CN105296644A - Isothermal amplification detection kit for chicken derived component nucleic acid and detection method - Google Patents
Isothermal amplification detection kit for chicken derived component nucleic acid and detection method Download PDFInfo
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- CN105296644A CN105296644A CN201510811692.2A CN201510811692A CN105296644A CN 105296644 A CN105296644 A CN 105296644A CN 201510811692 A CN201510811692 A CN 201510811692A CN 105296644 A CN105296644 A CN 105296644A
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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
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- 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
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Abstract
The invention discloses an isothermal amplification detection kit for chicken derived component nucleic acid and a detection method. The kit contains isothermal amplification reaction liquid, positive control and negative control. The detection kit provided by the invention has high specificity and high sensitivity. Nucleic acid amplification reaction is carried out at a same temperature, constant temperature amplification only needs 30min, nucleic acid test strip detection result can be obtained in 1-2min, the whole process from receiving a sample till getting the result needs only about 40min, and the whole reaction process only needs a constant temperature apparatus. Thus, the kit and the method are especially for field fast detection of chicken derived component nucleic acid.
Description
(1) technical field
The present invention relates to a kind of constant-temperature amplification Fast Detection Technique for chicken derived component nucleic acid, be applicable to detecting the on-the-spot fast qualitative of chicken derived component nucleic acid.
(2) background technology
China's production of meat increases year by year, and wherein chicken output occupies 13.97% of meat ultimate production.Meanwhile, the requirement of human consumer to Food Quality and Safety is also more and more higher, illegal retailer, enterprise, for obtaining illegal profit, often pretend to be ox, sheep, pork with chicken comparatively at a low price, serious infringement consumer's interests.This not only relates to the problems such as economy, nutritive value and food safety, more directly affects the health of human consumer, especially to the human consumer of some food anaphylaxis.For safeguarding consumers in general's legitimate rights and interests, order of stabilizing the market, qualification animal food being carried out to provenance seems very necessary.
At present, at the common detection methods of the qualitative detection of animal provenance, as electrophoresis, immunological technique, spectroscopic techniques etc. exist very large defect, as low in sensitivity, the cycle is grown, waste time and energy, and therefore its application still has some limitations.The technology of the present invention have developed constant-temperature amplification chicken derived component nucleic acid, and the colour developing of bind nucleic acid test strip judges detected result, and whole reaction process does not open reaction tubes lid, test strip flow detection process is airtight, has stopped nucleic acid amplification product and has polluted extraneous possibility.Research shows that the constant-temperature amplification-nucleic acid test strip detection method of this chicken derived component nucleic acid has the advantages such as high specificity, susceptibility is high, simple to instrument requirements, detection time is short, use so be useful in very much inspection body of basic unit, on the Site Detection of the market of farm produce, also there is good application prospect simultaneously.
(3) summary of the invention
The object of the invention is to provide a kind of constant-temperature amplification detection kit that is accurate, sensitive, detection chicken derived component nucleic acid rapidly and detection method thereof.
The technical solution used in the present invention is:
The invention provides a kind of constant-temperature amplification detection kit of chicken derived component nucleic acid, described test kit comprises following composition:
(1) isothermal amplification reactions liquid, comprising: primer, reverse peripheral primer, two probes, two intersections amplimer, 1 × Thermolbuffer, MgSO just to the periphery
4, dNTPs solution, BstDNA polysaccharase and aseptic double-distilled water; Wherein:
Described peripheral primer is respectively:
Primer is just to the periphery: 5 '-TCTCCCATGGGGCCAAAT-3 ' (SEQIDNO.2);
Reverse peripheral primer is: 5 '-TAGGAAGGTGAGGTGGATGA-3 ' (SEQIDNO.3);
The sequence of described two probes is respectively:
Forward 5 ' holds Biotin label probe: 5 '-Biotin-GTCCAATGTAGGGAATTGCTG-3 ' (SEQIDNO.4);
Reverse 5 ' end Fitc label probe: 5 '-Fitc-GTAAGGCGAAGAATCGGGA-3 ' (SEQIDNO.5);
Described two amplification cross primers are respectively:
Amplification forward primer:
5′-TCCCCCTCAGGCTCACTCTACTCTGAGGGGCCACCGTTAT-3′(SEQIDNO.6)；
Amplification reverse primer:
5′-TTCAGTCGACAACCCAACCCTTCGATTGCAAAGGGGAGGAG-3′(SEQIDNO.7)；
(2) positive control: the DNA plasmid belonging to specific cytochrome b gene (Cytb gene) fragment containing chicken;
(3) negative control: aseptic double-distilled water.
Further, 1 × Thermolbuffer of the present invention consists of: (the NH of KCl, 10mM of Tris-HCl, 10mM of 20mM
4)
2sO
4, 2mM MgSO
4and mass concentration is the TritonX-100 of 0.1%, pH7.8.
Further, positive control of the present invention is the fragment that the chicken containing long 205bp belongs to specific Cytb gene order, the nucleotide sequence following (SEQIDNO.1) of described fragment:
TCTCCCATGGGGCCAAATATCATTCTGAGGGGCCACCGTTATCACAAACCTATTCTCAGCAATTCCCTACATTGGACACACCCTAGTAGAGTGAGCCTGAGGGGGATTTTCAGTCGACAACCCAACCCTTACCCGATTCTTCGCCTTACACTTCCTCCTCCCCTTTGCAATCGCAGGTATTACTATCATCCACCTCACCTTCCTA。
Further, positive control of the present invention is prepared as follows:
Belong to specific Cytb gene fragment for template with the chicken comprising amplification region, carry out pcr amplification by two peripheral primers and obtain goal gene; PCR purification kit (Promega) is utilized to carry out purifying to pcr amplification product; Amplified production after purifying is built the complete plasmid sequence containing goal gene by T-easy plasmid transfection test kit (Promega), with spectrophotometer, A is surveyed to the plasmid of institute's extracting
280quantitatively and be diluted to 10 copy/μ l, acquisition positive control ,-20 DEG C of preservations.
Further, constant temperature pcr amplification reaction liquid of the present invention consists of: two peripheral primer 5.7nmol separately, two probe 11.4nmol separately, two cross primer 22.8nmol separately, 1 × Thermolbuffer, MgSO
4be 0.2 μm of ol, dNTPs solution each 35pmol, BstDNA polysaccharase 8U, aseptic double-distilled water composition complements to 25 μ l.
The present invention also provides a kind of detection method of constant-temperature amplification detection kit of described chicken derived component nucleic acid, and described detection method is:
A) extract specimen dna to be detected: clip is about the chicken meat sample of grain of rice size, vibrate after adding 500 μ L distilled waters 15s, in 95 DEG C of effect 5min, gets supernatant, namely obtain DNA after centrifugal;
B) step a) being extracted the DNA obtained joins in the PCR pipe that isothermal amplification reactions liquid is housed as template, amplified reaction 30 minutes at 65 DEG C; Standard positive template (i.e. positive control) adds in positive control PCR pipe, standard negative template (i.e. negative control) adds in negative control PCR pipe, described standard positive template is the DNA plasmid belonging to specific Cytb gene fragment containing chicken, and described standard negative template is aseptic double-distilled water;
C) nucleic acid test strip is adopted to carry out detecting (be preferably placed in the anti-pollution proofing unit of nucleic acid test strip (Hangzhou You Sida biotech company No. 3 devices) and detect) reacted PCR pipe, sentence read result after 2 minutes, when the detection line of test strip is positive, (test strip T line is for red, C line is red), containing chicken derived component nucleic acid in interpret sample.This test strip is included on the liner with non-setting adhesive and is provided with sample pad, coloured particle binding substances pad and absorbent filter pad in order, each part mentioned above partly overlaps at adjacent, tunica fibrosa is provided with detection line and nature controlling line, coloured particle wherein on coloured particle binding substances pad has anti-Fitc antibody bag quilt, detection line there is avidin bag quilt, nature controlling line has anti-Fitc antibody bag quilt, coloured particle is selected from colloid gold particle, latex particle.
In test kit provided by the invention, there are two peripheral primers, two cross primers and two detection probes.6 oligonucleotide sequences in this test kit rely on the highly active strand displacement characteristic of BstDNA polysaccharase, make strand displacement DNA synthesize continuous self-amplification cycles.In the constant-temperature amplification detection kit of chicken derived component nucleic acid provided by the invention, different reaction conditionss is optimized, as the concentration of primer and probe, Mg
2+concentration, the optimization of temperature of reaction etc., and the present invention is combined with nucleic acid detection test strip detection system, establish the method for the constant-temperature amplification qualitative detection of chicken derived component nucleic acid.The sensitivity of this test kit can detect 10 copies in each reaction system, can meet the requirement of rapid detection chicken derived component nucleic acid.
The present invention compared with prior art, has the following advantages and effect:
(1) this invention simplifies the extracting method of meat sample nucleic, reduce cost, and substantially reduce detection time.
(2) specificity of chicken constant-temperature amplification detection kit is good, highly sensitive, and step is simple, repeatable high;
(3) speed of response is fast, and single sample, from sample process to completing detection, only needs about 40 minutes;
(4) do not need to open pipe lid in whole amplification and testing process, decrease the chance that amplified production pollutes; Whole reaction process does not need complicated instrument.
(4) accompanying drawing explanation
Fig. 1 is nucleic acid detection test strip principle schematic.
Fig. 2 be test kit of the present invention for detecting the specificity of chicken derived component nucleic acid, 1-7 respectively chicken, duck, pork, beef, mutton, positive control, negative controls in figure.
Fig. 3 be test kit of the present invention for detecting the sensitivity of chicken derived component nucleic acid, in figure, 1-6 represents 10 respectively
4copy/microlitre, 10
3copy/microlitre, 10
2copy/microlitre, 10
1copy/microlitre, 10
0copy/microlitre, negative controls.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
The composition of embodiment 1 test kit of the present invention and preparation
A) the isothermal amplification reactions liquid of chicken derived component nucleic acid: two peripheral primers (5.7nmol), two probes (11.4nmol) and two cross primers (22.8nmol), 1 × Thermolbuffer, MgSO
4(0.2 μm of ol), dNTPs solution (each 35pmol), BstDNA polysaccharase (8U) and aseptic double-distilled water composition, it is 25 μ l that total reaction liquid amasss;
Primer is just to the periphery: 5 '-TCTCCCATGGGGCCAAAT-3 ';
Reverse peripheral primer is: 5 '-TAGGAAGGTGAGGTGGATGA-3 ';
Forward 5 ' holds Biotin label probe: 5 '-Biotin-GTCCAATGTAGGGAATTGCTG-3 ';
Reverse 5 ' end Fitc label probe: 5 '-Fitc-GTAAGGCGAAGAATCGGGA-3 ';
Amplification forward primer:
5′-TCCCCCTCAGGCTCACTCTACT-CTGAGGGGCCACCGTTAT-3′；
Amplification reverse primer:
5′-TTCAGTCGACAACCCAACCCTT-CGATTGCAAAGGGGAGGAG-3′；
1 described × Thermolbuffer consists of: (the NH of KCl, 10mM of Tris-HCl, 10mM of 20mM
4)
2sO
4, 2mM MgSO
4and mass concentration is the TritonX-100 of 0.1%, pH7.8.
All primers and probe are by the synthesis of Shanghai raw work biological company limited.
B) positive control: positive control is that the chicken containing long 205bp belongs to specific Cytb gene order, and sequence is as follows:
TCTCCCATGGGGCCAAATATCATTCTGAGGGGCCACCGTTATCACAAACCTATTCTCAGCAATTCCCTACATTGGACACACCCTAGTAGAGTGAGCCTGAGGGGGATTTTCAGTCGACAACCCAACCCTTACCCGATTCTTCGCCTTACACTTCCTCCTCCCCTTTGCAATCGCAGGTATTACTATCATCCACCTCACCTTCCTA。
Positive control is prepared as follows:
Belong to specific Cytb gene fragment for template with the chicken comprising amplification region, carry out pcr amplification by two peripheral primers and obtain goal gene; PCR purification kit is utilized to carry out purifying to pcr amplification product; Amplified production after purifying is built the complete plasmid sequence containing goal gene by T-easy plasmid transfection test kit, with spectrophotometer, A is surveyed to the plasmid of institute's extracting
280quantitatively and be diluted to 10 copy/μ l ,-20 DEG C of preservations.
C) negative control: aseptic double-distilled water.
Embodiment 2 test kit of the present invention detects the concrete grammar of chicken derived component nucleic acid
A) specimen dna to be detected is extracted.
B) get specimen dna as template, join in the PCR pipe of the isothermal amplification reactions liquid that chicken derived component nucleic acid is housed, wherein specimen dna 3 μ l, reaction solution 22 μ l, 65 DEG C of amplified reactions 30 minutes; Positive template and negative template is added respectively in the positive and negative control PCR pipe; Described positive template is the DNA plasmid belonging to specific Cytb gene fragment containing chicken, and described negative template is aseptic double-distilled water.
C) reacted PCR pipe is placed in the anti-pollution proofing unit of nucleic acid test strip (Hangzhou You Sida biotech company No. 3 devices) and detects, sentence read result after 2 minutes.When containing chicken derived component nucleic acid in sample, the detection line of test strip is positive.
Repeatedly repeat experiment 3 times, detected result there was no significant difference, the detected result between the different batches that this test kit is described has comparability, has good repeatability.Above-described embodiment illustrates, detects reproducible, and only needs just can complete for about 40 minutes to the detection of sample, greatly shorten detection time with test kit of the present invention.
Detect the reproducible of chicken derived component nucleic acid with test kit of the present invention, and only within about 40 minutes, just can complete the detection of sample, greatly shorten detection time.
Embodiment 3 test kit of the present invention detects the specificity of chicken derived component nucleic acid
Chicken, duck, pork, beef, mutton derived component nucleic acid is detected according to the method for embodiment 2, result display test kit of the present invention detects chicken derived component nucleic acid and has very strong specificity, except chicken derived component nucleic acid and test kit positive reference substance, (C line is red, T line is red), other meat detected results are feminine gender (C line is red, and T line does not develop the color).As shown in Figure 2, in figure, 1-7 represents the experimental result of chicken, duck, pork, beef, mutton, positive control, negative controls respectively.
Embodiment 4 test kit of the present invention detects the sensitivity of chicken nucleic acid
Carry out quantitatively to the plasmid DNA belonging to specific Cytb gene fragment containing chicken, being diluted to concentration is respectively 10
4copy/microlitre, 10
3copy/microlitre, 10
2copy/microlitre, 10
1copy/microlitre, 10
0copy/microlitre, adopts method described in embodiment 2 to determine that test kit of the present invention is for detecting the sensitivity of chicken derived component nucleic acid.As shown in Figure 3, in figure, 1-6 represents 10 to result respectively
4copy/microlitre, 10
3copy/microlitre, 10
2copy/microlitre, 10
1copy/microlitre, 10
0the experimental result of copy/microlitre, negative controls.Can find that this test kit can detect 10 copies in each reaction system, there is very high sensitivity, the requirement of rapid detection chicken derived component nucleic acid can be met.
Claims (6)
1. a constant-temperature amplification detection kit for chicken derived component nucleic acid, is characterized in that described test kit comprises following composition:
(1) isothermal amplification reactions liquid, comprising: primer, reverse peripheral primer, two probes, two increase cross primer, 1 × ThermolBuffer, MgSO just to the periphery
4, dNTPs solution, BstDNA polysaccharase and aseptic double-distilled water;
Described primer just is to the periphery: 5 '-TCTCCCATGGGGCCAAAT-3 ';
Reverse peripheral primer is: 5 '-TAGGAAGGTGAGGTGGATGA-3 ';
The sequence of described two probes is respectively:
Forward 5 ' holds Biotin label probe: 5 '-Biotin-GTCCAATGTAGGGAATTGCTG-3 ';
Reverse 5 ' end Fitc label probe: 5 '-Fitc-GTAAGGCGAAGAATCGGGA-3 ';
Described two amplification cross primers are respectively:
Amplification forward primer:
5′-TCCCCCTCAGGCTCACTCTACT-CTGAGGGGCCACCGTTAT-3′；
Amplification reverse primer:
5′-TTCAGTCGACAACCCAACCCTT-CGATTGCAAAGGGGAGGAG-3′；
(2) positive control: the DNA plasmid belonging to specific cytochrome b gene Cytb containing chicken;
(3) negative control: aseptic double-distilled water.
2. the constant-temperature amplification detection kit of chicken derived component nucleic acid as claimed in claim 1, is characterized in that described 1 × ThermolBuffer consists of: (the NH of KCl, 10mM of Tris-HCl, 10mM of 20mM
4)
2sO
4, 2mM MgSO
4and mass concentration is the TritonX-100 of 0.1%, pH8.8.
3. chicken derived component nucleic acid isothermal amplification detection kit as claimed in claim 1, it is characterized in that described positive control is the fragment that chicken containing long 205bp belongs to specific Cytb gene order, the nucleotides sequence of described fragment is classified as shown in SEQIDNO.1.
4. the constant-temperature amplification detection kit of chicken derived component nucleic acid as claimed in claim 1, is characterized in that described positive control is prepared as follows:
Belong to specific Cytb gene fragment for template with the chicken comprising amplification region, carry out pcr amplification by two peripheral primers and obtain goal gene; PCR purification kit is utilized to carry out purifying to pcr amplification product; Amplified production after purifying is built the complete plasmid sequence containing goal gene by T-easy plasmid transfection test kit, with spectrophotometer to the plasmid of institute's extracting quantitatively and be diluted to 10 copy/μ l, acquisition positive control ,-20 DEG C of preservations.
5. the constant-temperature amplification detection kit of chicken derived component nucleic acid as claimed in claim 1, it is characterized in that described isothermal amplification reactions liquid consists of: two peripheral primer 5.7nmol separately, article two, probe 11.4nmol separately, article two, cross primer 22.8nmol separately, 1 × Thermolbuffer, MgSO
4be 0.2 μm of ol, dNTPs solution each 35pmol, BstDNA polysaccharase 8U, aseptic double-distilled water composition complements to 25 μ l.
6. a detection method for the constant-temperature amplification detection kit of chicken derived component nucleic acid described in claim 1, is characterized in that described detection method is:
A) extract specimen dna to be detected: clip is about the chicken meat sample of grain of rice size, vibrate after adding 500 μ L distilled waters 15s, in 95 DEG C of effect 5min, gets supernatant, namely obtain DNA after centrifugal;
B) step a) is extracted the DNA that obtains as template, join in the PCR pipe that isothermal amplification reactions liquid is housed, amplified reaction 30 minutes at 65 DEG C; Standard positive template adds in positive control PCR pipe, and standard negative template adds in negative control PCR pipe, and described standard positive template is the DNA plasmid belonging to specific Cytb gene fragment containing chicken, and described standard negative template is aseptic double-distilled water;
C) reacted PCR pipe is placed in the anti-pollution proofing unit of nucleic acid test strip detects, sentence read result after 2 minutes, when the detection line of test strip is positive, containing chicken derived component nucleic acid in interpret sample.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108796088A (en) * | 2017-05-03 | 2018-11-13 | 杭州众测生物科技有限公司 | The kit and detection method of chicken derived components in a kind of detection food |
| CN109457034A (en) * | 2018-11-20 | 2019-03-12 | 昆明理工大学 | The application and its kit of a kind of gene Gcg in detection turkey derived component |
| CN110791568A (en) * | 2018-08-03 | 2020-02-14 | 上海市质量监督检验技术研究院 | LAMP primer group for rapidly detecting chicken-derived components in beef and mutton, detection kit, detection method and application |
| CN114752691A (en) * | 2022-05-31 | 2022-07-15 | 宁波大学 | Kit and method for detecting chicken adulteration |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110791568A (en) * | 2018-08-03 | 2020-02-14 | 上海市质量监督检验技术研究院 | LAMP primer group for rapidly detecting chicken-derived components in beef and mutton, detection kit, detection method and application |
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| CN114752691A (en) * | 2022-05-31 | 2022-07-15 | 宁波大学 | Kit and method for detecting chicken adulteration |
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Application publication date: 20160203 |