CN116064838A - Primer group, kit and method for detecting oyster components in food - Google Patents

Primer group, kit and method for detecting oyster components in food Download PDF

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CN116064838A
CN116064838A CN202211353498.0A CN202211353498A CN116064838A CN 116064838 A CN116064838 A CN 116064838A CN 202211353498 A CN202211353498 A CN 202211353498A CN 116064838 A CN116064838 A CN 116064838A
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徐亚维
赵杨
董丽华
张馨予
孙旭哲
王尧
魏晓通
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Jilin Agricultural Science and Technology College
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Abstract

The invention provides a primer group, a kit and a method for detecting oyster components in food, which belong to the technical field of food detection, wherein the primer group comprises a first primer group or a second primer group; the first primer set comprises F1 and R1; the nucleotide sequence of F1 is shown as SEQ ID No.1, the nucleotide sequence of R1 is shown as SEQ ID No.2, and the second primer group comprises F2 and R2; the nucleotide sequence of F2 is shown as SEQ ID No.3, and the nucleotide sequence of R2 is shown as SEQ ID No. 4. The primer group, the kit and the detection method for detecting oyster components in food provided by the invention have the advantages of simplicity and rapidness in operation; the method is independent of the advantage of high-end equipment, has higher specificity and sensitivity, and the detection limit can reach fg grade, which is higher than that of the common PCR method.

Description

Primer group, kit and method for detecting oyster components in food
Technical Field
The invention belongs to the technical field of food detection, and particularly relates to a primer group, a kit and a method for detecting oyster components in food.
Background
Marine products are common allergic foods, and allergic diseases caused by crustaceans such as shrimps and mollusks such as oysters are common. Oyster (oyster) is rich in nutrition and has complete protein types, and is often used as an important food for human nutrition supplement, but eating oyster can cause allergic symptoms of human beings [ Cheng Qingli, li Guoming, zhao Xiaohan, and the like ], separation, purification and identification of oyster allergen troponin [ J ]. Food and fermentation industry, 2021,47 (16)): 15-21]. Moreover, oyster is the first most cultivated shellfish in the world, and is one of four cultivated shellfish in China, and particularly food allergy caused by shellfish represented by oyster is receiving more and more attention and importance along with the rapid increase of the production and consumption quantity of the shellfish [ Carrera M, pazos M, gasset M.proteomics-Based Methodologies for the Detectionand Quantification of Seafood Allergens [ J ]. Foods,2020,9 (8): 1134-1148 ].
Currently, the detection of marine ingredients is mainly performed by polymerase chain reaction (polymerase chain reaction, PCR) technology [ Miao G, zhang L, zhang J, et al, free continuity PCR: fromprinciple study to commercial applications-A critical review [ J ]. Anal Chim Acta,2020,108:177-197; PENG M, LUO B, ZHU WL. Comparison and Identification of DNA Sequences of Oysters in the Genus Crassostrea [ J ]. J Guangdong Ocean Univ,2020,40 (1): 29-37 ] and its derivative technique are represented by [ Peng Min, robang, zhu Weilin, etc. ] comparison and identification of oyster DNA sequences of the genus oyster [ J ]. Guangdong university of ocean, 2020,40 (1): 29-37 ]. Suh et al [ Suh SM, kim MJ, kim HI, et al A multiplex PCR assay combined with capillary electrophoresis for the simultaneous detection of tropomyosin allergens from oyster, mussel, abalone, and clam mollusk species [ J ]. Food Chem,2020,317:126451-126457 ] detected mollusks in seafood using multiplex PCR with a detection limit of 16pg. Liu Kaikai et al [ Liu Kaikai, li Qi ] mitochondrial DNA PCR-RFLP rapid identification of Crassostrea gigas and Crassostrea gigas [ J ]. University of ocean in China (Nature science edition), 2018,48 (S2): 20-25.] restriction fragment length polymorphism analysis (Restriction Fragment Length Polymorphism, RFLP) and PCR combination established PCR-RFLP detection techniques for the identification of oyster species. However, these nucleic acid detection methods are not highly sensitive, limiting their application. The real-time fluorescent quantitative PCR technology is widely applied to the detection of marine product components as a national standard technology in recent years, but the method and the digital PCR relate to the problem of relying on high-end equipment and professionals, and are not suitable for the requirements of on-site detection and detection in areas with lack of resources [ Daga C, cau S, tilocca MG, et al detection of fish allergen by droplet digital PCR [ J ]. Ital J Food Sci,2018,7:222-225 ].
In recent years, isothermal amplification techniques, represented by loop-mediated isothermal amplification (loop-mediated isothermal amplification, LAMP) [ SENA-TORRALBA A, PALLAS-TAMARIT Y, MORAIS S, et al, remote advances and challenges in food-borne allergen detection [ J ]. Trac-Trend Anal Chem,2020,132:116050] and recombinase polymerase amplification (recombinase polymerase amplification, RPA) techniques, have become new orientations in the detection field due to simple and rapid detection methods and the lack of expensive precision equipment. The existing application of LAMP in Food detection mainly aims at detection of peanut, wheat and the like [ Shea, SC, tsou PC, lien YY.et al.development of loop-mediated isothermal amplification (LAMP) assays for the rapid detection of allergic peanut in processed Food [ J ]. Food Chem,2018,257:67-74 ], few cases of detection report of marine product components [ Zhangxiang, miss, song Chunhong, and the like ]. Loop-mediated isothermal amplification technology is used for rapidly detecting Food allergen oyster components [ J ]. Food safety quality detection report, 2019,10 (7): 1804-1810 ] ]. Compared with the RPA technology, the LAMP is complicated in primer design and is easy to generate false positive phenomenon. RPA has the advantages of simplicity and rapidness in primer design and reaction, and is particularly suitable for on-site immediate detection [ Huang Tao, li Dandan, gao Shenyang, and the like ]. The establishment of a rapid detection method for classical swine fever virus nucleic acid RT-RPA-LFD [ J ]. Veterinary journal, 2021 (05): 115-118 ].
The application of RPA in the marine product field is also mainly applied to the identification of pathogenic microorganisms and species, so that research on the detection of sea product components in food has important significance [ Poulton K, webster B.development of a lateral flow recombinase polymerase assay for the diagnosis of Schistosoma mansoni infections [ J ]. Anal Biochem,2018,546:65-71 ].
Disclosure of Invention
In view of the above, the present invention aims to provide a primer set, a kit and a method for detecting oyster components in food, which take oyster mitochondrial gene components as detection objects and establish products and methods for rapid detection of oyster components by using ERA technology.
The invention provides a primer group for detecting oyster components in food, which comprises a first primer group or a second primer group; the first primer set comprises F1 and R1; the nucleotide sequence of F1 is shown as SEQ ID No.1, the nucleotide sequence of R1 is shown as SEQ ID No.2, and the second primer group comprises F2 and R2; the nucleotide sequence of F2 is shown as SEQ ID No.3, and the nucleotide sequence of R2 is shown as SEQ ID No. 4.
The invention also provides a kit for detecting oyster components in food, which comprises the primer group.
Preferably, the kit further comprises a detection reagent.
The invention also provides a method for detecting whether oyster components exist in food or not, which comprises the following steps:
1) Extracting total DNA of a sample to be detected;
2) Taking the total DNA in the step 1) as a template, and carrying out enzymatic isothermal amplification by taking the primer group as a primer to obtain an amplification product;
3) Agarose gel electrophoresis is carried out on the obtained amplification product, if a band exists at the 286bp position, oyster components are considered to exist in the sample to be detected; if there is no band at 286bp, it is considered that oyster component is not present in the sample to be tested.
Preferably, the system for enzymatic isothermal amplification according to step 2) comprises, in 50. Mu.L, 20. Mu.L of a lysing agent,2.5. Mu.L of upstream primer, 2.5. Mu.L of downstream primer, 1. Mu. L, ddH of total DNA 2 O22. Mu.L and activator 2. Mu.L.
Preferably, the concentration of the total DNA is 80-120 ng/. Mu.L.
Preferably, the concentration of the upstream primer and the downstream primer is independently 8-12 mu mol/L.
Preferably, the temperature of the enzymatic isothermal amplification in step 2) is 38 to 39.5 ℃.
Preferably, the time of the enzymatic isothermal amplification in step 2) is 15 to 30 minutes.
Compared with the prior art, the invention has the following beneficial effects: the primer group, the kit and the detection method for detecting oyster components in food provided by the invention have the advantages of simplicity and rapidness in operation; the method is independent of the advantage of high-end equipment, has higher specificity and sensitivity, and has detection limit reaching fg grade which is higher than that of the common PCR method; provides a new means for rapid, constant temperature and on-site identification of oyster components in food.
Drawings
FIG. 1 is a gel electrophoresis chart of total DNA extracted by different oyster total DNA extraction methods, wherein M is DL 15000bp DNA Marker;1, extracting the obtained total DNA by an SDS method; 2, extracting the obtained total DNA by using a CTAB method as a sample; 3, extracting the obtained total DNA by using a CTAB+guanidine hydrochloride method as a sample; 4, extracting the total DNA obtained by the DNA extraction kit method;
FIG. 2 is a gel electrophoresis diagram of the total DNA of oyster amplified by different primers, wherein M is DL 500bp DNA Marker;1 is F1/R1;2 is DF1/DR1;3 is DF2/DR2;4 is DF3/DR3;5 is F2/R2;
FIG. 3 is a graph showing the result of gel electrophoresis at different reaction temperatures, wherein M is DL 500bp DNA Marker;1 is 37 ℃;2 is 38 ℃;3 is 39 ℃;4 is 40 ℃;5 is 41 ℃;6 is a negative control;
FIG. 4 is a graph showing the result of gel electrophoresis at different reaction times, wherein M is DL 500bp DNA Marker;1 is 20min;2 is 25min;3 for 30min;4 is 35min;5 is 40min;6 is a negative control;
FIG. 5 is a gel electrophoresis diagram for specificity detection of a reaction system, wherein M is DL 500bp DNA Marker;1 is oyster DNA;2 is clam DNA;3 is razor clam DNA;4 is conch DNA;5 is a negative control;
FIG. 6 is a gel electrophoresis chart of sensitivity detection of a reaction system, wherein M is DL 500bp DNA Marker;1 at a template concentration of 8X 10 7 fg/. Mu.L; 2 at a template concentration of 8X 10 6 fg/. Mu.L; 3 template concentration of 8X 10 5 fg/. Mu.L; 4 template concentration of 8X 10 4 fg/. Mu.L; 5 template concentration of 8X 10 3 fg/. Mu.L; 6 template concentration of 8X 10 2 fg/. Mu.L; 7 template concentration of 8X 10 1 fg/. Mu.L; 8 is a negative control;
FIG. 7 is a gel electrophoresis diagram for detecting actual samples of a reaction system, wherein M is DL 500bp DNA Marker;1 is a fresh oyster sample; 2 is oyster powder sample; 3 is oyster can sample; 4 is a horseshoe snail sample; 5, freezing oyster samples; 6 is a Qingdao shrimp sample; 7 is an oyster sauce sample; 8 is a negative control.
Detailed Description
The invention provides a primer group for detecting oyster components in food, which comprises a first primer group or a second primer group; the first primer set comprises F1 and R1; the nucleotide sequence of F1 is shown as SEQ ID No.1, the nucleotide sequence of R1 is shown as SEQ ID No.2, and the second primer group comprises F2 and R2; the nucleotide sequence of F2 is shown as SEQ ID No.3, and the nucleotide sequence of R2 is shown as SEQ ID No. 4. In the practice of the present invention, the second primer set is preferred.
The invention also provides a kit for detecting oyster components in food, which comprises the primer group. In the present invention, the kit preferably further comprises a detection reagent, wherein the detection reagent is preferably a reagent used for ERA detection, and specifically comprises a dissolving agent and an activating agent; the lytic reagent and the activator are derived from a basic nucleic acid amplification kit (ERA method), and more preferably from a basic nucleic acid amplification kit (ERA method) of limited biotechnology of first reach, su. In the present invention, the kit preferably further comprises a negative control, the negative control being ddH 2 O。
The invention also provides a method for detecting whether oyster components exist in food or not, which comprises the following steps:
1) Extracting total DNA of a sample to be detected;
2) Taking the total DNA in the step 1) as a template, and carrying out enzymatic isothermal amplification by taking the primer group as a primer to obtain an amplification product;
3) Agarose gel electrophoresis is carried out on the obtained amplification product, if a band exists at the 286bp position, oyster components are considered to exist in the sample to be detected; if there is no band at 286bp, it is considered that oyster component is not present in the sample to be tested.
In the invention, total DNA of a sample to be detected is extracted; the method for extracting the total DNA is not particularly limited, and the method for extracting the total DNA is conventional in the art, and in the specific implementation process of the invention, the method for extracting the total DNA adopts a CTAB method, a CTAB+guanidine hydrochloride method, an SDS method and a DNA extraction kit; preferably, CTAB+guanidine hydrochloride is used.
After the total DNA is obtained according to the present invention; and carrying out enzymatic isothermal amplification by taking the total DNA as a template and the primer group as a primer to obtain an amplification product. In the present invention, the system for enzymatic isothermal amplification comprises, in 50. Mu.L, 20. Mu.L of a lytic reagent, 2.5. Mu.L of an upstream primer F1 or F2, 2.5. Mu.L of a downstream primer R1 or R2, 1. Mu. L, ddH of total DNA 2 O22. Mu.L and activator 2. Mu.L. The concentration of the total DNA is preferably 80 to 120 ng/. Mu.L, more preferably 90 to 110 ng/. Mu.L, and still more preferably 100 ng/. Mu.L. In the present invention, the concentration of the upstream primer and the downstream primer is independently preferably 8 to 12. Mu. Mol/L, more preferably 9 to 11. Mu. Mol/L, and still more preferably 10. Mu. Mol/L.
In the present invention, the temperature of the enzymatic isothermal amplification is preferably 38 to 39.5 ℃, and more preferably 38.5 to 39 ℃; the time for the enzymatic isothermal amplification is preferably 15 to 30 minutes, more preferably 20 minutes.
After the amplification product is obtained, agarose gel electrophoresis is carried out on the obtained amplification product, and whether oyster components exist in a sample is judged according to an electrophoresis result: if a band exists at the 286bp position, the oyster component is considered to exist in the sample to be detected; if there is no band at 286bp, it is considered that oyster component is not present in the sample to be tested. The agarose gel electrophoresis method of the invention is not particularly limited, and the agarose gel electrophoresis method conventional in the art can be adopted. In the practice of the invention, the agarose gel concentration is preferably 3.5%.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Materials and methods
Materials and reagents
Pacific oyster (Crassostrea gigas), conch, sea snail, freshwater shrimp, razor clam and the like are purchased in the sea food wholesale market in the east market in Jilin; oyster cans (Guangxi Lian Hua), oyster powder (Yimen Zhenxin food in the milli-Zhou city) and sea oyster oil are purchased from Taobao.
Blood/cell/tissue genomic DNA extraction kit (DP 130227, tiangen biochemical technology (beijing) limited);
basic nucleic acid amplification kit (KS 101, soviet first reach Gene technologies Co., ltd.);
nucleic acid dye (SYBR@Green I), DL 15000DNA Marker, 10×Loading Buffer, rTaq enzyme (Takara doctor technology (Beijing)) was used;
DL 500DNA Marker, agarose, cetyl trimethylammonium bromide (cetyl trimethyl ammonium bromide, CTAB), tris-hydrochloric acid solution, disodium ethylenediamine tetraacetate, SDS (sodium dodecyl sulfonate) (engineering Shanghai bioengineering Co., ltd.);
the RIPA lysate (strong) (P0013B, shanghai Biyun Biotechnology Co., ltd.);
absolute ethanol (analytically pure, national pharmaceutical systems chemical company, inc.).
Apparatus and device
Centrifuge 5430R bench-top high-speed refrigerated Centrifuge (Ai Bende, germany);
HH-12468 thermostat water bath (Changzhou Lang Yue instruments Co., ltd.);
DYY-60 electrophoresis apparatus (Beijing six biotechnology Co., ltd.);
BioSpec-nano ultraviolet spectrophotometer (Shimadzu corporation);
MVS-83 autoclave (Beijing Guanpu technologies Co., ltd.);
GenoSens 2000 gel imager (Shanghai service glider science instruments limited);
BSC-1100 IIB 2-X biosafety cabinet (Jinan Xin Baxi Biotechnology Co., ltd.);
GS8 fluorescence isothermal amplification apparatus (Souzhou first reach gene technology Co., ltd.).
Experimental method
Extracting oyster total DNA by adopting a CTAB method, a CTAB+guanidine hydrochloride method, an SDS method and a blood/cell/tissue genome DNA extraction kit extraction method of Tiangen company respectively.
1. CTAB method for extracting oyster DNA
(1) 200mg of oyster sample ground into powder by liquid nitrogen is taken and added with 1mL of CTAB extract (20 g/L CTAB,1.4mol/L NaCL,0.1mol/L Tris-HCL,0.02mol/L Na) 2 EDTA, pH 8.0) and 30. Mu.L proteinase K solution (20 mg/mL), shaking at 56℃for 2h, and centrifuging at 12000r/min for 5min.
(2) The supernatant was placed in a new 1.5mL centrifuge tube, added with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) mixture and stirred for 1min with a vortex mixer, and centrifuged at 12000r/min for 10min at 4 ℃.
(3) Transferring the supernatant to a new 1.5mL centrifuge tube, adding chloroform/isoamyl alcohol (24:1) solution with the same volume as the supernatant again, shaking by a vortex mixer for 1min, uniformly mixing, and centrifuging at 4 ℃ for 10min at 12000 r/min.
(4) Transferring about 400 μL of the supernatant into a new 1.5mL centrifuge tube, adding 2 times volume of precooled absolute ethanol solution, standing on ice for 20min after reversing and mixing, centrifuging for 2min at 12000r/min, discarding the supernatant, adding 1mL of 70% ethanol solution to wash the precipitate for 2 times, air-drying, and adding 50 μL TE solution (1 mmol/L Tris-HCl,0.1mmol/L Na) 2 EDTA, pH 8.0) was dissolved well and 4. Mu.L of RNaseA solution was added on ice to remove RNA and stored at-20 ℃.
2. Extraction of oyster DNA by CTAB+guanidine hydrochloride method
Grinding liquid nitrogen into powderIs added with 0.9mL CTAB extract (20 g/L CTAB,1.4mol/L NaCL,0.1mol/L Tris-HCL,0.02mol/L Na) 2 EDTA, pH 8.0), 0.1mL guanidine hydrochloride (5 mol/L) and 30. Mu.L proteinase K solution (20 mg/mL), shaking at 56℃for 2h, centrifuging at 12000r/min for 5min, and extracting oyster DNA in the same manner as in the previous 1 and CTAB method.
3. SDS method for extracting oyster DNA
Taking 200mg of oyster sample ground into powder by liquid nitrogen, adding 1mL of 10% SDS, 4 mu L of RNaseA, carrying out water bath at 56 ℃ for 1h, taking out and shaking uniformly every 10min, carrying out centrifugation at 12000r/min for 10min at 4 ℃, and extracting oyster DNA by the rest steps by the method of 1 and CTAB.
4DNA extraction kit for extracting oyster DNA
(1) PMSF with a final concentration of 1mM is added to the RIPA lysate of Biyun Tian according to the specification, and the lysate is added in a proportion of 150-200 mu L of lysate per 20mg of oyster tissue sample. Weighing 0.1g of oyster powder ground by liquid nitrogen into a 1.5mL centrifuge tube, adding the lysate, oscillating by a vortex oscillator to suspend the cell powder, placing the cell powder into ice for cracking, and oscillating for 30s every 2min until the cracking is completed. (2) Oyster tissue DNA was extracted according to the blood/cell/tissue genomic DNA extraction kit instructions of the root of tendril.
ERA primer design
The ERA detection system comprises an upstream Primer (F) and a downstream Primer (R), according to a conserved segment of oyster mitochondrial gene sequence (AF 177226.1) on GenBank, 5 pairs of ERA primers with higher homology are designed for the segment by using software Primer Premier 5.0 software and are synthesized by the company of the biological engineering of the Shanghai, and the synthesized Primer sequences are shown in table 1.
TABLE 1 ERA primer sequences
Figure BDA0003919951480000081
Establishment of ERA reaction system and comparison of primer detection results
50. Mu.L of a premix (20. Mu.L of a lytic reagent) was prepared according to ERA-based kit instructionsL is; 10. Mu. Mol/L of each of the upstream and downstream primers was 2.5. Mu.L; 1 mu L of oyster DNA template with the concentration of 100 ng/. Mu.L; ddH 2 O22 μl), thoroughly mixed with shaking and briefly centrifuged. Transfer 48 μl of premix to each tube of base amplification reagent, mix well with shaking until the amplification reagents are resuspended, and centrifuge briefly. 2. Mu.L of activator was added to the reaction cap, the cap was carefully closed, the activator was put into the premix by brief centrifugation, mixed by brief shaking and centrifuged again rapidly. And respectively establishing amplification systems of different primers aiming at 5 pairs of designed primers. All the reaction tubes were incubated in a 39℃thermostat for 30min, 10. Mu.L of each sample was added with 1.5. Mu.L of Loading Buffer, incubated at 56℃for 5min, and 3.5% agarose gel electrophoresis was used to identify the amplification effect of the different primer pairs.
Detection results of different reaction temperatures of ERA reaction system
Taking oyster DNA with concentration of 80 ng/. Mu.L as template, selecting F2 and R2 as primers, preparing 50. Mu.L premix of system sample (20. Mu.L of dissolvent; 10. Mu. Mol/L of upstream and downstream primers each 2.5. Mu.L; 100 ng/. Mu.L oyster DNA template 1. Mu.L; ddH) 2 O22 μl), thoroughly mixed with shaking and briefly centrifuged. Transfer 48 μl of premix to each tube of base amplification reagent, mix well with shaking until the amplification reagents are resuspended, and centrifuge briefly. 2. Mu.L of activator was added to the reaction cap, the cap was carefully closed, the activator was put into the premix by brief centrifugation, mixed by brief shaking and centrifuged again rapidly. The reaction was placed in a temperature gradient of 37, 38, 39, 40, 41℃for 30min, and the results were detected by 3.5% agarose gel electrophoresis.
Detection results of ERA System at different reaction times
50 mu L of premix (20 mu L of dissolvent; 10 mu mol/L of upstream and downstream primers each 2.5 mu L;100 ng/. Mu.L of oyster DNA template 1 mu L; ddH) of system sample was prepared using 80 ng/. Mu.L of oyster DNA as template and F2 and R2 as primers 2 O22 μl), thoroughly mixed with shaking and briefly centrifuged. Transfer 48 μl of premix to each tube of base amplification reagent, mix well with shaking until the amplification reagents are resuspended, and centrifuge briefly. Adding 2 μl of activator on the reaction cap, carefully covering the cap, centrifuging briefly to make the activator enter the premix, and mixing by short shakingAnd again centrifuged rapidly. Setting the amplification temperature at 39 ℃ and the amplification time at 20, 25, 30, 35 and 40min respectively. After the reaction, the product was taken out and subjected to 3.5% agarose gel electrophoresis.
Specific detection of ERA detection systems
Diluting four genomes of oyster total DNA, clam total DNA, razor clam total DNA and conch total DNA to 80 ng/. Mu.L as templates, preparing 50 mu.L of premix of system samples (20 mu.L of dissolvent; 10 mu mol/L of upstream primer and downstream primer of 2.5 mu.L respectively; 100 ng/. Mu.L of oyster DNA template 1 mu.L; ddH) 2 O22 μl), thoroughly mixed with shaking and briefly centrifuged. Transfer 48 μl of premix to each tube of base amplification reagent, mix well with shaking until the amplification reagents are resuspended, and centrifuge briefly. 2. Mu.L of activator was added to the reaction cap, the cap was carefully closed, the activator was put into the premix by brief centrifugation, mixed by brief shaking and centrifuged again rapidly. Amplification with F2 and R2 as primers was performed at 39deg.C for 20min with ddH 2 O is a negative control, 3.5% agarose gel electrophoresis detection is carried out after amplification, and the specificity of the detection method is determined.
ERA detection system sensitivity detection
Taking oyster total DNA as a template, setting concentration gradients as follows: 8X 10 7 、8×10 6 、8×10 5 、8×10 4 、8×10 3 、8×10 2 、8×10 1 Amplifying the template with each concentration by using a reaction system with the concentration of 50 mu L and taking the oyster total DNA amplified product as a positive control and ddH (double-sided hydroxyl) 2 O was used as a negative control. Premix (20. Mu.L of lytic reagent; 10. Mu. Mol/L of upstream and downstream primers each 2.5. Mu.L; 100 ng/. Mu.L of oyster DNA template 1. Mu.L; ddH) for 50. Mu.L of system sample was prepared 2 O22 μl), thoroughly mixed with shaking and briefly centrifuged. Transfer 48 μl of premix to each tube of base amplification reagent, mix well with shaking until the amplification reagents are resuspended, and centrifuge briefly. 2. Mu.L of activator was added to the reaction cap, the cap was carefully closed, the activator was put into the premix by brief centrifugation, mixed by brief shaking and centrifuged again rapidly. Adopting F2 and R2 as primers, amplifying for 20min at 39 ℃, performing 3.5% agarose gel electrophoresis identification after amplification, and verifying the detection formulaSensitivity of the method.
Detecting actual samples
Taking total DNA of related products such as fresh oyster, oyster can, oyster powder, oyster sauce, water chestnut snail, frozen oyster, qingdao shrimp and the like as a template, ddH 2 O was used as a negative control, and 50. Mu.L of a premix (20. Mu.L of a lytic reagent; 10. Mu. Mol/L of each of the upstream and downstream primers 2.5. Mu.L; 100 ng/. Mu.L of oyster DNA template 1. Mu.L; ddH) was prepared 2 O22 μl), thoroughly mixed with shaking and briefly centrifuged. Transfer 48 μl of premix to each tube of base amplification reagent, mix well with shaking until the amplification reagents are resuspended, and centrifuge briefly. 2. Mu.L of activator was added to the reaction cap, the cap was carefully closed, the activator was put into the premix by brief centrifugation, mixed by brief shaking and centrifuged again rapidly. F2 and R2 are adopted as primers, amplification is carried out for 20min at 39 ℃,3.5% agarose gel electrophoresis identification is carried out after amplification, and the verification condition of the detection method on an actual sample is verified. Meanwhile, the comparison with the conventional PCR is carried out, and the proportion of detected oyster positives is verified.
Results and analysis
Agarose gel electrophoresis identification of oyster total DNA
Extracting oyster total DNA by the four methods, and identifying by 0.7% agarose gel electrophoresis. 1, the genome extracted by the SDS method has darker band brightness, lowest concentration and a little dispersion phenomenon; the extraction results of the lanes 2 and 3 are not different, no impurity band and tailing phenomenon occurs, but the purity of the DNA band extracted by the ultraviolet identification result CTAB+guanidine hydrochloride method is better, and the concentration is higher; the genome extracted by the DNA extraction kit has the highest price although the concentration and purity are good. In summary, CTAB+guanidine hydrochloride was determined to be the best extraction method (FIG. 1).
Comparison of detection results of different ERA primers
As a result, as shown in FIG. 2, it was revealed that the primers DF3 and DR3 in lane 4 were not amplified to obtain the target gene, that the non-specific bands were present in both lanes 2 (DF 1 and DR 1) and 3 (DF 2 were good DR 2), that the bands amplified in lanes 1 (F1 and R1) and 5 (F2 and R2) were the highest in brightness and the band specificity was strong, but that the repeated stability of the F2 and R2 primer pairs was the best, and that the amplified product was 286bp (FIG. 2). The difference in amplification results is related to the quality of primer design or the efficiency of annealing of the primer to the template.
Comparison of the detection results of different ERA reaction temperatures
The result of the amplification by the temperature gradient showed that the target fragment band of 286bp appeared at all temperatures (FIG. 3). Wherein, lane 1 shows that the strip brightness at 37 ℃ is the weakest, the reaction efficiency is low, the yield of amplified products is unstable at 40 ℃ and 41 ℃, the amplification efficiency of isothermal amplification enzyme is affected by the excessively high temperature, and the non-specific amplified products are more; the amplified bands at 38 ℃ and 39 ℃ have the best brightness, the target product is higher, the target product of the 39 ℃ reaction is relatively specific, and the amplification is incomplete or the high temperature influence is caused to generate nonspecific amplification below or above 39 ℃.
Comparison of detection results of different ERA reaction times
The results consistent with the size of the target band are obtained in all reaction time, the brightness of the band obtained in lane 1, 20min, is bright, the change of the target gene product is small along with the extension of time, and the target gene product is relatively specific, which shows that the influence of the change of the target gene product along with the time is small within 40min (figure 4) and the product is degraded after long-time reaction.
Detection of ERA reaction specificity
Respectively taking 4 genomes of oyster, clam, razor clam and conch as templates to prepare 50 mu L of premix of system samples (20 mu L of dissolvent, 2.5 mu L of 10 mu mol/L of upstream primer and 2.5 mu L of downstream primer, 1 mu L of 100 ng/mu L of oyster DNA template and ddH) 2 O22 μl), thoroughly mixed with shaking and briefly centrifuged. Transfer 48 μl of premix to each tube of base amplification reagent, mix well with shaking until the amplification reagents are resuspended, and centrifuge briefly. 2. Mu.L of activator was added to the reaction cap, the cap was carefully closed, the activator was put into the premix by brief centrifugation, mixed by brief shaking and centrifuged again rapidly. Adopting F2 and R2 as primers, amplifying for 20min at 39 ℃, carrying out 3.5% agarose gel electrophoresis identification after amplification, wherein the electrophoresis result only shows that the total DNA of the 2 nd oyster is used as a template to generate a specific band, the size of the specific band is consistent with 286bp of the target band, and no impurity band exist in a laneThe dispersion, no target band was present in all other three templates, nor in the negative control, indicating that the detection system was highly specific for the detection of oyster components (FIG. 5).
Detection of ERA response sensitivity
As shown in FIG. 6, the concentrations of the templates in lanes 1 to 7 are reduced by 10 times, the same size of 286bp of the target band appears in each lane, the brightness in each lane is nearly reduced in sequence, and as the concentration is continuously diluted, the detection limit of 800 fg/mu L,80 fg/mu L and the negative control are not appeared in lanes 6, which indicates that the reaction sensitivity is higher and the detection limit can reach fg level.
Actual sample detection results
As shown in FIG. 7, lanes 1-3 and 5 show bands with the same size as 286bp of the target band, and the amplified target bands of lanes 1, 3 and 5 are brighter, wherein lanes 1 and 5 are unprocessed oysters, and 3 is an oyster can, which shows that the oyster content in the oyster can is higher, but lane 2 shows that the band is darker and the dragging is more serious, which shows that the oyster component in oyster powder is slightly lower; the 7 lane is oyster sauce without target bands, so that the original components are destroyed or the oyster components are extremely low and are not amplified in the deep processing process; 4. lanes 6 and negative control did not have a band of interest (see FIG. 7).
And then, the established ERA method and the mature PCR method are adopted to detect samples of purchased oysters, oyster foods, oyster health products and seasonings. When the template concentration is higher than 80 pg/mu L, the oyster component can be detected by detecting oyster, dried oyster, canned oyster and oyster powder by two methods, and the original components can be destroyed due to extremely low oyster component content or deep processing. When the template concentration was reduced to 8 pg/. Mu.L (as shown in Table 2), the unprocessed oyster could be detected by both methods, and the oyster food and health care product could be detected by ERA method, but some of the oyster food and health care product could not be detected by PCR, and the sensitivity of detecting oyster components by ERA method was higher than that by PCR.
TABLE 2 practical sample verification of ERA method and ordinary PCR method in the present invention
Figure BDA0003919951480000131
As can be seen from the contents of the above examples, the primer set, the kit and the detection method based on the ERA enzymatic isothermal amplification technology provided by the invention can be used for rapidly detecting oyster components, and the dependence on a nucleic acid amplification instrument and a professional is avoided in the selection of instruments and equipment, and only one isothermal metal bath or water bath kettle is needed. In the aspect of reaction, the method does not need to undergo steps such as complex thermal cycle and the like in the conventional PCR, only one constant temperature is needed for reaction, the parameter design and optimization are simpler, the time is greatly shortened, and the whole isothermal amplification can be completed within about 20 minutes.
Compared with the previously reported fluorescence quantitative PCR detection method with the sensitivity reaching 0.01 ng/. Mu.L, the detection method provided by the invention not only improves the sensitivity by 100 times, but also does not need large-scale expensive equipment such as fluorescence quantitative PCR, is suitable for being used in areas with lack of resources such as remote areas, and is more suitable for the requirement of on-site detection. The detection of oyster components by LAMP combined with fluorescence color reaction has also been reported to have a limit DNA concentration level up to 0.01 ng/. Mu.L, and can detect samples with oyster component content of 0.1%, and the method is also based on isothermal amplification detection, and is also suitable for on-site detection with simple operation method and low requirements on equipment. However, one reaction of LAMP requires the design of 6 pairs of primers, which is complicated in primer design, and the detection limit of DNA concentration is two orders of magnitude higher than that of the method provided by the invention. The ERA technology provided by the invention has the advantages that the detection sensitivity and RPA can reach fg level, but the cost advantage is obvious compared with an imported reagent RPA system, and the ERA technology has larger popularization and application value of field detection. The detection system provided by the invention can detect oyster components with sensitivity reaching 800fg after reacting for 20min at 39 ℃, and has the characteristics of strong specificity, high sensitivity, simplicity and rapidness.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (9)

1. A primer set for detecting oyster components in food, which is characterized by comprising a first primer set or a second primer set; the first primer set comprises F1 and R1; the nucleotide sequence of F1 is shown as SEQ ID No.1, the nucleotide sequence of R1 is shown as SEQ ID No.2, and the second primer group comprises F2 and R2; the nucleotide sequence of F2 is shown as SEQ ID No.3, and the nucleotide sequence of R2 is shown as SEQ ID No. 4.
2. A kit for detecting oyster components in food, comprising the primer set of claim 1.
3. The kit of claim 2, further comprising a detection reagent.
4. A method for detecting the presence of oyster components in a food product, comprising the steps of:
1) Extracting total DNA of a sample to be detected;
2) Performing enzymatic isothermal amplification by taking the total DNA in the step 1) as a template and the primer set as the primer set of claim 1 to obtain an amplification product;
3) Agarose gel electrophoresis is carried out on the obtained amplification product, if a band exists at the 286bp position, oyster components are considered to exist in the sample to be detected; if there is no band at 286bp, it is considered that oyster component is not present in the sample to be tested.
5. The method according to claim 4, wherein the system for enzymatic isothermal amplification in step 2) comprises, in 50. Mu.L, 20. Mu.L of a lytic reagent, 2.5. Mu.L of an upstream primer, 2.5. Mu.L of a downstream primer, 1. Mu. L, ddH of total DNA 2 O22. Mu.L and activator 2. Mu.L.
6. The method according to claim 5, wherein the concentration of the total DNA is 80 to 120 ng/. Mu.L.
7. The method according to claim 5, wherein the concentration of the upstream primer and the downstream primer is independently 8 to 12. Mu. Mol/L.
8. The method according to claim 4, wherein the temperature of the enzymatic isothermal amplification in step 2) is 38-39.5 ℃.
9. The method according to claim 4, wherein the time for the enzymatic isothermal amplification in step 2) is 15 to 30 minutes.
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