CN113832238A - Primer and probe for detecting environmental DNA of Lasa naked nojirimus fish and application of primer and probe - Google Patents
Primer and probe for detecting environmental DNA of Lasa naked nojirimus fish and application of primer and probe Download PDFInfo
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Abstract
The invention relates to a primer and a probe for detecting environmental DNA of a rassa nojirimus fish and an application method thereof, belonging to the field of molecular ecology. The primer is as follows: ScYo-F: CTCTGCTCCATACCTCCAAGCT, ScYo-R: TGAGAAACAGTGCAAAGTACAGGG, wherein the probe is ScYo-P: ACTAACATTCCGCCCAATCACCC are provided. The method comprises the following steps: collecting a water sample and extracting eDNA; using the primer and the probe to carry out micro-drop digital PCR (ddPCR) amplification on the eDNA; reading the copy number of the target fragment in the reaction system, and calculating the environmental DNA concentration of the rassa naked nojirimus fish in the water sample. The primer has species specificity, and the method can be used for rapidly, efficiently and accurately detecting the distribution and abundance of the nojirimus rassa on the premise of not damaging the environment and fish population.
Description
Technical Field
The invention belongs to the field of molecular ecology, and particularly relates to a primer and a probe for detecting environmental DNA of Lasa naked nojirimus fish and application thereof.
Background
The Yalu Tibetan Bujiang is a river with the highest altitude in the world, and 4 schizothorax bigelovii types belong to 9 types in the water system, wherein most of the Schizothorax bigelovii types are specific to the Yalu Tibetan Bujiang. The Lassa nudus nojiri fish (Schizopygopsis yonghussbandi) is mainly distributed in the middle and upstream of Yalu Tibetan Yajbanjiang, has the characteristics of slow growth, late sexual maturity, low fertility and the like, and is difficult to recover once resources are damaged. In recent decades, due to the influences of water conservancy and hydropower engineering construction, over fishing, foreign species invasion and the like, the resource amount of the Lasa naked nojirimus fish is rapidly reduced, and effective protection is urgently needed. The monitoring of the resource conditions such as the distribution and the abundance of the resources can provide scientific basis for the resource protection and management. The common monitoring methods such as net catching and the like have great damage to fish populations, and are time-consuming, labor-consuming and low in efficiency.
Environmental DNA (edna) refers to DNA extracted from environmental samples such as water, soil or sediment. Free fish DNA in a body of water may originate from skin, excrement, sperm, eggs, etc. shed or shed from the fish body. The environmental DNA technology provides a new method for detecting fish species by extracting water sample DNA and detecting species distribution and abundance by using species specific molecular markers, and has the advantages of no damage to fish populations, simplicity, high efficiency, high sensitivity and the like. Successful implementation of this technique depends on efficient and specific amplification of the detection primers for a particular fish species. Generally, the fish species of the same family or even the same genus, which are relatively closely related, are distributed in the same water area, and the conventional DNA barcode primer is difficult to accurately detect one species.
The fish is distributed in the same domain as Lasa naked nojiri fish, and not only native cyprinid, Lasa Schizothorax prenanti, and Leptospira bicolor, but also other fishes of Cyprinus carpio, Carassius auratus, and Pseudorasbora parva. The similarity of mitochondrial gene sequences of the species is high, and if the common fish species identification primer is used for carrying out environmental DNA detection on the Lasa nudivijiri fish, the result is greatly influenced by non-specific amplification of other closely related species. Therefore, only molecular marker primers with efficient and specific amplification on the Lasa nudiflora need to be developed to achieve the purpose of accurate environmental DNA detection.
Disclosure of Invention
Aiming at the problems that the environmental DNA detection of fish species is easily influenced by homologous distribution and kindred species and the like, the invention develops a group of primers and probes for detecting the environmental DNA of the Lasa nudivijiri fish by utilizing a molecular marker technology, and the primers and the probes have optimal specificity, sensitivity and amplification efficiency, namely Scyo-F, ScYo-R and Scyo-P. The other purpose is to provide the application of the primer and the probe in detecting the distribution and abundance of the lasalopecuroides.
In order to solve the technical problem of the invention, the technical scheme is as follows: a primer and a probe for detecting environmental DNA of the Lasa naked nojirimus fish are disclosed, wherein the primer sequence is as follows: Scyo-F (SEQ ID NO:1): CTCTGCTCCATACCTCCAAGCT and Scyo-R (SEQ ID NO: 2): TGAGAAACAGTGCAAAGTACAGGG, the probe sequence is ScYo-P (SEQ ID NO: 3): ACTAACATTCCGCCCAATCACCC are provided.
In order to solve the technical problem of the invention, another technical scheme is provided as follows: the application of the primer and the probe in detecting distribution and abundance of the lasalopecuroides comprises the following steps:
(1) collecting a water sample and extracting eDNA;
(2) the primers and probes of claim 1 are used to perform digital micro-pcr (ddpcr) on eDNA by the following reaction scheme: 10.0ul 2 XddPCR Supermix for Probes, 0.5ul each forward and reverse primer, 0.3ul probe, 2.0ul DNA template, 6.7ul ddH 2O; the reaction parameters are as follows: pre-denaturation at 95 ℃ for 10 min; 40 cycles: denaturation at 95 ℃ for 30s, annealing and extension at 56 ℃ for 1 min; 10min at 98 ℃;
(3) and reading and analyzing the copy number of the target DNA fragment in the reaction system by using a microdroplet analyzer, and calculating the environmental DNA concentration (copy/ml) of the rassa naked nojirimfish.
In order to solve the technical problem of the invention, another technical scheme is provided as follows: a kit for rapid detection of lasalopecuroide comprising the primers and probe of claim 1.
In order to solve the technical problem of the invention, another technical scheme is provided as follows: the kit for rapidly detecting the lasalopecuroide comprises ddPCR buffer solution and ddH 2O.
The purpose of the invention is realized as follows: firstly, obtaining a potential fish list distributed in the same domain as the Lasa nudivijiri fish, determining the kindred species which may affect the detection effect, then designing primers and probes in the regions which are conserved in species but have large interspecies difference according to the comparison information of mitochondrial sequences of all species, obtaining the optimal primers and probes which have efficient and specific amplification on the Lasa nudivijiri fish through experimental screening, and then combining with a ddPCR technology to establish the environment DNA detection technology of the Lasa nudivijiri fish.
1. Primer and probe for detecting environmental DNA of lasalosijiri fish
The Lasa nudivijiri fish is mainly distributed in the middle and upstream of the Yalu Tibetan Bujiang, and the applicant collects the fish name list and the mitochondrial gene sequence of the distribution in the drainage basin through field investigation and literature reference. The gene sequences are linearly arranged by using ClustalW software, and primers and probes for detecting the rasa nojirimus are designed by using Primer5 software according to the following principle: (1) the forward and reverse primer sequences have at least two base differences compared with any other fish; (2) the forward and reverse primers and probe sequences are highly conserved in species and have no variation sites; (3) the length of the amplicon is 100-200 bp; (4) otherwise, the design rules of conventional primers and probes were referred to. Six groups of primers and probes are designed, and one group with optimal specificity, sensitivity and amplification efficiency is selected through a large amount of experimental screening, namely the forward primers and the reverse primers are as follows: CTCTGCTCCATACCTCCAAGCT and ScYo-R: TGAGAAACAGTGCAAAGTACAGGG, the probe is ScYo-P: ACTAACATTCCGCCCAATCACCC, the amplicon length is 177 bp. PCR amplification is carried out in various species by using the primer and the probe, and the primer and the probe are only suitable for environmental DNA detection of the rassa naked nojirimus fish.
2. Application of primer and probe in detection of distribution and abundance of lasalopecuroides
Based on the primers and the probes, the environment DNA detection method of the Lasa naked nojirimus fish is established by combining the micro-drop digital PCR (ddPCR) technology, and mainly comprises the following steps: (1) collecting a water sample and extracting eDNA; (2) ScYo-F and ScYo-R are used as primers, ScYo-P is used as a probe, a fluorescent reporter group and a quenching group are respectively marked on 5 'and 3' of the probe, water sample eDNA is used as a template for ddPCR amplification, and reaction parameters are set as follows: pre-denaturation at 95 ℃ for 10 min; 40 cycles: denaturation at 95 ℃ for 30s, annealing and extension at 56 ℃ for 1 min; 10min at 98 ℃; (3) after amplification is finished, putting the amplified product into a microdroplet analyzer, and reading and analyzing the copy number of target DNA in a reaction system by using QuantaSoft software; (4) calculating the DNA concentration (copy/ml) of the lasalopecuroide in the water sample.
Compared with the prior art, the invention has the following advantages:
(1) the existing method is to design primers and probes according to the DNA sequences of the species, detect the species through qPCR or ddPCR and ignore the influence of the homologous distributed sibling species. The primer and the probe of the rassa naked nojirimus fish obtained by the existing method have high amplification efficiency, but may have no specificity, namely the primer and the probe can still detect the existence of the rassa naked nojirimus fish in a water body without the distribution of the rassa naked nojirimus fish.
The invention creatively designs a primer and a probe in a region which is conserved in the fish species but has large interspecific difference by comparing DNA sequences of the Lasa naked nojiri fish and other fishes distributed in the same domain, and obtains a molecular marker primer and a probe which only have efficient and specific amplification to the Lasa naked nojiri fish through a large amount of experimental screening, thereby achieving the purpose of accurate environmental DNA detection. The primers and the probes are ineffective in amplifying other fishes distributed in the same domain, the amplification efficiency reaches 99.9%, and the lowest detection species DNA concentration reaches 0.001 ng/ul.
(2) The environmental DNA detection method of the Lasa naked nojirimus fish is established according to the ddPCR technology, the steps are simple and easy to operate, and the target DNA copy number of a water sample to be detected can be directly quantified without making a standard curve.
(3) Compared with the investigation methods such as net catching, electric catching and the like, the invention has the advantages of high sensitivity, high efficiency, no damage to the environment and the fish body and the like.
Drawings
FIG. 1 shows the design regions and sequences of primers and probes according to the present invention.
FIG. 2 shows the result of ddPCR amplification of primers and probes in the invention in the fish species of the rassa-naked nojirimus and the fish species distributed in the same domain.
FIG. 3 shows the result of ddPCR amplification of primers and probes in the DNA of Lasa naked nojirimus fish with 10-fold concentration gradient.
FIG. 4 shows the result of ddPCR detection of the primer and probe used for the Lalu wetland Lasa nojirimus fish in the embodiment of the present invention.
Detailed Description
The invention will be further elucidated by means of specific embodiments in conjunction with the accompanying drawings.
Example 1:
obtaining primers and probes for detecting environmental DNA of the Lasa naked nojirimus fish:
the applicant collected the fish name list and its mitochondrial gene sequence distributed in the same domain as that of lasasanojiri fish by field investigation and literature reference, including 12 cyprinid fishes: gymnocypris aurantiaca, schizothorax bigrahami, schizothorax heterodentatus, schizothorax rassa, schizothorax bigrahami, pseudorasbora parva, crucian carp, silver carp, bighead carp and grass carp. After alignment of the sequences, 6 sets of primers and probes were designed in regions conserved within species but widely different between species. Through a large number of experiments, the amplification specificity, sensitivity and amplification efficiency of the primers are evaluated, and the detection effect of most primers is still influenced by closely related species, such as primers CTGCCACCCTCGTTCCTATTATTAT and TTGATGAAACACCTGCTAGGTGTA which have weak amplification results in the closely related species Lassa Schizothorax bikino. Finally, an optimal group is screened, namely forward and reverse primers are ScYo-F: CTCTGCTCCATACCTCCAAGCT and ScYo-R: TGAGAAACAGTGCAAAGTACAGGG, the probe is ScYo-P: ACTAACATTCCGCCCAATCACCC located in the coding regions 914-935bp, 1080-1103bp and 942-964bp of Cytb gene (FIG. 1). The amplification effect of the primer on DNA of the lyrataria sinzakii and homologous distributed kindred species is shown in figure 2, and only amplification signals are obtained in the lyrataria sinzakii, so that the primer is ineffective for other fishes. Through the amplification of genome DNA of 10ng/ul, 1ng/ul, 0.1ng/ul, 0.01ng/ul and 0.001ng/ul of the Lasa naked nojirimus fish (figure 3), the lowest DNA concentration which can be detected by the primers of the group is 0.001ng/ul, and the amplification efficiency reaches 99.9 percent. Therefore, the set of primers and probes has high specificity, sensitivity, and extremely high amplification efficiency.
Example 2:
the application of the primer and the probe in the detection of the Lasa Laura wetland Lasa nojiri fish comprises the following steps:
(1) 1L of water was collected from each of 3 spots (A, B, C) on the Lasalalu wetland, and the DNA was collected on a mixed cellulose filter using a vacuum filtration pump.
(2) The DNA of the water sample is extracted by using a strong water sample DNA extraction kit and is diluted by 200ul of TE solution, and the concentrations of the DNA of the water samples of the sample points A, B and C are respectively 27.8ng/ul, 11.3ng/ul and 10.5 ng/ul.
(3) The method comprises the following steps of (1) marking a fluorescent reporter group and a quenching group on 5 'and 3' of a probe respectively by using water sample DNA as a template, ScYo-F and ScYo-R as primers and ScYo-P as the probe, and carrying out ddPCR amplification by using a QX200 Droplet Digital PCR system, wherein the reaction system is as follows: 10.0ul 2 XddPCR Supermix for Probes, 0.5ul each forward and reverse primer, 0.3ul probe, 2.0ul DNA template, 6.7ul ddH 2O; the reaction parameters are as follows: pre-denaturation at 95 ℃ for 10 min; 40 cycles: denaturation at 95 ℃ for 30s, annealing and extension at 56 ℃ for 1 min; 10min at 98 ℃. Each sample was set up for 3 technical replicates.
(4) After amplification was completed, the samples were placed in a microdroplet analyzer (Droplet Reader) and the average concentrations of target DNA in the spots A, B and C reaction were read and analyzed using QuantaSoft software as 160 copies/ul, 17 copies/ul and 0, respectively (FIG. 4).
(5) According to the water sampling amount and the ddPCR result, the environmental DNA concentrations of the lasalopecuroide in the sample points A, B and C are respectively calculated to be 320 copies/ml, 34 copies/ml and 0, and the environmental DNA concentrations are consistent with the fishing harvest amount data (660g, 90g and 0).
SEQUENCE LISTING
<110> institute of aquatic organisms of Chinese academy of sciences
Primer and probe for detecting environmental DNA of <120> Lasa naked nojirimus fish and application of primer and probe
<130> 20210929
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213> Artificial sequence
<400> 1
ctctgctcca tacctccaag ct 22
<210> 2
<211> 24
<212> DNA
<213> Artificial sequence
<400> 2
tgagaaacag tgcaaagtac aggg 24
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence
<400> 3
actaacattc cgcccaatca ccc 23
Claims (4)
1. A primer and a probe for detecting environmental DNA of the Lasa naked nojirimus fish are disclosed, wherein the primer sequence is as follows: Scyo-F (SEQ ID NO:1): CTCTGCTCCATACCTCCAAGCT and Scyo-R (SEQ ID NO: 2): TGAGAAACAGTGCAAAGTACAGGG, the probe sequence is ScYo-P (SEQ ID NO: 3): ACTAACATTCCGCCCAATCACCC are provided.
2. The use of primers and probes according to claim 1 for detecting distribution and abundance of lasalopecuroides, characterized in that: the method comprises the following steps:
(1) collecting a water sample and extracting eDNA;
(2) the primers and probes of claim 1 are used to perform digital micro-pcr (ddpcr) on eDNA by the following reaction scheme: 10.0ul 2 XddPCR Supermix for Probes, 0.5ul each forward and reverse primer, 0.3ul probe, 2.0ul DNA template, 6.7ul ddH 2O; the reaction parameters are as follows: pre-denaturation at 95 ℃ for 10 min; 40 cycles: denaturation at 95 ℃ for 30s, annealing and extension at 56 ℃ for 1 min; 10min at 98 ℃;
(3) and reading and analyzing the copy number of the target DNA fragment in the reaction system by adopting a microdroplet analyzer, and calculating the environmental DNA concentration copy/ml of the rassa naked nojirimfish.
3. A kit for rapidly detecting the nojirimus rassa is characterized in that: comprising the primer and probe of claim 1.
4. A kit for the rapid detection of lasalopecuroide according to claim 3, characterized in that: also included was ddPCR buffer and ddH 2O.
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| CN116814809A (en) * | 2023-08-04 | 2023-09-29 | 湖南农业大学 | Digital PCR detection method for DNA of environment of rana spinosa |
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