CN102634581B - Fluorescent quantitative PCR (Polymerase Chain Reaction) qualitative detection method for yak-derived fiber composition - Google Patents
Fluorescent quantitative PCR (Polymerase Chain Reaction) qualitative detection method for yak-derived fiber composition Download PDFInfo
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Abstract
The invention discloses a fluorescent quantitative PCR (Polymerase Chain Reaction) qualitative detection method for yak-derived fiber composition. The fluorescent quantitative PCR qualitative detection method comprises the following steps of: performing PCR amplification with the DNA (Deoxyribonucleic Acid) of yakwool as a template, and detecting fluorescent signals of amplification products, wherein a reaction system for the PCR amplification contains a specific primer pair for amplification of yak-derived fiber composition and specific probes for the yak-derived fiber composition. The fluorescent quantitative PCR qualitative detection method can be used for easily and quickly detecting and identifying whether the yak-derived fiber composition exists in textiles or not; and due to the good specificity and excellent sensitivity of the designed specific primer pairs and specific probes, the method can be used for detecting yakwool with proportion being 1%(wt).
Description
Technical field
The invention belongs to bionic technical field, specifically, is yak source property fibre composition quantitative fluorescent PCR qualitative checking method.
Background technology
Yak is a kind of Mammals that can survive under the severe environment of highland and severe cold, is also the unique member who has weaving utility value of bovid.Yak is divided into Bos mutus and family yak, and Bos mutus cries again wild ox, formal name used at school Bos mutus (Po ě phagrt mutrs), and English name wild yak, hides name transliteration Asia and returns.Yak Growth is at the extremely frigid zones of 3000 meters～5000m of height above sea level, and the severe cold that ability is subzero 30 ℃～40 ℃, is assembling fine and closely woven fine hair under the thick long batt of yak, and these fine hair provide for yak survives under high and cold weather conditions may.More than 1,400 ten thousand of the existing yaks in the whole world, are mostly distributed in China Qinghai-Tibet Platean and extremely frigid zones more than height above sea level 3000m around thereof.China is the maximum country of yak number in the world, accounts for 90% of whole world sum.
Yakwool is very similar in appearance to purple cashmere, and as shown in Figure 1, Fig. 1 is purple cashmere (left side) under microscope and the photo of yakwool (right side).Both all there is under the microscope pigment group, brought difficulty to discriminating work.Because itself is with pigment, purple goat and yakwool can be made into dark fabric conventionally, and just further to detection and identification, work has increased difficulty for this.Some lawless persons utilize this similarity, use yakwool as purple cashmere and lead into a trap to human consumer, get some advantage from it.
The standard GB/T 16988-1997 < < special animal fiber that China is existing and the description of the mensuration > > of sheep's wool mixture content to yakwool: yakwool fiber scale subtended angle is less, scale density is 100-130/mm, and hair color is black, brown.And under general opticmicroscope, scale is not very clear, detect to get up to waste time and energy.Electron microscopy cost is high, and the relatively more tired rope of sample preparation.
Summary of the invention
The present invention is a kind of qualitative identification method of the yakwool fiber based on DNA detection technology, overcome under existing Microscopy that scale is unintelligible, the shortcoming that wastes time and energy and; And it is high to have overcome Electronic Speculum inspection cost, and the loaded down with trivial details deficiency of sample preparation.
The object of the invention is to, a kind of yak source property fibre composition quantitative fluorescent PCR qualitative checking method is provided.
Another object of the present invention is to, the detection kit of a kind of yak source property fibre composition is provided, and the purposes of test kit.
A further object of the present invention is, primer and the probe of a kind of yak source property fibre composition quantitative fluorescent PCR is provided.
The technical problem that will solve required for the present invention, can be achieved through the following technical solutions:
As a first aspect of the present invention, a kind of yak source property fibre composition quantitative fluorescent PCR qualitative checking method, comprises the following steps:
The fluorescent signal of step 2, detection amplified production;
It is characterized in that, for the reaction system of pcr amplification contain amplification yak source property fibre composition Auele Specific Primer to yak source property fibre composition specific probe.
Wherein, property fibre composition Auele Specific Primer right sequence in described amplification yak source is as shown in SEQ ID NO:1 and SEQ ID NO:2.
Wherein, the nucleotide sequence of described yak source property fibre composition specific probe is as shown in SEQ ID NO:3.
Wherein, described method also comprises step: in the process of polymerase chain reaction or afterwards, detect the fluorescent signal that yak source property fibre composition specific probe sends.
Wherein, the condition of described pcr amplification is as follows:
Reaction system: ABI Taqman universal PCR master mix (2 *) 10 μ l, DNA profiling 4 μ l, primer 0.6 μ l, probe 2.5 μ l, ddH
2o 4.9 μ l;
Reaction conditions: 95 ℃, 5min; 95 ℃, 15s, 60 ℃, 45s, 40 circulations.
The concentration of primer is 0.3 μ M, and the concentration of probe is 0.25 μ M.
As a second aspect of the present invention, the detection kit of a kind of yak source property fibre composition, contains following reagent:
(a) amplification yak source property fibre composition Auele Specific Primer pair;
(b) yak source property fibre composition specific probe.
Further, described test kit also comprises:
(c) marker.
Wherein, described marker is yak source property fibre composition DNA, or the plasmid DNA that contains yak source property fibre composition amplification object fragment, or yakwool.
Wherein, the right sequence of described Auele Specific Primer is as shown in SEQ ID NO:1 and SEQ ID NO:2.
Wherein, the nucleotide sequence of described specific probe is as shown in SEQ ID NO:3.
Wherein, the fluorescent signal that described yak source property fibre composition specific probe sends.
As a third aspect of the present invention, the application of the detection kit of a kind of yak source property fibre composition, for detection of whether there is yak source property fibre composition in textiles.
As a fourth aspect of the present invention, Auele Specific Primer and the specific probe of a kind of yak as claimed in claim 1 source property fibre composition quantitative fluorescent PCR, is characterized in that:
The sequence of described Auele Specific Primer is as shown in SEQ ID NO:1 and SEQ ID NO:2;
The sequence of described specific probe is as shown in SEQ ID NO:3.
As a fifth aspect of the present invention, the Auele Specific Primer of yak source property fibre composition quantitative fluorescent PCR and a purposes for specific probe, described Auele Specific Primer and specific probe are for the preparation of detecting the test kit that whether has yak source property fibre composition in textiles.
Beneficial effect of the present invention:
1, can detect easy, fast and identify in textiles, whether have yak source property fibre composition.
2, the present invention is based on the sequence of yak mtDNA 12S rRNA gene, the specificity of design Auele Specific Primer and specific probe is good.
3, detection method sensitivity is good, the yakwool that proportioning is 1% (wt) can be detected.
Accompanying drawing explanation
Fig. 1: the purple cashmere (left side) under microscope and yakwool (right side).
Fig. 2: reaction system optimization result.
Fig. 3: the blast result of yak primer probe sequence and goat sequence.
Fig. 4: the blast result of yak primer probe sequence and sheep sequence.
Fig. 5: the real-time fluorescence PCR result of yak composition.
Fig. 6: the detection case after 10 times of gradient dilutions.
Fig. 7: the linear relationship of 10 times of gradient dilutions.
Fig. 8: the yakwool detection case of different proportionings.
Fig. 9: the doubtful cashmere sample that contains yakwool.
Figure 10: the detected result of sample A.
Figure 11: the detected result of sample B.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following examples are only for the present invention is described but not for limiting scope of the present invention.
The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, as < < molecular cloning: the condition that the conditioned disjunction manufacturer described in laboratory manual > > (New York:Cold Spring Harbor Laboratory Press, 1989) provides is carried out.
Related " % " in following examples, except special instruction, is all weight percentage.
1, primer and the probe design of the amplification of yak composition real-time fluorescence PCR
Relatively the sequence of goat, sheep, yak mtDNA 12S rRNA gene, chooses suitable sequence and carries out design of primers.Yak source property fibre composition primer amplified product size is 65bp.
The sequence of Auele Specific Primer and probe is as follows:
YakF：5’-AAGCATCTACACCCCAGTGAGAAT-3’(SEQ ID NO：1)
YakR：5’-GCTTGATGCCAGCTCCTCTT-3’(SEQ ID NO：2)
Pyak：FAM-CCCTCTAGGTTGTTAAAAT-MGB(SEQ ID NO：3)
Designed yak, goat, the total universal primer of sheep, universal primer amplified production size is 88bp simultaneously, and the sequence of universal primer and probe is as follows:
GsyF：5’-GGGTTTACGACCTCGATGTTG-3’(SEQ ID NO：4)
GsyR：5’-ACGTAGGACTTTAATCGTTGAACAAA-3’(SEQ ID NO：5)
Pgsy：FAM-ACCGCTATCAAAGGTT-MGB(SEQ ID NO：6)
Wherein, FAM represents fluorescence report group, and MGB represents quenching group.The present invention adopts fluorescent probe method, and it detects principle is to utilize fluorescent mark specific probe to carry out recognition template.Compare with SYBR dye method in prior art, the specificity of fluorescent mark specific probe of the present invention is stronger.
2, the optimization of the reaction system of yak composition
Get yakwool 5mg and use Progema hair extraction agent box extracting DNA, 50 μ l elutriant eluted dnas, get 4 μ l sample detection.
Reaction system: except ABI Taqman universal PCR master mix (2 *) 10 μ l, beyond DNA profiling 4 μ l, other compositions are as shown in table 1.
The concentration of primer is 0.3 μ M, and the concentration of probe is 0.25 μ M.
The condition of reaction is: 95 ℃, and 5min; 95 ℃, 15s, 60 ℃, 45s, 40 circulations.
The consumption of other compositions in table 1, optimizing reaction system
| System | Primer | Probe | ddH 2O |
| 1 | 0.4 | 1 | 6.6 |
| 2 | 0.4 | 2 | 5.6 |
| 3 | 0.6 | 1 | 6.4 |
| 4 | 0.6 | 2 | 5.4 |
| 5 | 0.6 | 2.5 | 4.9 |
Optimum result as shown in Figure 2.Consider fluorescence signal intensity, the factors such as Ct value, select system 5 for the real-time fluorescence PCR reaction system of amplification yak composition, be reaction system 5 (composition consumption ABITaqman universal PCR master mix (2 *) 10 μ l, DNA profiling 4 μ l, primer 0.6 μ l, probe 2.5 μ l, ddH
2o 4.9 μ l).
3, the primer of yak composition real-time fluorescence PCR amplification and probe specificity comparison and checking
3.1, the primer of yak composition real-time fluorescence PCR amplification and probe specificity comparison
The mtDNA 12S rRNA of yak and the similarity of goat are 89%, with the similarity of sheep be 87%.The situation of comparing of primer and probe and corresponding position is as Fig. 3 and Fig. 4:
The blast result of yak primer probe sequence and goat sequence, is shown in Fig. 3.
The blast result of yak primer probe sequence and sheep sequence, is shown in Fig. 4.
3.2, primer and the probe specificity experimental verification of the amplification of yak composition real-time fluorescence PCR
Get respectively yakwool, cashmere, continuous mountain hair 5mg, with progema hair extraction agent box extracting DNA, 70 μ l elutriant eluted dnas.The concentration of primer is 10 μ M, and the concentration of probe is 2 μ M.Respectively with ddH
2o, yakwool, cashmere, continuous mountain hair DNA are that template is carried out fluorescent PCR amplification.Annealing temperature is 60 ℃ of 45s.Reaction system is as follows, referring to table 2:
Table 2, yak primer probe specificity confirmatory experiment reaction system
The real-time fluorescence PCR result of yak composition, is shown in Fig. 5.
4, sensitivity detects
By the reaction system of having optimized, carry out the mensuration of sensitivity.By 10 times of gradient dilutions of the DNA of extracting gained, can detect 10
4the yak DNA doubly diluting.To easily obscure simultaneously, the indistinguishable yakwool of microscopy and purple cashmere mix with different proportionings, wherein the content of yakwool (weight percent) is respectively 100%, 10%, 1%, experimental results show that it is 1% yakwool that the method can detect proportioning.Result is as Fig. 6-Fig. 8, and Fig. 6 is 10 times of detection case after gradient dilution, and Fig. 7 is the linear relationship of 10 times of gradient dilutions, the yakwool detection case that Fig. 8 is different proportionings.
Fig. 7 shows, in weight percent levels, the linear relationship between Ct value and template copy number is good, can detect yak source property fibre composition DNA concentration.
5, detection in kind
Have in two doubtful cashmere samples that contain yakwool.
A sample is the two-layer hair of dark-grey light gray woolen material, and it is 100% cashmere product that client applies for checking and examination.Through microscopy, detect, A sample is containing cashmere 86.8%, yakwool 9.0%, wool 4.2%.
B sample is containing cashmere 78.1%, yakwool 19.1%, wool 2.8%.The apparent See Figure 9 of sample.
Respectively get A sample, B sample one fritter, shred and mix, accurate weighing 5mg is used Progema test kit extracting DNA.Two Duplicate Samples of each sample.Because of color sample darker, eluted dna repeatedly.Get 4 μ l sample detection, make Gsy simultaneously and detect, as positive control.
As shown in Figure 10 and table 3, the detected value of the Yak of A sample is respectively 25.55,24.97, and it is 22.65 and 22.11 that the Gsy of the DNA of respective amount detects.
As shown in Figure 11 and table 4, the detected value of black sample B is all bigger than normal.Black sample B may be subject to the impact of dyestuff.Certainly sample A contains yakwool, infers that sample B also contains yakwool.
The detected result of table 3, sample A
The detected result of table 4, sample B
According to above enlightenment of the present invention, those skilled in the art will readily understand, can utilize (a) amplification yak source property fibre composition Auele Specific Primer pair; (b) yak source property fibre composition specific probe, makes the detection kit of yak source property fibre composition.
Further, described test kit can also comprise (c) marker; Described marker can be yak source property fibre composition DNA, or the plasmid DNA that contains yak source property fibre composition amplification object fragment, or yakwool.
Obtained (a) amplification yak source property fibre composition Auele Specific Primer to the basis with (b) yak source property fibre composition specific probe on, utilize routine techniques means in the art just can make detection kit, repeat no more herein.
More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; that in above-described embodiment and specification sheets, describes just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.
Claims (7)
1. a yak source property fibre composition quantitative fluorescent PCR qualitative checking method, comprises the following steps:
Step 1, the DNA of yakwool of take are template, carry out pcr amplification;
The fluorescent signal of step 2, detection amplified production;
It is characterized in that, for the reaction system of pcr amplification contain amplification yak source property fibre composition Auele Specific Primer to yak source property fibre composition specific probe, property fibre composition Auele Specific Primer right sequence in described amplification yak source is as shown in SEQ ID NO:1 and SEQ ID NO:2, and the nucleotide sequence of described yak source property fibre composition specific probe is as shown in SEQ ID NO:3.
2. the method for claim 1, is characterized in that, the condition of described pcr amplification is as follows:
Reaction system: 2 * ABI Taqman universal PCR master mix10 μ l, DNA profiling 4 μ l, primer 0.6 μ l, probe 2.5 μ l, ddH
2o4.9 μ l;
Reaction conditions: 95 ℃, 5min; 95 ℃, 15s, 60 ℃, 45s, 40 circulations.
3. a detection kit for yak source property fibre composition, contains following reagent:
(a) amplification yak source property fibre composition Auele Specific Primer pair;
(b) yak source property fibre composition specific probe;
Wherein, the right sequence of described Auele Specific Primer is as shown in SEQ ID NO:1 and SEQ ID NO:2, and the nucleotide sequence of described specific probe is as shown in SEQ ID NO:3.
4. test kit according to claim 3, is characterized in that, described test kit also comprises:
(c) marker.
5. the application of the test kit as described in claim 3 or 4, is characterized in that, for detection of whether there is yak source property fibre composition in textiles.
6. the Auele Specific Primer in detection method as claimed in claim 1, is characterized in that, the sequence of described Auele Specific Primer is as shown in SEQ ID NO:1 and SEQ ID NO:2.
7. the specific probe in detection method as claimed in claim 1, is characterized in that, the sequence of described specific probe is as shown in SEQ ID NO:3.
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