CN113005208B - Universal macro-barcode amplification primer for mollusks and application method thereof - Google Patents
Universal macro-barcode amplification primer for mollusks and application method thereof Download PDFInfo
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention belongs to the technical field of DNA detection, and discloses a universal macro-barcode amplification primer for mollusks and an application method thereof. According to the method, 6 macro barcode amplification primers are designed according to a common soft 18S gene sequence, the primers comprise 3 upstream primers and 3 downstream primers, the coverage of the primers to a soft animal community is wide, the amplification efficiency is high, the primer combination can amplify products with the length of 200bp to 350bp, and the primers are compatible with different high-throughput sequencing platforms, so that the method for combining the multiple primers can reduce nonspecific amplification, improve the PCR success rate, and can be widely used as a characteristic barcode sequence for researches such as soft animal species identification, soft animal diversity analysis and the like.
Description
Technical Field
The invention belongs to the technical field of DNA detection, and discloses a universal macro-barcode amplification primer for mollusks and an application method thereof.
Background
Molluscs, also known as shellfish, are the collective term for mollusca phylum animals, the largest group of species other than arthropods, about 10 tens of thousands. The systems vary widely but share common features: the body is soft but not segmented, and is generally divided into two parts, namely the head-foot (some head degenerates or disappears; foot muscle mass) and the viscera-mantle (composed of viscera mass, mantle and coat cavity on the back side).
Mollusks have a very broad habitat, from tropical continents to the north and south ocean, from high-rise ponds above 7000 meters in elevation to deep seas below 800 meters. Mollusks are very diverse, being next to arthropods-more than 10 tens of thousands of existing species. They differ greatly in morphology but all have a soft body. Most of them live in the ocean, only part of bivalve and gastropod migrate to brackish water and fresh water for perching.
For DNA detection of mollusks, related applications are disclosed in the prior art through retrieval, for example, the application with Chinese patent application number 2012104811798 and publication date 2013 and 03 month 06 discloses a method for identifying forest shallot snails through PCR detection, wherein the method is to identify molecular characteristics of the forest shallot snails by applying specific primers; the upstream primer of the PCR reaction is CN- (P1): 5'-ACCTCCTTCCTTTCTACT-3', the downstream primer is CN- (P2): 5'-GTCAACATCTATCCCAAC-3'. The total volume of the PCR reaction system is 25uL, wherein 2×TaqPCRMasterMix12.5uL, each of the upstream primer and the downstream primer is 0.5uL, the DNA template is 2uL, and the rest is complemented by sterilized ddH 20; the PCR reaction procedure was: pre-denaturation at 5 ℃ for 5min;95 ℃ for 50s,52 ℃ for 30s,72 ℃ for 50s,35 cycles; the reaction was terminated by extension at 72℃for 10 min. Although the method can rapidly and accurately detect and identify the forest shallot snail, the patent is designed for a certain species, can only be used for detecting the species of the forest shallot snail, and cannot cover the whole mollusk.
For example, the application number of Chinese patent is 2019110516175, the publication date is 2020, 02 month and 04, discloses a universal primer for the transcription spacer in the ribosome of the opisthorchiaceae, and the forward primer sequence is as follows: ACAATGACGGTTTCAGCGAGT (SEQ ID No. 1), the reverse primer sequence is: CACAAACAACCCGACTCCAAGG (SEQ ID No. 2). The method of this application also discloses the design and amplification methods and uses of the universal primers. The universal primers of this application were designed based on 18SrDNA and 28SrDNA sequences conserved at both ends of the transcribed spacer (ITS) in the ribosome, and the resulting primers were extremely specific. Also, the method of this application can only be used for accurate detection of posttestosterone flukes, and cannot cover the whole mollusc species.
Mollusks are important indicator species for indicating the quality of water environment, and are one of key monitoring objects for evaluating the ecological health of watershed. At present, the monitoring flow of mollusks comprises the steps of sample collection, manual picking, morphological identification and the like, and has the defects of long time consumption, high labor cost, high dependence on species identification experts and various limitations in actual ecological health evaluation. The existing detection methods for mollusks can only detect specific species, cannot completely cover the whole mollusks, and cannot realize identification and classification of various mollusks through one-time detection.
Based on the defects of the prior art, a need exists for a method for detecting the DNA of mollusks which can cover the whole area.
Disclosure of Invention
1. Problems to be solved
At present, the monitoring flow of mollusks comprises the steps of sample collection, manual picking, morphological identification and the like, and has the defects of long time consumption, high labor cost, high dependence on species identification experts and various limitations in actual ecological health evaluation. Aiming at the problems, the invention provides a group of mollusc PCR amplification primers based on ribosome 18S genes and an application method thereof, wherein the primer pair has wide coverage and high amplification efficiency on mollusc communities, and the method adopting a multi-primer combination can reduce non-specific amplification and improve the PCR success rate, and can be widely used as a characteristic bar code sequence for researching mollusc species identification, mollusc diversity analysis and the like.
2. Technical proposal
In order to solve the problems, the technical scheme adopted by the invention is as follows:
the invention provides a mollusc universal macro-bar code amplification primer, which comprises at least one primer pair sequence in a), b) and c), wherein the primer sequences in each of the a), b) and c) are as follows:
a) M18-1F, wherein the nucleotide sequence of the M18-1F is GGATAACTGTGGNAATTCNAGA,
M18-1R, wherein the nucleotide sequence of the M18-1R is CCTGATTCCCCGNTACCCGT;
b) M18-2F, wherein the nucleotide sequence of the M18-2F is AAGCTCGTAGTTGGATCTC,
M18-2R, wherein the nucleotide sequence of the M18-2R is TCCAAGAATTTCACCTCT;
c) M18-3F, wherein the nucleotide sequence of the M18-3F is CCGATAACGAACGAGACTC;
M18-3R, wherein the nucleotide sequence of the M18-3R is GGGAATTCCTCGTTCANGGG.
Further, the mollusc universal macro barcode amplification primer application comprises the following (1) or (2) or (3) or (4):
(1) Identifying or aiding in identifying whether the test DNA is mollusc DNA;
(2) Preparing a kit for identifying or assisting in identifying whether the DNA to be tested is a mollusc;
(3) Detecting whether a sample to be detected contains mollusks or not;
(4) A kit for detecting whether a sample to be tested contains molluscs is prepared.
Further, the application of (3) further includes:
(5) Mollusc species identification and mollusc diversity analysis;
(6) Monitoring of mollusc endangered species and mollusc invasive species;
(7) Analysis of mollusc evolution, phylogenetic, and genetic diversity.
Further, the sample to be detected comprises environment source DNA and biological source DNA, and the environment source DNA comprises water sample DNA, sediment DNA and biological film DNA.
Further, the application of the step (3) includes the following steps:
(S1) extracting a DNA sample to be detected;
(S2) amplification of the fragment of interest: performing PCR amplification on the DNA to be detected extracted in the step (S1), wherein the amplification primer adopts the mollusc universal macro barcode amplification primer;
(S3) high throughput sequencing of the amplified product of step (S2); or analyzing the amplified product of step (S2) for a diversity of mollusc populations.
Further, in the PCR amplification process of the step (S2), if the M18-1F and the M18-1R primer are adopted for PCR reaction, the annealing temperature is 55-57 ℃ and the product length is 200-300bp; if the M18-2F and the M18-2R primer are adopted for PCR reaction, the annealing temperature is 53-56 ℃ and the product length is 280-350bp; if the M18-3F and the M18-3R primer are adopted for PCR reaction, the annealing temperature is 52-55 ℃ and the product length is 250-320bp.
Further, the step (S1) operates as follows: collecting sediment by a mud sampler, cleaning the sediment, picking out mollusks in the sediment, soaking the mollusks in 75% alcohol, preserving the mollusks for a plurality of days at normal temperature, adding the alcohol, centrifuging the mixture, then placing the mixture into a-80 refrigerator for precooling for a period of time, performing vacuum freeze-drying at the temperature of minus 40 ℃ to remove all liquid, and extracting DNA by using a tissue extraction kit.
Further, the primers for performing PCR amplification in the step (S2) include three pairs of primer sets a), b) and c):
a) M18-1F, wherein the nucleotide sequence of the M18-1F is GGATAACTGTGGNAATTCNAGA,
M18-1R, wherein the nucleotide sequence of the M18-1R is CCTGATTCCCCGNTACCCGT;
b) M18-2F, wherein the nucleotide sequence of the M18-2F is AAGCTCGTAGTTGGATCTC,
M18-2R, wherein the nucleotide sequence of the M18-2R is TCCAAGAATTTCACCTCT; and
c) M18-3F, wherein the nucleotide sequence of the M18-3F is CCGATAACGAACGAGACTC;
M18-3R, wherein the nucleotide sequence of the M18-3R is GGGAATTCCTCGTTCANGGG.
Further, in the step (S2), PCR amplification was performed using Platinum Taq enzyme from Invitrogen company, the volume of the amplification reaction system was 50. Mu.L, and the reaction system contained the following: 1 μL of 10 μM of the mollusc universal macro barcode amplification primer, 37.8 μL of deionized water, 5 μL of 10 XPCR Hifi buffer, 2 μL of MgSO 4 (50 mM), 1. Mu.L of dNTP Mix (10 mM), 0.2. Mu.LTaq DNA polymerase, 2. Mu.L of DNA sample to be tested.
Further, the invention provides a kit of the mollusc universal macro barcode amplification primer, and the kit is used as follows:
(a1) Identifying or aiding in identifying whether the test DNA is a mollusc;
(a2) Detecting whether a sample to be detected contains mollusks or not;
(a3) Mollusc species identification and mollusc diversity analysis;
(a4) Monitoring of mollusc endangered species and mollusc invasive species;
(a5) Analysis of mollusc evolution, phylogenetic, and genetic diversity.
3. Advantageous effects
Compared with the prior art, the invention has the beneficial effects that:
(1) The universal macro barcode amplification primer for molluscs does not use the traditional COI barcode gene region, and the 18S gene which is more conservative for molluscs is selected as the macro barcode primer region, so that the amplification capability of the primer in the research of macro barcode diversity on a mollusc community in environmental DNA is improved, and the amplification and recognition of the low-abundance mollusc DNA in the environment are improved; the invention discloses 3 pairs of mollusc macro-bar code amplification primers, wherein three pairs of primers are designed for molluscs, have good amplification capability for molluscs, can reduce the proportion of non-mollusc sequences in macro-bar code research, improve the data flux of molluscs, provide a novel method for monitoring mollusc macro-bar codes, and are beneficial to research on biological diversity of molluscs;
(2) The length of the designed primer is suitable for high-throughput sequencing, the traditional primer has excessively long amplified fragments (more than 500 bp) and can not be sequenced on a second-generation sequencing platform, and the designed primer has the amplification length of 200bp to 350bp and proper length and can be sequenced on the high-throughput sequencing platform;
(3) The universal macro barcode amplification primer for molluscs provided by the invention can be used for mollusc species identification and mollusc diversity analysis, and a product obtained by PCR amplification of sequences M18-1F and M18-1R, M18-2F and M18-2R and M18-3F and M18-3R is of great significance for researching species classification, system development, system geography and protecting mollusc germplasm resources of a mollusc community;
(4) The 18S primer obtained by screening is low in degeneracy, has strong amplification preference on soft communities, is suitable for amplifying low-abundance DNA in environmental samples, is mild in conditions during PCR amplification, is high in PCR success rate, has no severe requirement on a DNA polymerase (polymerase chain reaction) primer, has the fragment length of less than 500bp (200 bp to 350 bp), can be sequenced by using a high-throughput sequencing platform, can obtain a plurality of sequences at one time, is low in cost, and has good application prospect;
(5) The primers are designed based on the full length of the 18S gene of common molluscs in China, have the advantages of high universality and high sensitivity for molluscs, and 3 upstream primers and 3 downstream primers are designed, so that the primers can be well compatible with different sequencing platforms.
Drawings
FIG. 1 shows the amplification of Yangtze river molluscs using three pairs of primers of the present invention, FIG. a shows the amplification of Yangtze river molluscs using M18-1F and M18-1R, FIG. b shows the amplification of Yangtze river molluscs using M18-2F and M18-2R, and FIG. c shows the amplification of Yangtze river molluscs using M18-3F and M18-3R.
FIG. 2 shows the species information and the results of the diversity analysis in example 2, and FIG. 2 shows, from left to right, the results of the diversity analysis using M18-1F and M18-1R, the results of the diversity analysis using M18-2F and M18-2R, the results of the diversity analysis using M18-3F and M18-3R, and the results of the diversity analysis using the COI primer.
Detailed Description
The invention is further described below in connection with specific embodiments.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs; the term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. Moreover, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.
Example 1
(1) The mollusc 18S gene macro barcode universal primer list, table 1 shows the amplified fragment sizes of the upstream and downstream primer combinations.
TABLE 1 amplification fragment size for upstream and downstream primer combinations
The method for detecting molluscs in the environmental sample by using the primer combination comprises the following specific steps:
mollusc samples were collected in the bay (east longitude 120.036 °, north latitude 31.373 °). The 10 clip sediment was collected by peterson's dredger, and the sediment was preliminarily washed by 80 mesh screen, and then put into 10L self-sealing bags and transported back to the laboratory. The sediment was further washed in the laboratory, the mollusks inside were picked up, stored in a-80 refrigerator and DNA was extracted using Tissue DNA kit OMEGA, CHINA.
PCR amplification was performed on the extracted mollusk DNA using the primer pairs of M18-1F and M18-1R, M18-2F and M18-2R, and M18-3F and M18-3R, respectively.
The annealing temperature of the PCR reaction of the M18-1F and the M18-1R primer is 55-57 ℃, and the length of an amplified product is 200-300bp; the annealing temperature of the PCR reaction of the M18-2F and the M18-2R primer is 53-56 ℃, and the length of an amplified product is 280-250bp; the annealing temperature of the PCR reaction of the M18-3F and the M18-3R primer is 52-55 ℃, and the length of the amplified product is 250-320bp.
PCR amplification was performed using Platinum Taq enzyme from Invitrogen, the volume of the amplification reaction system was 50. Mu.L, and the reaction system contained the following: 1. Mu.L of 10. Mu.M of the 12S primer described above; 37.8 μl of deionized water; 5 μL of 10 XPCR Hifi buffer;2 mu L of MgSO 4 (50 mM); 1. Mu.L of dNTP Mix (10 mM); 0.2 mu LTaq DNA polymerase; 2. Mu.L of DNA template (sample of the species to be amplified or sample DNA of the environment). The PCR product obtained was detected with agarose gel at a concentration of 2%. The results show (FIG. 1), according to FIG. 1, that for the acquisition of environmental samples, amplification can be achieved for all three pairs of primers, indicating that the primers have good amplification ability for molluscs.
Example 2
Collecting 5 clamp sediment in Nanjing sheep mountain lake, cleaning the sediment with 80 mesh sieve, picking out the animal, soaking in 75% alcohol, and preserving at normal temperature for 14 days. 10 ml of ethanol was centrifuged at 3000 rpm for 30 minutes, then placed in a-80 refrigerator for 4 hours, vacuum freeze-dried at-40℃to remove all liquid, and then DNA was extracted using Tissue DNA Kit (accession number D3396, OMEGA).
The extracted mollusk DNA was PCR amplified using M18-1F in combination with M18-1R, M18-2F in combination with M18-2R, M18-3F, M18-3R in combination with the common COI primer (upstream primer: GGWACWGGWTGAACWGTWTAYCCYCC; downstream primer: TAAACYTCAGGRTGACCRAARAAYCA) as already disclosed. The annealing temperature of the PCR reaction of the M18-1F and the M18-1R primer is 55-57 ℃, and the length of an amplified product is 200-300bp; the annealing temperature of the PCR reaction of the M18-2F and the M18-2R primer is 53-56 ℃, and the length of an amplified product is 280-250bp; the annealing temperature of the PCR reaction of the M18-3F and the M18-3R primer is 52-55 ℃, and the length of an amplified product is 250-320bp; the annealing temperature of the PCR reaction of the COI primer is 48 ℃, and the length of an amplified product is 313bp.
PCR amplification was performed using Platinum Taq enzyme from Invitrogen, the volume of the amplification reaction system was 50. Mu.L, and the reaction system contained the following: 1. Mu.L of 10. Mu.M of the 12S primer described above; 37.8 μl of deionized water; 5 μL of 10 XPCR Hifi buffer;2 mu L of MgSO 4 (50 mM); 1. Mu.L of dNTP Mix (10 mM); 0.2 mu LTaq DNA polymerase; 2. Mu.L of DNA template (sample of the species to be amplified or sample DNA of the environment); the PCR product obtained was detected with agarose gel at a concentration of 2%.
High-throughput sequencing of the amplified product by using Ion Torrent S5 plus sequencer, and using a special kit Ion Xpress of the sequencer before sequencing TM Plus Region Library Kit (Life Technologies, USA) constructing a sequencing library, outputting the sequencing result in Fastq format, performing sequence pretreatment (the process is as follows) on a QIIME2 software platform based on a Linux system, filtering the sequence with low sequencing quality value and sequence length shorter than 200bp, and adding the sequencing result according to the corresponding Barcode (the self-contained reagent of the sequencing kit during sequencing and library establishment)According to different sample classifications, the classified sequences are respectively subjected to UCHIME software to remove chimeric subsequences, then Ucluster software is used for carrying out OTU typing according to the similarity among the sequences, the representative sequence of each OTU is extracted and BLAST annotation is carried out, the sequences with the BLAST sequence similarity being more than or equal to 97% are screened, and finally species information is determined and diversity analysis is carried out, wherein the analysis results are shown in figure 2.
This example shows that the three pairs of primers of the invention have better amplification for molluscs. The mollusk ratio in the amplified sequences of the M18-1F and M18-1R primers is 29.5 percent, and the arthropod ratio is 63.1 percent; mollusc ratio 19.3% and arthropod ratio 58.9% in the amplified sequences of M18-2F and M18-2R primers; the mollusc ratio in the amplified sequences of the M18-3F and M18-3R primers was 22.1% and the arthropod ratio was 57.4%. Whereas only 0.03% of the COI amplified sequences were from molluscs, the arthropod was 65.7% and the other 14.87% of the sequences could not be annotated.
The 18S primer related to the invention has better specificity to mollusc communities, most of amplified sequences are from molluscs, and the primer is more suitable for mollusc diversity research.
Sequence listing
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Claims (6)
1. A mollusc universal macro barcode amplification primer, characterized in that: comprising at least one primer pair sequence in groups a), b), c), wherein the primer sequences in each group a), b), c) are as follows:
a) M18-1F, wherein the nucleotide sequence of the M18-1F is GGATAACTGTGGNAATTCNAGA,
M18-1R, wherein the nucleotide sequence of the M18-1R is CCTGATTCCCCGNTACCCGT;
b) M18-2F, wherein the nucleotide sequence of the M18-2F is AAGCTCGTAGTTGGATCTC,
M18-2R, wherein the nucleotide sequence of the M18-2R is TCCAAGAATTTCACCTCT;
c) M18-3F, wherein the nucleotide sequence of the M18-3F is CCGATAACGAACGAGACTC;
M18-3R, wherein the nucleotide sequence of the M18-3R is GGGAATTCCTCGTTCANGGG.
2. The use of a mollusc universal macro barcode amplification primer according to claim 1, wherein: comprising the following (1) or (2) or (3) or (4):
(1) Identifying or aiding in identifying whether the test DNA is mollusc DNA;
(2) Preparing a kit for identifying or assisting in identifying whether the DNA to be tested is mollusc DNA;
(3) Detecting whether a sample to be detected contains mollusks or not;
(4) A kit for detecting whether a sample to be tested contains molluscs is prepared.
3. The use of a mollusc universal macro barcode amplification primer according to claim 2, wherein: the sample to be tested comprises environment source DNA and biological source DNA, and the environment source DNA comprises water sample DNA, sediment DNA and biological film DNA.
4. Use of a mollusc universal macro barcode amplification primer according to any of claims 2-3, characterized in that: the method comprises the following steps:
(S1) extracting DNA to be detected;
(S2) amplification of the fragment of interest: performing PCR amplification on the DNA to be detected extracted in the step (S1), wherein an amplification primer is the mollusc universal macro barcode amplification primer;
(S3) high throughput sequencing of the amplified product of step (S2); or performing a community diversity analysis on the mollusks amplified in step (S2).
5. The use of a mollusc universal macro barcode amplification primer according to claim 4, wherein: in the PCR amplification process of the step (S2), if the M18-1F and the M18-1R primer are adopted for PCR reaction, the annealing temperature is 55-57 ℃ and the product length is 200-300bp; if the M18-2F and the M18-2R primer are adopted for PCR reaction, the annealing temperature is 53-56 ℃ and the product length is 280-350bp; if the M18-3F and the M18-3R primer are adopted for PCR reaction, the annealing temperature is 52-55 ℃ and the product length is 250-320bp.
6. The use of a mollusc universal macro barcode amplification primer according to claim 4, wherein: the step (S1) is operated as follows: collecting sediment by a mud sampler, cleaning the sediment, picking out mollusks in the sediment, soaking in 75% alcohol, preserving for several days at normal temperature, adding alcohol, centrifuging, pre-cooling in a refrigerator at-80 ℃ for a period of time, vacuum freeze-drying at-40 ℃ to remove all liquid, and extracting DNA by using a tissue extraction kit.
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