CN104694632A - Real-time fluorescent PCR species composition detection method, primer, probe and kit for mice - Google Patents
Real-time fluorescent PCR species composition detection method, primer, probe and kit for mice Download PDFInfo
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Abstract
The invention relates to a real-time fluorescent PCR species composition detection method for mice for detecting species compositions of mice by virtue of a TaqMan probe real-time fluorescent PCR detection technique, belonging to the technical field of biology. The method comprises the following steps: (1) extracting DNA of samples; (2) carrying out real-time fluorescent PCR amplification detection; (3) carrying out result determination, namely determining the result to be positive when a Ct value is less than or equal to 35, and determining the result to be negative when the Ct value is more than 35. According to the method, a primer and a probe have very high specificity and sensitivity, and related mice are common species in China, so that related departments can be helped to effectively fight against disgusting conducts of the production and sale of fake meat and meat products.
Description
Technical field
The present invention relates to the real-time fluorescence PCR detection method of mouse, use TaqMan probe real-time PCR detection technology, Species composition detection is carried out to mouse, belongs to biological technical field.
Background technology
In recent years, the doping adulteration incident of meat and meat products is exposed by the media again and again, has become one of significant challenge that Chinese food quality control faces.Pork relatively honest and clean for price, chicken, duck pretend to be beef, mutton to sell by lawless person.Recently report, mouse meat mixes in beef and mutton and sells by illegal retailer.Mouse is as the propagating source of epidemic disease, and processing and eating is very large to health risk.Animal species composition detection hits meat product to manufacture the fake adulterated effective means, but the detection method of not a kind of efficiently and accurately for mouse composition at present.
Morphological method is traditional animal species authentication method, however by its accuracy and repeatability poor, can not meet a series of supervision of animals products adulteration and the demands of control such as meat products.The immunological method of the Species estimation protein ingredient mostly in Direct Identification sample, its ultimate principle is antibody antigen reaction, but immunological method exists sample material requirement high, the defects such as Detection accuracy is low, if if animal-derived food product is through process such as heating, pickle, its protein structure is once be destroyed, and immunological method is just no longer applicable.And molecular biology method, based on features such as the high stability of the genetic material such as nucleic acid and diversity, be widely used in species Species estimation, the nucleic acid detection method particularly based on round pcr is applied to the history that species differentiate existing 20 years.Real-time fluorescence PCR adds fluorophor in PCR reaction system, utilizes fluorescent signal to accumulate the whole PCR process of Real-Time Monitoring, analyze finally by amplification curve to result.
Summary of the invention
In order to solve above-mentioned technical problem, an object of the present invention is to provide the real-time fluorescence PCR Species composition detection method of a kind of mouse, second object of the present invention is to provide detection primer and the probe of aforesaid method, and second object of the present invention is to provide the test kit of aforesaid method.Present method has very high specificity and sensitivity due to its primer and probe, and involved mouse kind is all China's frequent species, and relevant department can be assisted effectively to hit the disgusting conduct of SDS in broiler chickens manufacturing and marketing fake.
In order to realize first above-mentioned object, present invention employs following technical scheme:
The real-time fluorescence PCR Species composition detection method of mouse, the method comprises the following steps:
1) sample DNA extracts;
2) real-time fluorescent PCR amplification detects;
Real-time fluorescence PCR reaction system is as follows: 10ul Premix Ex Taq, upstream primer 0.4ul, downstream primer 0.4ul, probe 0.4ul, and upstream primer, downstream primer and concentration and probe concentration are 10pmol/ μ l; The sequence of upstream primer is AAAY
*cCAACTTATATGTGAAAATTCATTGT, the sequence of downstream primer is AGTACCGCCAAGTCCTTTGA, and the sequence of probe is FAM-TACCCCACTATGCTTAGCC-Eclipse, described Y
*represent and annex base C or T; Get the DNA liquid 1ul of said extracted, supply volume to 20ul with water; Application quantitative real time PCR Instrument lightcycle 480 reacts, and response procedures is (1) 95 DEG C, 10sec; (2) 95 DEG C, 5sec; 60 DEG C, 23sec; 40 circulations;
3) result judges; When Ct value is less than or equal to 35, result is positive, is feminine gender when Ct value is greater than 35.
As preferably, the method that described sample DNA extracts is as follows: get 0.2g rat meat, and in liquid nitrogen fully after grinding, use DNA extraction agent box to carry out DNA extracting, the DNA after extracting is dissolved in 100ul water.
As preferably, described DNA extraction agent box adopts Promega FF3750.
In order to realize second above-mentioned object, present invention employs following technical scheme:
The real-time fluorescence PCR Species composition detection primer of mouse and probe, the sequence of upstream primer is AAAY
*cCAACTTATATGTGAAAATTCATTGT, the sequence of downstream primer is AGTACCGCCAAGTCCTTTGA, and the sequence of probe is FAM-TACCCCACTATGCTTAGCC-Eclipse, described Y
*represent and annex base C or T.
In order to realize the 3rd above-mentioned object, present invention employs following technical scheme:
The real-time fluorescence PCR Species composition detection test kit of mouse, this test kit comprises upstream primer, downstream primer and probe, and the sequence of described upstream primer is AAAY
*cCAACTTATATGTGAAAATTCATTGT, the sequence of downstream primer is AGTACCGCCAAGTCCTTTGA, and the sequence of probe is FAM-TACCCCACTATGCTTAGCC-Eclipse, described Y
*represent and annex base C or T.
The present invention is based on mouse plastosome 12S ribosome-RNA(rRNA) (the 12S ribosomal RNA) sequence (gene order number: KC663621.1 with height species specificity, FQ210435.1, AJ005780.1 and AJ311140.1) devise in Muridae (Muridae) Murinae (Murinae) there is house mouse (Mus musculus), Rattus norvegicus (Rattus norvegicus), rattus rattus (Rattus rattus), Rattusflauipectus (Rattus flavipectus), the primer of the species specificities such as Apodemus agrarius (Apodemus agrarius) and probe, realize carrying out Species composition detection with the method for Real-time PCR to above-mentioned mouse kind.Present method has very high specificity and sensitivity due to its primer and probe, and involved mouse kind is all China's frequent species, and therefore the present invention can assist relevant department effectively to hit the disgusting conduct of SDS in broiler chickens manufacturing and marketing fake.
Embodiment
Embodiment 1: the design of primer and probe
Based on mouse 12S ribosome-RNA(rRNA) (the 12S ribosomal RNA) sequence (gene order number: KC663621.1, FQ210435.1, AJ005780.1 and AJ311140.1) with height species specificity, with bioinformatics method, above-mentioned sequence is compared, and according to design of primers principle, utilize Primer Express design software, design Auele Specific Primer and fluorescent probe some groups.The primer obtain design and probe carry out BLAST checking (http://blast.ncbi.nlm.nih.gov/), one by one to ensure the high specific of primer.Through above-mentioned checking, finally have selected primer as shown in table 1 and probe, and marked fluorescent signal on probe, detect to realize real-time fluorescence.
The primer of the fluorescence Real-time PCR Species composition detection of table 1 mouse and probe
F: upstream primer; R: downstream primer; P: probe.
*y represents merger base C or T;
*quote from GB/T25165-2010.This primed probe can be used as internal reference primed probe, the quality of inspection institute extracting DNA.
Embodiment 2: sample DNA extracts
Get 0.2g meat, after fully grinding in liquid nitrogen, use DNA extraction agent box (Promega FF3750), carry out DNA extracting to specifications, the DNA after extracting is dissolved in 100ul water.Leaching process arranges the extraction blank substituting sample with water.
Embodiment 3: the detection of real-time fluorescence PCR to sample and the judgement to result
Real-time fluorescent PCR amplification is reacted.Primer probe sequence is as shown in table 1, real-time fluorescence PCR reaction system is as follows: 10ulPremix Ex Taq (Takara), upstream primer (10pmol/ μ l) 0.4ul, downstream primer (10pmol/ μ l) 0.4ul, probe (10pmol/ μ l) 0.4ul, get the DNA liquid 1ul of said extracted, supply volume to 20ul with water.Application real-time fluorescence PCR instrument lightcycle 480 (Roche) is reacted, and response procedures is (1) 95 DEG C, 10sec; (2) 95 DEG C, 5sec; 60 DEG C, 23sec; 40 circulations.
Testing process, except sample, arranges positive control, negative control, extraction blank.Detect with the detection primed probe of mouse and internal reference primed probe respectively.
If detected result display blank, negative control are without FAM fluorescent signal, when positive control Ct value is less than or equal to 35, can judges that Fluorescence PCR is effective, otherwise react invalid.Under the effective prerequisite of Fluorescence PCR, the Ct value of carrying out the sample detected with internal reference primed probe is less than or equal to 35, and when there is obvious amplification curve, represents that DNA extracting is effective, otherwise again should extract DNA, until Ct value is less than or equal to 35.Effective at Fluorescence PCR, and the DNA of extracting is also in effective situation, when the Ct value that the Auele Specific Primer probe in detecting of mouse obtains is less than or equal to 35, then judgement sample is positive, if Ct value is greater than 35, and judgement sample feminine gender.
Embodiment 4: real-time fluorescence PCR specificity verification
Respectively 21 species (table 2) such as different types of mouse and ox, sheep, pig, horse are carried out to the specificity verification of primed probe, reaction system and response procedures are as described in example 3.As shown in table 2, during Auele Specific Primer probe in detecting with mouse, only have the mouse of each kind to obtain positive findings, other species all show negative findings; And during by eukaryote universal primer probe in detecting, except negative control, other all obtain positive findings.The above results shows, the DNA quality that all species extractings obtain is good, and Fluorescence PCR is effective, can judge that the primed probe of the mouse that the present invention designs has good species specificity to house mouse, Rattus norvegicus, rattus rattus, Rattusflauipectus, Apodemus agrarius, small white mouse on this basis.
Table 2 real-time fluorescence PCR specificity verification result
* be the immune deficiency mutation of house mouse;
The mean value of repetition 3 times is got in all experiments; Undet.:Ct value is lower than detectability.
Embodiment 5: real-time fluorescence PCR sensitivity is verified
To extracting to the DNA of target mouse kind carry out 5 times of gradient dilutions, detect the sensitivity of primed probe.Reaction system and response procedures are as described in example 3.Result display (table 3), when DNA concentration is 0.02ng/ul, can obtain amplification curve (figure is slightly) and the strong positive Ct value (Ct value is less than 30) of standard, detected result is now reliable; Continue DNA concentration dilution 5 times, when the DNA concentration detected reaches 0.004ng/ul, result display cannot detect, exceeds detectability.
Table 3 real-time fluorescence PCR sensitivity the result
* the actual concentration/5 times gradient dilution that records of Nanodrop 1000 (Thermo) is used to calculate the concentration obtained;
-lower than sensing range;
The mean value of repetition 3 times is got in all experiments; Undet.:Ct value is lower than detectability.
Sequence table
<110> Zhejiang Entry-Exit Inspection and Quarantine Bureau
The real-time fluorescence PCR Species composition detection method of <120> mouse and detection primer, probe and test kit
<160>4
<210>1
<211>29
<212>DNA
<213> artificial sequence
<400>1
AAACCCAACT TATATGTGAA AATTCATTG 29
<210>2
<211>29
<212>DNA
<213> artificial sequence
<400>2
AAATCCAACT TATATGTGAA AATTCATTG 29
<210>3
<211>20
<212>DNA
<213> artificial sequence
<400>3
AGTACCGCCA AGTCCTTTGA 20
<210>4
<211>29
<212>DNA
<213> artificial sequence
<400>4
TACCCCACTA TGCTTAGCC 19
Claims (5)
1. the real-time fluorescence PCR Species composition detection method of mouse, is characterized in that the method comprises the following steps:
1) sample DNA extracts;
2) real-time fluorescent PCR amplification detects;
Real-time fluorescence PCR reaction system is as follows: 10ul Premix Ex Taq, upstream primer 0.4ul, downstream primer 0.4ul, probe 0.4ul, and upstream primer, downstream primer and concentration and probe concentration are 10 pmol/ μ l; The sequence of upstream primer is AAAY
*cCAACTTATATGTGAAAATTCATTGT, the sequence of downstream primer is AGTACCGCCAAGTCCTTTGA, and the sequence of probe is FAM-TACCCCACTATGCTTAGCC-Eclipse, described Y
*represent and annex base C or T; Get the DNA liquid 1ul of said extracted, supply volume to 20ul with water; Application quantitative real time PCR Instrument lightcycle 480 reacts, and response procedures is (1) 95 ° of C, 10 sec; (2) 95 ° of C, 5 sec; 60 DEG C, 23 sec; 40 circulations;
3) result judges; When Ct value is less than or equal to 35, result is positive, is feminine gender when Ct value is greater than 35.
2. the real-time fluorescence PCR Species composition detection method of mouse according to claim 1, it is characterized in that the method that sample DNA extracts is as follows: get 0.2g rat meat, after fully grinding in liquid nitrogen, use DNA extraction agent box to carry out DNA extracting, the DNA after extracting is dissolved in 100ul water.
3. the real-time fluorescence PCR Species composition detection method of mouse according to claim 2, is characterized in that DNA extraction agent box adopts Promega FF3750.
4. the real-time fluorescence PCR Species composition detection primer of mouse and probe, is characterized in that: the sequence of upstream primer is AAAY
*cCAACTTATATGTGAAAATTCATTGT, the sequence of downstream primer is AGTACCGCCAAGTCCTTTGA, and the sequence of probe is FAM-TACCCCACTATGCTTAGCC-Eclipse, described Y
*represent and annex base C or T.
5. the real-time fluorescence PCR Species composition detection test kit of mouse, is characterized in that: this test kit comprises upstream primer, downstream primer and probe, and the sequence of described upstream primer is AAAY
*cCAACTTATATGTGAAAATTCATTGT, the sequence of downstream primer is AGTACCGCCAAGTCCTTTGA, and the sequence of probe is FAM-TACCCCACTATGCTTAGCC-Eclipse, described Y
*represent and annex base C or T.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105177153A (en) * | 2015-09-30 | 2015-12-23 | 南通市疾病预防控制中心 | PCR (polymerase chain reaction) identification primer and identification method for rattus flavipectus |
| CN106591469A (en) * | 2017-01-10 | 2017-04-26 | 青岛正方元信公共卫生检测有限公司 | Specific primer for detecting mouse-derived components |
| CN109266755A (en) * | 2017-07-17 | 2019-01-25 | 成都市食品药品检验研究院 | It is a kind of for detecting the kit and method of mouse ingredient |
| CN110527712A (en) * | 2019-08-14 | 2019-12-03 | 深圳市检验检疫科学研究院 | The system and method for PCR reaction detection mouse ingredient in a kind of meat products |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102312011A (en) * | 2011-10-10 | 2012-01-11 | 侯东军 | PCR primer pair for identification or auxiliary identification of tissues and/or organs of mouse and applications thereof |
| CN103642917A (en) * | 2013-12-06 | 2014-03-19 | 南京市产品质量监督检验院 | Kit for identifying murine components in food, preparation method and detection method |
-
2015
- 2015-02-05 CN CN201510061450.6A patent/CN104694632A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102312011A (en) * | 2011-10-10 | 2012-01-11 | 侯东军 | PCR primer pair for identification or auxiliary identification of tissues and/or organs of mouse and applications thereof |
| CN103642917A (en) * | 2013-12-06 | 2014-03-19 | 南京市产品质量监督检验院 | Kit for identifying murine components in food, preparation method and detection method |
Non-Patent Citations (1)
| Title |
|---|
| I. MARTÍN等: "Detection of cat, dog, and rat or mouse tissues in food and animal feed using species-specific polymerase chain reaction", 《J. ANIM. SCI.》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105177153A (en) * | 2015-09-30 | 2015-12-23 | 南通市疾病预防控制中心 | PCR (polymerase chain reaction) identification primer and identification method for rattus flavipectus |
| CN105177153B (en) * | 2015-09-30 | 2018-04-24 | 南通市疾病预防控制中心 | A kind of Rattusflauipectus PCR identification primers and identification method |
| CN106591469A (en) * | 2017-01-10 | 2017-04-26 | 青岛正方元信公共卫生检测有限公司 | Specific primer for detecting mouse-derived components |
| CN109266755A (en) * | 2017-07-17 | 2019-01-25 | 成都市食品药品检验研究院 | It is a kind of for detecting the kit and method of mouse ingredient |
| CN109266755B (en) * | 2017-07-17 | 2022-01-28 | 成都市食品药品检验研究院 | Kit and method for detecting mouse-derived components |
| CN110527712A (en) * | 2019-08-14 | 2019-12-03 | 深圳市检验检疫科学研究院 | The system and method for PCR reaction detection mouse ingredient in a kind of meat products |
| CN110527712B (en) * | 2019-08-14 | 2024-02-09 | 深圳市检验检疫科学研究院 | System and method for detecting murine components by PCR (polymerase chain reaction) in meat product |
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