High-sensitivity large yellow croaker germplasm identification primer combination and application and identification method thereof

Abstract

The invention belongs to the technical field of molecular identification of aquatic organisms, and particularly relates to a high-sensitivity large yellow croaker germplasm identification primer combination as well as application and an identification method thereof. The primer composition is a first pair of primers LC-1, and the sequences of the first pair of primers LC-1 are as follows: an upstream primer LC-1F: 5 '-AATTCCACACGACTAATC-3'; the downstream primer LC-1R is 5 '-CTCAGCCGCCGTGCAATC-3', and the downstream primer LC-1R is 5 '- The sequences of the second pair of primers LC-2 are as follows: an upstream primer LC-2F: 5 '-AGGCATTAGGCACAAGG-3', a downstream primer LC-2F: 5 '-AGGCATTAGGCACAAGG-3', a downstream And the downstream primer LC-2R is 5 '-AAACCCCTAGCCTCGAAG-3'. The invention also discloses a kit for detecting the disease resistance of the traditional Chinese medicine. The two large yellow croaker germplasm identification primers provided by the invention have relatively high sensitivity and specificity, can be stably amplified in a DNA sample with extremely low concentration, and overcome the limitation that accurate identification cannot be realized due to low DNA concentration in a traditional method.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular identification of aquatic organisms, and particularly relates to a high-sensitivity large yellow croaker germplasm identification primer combination and an application and identification method thereof. Background Art

[0002] Large yellow croaker Larimichthys crocea ) is a fish of the genus Yellow Croaker in the family Sciaenidae in the order Perciformes. It is one of the important economic fish species along the coast of China. Its high-quality meat and rich nutritional value make it occupy an important position in aquaculture. However, due to factors such as overfishing, habitat destruction and global climate change, the population of wild Yellow Croaker has declined rapidly and has been listed as a critically endangered (CR) species by the IUCN. In addition, with long-term overfishing and inbreeding depression in the breeding process, the germplasm resources of wild and farmed Yellow Croaker have gradually degraded and are facing the risk of loss of genetic diversity. Therefore, accurate and efficient identification and investigation of the germplasm information of Yellow Croaker is of great significance for its protection, breeding and sustainable utilization.

[0003] At present, the germplasm identification method of large yellow croaker mainly relies on morphological characteristics, but due to the large differences between individuals of the same species at different ages, morphological identification has certain limitations. In addition, large yellow croaker and small yellow croaker belong to the same genus and are closely related species. The two species are extremely similar in morphology, and it is difficult to distinguish the two species morphologically. Therefore, the development of highly sensitive genetic markers and efficient molecular detection methods for identifying large yellow croaker has important scientific significance and practical application value. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a high-sensitivity large yellow croaker germplasm identification primer combination and its application and identification method.

[0005] To achieve the above object, the technical solution adopted by the present invention is a highly sensitive primer combination for identifying large yellow croaker germplasm, the primer combination is The sequences of the first pair of primers LC-1 are: Upstream primer LC-1F: 5′-AATTCCACACGACTAATC-3′; Downstream primer LC-1R: 5′-CTCAGCCGCCGTGCAATC-3′; The sequences of the second pair of primers LC-2 are: Upstream primer LC-2F: 5′-AGGCATTTAGGCACAAGG-3′; Downstream primer LC-2R: 5′-AAACCCCTAGCCTCGAAG-3′.

[0006] The primers are obtained based on the D-loop conserved sequence encoding the mitochondrial genome of large yellow croaker. Since the genome similarity between the two closely related species of small yellow croaker and large yellow croaker is very high, it is difficult to find bilateral segments on both sides of the amplified fragment that are both highly species-specific and meet the primer sequence requirements. Considering the principle that the PCR reaction requires the upstream and downstream primers to be effective at the same time, two reverse primer segments with four or more mutations at the 3' end between small yellow croaker and large yellow croaker are selected to design unidirectional large yellow croaker-specific primers, such as Figure 1 Then, based on the two reverse primers and according to other primer conditions such as annealing temperature, two pairs of large yellow croaker mitochondrial genome D-loop region specific amplification primers were designed using Primer Premier 5 software.

[0007] An application of the highly sensitive large yellow croaker germplasm identification primer combination, and an application of the primer combination in large yellow croaker germplasm identification.

[0008] A method for rapidly identifying large yellow croaker using the primer combination, (1) Extract the sample to be tested using the phenol-chloroform method; (2) using the 10,000-fold diluted DNA as a template, and using the large yellow croaker specific primers LC-1 and LC-2 for PCR amplification; (3) Identify the product through PCR amplification.

[0009] The amplified product obtains a clear specific identification band at 105bp (LC-1) and 153bp (LC-2), respectively, which means that the sample contains large yellow croaker; if multiple non-specific bands appear in the amplified product, the sample is small yellow croaker.

[0010] The PCR amplification reaction system in step (2) is as follows: a total volume of 25 μL, including 12.5 μL PCR Mix, 1 μL genomic DNA template (diluted 10,000 times), 0.5 μL each of upstream and downstream primers with a concentration of 10 μM / μL, and the volume is made up with sterile double distilled water.

[0011] The PCR amplification reaction conditions in step (2) are: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 50 s, 35 cycles, and finally extension at 72°C for 5 min.

[0012] The amplified product in step (3) is recorded with a gel imager to record the gel electrophoresis image, thereby identifying the sample, wherein the gel electrophoresis is 1.5% agarose gel electrophoresis; the electrophoresis buffer is 1×TAE, the voltage does not exceed 5V / cm, the electrophoresis time is 30-40min, and 2μL of the PCR amplified product is detected by agarose gel electrophoresis.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The two large yellow croaker germplasm identification primers provided by the present invention have high sensitivity and specificity, can achieve stable amplification in extremely low concentration DNA samples, and effectively solve the problem of inability to accurately identify due to low DNA concentration in the prior art. At the same time, the primers designed by the present invention amplify smaller fragments (105bp and 153bp), which overcomes the problem that large fragments cannot be amplified due to high degradation of genomic DNA caused by improper sample preservation in traditional methods, and greatly improves the applicability of the method. The method of the present invention is simple to operate and has a clear process. It can complete the accurate identification of large yellow croaker germplasm in a short time, significantly improves the experimental efficiency, and is suitable for rapid screening of large-scale samples. In addition, when the genomic DNA of large yellow croaker and small yellow croaker is mixed as a template, the primers designed by the present invention can amplify clear large yellow croaker-specific bands at the target position, avoiding the problem of multiple non-specific amplification bands appearing in the small yellow croaker DNA template, thereby ensuring the reliability of the identification results. In view of the actual needs of large yellow croaker germplasm identification, the present invention makes up for the shortcomings of traditional methods in sensitivity, specificity and ease of operation, and has important practical value and commercial promotion potential. More importantly, the method of the present invention is not only applicable to traditional germplasm identification in the laboratory, but can also be applied to the monitoring of the distribution dynamics and biomass of large yellow croaker populations in natural waters by combining the high efficiency and sensitivity of primer amplification with environmental DNA (eDNA) technology. This innovative expansion provides researchers with a reliable tool for real-time monitoring of large yellow croaker populations and resource assessment in complex environments, further enhancing the application value and prospects of the technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Sequence comparison of the species-specific reverse primer design segment of the D-Loop region of the mitochondrial genome of large yellow croaker and its closely related species, small yellow croaker.

[0015] Figure 2 Electrophoresis results of LC-1 primer PCR amplification products using large yellow croaker genomic DNA, equal amounts of large and small yellow croaker genomic DNA mixed, and small yellow croaker genomic DNA as templates.

[0016] Figure 3 Electrophoresis results of LC-2 primer PCR amplification products using large yellow croaker genomic DNA, equal amounts of large and small yellow croaker genomic DNA mixed, and small yellow croaker genomic DNA as templates. DETAILED DESCRIPTION

[0017] The present invention is further described below in conjunction with the accompanying drawings and specific application examples. It should be understood that these application examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0018] The identification method of the present invention comprises: (1) extracting genomic DNA of large yellow croaker and small yellow croaker samples; (2) using total DNA diluted to ten thousand times as a template and using two designed specific primers (LC-1 and LC-2) to perform PCR amplification respectively; (3) analyzing the two PCR amplification products by gel electrophoresis and recording the comparison results: when the large yellow croaker and small yellow croaker genomic DNAs are mixed in equal amounts as templates or the large yellow croaker genomic DNA is used as a template for amplification, clear large yellow croaker specific identification bands are obtained at 105 bp (LC-1) and 153 bp (LC-2), respectively, while when the small yellow croaker genomic DNA is used as a template for amplification, multiple non-specific amplification bands are obtained.

[0019] The two large yellow croaker germplasm identification primers provided by the present invention have high sensitivity and specificity, can stably amplify in extremely low concentration DNA samples, and overcome the limitation of the traditional method that cannot accurately identify due to low DNA concentration; at the same time, the two primers provided by the present invention amplify small fragments, which can overcome the problem of large fragment amplification failure caused by high degradation of genomic DNA due to sample preservation. The method of the present invention is simple to operate and has a clear process. It can complete the accurate identification of large yellow croaker germplasm in a relatively short time, greatly improve the experimental efficiency, is suitable for rapid screening of large-scale samples, and has good application prospects.

[0020] Example 1: The mitochondrial genome sequences of small yellow croaker and large yellow croaker were downloaded from the NCBI official website, and the D-loop region was selected for genome sequence comparison using BioEdit software. Due to the high genome similarity between closely related species, it is difficult to find bilateral segments that are highly specific and suitable as primers. Therefore, two reverse primer segments with four or more mutations at the 3' end were selected as unidirectional specific primers, such as Figure 1 Using Primer Premier 5 software, two pairs of primers were synthesized based on the two reverse primers according to other primer conditions such as annealing temperature, namely The sequences of the first pair of primers LC-1 are: Upstream primer LC-1F: 5′-AATTCCACACGACTAATC-3′ (SEQ NO. 1); Downstream primer LC-1R: 5′-CTCAGCCGCCGTGCAATC-3′ (SEQ NO. 2); The sequences of the second pair of primers LC-2 are: Upstream primer LC-2F: 5′-AGGCATTTAGGCACAAGG-3′ (SEQ NO. 3); Downstream primer LC-2R: 5'-AAACCCCTAGCCTCGAAG-3' (SEQ NO. 4).

[0021] Example 2: (1) Genomic DNA was extracted from muscle tissue samples of 3 large yellow croakers and 2 small yellow croakers, with a tissue amount of 0.5 g. Then, the DNA of large yellow croaker, small yellow croaker, and a mixture of large yellow croaker and small yellow croaker was obtained as a template (wherein the mixture was a mixture of DNA of equal mass of large yellow croaker and small yellow croaker).

[0022] (2) PCR amplification using specific primers LC-1 and LC-2: The PCR amplification system is as follows: total volume 25 μL, including 12.5 μL PCR Mix, 1 μL DNA template diluted 10,000 times, concentration 10 μM / μL, 0.5 μL each of upstream and downstream primers, and the volume is made up with sterile double distilled water.

[0023] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 50 s, 35 cycles, and a final extension at 72°C for 5 min.

[0024] (3) Take 2 μL of PCR amplification product and perform agarose gel electrophoresis. The gel electrophoresis is 1.5% agarose gel electrophoresis; the electrophoresis buffer is 1×TAE, the voltage does not exceed 5 V / cm, and the electrophoresis time is 30-40 min. The electrophoresis results are displayed under a UV gel imager (see Figure 3 ).

[0025] like Figure 2 and Figure 3 , M is DL2000 DNA Marker, A1-A3 are the bands amplified by LC-1 when the DNA of large and small yellow croakers was diluted to 1 / 10,000 with sterile deionized water after equal amounts of DNA were mixed, A4 and A5 are the bands amplified by LC-1 when the DNA concentration of large yellow croakers was 1 / 10,000; B1-B3 are the bands amplified by LC-2 when the DNA of large and small yellow croakers was diluted to 1 / 10,000 after equal amounts of DNA were mixed, B4 and B5 are the bands amplified by LC-2 when the DNA concentration of large yellow croakers was 1 / 10,000. It can be seen that under these two pairs of primers, large yellow croakers can be specifically amplified in the presence of small yellow croaker DNA, and there is a clear specific band at 105bp and 153bp respectively. And as Figure 3As shown, C1-C4 are the bands amplified by LC-1 in small yellow croaker DNA, and D1-D4 are the bands amplified by LC-2 in small yellow croaker DNA. It can be seen that the bands are messy, not single, and are not the target segment size.

[0026] In summary, the two pairs of specific primers LC-1 and LC-2 and the method described in the present invention can stably amplify a specific band of 105 bp and 153 bp respectively under the condition of low DNA concentration in large yellow croaker, while multiple non-specific amplification bands appear in small yellow croaker. Therefore, the method for identifying large yellow croaker germplasm resources of the present invention is suitable for large-scale identification of large yellow croaker and environmental DNA monitoring. This technology has broad application prospects, can effectively support the protection, breeding and sustainable utilization of large yellow croaker, and provide important molecular tools for aquatic resource management and species protection.

Claims

1. A highly sensitive primer combination for identifying germplasm of large yellow croaker, characterized in that: The primer composition is The sequences of the first pair of primers LC-1 are: Upstream primer LC-1F: 5′-AATTCCACACGACTAATC-3′; Downstream primer LC-1R: 5′-CTCAGCCGCCGTGCAATC-3′; The sequences of the second pair of primers LC-2 are: Upstream primer LC-2F: 5′-AGGCATTTAGGCACAAGG-3′; Downstream primer LC-2R: 5′-AAACCCCTAGCCTCGAAG-3′.

2. An application of the highly sensitive large yellow croaker germplasm identification primer combination according to claim 1, characterized in that: The primer combination is used in the identification of large yellow croaker germplasm.

3. A method for rapidly identifying large yellow croaker using the primer combination of claim 1, characterized in that: (1) Extract the sample to be tested using the phenol-chloroform method; (2) using the DNA diluted 10,000 times as a template and using the primer combinations LC-1 and LC-2 described in claim 1 to perform PCR amplification; (3) Identify the product through PCR amplification.

4. The method for identifying large yellow croaker according to claim 3, wherein: The amplified product obtains a clear specific identification band at 105 bp and 153 bp respectively, that is, the sample contains large yellow croaker; If multiple non-specific bands appear in the amplified product, the sample is small yellow croaker.

5. The method for identifying large yellow croaker according to claim 3, characterized in that: The PCR amplification reaction system in (2) is as follows: the total volume is 25 μL, including 12.5 μL PCR Mix, 1 μL genomic DNA template, 0.5 μL each of upstream and downstream primers with a concentration of 10 μM / μL, and the volume is made up with sterile double distilled water.

6. The method for identifying large yellow croaker according to claim 3, characterized in that: The PCR amplification reaction conditions in (2) were: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 50 s, 35 cycles, and a final extension at 72°C for 5 min.

7. The method for identifying large yellow croaker according to claim 3, characterized in that: (3) The amplified product is recorded by gel electrophoresis image using a gel imager to identify the sample, wherein the gel electrophoresis is 1.5% agarose gel electrophoresis; The electrophoresis buffer was 1×TAE, the voltage did not exceed 5 V / cm, the electrophoresis time was 30-40 min, and 2 μL of the PCR amplification product was detected by agarose gel electrophoresis.

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