CN112280869B - Method for detecting marine mammal components in feed by using gene chip technology - Google Patents

Method for detecting marine mammal components in feed by using gene chip technology Download PDF

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CN112280869B
CN112280869B CN202011163374.7A CN202011163374A CN112280869B CN 112280869 B CN112280869 B CN 112280869B CN 202011163374 A CN202011163374 A CN 202011163374A CN 112280869 B CN112280869 B CN 112280869B
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宦海霞
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Huai'an Customs Comprehensive Technical Service Center
Huaiyin Normal University
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Abstract

The invention discloses a method for detecting marine mammal components in feed by using a gene chip technology, which comprises the following steps: (1) extracting DNA in a sample; (2) taking the DNA extracted in the step (1) as a template, and carrying out PCR amplification reaction by using a universal primer shown as SEQ ID No. 20-21; (3) and (3) hybridizing the product of the PCR amplification reaction in the step (2) with a specific probe combination for detecting the marine mammal components in the feed, so as to realize the visual detection of the marine mammal components in the feed. The research establishes a rapid, specific, sensitive and stable detection method for the animal-derived gene chip, can be applied to detecting whether the import and export feed contains forbidden marine mammal components, can also be popularized to the detection of animal products, and can effectively guarantee the healthy development of the feed and food industry.

Description

Method for detecting marine mammal components in feed by using gene chip technology
Technical Field
The invention belongs to the field of biotechnology, and particularly relates to a method for detecting marine mammal components in feed by using a gene chip technology.
Background
The conventional detection methods for animal components in the feed comprise a microscope, an infrared spectrum, PCR, ELISA and the like, but the methods have the defects of small flux, easy generation of false positive results and the like, and are not suitable for the development trend of large-scale simultaneous detection of the current high-flux import and export feed. There is a need to establish a faster, accurate and economical method, and the gene chip assay is one that can meet these requirements. The aim of the method is to establish a method for detecting marine mammal components in feed by combining a pair of universal primers and a gene chip technology, which is rapid, simple, convenient, specific and low-cost, and ensures the safety of import and export feed.
Disclosure of Invention
The invention aims to provide a specific probe combination for detecting marine mammal components in feed.
Another object of the present invention is to provide a gene chip for detecting marine mammal components in a feed.
It is still another object of the present invention to provide a kit for detecting marine mammal components in a feed.
Still another object of the present invention is to provide a method for detecting marine mammal components in feed using gene chip technology.
The purpose of the invention can be realized by the following technical scheme:
the specific probe combination for detecting the marine mammal components in the feed comprises 1 positive control probe and 10 marine mammal family probes, wherein the sequence of the positive control probe is shown as SEQ ID No.6, and the sequences of the 10 marine mammal family probes are shown as SEQ ID Nos. 1-5 and 7-19.
Figure BDA0002745060120000011
Figure BDA0002745060120000021
A primer and probe combination for detecting marine mammal components in feed comprises a universal primer shown as SEQ ID No. 20-21 and the specific probe combination.
Primer Sequence Sequence ID
F AAGACGAGGAAGACCCTATGGAGCTTHA (SEQ ID No.20)
R GATTGCGCTGTTATCCCTAGGGTA (SEQ ID No.21)
A gene chip for detecting the components of marine mammals in feed contains chip carrier and the specific probe combination.
As a preferred technical scheme, the gene chip also comprises a quality control probe system, wherein the quality control probe system comprises a positive positioning Probe (PC) shown as SEQ ID No.22, a negative quality control probe (NC) shown as SEQ ID No.23, a fluorescein labeled complementary strand (QC) shown as SEQ ID No.24 and a Blank Control (BC), and the Blank Control (BC) is 50% (v/v) DMSO.
Probe name Probe sequence (5 'one 3') Sequence ID
PC CTGGAACAGCCAGAAGGAC SEQ ID No.22
NC GGGTGGGATCAATTTGG SEQ ID No.23
QC CCAAATTGATCCCACCC SEQ ID No.24
BC 50％DMSO
A kit comprising the specific probe combination, the primer and probe combination or the gene chip.
The specific probe combination, the primer and probe combination, the gene chip or the kit are applied to the detection of the components of the marine mammals in the feed.
A method for detecting marine mammal components in feed by using gene chip technology comprises the following steps:
(1) extracting DNA in a sample;
(2) taking the DNA extracted in the step (1) as a template, and carrying out PCR amplification reaction by using a universal primer shown as SEQ ID No. 20-21;
(3) hybridizing the product of the PCR amplification reaction in the step (2) with the specific probe combination of claim 1 to realize the visual detection of the marine mammal components in the feed.
The invention constructs a detection gene chip by carrying out sequence analysis on marine mammal marker genes, selecting mitochondrial DNA (mtDNA)16SrRNA genes as target genes, and designing specific gene chip detection probes and 1 positive probe (oligo6) aiming at 10 marine mammals in a primer amplification region by utilizing a pair of universal primers. The gene chip can be used for simultaneously and rapidly and accurately detecting at least 5 marine mammal (such as lions, seals, dolphins, porpoises and whales), and has good specificity and sensitivity reaching 10 pg. The gene chip detection technology provides a new detection method and technical support for the identification and detection of import and export feeds in China. When the method is used for detecting 5 known positive samples, the detection results are positive, and the coincidence rate with the conventional PCR result reaches 100%.
The invention has the beneficial effects that:
the research establishes a rapid, specific, sensitive and stable detection method for the animal-derived gene chip, can be applied to detecting whether the import and export feed contains forbidden marine mammal components, can also be popularized to the detection of animal products, and can effectively guarantee the healthy development of the feed and food industry.
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FIG. 1 shows chip sample hybridization.
Wherein, I: hybridizing the whale samples in the microarray; II: hybridization of the dolphin sample on the microarray; III: hybridization of the micro-array finless porpoise; IV: hybridization of seal samples of the microarray; v: hybridization of the micro-arrayed sea lion samples; VI: fish meal microarray hybridization from russia; VII: fish meal microarray hybridization in peru; VIII: fish meal microarray of chile; IX: NC (PCR negative product); x: NC (water).
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Materials and methods
1. Design of the Probe
Search of DNA sequence in oligonucleotide Probe design: the mitochondrial gene 16SrRNA of the species of interest and closely related non-species of interest was retrieved from GenBank, all relevant DNA sequences were downloaded and analyzed by alignment using Megalign (software) to screen for specific fragments of 16SrDNA that could be amplified using a pair of universal primers for identification.
After alignment with Megalign (software), for each target species, probe sequences were selected, with the emphasis on their ability to distinguish between the target species and related non-target species. Other probes were selected from conserved regions of the fragment of interest as positive controls. All selected probe sequences were approximately 20 nucleotides in length and were evaluated using primer Premier5 (software). The probe annealing temperature (Tm) was adjusted to a similar value. Finally, the probes were screened against GenBank sequences using a BLAST search to determine specific fragments in species identification.
2. Collection of samples of five marine mammals
The test samples (see table 1) were provided by Nanjing ocean world, Yangzhou university, and Huaian Longgong. Some samples were blood, some were nasal swabs, and two DNA samples of finless porpoise were given by the Wuxi aquatic institute.
TABLE 1 test samples
Figure BDA0002745060120000041
DNA extraction and amplification
DNA extraction was performed on blood and nasal swabs of 5 animals using a DNA extraction kit (Tigen). The fluorescence labeling universal primer PCR amplifies the specific segment. 3 fish meals from different countries (Russia, Peru, Chilean) were DNA extracted with DNA extraction kit (Tigen) and tested as negative controls. And performing species identification by conventional PCR and Sanger sequencing and query in GenBank to determine the accuracy of the chip hybridization result.
PCR was performed using a PCR mixture (Promega Gotaq) consisting of 2. mu.L of DNA template, 4. mu.L of 5 Xbuffer, 0.5. mu.L of primer F (10. mu.M), 1. mu.L of primer R (10. mu.M), MgCl21.2. mu.L, 1.6. mu.L of dNTPs (2.5mM), 0.4. mu.L of Go Taq, and ddH2O to 20 ul. The RCR procedure is shown in table 2.
TABLE 2 RCR procedure
Figure BDA0002745060120000042
Figure BDA0002745060120000051
4. Construction and hybridization of chips
10 kinds of marine mammal oligonucleotide probes (shown as SEQ ID No. 1-5 and SEQ ID No. 7-19, synthesized by Beijing Boo organism Co., Ltd., China) and 1 positive control probe (shown as SEQ ID No. 6) were located on the gene chip. Each probe concentration was 15. mu.M, and the spots were repeated 3 times (see Table 3). The gene chip also comprises a quality control probe system, namely a negative quality control probe (NC) shown as SEQ ID No.23, a positive positioning Probe (PC) shown as SEQ ID No.22, a fluorescein labeled complementary chain (QC) and a blank control (BC, 50% DMSO) shown as SEQ ID No.24, wherein the positive positioning probe and the Cys labeled complementary chain used in the research are shown in a table 4, and the quality control probe has no homology with the detected 16srRNA gene sequence of the marine mammal. The positive positioning point probe and the fluorescein labeled complementary strand are used for monitoring whether the amplification label, the hybridization and the color development system are effective or not; the negative quality control probe is used as a monitoring index of non-specific hybridization in the hybridization process; the blank was 50% DMSO, no oligonucleotide, and was used as an indicator for contamination monitoring during chip preparation.
TABLE 3 microarray matrix map
1 2 3 4 5 6 7 8 9
1 QC QC QC BC BC BC PC PC PC
2 NC NC NC OLIGO1 OLIGO1 OLIGO1 OLIGO2 OLIGO2 OLIGO2
3 OLIGO3 OLIGO3 OLIGO3 OLIGO4 OLIGO4 OLIGO4 OLIGO5 OLIGO5 OLIGO5
4 OLIGO6 OLIGO6 OLIGO6 OLIGO7 OLIGO7 OLIGO7 OLIGO8 OLIGO8 OLIGO8
5 OLIGO9 OLIGO9 OLIGO9 OLIGO10 OLIGO10 OLIGO10 OLIGO11 OLIGO11 OLIGO11
6 OLIGO12 OLIGO12 OLIGO12 OLIGO13 OLIGO13 OLIGO13 OLIGO14 OLIGO14 OLIGO14
7 OLIGO15 OLIGO15 OLIGO15 OLIGO16 OLIGO16 OLIGO16 OLIGO17 OLIGO17 OLIGO17
8 OLIGO18 OLIGO18 OLIGO18 OLIGO19 OLIGO19 OLIGO19 NC NC NC
9 PC PC PC BC BC BC QC QC QC
QC:quality control；BC:blank control；PC:positive control；NC:negative control
TABLE 4 Gene chip quality control probe
Figure BDA0002745060120000052
Some purified water is added at the bottom of the hybridization box, the chip is put into the box, and the slide cover is covered for use. 10. mu.L of the hybridization solution (2 х SSPE) 10. mu.L of the mixed PCR product was denatured at 95 ℃ for 3min, immediately cooled on ice for 3min, and hybridized at 50 ℃ for 60minAt 42 ℃ with solution I: 15ml SSC +10ml SDS +975ml H2O (0.3 XSSC, 0.1% SDS) and washed once with solution II: 3ml SSC +997ml H2O wash twice.
The arrays were scanned with a microarray scanner (LuxScan10K) and the fluorescence signal intensity was analyzed with LuxScan10K software.
Second, result and discussion
1. Universal primer and probe design
Although degenerate bases were used in the universal primers (as shown in Table 5), the efficiency and accuracy of primer application was still high. All correct target sequences were obtained by PCR.
TABLE 516 s rRNA Universal primers
Figure BDA0002745060120000061
Since some family members show close relativity in the 16srDNA sequence, there is no specific difference in this study in the range of amplified fragments that can be used to distinguish members of similar families such as oligonucleotides 2, 3. A total of 19 probes (see Table 6) were designed, including 1 positive control probe, 10 marine mammal family probes (more than one probe in some families), with probe lengths between 21bp and 25bp, GC contents between 40% and 65%, and annealing temperatures between 50 ℃ and 58 ℃.
TABLE 6 microarray Probe sequences
No sequence(53‘) species
OLIGO
1 CAACCCAATAAATCAAAACC Cedar
OLIGO
2 CAACCCAATAAATCAAAACC Whale beard
OLIGO
3 GCATAAAATAATACCACCAAGGG Whale tiger dolphin
OLIGO
4 CCTACCAGCCAAAGTATAGTATCAC Dolphin
OLIGO
5 GGTTGGGGTGACCTCGGAGTAT Dolphin
OLIGO
6 TTACATGAGCTGACAATTTCGG Positive probe
OLIGO
7 AATAAAACAGTACCACCTAAGGGATA Jiangtong pig
OLIGO
8 CACAAAACAGCACCACCAAGGGA White whale
OLIGO
9 ATCAACCCAATAACCCACAAC Sperm whale
OLIGO 10 TCAACCCAATAACCCACAAC Sperm whale
OLIGO 11 CCAACCCAAAAACCACAATCT Sperm whale
OLIGO 12 CCAACCCAAAAACCACAATCT Sea cattle
OLIGO 13 CCACCCTACGGGCTTAACCTA Sea cattle
OLIGO 14 GTTCAACCACAAAACATACCCTAC Chinese painting of chinese painting
OLIGO 15 GCCACCCAACAGTTTAATGAA Chinese painting of chinese painting
OLIGO 16 TACTCAAGTCAACCAAAACGGGAT Sea lion
OLIGO 17 CAGTCAACCAACAGGGAATAAA Seal
OLIGO 18 CTAAGACAAACCAGTCAAAGTGC Seal
OLIGO 19 AGAGCAAATCCAGTCAACCAAC Seal
2. Target species selection and sample collection
The microarray was designed for as many marine mammals as possible, with a total of 19 probes of 10 species. However, due to the difficulty of sample collection, we collected only 5 marine mammals (see table 1). From these five samples, we can verify the efficiency, specificity and stability of the five species probes on the chip. For probes of other species on the chip, we need to collect positive samples for validation in further studies.
3. Sample DNA amplification and sequencing
The selection of appropriate universal primers is an essential step in microarray analysis. Samples from 5 marine mammals were PCR amplified using 16SrDNA universal primers to generate specific sequences, the amplified fragments were sequenced, and BLAST was used for species identification (Table 7). The three fish meal are also amplified to obtain target fragments, sequenced and verified with the chip hybridization result. BLAST search fish meal sequencing results show that all fish meal contains no illegally added components.
TABLE 75 Marine mammal 16SrDNA specific sequence Blast results
Figure BDA0002745060120000071
4. Hybridization with chips
As shown in fig. 1, the 5 marine mammals target 16SrDNA fragment hybridization chips showed positive signals after scanning, 3 fish meal samples showed negative results, and 2 negative controls (PCR negative product, water) showed no signal in any probe of the microarray. To check the reproducibility of the detection, the amplified fragments of the different PCR reactions were hybridized twice to the chip at different times. The two repeated hybridization results show that the signal patterns of the positive probes are very similar and are consistent with the detection results of corresponding species, and the positive probes show good specificity and stability.
For the detection of non-compliant marine mammal components in food or feed, the method has the advantages of high throughput detection of samples, minimal reagent consumption and simultaneous identification of multiple targets.
This study describes the design of a DNA microarray containing 19 16SrDNA probes and demonstrates its specificity, with at least 5 species (whale, dolphin, no-wing, seal lion) confirming their specificity, and others to be further collected to generate definitive results.
As a proof of principle for the detection of non-compliant marine mammal components in seafood or feed based on DNA microarrays, it is simple and feasible to design DNA chips of more species of a particular genus or family with economically viable probes. In combination with the rapid DNA extraction method, the detection of food and feed products can be reduced to within one day. The application of the DNA microarray technology in a laboratory can greatly improve the working efficiency of the quality inspection department for monitoring and managing the composite food and feed.
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
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Claims (8)

1. The specific probe combination for detecting the marine mammal components in the feed is characterized by comprising 1 positive control probe and 10 marine mammal family probes, wherein the sequence of the positive control probe is shown as SEQ ID No.6, and the sequences of the 10 marine mammal family probes are shown as SEQ ID Nos. 1-5 and 7-19; the specific probe combination is used for detecting 5 marine mammal animals, namely sea lion, dolphin, porpoise, white whale and seal, in the feed.
2. A primer and probe combination for detecting marine mammal components in feed, which is characterized in that the primer and probe combination comprises a general primer shown as SEQ ID No. 20-21 and a specific probe combination as claimed in claim 1.
3. A gene chip for detecting marine mammal components in feed, comprising a chip carrier and the specific probe combination of claim 1.
4. The gene chip of claim 3, further comprising a quality control probe system, wherein the quality control probe system comprises a positive localization probe shown in SEQ ID No.22, a negative quality control probe shown in SEQ ID No.23, a fluorescein-labeled complementary strand shown in SEQ ID No.24, and a blank control, and the blank control is 50% DMSO.
5. A kit comprising the specific probe set according to claim 1, the primer and probe set according to claim 2, or the gene chip according to any one of claims 3 to 4.
6. Use of the specific probe set according to claim 1, the primer and probe set according to claim 2, the gene chip according to any one of claims 3 to 4, or the kit according to claim 5 for detecting marine mammal components in feed.
7. Use according to claim 6, characterized in that it comprises the following steps:
(1) extracting DNA in a sample;
(2) taking the DNA extracted in the step (1) as a template, and carrying out PCR amplification reaction by using a universal primer shown as SEQ ID No. 20-21;
(3) hybridizing the product of the PCR amplification reaction in the step (2) with the specific probe combination of claim 1 to realize the visual detection of the marine mammal components in the feed.
8. A method for detecting marine mammal components in feed by using gene chip technology is characterized by comprising the following steps:
(1) extracting DNA in a sample;
(2) taking the DNA extracted in the step (1) as a template, and carrying out PCR amplification reaction by using a universal primer shown as SEQ ID No. 20-21;
(3) hybridizing the product of the PCR amplification reaction in the step (2) with the specific probe combination of claim 1 to realize the visual detection of the marine mammal components in the feed.
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CN102758002A (en) * 2011-04-29 2012-10-31 宗卉 Method of using gene chip technology to identify and detect 16 animal-derived compositions
CN103397101B (en) * 2013-08-22 2016-01-13 山东省农业科学院生物技术研究中心 A kind of fluorescent marker gene composite amplification method simultaneously identifying goat, sheep, pig and duck source property
CN107541566B (en) * 2016-06-27 2021-04-23 中华人民共和国上海出入境检验检疫局 Method and kit for detecting mammal and bird animal derived components

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