CN113801942A - LAMP technology-based method for rapidly identifying puffer fish and products thereof - Google Patents

Abstract

The invention relates to the field of biological detection, in particular to a rapid identification method of puffer fish and products thereof based on LAMP technology, and a primer pair and a kit used by the method. The invention is based onCOITwo specific LAMP primer groups (primers LAMPCoi and LAMPCR) are respectively designed according to the sequence and the D-loop sequence. The experimental results show that: the primer LAMPCoi can amplify 6 globefish species in the genus of eastern globefish, and does not react with 2 globefish species and 7 globefish samples in the genus of non-eastern globefish; the primer LAMPCR only reacts specifically with the species of Takifugu obscurus and does not react with other samples of Takifugu obscurus and non-Takifugu obscurus. The sensitivity of both sets of primers was 0.1% with a limit of detection of 10 pg. Two sets of primers were used for species identification of commercial products: in 30 kinds of Fugu obscurus preparations, the content of Fugu obscurus species was detected, and No. 12-14 and No. 21-30 samples were usedIdentifying as Fugu obscurus; of the 20 unlabeled fish products, only sample Nos. 17-20 detected a component containing Takifugu species.

Description

LAMP technology-based method for rapidly identifying puffer fish and products thereof

Technical Field

The invention relates to the field of biological detection, in particular to a primer pair and a kit used in a rapid identification method of puffer fish and products thereof based on LAMP technology.

Background

The puffer fish (Pufferfish) is used as a traditional food in coastal areas of Asia such as China and has very high nutritional value and economic value. However, the wild puffer fish has strong toxicity, and serious poisoning and even death can be caused by improper eating. The phenomenon that the puffer fish fills other aquatic products for sale exists in the market, and the life health of consumers is seriously damaged. The identification of puffer fish products is difficult only by morphological methods, so that a rapid and accurate molecular identification method of puffer fish species and products thereof is urgently needed to be established.

The food identification mainly comprises the traditional morphological method, physicochemical analysis and molecular biological method. After the deep-processed food has no morphology in the living state, the method of morphological identification cannot be used at all. The physicochemical analysis of food refers to the analysis of properties such as protein and fat by means of measuring tools or instruments, and common methods include enzyme-linked immunosorbent assay (ELISA), immunofluorescence technique (IFT), high performance liquid chromatography and the like. However, these methods often require relatively fresh samples, which can affect the results of the assay once the food is spoiled. In addition, non-destructive inspection techniques that are emerging in recent years are receiving increasing attention. The technology can detect the structure and properties of the surface or the interior of the food by utilizing optical, electromagnetic and acoustic technologies. For example: x-ray methods, terahertz techniques and infrared spectroscopy. Although these methods are fast, lossless and large in information quantity, the maintenance cost of the instruments and the later cost and technical threshold required for establishing and improving the models are too high.

Therefore, the Chinese invention patent application (CN108950007A, publication No. 20181207) applied by the applicant takes 4 kinds of common fugu obscurus in coastal areas of eastern China as research objects, namely, the fugu flavidus (Takifugu xanthopterus), the Takifugu obscurus (Takifugu fasciatus), the Takifugu flavidus (Takifuguflavidus) and the Takifugu rubripes (Takifugurubripes), selects and constructs a suitable DNA barcode COI sequence, and designs 2 pairs of HRM primers on the basis of the sequence, thereby providing the HRM rapid identification method of the fugu obscurus and other fish products based on the COI sequence.

Loop-mediated isothermal amplification (LAMP), proposed by Notomi et al in 2000, is a novel isothermal amplification technique for nucleic acids, i.e., rapid amplification of nucleic acids using a DNA polymerase with strand displacement function (Bst DNA polymerase) under isothermal conditions. The primer design mainly aims at 6 regions of the 5 'end and the 3' end of the target fragment to design 2 pairs of specific primers, including 1 pair of inner primers (FIP and BIP) and 1 pair of outer primers (F3 and B3). At a constant temperature of 60-65 ℃, the inner primer recognizes and extends a corresponding site, and simultaneously the outer primer displaces a strand formed by the inner primer under the action of Bst DNA polymerase to form a DNA fragment with a dumbbell-shaped structure, so as to provide a template for subsequent reaction. Then the DNA fragments are identified and extended by the inner primer, and finally a series of DNA fragment mixtures with stem-loop structures and multi-loop cauliflower structures are formed.

In the field of foods of animal origin, particularly foods such as meat of livestock and poultry, a large number of studies have confirmed that the LAMP technique is practicable. Wang et al (WANG J, WAN Y, CHEN G, et al. colorimetric Detection of Horse Meat composition, LAMP) J. Food Analytical Methods, 2019,12(11): 2535-. Zhang et al (ZHANG C, ZHANG X, LIAO G, et al, Specifics-specific TM-LAMP and Trident-like fluorescence biosensor for on-site automation detection of horse and dog mean [ J ]. Sensors and actors B-Chemical,2019,301:127039.) established a method for field detection of horse and donkey meat based on TM-LAMP (tag-labeled multiplex loop-mediated isothermal amplification) and LFB (linear flow biosensor), with detection limits as low as 40pg, which can be done at 40 min. Specific LAMP primers are respectively designed for Cytb genes of pig, cattle, chicken and duck in Tanga, and the like, an LAMP detection method for animal-derived components in edible vegetable oil and kitchen waste oil is established, and animal-derived components with the content of 1% and 5% in animal fat and kitchen waste oil can be respectively detected. Girish et AL (Girish P S, Barbuddhe S B, Kumari A, et AL. Rapid detection of position using alkali soluble-Loop medical Isothermal Amplification (AL-LAMP) detection [ J ]. Food Control, 2020,110:107015.) established a LAMP detection method of pork components based on D-Loop sequence, with a sensitivity of 0.5ng/μ L, which can detect 0.1% of pork components in a mixed sample, rapidly extract DNA by an alkaline lysis method, and shorten the detection time to 120min in combination with LAMP detection, even if the sample is subjected to high temperature treatment, the method is also applicable. Xiyu Yin, etc. (Xian Yu Yin, Yixue Ying, Zhanghuang, etc.. Loop-mediated isothermal amplification technology for quickly detecting bovine-derived components [ J ] in meat and meat products, 2016, 37(7):278 and 282.) food industry science and technology, an LAMP detection method for detecting bovine-derived components in meat and meat products is established by taking bovine-derived mitochondrial Cytb gene as a template, 12 kinds of beef and beef products in the market are detected, and the detection limit is 0.1 percent. In the aspect of aquatic products, the application of LAMP technology is less. The LAMP rapid identification method of Atlantic cod is developed by Saull et al (Saull J, Duggan C, Hobbs G, et al. the detection of the use of loop-mediated isothermal amplification in connection with a simplified DNA extraction process [ J ]. Food Control,2016,59: 306-313.), and the detection time of samples is greatly shortened by combining with simple DNA pretreatment. And the sensitivity is high, and 0.1% of cod components in the mixed sample can be detected. Spielmann et al (Spielmann G, Ziegler S, Haszpranar G, et al. use loop-mediated isothermal amplification for fast reactions amplification in oils, with specific reference to the European animals [ J ]. Food Control,2019,101: 156. 162.) established a rapid identification method for eel species and eel LAMP based on Cytb and D-loop sequences, respectively, which can be used for identification of eel eggs. The detection limit of both methods reaches 500pg without false positive reaction. Tatuli et al (Tatuli G, center P, Magnetii D, et al. A Rapid Colorimetric analysis for On-Site Authentication of centrifugal specifices [ J ]. Biosensors-Basel,2020,10(12):1-8.) combine Rapid DNA extraction with LAMP technology to achieve Rapid identification of Cephalopod Species at 30 min. There are no reports on puffer fish species.

Disclosure of Invention

In order to solve the above technical problems, a first object of the present application is to provide a method for rapidly identifying puffer fish and products thereof based on LAMP technology, which is different from DNA barcode and HRM analysis, has simple LAMP reaction conditions, high speed and high sensitivity, can be completed without using complicated large-scale instruments, and can be widely applied to field detection of puffer fish and products thereof.

In order to achieve the above object, the present application adopts the following technical solutions:

a method for rapidly identifying puffer fish and products thereof based on LAMP technology comprises the following steps:

1) extracting DNA of the puffer fish sample by using a marine animal tissue genome DNA extraction kit, and storing the prepared genome solution at-20 ℃;

2) designing 1 group of primers aiming at the takifugu species based on the COI sequence; or 1 group of primers aiming at the takifugu obscurus are designed based on the D-loop sequence;

primer sequences based on the COI sequence are as follows:

the primer sequences based on the D-loop sequence are as follows:

3) the LAMP reaction adopts a 20-mu-L reaction system, and comprises the following specific components:

4) before the reaction, 20 microliter of paraffin oil is added on the reaction liquid surface; the reaction conditions are as follows: reacting at 65 deg.C for 60min, and inactivating at 80 deg.C for 10 min;

5) detection of the LAMP reaction product takes one of two modes:

firstly, electrophoresis detection; preparing 2% agarose gel with 1 × TAE buffer solution, mixing 10 μ L reaction product with 2 μ L6 × locking buffer, adding into sample loading hole, and performing 120V electrophoresis for 30 min; after electrophoresis is finished, observing by a gel imaging system, and if a gradient band exists, the reaction is positive;

detection by a dye method: before LAMP reaction, 5 mu L of 100 XSSYBR Green I dye is dripped on the tube cover or the tube wall of the PCR tube, and the mixture is uniformly mixed after the reaction is finished, wherein the reaction is positive if the color is changed from orange to Green.

Further, the invention also provides a primer pair for quickly identifying the puffer fish and the products thereof by the LAMP technology, wherein the primer sequence of the primer pair is as follows:

the primer pair is designed based on a COI sequence and aims at the Takifugu spp.

Further, the invention also provides a primer pair for quickly identifying the puffer fish and the products thereof by the LAMP technology, wherein the primer sequence of the primer pair is as follows:

the primer pair is designed according to a D-loop sequence and aims at the fugu obscurus.

Further, the invention also provides a kit for quickly identifying the puffer fish and the products thereof by the LAMP technology, and the kit comprises the primer pair.

According to the invention, due to the adoption of the technical scheme, two groups of specific LAMP primer groups (primers LAMPCoI and LAMPCR) are respectively designed based on the COI sequence and the D-loop sequence. The experimental results show that: the primer LAMPCoi can amplify 6 species of Takifugu species, and does not react with 2 species of Takifugu and 7 species of Takifugu; the primer LAMPCR only reacts specifically with the species of Takifugu obscurus and does not react with other samples of Takifugu obscurus and non-Takifugu obscurus. The sensitivity of both sets of primers was 0.1% with a limit of detection of 10 pg. Two sets of primers were used for species identification of commercial products: in 30 kinds of fugu preparations, the content of the fugu species was detected, and the samples No. 12-14 and No. 21-30 were identified as fugu obscurus; of the 20 unlabeled fish products, only sample Nos. 17-20 detected a component containing Takifugu species. In addition, SYBR Green I dye is adopted to visualize the LAMP reaction result. In order to avoid the influence of uncapping detection and dye on the reaction, the dye is dripped on the tube cap or the tube wall before the reaction, and is mixed with the reaction liquid after the reaction is finished to observe the color change. The result proves that the method is simple to operate, obvious in visualization effect and very suitable for detecting field conditions.

Drawings

FIG. 1 shows the results of the specificity test of the primer LAMPCoi; note: A. b is an electropherogram, and C, D is a dye visualization result. M: 100bp DNA Ladder; n: negative control; 1-8 are Fugu obscurus, Fugu rubripes, Fugu flavipes, Fugu asteriskei, Fugu arcuatus, Fugu bimaculatus, Fugu flavipes, and Fugu huashanensis, respectively; 9-15 are pork, beef, mutton, chicken, duck meat, shrimp meat and large yellow croaker meat respectively.

FIG. 2 shows the results of the specificity test of the primer LAMPCR. Note: A. b is an electropherogram, and C, D is a dye visualization result. M: 100bp DNA Ladder; n: negative control; 1-8 are Fugu obscurus, Fugu rubripes, Fugu flavipes, Fugu asteriskei, Fugu arcuatus, Fugu bimaculatus, Fugu flavipes, and Fugu huashanensis, respectively; 9-15 are pork, beef, mutton, chicken, duck meat, shrimp meat and large yellow croaker meat respectively.

FIG. 3 shows the results of sensitivity tests of primers LAMPCoi and LAMPCR; note: m: 100bp DNA Ladder; n: negative control; 1-9, DNA content is 100%, 50%, 25%, 10%, 5%, 1%, 0.5%, 0.1%, 0.01%.

FIG. 4 shows the results of limit tests for primers LAMPCoi and LAMPCR; note: m: 100bp DNA Ladder; n: negative control; the DNA content of 1-7 is 100ng, 10ng, 1ng, 100pg, 10pg, 1pg, 0.1pg, respectively.

FIG. 5 shows the experimental results of the rapid crude DNA extraction method; note: m: 100bp DNA Ladder; n: the negative control was undiluted at 1X, diluted 10X 10 times, diluted 100X 100 times and diluted 1000X 1000 times.

FIG. 6 shows LAMP detection results of commercially available puffer fish products; note: n: negative control; 1-30: 30 kinds of pufferfish products.

FIG. 7 shows LAMP detection results of 20 unlabeled fish products; note: n: negative control; 1-20: 20 samples of unlabeled fish products.

Detailed Description

Materials and methods

1. Materials, reagents and apparatus

Experimental materials: 8 kinds of Fugu ocellatus samples (Fugu obscurus, Fugu rubripes, Fugu flavipes, Fugu asteroides, Fugu arcuatus, Fugu bimaculatus, Fugu flavipes and Fugu huashanensis) and 30 kinds of commercial Fugu ocellatus products (2.1.1) and 7 kinds of non-Fugu ocellatus meat (pork, beef, mutton, chicken, duck, shrimp and large yellow croaker meat) and 20 kinds of no-mark fish products (3.1.1). The instrument comprises the following steps: the biological sample homogenizer (Bioprep-24) was purchased from Osheng instruments, Hangzhou, and the other related instruments were 2.1.1. Reagent: CTAB lysate (2% CTAB, 0.02M Na2EDTA, 1.4M NaCl, 2% PVP, 0.1M Tris-HCl pH 8.2), 0.5M NaOH lysate (0.5M NaOH, 10mM Na2EDTA), 3M guanidine hydrochloride lysate (3M guanidine hydrochloride, 0.1M Tris-HCl, 0.05M Na2EDTA, pH 6.4), Bst DNA Polymerase Large Fragment from Nanjing Nozan Biotech GmbH, SYBR green I (PCR grade) from Beijing Sorbero Tech Co., Ltd., dNTP Mixture from Takara Japan, betaine, paraffin oil, etc.

Second, Experimental methods

2.1 Rapid extraction of DNA

The DNA extraction of the puffer fish sample is carried out by using a marine animal tissue genome DNA extraction kit. Taking 0.05g of the flesh on the back of the puffer fish, grinding the puffer fish into powder by using liquid nitrogen, and extracting by referring to the kit use instruction; the DNA extraction of the puffer fish product adopts CTAB reagent cracking and phenol-chloroform extraction, and the specific method is as follows:

1) taking 0.05g of a sample, grinding the sample into powder in a mortar by using liquid nitrogen, and transferring the powder into a 2mL centrifuge tube;

2) adding 1mL of CTAB lysate, 20 mu L of protease K (20mg/mL) solution and 4 mu L of RNase A (100mg/mL) solution into a centrifuge tube, uniformly mixing, and placing in a 56 ℃ water bath until the tissues are completely lysed;

3) to the well-lysed solution was added 500. mu.L of phenol/chloroform iso/pentanol (25:24:1), shaken and centrifuged at 12500rpm for 10min at 4 ℃. The centrifuged solution is divided into three layers, the upper layer solution is carefully transferred into another sterile centrifuge tube, 500 mu L chloroform/isoamylol (24:1) is added, the mixture is evenly stirred and centrifuged at 12500rpm for 10min at 4 ℃;

4) transferring the supernatant into another sterile centrifuge tube, adding isopropanol with the same volume, shaking and mixing uniformly, centrifuging at 4 ℃ and 12500rpm for 10min, and removing the supernatant;

5) adding 500 μ L70% ethanol solution into the precipitate, oscillating, centrifuging at 4 deg.C and 12500rpm for 5min, and removing the supernatant; adding 500 μ L of anhydrous ethanol, oscillating, centrifuging at 4 deg.C and 12500rpm for 5min, and removing supernatant;

6) placing the centrifugal tube on absorbent paper upside down, and drying at room temperature;

7) after drying, 50-100. mu.L of sterile water or TE solution is added to dissolve the DNA.

The prepared genome solution is detected by a nucleic acid protein analyzer and stored at the temperature of minus 20 ℃.

In order to further shorten the time required by the detection of the commercial puffer fish products, a DNA rapid extraction method is adopted to simplify the flow of pretreatment of samples. Firstly, screening a lysate for quickly extracting puffer fish DNA:

weighing 0.05g of Fugu obscurus muscle in a 2mL centrifuge tube, and respectively adding 500 μ L of 0.5M NaOH lysate, 3M guanidine hydrochloride lysate and CTAB lysate;

adding 20 grinding beads of 1mm into a centrifuge tube, placing in an automatic homogenizer, shaking at 4m/s for 1min, and standing for 9 min;

after brief centrifugation, 50. mu.L of the supernatant was aspirated, and ddH2O was added for 10-fold gradient dilution (1X, 10X, 100X, 1000X);

the resulting dilution was immediately subjected to LAMP amplification (reaction system and conditions were the same as 4.1.2.2) and the results were observed by electrophoresis.

2.2 LAMP primer design

The invention designs 1 group of primers aiming at the takifugu species based on the COI sequence, and designs 1 group of primers aiming at the takifugu obscurus based on the D-loop sequence, wherein the primer sequences are shown in a table 1.

TABLE 1 LAMP primer Table

2.3 LAMP reaction System and product detection

The LAMP reaction adopts a 20-mu-L reaction system, and the specific components are shown in Table 2. Before starting the reaction, 20. mu.L of paraffin oil was added to the reaction solution. The reaction conditions are as follows: reacting at 65 deg.C for 60min, and inactivating at 80 deg.C for 10 min. The detection of LAMP reaction products adopts two modes: firstly, electrophoresis detection; prepare 2% agarose gel with 1 × TAE buffer solution, mix 10 μ L reaction product with 2 μ L6 × locking buffer, add to the loading well, and perform 120V electrophoresis for 30 min. After electrophoresis is finished, observing by a gel imaging system, and if a gradient band exists, the reaction is positive; detection by a dye method: before LAMP reaction, 5 mu L of 100 XSSYBR Green I dye is dripped on the tube cover or the tube wall of the PCR tube, and the mixture is uniformly mixed after the reaction is finished, wherein the reaction is positive if the color is changed from orange to Green.

TABLE 2 LAMP reaction System

2.4 specificity of LAMP reaction

To verify whether the primers can specifically detect the target species without generating false positive amplification on other DNAs, LAMP specificity tests were performed on DNAs of 8 kinds of Fugu samples (Fugu obscurus, Fugu rubripes, Fugu flavipes, Fugu asteriskei, Fugu arcuatus, Fugu bimaculatus, Fugu ventralis, and Fugu huashanensis) and 7 kinds of non-Fugu meat samples (pork, beef, mutton, chicken, duck, shrimp, and large yellow croaker meat). The two sets of primers were used to perform LAMP reactions on the DNA of 15 species with the reaction system and conditions described in 2.3, and the results were verified by fluorescence and electrophoresis, respectively.

2.5 sensitivity and detection Limit of LAMP reaction

The LAMP reaction sensitivity test is carried out by taking a mixed DNA solution of the takifugu obscurus and the large yellow croaker as a detection object. The takifugu obscurus DNA with the concentration of 50 ng/. mu.L and the large yellow croaker DNA are mixed according to the proportion of 100%, 50%, 25%, 10%, 5%, 1%, 0.5%, 0.1% and 0.01%, and are respectively reacted with two groups of LAMP primers, and the amplification result is verified by an electrophoresis method.

The LAMP reaction detection limit is tested by taking the DNA of the takifugu obscurus as a detection object. After the standard DNA solution is diluted by a 10-fold gradient, two groups of primers are respectively used for LAMP reaction, so that the DNA content in the LAMP reaction system is respectively 100ng, 10ng, 1ng, 100pg, 10pg, 1pg and 0.1 pg. The SYBR Green I dye method is not obvious in the LAMP reaction of the DNA template with extremely low concentration, and is easy to cause misjudgment, so the result of the detection limit experiment is verified by the electrophoresis method.

2.6. LAMP detection of commercial puffer fish products and unlabeled fish products

In order to simplify the process of DNA extraction, the method is more suitable for field detection. The DNA rapid extraction method obtained by screening in 2.1 is used for roughly extracting the DNA of 30 kinds of commercial puffer fish products and 20 kinds of unlabelled fish products, LAMP reaction is carried out by adopting the LAMP reaction conditions and the primers, the result is checked by a dye visualization method, and whether puffer fish components are contained or whether labels are not matched is judged according to the reaction result.

Third, results and analysis

3.1 specificity test results of LAMP

The experiment uses DNA extracted from 8 kinds of puffer fish species and 7 kinds of non-puffer fish meat as template, and verifies the specificity of two sets of LAMP primers LAMPCoi and LAMPCR, the result is shown in figure 1 and figure 2. Electrophoresis and dye visualization results show that the reaction product of the primer LAMPCoi and 6 Takifugu species presents gradient-shaped bands in gel electrophoresis, the color of the dye visualization result changes from orange yellow to yellow green, and the dye visualization result does not react with the Fugu flavidus and the Fugu huashanensis, and the result is negative; in the reaction of 7 non-puffer fish meats, no positive reaction occurred in all samples. The primer LAMPCR only shows positive results in the DNA of the Fugu obscurus, and the primer LAMPCR does not show positive results in other 7 kinds of Fugu obscurus and 7 kinds of non-Fugu obscurus meat. The primer LAMPCoi has good specificity to the species of the Takifugu genus, and can not amplify the Takifugu other than the Takifugu genus or other species; the primer LAMPCoi can specifically amplify the species of the takifugu obscurus.

3.2 sensitivity test results of LAMP

In order to verify the detection sensitivity of the method, the LAMP reaction is carried out by taking mixed DNA of the takifugu obscurus and the large yellow croaker in different proportions as a simulation sample, and the experimental result is shown in figure 3. The reaction results of the two groups of primers are similar, and obvious gradient-shaped bands appear in the reaction with the DNA content of the takifugu obscurus of 100-0.5 percent. At a DNA content of 0.1%, the intensity of the band decreased, but a positive reaction could still be observed. On the other hand, when the DNA content was 0.01%, almost no electrophoretic band was observed. Therefore, the sensitivity of this method is considered to be 0.1%.

3.3 detection Limit test results for LAMP

In order to determine the detection limit of the two groups of LAMP primers, the DNA of the takifugu obscurus is taken as a detection object, a standard DNA solution is diluted by 10 times of gradient, and then the two groups of primers are respectively used for LAMP reaction, wherein the DNA content in each reaction system is respectively 100ng, 10ng, 1ng, 100pg, 10pg, 1pg and 0.1 pg. The results of the reaction were detected by electrophoresis, and are shown in FIG. 4. Both sets of primers showed similar reaction results, and a gradient-like band was observed at a DNA content of 100ng-10 pg. Whereas when the DNA content is less than 10pg, no significant amplification occurs. Therefore, the detection limit of the method on the Takifugu species and the Fugu obscurus species is 10 pg.

3.4 screening results of Rapid DNA crude extraction method

In order to simplify the DNA extraction process and shorten the detection time of commercial products, the dilution ratio of the lysate and the crude extract from the rapid crude extraction of DNA was investigated, and the results are shown in FIG. 5. LAMP positive reaction can be observed by comparing the crude extract and 10-fold diluent of the three lysates. The bands were lighter in the 0.5M NaOH group and the 3M guanidine hydrochloride group, and darker in the CTAB group. When the dilution factor is increased to 100 times or 1000 times, almost no electrophoretic band is observed in the CTAB group, while an electrophoretic band is also observed in the NaOH group or the guanidine hydrochloride group, but the brightness of the guanidine hydrochloride group is obviously reduced. Therefore, 0.5M NaOH solution was selected as the lysis solution for rapid crude DNA extraction from commercial products.

3.5 LAMP detection result of Fugu products on the market

30 kinds of commercial puffer fish products were tested by primers LAMPCoi and LAMPCR (see Table 3), and the visualization result of the dye is shown in FIG. 6. In the detection result of the primer LAMPCoi, all samples have color changes, and the reaction shows positive property, which indicates that all the puffer fish products contain the puffer fish components. In the detection result of the primer LAMPCR, only the samples No. 12-14 and No. 21-30 have positive reactions, which indicates that the samples contain the takifugu obscurus component; although the samples No. 1-11 and No. 17-20 did not contain the Fugu obscurus component, they contained other Fugu obscurus components except for Fugu obscurus as seen from the reaction result of the primer LAMPCoi. The above results are similar to the conclusions of the DNA barcode method and HRM analysis, and both are consistent with their commercial labels. Therefore, the LAMP method combined with the rapid extraction of DNA can be used for detecting the puffer fish ingredients in the puffer fish products.

3.6 LAMP detection result of tag fish product

20 commercially available unlabeled fish products were tested with primers LAMPCoi and LAMPCR (see Table 4), and the visualization results of the dyes are shown in FIG. 7. Wherein, the primer LAMPCR has no color change in all samples, and the result is negative, so that the 20 fish products do not contain the Fugu obscurus component. On the other hand, the sample No. 17-20 showed a positive result in the reaction with the primer LAMPCoi, indicating that it contains a component of Takifugu species, but the species contained therein cannot be determined. DNA barcode and HRM analysis verified that sample No. 17-20 was actually Fugu flavidus (CN108950007A, published as 20181207).

TABLE 330 LAMP reaction results of commercially available puffer fish products

Note: "+" indicates a positive result, and "-" indicates a negative result.

LAMP detection results of table 420 kinds of tag-free fish products

Note: "+" indicates a positive result, and "-" indicates a negative result.

Fourth, discuss

The application is different from DNA bar codes and HRM analysis, the LAMP reaction has simple conditions, high speed and high sensitivity, and can be completed without a complex large instrument, so the LAMP reaction is widely applied to the field detection of various meat-source foods. The application designs and verifies that two sets of LAMP primers (LAMPCII and LAMPCR) capable of respectively detecting Takifugu species and Takifugu obscurus species have strong specificity, the sensitivity is 0.1 percent, and the detection limit is 10 pg. In addition, in order to further shorten the detection time of commercial samples, the LAMP reaction has the characteristic of strong tolerance to the inhibitor, and the LAMP reaction is used with a DNA rapid extraction method, so that the puffer fish components in the puffer fish products can be successfully detected within 1.5 h. Detecting 30 kinds of commercial puffer fish products and 20 kinds of fish products without labels by using the method, wherein the puffer fish products detect the species components of the eastern puffer, and the No. 12-14 and No. 21-30 samples are determined to contain the species components of the puffer fish; in the unlabeled fish meat product, sample No. 17-20 was detected to contain the species of Takifugu.

The LAMP reaction result judgment method also has great influence on the detection time and the detection efficiency. The current LAMP reaction result judging method comprises the following steps: electrophoresis, dye method, turbidity method, transverse flow test paper, micro-fluidic chip, etc. Although the electrophoresis method has reliable results, the electrophoresis method is not suitable for detecting the environment outside a laboratory, and aerosol pollution is easily caused because the cover is required to be opened for detection. Although simple, the turbidity method is difficult to compare the slight difference in turbidity with the naked eye, and often requires a turbidimeter for judgment. The invention adopts SYBR Green I dye to visualize the LAMP reaction result. In order to avoid the influence of uncapping detection and dye on the reaction, the dye is dripped on the tube cover or the tube wall before the reaction, and is mixed with the reaction liquid after the reaction is finished, and the color change is observed. The result proves that the method is simple to operate, obvious in visualization effect and very suitable for detecting field conditions.

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Claims (4)

1. A rapid identification method of puffer fish and products thereof based on LAMP technology is characterized by comprising the following steps:

1) extracting DNA of the puffer fish sample by using a marine animal tissue genome DNA extraction kit, and storing the prepared genome solution at-20 ℃;

2) designing 1 group of primers aiming at the takifugu species based on the COI sequence; or 1 group of primers aiming at the takifugu obscurus are designed based on the D-loop sequence;

primer sequences based on the COI sequence are as follows:

the primer sequences based on the D-loop sequence are as follows:

3) the LAMP reaction adopts a 20-mu-L reaction system, and comprises the following specific components:

4) before the reaction, 20 microliter of paraffin oil is added on the reaction liquid surface; the reaction conditions are as follows: reacting at 65 deg.C for 60min, and inactivating at 80 deg.C for 10 min;

5) detection of the LAMP reaction product takes one of two modes:

firstly, electrophoresis detection; preparing 2% agarose gel with 1 × TAE buffer solution, mixing 10 μ L reaction product with 2 μ L6 × locking buffer, adding into sample loading hole, and performing 120V electrophoresis for 30 min; after electrophoresis is finished, observing by a gel imaging system, and if a gradient band exists, the reaction is positive;

detection by a dye method: before LAMP reaction, 5 mu L of 100 XSSYBR Green I dye is dripped on the tube cover or the tube wall of the PCR tube, and the mixture is uniformly mixed after the reaction is finished, wherein the reaction is positive if the color is changed from orange to Green.

2. A primer pair for quickly identifying puffer fish and products thereof by LAMP technology is characterized in that the primer sequence of the primer pair is as follows:

the primer pair is designed based on a COI sequence and aims at the Takifugu spp.

3. A primer pair for quickly identifying puffer fish and products thereof by LAMP technology is characterized in that the primer sequence of the primer pair is as follows:

the primer pair is designed according to a D-loop sequence and aims at the fugu obscurus.

4. A kit for rapid identification of puffer fish and products thereof by LAMP technology, comprising the primer set according to claim 2 and/or 3.

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