Primer, probe, kit and method for identifying giant-bone glossogyne and application of primer, probe, kit and method

Abstract

The invention relates to the technical field of identification of giant-bone tongue fish, in particular to a primer, a probe, a kit and a method for identifying the giant-bone tongue fish and application of the primer, the probe, the kit and the method. The primer pair and the probe are designed aiming at a target sequence as shown in SEQ ID NO: 4, are high in specificity for identifying genome DNA (Deoxyribose Nucleic Acid) of the giant-bone glossogyne, can realize specific amplification, and have good reproducibility and stable and reliable results. According to the invention, not only is the detection sensitivity of the giant-bone tongue fish improved, but also the detection efficiency is greatly improved.

Description

Technical Field

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

[0002] Arapaimagigas, commonly known as the "walrus fish", belongs to the order Osteoglossiformes, family Osteoglossidae, genus Arapaima. Arapaima is a high-priced commercial fish. Unfortunately, the sharp decline in natural resources and genetic bottlenecks have made Arapaima endangered. It is listed in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), a trade-restricted species.

[0003] As one of the ornamental fish, Arapaima is similar in morphology to other ornamental fish (such as silver arowana), and it is difficult to identify them by morphology. Therefore, an accurate detection method that can identify these fish is urgently needed. Summary of the invention

[0004] In view of the difficulty in identifying Arapaima, the primary purpose of the present invention is to provide a primer, a probe, a kit, a method and an application for sex identification of Arapaima.

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

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

[0007] In a second aspect, the embodiment discloses a probe for identifying Arapaima, comprising a DNA molecule as shown in SEQ ID NO:3.

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

[0009] In a fourth aspect, an embodiment discloses a method for identifying Arapaima, comprising: obtaining genomic DNA of a sample to be tested; preparing a fluorescent 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 based on the signal of the reaction.

[0010] In a 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, this application has at least the following technical effects:

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

[0013] Figure 1 The fluorescence amplification curves for identifying Arapaima genomic DNA at different concentrations provided in the examples are, from left to right, 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 of genomic DNA.

[0014] Figure 2 The fluorescence amplification curves of the Arapaima (blue), the beautiful hard bone tongue fish, the double-bearded bone tongue fish, the Australian hard bone tongue fish, the Channa family thunder dragon fish, the Cobitidae snake fish, the Cobitidae three-pointed cory, the Cichlidae ten-banded Luoli sea bream, the Cichlidae crown six, the Cyprinidae one-browed Taoist, the Cyprinidae panda head and the osteoclastidae provided in the embodiment. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0016] The experimental methods in the following examples where specific conditions are not specified 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 conditions provided by the manufacturer.

[0017] In one aspect, the embodiment discloses a primer pair for identifying Arapaima, comprising a DNA molecule as shown in SEQ ID NO:1 and a DNA molecule as shown in SEQ ID NO:2.

[0018] In one aspect, the embodiment discloses a probe for identifying Arapaima, comprising a DNA molecule as shown in SEQ ID NO:3.

[0019] In one aspect, the embodiment discloses a kit for identifying Arapaima, which comprises: a DNA molecule as shown in SEQ ID NO:1, a DNA molecule as shown in SEQ ID NO:2, and a DNA molecule as shown in SEQ ID NO:3.

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

[0021] On the other hand, the embodiment discloses a method for identifying Arapaima, comprising: obtaining genomic DNA of a sample to be tested; preparing a fluorescent PCR reaction system with the genomic DNA, a DNA molecule as shown in SEQ ID NO:1, a DNA molecule as shown in SEQ ID NO:2, and a DNA molecule as shown in SEQ ID NO:3 for reaction; and identifying the Arapaima based on the reaction signal.

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

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

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

[0025] In addition, the embodiments also disclose the use of the primer pairs described in the above embodiments, the probes provided in the above embodiments, or the kits provided in the above embodiments in identifying Arapaima.

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

[0027] 1. Primer pairs and probes

[0028] The embodiment synthesizes the primer pairs and probes shown in Table 1. The synthesis method can be PCR amplification or chemical synthesis, and the synthesis method is not limited. 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 adopts a fluorescent 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 fluorescently labeled specific 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] The animal sample DNA was extracted using an animal genome extraction kit (Tiangen Biochemical Technology Co., Ltd.; catalog number: DP323). The extraction method is detailed in the kit operating instructions. The DNA solution was stored at -20°C for future use.

[0034] 3. PCR amplification

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

[0036] In some embodiments, the fluorescent PCR reaction steps include: 50°C treatment for 2 min (1 cycle); 95°C treatment for 10 min (1 cycle); 95°C treatment for 20 s and 60°C treatment for 30 s, for a total of 40 cycles, and simultaneous detection of fluorescent signals.

[0037] 4. Repeatability test

[0038] The genomic DNA of Arapaima was extracted (concentration 42.29 ng / μL), and 10-fold dilutions were used to obtain genomic DNA of 4.229 ng / μL, 0.4229 ng / μL, 0.04229 ng / μL, and 0.004229 ng / μL, respectively. The above-mentioned fluorescent PCR reaction system and steps were used, and 3 parallels were tested for each dilution. The results are shown in Table 2, indicating that the primer pairs, probes, and kits provided in the embodiment have high repeatability for detecting the genomic DNA of Arapaima.

[0039] Table 2

[0040] Genomic DNA (ng / μL) Mean 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] The genomic DNA of Arapaima was extracted (concentration 42.29 ng / μL), and 10-fold dilution was used to obtain genomic DNA 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. The above-mentioned fluorescent PCR reaction system and steps were used, and 3 parallel tests were performed for each dilution.

[0043] like Figure 1 It can be seen that the primer pairs, probes and kits provided in the examples can detect 0.00004229 ng / μL of Arapaima genomic DNA, with high sensitivity.

[0044] 6. Specificity

[0045] The genomic DNA of Arapaima (42.29ng / μL), Beautiful Bone Tongue (47.8ng / μL), Double-bearded Bone Tongue (53.93ng / μL), Australian Bone Tongue (51.73ng / μL), Channa thunbergii (57.02ng / μL), Cobitidae Snake Fish (46.82ng / μL), Cobitidae Triceratops (46.77ng / μL), Cichlidae Ten-banded Lolita (54.23ng / μL), Cichlidae Crown Six (51.67ng / μL), Cyprinidae One-browed Taoist (49.29ng / μL), Cyprinidae Panda Head (57.36ng / μL) and Osteochondria (55.36ng / μL) were extracted. These ornamental fish were all from commercial samples. Figure 2 As shown, only Arapaima showed an "S"-shaped amplification curve, while other fish had no obvious "S"-shaped amplification curve, indicating that the primer pairs, probes and kits provided in the examples have high specificity for Arapaima.

[0046] 7. Contrast

[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) Mean 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. A primer pair for identifying Arapaima, comprising a DNA molecule as shown in SEQ ID NO: 1 and a DNA molecule as shown in SEQ ID NO:

2.

2. A probe for identifying Arapaima, comprising a DNA molecule as shown in SEQ ID NO:

3.

3. A kit for identifying Arapaima, comprising a DNA molecule as shown in SEQ ID NO: 1, a DNA molecule as shown in SEQ ID NO: 2, and a DNA molecule as shown in SEQ ID NO:

3. The kit according to claim 3 , further comprising TaqMan Mastermix.

5. Methods for identifying Arapaima, including: Obtaining genomic DNA of the sample to be tested; The genomic DNA, the primer pair according to claim 1, claim 2 and the probe are prepared into a fluorescent PCR reaction system for reaction; The Arapaima is identified based on the reaction signal.

6. The method according to claim 5, wherein the fluorescent PCR reaction system comprises, in 25 μL, 12.5 μL 2×TaqMan Mastermix, 1 μL, 10 μM of the DNA molecule shown in SEQ ID NO: 1, 1 μL, 10 μM of the DNA molecule shown in SEQ ID NO: 2, 1 μL, 5 μM of the DNA molecule shown in SEQ ID NO: 3, and 5 μL 42.29 ng / μL genomic DNA.

7. The method according to claim 5, wherein the fluorescent PCR reaction step comprises: 50°C for 2 min, 1 cycle; 95°C for 10 min, 1 cycle; as well as The cells were treated at 95°C for 20 s and 60°C for 30 s for a total of 40 cycles, and the fluorescence signal was detected simultaneously.

8. The method according to claim 5, wherein the step of identifying the Arapaima according to the reaction signal comprises: Detecting the fluorescent signal of the amplified product; The Ct value was calculated based on the fluorescence signal; Determine whether the sample to be tested is Arapaima according to the Ct value.

9. Use of the primer pair according to claim 1, the probe according to claim 2 or the kit according to claim 3 in identifying Arapaima.

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