CN105296643A - Isothermal amplification detection kit and detection method for duck meat derived component nucleic acid - Google Patents
Isothermal amplification detection kit and detection method for duck meat derived component nucleic acid Download PDFInfo
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- CN105296643A CN105296643A CN201510811223.0A CN201510811223A CN105296643A CN 105296643 A CN105296643 A CN 105296643A CN 201510811223 A CN201510811223 A CN 201510811223A CN 105296643 A CN105296643 A CN 105296643A
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- nucleic acid
- duck
- derived component
- amplification
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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
Abstract
The invention discloses an isothermal amplification detection kit and a detection method for duck meat derived component nucleic acid. The kit includes an isothermal amplification reaction solution, positive control and negative control. The detection kit provided by the invention has high specificity and high sensitivity. Nucleic acid amplification reaction is all carried out at a same temperature, isothermal amplification only needs 30min, a nucleic acid test strip detection result needs 1-2min, and the whole detection process from receiving the sample to acquiring the result only needs about 40min. Also, the whole reaction process only needs one constant temperature apparatus, and the kit is especially for field fast detection of duck meat derived component nucleic acid.
Description
(1) technical field
The present invention relates to a kind of constant-temperature amplification Fast Detection Technique for duck derived component nucleic acid, be applicable to detecting the on-the-spot fast qualitative of duck derived component nucleic acid.
(2) background technology
In recent years, the adulterated problem of meat product causes great concern socially.2013, " the horseflesh disturbance " that relate to European 16 states shocked whole Europe.In China, meat adulteration doping event is in recent years by media multiple-exposure.In Shandong, the ground such as Shaanxi and Henan occurs using duck to pretend to be the report of beef and mutton successively, mixes a large amount of duck and a small amount of cattle and sheep fat, tankage as beef and mutton and sells, earning juice the market of farm produce and indivedual illegal businessman or individual.
At present, regular-PCR or real-time fluorescence quantitative PCR are the most frequently used methods of meat product Variety identification, and regular-PCR method has length consuming time, complex operation, insufficient sensitivity, easily produce the shortcomings such as crossed contamination.Real-time fluorescence PCR rule has cost intensive, length consuming time, and technical requirements is high, and need the shortcomings such as large-scale plant and instrument, therefore its application still has some limitations.Isothermal amplification technology is a kind of new isothermal DNA amplification that developed recently gets up after round pcr, the technology of the present invention have developed constant-temperature amplification duck 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 duck 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 duck 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 duck 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:
Described primer just is to the periphery: 5 '-TGTCCGACGTGACTAGCT-3 ' (SEQIDNO.2);
Reverse peripheral primer is: 5 '-CAGAGGCGCCAAAAAGCT-3 ' (SEQIDNO.3);
The sequence of described two probes is respectively:
Forward 5 ' holds Biotin label probe: 5 '-Biotin-GGCGCAAAAATGTGAGGAGG-3 ' (SEQIDNO.4);
Reverse 5 ' end Fitc label probe: 5 '-Fitc-AAAGTGGCATCTGTGGAATACTTC-3 ' (SEQIDNO.5);
Described two amplification cross primers are respectively:
Amplification forward primer:
5′-GCCCTGACCGAGGAACCAGAGCCCATACGTTCCCCCTA-3′(SEQIDNO.6)；
Amplification reverse primer:
5′-TTCACTCACCTCTCCTTGCCCTGCCGCGATTACGCATTGA-3′(SEQIDNO.7)；
(2) positive control: the DNA plasmid belonging to specific D-Loop gene fragment containing duck;
(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 duck containing long 199bp belongs to specific D-Loop gene order, the nucleotide sequence following (SEQIDNO.1) of described fragment: TGTCCGACGTGACTAGCTTCAGGCCCATACGTTCCCCCTAAACCCCTCGCCCTCCT CACATTTTTGCGCCTCTGGTTCCTCGGTCAGGGCCATCAATTGGGTTCACTCACCT CTCCTTGCCCTTCAAAGTGGCATCTGTGGAATACTTCCACCATCTCAATGCGTAAT CGCGGCATCTTCCAGCTTTTTGGCGCCTCTG.
Further, positive control of the present invention is prepared as follows:
Belong to specific D-Loop gene fragment for template with the duck 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, isothermal amplification reactions 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 application of constant-temperature amplification detection kit of described duck derived component nucleic acid, described in be applied as:
A) extract DNA in sample to be detected: clip is about the duck 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 D-Loop gene fragment containing duck, 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 (Hangzhou You Sida biotech company No. 3 devices) and detects, sentence read result after 2 minutes, when the detection line of test strip is positive (C, T line is redness), containing duck derived component nucleic acid in interpret sample.This test strip is included on the liner with non-setting adhesive 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, 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 duck 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 duck derived component nucleic acid.The sensitivity of this test kit can detect 1 copy in each reaction system, can meet the requirement of rapid detection duck 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 the constant-temperature amplification detection kit of described duck derived component nucleic acid 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 PCR 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 duck derived component nucleic acid, in figure, 1-7 represents duck, chicken, pork, beef, mutton, positive reference substance, negative controls respectively.
Fig. 3 be test kit of the present invention for detecting the sensitivity of duck derived component nucleic acid, in figure, 1-7 represents 10 respectively
4copy/microlitre, 10
3copy/microlitre, 10
2copy/microlitre, 10
1copy/microlitre, 10
0copy/microlitre, 10
-1copy/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 duck 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 '-TGTCCGACGTGACTAGCT-3 ';
Reverse peripheral primer is: 5 '-CAGAGGCGCCAAAAAGCT-3 ';
Forward 5 ' holds Biotin label probe: 5 '-Biotin-GGCGCAAAAATGTGAGGAGG-3 ';
Reverse 5 ' end Fitc label probe: 5 '-Fitc-AAAGTGGCATCTGTGGAATACTTC-3 ';
Amplification forward primer:
5’-GCCCTGACCGAGGAACCAGAGCCCATACGTTCCCCCTA-3’；
Amplification reverse primer:
5’-TTCACTCACCTCTCCTTGCCCTGCCGCGATTACGCATTGA-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 duck containing long 199bp belongs to specific Cytb gene order, and sequence is as follows:
TGTCCGACGTGACTAGCTTCAGGCCCATACGTTCCCCCTAAACCCCTCGCCCTCCTCACATTTTTGCGCCTCTGGTTCCTCGGTCAGGGCCATCAATTGGGTTCACTCACCTCTCCTTGCCCTTCAAAGTGGCATCTGTGGAATACTTCCACCATCTCAATGCGTAATCGCGGCATCTTCCAGCTTTTTGGCGCCTCTG。
Positive control is prepared as follows:
Belong to specific Cytb gene fragment for template with the duck 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 duck derived component nucleic acid
A) DNA in sample to be detected is extracted.
B) get specimen dna as template, join in the PCR pipe of the isothermal amplification reactions liquid that duck 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 (duck containing long 199bp belongs to specific Cytb gene order) and negative template (aseptic double-distilled water) is added respectively in the positive and negative control PCR pipe.
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 duck 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 duck 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 duck derived component nucleic acid
Duck, chicken, pork, beef, mutton derived component nucleic acid and detection kit positive reference substance is detected according to the method for embodiment 2, result display test kit of the present invention detects duck derived component nucleic acid and has very strong specificity, except duck derived component nucleic acid and test kit positive reference substance, other meat detects and is feminine gender.As shown in Figure 2, in figure, 1-7 represents the experimental result of duck, chicken, pork, beef, mutton, positive reference substance, negative controls respectively.
Embodiment 4 test kit of the present invention detects the sensitivity of duck derived component nucleic acid
Carry out quantitatively to the plasmid DNA belonging to specific D-Loop gene fragment containing duck, being diluted to concentration is respectively 10
3copy/microlitre, 10
2copy/microlitre, 10
1copy/microlitre, 10
0copy/microlitre, 10
-1copy/microlitre, adopts method described in embodiment 2 to determine that test kit of the present invention is for detecting the sensitivity of duck derived component nucleic acid.As shown in Figure 3, in figure, 1-7 represents 10 to result respectively
4copy/microlitre, 10
3copy/microlitre, 10
2copy/microlitre, 10
1copy/microlitre, 10
0copy/microlitre, 10
-1the experimental result of copy/microlitre, negative controls, can find that this test kit can detect 1 copy in each reaction system, have very high sensitivity, can meet the requirement of rapid detection duck derived component nucleic acid.
Claims (6)
1. a constant-temperature amplification detection kit for duck 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 '-TGTCCGACGTGACTAGCT-3 ';
Reverse peripheral primer is: 5 '-CAGAGGCGCCAAAAAGCT-3 ';
The sequence of described two probes is respectively:
Forward 5 ' holds Biotin label probe 5 '-Biotin-GGCGCAAAAATGTGAGGAGG-3 ';
Reverse 5 ' end Fitc label probe 5 '-Fitc-AAAGTGGCATCTGTGGAATACTTC-3 ';
Described two amplification cross primers are respectively:
Amplification forward primer:
5′-GCCCTGACCGAGGAACCAGA-GCCCATACGTTCCCCCTA-3′；
Amplification reverse primer:
5′-TTCACTCACCTCTCCTTGCCCT-GCCGCGATTACGCATTGA-3′；
(2) positive control: the DNA plasmid belonging to specific D-Loop gene containing duck;
(3) negative control: aseptic double-distilled water.
2. the constant-temperature amplification detection kit of duck 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. the constant-temperature amplification detection kit of duck derived component nucleic acid as claimed in claim 1, it is characterized in that described positive control is the fragment that duck containing long 199bp belongs to specificity D-Loop gene order, the nucleotides sequence of described fragment is classified as shown in SEQIDNO.1.
4. constant-temperature amplification detection kit as claimed in claim 1, is characterized in that described positive control is prepared as follows:
Belong to specificity D-Loop gene fragment for template with the duck 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 duck 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 duck derived component nucleic acid described in claim 1, is characterized in that described detection method is:
A) extract DNA in sample to be detected: clip is about the duck 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 belonging to specific D-Loop gene fragment containing duck, 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 duck derived component nucleic acid in interpret sample.
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| CN107937562A (en) * | 2017-12-05 | 2018-04-20 | 厦门出入境检验检疫局检验检疫技术中心 | Detect the primer and its design method of the LAMP method of duck derived component |
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Application publication date: 20160203 |