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CN-122012733-A - Fluorescent quantitative PCR primer probe composition for long spike fish and application thereof

CN122012733ACN 122012733 ACN122012733 ACN 122012733ACN-122012733-A

Abstract

The invention belongs to the field of biotechnology detection, and discloses a fluorescent quantitative PCR primer probe composition for long pseudorasbora parva and application thereof, wherein specific upstream primers, specific downstream primers and specific TaqMan probes are designed according to COI gene sequences in mitochondrial DNA sequences of the long pseudorasbora parva, and the fluorescent quantitative PCR probe composition is obtained through comparison and screening, has the characteristic of strong specificity, can efficiently, accurately and specifically identify the long pseudorasbora parva through genome DNA, eliminates the influence of subjective factors, has high sensitivity and good repeatability, can quantitatively analyze eDNA of the long pseudorasbora parva, realizes the harmless fish body, still efficiently, accurately and specifically identifies the long pseudorasbora parva, and can provide technical support for long pseudorasbora parva species resource monitoring and protection management of special and narrow-domain distribution in China.

Inventors

  • YANG XIAOGE
  • LIAN YUXI
  • LI YIJUN
  • YANG WENXIAO
  • WANG KEHANG
  • ZHONG ZHIHUA

Assignees

  • 安庆师范大学

Dates

Publication Date
20260512
Application Date
20260225

Claims (7)

  1. 1. A fluorescent quantitative PCR primer probe composition for pseudorasbora parva is characterized in that the probe composition is based on a COI gene sequence of pseudorasbora parva mitochondria as a target detection gene, the probe composition comprises an upstream primer CMS-F2, a downstream primer CMS-R2 and a TaqMan probe CMS-P2, wherein the nucleotide sequence of the upstream primer CMS-F2 is shown as SEQ ID NO.1, the nucleotide sequence of the downstream primer CMS-R2 is shown as SEQ ID NO.2, and the probe composition also comprises a TaqMan probe CMS-P2, and the nucleotide sequence of the TaqMan probe CMS-P2 is shown as SEQ ID NO. 3.
  2. 2. The application of the fluorescent quantitative PCR primer probe composition for the long spike fish is characterized by comprising the following steps of: (1) Extracting genome DNA or water sample eDNA of a fish sample to be detected as a template to be detected; (2) Establishing a fluorescent quantitative PCR amplification system based on the primer probe composition of claim 1, performing a reaction and recording an amplification curve; The fluorescent quantitative PCR reaction system comprises 10 mu L of 2X TAQMAN FAST QPCR premix, 0.4 mu L of each of the upstream primer CMS-F2 and the downstream primer CMS-R2, 0.2 mu L of TaqMan probe CMS-P2, 2 mu L of the template to be detected obtained in the step (1) and 7 mu L of sterilized ultrapure water; The fluorescent quantitative PCR reaction is carried out by pre-denaturing at 93-95 ℃ for 3min, then three-step amplifying at 94 ℃ for 5sec,58 ℃ for 15sec,72 ℃ for 30sec, cycling for 35-50 times, carrying out fluorescent quantitative PCR reaction, and recording fluorescent growth curve; (3) Based on the amplification curve obtained in the step (2), when a strong fluorescence growth curve appears and the Ct value is smaller than 35, judging that the fish sample to be detected extracted in the step (1) is a long pseudorasbora parva sample or an eDNA sample contains a long pseudorasbora parva gene.
  3. 3. The use according to claim 2, wherein the final concentration of the upstream primer is 0.3 to 0.5. Mu.M, the final concentration of the downstream primer is 0.3 to 0.5. Mu.M, and the final concentration of the TaqMan probe is 0.2 to 0.4. Mu.M in the fluorescent quantitative PCR amplification system.
  4. 4. The use according to claim 3, wherein the final concentration of the upstream primer CMS-F2 and the downstream primer CMS-R2 is 1.25 to 2.5 times the final concentration of the TaqMan probe CMS-P2.
  5. 5. The method according to claim 2, 3 or 4, wherein the real-time fluorescent PCR is performed at 94℃for 3min, 94℃for 5sec,58℃for 15sec, and 72℃for 30sec, and the amplification procedure is performed for 40 cycles.
  6. 6. The use according to claim 2, wherein the primer probe combination detects a minimum concentration of 6.45 x 10 -7 ng/. Mu.l of long ear fish target genes.
  7. 7. A kit for identifying a variety of spiked fish, comprising at least the fluorescent quantitative PCR primer probe composition of claim 1.

Description

Fluorescent quantitative PCR primer probe composition for long spike fish and application thereof Technical Field The invention relates to the field of biotechnology detection, in particular to a fluorescent quantitative PCR primer probe composition for pseudorasbora parva and application thereof. Background Long pseudorasbora parva (Pseudorasboraelongata) belongs to the order of Cyprinus, the family of Cypridae, the genus pseudorasbora, is a special small fish in China, and has been listed as an "easy-to-risk" species by the "Chinese endangered animal red book: fish" and "Chinese species red directory (first volume)". Long ear fish has severe requirements on habitat, belongs to typical narrow-range distribution fish, and is historically only distributed in the Western river system and the middle and downstream water system of the Yangtze river. In recent years, the distribution area of long spike fishes is continuously withered and the number of wild species is also in a decreasing trend under the influence of the change of habitat and the human activities. The accurate evaluation of the wild resource quantity of the fish is the basis for formulating the protection strategy, however, the data of the long spike fish published at present are extremely limited, the existing distribution area and resource quantity of the long spike fish are not clear, and therefore, the management and the protection strategy are difficult to formulate. Because the individual long spike fish is small, the fish is loved to live in mountain stream with aquatic weed and egg gravel substrate habitat, and is not easy to distinguish from other small-sized stream fish in the same habitat in appearance, and the fish is required to be identified by professional personnel according to the appearance characteristics. Therefore, the resource amount of the long spike fish is investigated and evaluated by means of the traditional fish monitoring and sampling technology, so that a certain difficulty exists. In addition, for long spike fish, which is a narrow-range 'Yi Wei', traditional fish sampling technology is often invasive and can cause secondary damage to the fish. Therefore, development of a method for detecting and evaluating the biomass of long spike fish with strong specificity, convenient operation, high efficiency and non-invasiveness is needed, and technical support is provided for formulating the protection strategy of the long spike fish. Disclosure of Invention The invention aims to provide a fluorescent quantitative PCR primer probe composition for long pseudorasbora parva, and provides application of the primer probe composition, and the long pseudorasbora parva is specifically detected by establishing a fluorescent quantitative PCR method, and has the characteristics of non-invasiveness, strong specificity and high sensitivity. In order to achieve the above object, the present invention provides the following technical solutions: In a first aspect, the invention provides a fluorescent quantitative PCR primer probe composition for long pseudorasbora parva, which comprises an upstream primer CMS-F2, a downstream primer CMS-R2 and a TaqMan probe CMS-P2, wherein the nucleotide sequence of the upstream primer CMS-F2 is shown as SEQ ID NO.1, the nucleotide sequence of the downstream primer CMS-R2 is shown as SEQ ID NO.2, and the nucleotide sequence of the TaqMan probe CMS-P2 is shown as SEQ ID NO. 3. The probe composition adopts COI gene sequence of long pseudorasbora parva mitochondria (namely, 5492 th to 7042 th positions in genome sequence with accession number NC 021769) as target detection genes, and primer probe screening is carried out in a hypervariable region. Specifically, the TaqMan probe CMS-P2 is labeled with FAM as a fluorescent reporter group at the 5 'end and with MGB as a fluorescent quenching group at the 3' end. The fluorescent signal of the reporter group is inhibited by the quencher group while the probe CMS-P2 remains intact, and the fluorescent signal of the reporter group can be detected once the probe is cleaved and the inhibition is lost. Furthermore, the primer probe composition is added in the fluorescent quantitative PCR reaction, and in the reaction low-temperature renaturation extension stage, the 5'-3' exonuclease activity of Taq enzyme in the system is used for enzyme cutting of the probe CMS-P2, so that a report fluorescent group and a quenching fluorescent group are separated, a fluorescent monitoring system can receive fluorescent signals, and the accumulation of the fluorescent signals and the formation of PCR products are completely synchronous. In a second aspect, the invention provides the use of the composition described above for identification and detection of species of pseudorasbora parva, based on fluorescent quantitative PCR, comprising the steps of: (1) Extracting genome DNA or water sample eDNA of a fish sample to be detected as a template to be detected; (2) Preparing a fluorescen