CN-121992119-A - Primer, probe and kit for identifying round-mouth copper fish based on environmental DNA

Abstract

The invention provides a primer, a probe and a kit for identifying round-mouth copper fish based on environmental DNA, which are designed according to a COI gene sequence in mtDNA of round-mouth copper fish, namely the primer probe can specifically identify round-mouth copper fish in a plurality of fishes, the detection time is greatly shortened, the detection can be completed within 6 hours, the detection result is accurate, the sensitivity is high, the minimum DNA concentration of the real-time fluorescence PCR detection can reach pg level, and the primer and the probe are more suitable for the actual requirements of the field detection of eDNA on the upstream of Yangtze river.

Inventors

• HE WENPING
• ZHOU YUE
• YAO WEIZHI

Assignees

• 西南大学

Dates

Publication Date

20260508

Application Date

20260408

Claims (4)

1. 1. A primer and a probe for identifying the round-mouth copper fish based on environmental DNA are characterized in that an upstream primer of the primer is CG-COI-F1, the nucleotide sequence of the upstream primer is shown as SEQ ID No.1, a downstream primer is CG-COI-R1, the nucleotide sequence of the downstream primer is shown as SEQ ID No.2, the probe is CG-COI-MGB, and the nucleotide sequence of the probe is shown as SEQ ID No. 3.
2. 2. A detection kit for identifying round-mouth copper fish based on environmental DNA is characterized by comprising the primer probe of claim 1.
3. 3. A method of detecting primers and probes based on the identification of round-mouth copper fish of the environmental DNA of claim 1, the steps comprising: (1) Extracting DNA of the copper fish with round mouth; (2) Performing fluorescent PCR amplification; The real-time fluorescence PCR amplification system is 20 mu L, which comprises 0.4 mu L of each of the above-mentioned upstream and downstream primers CG-COI-F1/CG-COI-R1, 0.2 mu L of the above-mentioned CG-COI-MGB probe and 1.0 mu L of DNA template; The PCR reaction conditions, the two-step amplification reaction procedure, were pre-denatured 94℃3 mins, then 94℃5 sec,55℃30 sec cycles 40 times.
4. 4. Use of the primers and probes of claim 1 for identifying round-mouth copper fish.

Description

Primer, probe and kit for identifying round-mouth copper fish based on environmental DNA Technical Field The invention belongs to the technical field of molecular biological detection, and particularly relates to a primer, a probe and a kit for identifying round-mouth copper fish based on environmental DNA. Background Round-mouth copper fish Coreius guichenoti belongs to the genus (Coreius) of the genus (Gobioninae) of the subfamily (CYPRINIDAE) of the order cyprinid (Cypriniformes) of the order cyprinid, commonly called Watertight, and is mainly distributed on the upstream and the downstream of the Yangtze river, the middle and downstream of the Jinshajiang, the branches of the Mingjiang, jiang river, wujiang and the like, is one of the special species of the Yangtze river in China, and is an important economic fish in the Yangtze river basin because of fine and smooth meat, rich fat. In recent years, the wild resource amount is drastically reduced due to the reasons of overdrawing, dam construction and the like, and the wild resource amount is listed as a second-level protection wild animal in China 'national important protection wild animal directory', and reaches the extremely dangerous (CR) grade in IUCN grades. According to the recent relevant investigation records, in the round-mouth copper fish cluster area, round-mouth copper fish cannot overcome the main stream to finish tracing in most cases, natural survival and reproduction of the round-mouth copper fish are seriously affected, the population number is greatly limited, and the importance and urgency of population number and space-time distribution research of the round-mouth copper fish are further described. However, the traditional fish investigation is generally completed through morphological identification, which is invasive and disturbing to the biological community, and is contrary to the original purpose of protecting the biological diversity in the 'banned fishing in the Yangtze river for ten years', the water flow rate of the environment where the round-mouth copper fish inhabits is fast, the species number is very small, the fish is not easy to catch, and the biological identification of the round-mouth copper fish is limited by the professional background of the investigator, so that the investigation of the population resources of the round-mouth copper fish is difficult to break through by rapidly and accurately distinguishing the round-mouth copper fish from the similar species such as the copper fish (Coreius heterodon). Therefore, it is important to establish a harmless rapid and accurate identification method. Environmental DNA (eDNA) refers to free DNA molecules released from organisms. And (3) extracting, amplifying, sequencing, analyzing and the like the eDNA sample captured in the environmental sample to obtain a large amount of biological community data information. Currently, eDNA has proven to have great potential in biological monitoring and wide application in detecting low density species. Compared with the traditional method which is time-consuming and labor-consuming, has low capturing rate of rare target organisms and causes damage to fishes, the eDNA technology has the advantages of time and labor saving, cost saving, high sensitivity, environmental friendliness, no damage to the investigation object and the like. The eDNA technology makes up the defects of the traditional investigation method to a great extent, and plays an important role in the aspects of rare species protection, biomass evaluation and the like. No disclosures of patent literature and non-patent literature related to real-time fluorescent identification of environmental DNA of round-mouth copper fish are found so far. The biological information of the round-mouth copper fish is quite limited by the existing observation and sampling technology, and the distribution range and population condition of the round-mouth copper fish are not clear. The number of species of the Yangtze river fish is large, the difference of molecular levels of species with close relativity is very small, and the design of the real-time fluorescence specificity Taqman probe is difficult to a certain extent. Disclosure of Invention The invention aims to provide a primer and a probe for identifying round-mouth copper fish based on environmental DNA. The invention further aims at providing a kit for identifying the round-mouth copper fish based on the environmental DNA. The invention aims at realizing the following technical scheme: The primer and probe for identifying the round-mouth copper fish based on the environmental DNA are characterized in that an upstream primer of the primer is CG-COI-F1, the nucleotide sequence of the upstream primer is shown as SEQ ID No.1, a downstream primer is CG-COI-R1, the nucleotide sequence of the downstream primer is shown as SEQ ID No.2, the probe is CG-COI-MGB, the nucleotide sequence of the probe is shown as SEQ ID No.3, and a fluoresc