide primers are to SEQ ID No.1 ~ SEQ ID No.2 and probe SEQ ID
No.3 sequences；
（2）Sheep specific oligonucleotide primer pair SEQ ID No.4 and SEQ ID No.5 and probe SEQ ID No.6 sequences
Row；
（3）Goat specific oligonucleotide primer pair SEQ ID No.7 and SEQ ID No.8 and probe SEQ ID No.9
Sequence；
（4）Bovine Oligonucleolide primers are to SEQ ID No.10 and SEQ ID No.11 and probe SEQ ID No.12
Sequence.
In one embodiment, mammal internal reference, sheep, goat real-time fluorescent PCR amplification condition are 50 DEG C of 2 min；
95 DEG C of min of pre-degeneration 10；95 DEG C of 15 s, 60 DEG C of 1 min, 45 circulations.Ox real-time fluorescent PCR amplification condition is 50 DEG C 2
min；95 DEG C of min of pre-degeneration 10；95 DEG C of 15 s, 60 DEG C of 1 min, 40 circulations, it is preferably reality used in the present invention
When fluorescence PCR method be Taqman fluorescence probe method.
In the present invention, sheep is containing two category of goat and sheep, and ox is containing three ox, yak, buffalo kinds.
In another aspect of the present invention, there is provided for mammal composition detection kit, sheep composition real-time fluorescence
PCR specific detection agents box, goat composition real-time fluorescence PCR specific detection agents box, Niu Chengfen real-time fluorescence PCRs are special
Property detection kit, the oligonucleotide sequence or the composition are included in the kit.
Kit provided by the invention is used for real-time PCR detection mammal, sheep, goat, ox including the present invention
Composition specific primer and operation instructions.
In one embodiment, universal for mammals internal reference primer of the invention is respectively according to mammal GH genes
Conserved sequence designs.In one embodiment, the general internal reference amplification goat of lactation, sheep, ox are included in the kit
Target sequence is: CTCAGCAGAGTCTTCACCAACAGCCTGGTGTTTGGCACCTCGGACCGTGTCTATGAGAAGCTGAA
GGACCTGGAGGAAGGCA (SEQ No.13), in a preferred embodiment, in the operation instructions of the kit
Including to the description with then real-time PCR detection mammal internal reference universal primer and amplification condition.It is specific at one
Embodiment in, the of the invention kit for being used to detect mammal composition also includes reference substance.Preferably, reference substance bag
Include positive reference substance and negative controls.In one embodiment, negative control is aseptic double-distilled water.
In another embodiment, sheep specific primer of the invention is respectively according to sheep（Goat and sheep）GH sequences are set
Meter.In one embodiment, it is comprising sheep specific amplification target sequence in the kit: TGCCAGCCATCTGTTGTTA
CCCCTCCCCGTGCCTTCCTAGACCCTGGAAGGTGCCACTCCAGTGCCCACTGTCCT TTCC (SEQ No.14), one
In individual preferred embodiment, the operation instructions of the kit are included to special with then real-time PCR detection sheep
The description of property primer and amplification condition.In a specific embodiment, the kit for being used to detect sheep composition of the invention
Also include reference substance.Preferably, reference substance includes positive reference substance and negative controls.In one embodiment, it is negative right
According to for aseptic double-distilled water.
In another embodiment, goat specific primer of the invention is respectively according to goat GH sequences Designs.One
In individual embodiment, it is comprising goat specific amplification target sequence in the kit: CAATGGGAGGGAACTGAGGACCTC
AGTGGTATTTTATCCAAGTAAGGATGTGGTCAGGGGAGTAGAAATGGGGGTGTGTGGGGTGGGGAGGGTTCCGAATA
AGGCAGTGAGGGGAACCACACACCAGCTTAGACCCGGG (SEQ No.15), in a preferred embodiment, institute
Stating the operation instructions of kit is included to being retouched with then real-time PCR detection goat specific primer and amplification condition
State.In a specific embodiment, the kit for being used to detect goat composition of the invention also includes reference substance.It is preferred that
Ground, reference substance include positive reference substance and negative controls.In one embodiment, negative control is aseptic double-distilled water.
In another embodiment, bovine primer of the invention is respectively according to ox GH sequences Designs.In a reality
Apply in scheme, be comprising bovine amplification target sequence in the kit: GTTGCCAGCCATCTGTTGTTTGCCCCTCCC
CCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAAT (SEQ No.16), it is preferable at one
In embodiment, the operation instructions of the kit are included to then real-time PCR detection goat specific primer
With the description of amplification condition.In a specific embodiment, the kit for being used to detect goat composition of the invention also wraps
Include reference substance.Preferably, reference substance includes positive reference substance and negative controls.In one embodiment, negative control is
Aseptic double-distilled water.
In one embodiment, mammal in the real-time fluorescence PCR detection method（By taking ox as an example）Composition it is exhausted
10 pg/ μ L minimum to sensitivity, sheep（By taking goat as an example）The minimum 10 pg/ μ L of absolute sensitivity of composition, goat composition
Absolute sensitivity minimum 1 pg/ μ L, Niu Chengfen the minimum 10 pg/ μ L of absolute sensitivity.
In one embodiment, the minimum detectability of the real-time fluorescence PCR detection method is volume ratio 1% in sheep breast
Niu Chengfen, including skimmed milk, nougat and bovine whey.
Again on the one hand, the invention provides the composition or the kit in sheep breast and its product is differentiated sheep,
Goat, Niu Chengfen application.
Real-time fluorescence quantitative PCR adds the probe or fluorescence dye of fluorescence labeling i.e. on the basis of conventional PCR method
Material, with the accumulation of PCR primer, the fluorescence signal that probe or dyestuff are sent strengthens, and fluorescence monitoring system can receive fluorescence
Signal, i.e., a DNA is often produced, just has a fluorescence molecule to be formed, whole PCR courses of reaction are with the increasing of cycle-index
Add, exponentially rule increases the target gene fragment being amplified, and corresponding changes fluorescence with amplification by detecting in real time
Signal intensity, try to achieve Ct (cycle threshold, Ct) value.The fluorescence letter of amplified production in Ct values, i.e. PCR amplification procedures
Number reach the amplification cycles number passed through during the threshold value of setting, the logarithm of it and the starting copy number of template has linear close
System, template DNA amount is more, and fluorescence is fewer up to the period of threshold value, i.e. Ct values are smaller, quantitative to starting template and fixed so as to realize
Property analysis, so as to can detect composition to be measured.
The present invention's differentiates that the real-time fluorescence PCR of template detects by primer or probe and the specific hybrid of template
Method specificity is high, and false positive is low；Complete stopped pipe can be used to detect, be post-processed without PCR, avoid cross pollution and vacation sun
Property, shorten the reaction time.The fluorescence after PCR amplifications is detected, is exaggerated reaction signal, sensitivity greatly improves.The present invention's
Method dexterously used the DNA efficient amplifications of PCR technologies, the specificity of nucleic acid hybridization and detection technique of fluorescence quick and
Sensitiveness, there are simple to operate, time saving and energy saving, reliable results and accurate sensitive.Use the real-time fluorescence PCR of the present invention
Detection method, the characteristics of its is simple, quick, special and sensitive, are suitable on domestic and international market sheep, mountain in sheep milk and milk productses
Sheep, Niu Chengfen qualitative detection.
Brief description of the drawings
Fig. 1 is the typical case that mammal occurs when utilizing real-time PCR detection mammal internal reference primed probe versatility
Amplification curve.1-16 is respectively：Pig, horse, donkey, camel, red deer, goat, sheep, ox, buffalo, yak, dog, rabbit, fox,
Mink, roe deer, rat, 17-22 are negative control and blank control, are sequentially：Chicken, duck, quail, turkey, fish, sterilized water, each
2, sample is parallel.
Fig. 2 is sheep primed probe specificity real-time PCR detection result.1 is goat in figure, and 2 be sheep, and 3-22 is successively
For pig, horse, donkey, camel, red deer, ox, buffalo, yak, dog, rabbit, fox, mink, roe deer, rat, chicken, duck, quail, turkey,
Fish, sterilized water, each 2, sample are parallel.
Fig. 3 is goat primed probe specificity real-time PCR detection result.1 is goat in figure, and 2-22 is followed successively by silk floss
Sheep, pig, horse, donkey, camel, red deer, ox, buffalo, yak, dog, rabbit, fox, mink, roe deer, rat, chicken, duck, quail, turkey,
Fish, sterilized water, each 2, sample are parallel.
Fig. 4 is ox primed probe specificity real-time PCR detection result.1 is ox in figure, and 2 be yak, and 3 be buffalo,
4-22 is followed successively by pig, horse, donkey, camel, red deer, goat, sheep, dog, rabbit, fox, mink, roe deer, rat, chicken, duck, quail, fire
Chicken, fish, sterile, each 2, sample is parallel.
Fig. 5 is mammal internal reference primed probe real-time fluorescence PCR absolute sensitivity testing result（Using ox DNA as mould
Plate）.1-5 represents that DNA concentration is 100 ng/ μ L, 10 ng/ μ L, 1 ng/ μ L, 0.1 ng/ μ L, 0.01ng/ μ L respectively, each dilute
Degree of releasing 3 is parallel.
Fig. 6 is sheep primed probe real-time fluorescence PCR absolute sensitivity testing result, and wherein Fig. 6 A are using goat DNA as mould
The amplification curve that plate obtains, Fig. 6 B are the amplification curves obtained using sheep DNA as template.1-6 represents that DNA concentration is 100 respectively
Ng/ μ L, 10 ng/ μ L, 1 ng/ μ L, 0.1 ng/ μ L, 0.01ng/ μ L, 0.001ng/ μ L, each dilution factor 3 are parallel.
Fig. 7 is goat primed probe real-time fluorescence PCR absolute sensitivity testing result.1-5 represents that DNA concentration is respectively
100 ng/ μ L, 10 ng/ μ L, 1 ng/ μ L, 0.1 ng/ μ L, 0.01ng/ μ L, each dilution factor 3 are parallel.
Fig. 8 is ox primed probe real-time fluorescence PCR absolute sensitivity testing result.1-5 represents that DNA concentration is 100 respectively
Ng/ μ L, 10 ng/ μ L, 1 ng/ μ L, 0.1 ng/ μ L, 0.01ng/ μ L, each dilution factor 3 are parallel.
Fig. 9 is the relative sensitivity to doping milk powder ox composition detection in sheep milk powder using ox primed probe.1-5 distinguishes
It is 0.1%, 1%, 10%, 50% to represent volume ratio, ox skimmed milk powder/goat skimmed milk powder aggregate sample of 100% ratio mixing
Product.
Figure 10 is the relative sensitivity to addition nougat composition detection in sheep milk powder using ox primed probe.1-5 distinguishes
It is 0.1%, 1%, 10%, 50% to represent volume ratio, nougat/goat whole-fat milk powder biased sample of 100% ratio mixing.
Figure 11 is the relative sensitivity to addition bovine whey composition detection in sheep milk powder using ox primed probe.1-5 distinguishes
It is 0.1%, 1%, 10%, 50% to represent volume ratio, bovine whey/goat whey powder biased sample of 100% ratio mixing.
Embodiment
The present invention is further illustrated by way of embodiment, but the present invention is not limited only to following implementation
Example.
Embodiment 1
Although specific embodiments of the present invention are described, those skilled in the art will appreciate that not
The present invention can be variously changed and be modified on the premise of deviateing the scope or spirit of the invention.Thus, this invention is intended to
Cover to fall all these changes and modification in claims and its range of equivalency.
Claims (8)
1. the composition for being used as mammal internal reference by the detection of real-time fluorescence PCR method includes mammal internal reference few nucleosides
Sour primer pair SEQ ID No.1 ~ SEQ ID No.2 and probe SEQ ID No.3 sequences.
2. sheep composition is detected by real-time fluorescence PCR method（Sheep, goat）Composition, the composition includes sheep（Sheep, mountain
Sheep）Specific oligonucleotide primer pair SEQ ID No.4 and SEQ ID No.5 and probe SEQ ID No.6 sequences.
3. detecting the composition of goat composition by real-time fluorescence PCR method, the composition includes goat specific oligonucleotide
Primer pair SEQ ID No.7 and SEQ ID No.8 and probe SEQ ID No.9 sequences.
4. Niu Chengfen is detected by real-time fluorescence PCR method（Ox, yak, buffalo）Composition, the composition includes ox（It is yellow
Ox, yak, buffalo）Specific oligonucleotide primer pair SEQ ID No.10 and SEQ ID No.11 and probe SEQ ID
No.12 sequences.
5. according to the composition described in claim 1 and 4,3 ' ends of wherein specific probe are connected with a fluorescent quenching group
TAMRA, 5 ' ends are connected with a fluorescent reporter group FAM.
6. differentiate mammal, sheep in sheep breast for real-time fluorescence PCR detection method（Sheep, goat）, goat, ox（Ox, yak
Ox, buffalo）The kit of composition, the kit include the composition described in claim any one of 1-5.
7. mammal, sheep in sheep breast（Sheep, goat）, goat and ox（Ox, yak, buffalo）The real-time fluorescence that composition differentiates
PCR detection methods, methods described is including the use of described in the composition described in claim any one of 1-5 or claim and 6
Kit.
8. specific oligonucleotide primer pair and the probe lactation in milk and milk productses any one of claim 1-7 are moved
Thing, sheep（Sheep, goat）, goat and ox（Ox, yak, buffalo）Application in composition discriminating.
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| CN109825612A (en) * | 2019-04-11 | 2019-05-31 | 中国农业大学 | Bovine material quick detection kit and its application in a kind of food |
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Cited By (11)
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| CN108342495A (en) * | 2018-05-18 | 2018-07-31 | 锡林郭勒职业学院 | Sheep and goat source property synchronizes the primer and probe and kit of detection in meat products |
| CN108467895A (en) * | 2018-05-18 | 2018-08-31 | 锡林郭勒职业学院 | The primer and probe and kit of goat detection synchronous with milk cow source property in former milk |
| CN108467895B (en) * | 2018-05-18 | 2020-08-04 | 锡林郭勒职业学院 | Primer, probe and kit for synchronously detecting sources of goats and cows in raw milk |
| CN109868321A (en) * | 2019-04-01 | 2019-06-11 | 陕西科技大学 | Identify the multiple fluorescence PCR method and kit of animal derived materials |
| CN109868322A (en) * | 2019-04-01 | 2019-06-11 | 陕西科技大学 | Identify the high-resolution melting method and kit of animal derived materials |
| CN109868321B (en) * | 2019-04-01 | 2023-03-17 | 陕西科技大学 | Multiple fluorescence PCR method and kit for identifying animal-derived components |
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