Primers, probes, kits, methods and applications for identifying Arapaima gigas

Abstract

This application relates to the technical field of arapaima identification, specifically to primers, probes, kits, methods and applications for identifying arapaima. The primer pair and probe are designed for the target sequence shown in SEQ ID NO:4, with high specificity for identifying the genomic DNA of arapaima, capable of achieving specific amplification, and having good reproducibility and stable and reliable results. This application not only improves the detection sensitivity of arapaima, but also greatly improves the detection efficiency.

Description

Technical Field

[0001] This application relates to the technical field of arapaima identification, specifically to primers, probes, kits, methods and applications for identifying arapaima. Background Art

[0002] The arapaima (Arapaima gigas), commonly known as the "pirarucu", belongs to the order Osteoglossiformes, family Osteoglossidae, and genus Arapaima. The arapaima is a high-value commercial fishery fish. Unfortunately, the sharp decline in natural resources and genetic bottlenecks have put the arapaima on the verge of extinction. It is listed in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) and is a restricted trade species.

[0003] As one of the ornamental fish, the arapaima is morphologically very similar to other ornamental fish (such as the silver arowana), and it is very difficult to identify it morphologically. Therefore, there is an urgent need for an accurate detection method to identify these fish. Summary of the Invention

[0004] Aiming at the difficulties in arapaima identification, the primary objective of the present invention is to provide primers, probes, kits, methods and applications for arapaima sex identification.

[0005] To this end, the embodiments of this application at least disclose the following technical solutions:

[0006] In the first aspect, the embodiments disclose a primer pair for identifying arapaima, including a DNA molecule shown in SEQ ID NO:1 and a DNA molecule shown in SEQ ID NO:2.

[0007] In the second aspect, the embodiments disclose a probe for identifying arapaima, including a DNA molecule shown in SEQ ID NO:3.

[0008] In the third aspect, the embodiments disclose a kit including the primer pair described in the first aspect and the probe described in the second aspect.

[0009] In the fourth aspect, the embodiments disclose a method for identifying arapaima, including: obtaining the genomic DNA of the sample to be tested; preparing a fluorescence PCR reaction system with the genomic DNA, the primer pair described in the first aspect, the second aspect and the probe for reaction; and identifying the arapaima according to the signal of the reaction.

[0010] In the fifth aspect, the embodiments disclose the use of the primer pair described in the first aspect, the probe described in the second aspect or the kit described in the third aspect in identifying arapaima.

[0011] Compared with the prior art, the present application has at least the following technical effects:

[0012] The primer pairs and probe pairs provided by the embodiments have high specificity for identifying the genomic DNA of Arapaima gigas, can achieve specific amplification, and have good reproducibility and stable and reliable results. In addition, the primer pairs, probes, and real-time fluorescence PCR detection method provided by the embodiments have a wide range of applications, not only improving the detection sensitivity but also greatly improving the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 FIG. 9 shows the fluorescence amplification curves for identifying the genomic DNA of Arapaima gigas at different concentrations provided by the embodiments. From left to right, they are genomic DNA at 42.29 ng / μL, 4.229 ng / μL, 0.4229 ng / μL, 0.04229 ng / μL, 0.004229 ng / μL, 0.0004229 ng / μL, 0.00004229 ng / μL, and 0.000004229 ng / μL.

[0014] Figure 2 FIG. 13 shows the fluorescence amplification curves of Arapaima gigas (blue), Osteoglossum bicirrhosum, Osteoglossum ferreirai, Scleropages jardinii, Channa asiatica, Cobitidae, Botiidae, Cichlidae, Cichlidae, Cyprinidae, and Loricariidae provided by the embodiments. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0015] In order to make the objectives, technical solutions, and advantages of the present application clearer, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application. The reagents not specifically described in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0016] For the experimental methods without specific conditions indicated in the following embodiments, they are usually carried out according to conventional conditions, such as those described in "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 1989) or the conditions provided by the manufacturer.

[0017] On the one hand, the embodiments disclose primer pairs for identifying Arapaima gigas, including a DNA molecule shown in SEQ ID NO: 1 and a DNA molecule shown in SEQ ID NO: 2.

[0018] On the one hand, the embodiments disclose a probe for identifying Arapaima gigas, including a DNA molecule shown in SEQ ID NO: 3.

[0019] On the one hand, the embodiments disclose a kit for identifying Arapaima gigas. The kit includes: a DNA molecule shown in SEQ ID NO:1, a DNA molecule shown in SEQ ID NO:2, and a DNA molecule shown in SEQ ID NO:3.

[0020] In some embodiments, the kit further includes other fluorescent PCR reagents. The other fluorescent PCR reagents may be TaqMan Mastermix.

[0021] On the other hand, the embodiments disclose a method for identifying Arapaima gigas, including: obtaining genomic DNA of a sample to be tested; preparing a fluorescent PCR reaction system with the genomic DNA, a DNA molecule shown in SEQ ID NO:1, a DNA molecule shown in SEQ ID NO:2, and a DNA molecule shown in SEQ ID NO:3 for reaction; and identifying the Arapaima gigas according to the signal of the reaction.

[0022] In some embodiments, the fluorescent PCR reaction system contains, based on 25 μL: 12.5 μL of 2× TaqMan Mastermix, 1 μL of 10 μM DNA molecule shown in SEQ ID NO:1, 1 μL of 10 μM DNA molecule shown in SEQ ID NO:2, 1 μL of 5 μM DNA molecule shown in SEQ ID NO:3, and 5 μL of 42.29 ng / μL genomic DNA.

[0023] In some embodiments, the steps of the fluorescent PCR reaction include: treating at 50 °C for 2 min (1 cycle); treating at 95 °C for 10 min (1 cycle); treating at 95 °C for 20 s and 60 °C for 30 s, for a total of 40 cycles, and simultaneously detecting the fluorescent signal.

[0024] In some embodiments, the step of identifying the Arapaima gigas according to the signal of the reaction includes: detecting the fluorescent signal of the amplification product; calculating the Ct value according to the fluorescent signal; and determining whether the sample to be tested is Arapaima gigas according to the Ct value.

[0025] In addition, the embodiments also disclose the use of the primer pair, the probe provided in the above embodiments, or the kit provided in the above embodiments in identifying Arapaima gigas.

[0026] The embodiments of the present application will be described in more detail below, but do not constitute a limitation on the implementation manners of the present application.

[0027] 1. Primer pair and probe

[0028] In the examples, primer pairs and probes as shown in Table 1 were synthesized. The synthesis method can be PCR amplification or chemical synthesis, and there is no limitation on the synthesis method. In Table 1, F and R are primer pairs, and P is a probe. Among them, FAM represents a fluorescent reporter group, and TAMRA represents a quenching group. The present application uses the fluorescence probe method, and its detection principle is to use a fluorescently labeled specific probe to identify the template. Compared with the SYBR dye method in the prior art, the specific fluorescently labeled probe of the present invention has stronger specificity and lower background interference. In addition, Table 1 shows the target sequence SEQ ID NO:4 amplified by the F and R primer pairs, and the target sequence SEQ ID NO:7 amplified by the F1 and R1 primer pairs (control).

[0029] Table 1

[0030]

[0031]

[0032] 2. Genomic DNA extraction

[0033] An animal genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd.; catalog number: DP323) was used to extract animal sample DNA. The extraction method is detailed in the kit operation manual. The DNA solution was stored at -20 °C for later use.

[0034] 3. PCR amplification

[0035] The fluorescence PCR reaction system was 25 μL in total and contained: 12.5 μL of 2×TaqMan Mastermix, 1 μL of 10 μM DNA molecule as shown in SEQ ID NO:1, 1 μL of 10 μM DNA molecule as shown in SEQ ID NO:2, 1 μL of 5 μM DNA molecule as shown in SEQ ID NO:3, and 5 μL of 42.29 ng / μL genomic DNA.

[0036] In some examples, the fluorescence PCR reaction steps included: treating at 50 °C for 2 min (1 cycle); treating at 95 °C for 10 min (1 cycle); treating at 95 °C for 20 s and 60 °C for 30 s, for a total of 40 cycles, and simultaneously detecting the fluorescence signal.

[0037] 4. Repeatability test

[0038] Extract the genomic DNA of Arapaima gigas (concentration 42.29 ng / μL), and perform 10-fold dilutions to obtain genomic DNA with concentrations of 4.229 ng / μL, 0.4229 ng / μL, 0.04229 ng / μL, and 0.004229 ng / μL respectively. Using the above fluorescence PCR reaction system and steps, test 3 parallels for each dilution. The results are shown in Table 2, indicating that the primer pair, probe, and kit provided in the examples have high repeatability for detecting the genomic DNA of Arapaima gigas.

[0039] Table 2

[0040] Genomic DNA (ng / μL) Average Ct value X±SD 4.229 22.30±0.540 0.4229 25.55±1.068 0.04229 26.52±3.35 0.004229 36.12±2.25

[0041] 5. Sensitivity test

[0042] Extract the genomic DNA of Arapaima gigas (concentration 42.29 ng / μL), and perform 10-fold dilutions to obtain genomic DNA with concentrations of 4.229 ng / μL, 0.4229 ng / μL, 0.04229 ng / μL, 0.004229 ng / μL, 0.0004229 ng / μL, 0.00004229 ng / μL, and 0.000004229 ng / μL respectively. Using the above fluorescence PCR reaction system and steps, test 3 parallels for each dilution.

[0043] As Figure 1 can be seen, the primer pair, probe, and kit provided in the examples can detect the genomic DNA of Arapaima gigas at a concentration of 0.00004229 ng / μL, showing high sensitivity.

[0044] 6. Specificity

[0045] Extract the genomic DNA of Arapaima gigas (42.29 ng / μL), Osteoglossum bicirrhosum (47.8 ng / μL), Osteoglossum ferreirai (53.93 ng / μL), Scleropages jardinii (51.73 ng / μL), Channa asiatica (57.02 ng / μL), Cobitidae kuhlii (46.82 ng / μL), Corydoras trilineatus (46.77 ng / μL), Cichlidae Pelvicachromis pulcher (54.23 ng / μL), Cichlidae Cryptoheros sajica (51.67 ng / μL), Cyprinidae Acrossocheilus cantonensis (49.29 ng / μL), Cyprinidae Garra pingi pingi (57.36 ng / μL), and Loricariidae (55.36 ng / μL). These ornamental fish are all from commercially available samples. As Figure 2 shown, only Arapaima gigas shows an "S"-shaped amplification curve, while the other fish do not have an obvious "S"-shaped amplification curve, indicating that the primer pair, probe, and kit provided in the examples have high specificity for Arapaima gigas.

[0046] 7. Comparison

[0047] In addition, the genomic DNA of Arapaima was amplified using F1, R1 and P1 in Table 1, and the amplification reaction system and amplification procedure were the same as those in the above embodiment, and the results were shown in Table 3. In Table 3, "--" indicates that the fluorescence curve is not "S" shaped. This shows that the genomic DNA of Arapaima can only be detected at 4.229 ng / μL by amplifying the genomic DNA of Arapaima using F1, R1 and P1, and its detection sensitivity is far less than that of F, R and P, which shows that the primer pairs, probes and kits provided in the embodiment have better sensitivity.

[0048] Table 3

[0049] Genomic DNA (ng / μL) Average Ct value X±SD 4.229 36.96±0.324 0.4229 -- 0.04229 -- 0.004229 --

[0050] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. Kit for identifying Arapaima gigas, comprising primer pairs, said primer pairs including a DNA molecule shown in SEQ ID NO:1, a DNA molecule shown in SEQ ID NO:2, and further including a probe, said probe being a DNA molecule shown in SEQ ID NO:

3.

2. The kit according to claim 1, further comprising TaqMan Mastermix.

3. Method for identifying Arapaima gigas, comprising: obtaining genomic DNA of a sample to be tested; formulating a fluorescence PCR reaction system with the genomic DNA, the primer pairs described in claim 1, and the probe for reaction; identifying the Arapaima gigas according to the signal of the reaction.

4. The method according to claim 3, the fluorescence PCR reaction system contains, based on 25 μL: 12.5 μL of 2× TaqMan Mastermix, 1 μL of 10 μM DNA molecule shown in SEQ ID NO:1, 1 μL of 10 μM DNA molecule shown in SEQ ID NO:2, 1 μL of 5 μM DNA molecule shown in SEQ ID NO:3, 5 μL of 42.29 ng / μL genomic DNA.

5. The method according to claim 3, the fluorescence PCR reaction steps include: treating at 50 °C for 2 min, 1 cycle; treating at 95 °C for 10 min, 1 cycle; and treating at 95 °C for 20 s and 60 °C for 30 s, 40 cycles in total, and detecting fluorescence signals simultaneously.

6. The method according to claim 3, the step of identifying the Arapaima gigas according to the signal of the reaction includes: detecting the fluorescence signal of the amplification product; calculating the Ct value according to the fluorescence signal; judging whether the sample to be tested is Arapaima gigas according to the Ct value.

7. Use of the kit according to claim 1 in identifying Arapaima gigas.

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