Primer, probe, kit, method and application for identifying osteoglossus dimaculata

Abstract

The invention relates to the technical field of identification of ligustrum dibarbatum, in particular to a primer, a probe, a kit and a method for identifying ligustrum dibarbatum and application of the primer, the probe, the kit and the method. The primer pair and the probe are designed according to a target sequence as shown in SEQ ID NO: 4, have high specificity for identifying the genome DNA of the acorus distraction, can realize specific amplification, and have good reproducibility and stable and reliable results. The detection sensitivity is improved, the detection efficiency is improved, and the customs clearance speed of an enterprise is increased.

Description

Technical Field

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

[0002] Osteoglossum bicirrhosum, belonging to the order Osteoglossiformes, family Osteoglossidae and genus Osteoglossum, is a medium-sized freshwater fish. In recent years, the market economic value of Osteoglossum bicirrhosum has been increasing. As one of the ornamental fish, Osteoglossum bicirrhosum is morphologically similar to other ornamental fish (such as silver arowana), and it is difficult to identify it by morphology. Therefore, an accurate detection method that can identify these fish is urgently needed. Summary of the invention

[0003] In view of the difficulty of non-destructive identification of B. brevis, 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 B. brevis.

[0004] To this end, the embodiments of the present application disclose at least the following technical solutions:

[0005] In a first aspect, the embodiments disclose a primer pair for identifying the bivalve, comprising a DNA molecule as shown in SEQ ID NO:1 and a DNA molecule as shown in SEQ ID NO:2.

[0006] In a second aspect, the embodiment discloses a probe for identifying the bivalve, comprising a DNA molecule as shown in SEQ ID NO:3.

[0007] 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.

[0008] In a fourth aspect, an embodiment discloses a method for identifying the broadbone tongue fish, 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 broadbone tongue fish based on the reaction signal.

[0009] 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 the broadbill fish.

[0010] Compared with the prior art, this application has at least the following technical effects:

[0011] The primer pair and probe pair provided in the embodiment have high specificity for identifying the genomic DNA of the fish, can achieve specific amplification, and have good reproducibility, and the results are stable and reliable.

[0012] In addition, the primer pairs and probes provided in the embodiment, and the real-time fluorescence PCR detection method, not only improve the detection sensitivity, but also greatly improve the detection efficiency. The detection and identification work is fast, the application range is wide, and the customs clearance speed for these ornamental fish is accelerated. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The fluorescence amplification curves for identifying the genomic DNA of the broadbill fish at different concentrations provided in the example are, from left to right, 53.9 ng / μL, 5.393 ng / μL, 0.5393 ng / μL, 0.05393 ng / μL, 0.005393 ng / μL, 0.0005393 ng / μL, 0.00005393 ng / μL, and 0.000005393 ng / μL of genomic DNA.

[0014] Figure 2 The fluorescence amplification curves of the double-barbed bone tongue fish, beautiful hard bone tongue fish, giant arapaima, Australian hard bone tongue fish, Channa family thunder dragon fish, Cobitidae snake fish, Cobitidae three-pointed cory, Cichlidae ten-banded Luoli sea bream, Cichlidae crown six, Cyprinidae one-browed Taoist, Cyprinidae panda head and bone armored catfish provided in the embodiments. 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 embodiments disclose a primer pair for identifying the bivalve, 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 the fish, comprising a DNA molecule as shown in SEQ ID NO:3.

[0019] In one aspect, the embodiment discloses a kit for identifying Osteoglossus bisporus. The kit 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 the bony tongue fish, 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 bony tongue fish 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 53.9 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 fish according to the reaction signal 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 the fish 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 Osteoglossus bicolor.

[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 synthesized 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 MGB 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 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 53.93 ng / μL 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 the fish was extracted (concentration 53.93 ng / μL), and 10 times dilution was performed to obtain genomic DNA of 5.393 ng / μL, 0.5393 ng / μL, 0.05393 ng / μL, and 0.005393 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 the fish.

[0039] Table 2

[0040] Genomic DNA (ng / μL) Mean Ct value X±SD 5.393 20.53±0.109 0.5393 23.66±0.114 0.05393 27.36±0.128 0.005393 32.38±0.242

[0041] 5. Sensitivity test

[0042] The genomic DNA of the broadbill fish was extracted (concentration 53.9 ng / μL), and 10-fold dilution was used to obtain genomic DNA of 5.393 ng / μL, 0.5393 ng / μL, 0.05393 ng / μL, 0.005393 ng / μL, 0.0005393 ng / μL, 0.00005393 ng / μL, and 0.000005393 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 pair, probe and kit provided in the embodiment can detect 0.00005393 ng / μL of genomic DNA of Osteoglossus bicolor, with high sensitivity.

[0044] 6. Specificity

[0045] The genomic DNA of the beautiful hard-boiled bone tongue fish (47.8ng / μL), the double-bearded bone tongue fish (53.93ng / μL), the giant arapaima (42.29ng / μL), the Australian hard-boiled bone tongue fish (51.73ng / μL), the brontosaurus of Channa family (57.02ng / μL), the snake fish of Cobitidae family (46.82ng / μL), the three-pointed corydoras of Cobitidae family (46.77ng / μL), the ten-banded sea bream of Cichlidae family (54.23ng / μL), the crown six of Cichlidae family (51.67ng / μL), the one-browed Taoist of Cyprinidae family (49.29ng / μL), the panda head of Cyprinidae family (57.36ng / μL) and the bone armored catfish family (55.36ng / μL) were extracted. These ornamental fish were all from commercial samples. Figure 2 As shown, only the two-barbed tongue fish 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 embodiment have high specificity for two-barbed tongue fish.

[0046] 7. Contrast

[0047] In addition, the genomic DNA of the fish 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 F1, R1 and P1 cannot form an S-shaped curve within 40 cycles, and their 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 5.393 -- 0.5393 -- 0.05393 -- 0.005393 --

[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 Osteoglossus bicolor, 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 the fish, comprising a DNA molecule as shown in SEQ ID NO:

3.

3. A kit for identifying Osteoglossus bicolor, 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 the fish include: 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 Bone Tongue is identified based on the signal of the reaction.

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 53.9 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 fish 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 Osteoglossus bicolor based on 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 Osteoglossus bicinctus.

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