CN107988380B

CN107988380B - Method for inter-species molecular identification of Mytilus edulis, Mytilus coruscus and perna viridis

Info

Publication number: CN107988380B
Application number: CN201711159914.2A
Authority: CN
Inventors: 陈兴强; 李荣华; 王春琳; 母昌考; 宋微微; 刘磊; 詹萍萍; 史策
Original assignee: Ningbo University
Current assignee: Ningbo University
Priority date: 2017-11-20
Filing date: 2017-11-20
Publication date: 2021-06-29
Anticipated expiration: 2037-11-20
Also published as: CN107988380A

Abstract

The invention relates to a method for molecular identification among Mytilus edulis, Mytilus coruscus and perna viridis species, and particularly relates to a pair of specific primers ZHF-18S-F/R which are designed according to SNP site differences of ribosomal gene 18S rRNA of three common Mytilus edulis, Mytilus coruscus and perna viridis in China coastal region, and the three Mytilus coruscus can be genotyped by utilizing high resolution melting curve (HRM) technology. The method gets rid of the limitation of identifying by depending on the phenotype, identifies the mussel variety from the molecular level, has the advantages of rapidness, high efficiency and accuracy, and shows that the success rate of identification is 100% by a sequencing result.

Description

Method for inter-species molecular identification of Mytilus edulis, Mytilus coruscus and perna viridis

Technical Field

The invention belongs to the technical field of shellfish identification, and particularly relates to a method for identifying common mussels, thick-shell mussels and emerald mussels.

Background

Mussels belong to the phylum Mollusca (molllusca), class Bivalvia (Bivalvia), order heterophylla (Anisomyaria), family mytiludae (Mytilidae), and are widely distributed in the temperate zone and the intertidal zone of the subarctic region. China is a large mussel culture country, the total yield reaches 84.5 thousand tons in 2015, and the front row of the mussel culture yield is located, wherein three economic mussels, namely cultured and exported Mytilus edulis (Mytilus edulis), Mytilus coruscus (Mytiloida) and perna viridis (Linnaeus), are mainly used. However, more than 50 kinds of mussels exist in coastal areas of China, the shapes of the mussel varieties are similar in the floating period and the juvenile period, and natural hybridization individuals, unshelling vending individuals and individuals with phenotypes subjected to environmental modeling are difficult to distinguish by the shapes alone. A set of efficient and practical mussel variety identification method is developed, and powerful basis is provided for production seedling culture, scientific research and market consolidation of mussels.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for identifying molecules among species of common mussels, thick-shell mussels and perna viridis aiming at the current situation of the prior art.

The technical scheme adopted by the invention for solving the technical problems is that the genomic DNA of the perna canaliculus, the perna canaliculus and the perna viridis to be identified is taken as a template, primers are designed according to the gene sequences of the perna canaliculus, the perna canaliculus and the perna viridis to carry out PCR amplification, the Tm value difference of amplification products in a high-resolution melting curve is utilized to distinguish the similar species of the perna canaliculus, and the sequence amplification primers are as follows:

ZHF-18S-F:CGTAGTTGGATCTCGGGT

ZHF-18S-R:AAACCGGGAGGTAGGTCAGG。

further, the SNP target amplification fragment of perna viridis is CGTAGTTGGATCTCGGGTCCCAGGCTTGCGGTCCGACGCACGTCGGTTTACTGCTTGT CCTGACCTACCTCCCGGTTT.

Further, the SNP target amplified fragment of the Mytilus edulis is CGTAGTTGGATCTCGGGTCCAGGCTGGCGGTCCGACGCCTGTCGGTTACTGCCTGCTC CTGACCTACCTCCCGGTTT.

Further, the SNP target amplification fragment of the mytilus coruscus is

CGTAGTTGGATCTCGGGTCCAGGCTTGCGGTCCGACGCCTGTCGGTTACTGCTCG TCCTGACCTACCTCCCGGTTT。

Compared with the prior art, the invention has the following advantages:

1. the operation is simple and flexible: only one PCR reaction system added with saturated fluorescent dye and one conventional real-time fluorescent quantitative PCR instrument (a high-resolution melting analysis module can be additionally arranged) are needed, separation of products after PCR is not needed, closed-tube operation is really realized, and the possibility of pollution is avoided;

2. high flux: the identification work of 96 or even 384 unknown samples can be realized within 2 hours on one fluorescence quantitative PCR instrument;

3. the result is accurate and intuitive: the steps of sequencing, sequence comparison and the like are not needed, the difference of the DNA levels among different species is expressed in the form of an intuitive peak diagram or a dissolution curve, the discrimination is easy to distinguish, and the identification result is consistent with the sequencing result.

Drawings

FIG. 1 is a high-resolution melting curve diagram for identification of closely related species of shellfish (wherein the abscissa is temperature) and the ordinate is a logarithmic value of fluorescence intensity; the abscissa corresponding to the highest point of each curve is the Tm value of the PCR product corresponding to that curve).

Detailed Description

The invention is further described by the following embodiments in conjunction with the drawings.

The invention introduces a high-resolution melting curve method in the process of species identification by means of nucleic acid sequence distinction, and only one pair of PCR primers is used for converting the difference of different species on the DNA level into a more visual melting curve.

The method comprises the following steps:

a. species identification was based on the acquisition of sequences: obtaining a reference sequence (such as a biological tag sequence such as a COI gene sequence) capable of distinguishing target species from an existing database or other sources;

b. designing a primer: comparing different species according to sequence differences, searching a region capable of obviously distinguishing target species, and designing a PCR amplification primer in the region by using primer premier5 software to ensure that amplification products have obvious sequence differences (namely different Tm values) among different species, which is mainly reflected in different G, C contents;

c. and (3) PCR amplification: performing PCR amplification by using the primer and taking the genomic DNA of the target mussel to be identified as a template;

d. product detection and treatment: detecting the PCR amplification products by using 1% agarose gel electrophoresis, after determining that the amplification products are single and have no non-specific amplification, adding 1 mul of nucleic acid saturation dye LC-green (Idaho, USA) into each reaction, running the reaction on a common PCR instrument for 10min at 95 ℃, and then cooling to room temperature.

e. High resolution melting curve (HRM) analysis of the above products in

And (3) running HRM on a 480 real-time quantitative analyzer (Roche Diagnostics), obtaining a product melting curve by using analysis software on a platform after the HRM is finished, and distinguishing different species by using a welding curve of the product.

The annealing temperature of the PCR amplification primer is between 50 and 60 ℃, and the length of the PCR product is controlled between 40bp and 100 bp. SNP sites and In/del are avoided as much as possible In the PCR amplification primers, and the sequences of PCR products have obvious Tm value differences among different target species to be identified. The PCR amplification procedure was: 95 ℃ (pre-denaturation for 10min), 95 ℃ (denaturation for 15sec), Tm (renaturation for 15sec), 72 ℃ (extension for 30sec), and after 44 times of co-circulation, extension for 10min at 72 ℃, and finishing at 16 ℃ (cooling). The reference sequence for species identification can be selected freely according to the need, and can be COI gene sequence or other sequence conforming to the characteristics of biological label.

Example 1

The identification of common mussels, thick-shell mussels and emerald mussels in the mussels:

a. species identification by sequence acquisition and alignment analysis

The 18S rRNA sequences of the three mussels were obtained in the NCBI database, and aligned using AlignX (a component of Vector NTI Suite 7.1) software to find regions where there was a base difference among the three mussels.

b. Primer design and screening

The primer design should satisfy the following conditions: the target amplification sequence is between 50 and 150 bp; in order to make the melting temperatures different, there is a difference in the GC base content between the sequences; the annealing temperature (Tm) should be between 50 and 60 ℃; mismatch, hairpin structure and primer dimer should be avoided as much as possible between the positive and negative primers; the primers were synthesized by Shanghai Biotech, Inc. Randomly selecting 5 corresponding mussel DNA samples as templates, primarily screening primers by using an agarose gel electrophoresis technology, and selecting a primer combination capable of amplifying a bright single band for HRM analysis.

c. HRM analysis

Use of

480 saturated fluorescent dye HRM kit comprising: 10 μ L1 is prepared from

480 HRM Master Mix with

dye (Roche diagnostics), forward and reverse primers 10. mu. mol each, 30ng DNA template, 1.6. mu.L Mgcl2, and water to 20. mu.L.

The PCR reaction and the melting curve analysis of the product are carried out simultaneously

480 real-time quantitative analyzer (Roche Diagnostics).

CGTAGTTGGATCTCGGGT as a forward primer ZHF-18S-F;

the reverse primer ZHF-18S-R: AAACCGGGAGGTAGGTCAGG.

The reaction procedure was as follows: the PCR amplification procedure was as follows: 95 ℃ (pre-denaturation 30sec), 95 ℃ (denaturation 30sec), Tm (renaturation 30sec), 72 ℃ (extension 30sec), after 45 times of co-circulation of denaturation, renaturation and extension, extension 10min after 72 ℃, and after 16 ℃ (cooling) is finished.

After PCR amplification is finished, the program automatically runs a high-resolution melting curve program, and fluorescence is collected for 25 times at 1 ℃ per liter. Use of

Tm value analysis and genotyping were carried out using Tm Calling and Gene Scanning Software 1.5 carried on 480.

d. Product detection and treatment: detecting the PCR amplification product by using 1% agarose gel electrophoresis, adding 1ul LC-green into each reaction after determining that the amplification product is single, running the reaction on a common PCR instrument at 95 ℃ for 5sec, and cooling to room temperature.

e. High resolution melting curve (HRM) analysis: the above products were subjected to HRM on the Lightscanner96 platform and fluorescence signal data were collected between 55 ℃ and 95 ℃. After the reaction is finished, converting the fluorescence signal into a melting curve peak diagram by using self-contained analysis software on the platform; different shellfish similar species are distinguished according to the difference of Tm values corresponding to the peak values of different curves (see figure 1).

f. Respectively get 8 purple mussels, thick shell mussel, emerald mussel as the sample respectively, detect after extracting DNA, the experimental result is as follows:

as can be seen from fig. 1: the melting curves are respectively gathered into 3 bundles, and the 3 bundles respectively present 3 different Tm values and correspond to common mussels, thick-shell mussels and emerald mussels in the sample; the TM value of the Mytilus edulis of type A is 81.0, the TM value of the Mytilus edulis of type B is 81.8, the TM value of the Mytilus edulis of type C is perna viridis, and three groups of samples are remarkably distinguished, so that the site is proved to be effective in identifying the three species.

g. Sequencing validation

For the SNP sites capable of obviously typing, PCR products corresponding to 5 curves of each genotype are randomly selected for sequencing, the sequencing results of various genotypes are compared and analyzed, the sequences are respectively SEQ1, SEQ2 and SEQ3, and the difference of the base compositions among the sequences is observed, as shown in the following table 1.

Table 1 sequence sequencing and alignment results: SNPs present in the three mussel target amplified fragments are shown.

Jadeite musselCGTAGTTGGATCTCGGGTCCCAGGCTTGCGGTCCGACGCACGTCGGTTTACTGCTTG-TCCTGACCTACCTCCCGGTTT

Common musselCGTAGTTGGATCTCGGGTCC-***** G************ CT******* -***** CT*CTCCTGACCTACCTCCCGGTTT

Thick shell musselCGTAGTTGGATCTCGGGTCC-***** T************ CT******* -***** TC*-TCCTGACCTACCTCCCGGTTT

Sequence listing

<110> Ningbo university

<120> method for identifying molecules among species of common mussels, thick-shell mussels and perna viridis

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 78

<212> DNA

<213> Mytilus edulis

<400> 2

cgtagttgga tctcgggtcc caggcttgcg gtccgacgca cgtcggttta ctgcttgtcc 60

tgacctacct cccggttt 78

<210> 2

<211> 77

<212> DNA

<213> Mytiloida

<400> 2

cgtagttgga tctcgggtcc aggctggcgg tccgacgcct gtcggttact gcctgctcct 60

gacctacctc ccggttt 77

<210> 3

<211> 76

<212> DNA

<213> Linnaeus

<400> 3

cgtagttgga tctcgggtcc aggcttgcgg tccgacgcct gtcggttact gctcgtcctg 60

acctacctcc cggttt 76

<210> 4

<211> 18

<212> DNA

<213> ZHF-18S-F

<400> 4

cgtagttgga tctcgggt 18

<210> 5

<211> 20

<212> DNA

<213> ZHF-18S-R

<400> 5

aaaccgggag gtaggtcagg 20

Claims

1. A method for identifying molecules among species of Mytilus edulis, Mytilus coruscus and perna viridis is characterized by comprising the following steps: the method is characterized in that genomic DNA of the common mussels, the thick-shell mussels and the perna viridis to be identified is used as a template, primers are designed according to gene sequences of the common mussels, the thick-shell mussels and the perna viridis to perform PCR amplification, Tm value differences of amplification products in a high-resolution melting curve are utilized to distinguish similar species of the common mussels, and the sequence amplification primers are as follows:

ZHF-18S-F: CGTAGTTGGATCTCGGGT；

ZHF-18S-R: AAACCGGGAGGTAGGTCAGG。

2. the method for molecular inter-species molecular characterization of perna canaliculus, perna viridis according to claim 1, wherein the SNP target amplified fragment of perna viridis is CGTAGTTGGATCTCGGGTCCCAGGCTTGCGGTCCGACGCACGTCGGTTTACTGCTTGTCCTGACCTACCTCCCGGTTT.

3. The method for molecular inter-species molecular identification of Mytilus edulis, Mytilus coruscus and perna viridis as claimed in claim 1, wherein the SNP target amplified fragment of Mytilus edulis is CGTAGTTGGATCTCGGGTCCAGGCTGGCGGTCCGACGCCTGTCGGTTACTGCCTGCTCCTGACCTACCTCCCGGTTT.

4. The method for molecular inter-species molecular characterization of Mytilus edulis, Mytilus coruscus, and perna viridis as claimed in claim 1, wherein the SNP target amplified fragment of Mytilus coruscus is CGTAGTTGGATCTCGGGTCCAGGCTTGCGGTCCGACGCCTGTCGGTTACTGCTCGTCCTGACCTACCTCCCGGTTT.