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CN-121992084-A - Primer probe combination for detecting red ear tortoise based on environment DNA and application thereof

CN121992084ACN 121992084 ACN121992084 ACN 121992084ACN-121992084-A

Abstract

The invention belongs to the field of biological detection, and particularly relates to a primer probe combination for detecting red ear tortoise based on environmental DNA and application thereof. The invention provides a composition of a specific primer and a TaqMan probe for detecting red ear tortoise in an environment. The TaqMan real-time fluorescent quantitative PCR detection system for the red-ear tortoises is constructed by screening the high-specificity fragment of the red-ear tortoises mitochondrial COI gene as a target. The combination shows excellent species specificity and detection sensitivity in complex environmental samples, and can realize accurate identification and quantitative analysis of genetic materials of target species. The invention obviously improves the reliability of environmental DNA detection, can be widely applied to the field distribution investigation of red-ear turtles, the intrusion diffusion early warning and the dynamic monitoring of aquatic ecosystems, and provides an important technical means for preventing and controlling foreign invasive species and maintaining biodiversity.

Inventors

  • Mou Chaosheng
  • CHEN YOUHUA

Assignees

  • 中国科学院成都生物研究所

Dates

Publication Date
20260508
Application Date
20260325

Claims (7)

  1. 1. The composition is characterized by comprising an upstream primer, a downstream primer and a TaqMan probe, wherein the sequence of the upstream primer is shown as SEQ ID NO. 1, the sequence of the downstream primer is shown as SEQ ID NO. 2, and the sequence of the TaqMan probe is shown as SEQ ID NO. 3.
  2. 2. The composition of claim 1, wherein the TaqMan probe has a5 '-end modified FAM and a 3' -end modified MGB.
  3. 3. A kit, reagent or test strip comprising the composition of claim 1 or 2.
  4. 4. Use of a composition according to claim 1 or 2 or a kit, reagent or test strip according to claim 3 for detecting or monitoring red-ear tortoise.
  5. 5. A method for detecting or monitoring red ear tortoise is characterized by comprising the following steps: (1) Extracting DNA in a sample to be detected; (2) Taking the DNA extracted in the step (1) as a template, and carrying out TaqMan real-time fluorescence quantitative PCR reaction by using a composition, wherein the composition comprises an upstream primer, a downstream primer and a TaqMan probe, the sequence of the upstream primer is shown as SEQ ID NO. 1, the sequence of the downstream primer is shown as SEQ ID NO. 2, and the sequence of the TaqMan probe is shown as SEQ ID NO. 3; (3) And judging that the red ear tortoise exists in the detected environment if the detection result shows a typical amplification curve and the Ct value is less than or equal to 40, and judging that the red ear tortoise does not exist in the detected environment if the detection result shows no amplification curve, no Ct value is obtained or the amplification curve shows a Ct value of > 40.
  6. 6. The method according to claim 5, wherein in the step (2), the TaqMan real-time fluorescent quantitative PCR reaction system is 2×T5Fast qPCR Mix (probe) 10. Mu.L, the concentration of the upstream primer and the downstream primer is 10. Mu.M each of 0.7. Mu.L, the concentration of the probe is 10. Mu.M of 0.6. Mu.L, and the concentration of the DNA template is 1. Mu.L, and the reaction system is supplemented to 20. Mu.L with dd H 2 O.
  7. 7. The method according to claim 5, wherein in the step (2), the TaqMan real-time fluorescent quantitative PCR reaction is performed by pre-denaturing at 95℃for 2min, followed by 40 cycles of denaturation at 95℃for 15s and annealing/extension at 60℃for 30s, and fluorescence signals are collected at 60 ℃.

Description

Primer probe combination for detecting red ear tortoise based on environment DNA and application thereof Technical Field The invention belongs to the field of biological detection, and particularly relates to a primer probe combination for detecting red ear tortoise based on environmental DNA and application thereof. Background Red ear tortoise (TRACHEMYS SCRIPTA ELEGANS) is listed by the world's natural protection consortium as one of the most threatening invasive species worldwide. Because the red ear tortoise has extremely strong environmental adaptability, the red ear tortoise has a wide range of potential suitable areas and is in a dynamic expansion state, so that the direct competition with native tortoises on food resources and nesting sites is aggravated, and the serious retraction of native species habitats is further caused. Therefore, field monitoring of red ear turtles is particularly important. Traditional red-ear turtle field monitoring relies on visual observation and physical capture, and the entity-based investigation mode faces multiple challenges in practical application. The traditional direct observation method is extremely easy to be interfered by environmental noise in complex habitats such as aquatic vegetation flourishing or water quality cloudiness and the like under the influence of target species hiding habit and environmental heterogeneity, and the detection success rate fluctuates severely. Moreover, the red ear tortoise population at the initial stage of invasion is usually in a low abundance state, the sensitivity of the traditional means is difficult to cross the detection limit threshold value, and early-stage accurate capture of invasion risks cannot be realized. Furthermore, the traditional investigation mode has natural bottlenecks such as high labor cost, low monitoring frequency, difficult lateral expansion of spatial dimensions and the like, and is difficult to meet the technical requirements of a modern biosafety prevention and control system on real-time and dynamic early warning of a large-range area. Physical contact investigation may also cause artificial disturbance to the monitored sensitive area, which does not conform to the technological trend of non-invasive monitoring. The rise of Environmental DNA (eDNA) technology provides a breakthrough means for non-invasive monitoring of invasive species. The technology realizes the accurate identification of the biodistribution information by capturing and analyzing trace genetic materials such as skin fragments, mucus, excrement, gametes and the like of target species falling into environmental media (such as water bodies, sediments and the like). The technology can sharply capture genetic residues with extremely low concentration at a low density stage at the initial stage of the invasion of the red ear tortoises by virtue of extremely high detection sensitivity, effectively overcomes the monitoring leakage caused by strong species concealment in the traditional means, simultaneously, through non-invasive sampling, the eDNA technology can perfectly adapt to complex habitats such as dense vegetation, turbid water quality and the like, remarkably improves the detection efficiency in complex environments while avoiding the disturbance of an ecological system, and provides scientific technical support for dynamic monitoring, distribution evaluation and early warning system construction of foreign species in a large-scale flow. In the field of molecular detection for red-ear turtle environmental DNA (eDNA), currently common technical paths include conventional PCR, digital droplet PCR (ddPCR), and real-time fluorescent quantitative PCR (qPCR). Although the conventional PCR technology is simple and convenient to operate, qualitative judgment of 'detection or not' can be realized, the detection limit is higher, the amplification efficiency is extremely unstable when facing to complex chemical inhibitors in environmental samples, and false negative deviation is easily caused. Although ddPCR technology has higher sensitivity and absolute quantification capability, the expensive instrument and equipment, complex operation flow and extremely low detection flux make it difficult to realize low-cost popularization in basic monitoring units or large-scale field investigation. The real-time fluorescent quantitative PCR technology is considered to be a scheme with more application potential in species identification because of the high efficiency and economy. However, most of the existing qPCR detection methods of red-ear tortoises still stay in a detection mode of combining only an upstream primer and a downstream primer with a fluorescent dye method, and are extremely susceptible to signal interference of nonspecific amplification of primer dimers or kindred species when a trace environmental sample with complex components is processed. The specificity of the detection system is limited, false positive judgment and obvious quant