CN109811066B - Method and kit for rapidly detecting sheep-derived components in food - Google Patents
Method and kit for rapidly detecting sheep-derived components in food Download PDFInfo
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Abstract
The invention provides a kit for rapidly detecting sheep-derived ingredients in food, and relates to the technical field of biological species identification. According to the invention, a sheep-derived universal internal standard gene, namely, a Calponin, is screened out firstly, the nucleotide sequence of the Calponin is shown as SEQ ID No.1, the copy number of the Calponin is constant in sheep species, allelic variation does not exist, and the Calponin can be used as a target gene for identifying sheep sources. An LAMP amplification primer is designed by taking the gene as a target sequence, and is subjected to constant-temperature amplification together with LAMP reaction liquid, and an LAMP reaction product is used for detecting the colloidal gold nucleic acid test strip. The LAMP reaction is combined with the colloidal gold nucleic acid test strip for detection to form a rapid detection kit, sheep-derived components in food can be rapidly and sensitively detected, and the detection sensitivity can reach 0.16% (w/w). The kit has the advantages of simple use method, low cost, easy observation of reaction results and good specificity, and is very suitable for field real-time detection.
Description
Technical Field
The invention relates to the technical field of biological species identification, in particular to a rapid detection kit for detecting sheep-derived components in food by a loop-mediated isothermal amplification (LAMP) technology combined with a colloidal gold nucleic acid test strip.
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, the incorporation of beef or pork in mutton, the incorporation of other meats in sausages, and so on, for cost reduction. At present, the detection method for food adulteration is mainly a PCR method. The PCR method needs to depend on a special instrument, the judgment of the final result needs to be finished by agarose gel electrophoresis, and the whole process takes longer time. Therefore, the invention aims to provide a rapid and sensitive detection kit for sheep-derived ingredients 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.
Loop-mediated isothermal nucleic acid amplification (LAMP) is a novel isothermal nucleic acid amplification method developed by Notomi in 2000, and the principle is that a strand displacement DNA polymerase (BstDNA polymerase) and two pairs of special primers are used to specifically identify 6 independent regions on a target sequence, and the amplification reaction of nucleic acid can be completed by keeping the temperature at isothermal condition (about 65 ℃) for tens of minutes.
The colloidal gold nucleic acid test strip depends on the specific combination of antigen and antibody, so that the colloidal gold nucleic acid test strip has extremely high sensitivity. The invention combines LAMP technology with colloidal gold nucleic acid test strip detection, and provides a rapid and sensitive detection kit for sheep-derived components in food. The invention does not need complex instruments, has short time, simple operation and high sensitivity, and can completely meet the requirements of field detection.
Disclosure of Invention
The invention aims to provide a sheep-derived universal internal standard gene for detecting sheep-derived ingredients in food.
The invention also aims to provide a rapid detection kit for LAMP combined colloidal gold nucleic acid test strip detection for detecting sheep-derived components in food, which has the advantages of high sensitivity, high specificity and simple operation.
A gene for detecting sheep-derived components in food is an internal standard gene, namely, Calponin, and has a sequence shown in SEQ ID NO. 1.
The invention provides application of the internal standard gene calcinin in detection of sheep-derived components.
The invention provides application of the internal standard gene calcinin in identification of sheep-derived ingredients in food.
The invention provides a specific LAMP primer combination for detecting the internal standard gene calcinin, which comprises the following 4 primers:
F3：5’-CTCACTCCTCTGACAGTCCT-3’(SEQ ID NO.2)；
B3：5’-CCTGGGGCAACAGAATGG-3’(SEQ ID NO.3)；
FIP：5’-GCTAACCTCCAGGCCTCAGTTTGGTGGGTACTGTTACCGTCA-3’(SEQ ID NO.4)；
BIP：5’-AGCTGAACCCTGGAAATCAGGCACTGAGGCTCAGAGAAGGT-3’(SEQ ID NO.5)。
wherein the 5 'end of the inner primer FIP is labeled with Biotin (Biotin), and the 5' end of the BIP is labeled with fluorescein (Cy 5).
The invention provides application of the specific LAMP primer combination in sheep source component identification.
The invention provides application of the specific LAMP primer combination in preparation of a sheep-derived component detection kit or detection reagent.
Furthermore, the invention provides a rapid detection kit containing the specific LAMP primer combination for detecting sheep-derived components.
The invention provides a method for detecting sheep-derived components in food, which comprises the following steps:
(1) extracting DNA from a sample to be detected;
(2) performing LAMP detection by using the specific LAMP primer combination according to claim 3 by using the extracted DNA as a template;
(3) and (5) judging a result: adopting a colloidal gold nucleic acid test strip to judge the result, wherein the detection T line and the quality control C line both have red strips and contain target genes; the quality control C line has a red band, and the detection T line has no band and does not contain a target gene.
In the above method, the LAMP detection in step (2) is performed in a 25 μ L LAMP detection system configured specifically as follows: 1 XThermopol buffer, 0.4mM dNTP, 3mM MgSO41.0M betaine, 1.6. mu.M primer FIP, 1.6. mu.M primer BIP, 0.2. mu.M primer F3, 0.2. mu.M primer B3, 8U Bst DNA polymerase large fragment.
In the method, the LAMP detection reaction conditions are as follows: keeping the temperature at 60-65 ℃ for 20min and 85 ℃ for 5min, and then stopping the reaction.
In the above method, the colloidal gold nucleic acid test strip in step (3) comprises a test line and a quality control line of the colloidal gold nucleic acid test strip, wherein the test line is labeled with a Cy5 antibody, the quality control line is labeled with a biotin secondary antibody, and a colloidal gold-biotin antibody marker is bound and padded.
The invention screens sheep-derived internal standard genes on chromosomes for the first time. The invention uses a plurality of varieties of sheep 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 is a schematic diagram of a method for loop-mediated isothermal amplification and colloidal gold nucleic acid test strip detection. The invention designs 4 primers aiming at 6 specific regions of a sheep specific internal standard gene target sequence by utilizing LAMP reaction to carry out isothermal amplification. The inner primers FIP and BIP of LAMP are labeled with Biotin (Biotin) and fluorescein (Cy5), respectively. A large amount of double-stranded DNA target substances with biotin and fluorescein can be generated through LAMP reaction. And detecting the LAMP reaction product by using a colloidal gold nucleic acid test strip. The Test Line (TL) and the quality Control Line (CL) of the colloidal gold nucleic acid test strip are respectively marked with a Cy5 antibody and a biotin secondary antibody, and a colloidal gold-biotin antibody marker is combined and padded. When the LAMP reaction product is dripped into the test strip sample pad, the product sequentially passes through the combination pad, the detection line (TL) and the quality Control Line (CL) due to capillary action, and when the LAMP reaction product is positive due to the specific combination action of the antigen-antibody and the coagulation action of the colloidal gold, the detection line (TL) and the quality Control Line (CL) are both provided with red strips; when the reaction product was negative, only the Control Line (CL) had a red band.
The colloidal gold nucleic acid test strip depends on the specific combination of antigen and antibody, so that the colloidal gold nucleic acid test strip has extremely high sensitivity. According to the invention, the sheep mutton minced meat and the non-sheep mutton minced meat are mixed in a gradient manner by 5 times in quality, and the sensitivity of the detection method is researched by combining LAMP reaction and colloidal gold nucleic acid test strip detection. The results show that the detection limit of the detection kit of the invention is 0.16% (w/w).
Based on the internal standard gene Calponin for detecting sheep-derived components in food, which is determined by the invention, the invention designs the LAMP primer combination for detecting the gene, and can detect whether sheep-derived components exist in a sample to be detected by combining LAMP reaction with a colloidal gold nucleic acid test strip.
Drawings
FIG. 1 is a schematic diagram of a rapid detection kit for loop-mediated isothermal amplification and colloidal gold nucleic acid test strip detection;
FIG. 2 shows LAMP amplification of sheep specific internal standard genes in 16 animals, lane 1 shows LAMP products from sheep, 1: sheep; 2: cattle; 3: a pig; 4: goat: 5: chicken; 6: a duck; 7: goose: 8: a horse; 9: donkey; 10: deer; 11: a yak; 12: buffalo; 13: mink; 14: a camel; 15: fish; 16: a rat; m is marker DL 2000;
FIG. 3 shows the judgment of positive and negative results in the detection of a sheep-derived LAMP product colloidal gold nucleic acid test strip, wherein a sample 1 is proved to be a positive sample if the Test Line (TL) and the quality Control Line (CL) both have red strips, and a sample 2 is proved to be a negative sample if only the quality Control Line (CL) has red strips;
fig. 4 is a test of detection sensitivity of the LAMP-colloidal gold nucleic acid test strip, 1: the mixing gradient is 0 time, namely 100 percent of the original mass; 2: the mixing gradient is 5 times, namely 20 percent of the original mass; 3: the mixing gradient is 25 times, namely the original mass is 4%; 4: the mixed gradient is 125 times, namely the original mass is 0.8%; 5: the mixed gradient is 625 times, namely the original mass is 0.16%; 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.
Pigs (Sus scrofa), cattle (Bos taurus), sheep (Ovis aries), general chickens (Gallus gallous), pheasants (Phasianus colchicus), turkeys (Meleagris gallopavo), black-boned chickens (Gallus domesticus brisson), ducks (Anas platyrhynchos), geese (Goose calicivirus), dogs (Canis auricularis), rabbits (Oryctolagus cuniculus), yaks (Bos mutus), and yellow croakers (Pseudosciaena 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 sheep-derived Universal internal Standard Gene, Calponin
The target genome was downloaded from NCBI by searching GenBank for genetic information on sheep and saved in ". FASTA" format. The sheep whole genome information is analyzed, and homology analysis is carried out by utilizing BLAST and DNAMAN Version 4.0 software, so that a calcinin gene is screened out, is positioned on a chromosome and encodes a calcium opsonin gene. 20 meats (respectively, chicken of the common family (Gallus gallicus), pheasant (Phasianus colicuus), turkey (Meleagris galliphacopavo), black-bone chicken (Gallus domesticus brisson), pig (Sus scrofa), cow (Bos taurus), sheep (Ovis aries), duck (Anas platyrhynchos), Goose (Goose calicivirus), dog (Canis lupis farians), rabbit (Orycolagus cunicus), yak (Bos mutus), yellow croaker (Pseudosciaena polyactis), horse (Equus caballus), donkey (Equus asinus), mouse (Mus musculus), buffalo (Bubalus), mink (Martes zibellis), camel (Camellia), and DNA (California fusion), and the result is obtained by comparing the sequence of 0. v.q.4. the sequence is analyzed. And selecting the fragments with high specificity, and performing BLAST analysis to search sequence homology and specificity in the database. And finally, integrating the sequence to determine that the final specific target gene, namely the gene calcinin, can be used as an internal standard gene. The nucleotide sequence of the Calponin fragment is shown in SEQ ID NO. 1.
Example 2 establishment of sheep derived component LAMP detection method
LAMP primers were designed for the Calponin gene determined in example 1 using LAMP primer designing software of Loop-mediated primer design software, Rongy, Japan, Inc. V5.0 (http:// primer. jp/elamp5.0.0/index. html), including 2 outer primers F3, B3, and 2 inner primers FIP, BIP, see Table 1. The 5 'end of the inner primer FIP was labeled with Biotin (Biotin), and the 5' end of BIP was labeled with fluorescein (Cy 5).
TABLE 1 LAMP primer sequences
LAMP is used for quickly detecting sheep samples, and a reaction system is 25 mu L and comprises 1 XThermopol buffer, 0.4mM dNTP and 3mM MgSO41.0M betaine, 1.6. mu.M primer FIP, 1.6. mu.M primer BIP, 0.2. mu.M primer F3, 0.2. mu.M primer B3, 8U Bst DNA polymerase large fragment. The reaction program is constant temperature at 65 ℃ for 1h, and 85 ℃ for 5 min. After amplification is finished, product judgment is carried out by using 2% agarose gel electrophoresis, and a ladder-shaped band is formed to prove that amplification is successful and contains a target gene. As shown in fig. 2, the sheep specific internal standard gene was amplified by LAMP in 16 animals, lane 1 is the sheep LAMP product, 1: sheep; 2: cattle; 3: a pig; 4: goat: 5: chicken; 6: a duck; 7: goose: 8: a horse; 9: donkey; 10: deer; 11: a yak; 12: buffalo; 13: mink; 14: a camel; 15: fish; 16: a rat; m is Maker DL 2000. Only the sheep sample shows a bright band, which indicates that the calcinin gene and the corresponding LAMP system can be used for the rapid detection of sheep species.
Example 3 establishment of test paper strip for detection of colloidal gold nucleic acid of LAMP product of sheep-derived component
Preparing colloidal gold labeled antibody by trisodium citrate modification method, purifying the gold labeled antibody by high-speed centrifugation method, and storing the prepared gold labeled antibody at 4 deg.C for use.
The Cy5 antibodies were each diluted with buffer to optimal concentrations. The distance between the Test Line (TL) and the quality Control Line (CL) was 4.5mm, and the Test Line (TL) and the quality Control Line (CL) were sprayed on the NC film at 1.0. mu.L/cm, respectively. And drying the sprayed NC membrane at 37 ℃ overnight for later use. The test strip was cut to a width of 3.8 mm.
And (3) fully mixing the LAMP reaction product with a buffer solution, then dropwise adding the mixture on a sample pad of the colloidal gold nucleic acid test strip, allowing the mixed solution to pass through the combination pad and the NC membrane under the capillary power, and continuously moving towards the water absorption pad, and observing the detection result after 3 min. As shown in FIG. 3, the Test Line (TL) and the Control Line (CL) of sample 1 both have red bands, which is considered as a positive sample, and the Control Line (CL) of sample 2 only has red bands, which is considered as a negative sample.
The colloidal gold nucleic acid test strip depends on the specific combination of antigen and antibody, so that the colloidal gold nucleic acid test strip has extremely high sensitivity. And (3) pre-blocking the colloidal gold nucleic acid test strip by using a BSA (bovine serum albumin) solution with the concentration of 3% so as to avoid the occurrence of false positive on the premise of not influencing the normal positive color development of the colloidal gold nucleic acid test strip. The method is characterized in that sheep mutton minced meat and non-sheep mutton minced meat (mixed minced meat of cattle, pigs, mice and the like) are mixed in a gradient manner by 5 times and the like, LAMP reaction is carried out, and the LAMP reaction is combined with a colloidal gold nucleic acid test strip to explore the sensitivity of the detection method of the colloidal gold nucleic acid test strip. The results are shown in FIG. 4, 1: the mixing gradient is 0 time, namely 100 percent of the original mass; 2: the mixing gradient is 5 times, namely 20 percent of the original mass; 3: the mixing gradient is 25 times, namely the original mass is 4%; 4: the mixed gradient is 125 times, namely the original mass is 0.8%; 5: the mixed gradient is 625 times, namely the original mass is 0.16%; 6: and (4) negativity. When the mixing gradient is 625 times (the mass of the mixed minced meat is 625 times of that of the minced mutton) which is 0.16 percent of the initial mass, the detection T line is particularly shallow and almost similar to a negative control, so that the detection limit of the detection kit of the invention is 0.16 percent (w/w).
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 technical 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> method and kit for rapidly detecting sheep-derived components in food
<130> MP1907457Z
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<213> sheep Calponin (ovis aries calponin)
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aggtatggcc ctttcctcgc ctgggtcttg ctgatgagct gtggttgagg agggcaccct 60
ggtccctgca ggctttggtg ggttggagcg ggtacagaat atgtggtctt taggaaaccc 120
tatgcttagc attttcggta ccttaactca ctcctctgac agtcctaggg ggtgggtact 180
gttaccgtca tgccctttat acagatggga aaactgaggc ctggaggtta gcacacttgc 240
taggattgct caggtgatga gtagctgaac cctggaaatc aggccactta gcagtcatgt 300
gacacacctt ctctgagcct cagtttccat tctgttgccc caggggggtg gaaacagggc 360
ctcattagag ggccatgtga ctgctgacat cagtagtgtc tggtcgcttt cggtgggatc 420
ct 422
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ctcactcctc tgacagtcct 20
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cctggggcaa cagaatgg 18
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gctaacctcc aggcctcagt ttggtgggta ctgttaccgt ca 42
<210> 5
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
agctgaaccc tggaaatcag gcactgaggc tcagagaagg t 41
Claims (5)
1. A rapid detection method for detecting sheep-derived components in food is characterized by comprising the following steps:
(1) extracting DNA from a sample to be detected;
(2) performing LAMP detection by using the extracted DNA as a template and adopting a specific LAMP primer combination;
(3) and (5) judging a result: adopting a colloidal gold nucleic acid test strip to judge the result, wherein the detection T line and the quality control C line both have red strips and contain target genes; the quality control C line has a red band, and the detection T line has no band and does not contain a target gene;
the gene for detecting the sheep-derived component in the food is an internal standard gene and has a sequence shown in SEQ ID NO. 1;
the specific LAMP primer combination comprises the following 4 primers:
the method is characterized in that the colloidal gold nucleic acid test strip in the step (3) comprises a test line and a quality control line of the colloidal gold nucleic acid test strip, wherein the test line is marked with a Cy5 antibody, the quality control line is marked with a biotin secondary antibody, and a colloidal gold-biotin antibody marker is bound and padded.
2. Use of the specific LAMP primer combination of claim 1 in identification of sheep derived components.
3. The application of the specific LAMP primer combination in claim 1 in preparing a sheep-derived component detection kit or detection reagent.
4. The method of claim 1, wherein the LAMP assay of step (2) is a 25 μ L LAMP assay system specifically configured to: 1X Thermopol buffer, 0.4mM dNTP, 3mM MgSO41.0M betaine, 1.6. mu.M primer FIP, 1.6. mu.M primer BIP, 0.2. mu.M primer F3, 0.2. mu.M primer B3, 8U Bst DNA polymerase large fragment.
5. The method of claim 1, wherein the LAMP detection reaction conditions are: keeping the temperature at 60-65 ℃ for 20min and 85 ℃ for 5min, and then stopping the reaction.
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Citations (2)
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|---|---|---|---|---|
| CN105296647A (en) * | 2015-11-20 | 2016-02-03 | 华中农业大学 | Detection kit for sheep origin component identification and detection of multi-species origin components in products |
| CN109504786A (en) * | 2018-12-29 | 2019-03-22 | 博奥生物集团有限公司 | Primer combines the application in Species estimation and/or the meat of a sheep identification of sheep |
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Patent Citations (2)
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|---|---|---|---|---|
| CN105296647A (en) * | 2015-11-20 | 2016-02-03 | 华中农业大学 | Detection kit for sheep origin component identification and detection of multi-species origin components in products |
| CN109504786A (en) * | 2018-12-29 | 2019-03-22 | 博奥生物集团有限公司 | Primer combines the application in Species estimation and/or the meat of a sheep identification of sheep |
Non-Patent Citations (4)
| Title |
|---|
| A highly specific PCR assay for identification of raw and heat treated mutton (Ovis aries);Nagappa S. Karabasanavar等;《Small Ruminant Research》;20110903;第100卷;153-158 * |
| A novel common primer multiplex PCR (CP-M-PCR) method for the simultaneous detection of meat species;Wentao Xu等;《Journal of the Science of Food and Agriculture》;20081231;第88卷;2631-2637 * |
| AH0143872.2;Yue Z.等;《GenBank》;20160825;全文 * |
| 利用多重PCR和荧光定量PCR对羊肉进行定性定量鉴定方法的建立;王思伟;《中国学位论文全文数据库》;20160127;全文 * |
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