CN111793696A - RPA primer, probe and detection method for detecting naked gesso - Google Patents
RPA primer, probe and detection method for detecting naked gesso Download PDFInfo
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Abstract
The invention provides an RPA primer, a probe and a detection method for detecting naked capers, which are based on the naked capersNADH 2 The method is characterized in that the length of an RPA probe is 46-54bp, a dSpacer is adopted to modify a position 32bp away from a 5 ' end in the probe, thymine (dT) at positions 31bp and 34bp away from the 5 ' end on two sides of dSpacer molecules are respectively replaced by a fluorescent group FAM and a quenching group BHQ1, and a blocking group C3Spacer is also required to modify the 3 ' end of the probe. The detection method of the invention can complete detection within 15-20 minutes, and can rapidly determine whether the sample is nakedAnd (7) covering the fish.
Description
Technical Field
The invention relates to the field of detection, in particular to an RPA primer, a probe and a detection method for detecting naked-cover fishes.
Background
Bare fish (Anoplopoma fimbria), also commonly known as haddock, belongs to the class of the spoke fin fish, the order of sebastes, the family of sebastes. The fishing amount of the fish is only ten thousand tons per year, the price is high, and about 200 yuan and 300 yuan are used. It is rich in omega-3 fatty acid and easy to artificially breed, so it is an important economic fish. Research shows that omega-3 fatty acid is beneficial to human body, and can improve cardiovascular health, prevent fatal arrhythmia and relieve inflammation for normal people, is also important for brain development and normal retina development of fetuses, and influences cognitive, language or motor ability of children to a certain extent. In 2014, recommendations in a draft commonly issued by the U.S. Food and Drug Administration (FDA) and the U.S. Environmental Protection Agency (EPA): women during pregnancy and lactation should eat more fish with relatively high DHA and EPA contents. Therefore, naked-covered fish is favored.
Naked cover fish belongs to the large-scale fish in deep sea, generally gets rid of the head and the tail when selling, cuts apart the parcel and sells with modes such as fish piece, fish are taken off, consequently can't distinguish adulterated fish through the morphology. The labeling of naked caper fish with "silver cod" is allowed in the "guide on identification and labeling of oil fish/cod issued by hong Kong food safety center. According to market survey data, 85.7% of naked gecko fishes sold in the market of China are identified as Antarctic canine odontopathy. Antarctic canine, belonging to the class Philippines, order Perciformes, family Antarctic Fish, genus Antarctic Canine, two economic species containing Lepidoporus Canine (Dissotrichus eleginoides) and Lepidopodium Canine (Dissotrichus mawsoni). The Antarctic canine odontophagous carps mainly have the problem of over-fishing, only a few countries and regions can carry out limited fishing due to the protection of the Antarctic canine odontophagous carps, but illegal fishing still exists, and in view of the fact that the fish traceability system of China is not sound in other European and American countries, the illegally-fished Antarctic canine odontophagous carps are reported to be sold in China, so that the current situation that most of the naked-capped carps sold in China are actually Antarctic canine odontophagous carps is caused. Labeling of Antarctic Canine Fish with "white cod" was allowed in the guidelines issued in hong Kong.
Disclosure of Invention
The State general administration of market supervision and management in China issued 2019 a food supplement inspection method of detection of sources of naked capers, oil fishes and Antarctic canine odontophagus in cod and products thereof (BJS 201907), and the use method is a real-time fluorescence PCR detection method. In addition, a general identification method for fish based on gene barcode sequencing is specified in a national standard method GB/T35918-2018 Sanger sequencing method for animal origin detection gene barcode technology in animal products, and can be used for detecting the naked-cover fish. However, these methods require expensive precision equipment such as sequencers and fluorescence PCR instruments, which have high requirements for operator operation and environmental conditions, and have high costs for equipment calibration and maintenance, and thus do not meet the operational requirements in mobile laboratories, rapid field testing, factory acceptance and inspection, and enterprise self-inspection. In addition, Sanger sequencing cannot be applied to detection of adulterated and mixed samples because it is limited by the detection principle (a single peak per base is required to recognize base ATGC) and cannot detect multi-source samples.
Therefore, the invention provides an RPA primer, a probe and a detection method for detecting the naked-head carp.
Recombinase Polymerase Amplification (RPA) is known as a novel nucleic acid Amplification technique, and is most likely to replace PCR. The technology has the advantages of simple and convenient operation, quick reaction, high sensitivity, strong specificity, no need of precise instruments and the like, and has great potential in the field quick detection aspects of clinical diagnosis, disease prevention and control and the like. Compared with other technologies, the RPA technology has more flexibility, can detect not only DNA but also RNA, and can display experimental results in different modes through different modifications. If the kit is combined with a portable amplification instrument, the experimental result can be monitored on line in real time, the product can be recovered by combining with gel electrophoresis, and the experimental result can be rapidly read without instrument equipment by combining with a test strip.
The RPA technology was invented by TwistDx Biotech, Inc., established at Babraham, Cambridge, England. The T4 phage nucleic acid replication mechanism is used as a principle, and the proteins required by the reaction comprise recombinase proteins (uvsX and uvsY) coded by T4 phage, single-chain binding protein (gp32 protein) and Bsu DNA polymerase; the reaction primer is oligonucleotide; meanwhile, nucleic acid, a primer probe, a magnesium ion solution and the like are required. The recombinase is combined with the oligonucleotide primer to form a protein-nucleotide complex, the primer is combined with the homologous target sequence under the action of the recombinase, the recombinase can open the double-stranded DNA once finding a specific binding site by scanning the template sequence in two directions, the primer and the homologous sequence generate a strand exchange reaction at the same time, the DNA synthesis reaction is started, and the specific target DNA fragment on the template is amplified under the constant temperature condition. The parent DNA strand displaced by the primer binds to the single-stranded binding protein, preventing further displacement from occurring. In this reaction, a synthesis process is initiated by a pair of opposing primers, the reaction can be completed rapidly in a short time, and the synthesized progeny DNA can still proceed rapidly, which is the basis for the exponential amplification of the RPA technique. The RPA technology utilizes recombinase, single-strand binding protein and DNA polymerase to replace the thermal cycle melting process of the traditional PCR, realizes the rapid amplification of nucleic acid at the constant temperature of 37 ℃, can obtain an amplification product after reacting for about 20min, does not need special auxiliary instruments, has low requirements on operators, has the characteristics of simplicity, portability, rapidness and the like, and is very suitable for the rapid detection in basic-level mechanisms.
In view of the above, the present invention provides an RPA primer probe for detecting naked-cap fish, the primer of which is based on NADH of naked-cap fish2The gene is designed, and the sequences of the primer and the probe are respectively as follows:
(1) the upstream primer SEQ ID No. 1:
5’-TTCGCCAGCTCTCACTGACTACTCGCATGAATGGG-3’
(2) the downstream primer is SEQ ID No. 2:
5’-TCCTGTGGCTTGTGTGAGGAAGTACTTGGTGGAGG-3’
(3) probe SEQ ID No. 3:
5’-TCGAAATGAACACGCTTGCTATTATCCCCTTAATAGCACAGCATCACCACCCAC-3’。
the prior detection method for identifying the authenticity of fishes in a laboratory mainly adopts a Taqman probe method in a real-time fluorescence PCR technology. The reaction process comprises three core reagents: two specific primers and one specific probe. The Taqman probe is an oligonucleotide probe, the 5 ' end of the Taqman probe is marked with a luminescent group, the 3 ' end of the Taqman probe is marked with a quenching group, and when the complete probe is matched with a target sequence, the fluorescence emitted by the luminescent group is quenched because of the proximity of the luminescent group and the quenching group at the 3 ' end. When the PCR reaction reaches the extension step, the probe is cut by enzyme by the 5' exonuclease activity of the DNA polymerase, so that the luminescent group is separated from the quenching group, and a fluorescent signal is emitted. The released fluorophores accumulate as the number of amplification cycles increases. The fluorescence intensity is thus proportional to the amount of amplification product.
Preferably, the length of the RPA probe is 46-54bp, dSpacer is adopted for modification at a position 32bp away from the 5 ' end in the probe, thymine (dT) at positions 31bp and 34bp away from the 5 ' end on both sides of dSpacer molecules are respectively replaced by a fluorescent group and a quenching group, and the 3 ' end of the probe also needs to be modified by a blocking group.
Preferably, FAM is used as the fluorescent group, BHQ1 is used as the quenching group, and C3Spacer is used as the blocking group.
The invention also provides a method for using the RPA primer probe for detecting the naked-head fish for non-disease diagnosis and treatment purposes, which comprises the following steps:
step A, adding an RPA primer probe of the naked goby and nucleic acid extracted from a sample into an RPA reaction solution; b, amplifying by using a constant-temperature amplification instrument, simultaneously carrying out real-time fluorescence acquisition, and obtaining the detection condition of the components of the uncaria esculenta of the sample according to the change curve of the real-time fluorescence value;
step C, judging a result: fixing an upstream primer, combining the upstream primer with a downstream primer respectively, combining the upstream primer and the downstream primer with a probe, carrying out amplification reaction, and setting a blank control; and (3) judging and selecting a downstream primer by combining with an amplification result, then respectively combining with the three upstream primers, combining with a probe, carrying out amplification reaction again, setting a blank control, and finally determining the optimal upstream primer and downstream primer combination.
Preferably, the RPA reaction system comprises: 1 mu L of DNA template, 2.1 mu L of each of the upstream primer and the downstream primer of 10 mu mol/L, 0.6 mu L of probe of 10 mu mol/L, 29.5 mu L of hydration solution, 2.5 mu L of magnesium acetate solution of 280mmol/L, 12.2 mu L of sterilized deionized water and 50 mu L of total volume.
Preferably, in the step A, the reaction temperature in the RPA reaction liquid is 37-42 ℃ and the time is 15-25 min.
The RPA primers and probes are designed based on the NADH2 gene sequence of the bare-capped fish downloaded from an NCBI database, compared with the prior art, the detection method disclosed by the invention can complete detection within 15-20 minutes, can quickly detect whether a sample is the bare-capped fish, has strong specificity, and has important application value in the fields of entry and exit inspection, marine product supervision, food safety risk monitoring, food material authenticity identification, market supervision, on-site quick detection, factory incoming inspection and acceptance, enterprise self-inspection and the like. The application mode is that a sample is subjected to nucleic acid extraction, added into a prepared reaction system containing the specific RPA primer probe of the oil fish, and subjected to constant-temperature amplification at 37-42 ℃, so that the component of the naked-capped fish in the sample can be rapidly detected.
Drawings
FIG. 1 shows the amplification of positive control in the RPA detection method of naked gesso.
FIG. 2 schematic diagram of blank control amplification of bare lid RPA detection method.
FIG. 3 is a graph showing the results of 100% naked gaeumannomyces detection.
FIG. 4 is a graph showing the results of the detection of the content of naked gesso 30%.
FIG. 5 is a graph showing the results of 10% naked gaeumannomyces detection.
FIG. 6 is a graph showing the results of 5% naked gaeumannomyces detection.
FIG. 7 is a graph showing the results of 2% detection of naked gaeumannomyces.
FIG. 8 is a graph showing the results of 1% detection of naked gaeumannomyces.
FIG. 9 is a graph showing the results of 0.5% detection of naked gaeumannomyces.
Detailed Description
Preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings:
example 1
1. Naked goby detection RPA primer probe
The RPA primer probe for detecting the naked capers is designed based on NADH2 gene of the naked capers, the length of the RPA probe is 54bp, dSpacer modification is adopted at a position 32bp apart from a 5 ' end in the probe, thymine (dT) at positions 31bp and 34bp apart from the 5 ' end on two sides of dSpacer molecules are respectively replaced by a fluorescent group FAM and a quenching group BHQ1, and the 3 ' tail end of the probe also needs to be modified by a blocking group C3Spacer, and the sequence is as follows:
(1) the upstream primer SEQ ID No. 1:
5’-TTCGCCAGCTCTCACTGACTACTCGCATGAATGGG-3’
(2) the downstream primer is SEQ ID No. 2:
5’-TCCTGTGGCTTGTGTGAGGAAGTACTTGGTGGAGG-3’
(3) probe SEQ ID No. 3:
5’-TCGAAATGAACACGCTTGCTATTATCCCCTTAATAGCACAGCATCACCACCCAC-3’
2. working mode
2.1 operating procedure
(1) Adding an RPA primer probe of the naked goby and nucleic acid extracted from a sample into the RPA reaction solution;
(2) amplifying by a constant temperature amplification instrument, simultaneously carrying out real-time fluorescence acquisition, and obtaining the detection condition of the components of the uncaria in the sample within 20min according to the change curve of the real-time fluorescence value.
2.2 RPA reaction System, as shown in Table 1
TABLE 1
2.3 RPA reaction procedure
37-42 deg.C (preferably 39 deg.C), and 15-25min (preferably 20 min).
3. Specific primer probe design and screening
Based on the gene sequence (JQ088484.1) of the uncaria fries in the NCBI website, a conserved nucleic acid sequence is searched by comparing with the common adulterated fish sequence, and the nucleic acid sequence is compared in the NCBI website GenBank database to verify the specificity of the nucleic acid sequence. Probes are designed on the specific sequences, and 3 upstream primers and 3 downstream primers are respectively designed around the probe sequences. The length of the probe is 54bp, dSpacer modification is adopted at a position 32bp away from the 5 ' end in the probe, thymine (dT) at positions 31bp and 34bp away from the 5 ' end on both sides of dSpacer molecules are respectively replaced by a fluorescent group FAM and a quenching group BHQ1, and the 3 ' end of the probe also needs to be modified by a blocking group C3 Spacer.
Screening was performed in the following manner: fixing the upstream primer, combining with the downstream primers, combining with the probe, performing amplification reaction, and setting blank control. And (3) judging and selecting a downstream primer by combining with an amplification result, then respectively combining with the three upstream primers, combining with a probe, carrying out amplification reaction again, setting a blank control, and finally determining the optimal upstream primer and downstream primer combination.
Optimal combined positive control and blank amplification plots are shown in FIGS. 1 and 2.
4. Experimental testing of specificity
The specificity of the primer probe for the RPA primer of the naked gesso is tested by respectively using commonly confused or adulterated 7 fish (naked gesso, Antarctic canine, oily fish, scaly and strong cod, long tail without cod, pollock and pollock) and DNA extracted from 28 fishes, 6 shrimps, 5 crabs and sandworms. The test result shows that the naked-covered fish can be specifically detected.
TABLE 2 List of specific experimental species
5. Detection limit test
Samples with the content of the naked gesso being 100%, 30%, 10%, 5%, 2%, 1% and 0.5% respectively are prepared and tested for detection limit. Through detection, when the content is 1%, a typical amplification curve is provided, and the fluorescence intensity can reach more than 1000 mv. As shown in fig. 3-9.
The RPA technology has the advantages of simple and convenient operation, quick reaction, high sensitivity, strong specificity, no need of precise instruments and the like. Compared with the prior art, the detection method provided by the invention can complete detection within 15-20 minutes, and can quickly detect whether the sample is naked-covered fish. The method has the advantages that the production enterprises can monitor and detect the incoming materials, the benefits of the enterprises are maintained, the food safety risks of the enterprises are reduced, and the loss of the benefits of the enterprises is reduced. Meanwhile, the method is convenient for on-site law enforcement and rapid detection, and provides technical support for government supervision.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Sequence listing
<110> Shenzhen city measurement quality inspection research institute (national high and new technology measurement station, national digital electronic product quality supervision and inspection center)
<120> RPA primer, probe and detection method for detecting naked gesso
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>35
<212>PRT
<213> bare fish (Anoplopoma fimbria)
<400>1
Thr Thr Cys Gly Cys Cys Ala Gly Cys Thr Cys Thr Cys Ala Cys Thr
1 5 10 15
Gly Ala Cys Thr Ala Cys Thr Cys Gly Cys Ala Thr Gly Ala Ala Thr
20 25 30
Gly Gly Gly
35
<210>2
<211>35
<212>PRT
<213> bare fish (Anoplopoma fimbria)
<400>2
Thr Cys Cys Thr Gly Thr Gly Gly Cys Thr Thr Gly Thr Gly Thr Gly
1 5 10 15
Ala Gly Gly Ala Ala Gly Thr Ala Cys Thr Thr Gly Gly Thr Gly Gly
20 25 30
Ala Gly Gly
35
<210>3
<211>54
<212>PRT
<213> bare fish (Anoplopoma fimbria)
<400>3
Thr Cys Gly Ala Ala Ala Thr Gly Ala Ala Cys Ala Cys Gly Cys Thr
1 5 10 15
Thr Gly Cys Thr Ala Thr Thr Ala Thr Cys Cys Cys Cys Thr Thr Ala
20 2530
Ala Thr Ala Gly Cys Ala Cys Ala Gly Cys Ala Thr Cys Ala Cys Cys
35 40 45
Ala Cys Cys Cys Ala Cys
50
Claims (6)
1. An RPA primer probe for detecting naked-cap fish, characterized in that the primer is based on the naked-cap fishNADH 2 The gene is designed, and the sequences of the primer and the probe are respectively as follows:
(1) the upstream primer SEQ ID No. 1:
5’- TTCGCCAGCTCTCACTGACTACTCGCATGAATGGG -3’
(2) the downstream primer is SEQ ID No. 2:
5’- TCCTGTGGCTTGTGTGAGGAAGTACTTGGTGGAGG -3’
(3) probe SEQ ID No. 3:
5’-TCGAAATGAACACGCTTGCTATTATCCCCTTAATAGCACAGCATCACCACCCAC -3’ 。
2. the RPA primer probe for detecting naked goby fish according to claim 1, wherein the RPA probe has a length of 46-54bp and is modified with dSpacer at a position 32bp apart from the 5 ' end in the probe, thymine (dT) at positions 31bp and 34bp apart from the 5 ' end on both sides of the dSpacer molecule is substituted with a fluorescent group and a quencher group, respectively, and is modified at the 3 ' end of the probe with a blocking group.
3. The RPA probe for detecting naked goby according to claim 2, wherein the fluorescent group is FAM, the quencher group is BHQ1, and the blocking group is C3 Spacer.
4. A method of use for non-disease diagnostic and therapeutic purposes using the RPA primer probe for the detection of decapitation fish according to claim 1, comprising the steps of:
step A, adding an RPA primer probe of the naked goby and nucleic acid extracted from a sample into an RPA reaction solution;
b, amplifying by using a constant-temperature amplification instrument, simultaneously carrying out real-time fluorescence acquisition, and obtaining the detection condition of the components of the uncaria esculenta of the sample according to the change curve of the real-time fluorescence value;
step C, judging a result: fixing an upstream primer, combining the upstream primer with a downstream primer respectively, combining the upstream primer and the downstream primer with a probe, carrying out amplification reaction, and setting a blank control; and (3) judging and selecting a downstream primer by combining with an amplification result, then respectively combining with the three upstream primers, combining with a probe, carrying out amplification reaction again, setting a blank control, and finally determining the optimal upstream primer and downstream primer combination.
5. The method of claim 4, wherein said RPA reaction system comprises: 1 mu L of DNA template, 2.1 mu L of each of the upstream primer and the downstream primer of 10 mu mol/L, 0.6 mu L of probe of 10 mu mol/L, 29.5 mu L of hydration solution, 2.5 mu L of magnesium acetate solution of 280mmol/L, 12.2 mu L of sterilized deionized water and 50 mu L of total volume.
6. The method of claim 4, wherein in step A, the reaction temperature in the RPA reaction solution is 37-42 ℃ and the reaction time is 15-25 min.
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Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967816A (en) * | 2017-04-25 | 2017-07-21 | 暨南大学 | Detect the RPA specific primers of vibrio parahaemolytious and kit and application in food |
| WO2017210542A1 (en) * | 2016-06-03 | 2017-12-07 | The Children's Medical Center Corporation | Cross-genera target genome capture and analysis |
| CN107746879A (en) * | 2017-12-01 | 2018-03-02 | 深圳市计量质量检测研究院 | Detect RPA primers, probe, kit and the detection method of staphylococcus aureus |
| CN108034737A (en) * | 2017-12-26 | 2018-05-15 | 深圳市计量质量检测研究院 | For detecting the RPA primer and probes and detection method of Listeria monocytogenes |
| CN108588251A (en) * | 2018-06-29 | 2018-09-28 | 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） | RPA primers, probe and detection method for detecting salmonella |
| CN109735657A (en) * | 2019-02-02 | 2019-05-10 | 深圳出入境检验检疫局动植物检验检疫技术中心 | A kind of reagent, detection method and application for African swine fever virus detection |
| CN110564870A (en) * | 2019-10-22 | 2019-12-13 | 中国水产科学研究院黄海水产研究所 | Primer, probe and method for identifying naked-capped fish based on Cytb gene |
| CN110564869A (en) * | 2019-10-22 | 2019-12-13 | 中国水产科学研究院黄海水产研究所 | Primer, probe and method for identifying naked-cap fish based on 16S rRNA gene |
| CN110592239A (en) * | 2019-10-22 | 2019-12-20 | 中国水产科学研究院黄海水产研究所 | Primer, probe and method for identifying Antarctic canis based on Cyt b gene |
| CN110607377A (en) * | 2019-10-22 | 2019-12-24 | 中国水产科学研究院黄海水产研究所 | Primer, probe and method for identifying Antarctic canine fish based on 16S rRNA gene |
-
2020
- 2020-06-18 CN CN202010559761.6A patent/CN111793696B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017210542A1 (en) * | 2016-06-03 | 2017-12-07 | The Children's Medical Center Corporation | Cross-genera target genome capture and analysis |
| CN106967816A (en) * | 2017-04-25 | 2017-07-21 | 暨南大学 | Detect the RPA specific primers of vibrio parahaemolytious and kit and application in food |
| CN107746879A (en) * | 2017-12-01 | 2018-03-02 | 深圳市计量质量检测研究院 | Detect RPA primers, probe, kit and the detection method of staphylococcus aureus |
| CN108034737A (en) * | 2017-12-26 | 2018-05-15 | 深圳市计量质量检测研究院 | For detecting the RPA primer and probes and detection method of Listeria monocytogenes |
| CN108588251A (en) * | 2018-06-29 | 2018-09-28 | 深圳市计量质量检测研究院（国家高新技术计量站、国家数字电子产品质量监督检验中心） | RPA primers, probe and detection method for detecting salmonella |
| CN109735657A (en) * | 2019-02-02 | 2019-05-10 | 深圳出入境检验检疫局动植物检验检疫技术中心 | A kind of reagent, detection method and application for African swine fever virus detection |
| CN110564870A (en) * | 2019-10-22 | 2019-12-13 | 中国水产科学研究院黄海水产研究所 | Primer, probe and method for identifying naked-capped fish based on Cytb gene |
| CN110564869A (en) * | 2019-10-22 | 2019-12-13 | 中国水产科学研究院黄海水产研究所 | Primer, probe and method for identifying naked-cap fish based on 16S rRNA gene |
| CN110592239A (en) * | 2019-10-22 | 2019-12-20 | 中国水产科学研究院黄海水产研究所 | Primer, probe and method for identifying Antarctic canis based on Cyt b gene |
| CN110607377A (en) * | 2019-10-22 | 2019-12-24 | 中国水产科学研究院黄海水产研究所 | Primer, probe and method for identifying Antarctic canine fish based on 16S rRNA gene |
Non-Patent Citations (4)
| Title |
|---|
| "《中国兽医学报》2019年第39卷总目次", 《中国兽医学报》, no. 12, 15 December 2019 (2019-12-15), pages 211 - 218 * |
| CENK GÜREVIN等: "Predicting the effects of sediment based internal nutrient loads on eutrophication in Kücükcekmece Lagoon for rehabilitation planning", 《INTERNATIONAL JOURNAL OF SEDIMENT RESEARCH》, no. 04, 15 December 2017 (2017-12-15), pages 67 - 94 * |
| NULL: ""鳕鱼及其制品中裸盖鱼、油鱼和南极犬牙鱼源性成分检测BJS201907"", 《中国食品卫生杂志》, no. 02, 30 March 2019 (2019-03-30), pages 105 * |
| 李婷婷等: "肉及肉制品掺假鉴别技术研究进展", 《食品安全质量检测学报》, no. 02, 25 January 2018 (2018-01-25), pages 409 - 415 * |
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