Probe primer combination for identifying real-time fluorescent quantitative PCR of egret TaqMan, kit and application

Abstract

The embodiment of the invention discloses a probe primer combination for real-time fluorescent quantitative PCR (Polymerase Chain Reaction) of an egret TaqMan, a kit and application. The fluorescent primer combination comprises a probe, an upstream primer and a downstream primer, the nucleotide sequence of the probe is 5 '-VIC-AATGTGATTGTTACTGCC-MGB-3', the nucleotide sequence of the upstream primer is 5 '-AACTTGGACAACCAGGGACG-3', and the nucleotide sequence of the downstream primer is 5 '-ATCCTCCGATTATGCGGTATTAC-3'. The fluorescent primer combination comprises a probe, an upstream primer and a downstream primer. The TaqMan probe real-time fluorescent PCR detection method established by the invention can effectively distinguish the egret from other birds (covering near-source species and distant birds), has excellent specificity and sensitivity in the aspect of identifying the egret species, greatly improves the detection accuracy, shortens the detection time, and has good application prospects. The effects of stability, reliability, short period, low cost and the like are achieved, and the method has important significance on egret protection and related ecological research.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of molecular biomedical technology, and specifically to a probe and primer combination, a kit, and an application thereof for identifying herons using TaqMan real-time fluorescence quantitative PCR. Background Art

[0002] The grey heron (Ardea cinerea) is a flagship species of wetland ecosystems, and its population dynamics directly reflect the health of its habitat. Accurately identifying grey herons and their subspecies facilitates targeted habitat protection and prevents misidentification that could render conservation measures ineffective. Furthermore, distinguishing grey herons from other heron species prevents confusion in ecological data and ensures accurate biodiversity monitoring. Species identification of grey herons is not only fundamental to taxonomy but also provides scientific support for ecological conservation, environmental monitoring, and policymaking.

[0003] Traditional morphological identification relies on features such as beak length, body color, and feather ornamentation, but the error rate in identifying incomplete specimens (such as fallen feathers, digestive remains) or subadults is high, and they are easily confused with closely related species, especially in samples that have faded or been artificially processed.

[0004] In recent years, real-time fluorescence quantitative PCR (qPCR) has gradually become the preferred technology for species identification due to its high sensitivity and rapid detection capabilities. However, the ordinary SYBRGreen method relies on melting curve analysis, which requires extremely high primer specificity and is prone to false positive signals due to non-specific amplification. The TaqMan probe method can accurately identify single nucleotide polymorphisms (SNPs) through the complementary binding of specific probes to target sequences. It is particularly suitable for distinguishing highly homologous sequences, but its successful application depends on the design of probes and primers for the unique genetic markers of the target species. At present, there has been no report on the TaqMan detection system for herons. The existing universal primers for herons are easily interfered with by non-target DNA in complex samples (such as environmental DNA, old specimens or forensic degraded samples), resulting in insufficient sensitivity and specificity. Summary of the Invention

[0005] To this end, the present invention provides a probe and primer combination, a kit and an application for identifying herons using TaqMan real-time fluorescence quantitative PCR.

[0006] In order to achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] According to a first aspect of an embodiment of the present invention, the present invention provides a probe primer combination for identifying herons by TaqMan real-time fluorescence quantitative PCR, comprising a probe, an upstream primer and a downstream primer, the nucleotide sequence of the probe being 5'-VIC-AATGTGATTGTTACTGCC-MGB-3', the nucleotide sequence of the upstream primer being 5'-AACTTGGACAACCAGGGACG-3', and the nucleotide sequence of the downstream primer being 5'-ATCCTCCGATTATGATCGGTATTAC-3'.

[0008] According to a second aspect of the embodiments of the present invention, the present invention provides a kit for identifying herons using TaqMan real-time fluorescence quantitative PCR, comprising the probe and primer combination described above.

[0009] According to a third aspect of the embodiments of the present invention, the present invention provides the use of the probe and primer combination as described above, or the kit as described above, in the identification of heron species.

[0010] According to a fourth aspect of the embodiments of the present invention, the present invention provides a method for identifying heron species, the method comprising:

[0011] (1) Extracting cDNA or genomic DNA from the sample to be tested;

[0012] (2) performing TaqMan real-time fluorescence quantitative PCR reaction using the probe, upstream primer, and downstream primer described above to obtain a Ct value and an amplification curve;

[0013] (3) Determination of test results: If the Ct value is ≤35 and a typical amplification curve appears, the result is positive; if there is no Ct value or no amplification curve, the result is negative; if the Ct value is >35, the sample is repeated. If there is no Ct value in the repeated result, it is negative; otherwise, it is positive.

[0014] Furthermore, the TaqMan real-time fluorescence quantitative PCR reaction system was as follows: 2xT5 Fast qPCR Mix 10 μL, upstream primer 10 μM 0.7 μL, downstream primer 10 μM 0.7 μL, TaqMan probe 10 μM 0.6 μL, DNA template 1 μL, supplemented with ddH2O to a total volume of 20 μL.

[0015] Furthermore, the TaqMan real-time fluorescence quantitative PCR reaction conditions were as follows: pre-denaturation at 95°C for 2 min; 95°C for 15 s, 60°C for 30 s, and 40 cycles.

[0016] The embodiments of the present invention have the following advantages:

[0017] Based on the differences in the mitochondrial control regions of different ardine species, the present invention designs specific oligonucleotide primers and probes to establish a TaqMan probe real-time fluorescence PCR detection method that can effectively distinguish herons from other poultry (including closely related species and distantly related birds). It has excellent specificity and sensitivity in identifying heron species, greatly improving the accuracy of detection, while shortening the detection time, achieving stable, reliable, short cycle, and low cost effects, which is of great significance to heron protection and related ecological research. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0019] Figure 1 A specificity verification amplification curve provided by the present invention;

[0020] Figure 2 Amplification curve diagram of different copy numbers of the heron gene provided by the present invention;

[0021] Figure 3 The standard curve diagram of the heron gene detection provided by the present invention;

[0022] Figure 4 This is the amplification curve diagram of the heron gene detected by a single probe provided by the present invention;

[0023] Figure 5 This is the amplification curve diagram of the hybrid probe detection of the heron gene provided by the present invention;

[0024] Figure 6 This is the NTC detection spectrum provided by the present invention. DETAILED DESCRIPTION

[0025] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0026] Example 1 Design of primers and probes

[0027] Mitochondrial sequences of the target species and related species were downloaded from the NCBI database and aligned using MEGA6 software. Species-specific regions were selected and primers and probes were designed using Primer Express 3.0.1 software to ensure specific detection of the target species. Primer and probe design and synthesis were performed by Beijing Qingke Biotechnology Co., Ltd. Primer and probe information is provided in Table 1 below.

[0028] Table 1

[0029] name Sequence (5'→3') Upstream primer 5'-AACTTGGACAACCAGGGACG-3'(SEQ ID NO:1) Downstream primer 5'-ATCCTCCGATTATGATCGGTATTAC-3'(SEQ ID NO:2) probe 5'-VIC-AATGTGATTGTTACTGCC-MGB-3'(SEQ ID NO:3)

[0030] Example 2 Specificity Detection

[0031] DNA templates were extracted from herons as positive samples, and DNA templates were extracted from little egrets, night herons, pond herons, cattle egrets, white wagtails, yellow-rumped bulbuls, house sparrows, and black-bellied whistling pheasants as negative samples. ddH2O was used instead of the nucleic acid in the system as a no-template control (NFC). Amplification was performed using the upstream and downstream primers and probes listed in Table 1, with each sample assayed in triplicate. DNA templates were extracted from feathers using a commercially available kit.

[0032] The real-time fluorescence PCR reaction system was as follows: 2xT5 Fast qPCR Mix (Qingke, TSE301) 10 μL, upstream primer 10 μM 0.7 μL, downstream primer 10 μM 0.7 μL, probe 10 μM 0.6 μL, DNA template 1 μL, and ddH2O was added to make up the total volume to 20 μL.

[0033] The real-time fluorescence amplification program was as follows: pre-denaturation at 95°C for 2 min, 1 cycle; 95°C for 15 s, 60°C for 30 s, 40 cycles; and collection of fluorescence signals during annealing and extension (60°C).

[0034] The results are shown in Table 2 and Figure 1 , the positive sample showed a typical S-shaped amplification curve (see Figure 1 ), while other negative samples and no-template controls showed a straight line (see Figure 1 There was no increase in fluorescence value, indicating that the primers and probes provided by the present invention can specifically detect heron DNA.

[0035] Table 2

[0036]

[0037]

[0038] Example 3 Probe amplification efficiency detection

[0039] The target product obtained by amplification in Example 2 was cloned into a vector (Qingke pClone007 Versatile SimpleVector). The resulting plasmid was sequenced and verified and used as a standard plasmid for amplification efficiency testing. The nucleotide sequence of the Heron standard plasmid is as follows:

[0040]

[0041] The Heron standard plasmid was gradiently diluted as a positive standard and detected according to the real-time fluorescence PCR reaction system and amplification procedure of Example 2. Each dilution factor was repeated 3 times to obtain the probe gradient amplification curve ( Figure 2 , 1: dilution factor 10^3, 2: dilution factor 10^4, 3: dilution factor 10^5, 4: dilution factor 10^6, 4: dilution factor 10^7), CT and copy number LOG values ​​were fitted to obtain the standard curve equation ( Figure 3 ), detect R 2 The Ct value reached 0.9999, indicating a good linear relationship between the Ct and the logarithmic copy number within the range of 5.08E+7 to 5.08E+3 copies / µL for the Heron gene. Based on the established standard curve, the amplification efficiency was calculated to be 94.0%. The test data are shown in Table 3.

[0042] Table 3

[0043]

[0044] After the plasmid was diluted 10^7 times, the detection repeatability was good, and the probe detection efficiency was within the normal range (90%-110%). The sensitivity of the primers and probes provided by the present invention met the detection requirements.

[0045] Example 4 Interference Detection of Multiplex qPCR

[0046] Another positive sample was amplified using the upstream primers, downstream primers, and probes in Table 1 using the real-time fluorescence PCR reaction system and real-time fluorescence amplification program in Example 2. The experiment was repeated three times.

[0047] In addition, multiple primers and probes were mixed in the same reaction system and multiplex PCR was performed on positive samples of the same concentration to test whether they would interfere with each other. The information of the primers and probes used in the multiplex reaction system is as follows:

[0048] Upstream primer:

[0049] Upstream primer 1: SEQ ID NO: 1;

[0050] Upstream primer 2: 5′-GGATTCGGAAACTGACTAGTACCC-3′ (SEQ ID NO: 5);

[0051] Upstream primer 3: 5′-CGCCTTYGTAATAATCTTCTTTATAGTG-3′ (SEQ ID NO: 6);

[0052] Upstream primer 4: 5′-GGCCAACCCGGAACACTG-3′ (SEQ ID NO: 7);

[0053] Upstream primer 5: 5′-GAGCCGGCATAATTGGAACT-3′ (SEQ ID NO: 8);

[0054] Downstream primer:

[0055] Downstream primer 1: SEQ ID NO: 2

[0056] Downstream primer 2: 5′-GGTGGAAGGAGTCAGAAACTTATG-3′ (SEQ ID NO: 9);

[0057] Downstream primer 3: 5′-CAGAAGCTCATGTTGTTTATACGG-3′ (SEQ ID NO: 10);

[0058] Downstream primer 4: 5′-AAGAAAATTATTACGAAGGCATGG-3′ (SEQ ID NO: 11);

[0059] Downstream primer 5: 5′-CATGGGCGGTGACAATTACG-3′ (SEQ ID NO: 12);

[0060] Probe:

[0061] Probe 1: SEQ ID NO: 3

[0062] Probe 2: 5′-FAM-TGGAAATGCTATATCAGG-MGB-3′ (SEQ ID NO: 13);

[0063] Probe 3: 5′-ROX-TGACTAGTACCACTAATAA-MGB-3′ (SEQ ID NO: 14);

[0064] Probe 4: 5′-TAMRA-CGGTGACGACTACA-MGB-3′ (SEQ ID NO: 15);

[0065] Probe 5: 5'-CY5-CCGAGCCGAGCTT-MGB-3' (SEQ ID NO: 16).

[0066] The multiplex PCR reaction system was as follows: 15 μL of premixed reagent 2xT5 Fast qPCR Mix (Qingke, TSE301), 3.5 μL of 10 μM upstream primer (0.7 μL × 5), 3.5 μL of 10 μM downstream primer (0.7 μL × 5), 3 μL of 10 μM probe (0.6 μL × 5), and 1 μL of DNA template, which was supplemented with ddH2O to a total volume of 30 μL.

[0067] The amplification program was as follows: pre-denaturation at 95°C for 2 min, 1 cycle; 95°C for 15 s, 60°C for 1 min, 40 cycles; and collecting fluorescence signals during annealing and extension (60°C).

[0068] The results are shown in Table 4 and Figure 4-6 .

[0069] Table 4

[0070]

[0071] The results showed that the Ct values ​​of the mixed probe and the single probe for detecting samples of the same concentration had little difference, indicating that the primer probe provided by the present invention has strong anti-interference ability, ensuring the accuracy and reliability of the detection results.

[0072] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

[0073] SEQUENCE LISTING

[0074] <110> Kunming Institute of Zoology, Chinese Academy of Sciences

[0075] <120> Probe and primer combinations, kits, and applications for identifying herons using TaqMan real-time fluorescence quantitative PCR

[0076] <130> GG241439720A

[0077] <160> 16

[0078] <170> PatentIn version 3.5

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Claims

1. A probe and primer combination for identifying herons using TaqMan real-time fluorescence quantitative PCR, characterized in that: The method comprises a probe, an upstream primer and a downstream primer, wherein the nucleotide sequence of the probe is 5'-VIC-AATGTGATTGTTACTGCC-MGB-3', the nucleotide sequence of the upstream primer is 5'-AACTTGGACAACCAGGGACG-3', and the nucleotide sequence of the downstream primer is 5'-ATCCTCCGATTATGATCGGTATTAC-3'.

2. A kit for identifying herons using TaqMan real-time fluorescence quantitative PCR, characterized in that: Comprising the probe primer combination as claimed in claim 1.

3. Use of the probe-primer combination according to claim 1, or the kit according to claim 2, in identification of heron species.

4. A method for identifying heron species, characterized in that: The method comprises: (1) Extracting cDNA or genomic DNA from the sample to be tested; (2) performing a TaqMan real-time fluorescence quantitative PCR reaction using the probe, upstream primer, and downstream primer as claimed in claim 1 to obtain a Ct value and an amplification curve; (3) Determination of test results: If the Ct value is ≤35 and a typical amplification curve appears, the result is positive; if there is no Ct value or no amplification curve, the result is negative; if the Ct value is >35, the sample is repeated. If there is no Ct value in the repeated result, it is negative; otherwise, it is positive.

5. The heron species identification method according to claim 4, wherein: The TaqMan real-time fluorescence quantitative PCR reaction system was as follows: 2xT5 Fast qPCR Mix 10 μL, upstream primer 10 μM 0.7 μL, downstream primer 10 μM 0.7 μL, TaqMan probe 10 μM 0.6 μL, DNA template 1 μL, and supplemented with ddH2O to a total volume of 20 μL.

6. The heron species identification method according to claim 4, wherein: The reaction conditions of TaqMan real-time fluorescence quantitative PCR were as follows: pre-denaturation at 95°C for 2 min; 40 cycles of 95°C for 15 s and 60°C for 30 s.

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