CN111621576A - RPA primer and probe for detecting oil fish and detection method - Google Patents
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Abstract
The invention provides a set of RPA primers and probes for detecting oil fish, wherein the set of the primers and probes is based on the oil fishCytbThe gene design, its forward, reverse primer and probe are: (1) the upstream primer SEQ ID No. 1: 5'-CCCCCTCCAATATCTCAGTATGGTGAAATTTTGGG-3' (2) downstream primer SEQ ID No. 2: 5'-GACAGATGTGGGCTACGGAGGCAAAGGCTGATCCT-3' are provided. (3) Probe SEQ ID No. 3: 5'-GGTGAAATTTTGGGTCCCTACTAGGCCTCTGCCTAATTTCCCAAATCCTTAC-3' compared with the prior art, the detection method of the invention can complete detection within 15-20 minutes, and can rapidly detect whether the oil fish exists in the sample, so that the production enterprises can realize monitoring detection of the incoming materials by the method, the benefits of the enterprises are maintained, the food safety risk of the enterprises is reduced, and the loss of the benefits of the enterprises is reduced. Convenient on-site law enforcementAnd (4) quick detection, which provides technical support for government supervision.
Description
Technical Field
The invention belongs to the fields of biosafety technology and molecular biology, and particularly relates to an RPA primer and a probe for detecting oil fish and a detection method.
Background
The oil fish contains more waxy substances which are difficult to digest by human bodies, easily causes diarrhea symptoms, has larger food safety risk particularly for infants, and is particularly named as Keriorrhae abroad. The common names of the oil fishes are also called round cod, wax oil fish, dragon cod, water cod and white dolphin. Scomber belonging to family Scombridae of order Perciformes of class Finciformes in taxonomic order includes Epichthyes punctatus (Lepidocybium flavobrucinum) and Epichthyes heterophyllus (Ruvettus prediotis).
The oil fish belongs to large-scale fish in deep sea, generally removes the head and the tail when selling, cuts apart the small package and sells with modes such as fish piece, fish are taken off, and the oil fish is almost the same with cod, naked lid fish after the cutting, therefore can't distinguish through the sense organ morphology. The price of the oil fish is far lower than that of the cod and the naked-covered fish, so the adulterated sale is easy to carry out.
In view of the nature of the oil fish, japan and italy require that the sale and import of the fish be prohibited; in the guidance on identification and labeling of oil fish/Cod issued by hong Kong food safety center, all entrepreneur shall be entitled "wax oil fish" and "oil fish" for scombroid and heteroscombroid, and other common names including "Cod" or "Cod" cannot be used.
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 oil 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.
Disclosure of Invention
In order to solve the technical problems, the invention provides an RPA primer, a probe and a detection method for detecting oil fish.
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.
Specifically, the invention provides an RPA primer for detecting oil fish, which is designed based on Cytb gene of oil fish, and the forward primer and the reverse primer are respectively as follows:
(1) the upstream primer SEQ ID No. 1:
5’-CCCCCTCCAATATCTCAGTATGGTGAAATTTTGGG-3’
(2) the downstream primer is SEQ ID No. 2:
5’-GACAGATGTGGGCTACGGAGGCAAAGGCTGATCCT-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.
The invention provides an RPA probe for detecting oil fish, which is designed based on Cytb gene of oil fish, the length of the RPA probe of oil fish is 46-52bp, dSpacer is adopted to modify the position of 35bp apart from the 5 ' end in the probe, thymine (dT) at the positions 34bp and 37bp apart from the 5 ' end on both sides of dSpacer molecule are respectively replaced by fluorescent group FAM and quenching group BHQ1, and the 3 ' end of the probe is modified by blocking group C3 Spacer.
The RPA probe sequence was: SEQ ID No. 3:
5’-GGTGAAATTTTGGGTCCCTACTAGGCCTCTGCCTAATTTCCCAAATCCTTAC-3’。
the invention provides a method for using an RPA primer probe for non-disease diagnosis and treatment purposes, which comprises the following steps:
step A, adding an RPA primer probe of the oil fish 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 oily fish components of the sample according to the change curve of the real-time fluorescence value;
and C: and (4) judging a result: 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.
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.
The invention brings the following effects: 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 be used for rapidly detecting whether the oily fish exists in the sample. 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.
Drawings
FIG. 1 shows the amplification of positive control in the RPA detection method of oil fish.
FIG. 2 is a schematic diagram of blank control amplification of the oil fish RPA detection method.
FIG. 3 is a graph showing the results of the detection of the oil fish content of 100%.
FIG. 4 is a graph showing the results of 10% detection of the oil fish content.
FIG. 5 is a diagram showing the results of 5% detection of the oil fish content.
FIG. 6 is a graph showing the results of 2% detection of the oil fish content.
FIG. 7 is a graph showing the results of the detection of 1% of the oil fish content.
FIG. 8 is a graph showing the results of the detection of the oil fish content of 0.5%.
Detailed Description
Preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings:
example 1
1. Procedure for the preparation of the
(1) Adding an RPA primer probe of the oil fish and nucleic acid extracted from a sample into an RPA reaction solution;
(2) and amplifying by using a constant-temperature amplification instrument, simultaneously carrying out real-time fluorescence acquisition, and obtaining the detection condition of the oil fish components of the sample within 20min by using the change curve of the real-time fluorescence value.
RPA reaction system, as shown in table 1:
TABLE 1
RPA reaction procedure: 37-42 deg.C (preferably 39 deg.C), and 15-25min (preferably 20 min).
4. Specific primer probe design and screening
Based on the Channa Argus gene sequence (DQ197960.1, AP012506.1) in NCBI website, through the comparison with the common adulterated fish sequence, look for the conserved nucleic acid sequence, compare the nucleic acid sequence in NCBI website GenBank database in order to verify its specificity. 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 46-52bp, dSpacer is adopted for modification at a position 35bp away from the 5 ' end in the probe, thymine (dT) at positions 34bp and 37bp 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.
5. Experimental testing of specificity
The probe specificity of the primer RPA of the oil fish was tested by using commonly confused or adulterated 7 fish (naked-cover fish, Antarctic canine, oil fish, scaly and strong cod, pollock and pollock) and DNA extracted from 28 fish, 6 shrimp, 5 crab and sandworm, respectively. The test result shows that the oil fish can be specifically detected.
TABLE 2 List of specific experimental species
6. Detection limit test
Samples with oil fish contents of 100%, 10%, 5%, 2%, 1%, 0.5% were prepared and tested for detection limits. Through detection, when the content is 5%, a typical amplification curve is provided, and the fluorescence intensity can reach more than 4000 mv. As shown in fig. 3-8.
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 and probe for detecting oil fish and detection method
<160>3
<170>SIPOSequenceListing 1.0
<210>1
<211>35
<212>PRT
<213> oil fish (Keriorrhae)
<400>1
Cys Cys Cys Cys Cys Thr Cys Cys Ala Ala Thr Ala Thr Cys Thr Cys
1 5 10 15
Ala Gly Thr Ala Thr Gly Gly Thr Gly Ala Ala Ala Thr Thr Thr Thr
20 25 30
Gly Gly Gly
35
<210>2
<211>35
<212>PRT
<213> oil fish (Keriorrhae)
<400>2
Gly Ala Cys Ala Gly Ala Thr Gly Thr Gly Gly Gly Cys Thr Ala Cys
1 5 10 15
Gly Gly Ala Gly Gly Cys Ala Ala Ala Gly Gly Cys Thr Gly Ala Thr
20 25 30
Cys Cys Thr
35
<210>3
<211>52
<212>PRT
<213> oil fish (Keriorrhae)
<400>3
Gly Gly Thr Gly Ala Ala Ala Thr Thr Thr Thr Gly Gly Gly Thr Cys
1 5 10 15
Cys Cys Thr Ala Cys Thr Ala Gly Gly Cys Cys Thr Cys Thr Gly Cys
20 25 30
Cys Thr Ala Ala Thr Thr Thr Cys Cys Cys Ala Ala Ala Thr Cys Cys
35 40 45
Thr Thr Ala Cys
50
Claims (6)
1. An RPA primer for detecting oil fish, which is characterized in that the primer is based on oil fishCytbDesigned by gene, the forward primer and the reverse primer are respectively as follows:
(1) the upstream primer SEQ ID No. 1:
5’-CCCCCTCCAATATCTCAGTATGGTGAAATTTTGGG-3’
(2) the downstream primer is SEQ ID No. 2:
5’-GACAGATGTGGGCTACGGAGGCAAAGGCTGATCCT-3’。
2. an RPA probe for detecting oil fish, characterized in that the probe is based on oil fishCytbThe RPA probe of the oil fish is 46-52bp in length, dSpacer modification is adopted at a position 35bp apart from a 5 ' end in the probe, thymine (dT) at positions 34bp and 37bp 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 a blocking group C3Spacer modification is carried out at the 3 ' end of the probe.
3. The RPA probe of claim 2, having the sequence: probe SEQ ID No. 3:
5’-GGTGAAATTTTGGGTCCCTACTAGGCCTCTGCCTAATTTCCCAAATCCTTAC -3’。
4. a method of using the RPA primer probe of claim 2 for non-disease diagnostic and therapeutic purposes, comprising the steps of:
step A, adding an RPA primer probe of the oil fish 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 oily fish components of the sample according to the change curve of the real-time fluorescence value;
and C: and (4) 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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