Fluorescent quantitative PCR (Polymerase Chain Reaction) primer probe composition for pseudorasbora parva and application of fluorescent quantitative PCR primer probe composition

Abstract

The invention belongs to the field of biotechnology detection, and discloses a fluorescent quantitative PCR (polymerase chain reaction) primer probe composition for pseudorasbora parva and application. A specific upstream primer, a specific downstream primer and a TaqMan probe are designed according to a COI gene sequence in a mitochondrial DNA sequence of the pseudorasbora parva, the primer is obtained through comparison and screening, the primer has the characteristic of high specificity, the fluorescent quantitative PCR method established based on the primer can efficiently, accurately and specifically identify the pseudorasbora parva through genome DNA, the influence of subjective factors is eliminated, and the primer is high in sensitivity and high in specificity. The repeatability is good; and the method can be used for quantitatively analyzing the eDNA of the pseudorasbora parva, so that the pseudorasbora parva can be efficiently, accurately and specifically identified without damaging the pseudorasbora parva, and technical support can be provided for monitoring, protecting and managing population resources of the pseudorasbora parva which are specifically distributed in narrow areas in China.

Description

Technical Field

[0001] This invention relates to the field of biotechnology detection, specifically to a fluorescent quantitative PCR primer and probe composition for longfin gudgeon and its application. Background Technology

[0002] long-necked gudgeon ( Pseudorasboraelongata The long-necked gudgeon (Gudgeon spp.) belongs to the order Cypriniformes, family Cyprinidae, and genus Gudgeon. It is a small fish endemic to my country and is listed as "Vulnerable" in both the *China Red Data Book of Endangered Animals: Fishes* and the *China Species Red List (Volume 1)*. The long-necked gudgeon has strict habitat requirements and is a typical example of a narrow-range distribution fish, historically found only in the Xijiang River system and the middle and lower reaches of the Yangtze River. In recent years, due to habitat changes and human activities, its distribution area has been shrinking, and its wild population is declining. Accurate assessment of wild fish resources is fundamental to developing conservation strategies; however, currently published data on the long-necked gudgeon is extremely limited, and its existing distribution area and resource quantity remain unclear, leading to difficulties in formulating management and conservation strategies.

[0003] Because of their small size, preference for inhabiting mountain streams with aquatic plants and gravelly bottoms, and difficulty in distinguishing them from other small stream fish in the same habitat, identification by professionals based on their physical characteristics is essential. Therefore, relying on traditional fish monitoring and sampling techniques to investigate and assess the population size of the topmouth gudgeon presents certain challenges. Furthermore, for this narrowly defined and vulnerable species, traditional fish sampling techniques are often invasive and can cause secondary damage. Therefore, there is an urgent need to develop a highly specific, convenient, efficient, and non-invasive method for detecting and assessing the biomass of the topmouth gudgeon, providing technical support for the formulation of conservation strategies. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention aims to provide a primer and probe composition for quantitative real-time PCR of gudgeon; and to provide the application of the primer and probe composition, which establishes a quantitative real-time PCR method to specifically detect gudgeon, and is characterized by non-invasiveness, high specificity, and high sensitivity.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a primer and probe composition for quantitative real-time PCR of longmouth gudgeon, comprising an upstream primer CMS-F2, the nucleotide sequence of which is shown in SEQ ID NO.1; a downstream primer CMS-R2, the nucleotide sequence of which is shown in SEQ ID NO.2; and a TaqMan probe CMS-P2, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0007] The probe composition described above uses the COI gene sequence of the mitochondria of the long-tailed minnow (i.e., positions 5492 to 7042 in the genome sequence with accession number NC021769) as the target detection gene, and primers and probes are screened in the hypervariable region.

[0008] Specifically, the 5' end of the TaqMan probe CMS-P2 is labeled with FAM as a fluorescent reporter group, and the 3' end is labeled with MGB as a fluorescent quencher group. When the probe CMS-P2 remains intact, the fluorescence signal of the reporter group is suppressed by the quencher group. Once the probe is cut, the suppression disappears, and the fluorescence signal of the reporter group can be detected.

[0009] Furthermore, in this invention, the above-mentioned primer and probe composition is added to the real-time PCR reaction. During the low-temperature annealing and extension stage of the reaction, the 5'-3' exonuclease activity of Taq enzyme in the system digests the probe CMS-P2, separating the reporter fluorescent group and the quencher fluorescent group. The fluorescence monitoring system can receive the fluorescence signal, realizing complete synchronization between the accumulation of the fluorescence signal and the formation of PCR products.

[0010] Secondly, this invention provides the application of the above composition in the identification and detection of longmouth gudgeon species, based on quantitative real-time PCR, including the following steps: (1) Extract genomic DNA from the fish sample or eDNA from the water sample as the template for testing; (2) Prepare a real-time PCR amplification system containing the primer and probe composition described above, perform the reaction, and plot the amplification curve; The real-time PCR reaction system is 20 μL, including: 10 μL of 2×TaqMan Fast qPCR premix, 0.4 μL each of upstream primer CMS-F2 and downstream primer CMS-R2, 0.2 μL of TaqMan probe CMS-P2, 2 μL of the template to be tested obtained in step (1), and 7 μL of sterile ultrapure water; The procedure for quantitative real-time PCR is as follows: pre-denaturation at 93-95℃ for 3 min, followed by a three-step amplification: 94℃ for 5 sec, 58℃ for 15 sec, and 72℃ for 30 sec, repeated 35-50 times. The fluorescence growth curve is then recorded. (3) Based on the amplification curve obtained in step (2), when a strong fluorescence growth curve appears and the Ct value is less than 35, it can be determined that the fish sample to be tested extracted in step (1) is a long-tailed gudgeon sample or that the eDNA contains the long-tailed gudgeon gene.

[0011] Furthermore, in the PCR amplification system described in step (2), the final concentration of the upstream primer is 0.3-0.5 µM, the final concentration of the downstream primer is 0.3-0.5 µM, and the final concentration of the TaqMan probe is 0.2-0.4 µM. The final concentrations of the upstream and downstream primers should be 1.5-2.5 times the final concentration of the TaqMan probe.

[0012] Preferably, the pre-denaturation conditions are 94°C for 3 minutes.

[0013] Preferably, the specific conditions for the three-step amplification method of the real-time PCR reaction program are 94℃ for 5 seconds, 58℃ for 15 seconds, 72℃ for 30 seconds, and 40 cycles.

[0014] Furthermore, the minimum target gene concentration of the primer-probe composition for detecting genomic DNA in fish samples or eDNA in water samples is 6.45 × 10⁻⁶. -7 ng / µL.

[0015] Thirdly, the present invention provides a kit for detecting gudgeon, the kit comprising the aforementioned gudgeon fluorescent quantitative PCR primer and probe composition.

[0016] Compared with the prior art, the advantages and beneficial effects of the present invention are: Conventional species identification of the common topmouth gudgeon relies on manual judgment by professionals based on its appearance characteristics, thus the identification method is limited by subjective factors. The mitochondrial COI gene in fish exhibits relatively small differences at the molecular level. To ensure the specificity of primer and probe sequences, the region within the target gene sequence suitable for primer-probe design is very limited. The probe CMS-P2 sequence provided in this invention is located at positions 1191-1280 of the topmouth gudgeon COI gene sequence; furthermore, to ensure specificity, the probe CMS-P2 also contains three or more mismatched bases.

[0017] The primer and probe composition disclosed in this invention was obtained by screening based on the mitochondrial COI gene sequence of the gudgeon, exhibiting high specificity. The quantitative real-time PCR method established based on this composition can efficiently, accurately, and specifically identify gudgeon samples, eliminating the influence of subjective factors, and demonstrating high sensitivity and good reproducibility. Furthermore, experimental verification shows that the minimum detection limit of the gudgeon-specific primers CMS-F2 and CMS-R2 and probe CMS-P2 composition provided by this invention for the gudgeon target gene is 6.45 × 10⁻⁶. -7 It has a high sensitivity of ng / μL.

[0018] Based on the above probes, the fluorescence quantitative PCR detection method established in this invention can also quantitatively analyze the DNA material, namely eDNA, released by the gudgeon into the aquatic environment. This method can identify the gudgeon without damaging the fish, while still being efficient, accurate, and specific. Furthermore, it is simple to operate and low in cost, which is beneficial for the detection of gudgeon population resources in the wild and provides guidance for its management and protection. Attached Figure Description

[0019] Figure 1 The image shows the location, sequence, and alignment results of the TaqMan probe from the long-necked gudgeon in Example 1 with the corresponding regions of closely related species; the shaded areas represent mismatched base positions in the probe sequence. Figure 2 The image shows an agarose gel electrophoresis result of conventional PCR amplification of the long-eared minnow sample in Example 1. Figure 3 The quantitative real-time PCR amplification curves of longmouth gudgeon primer and probe composition CMS-F2 / CMS-R2 and probe CMS-P2 in Example 2 for longmouth gudgeon samples (curves CMS-1 and CMS-2) and other fish samples (fish in Table 1); Figure 4 The sensitivity test results of the long-tailed gudgeon primer and probe composition CMS-F2 / CMS-R2 and probe CMS-P2 in Example 3 are shown in curves 1-8, which are the amplification results of the recombinant plasmid constructed from the long-tailed gudgeon sample DNA after 10-fold serial dilution. Figure 5 This is the fluorescence quantitative PCR amplification curve of the water sample eDNA from the habitat of the long-tailed minnow in Example 4. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to specific embodiments, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0021] Unless otherwise specified, the experimental methods involved in the embodiments of this invention are all conventional molecular biology experimental methods. Unless otherwise specified, the reagents, consumables and other experimental materials used in the embodiments can be obtained from the market. The long-necked gudgeon and 11 other closely related or common fish species in the habitat of the long-necked gudgeon (long-necked gudgeon, Chinese gudgeon, glossy gudgeon, broadfin gudgeon, glossy barbel, chub, silver gudgeon, high-bodied bitterling, goby, loach, mud loach) used in the embodiments were all collected from the upper reaches of the Qiupu River in Anhui Province.

[0022] Example 1: Design and Screening of Primers and Probes for the Longmouth Gudgeon 1.1 Primer and probe design The COI gene (positions 5492 to 7042 in the genome sequence of accession number NC021769) from the mitochondrial genomic DNA sequence of the long-necked gudgeon was used as the target gene. Highly specific sequence regions were selected through sequence alignment, and the following principles were followed for the design of quantitative PCR primers and probes: the upstream and downstream primers were approximately 18-22 bp in length, the TM difference did not exceed 2°C, the GC content was 40%-60%, and special structures (such as hairpin structures, secondary structures, etc.) were avoided. The TaqMan oligonucleotide probe was located between the upstream and downstream primers, close to the upstream primer. The fluorescent group FAM was located at the 5' end of the TaqMan probe, and the quencher group MGB was located at the 3' end. Both primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0023] The mitochondrial genome sequence of the long-tailed gudgeon described in this embodiment is from the NCBI database, accession number: NC021769. The COI gene (i.e., positions 5492 to 7042 in the genome sequence of accession number: NC021769) is used as the target gene for primer and probe design.

[0024] The mitochondrial COI gene in fish exhibits relatively small differences at the molecular level. To ensure the specificity of primer and probe sequences, the region within the target gene sequence suitable for primer-probe design is very limited. Based on comparisons, this embodiment designed two primer-probe sets for detecting *Gnaphalium affine* for subsequent screening. The nucleic acid information of the two primer-probe sets is shown in Table 1. In this embodiment, the sequences of the two probes, CMS-P1 and CMS-P2, are located at positions 331-345 and 1191-1280 of the *Gnaphalium affine* COI gene sequence, respectively (e.g., ...). Figure 1 (As shown). In addition to meeting the above-mentioned probe design conditions, to ensure their specificity, the probe CMS-P1 designed in this embodiment has two or more mismatched bases in its corresponding sequence with closely related species, while the probe CMS-P2 has three or more mismatched bases. Figure 1 Medium shading indicates mismatched base positions.

[0025] Table 1. Nucleotide information of primers and probes used for detecting gudgeon.

[0026] 1.2. Genomic DNA extraction from the long-necked gudgeon A 0.5cm (<30mg) piece of the tail fin of a long-tailed minnow was cut off, and genomic DNA was extracted according to the instructions of the DP304 tissue genome extraction kit (Tiangen, Beijing).

[0027] 1.3. Conventional PCR Amplification The PCR reaction system used in this embodiment is based on a 25 μL volume: 12.5 μL of 2× Super PCR Mix (Beijing Qingke Biotechnology), 1 μL of upstream primer (10 μM concentration), 1 μL of downstream primer (10 μM concentration), 2 μL (approximately 40 ng) of longmouth gudgeon genomic DNA template, and sterile ultrapure water to a final volume of 25 μL. The conventional PCR amplification described in this invention was performed on an instrument (Dongsheng, ETC811) with the following optimized program: 94℃ pre-denaturation for 3 min, 94℃ denaturation for 30 sec, 53-60℃ annealing (adjusted to be 3-5℃ below the primer TM value) for 30 sec, 72℃ extension for 30 sec, for 30 cycles. The amplified products were subjected to 1.5% agarose gel electrophoresis, and the electrophoresis results are as follows: Figure 2 As shown, the amplification products of both primer pairs were in line with expectations. The amplification product of primer CMS-F1 / R1 was a 234 bp COI gene fragment, while the amplification product of primer CMS-F2 / R2 was a 191 bp COI gene fragment. Furthermore, the electrophoresis results showed that the amplification bands of primer CMS-F2 / R2 were brighter than those of CMS-F1 / R1 in both longfin gudgeon samples.

[0028] 1.4. Quantitative Real-Time PCR Detection The TaqMan quantitative PCR reaction used in this embodiment was performed on a LightCycler 96 quantitative PCR instrument (Roche, Switzerland). The quantitative PCR reaction system used in this embodiment, in 20 μL volume, included: 10 μL of 2×TaqManFast qPCR premix (Sangon Biotech, Shanghai), 0.4 μL each of the upstream primer CMS-F2 and the downstream primer CMS-R2, 0.2 μL of the TaqMan probe CMS-P2, 2 μL of *Gnaphalium affine* genomic DNA template, and 7 μL of sterile ultrapure water. Following the conventional PCR screening in step 1.3 above, the annealing temperatures of both primer pairs CMS-F1 / R1 and CMS-F2 / R2 in this embodiment were both below 60℃. Therefore, the optimal three-step quantitative PCR reaction program was as follows: denaturation at 94℃ for 3 min, followed by 94℃ for 5 sec, 58℃ for 15 sec, and 72℃ for 30 sec, repeated 40 times, and the fluorescence growth curve was recorded. The blank control group was identical to the above system except that the template was replaced with sterile ultrapure water.

[0029] After quantitative real-time PCR amplification, the combination of primers CMS-F2, CMS-R2 and probe CMS-P2 provided in this embodiment produced a clear fluorescence growth curve for the long-eared gudgeon sample (e.g., Figure 3As shown in the figure), and a Ct value less than 35, indicating positive amplification. No fluorescence growth curve was detected in the blank control group, indicating negative amplification. It is noteworthy that this example found that in the quantitative real-time PCR reaction, the amplification results of the primer CMS-F1 / R1 and probe CMS-P1 combination on long wheat ear samples were similar to those of the blank control group (as shown in the figure). Figure 3 As shown in the figure, it also showed a negative result, indicating that the probe CMS-P1 did not effectively bind to the template in the quantitative real-time PCR, and therefore no fluorescence growth signal was detected. This primer-probe set cannot be used for the detection of gudgeon. Therefore, the combination of gudgeon primers CMS-F2, CMS-R2 and probe CMS-P2 provided in this embodiment was selected for subsequent gudgeon-specific detection.

[0030] Example 2: Specificity verification of the combination of primers CMS-F2, CMS-R2 and probe CMS-P2 for the long-eared minnow 2.1. Genomic DNA Extraction Genomic DNA was extracted from the caudal fin rays of the gudgeon used in the specificity verification experiment and from the following 11 closely related or common fish species found in the gudgeon's habitat. The extraction method was the same as in Example 1. Information on the fish samples and their origins for the specificity verification experiment is shown in Table 2.

[0031] Table 2. Information on fish species participating in the specificity validation experiment.

[0032] 2.2. Specificity Validation of Primer-Probe Composition Two gudgeon and the genomic DNA of the above 11 fish species were used as templates for real-time PCR amplification (reaction system and procedure as described in Example 1). The results showed that the gudgeon-specific primers CMS-F2, CMS-R2 and probe CMS-P2 were effective against the two gudgeon tested. Figure 3 The amplification curves of CMS-1 and CMS-2 both showed strong fluorescence growth, with Ct values ​​of 16.96 and 18.93 respectively, indicating positive amplification. Figure 3 As shown; however, the other 11 fish species tested and the blank control group showed no fluorescence growth curves, indicating a negative result. Figure 3 As shown in the figure. The specificity experimental results of this invention show that the sample detected by real-time quantitative PCR is *Gnaphalium affine*, indicating that the real-time quantitative primer and probe composition for *Gnaphalium affine* designed and screened in this invention, namely CMS-F2 / R2 and CMS-P2, can specifically amplify *Gnaphalium affine*, and there is no cross-reaction with closely related species or other fish in the same habitat as those in Table 2. This indicates that the real-time quantitative primer and probe composition provided by this invention has good specificity and can be used for the specific detection of *Gnaphalium affine*.

[0033] Example 3: Sensitivity test of the combination of primers CMS-F2, CMS-R2 and probe CMS-P2 for the long-eared minnow Using the genomic DNA of the long-necked gudgeon as a template, conventional PCR amplification was performed using primers CMS-F2 / R2 (the reaction system and procedure are as described in the conventional PCR amplification section of Example 1) to obtain a specific fragment of the target gene with a COI length of 191 bp, which was then used as the target detection gene. After purification using the AP-PCR-50 clean kit (Axygen), this specific fragment was ligated into the pMD19-T vector (Takara) to construct a recombinant plasmid ( pMD19 - T - peCOI The DNA was transformed into Escherichia coli DH5α (Weidi Biotechnology), and positive clones were selected for sequencing (Sangon Biotech, Shanghai) for verification. The target recombinant plasmid DNA was then extracted by plasmid extraction.

[0034] The initial concentration of the recombinant plasmid was determined to be 6.45 ng / μL. After sequential 10-fold dilutions, it was used as a template for TaqMan quantitative PCR. Sensitivity tests were performed on the primer CMS-F2 / R2 and probe CMS-P2 combination. The reaction system and procedure were the same as described in Example 1, with three replicates at the same concentration. The fluorescence signal amplification curve after the quantitative PCR reaction is shown below. Figure 4 As shown, the mean Ct values ​​for curves 1 to 8 are 14.15, 16.06, 19.92, 23.13, 25.86, 29.97, 32.77, and 35.11, respectively. The Ct value for plasmid sample 8 is greater than 35, making it impossible to determine whether it represents a positive amplification. Therefore, the minimum detection limit for the target gene in *Gnaphalium affine* provided by this invention, using the specific primers CMS-F2 and CMS-R2 and the probe CMS-P2, is 6.45 × 10⁻⁶. -7 It has a high sensitivity of ng / μL.

[0035] Example 4: Detection of topmouth gudgeon based on environmental DNA (eDNA) 4.1. Water sample collection and eDNA extraction In the upper reaches of the Qiupu River, a habitat of the gudgeon, a 2L sterile water sampler (sterilized with 0.1% sodium hypochlorite solution) was used to collect 2L of water samples at a depth of 0.5m. Immediately afterward, eDNA was enriched on-site using a 47mm diameter, 0.45μm cellulose membrane (Xingya, Shanghai). The eDNA-enriched membrane was placed in a sterile centrifuge tube and transported back to the laboratory immediately using a -20℃ vehicle refrigerator. eDNA was immediately extracted using the EZNA® Water DNA Kit (Omega) according to the instructions. The extracted total eDNA was stored at -80℃ for later use. Water samples were collected from a confirmed area without gudgeon habitat (Shuanglong Lake on the campus of Anqing Normal University) using the same method, and eDNA was extracted as a control group for subsequent quantitative PCR.

[0036] 4.2. Detection of eDNA in water samples by real-time quantitative PCR Using the two types of total eDNA samples mentioned above as templates, a real-time PCR reaction was performed; the system and procedure were the same as in Example 1, with the same water sample repeated three times. The real-time PCR amplification curves are shown below. Figure 5 As shown, curves 1 and 2 represent two parallel water samples collected from the habitat of the gudgeon, with Ct values ​​of 33.49 and 34.24, respectively, indicating positive amplification, while the control group ( Figure 5 Curve C) shows no fluorescence growth, indicating negative amplification. These results demonstrate that the combination of the longfin gudgeon-specific primers CMS-F2, CMS-R2, and probe CMS-P2 provided by this invention can be used for non-invasive detection of longfin gudgeon based on eDNA.

[0037] The above description is merely a preferred embodiment of the present invention, used to illustrate the technical solution of the present invention, and is not intended to limit the present invention. Those skilled in the art can make appropriate improvements or equivalent substitutions without departing from the principles and spirit of the present invention, but these improvements and substitutions should also be considered within the scope of protection of the present invention.

Claims

1. A fluorescent quantitative PCR primer and probe composition for gudgeon, characterized in that, The probe composition is based on the COI gene sequence of the mitochondria of the minnow as the target detection gene; the probe composition includes an upstream primer CMS-F2, the nucleotide sequence of which is shown in SEQ ID NO.1; a downstream primer CMS-R2, the nucleotide sequence of which is shown in SEQ ID NO.2; the probe composition also includes a TaqMan probe CMS-P2, the nucleotide sequence of which is shown in SEQ ID NO.

3.

2. The application of a primer and probe composition for quantitative PCR of longmouth gudgeon, characterized in that, Includes the following steps: (1) Extract genomic DNA from the fish sample or eDNA from the water sample as the template for testing; (2) Establish a real-time PCR amplification system based on the primer and probe composition of claim 1, perform the reaction and record the amplification curve; The real-time PCR reaction system is 20 μL, including: 10 μL of 2×TaqMan Fast qPCR premix, 0.4 μL each of upstream primer CMS-F2 and downstream primer CMS-R2, 0.2 μL of TaqMan probe CMS-P2, 2 μL of the template to be tested obtained in step (1), and 7 μL of sterile ultrapure water; The procedure for quantitative real-time PCR is as follows: pre-denaturation at 93-95℃ for 3 min, followed by a three-step amplification process: 94℃ for 5 sec, 58℃ for 15 sec, and 72℃ for 30 sec, repeated 35-50 times. The fluorescence growth curve is then recorded. (3) Based on the amplification curve obtained in step (2), when a strong fluorescence growth curve appears and the Ct value is less than 35, it is determined that the fish sample to be tested extracted in step (1) is a long-tailed gudgeon sample or the eDNA sample contains the long-tailed gudgeon gene.

3. The application according to claim 2, characterized in that, In the aforementioned real-time PCR amplification system, the final concentration of the upstream primer is 0.3-0.5 µM, the final concentration of the downstream primer is 0.3-0.5 µM, and the final concentration of the TaqMan probe is 0.2-0.4 µM.

4. The application according to claim 3, characterized in that, The final concentrations of the upstream primer CMS-F2 and the downstream primer CMS-R2 should be 1.25-2.5 times the final concentration of the TaqMan probe CMS-P2.

5. The application according to claim 2, 3, or 4, characterized in that: The amplification program for the real-time fluorescence PCR reaction is as follows: pre-denaturation at 94℃ for 3 min, followed by cycles of 94℃ for 5 sec, 58℃ for 15 sec, and 72℃ for 30 sec, for a total of 40 cycles.

6. The application according to claim 2, characterized in that, The primer-probe combination can detect a minimum concentration of the target gene in the long-eared gudgeon at 6.45 × 10⁻⁶. -7 ng / µL.

7. A kit for identifying species of topmouth gudgeon, characterized in that, The kit contains at least the fluorescent quantitative PCR primer and probe composition of claim 1.

Images