CN115961059B - Primer composition for detecting or assisting in detecting insect source in animal-derived feed raw material and application of primer composition - Google Patents
Primer composition for detecting or assisting in detecting insect source in animal-derived feed raw material and application of primer composition Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
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- Y02P60/87—Re-use of by-products of food processing for fodder production
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The application discloses a primer composition for detecting or assisting in detecting an insect source in animal-derived feed raw materials, a biological material and application thereof. The application aims to solve the technical problem of how to detect insect sources in animal source feed. The application discloses a primer composition, which comprises one or more of a primer pair A, a primer pair B and a primer pair C; the primer pair A specifically detects yellow meal worm, the primer pair B specifically detects hermetia illucens, and the primer pair C specifically detects tussah. By using one or more of the primer pair A, the primer pair B and the primer pair C to carry out in-vitro thermal cycle amplification on animal source feed, whether the animal source feed contains yellow mealworms, black soldier flies or tussah can be accurately judged according to the amplification result.
Description
Technical Field
The invention relates to a primer composition for detecting or assisting in detecting insect sources in animal-derived feed raw materials in the technical field of molecular biology, a biological material and application thereof.
Background
The rapid development of animal husbandry and aquaculture makes the shortage of feed resources, especially protein feed resources, restrict the production of animal husbandry and aquaculture. Wherein the animal-derived feed raw material is the main source of feed protein, and comprises fish meal, meat meal, blood meal, plasma protein powder, feather meal, meat and bone meal, leather powder, insect raw material and the like. Insect raw materials are widely used in the feed industry in recent years as high-quality protein sources due to the advantages of high protein and fat content, high growth speed, high culture efficiency and the like, and mainly comprise yellow mealworms, black soldier flies and tussah silkworms. Tenebrio molitor (Tenebrio molitor) is commonly known as a bread worm, an insect belonging to the genus Leptoradix Tripterygii of the family Coleoptera. The yellow meal worm is rich in nutritional ingredients such as protein, vitamins, minerals and the like, has the characteristic of easy digestion and absorption, is an excellent feed additive for livestock and poultry, and can improve the yield and quality of products. The black soldier fly (Hermetia illucens L.), the rotten soldier fly can eat the livestock manure and the household garbage, and the high-value animal protein feed is produced, so that the resource utilization is carried out due to the characteristics of rapid propagation, large biomass, wide feeding range, high absorption and conversion rate, easy management, low feeding cost, good palatability of animals and the like, the larvae are called as 'phoenix insects', and the larvae become resource insects with the same names as fly maggots, yellow mealworms, barley insects and the like, and are popularized worldwide. Tussah (ANTHERAEAPERNYI) is an animal of the genus tussah of the family Lepidoptera, the family Fabricius. The tussah is widely distributed, the breeding rate is high, the tussah is wild and bred, and the resources are very rich. The tussah body not only accumulates a large amount of nutrient substances, but also contains extremely rich physiological active substances, and has considerable prospect in the feed industry. In order to better serve the feed industry, it is particularly critical to distinguish the types of common insect raw materials.
The insect raw materials are similar in morphology, have no obvious characteristics, and have poor effects in detection of the insect raw materials by using conventional sensory identification, chemical detection and optical detection.
The gene detection technology is based on the identification of genetic material DNA, can distinguish different species from the molecular level, and has the characteristics of small sample amount, more accuracy, sensitivity and the like. The method has relevant reports on aspects of fish meal, meat meal and the like, but has reports on fresh detection of insect raw materials, and the method needs to make up for the gap of insect raw material gene detection.
Disclosure of Invention
Based on the above, the technical problem to be solved by the present application is how to provide a primer composition for identifying and detecting or assisting in detecting insect sources in animal-derived feed materials, especially yellow mealworms, black soldier flies and tussah.
In order to solve the above problems, the present application provides a primer composition for identifying or assisting in identifying the source of insect raw material components in animal-derived feed raw materials.
The primer composition comprises three, two or one of three primer pairs, namely a primer pair A specific to yellow mealworms, a primer pair B specific to black soldier flies and a primer pair C specific to tussah;
The primer pair A consists of A-F and A-R, wherein A-F is a single-stranded DNA molecule with a nucleotide sequence of SEQ ID No.1, and A-R is a single-stranded DNA molecule with a nucleotide sequence of SEQ ID No. 2;
The primer pair B consists of B-F and B-R, wherein B-F is a single-stranded DNA molecule with a nucleotide sequence of SEQ ID No.3, and B-R is a single-stranded DNA molecule with a nucleotide sequence of SEQ ID No. 4;
The primer pair C consists of C-F and C-R, wherein the C-F is a single-stranded DNA molecule with a nucleotide sequence of SEQ ID No.5, and the C-R is a single-stranded DNA molecule with a nucleotide sequence of SEQ ID No. 6.
In order to solve the above problems, the present application also provides a method for identifying or assisting in identifying a biological material from which insect raw material components in animal-derived feed raw materials are derived.
The biological material comprises any one of the following:
D1 An in vitro nucleic acid amplification reagent comprising the above primer composition;
D2 A kit comprising the above primer composition or D1) the in vitro nucleic acid amplification reagent;
d3 A detection device comprising the primer composition, D1) the in vitro nucleic acid amplification reagent or D2) the kit.
The use of the above primer composition or the above biological material in any of the following;
E1 For aiding in the detection or detection of the presence or absence of insects in the sample;
E2 For preparing products for aiding in the detection or detection of insects in a sample.
In the above application, the sample is animal-derived feed.
In the present application, the animal source may be a artiodactyla animal. The artiodactyla may be one or more of pigs, cattle and sheep.
The animal source may also be a Jinguanoidea animal. The Jinguanoides animal may be one or more of chicken and duck.
The animal source may also be an arthropoda animal. The arthropoda animal may be one or more of crabs, crayfish, antarctic krill, and shrimp.
The animal source may also be a zoo subanimal. The animal of the animal subclass can be one or more of fox, rabbit and raccoon dog.
The animal source may also be a vertebrate subgenera. The vertebrate subgenera animal can be one or more of BASHASHON, tilapia, sardine, engraulis japonicus Temminck et Schlegel, plaice, cod and fish.
In the application, the insects are one or more of yellow meal worm, black soldier fly and tussah.
In the application, whether the yellow mealworms, the black soldier flies or the tussah are present or not is judged according to the existence of the in-vitro nucleic acid amplification bands. Specifically, the existence of the amplified band is that the sample contains yellow meal worm, black soldier fly or tussah, and the nonexistence of the amplified band is that the sample does not contain yellow meal worm, black soldier fly or tussah.
In order to solve the above problems, the present application also provides a method for identifying or aiding in identifying the source of insect feedstock ingredients in animal-derived feed materials.
The method comprises the steps of carrying out in vitro nucleic acid amplification on the sample by using the primer composition or the biological material, and judging whether insects exist in the sample according to the specific products of the in vitro nucleic acid amplification.
The in vitro nucleic acid amplification technique may be Polymerase Chain Reaction (PCR), strand Displacement Amplification (SDA), ligase Chain Reaction (LCR) and nucleic acid sequence dependent amplification (NASBA), rolling circle nucleic acid amplification (RCA), loop-mediated isothermal amplification (lamp), helicase dependent isothermal amplification technique (HDA) or qβ replication technique.
The application uses Polymerase Chain Reaction (PCR) as an amplification means to carry out specific amplification.
In the above, the specific product of in vitro nucleic acid amplification may be the sequence or size or presence of the specific product of in vitro nucleic acid amplification.
In the present application, the feed may be an animal-derived feed.
In the application, the sample contains one or more of pig, cow, sheep, chicken, duck, shrimp, balsa fish, tilapia, sardine, engraulis hance, engraulis japonicus Temminck et Schlegel, fox, raccoon dog, plaice, cod, fish, crab, crayfish, antarctic krill, rabbit, yellow meal worm, black soldier fly or tussah. The sample may be a genomic DNA template.
In the application, the pig, cow, sheep, chicken, duck, shrimp, babassu, tilapia, sardine, engraulis japonicus Temminck et Schlegel, fox, raccoon dog, plaice, cod, fish, crab, crayfish, antarctic krill and rabbit can be-20deg.C frozen and stored samples.
In the application, the insects are yellow meal worms, black soldier flies or tussah silkworms.
In the method, the reaction condition of the in vitro nucleic acid amplification is that the reaction is pre-denatured for 5min at 93-97 ℃; denaturation at 92-96℃for 30sec, annealing at 55-65℃for 30sec, elongation at 72℃for 0.5-1.5min,30 cycles.
In the present application, after 30 cycles, a final extension at 72℃may be performed for 5-15min. The terminal extension may be 10 minutes.
In the application, the reaction condition of the in vitro nucleic acid amplification is that the reaction is pre-denatured for 5min at 95 ℃; denaturation at 94℃for 30sec, annealing at 60℃for 30sec, elongation at 72℃for 1min,30 cycles; and finally extending at 72 ℃ for 10min.
In the method, the insects are one or more of yellow meal worm, black soldier fly and silkworm.
In the above application or method of any one of the above, the sample comprises one or more of artiodactyla, nikkera, arthropoda, zoo, and vertebrate animals.
The artiodactyla may be one or more of pigs, cattle and sheep.
The Jinguanoides animal may be one or more of chicken and duck.
The arthropoda animal may be one or more of crabs, crayfish, antarctic krill, and shrimp.
The animal of the animal subclass can be one or more of fox, rabbit and raccoon dog.
The vertebrate subgenera animal can be one or more of BASHASHON, tilapia, sardine, engraulis japonicus Temminck et Schlegel, plaice, cod and fish.
Use of a method as described in any of the preceding claims in the auxiliary detection or testing of animal-derived feed for the presence of an insect source.
In the above, the insect source may be one or more of yellow meal worm, black soldier fly and tussah.
Advantageous effects
The application discloses a primer composition for detecting or assisting in detecting an insect source in animal-derived feed raw materials, a biological material and application thereof. The technical problem to be solved by the application is how to detect insect sources in animal source feeds.
The application designs 3 pairs of primers, namely a primer pair A, a primer pair B and a primer pair C. The primer pair A specifically detects yellow meal worm, the primer pair B specifically detects hermetia illucens, and the primer pair C specifically detects tussah. The primer pair A comprises a nucleic acid molecule with A-F and A-R, wherein the sequence of A-F is 5'-TAAGAAGAATTGTAGAAAACGGGGC-3', and the sequence of A-R is 5'-TGTGGTCGTATGTTGATTACTGTTG-3'. The primer pair B comprises a nucleic acid molecule with a B-F sequence of 5'-TCATCTTGATGAAATTTCGGGTCAC-3' and a B-R sequence of 5'-TAGGTATGGGATGGCTGATAAAAGG-3'. The primer pair C comprises a nucleic acid molecule with a C-F sequence of 5'-TTCCAACAGCTCAGACAAATAAAGG-3' and a C-R sequence of 5'-CCCCTCTCTCTTCAAATATTGCTCA-3'. PCR amplification was performed on samples containing swine, cattle, sheep, chickens, ducks, shrimps, balanus, tilapia, sardine, engraulis hance, engraulis japonicus Temminck et Schlegel, foxes, raccoon dogs, plaice, cod, fish, crabs, crayfish, antarctic krill, rabbits, yellow meal worms, black soldiers and tussahs using the above primers. Whether the yellow meal worm, the black soldier fly or the tussah exists in the sample can be judged through the existence of the amplified product. Therefore, the application provides a primer composition and a detection method capable of detecting insect raw material genes, and the primer composition and the detection method can effectively judge insect sources in animal-derived feed raw materials.
Drawings
FIG. 1 is an agarose gel electrophoresis of a PCR product verified by a yellow meal worm primer in the present invention. 1-10 represents a yellow meal worm primer 1-primer 10, wherein yellow meal worm genome DNA is taken as a template, 10 pairs of primers of yellow meal worm are utilized for PCR amplification, and the primers 4-10 on the yellow meal worm DNA template are all amplified to form a band.
FIG. 2 is an agarose gel electrophoresis chart of a PCR product verified by the hermetia illucens primer in the invention. 1-10 represent hermetia illucens primer 1-primer 10, the genome DNA of hermetia illucens is used as a template, PCR amplification is carried out by using 10 pairs of the primer of hermetia illucens, and the primers 1-10 on the DNA template of hermetia illucens are all amplified to form a strip.
FIG. 3 is an agarose gel electrophoresis chart of a PCR product verified by tussah primers in the present invention. 1-10 represents tussah primer 1-primer 10, the genome DNA of silkworm chrysalis is used as a template, 10 pairs of primers of silkworm chrysalis are used for PCR amplification, and the primers 1-10 on the DNA template of tussah are all amplified to form a strip.
FIG. 4 is an agarose gel electrophoresis of a Tenebrio molitor primer specificity verification PCR product according to the present invention. The PCR amplification is carried out by taking pig, cattle, sheep, chicken, duck, shrimp, balsa fish, tilapia, sardine, engraulis japonicus Temminck et Schlegel (Engraulis japonicus Temminck et Schlegel), fox, raccoon dog, plaice, cod, fish, crab, crayfish, antarctic krill, rabbit, yellow meal worm, black soldier fly and tussah genomic DNA as templates and utilizing the yellow meal worm specific primer 7, wherein the strips are only amplified in the yellow meal worm, and the strips are not amplified in the non-yellow meal worm component.
FIG. 5 is an agarose gel electrophoresis chart of the PCR product for verifying the specificity of the hermetia illucens primer in the invention. The PCR amplification is carried out by taking genomic DNA of pigs, cattle, sheep, chickens, ducks, shrimps, balanus, tilapia, sardine, engraulis japonicus Temminck et Schlegel (Engraulis japonicus Temminck et Schlegel), red-nose Engraulis japonicus Temminck et Schlegel (Engraulis japonicus Temminck et Schlegel), foxes, raccoon dogs, plaice, codfish, crabs, crayfish, antarctic krill, rabbits, yellow meal worm, black soldier fly and tussah as templates and utilizing a black soldier fly specific primer 9, wherein the bands are only amplified in the black soldier fly, and the bands are not in the non-black soldier fly components in all samples.
FIG. 6 is an agarose gel electrophoresis chart of a PCR product for verifying the specificity of a silkworm chrysalis primer in the invention. The genome DNA of pigs, cattle, sheep, chickens, ducks, shrimps, balsa fish, tilapia, sardine, engraulis hance, engraulis japonicus Temminck et Schlegel, foxes, raccoon dogs, plaice, codfish, fish, crabs, crayfish, antarctic krill, rabbits, yellow meal worm, black soldier fly and tussah is used as a template, PCR amplification is carried out by utilizing tussah specific primers 6, only bands are amplified in silkworm chrysalis in all samples, and no bands exist in non-silkworm chrysalis components.
FIG. 7 is a sequence (SEQ ID No. 10) amplified by Tenebrio molitor primer 7 according to the present invention as compared to similarity at NCBI.
FIG. 8 shows the similarity of the sequence (SEQ ID No. 11) amplified by the hermetia illucens primer 9 of the present invention at NCBI.
FIG. 9 shows the alignment similarity of the sequences amplified by tussah primer 6 (SEQ ID No. 12) at NCBI according to the present invention.
FIG. 10 is a diagram showing agarose gel electrophoresis of a yellow meal worm primer 7 detection mix according to the present invention. And (3) taking the DNA of the mixed sample 1, the mixed sample 2 and the yellow meal worm as templates, carrying out PCR amplification by using the yellow meal worm specific primer 7, and amplifying strips in the mixed sample 1 and the yellow meal worm, wherein no strip exists in the mixed sample 2.
FIG. 11 shows the agarose gel electrophoresis of the mixed sample detected by the hermetia illucens primer 9. And (3) taking the DNA of the mixed sample 1, the mixed sample 2 and the hermetia illucens as templates, carrying out PCR amplification by using the hermetia illucens specific primer 9, and amplifying strips in the mixed sample 1 and the hermetia illucens, wherein no strip exists in the mixed sample 3.
FIG. 12 is a diagram showing agarose gel electrophoresis of a sample mixture for detection of tussah primer 6 according to the present invention. And (3) taking the DNA of the mixed sample 1, the mixed sample 2 and the tussah as templates, carrying out PCR amplification by using the tussah specific primer 6, amplifying strips in the mixed sample 1 and the tussah, and amplifying strips in the mixed sample 4.
Detailed Description
The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless specified, were purchased from conventional biochemical reagent stores, were purchased from markets for swine, cattle, sheep, chickens, ducks, shrimps, bassars, tilapia, sardine, engraulis japonicus Temminck et Schlegel, foxes, raccoon dogs, plaice, codfish, crabs, crayfish, antarctic krill, rabbits, yellow meal worm, black soldier flies, tussah silkworms, and were pure samples. In the examples described below, all experimental results were repeated three times unless otherwise specified.
Embodiment 1, yellow mealworm, hermetia illucens, silkworm chrysalis primer screening
1. Sample collection and preservation
1.1 Sampling
Freezing and preserving the yellow meal worm, the hermetia illucens and the tussah sample at-20 ℃.
1.2 Preparation of DNA templates
Taking a proper amount of 1.1 sample, adding 1ml ddH 2 O into a 1.5ml centrifuge tube, homogenizing with a homogenizer at 12000rpm, centrifuging for 2min, discarding supernatant, and extracting kit with animal tissue genome DNAExtracting genome DNA, which comprises the following steps:
(1) To the homogenized sediment sample 320. Mu.l of buffer GA was added and shaken until thoroughly suspended.
(2) Mu.l of protease K solution was added and mixed well. The mixture was left at 65℃for 1h, during which time the samples were mixed 2-3 times.
(3) 340 Μl of buffer GB is added, the mixture is fully and reversely mixed, and the mixture is placed at 65 ℃ for 20-30min, and the samples are mixed for 2-3 times.
(4) Centrifuge at 12,000rpm for 2min, take 440 μl supernatant into a new tube.
(5) 220 Μl of absolute ethanol was added to the supernatant, and mixed well upside down 6-8 times, at which time flocculent precipitate may appear.
(6) Adding all the solution and flocculent precipitate obtained in the last step into an adsorption column CB3, centrifuging at 12,000rpm for 2min, pouring out waste liquid, and placing the adsorption column CB3 into a collecting pipe.
(7) To the adsorption column CB3, 500. Mu.l of the buffer solution GD was added, and the mixture was centrifuged at 12,000rpm for 30sec, and the waste liquid was poured off, and the adsorption column CB3 was returned to the collection tube.
(8) 600. Mu.l of the rinse PW was added to the adsorption column CB3, centrifuged at 12,000rpm for 30sec, and the waste liquid was poured off, and the adsorption column CB3 was placed in a collection tube.
(9) The operation 8 is repeated.
(10) The adsorption column CB3 was put back into the collection tube and centrifuged at 12,000rpm for 2min, and the waste liquid was discarded. The adsorption column CB3 was placed in a new EP tube and left at room temperature for several minutes to thoroughly dry the residual rinse liquid in the adsorption material.
(11) And (3) suspending and dripping 100 mu lddH 2 O into the middle part of the adsorption film, standing for 2-5min at room temperature, and centrifuging at 12,000rpm for 2min.
1.3 Concentration detection
DNA concentration and purity detection: a concentration measurement was performed by taking 2.5. Mu.l of DNA sample.
TABLE 1 detection results of insect source DNA sample concentration
2. Primer screening assay
2.1 Primer design
10 Pairs of primers, namely, yellow meal worm primers 1-10 are designed according to the conserved region of the yellow meal worm mitochondrial gene, the sequences are shown in table 2, and PCR amplification verification is carried out by taking yellow meal worm genome DNA as a template; 10 pairs of primers, namely 1-10 pairs of hermetia illucens primers, are designed according to a conserved region of hermetia illucens mitochondrial genes, and verification is carried out; 10 pairs of primers, tussah primers 1-10, were designed according to the conserved region of tussah mitochondrial gene, and verified, the primers are shown in tables 2-4.
TABLE 2 primer sequences for Tenebrio molitor
TABLE 3 primer sequences of hermetia illucens
TABLE 4 primer sequences of tussah
2.2 Preparation of PCR System
TABLE 5PCR reaction System
According to experimental design, 10 pairs of primers are respectively verified on DNA templates of yellow meal worm, black soldier fly and tussah, and ddH 2 O is used as a blank control to prepare the system.
2.3 PCR reaction
The reaction procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30sec, annealing at 60℃for 30sec, elongation at 72℃for 1min,30 cycles; and finally extending at 72 ℃ for 10min, and cooling at 4 ℃ for 10min.
PCR amplified product detection and result determination
3.1 Detection of PCR products by electrophoresis
Weighing 1g agarose, adding 100ml of 1 xTAE buffer solution, heating and boiling for 30s, uniformly mixing, adding 10 mu L GENE GREEN nucleic acid dye after slightly cooling, standing for 30min, placing in an electrophoresis tank containing 1 xTAE after solidification for sample application, adding 5 mu L of PCR amplification product in 2.2 into each gel hole, carrying out electrophoresis for 30-40min at 110V, taking out gel blocks after electrophoresis is completed, and imaging by a gel imaging system.
3.2 Result determination
As shown in the results of FIG. 1-3, the yellow meal worm genome DNA is used as a template for PCR amplification, the primers 4-10 are all amplified to form a band, and the primers 4-10 can be used for primer specificity verification (FIG. 1); PCR amplification by using hermetia illucens genome DNA as a template, amplifying the bands by the primers 1-10, and verifying the specificity of the primers 1-10 (figure 2); PCR amplification using tussah genomic DNA as template, the primers 1-10 all amplify the bands, and the primers 1-10 can perform primer specificity verification (FIG. 3).
Example 2 primer-specific detection
Screening out the yellow meal worm primer 7, the black soldier fly primer 9 and the tussah primer 6 in the embodiment 1 according to the brightness and the size of insect strips, and respectively carrying out specificity verification on the primers in the sample pigs, cattle, sheep, chickens, ducks, shrimps, balsa, tilapia, sardine, engraulis hance, engraulis japonicus Temminck et Schlegel, foxes, raccoon dogs, plaice, codfish, fish, crabs, crayfish, antarctic krill, rabbits, yellow meal worms, black soldier flies and tussah.
1. Sample collection and preservation
1.1 Sampling
And (3) freezing and preserving pig, cattle, sheep, chicken, duck, shrimp, ba sard, tilapia, sardine, engraulis japonicus Temminck et Schlegel, fox, raccoon, plaice, cod, fish, crab, crayfish, antarctic krill and rabbit at-20deg.C.
1.2 Preparation of DNA templates
The DNA extraction method in example 1 was used.
1.3 Concentration detection
DNA concentration and purity detection: a concentration measurement was performed by taking 2.5. Mu.l of DNA sample.
TABLE 6 detection results of animal-derived DNA sample concentration
PCR detection
2.1 Preparation of PCR System
TABLE 7PCR reaction System
PCR amplification is performed on systems with genomic DNAs of pigs, cattle, sheep, chickens, ducks, shrimps, ba, tilapia, sardine, engraulis japonicus, foxes, raccoons, platichthys, codfish, fish, crabs, crayfish, antarctic krill, rabbits, yellow meal worm, black soldier fly, and silkworm chrysalis as templates, respectively, using ddH 2 O as a blank control, and primers of yellow meal worm, black soldier fly, and silkworm chrysalis, respectively. The upstream primer F in the PCR system for PCR amplification using yellow meal worm was A-F described below, and the downstream primer R was A-R described below.
TABLE 8 primers for insect feedstock
TABLE 9 theoretical amplified sequences
2.2 PCR reactions
The reaction procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 94℃for 30sec, annealing at 60℃for 30sec, elongation at 72℃for 1min,30 cycles; and finally extending at 72 ℃ for 10min, and cooling at 4 ℃ for 10min.
PCR amplified product detection and result determination
3.1 Detection of PCR products by electrophoresis
Weighing 1g agarose, adding 100ml of 1 xTAE buffer solution, heating and boiling for 30sec, uniformly mixing, adding 10 mu L GENEGREEN nucleic acid dye after slightly cooling, standing for 30min, placing in an electrophoresis tank containing 1 xTAE after solidification for sample application, adding 5 mu L of PCR amplification product in 2.2 into each gel hole, carrying out electrophoresis for 30-40min at 110V, taking out gel blocks after electrophoresis is completed, and imaging by a gel imaging system.
3.2 Result determination
As shown in the graph, primer 7 (primer pair A) of the yellow mealworm can only amplify a band in the DNA of the yellow mealworm, a PCR product is a DNA fragment with the size of 100-200bp, the DNA fragment is recovered for sequencing, the result shows that the DNA fragment is SEQ ID No.10 with the nucleotide sequence in a sequence table, the nucleotide sequence is consistent with SEQ ID No.7 in a theoretical amplification sequence table, the sizes of the amplification fragments are 181bp, and the primer has specificity (shown in figure 4); primer 9 (primer pair B) of hermetia illucens can only amplify the DNA of hermetia illucens, the PCR product is a DNA fragment (specific PCR product) with the size of 300-400bp, the DNA fragment is recovered for sequencing, the result shows that the DNA fragment is SEQ ID No.11 with the nucleotide sequence in a sequence table, the nucleotide sequence is consistent with SEQ ID No.8 in a theoretical amplification sequence table, the amplified fragments are 387bp in size, and the primer has specificity (figure 5); primer 6 (primer pair C) of tussah can only amplify DNA of silkworm chrysalis, the PCR product is a DNA fragment (specific PCR product) with the size of 100-200bp, the DNA fragment is recovered for sequencing, the result shows that the DNA fragment is SEQ ID No.12 with the nucleotide sequence in a sequence table, the nucleotide sequence is consistent with SEQ ID No.9 in a theoretical amplified sequence table, the amplified fragment size is 180bp, and the primer has specificity (figure 6).
TABLE 10 amplified DNA sequences
3.3 Species accuracy verification of PCR amplified bands
As shown in the results, the sequence (SEQ ID No. 10) amplified by the yellow meal worm primer 7 and the gene accession number MN176158-1 were 100% similar (FIG. 7), and it was judged as yellow meal worm; the similarity between the sequence (SEQ ID No. 11) amplified by the hermetia illucens primer 9 and the gene accession number KC177589-1 is 97% (figure 8), and the hermetia illucens is judged; the tussah primer 6 has 100% similarity with the sequence (SEQ ID No. 12) amplified by the gene accession NC-044744-1 (FIG. 9), and is judged to be tussah.
Example 3, mixed sample detection
Mixing genome DNA of different animals according to equal volume to prepare mixed sample 1-4, and uniformly mixing by vortex instrument.
Mixed sample 1 (full species) pigs (13.57 ng/. Mu.l), cattle (3.78 ng/. Mu.l), sheep (4.17 ng/. Mu.l), chickens (9.78 ng/. Mu.l), ducks (3.30 ng/. Mu.l), shrimps (6.65 ng/. Mu.l), babassu (22.00 ng/. Mu.l), tilapia (20.22 ng/. Mu.l), sardine (4.30 ng/. Mu.l), engraulis japonicus Temminck et Schlegel (2.91 ng/. Mu.l), engraulis japonicus Temminck et Schlegel (3.87 ng/. Mu.l), foxes (10.04 ng/. Mu.l), raccoon dogs (15.74 ng/. Mu.l), plaice fish (18.52 ng/. Mu.l), cod (27.17 ng/. Mu.l), fish (7.17 ng/. Mu.l), crabs (18.39 ng/. Mu.l), crayfish (27.48 ng/. Mu.l), sorrel (20.78 ng/. Mu.l), rabbits (2.74/. Mu.l), yellow meal (39.g/. Mu.l), and black solenosis (2.74 ng/. Mu.l).
Mixed sample 2 (without yellow meal worm) is prepared from pig (14.18 ng/. Mu.l), cow (3.95 ng/. Mu.l), sheep (4.36 ng/. Mu.l), chicken (10.23 ng/. Mu.l), duck (3.45 ng/. Mu.l), shrimp (6.95 ng/. Mu.l), babassu (23.00 ng/. Mu.l), tilapia (21.14 ng/. Mu.l), sardine (4.50 ng/. Mu.l), engraulis hance (3.05 ng/. Mu.l), engraulis japonicus Temminck et Schlegel (4.05 ng/. Mu.l), fox (10.50 ng/. Mu.l), raccoon dog (16.45 ng/. Mu.l), plaice (19.36 ng/. Mu.l), fish (28.41 ng/. Mu.l), fish (7.50 ng/. Mu.l), crab (19.23 ng/. Mu.l), crayfish (28.73 ng/. Mu.l), antarctica (21.73 ng/. Mu.l), rabbit (2.86 ng/. Mu.l), black-worm (2.82 ng/. Mu.l) and black-water shrimp (29 ng/. Mu.l).
Mixed 3 (without black soldier flies) pigs (14.18 ng/. Mu.l), cattle (3.95 ng/. Mu.l), sheep (4.36 ng/. Mu.l), chickens (10.23 ng/. Mu.l), ducks (3.45 ng/. Mu.l), shrimps (6.95 ng/. Mu.l), babassu (23.00 ng/. Mu.l), tilapia (21.14 ng/. Mu.l), sardine (4.50 ng/. Mu.l), engraulis hance (3.05 ng/. Mu.l), engraulis japonicus Temminck et Schlegel (4.05 ng/. Mu.l), foxes (10.50 ng/. Mu.l), raccoon dogs (16.45 ng/. Mu.l), plaice fish (28.41 ng/. Mu.l), fish (7.50 ng/. Mu.l), rabbits (19.23 ng/. Mu.l), crayfish (28.73 ng/. Mu.l), antarctica (21.73 ng/. Mu.l), yellow silkworms (2.86 ng/. Mu.l), and (41 ng/. Mu.73 ng/mu.l).
Mixed sample 4 (without tussah) is prepared from pig (14.18 ng/. Mu.l), cow (3.95 ng/. Mu.l), sheep (4.36 ng/. Mu.l), chicken (10.23 ng/. Mu.l), duck (3.45 ng/. Mu.l), shrimp (6.95 ng/. Mu.l), babassu (23.00 ng/. Mu.l), tilapia (21.14 ng/. Mu.l), sardine (4.50 ng/. Mu.l), engraulis hance (3.05 ng/. Mu.l), engraulis japonicus Temminck et Schlegel (4.05 ng/. Mu.l), fox (10.50 ng/. Mu.l), raccoon dog (16.45 ng/. Mu.l), plaice fish (19.36 ng/. Mu.l), cod (28.41 ng/. Mu.l), fish (7.50 ng/. Mu.l), crab (19.23 ng/. Mu.l), crayfish (28.73 ng/. Mu.l), antarctica (21.73 ng/. Mu.l), rabbit (2.86/. Mu.l), and black-water shrimp (41 ng/. Mu.l).
According to the method of example 1, PCR was performed on each of mix 1, mix 2, mix 3 and mix 4 using specific primers of Tenebrio molitor primer 7 (specifically shown in Table 2), black soldier Tabanus primer 9 (specifically shown in Table 3) and tussah primer 6 (specifically shown in Table 4), with a template amount of 5. Mu.L and ddH 2 O as a blank. The PCR products were detected by 1% agarose gel electrophoresis at 110V for 30min.
As shown in the graph, primer 7 of yellow meal worm (primer pair A) amplified a band in mixed sample 1 and yellow meal worm DNA, and the sizes of the bands are 181bp, and the bands in mixed sample 2 and blank control are not provided (FIG. 10), which indicates that primer 7 of yellow meal worm has specificity and can be used in mixed sample detection; the hermetia illucens primer 9 (primer pair B) amplifies a band in the DNA of the mixed sample 1 and the hermetia illucens, the sizes of the bands are 387bp, and the mixed sample 3 and the blank control have no band (figure 11), which shows that the hermetia illucens primer 9 has specificity and can be used in mixed sample detection; the tussah primer 6 (primer pair C) amplifies bands in the DNA of the mixed sample 1 and the tussah, the sizes of the bands are 180bp, and the bands are not arranged in the mixed sample 4 and the blank control (figure 12), which shows that the tussah primer 6 has specificity and can be used in mixed sample detection.
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (8)
1. The primer composition for identifying the source of insect raw material components in animal-derived feed raw materials is characterized by comprising three primer pairs, namely a primer pair A specific for yellow mealworms, a primer pair B specific for black soldier flies and a primer pair C specific for tussah silkworms;
The primer pair A consists of A-F and A-R, wherein A-F is a single-stranded DNA molecule with a nucleotide sequence of SEQ ID No.1, and A-R is a single-stranded DNA molecule with a nucleotide sequence of SEQ ID No. 2;
The primer pair B consists of B-F and B-R, wherein B-F is a single-stranded DNA molecule with a nucleotide sequence of SEQ ID No.3, and B-R is a single-stranded DNA molecule with a nucleotide sequence of SEQ ID No. 4;
The primer pair C consists of C-F and C-R, wherein the C-F is a single-stranded DNA molecule with a nucleotide sequence of SEQ ID No.5, and the C-R is a single-stranded DNA molecule with a nucleotide sequence of SEQ ID No. 6.
2. An in vitro nucleic acid amplification reagent for identifying the source of insect feedstock components in an animal-derived feed stock, wherein said in vitro nucleic acid amplification reagent comprises the primer composition of claim 1.
3. A kit for identifying the source of insect feed ingredient in an animal feed stock, said kit comprising the primer composition of claim 1.
4. Use of the primer composition of claim 1, the in vitro nucleic acid amplification reagent of claim 2 or the kit of claim 3 in any of the following;
E1 For detecting whether an insect is contained in the sample;
e2 For preparing a product for detecting whether the sample contains insects;
the insect is one or more of yellow meal worm, black soldier fly and tussah.
5. The use according to claim 4, wherein the sample is an animal-derived feed.
6. A method for identifying the source of an insect feedstock component in an animal-derived feed feedstock, the method comprising subjecting the animal-derived feed to in vitro nucleic acid amplification using the primer composition of claim 1, the in vitro nucleic acid amplification reagent of claim 2 or the kit of claim 3, and determining the presence or absence of an insect in the animal-derived feed based on the specific product of the in vitro nucleic acid amplification;
the insect is one or more of yellow meal worm, black soldier fly and tussah.
7. The method of claim 6, wherein the in vitro nucleic acid amplification is performed under reaction conditions of 93-97 ℃ pre-denaturation 5 min; denaturation at 92-96℃for 30 sec, annealing at 55-65℃for 30 sec, elongation at 72℃for 0.5-1.5 min,30 cycles.
8. Use of the method of claim 6 or 7 for detecting whether an insect source is contained in an animal-derived feed.
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