CN109852708B - Kit for rapidly identifying goose-derived components in food and application thereof - Google Patents
Kit for rapidly identifying goose-derived components in food and application thereof Download PDFInfo
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Abstract
The invention provides a kit for rapidly identifying goose-derived components in food, and relates to the technical field of biological species identification. The invention firstly screens out a goose-origin universal internal standard gene CCNB1, the nucleotide sequence of which is shown in SEQ ID NO.1, the copy number of which is constant in goose species, no allelic variation exists, and the gene can be used as a target gene for identifying goose origin. An asymmetric amplification primer is designed by taking the gene as a target sequence, and an asymmetric PCR reaction product is used for detecting DNA silver nanoclusters. The asymmetric PCR reaction is combined with the detection of the DNA silver nanocluster to form a rapid detection kit, so that the goose-source components in food can be rapidly and sensitively detected, and the detection sensitivity can reach 2% (w/w). The asymmetric PCR-DNA silver nanocluster universal fluorescence kit is simple in use method, low in cost, easy to observe reaction results, good in specificity and very suitable for field real-time detection.
Description
Technical Field
The invention relates to the technical field of biological species identification, in particular to an asymmetric PCR-DNA silver nanocluster rapid detection kit for detecting goose-derived components in food.
Background
With the rapid development of economy and the improvement of the living standard of people, the demand of residents in China on meat food is increased year by year. Although many countries have clear regulations that require food labels to truly and clearly identify the type and source of meat, and prohibit adulteration, there are many incidents of meat adulteration in the market, such as the incorporation of pork in donkey meat burns, pork in mutton, other meat in geese, and the like, for cost reduction. At present, the detection method for food adulteration is mainly a method of PCR plus agarose gel electrophoresis, the judgment of the final result needs to be completed by means of large-scale instruments such as a gel imaging system, and the process is complex and consumes a long time. Therefore, the invention aims to provide a rapid and sensitive detection kit for goose-derived components in food.
At present, the internal standard gene is widely used for identifying food adulteration, but how to screen out the proper internal standard gene is very important. At present, meat product adulteration detection technologies at home and abroad are mainly designed aiming at genes on mitochondria to carry out real-time fluorescent quantitative PCR amplification, and because the mitochondria genes are multicopy genes, the detection sensitivity is high, but simultaneously, the detection technology has trouble in distinguishing unconscious cross contamination and conscious illegal addition generated in the processes of sale, transportation and the like. In addition, the concentration of high copy number mitochondrial DNA cannot correspond to the concentration of genomic DNA, so that accurate quantitative analysis of a sample cannot be performed, and qualitative detection by ordinary PCR is difficult because of extremely high homology of mitochondrial genes. Therefore, the method can only realize screening and identification of meat adulteration by means of fluorescent quantitative PCR. If the low-concentration unintentional cross contamination and intentional illegal addition are identified, and the adulteration ratio is determined to realize rapid screening, the low-copy gene on the chromosome is selected as the standard gene in the meat.
Asymmetric PCR is a technology that double-stranded DNA is used as a template, a pair of primers with different amounts are added into a system, and a large amount of single-strand DNA (ssDNA) is generated through cyclic amplification. This technique is an important method for obtaining single-stranded target DNA. The silver nanocluster synthesis template comprises an organic template and an inorganic template, wherein the organic template mainly comprises DNA, polypeptide and the like. The silver nanocluster taking the DNA as the template forms a DNA-Ag + compound under high binding force through cytosine and silver ions (Ag +), and then reduces the silver ions by utilizing sodium borohydride, so that the micromolecule fluorescent probe with high quantum yield and adjustable excitation wavelength is formed. Compared with the traditional fluorescent probe, the silver nanocluster probe has the greatest characteristics of no need of fluorescent group labeling, specific binding effect on a DNA nucleation sequence and huge application potential in the field of molecular biology.
The invention combines asymmetric PCR with DNA silver nanoclusters, utilizes asymmetric PCR to amplify a specific sequence of a screened specific internal standard gene, then designs a DNA silver nanocluster probe which is specifically complementary with a single-chain target, and obtains a fluorescent product which can be seen under the ultraviolet (365nm) condition so as to detect species components and identify species sources. The method is simple to operate, does not need complicated detection such as gel electrophoresis and the like, has high sensitivity, and completely meets the requirement of rapidly detecting the goose-source components in the food.
Disclosure of Invention
The invention aims to provide a goose-derived universal internal standard gene for detecting goose-derived components in food.
The invention also aims to provide the asymmetric PCR-DNA silver nanocluster rapid detection kit for detecting the goose-derived component in the food, which has the advantages of high sensitivity, high specificity and simple operation.
A gene for detecting goose-origin components in food is an internal standard gene CCNB1 and has a sequence shown in SEQ ID NO. 1.
The invention provides application of the internal standard gene CCNB1 in detection of goose-derived components.
The invention provides application of the internal standard gene CCNB1 in identification of goose-origin components in food.
The invention provides a specific asymmetric PCR primer combination for detecting the internal standard gene CCNB1, which comprises the following two primers, wherein F is a non-limiting primer, and R + poly C is a reverse complementary sequence poly C of a 5' end connecting DNA-silver nano cluster excitation sequence poly G of a limiting primer R:
F：TTCCTCAACTGAAAACGCTA(SEQ ID NO.2)；
R+poly C：CCCCACCCCACCCCACCCTTGGCTCAAACAATTACAATG(SEQ ID NO.3)。
the invention provides application of the specific asymmetric PCR primer combination F and R + poly C in goose source component identification.
The invention provides application of the specific asymmetric PCR primer combination F and R + poly C in preparation of a goose-derived component detection kit or detection reagent.
Further, the invention provides a rapid detection kit for detecting goose-derived components, which contains the specific asymmetric PCR primer combination F and R + poly C.
The invention also provides a DNA-silver nanocluster probe which comprises a DNA nucleation sequence, silver ions (Ag +) and sodium borohydride (NaBH)4) The DNA nucleation sequence is: CCCCCTTAAT CCCCC (SEQ ID NO. 4); the DNA probe sequence is R: TTGGCTCAAACAATTACAATG (SEQ ID NO. 5).
The invention provides a method for detecting goose-source components in food, which comprises the following steps:
(1) extracting DNA from a sample to be detected;
(2) performing PCR amplification by using the extracted DNA as a template and the specific asymmetric PCR primer combination of the non-limiting primer F and the limiting primer R + poly C according to claim 4;
(3) hybridizing the asymmetric PCR product with a DNA silver nanocluster probe;
(4) and (5) judging a result: the specific single-stranded target can be complementarily combined with the specific base of the probe and can excite the probe, the yellow fluorescence can be generated under the ultraviolet (365nm), and the sample is positive.
In the above method, the asymmetric PCR assay in step (2) has a specific configuration of 25 μ L assay system: 1 XPCR buffer, 0.4mM dNTP, 0.8. mu.M primer F, 0.08. mu.M primer R + poly C, 1.5U rTaq DNA polymerase.
In the method, the asymmetric PCR detection reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; 45 cycles: 30min at 95 ℃, 30s at 53 ℃ and 30s at 72 ℃; extension at 72 ℃ for 10 min.
The invention screens the goose-origin internal standard gene on the chromosome for the first time. The invention uses a plurality of varieties of geese to verify the internal standard gene, and proves that the internal standard gene is stable and has no allelic variation. The selection of the internal standard gene generally requires low and stable copy number, and the internal standard gene of a general animal is often selected on mitochondria, so that the copy number of the internal standard gene is large and the internal standard gene is not easy to quantify. The gene on the chromosome is selected as the internal standard gene, the copy number is low, the quantification is easy, and the mutation rate is lower compared with the gene on the mitochondria.
FIG. 1 shows the principle of a silver nanocluster fluorescence visual sensor. The genome of the species is first extracted (step 1). The invention selects a C5-C5 type DNA nucleating sequence, and DNA-silver nanoclusters formed by the nucleating sequence can generate yellow fluorescence under the condition of ultraviolet (365nm) under the excitation of an excitation sequence poly G. The concentration of non-limiting primers in the asymmetric PCR is far greater than that of limiting primers, and in the first 10-15 cycles of the reaction, the asymmetric PCR is the same as that of ordinary qualitative PCR, so that DNA double chains are generated. When the restriction primers are consumed in the system, the non-restriction primers generate a large amount of single DNA strands using the double DNA strands generated in the previous 10-15 cycles as templates. By connecting the 5 ' end of the limiting primer R with the reverse complementary sequence poly C of the DNA-silver nanocluster excitation sequence poly G in the asymmetric PCR experiment, a large number of single-stranded targets with the 3 ' ends serving as the reverse complementary sequence R ' of the downstream primer R and the excitation sequence poly G can be generated by utilizing the asymmetric PCR experiment (step 2). And step 3 is a hybridization process of the asymmetric PCR product and the DNA-silver nanocluster probe. And (3) designing a hybrid chain of the DNA-silver nanocluster probe into a sequence identical to a downstream primer R, and if the sample is positive, complementarily combining the specific single-stranded target generated in the step (2) with a specific base of the probe and exciting the probe to generate yellow fluorescence under ultraviolet (365 nm).
The goose minced meat and the non-goose minced meat are mixed in equal mass, and the sensitivity of the asymmetric PCR-DNA silver nanocluster method is detected. The result shows that when the mixing gradient is 50 times (the mass of the mixed minced meat is 50 times of that of the minced goose meat), namely 2% of the initial mass, the color is light red, so that the detection limit of the DNA silver nanocluster universal fluorescence kit is 2% (w/w).
Based on the internal standard gene CCNB1 for detecting goose-origin components in food, the invention designs an asymmetric PCR primer combination for detecting the gene, applies non-specific PCR reaction combined with DNA silver nanocluster to detect whether goose-origin components exist in a sample to be detected, the reaction is rapid, less time is consumed, the specificity is good, the sensitivity is high, the operation is simple, no professional operation is needed, the result is easy to observe, and the method is very suitable for basic-level food supervision and inspection.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is the principle of a silver nanocluster fluorescence visual sensor;
FIG. 2 shows the verification of the specificity of asymmetric PCR primers; the length of the goose amplification product is 243bp, 1: goose meat; 2: a pig; 3: sheep; 4: cattle: 5: chicken; 6: a duck; 7: goat: 8: a horse; 9: donkey; 10: deer; 11: a yak; 12: buffalo; 13: mink; 14: a camel; 15: fish; 16: a rat; 17: a dog; m is marker DL 2000;
FIG. 3 is a specificity study of a universal fluorescence visual sensor for DNA silver nanoclusters; 1: positive; 2: goose meat; 3: a pig; 4: sheep; 5: cattle: 6: chicken; 7: a duck; 8: goat: 9: a horse; 10: donkey; 11: deer; 12: a yak; 13: negative;
FIG. 4 is a sensitivity study of a universal fluorescence visual sensor for DNA silver nanoclusters; 1: the mixing gradient is 0 time, namely 100 percent of the original mass; 2: the mixing gradient is 2 times, namely 50 percent of the original mass; 3: the mixing gradient is 10 times, namely 10 percent of the original mass; 4: the mixing gradient is 50 times, namely 2 percent of the original mass; 5: the mixing gradient is 250 times, namely 0.4 percent of the original mass; 6: and (4) negativity.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Cattle (Bos taurus), pigs (Sus scrofa), sheep (Ovis aries), goats (Capra hircus), chickens (Gallus Gallus), ducks (Anas platyrhynchos), geese (Goose calicivirus), yaks (Bos mutus) and yellow croakers (Pseudoscia polyactis) are purchased from supermarkets. Horse (Equus caballus), donkey (Equus asinus) was purchased from the farmer market in beijing. Mice (Mus musculus) were provided by the food safety and molecular biology laboratory of the university of agriculture, china. Buffalo (Bubalus bubalis), mink (Martes zibellina), camel (Camelus ferus), deer (Cervus) were provided by Dr Limon of the Tianjin Ex-in-Place laboratory.
Example 1 screening of goose-derived Universal internal Standard Gene CCNB1
The target genome was downloaded from NCBI by searching GenBank for genetic information about geese and saved in ". FASTA" format. The goose genome information was analyzed, and homology analysis was performed using BLAST and DNAMAN Version 4.0 software to screen the CCNB1 gene. 21 meats (respectively, chicken of the common family (Gallus gallicus), pheasant (Phasianus colicus), turkey (Melegagris galliphacopavo), black-bone chicken (Gallus domesticus brisson), pig (Sus scrofa), cow (Bos taurus), sheep (Ovis aries), goat (Capra hircus), duck (Anas platyrhynchos), goose (Gooselirus), dog (Canis lupis fariaris), rabbit (Orycolagus cunicus), yak (Bos mutus), yellow croaker (Pseudosciaena polyactis), horse (Equus callus), donkey (Equus asinus), mouse (Mus musulus), buffalo (Bululus bulus), mink (Martezilla polyactis), Camelus (Catauus), sequence of the horse (Catauus calis gallis), and sequence of Camellia sinensis (Camellia sinensis), and the result of the comparison is expressed in a format of 25.25.25. And selecting the fragments with high specificity, and performing BLAST analysis to search sequence homology and specificity in the database. Finally, the final specific target gene CCNB1 gene can be determined to be an internal standard gene by integrating the sequence. The nucleotide sequence of the CCNB1 fragment is shown as SEQ ID NO. 1.
Example 2 establishment of asymmetric PCR detection method of goose-derived component
An asymmetric PCR primer is designed aiming at the CCNB1 gene determined in example 1 by using primer premier5.0 software, and comprises two primers, wherein F is an unlimited primer, and R + poly C is a reverse complementary sequence poly C of a 5' end of a limited primer R connected with a DNA-silver nanocluster excitation sequence poly G, and the reverse complementary sequence poly C is shown in Table 1.
TABLE 1 asymmetric PCR primer sequences
The goose sample is rapidly detected by asymmetric PCR reaction, and the reaction system is 25 mu L and comprises 1 XPCR buffer, 0.4 MdNTP, 0.8 mu M primer F, 0.08 mu M primer R + poly C and 1.5U rTaq DNA polymerase. The reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min; 45 cycles: 30min at 95 ℃, 30s at 53 ℃ and 30s at 72 ℃; extension at 72 ℃ for 10 min. After the amplification is finished, the product is judged by using 2% agarose gel electrophoresis, and a 243bp specific band appears to prove that the amplification is successful and contains the target gene. Results are shown in fig. 2, asymmetric PCR amplification of goose-specific internal standard gene in 17 animals, 1: goose meat; 2: a pig; 3: sheep; 4: cattle: 5: chicken; 6: a duck; 7: goat: 8: a horse; 9: donkey; 10: deer; 11: a yak; 12: buffalo; 13: mink; 14: a camel; 15: fish; 16: a rat; 17: a dog; m is Maker DL 2000. Only goose samples show bright bands, and other species have no bands, so that the designed asymmetric PCR primers are proved to have high specificity.
Example 3 establishment of goose-derived component asymmetric PCR-DNA silver nanocluster detection method
The specificity of the universal fluorescent visual kit for the DNA silver nanoclusters constructed by the invention is verified by 11 animal species. The results showed that only the positive samples and the positive experimental group (species goose) had bright red-yellow color, and the remaining 10 samples were similar in color to the negative color, indicating that the present invention has excellent specificity. The specific results are shown in FIG. 3.
The sensitivity of the goose minced meat is researched by mixing the goose minced meat and non-goose minced meat (mixed minced meat of pigs, sheep, cattle and the like) in a gradient manner. The results show that when the mixing gradient is 50 times (the mass of the mixed minced meat is 50 times of that of the minced goose meat), namely 2% of the initial mass, the color is light red; and when the mixing gradient is 250 times, namely 0.4% of the initial mass, the color is similar to the negative color. Therefore, the detection limit of the universal fluorescent visual kit for the DNA silver nanoclusters provided by the invention is 2% (w/w). The specific results are shown in FIG. 4.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> university of agriculture in China
<120> kit for rapidly identifying goose-origin components in food and application thereof
<130> MP1907467Z
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 311
<212> DNA
<213> Goose CCNB1(Goose Calicivirus CCNB1)
<400> 1
ggcttggaga tggcacccat tttcatctgt ggattttcag gaggcactga taagtgattc 60
ctcaactgaa aacgctagag taacctgaga tgataacaga taatggtttt tagaccaaca 120
gatcagcagt agtggtacca tgctcttcat agcctgtctc tagtaaacca agaaacctta 180
tacacacagg cctagtactg tgatccatag tatactggta cggacctctt tcaaaagcct 240
taagatgaaa caggggctgg gcattgtaat tgtttgagcc aaatgattag atataataat 300
ctgattcctt c 311
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ttcctcaact gaaaacgcta 20
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ccccacccca ccccaccctt ggctcaaaca attacaatg 39
<210> 4
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ttggctcaaa caattacaat g 21
Claims (6)
1. A gene for detecting goose-origin components in food is an internal standard gene, and the nucleotide sequence of the gene is shown in SEQ ID NO. 1.
2. The use of the gene of claim 1 in detecting goose-derived components.
3. The use of the gene of claim 1 in the identification of goose components.
4. A specific asymmetric PCR primer combination for detecting the gene as claimed in claim 1, which comprises the following two primers:
f is a non-limiting primer, and the sequence is shown as SEQ ID NO. 2;
the sequence of the R + poly C primer is shown as SEQ ID NO. 3.
5. The use of the specific asymmetric PCR primer combination of claim 4 in goose-derived component identification.
6. The application of the specific asymmetric PCR primer combination of claim 4 in preparing goose-derived component detection reagents.
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| KR100951773B1 (en) * | 2008-05-26 | 2010-04-08 | 한국전력공사 | Apparatus and method for goose message detecting |
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| CN103492590A (en) * | 2011-02-22 | 2014-01-01 | 卡里斯生命科学卢森堡控股有限责任公司 | Circulating biomarkers |
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|---|---|---|---|---|
| KR100951773B1 (en) * | 2008-05-26 | 2010-04-08 | 한국전력공사 | Apparatus and method for goose message detecting |
| CN103492590A (en) * | 2011-02-22 | 2014-01-01 | 卡里斯生命科学卢森堡控股有限责任公司 | Circulating biomarkers |
| CN102337337A (en) * | 2011-09-28 | 2012-02-01 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Polymerase chain reaction (PCR) detection primer for goose-derived component |
| CN103773846A (en) * | 2014-03-05 | 2014-05-07 | 天津出入境检验检疫局动植物与食品检测中心 | Sidewise-flow test strip detection kit for detecting geese origin component in food and feed and application of kit |
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