A molecular identification primer set, kit and method for zhangxie and acanthopagrus acanthopagrus

Abstract

This invention discloses a primer set, kit, and identification method for molecular identification of *Sinocyclocheilus spp.* and *Sinocyclocheilus spinosa*, belonging to the field of molecular identification technology for aquatic organisms. By comparing the mitochondrial genomes of *Sinocyclocheilus spinosa* and *Sinocyclocheilus spp.*, sequence differences of TGGC and GAAT were found at positions 12331-12334, respectively. Based on this, *Sinocyclocheilus spinosa*-specific primers JF / JR were designed, with an amplification product length of 385 bp. Simultaneously, internal control primers CF / CR were designed using the common sequence at positions 14354-14667 of the mitochondrial genomes of both species, with an amplification product length of 314 bp. During detection, DNA was extracted from the sample, and duplex PCR amplification was performed using the above two primer sets, followed by electrophoresis. The presence of only a 314 bp internal control band indicated *Sinocyclocheilus spp.*, while the presence of both a 314 bp internal control band and a 385 bp specific amplification band indicated *Sinocyclocheilus spinosa*. This invention forms intuitive judgment rules by combining specific amplification with common internal references, which can be used for species verification in aquaculture seedlings, germplasm resource surveys, and stock enhancement and release.

Description

Technical Field

[0001] This invention relates to the field of molecular identification and molecular marker detection technology for aquatic organisms, specifically to a molecular identification primer set, kit, and identification method for *Sinocyclocheilus spicata* and *Sinocyclocheilus spinosa*. Background Technology

[0002] Spiny-cheeked stag ( Zacco acanthogenys *Ctenophora* is a stream-dwelling fish belonging to the genus *Ctenophora*. Its type locality is Ningbo, and it is distributed in the middle and lower reaches of the Yangtze River, as well as in southern regions such as Zhejiang, Anhui, Jiangxi, Hunan, and Fujian. *Ctenophora tauren* (also known as the Tiaoxi Ctenophora) Zacco tiaoxiensis This is a new species of the genus *Cyprinus*, officially reported in 2023, with its type locality located in the Tiaoxi River Basin, Zhejiang Province. It was identified in the article "A New Species of Genus" published by Zhang Yan, Zhou Jiajun, and Yang Jinquan in the *Journal of Shanghai Ocean University*, Vol. 32, No. 3, 2023, pp. 544-552. Zacco The study from Southern China (Cypriniformes: Cyprinidae) used morphological comparisons combined with genetic analysis of the mitochondrial cytochrome b gene to support the identification of *Cypriniformes tiaoxiensis* as a separate species. This study also indicated that *Cypriniformes tiaoxiensis* and *Cypriniformes spinosa* are morphologically similar. These studies primarily address the description, classification, and phylogenetic issues of the new species, but do not provide specific PCR primers for identifying *Cypriniformes tiaoxiensis* / *Cypriniformes spinosa* in scenarios such as aquaculture seedlings, germplasm resource surveys, or verification of stock enhancement and release, nor do they provide a system for determining internal controls within the same tube.

[0003] In the field of molecular identification technology for fish, Chinese patent application CN103131766A discloses a molecular biological method for identifying spotted sea bass. This method designs universal primers for perciformes fish and further designs specific primers for spotted sea bass. The universal and specific primers are combined into a mixed primer for PCR amplification, and the results are then determined based on agarose gel electrophoresis bands: spotted sea bass samples show both universal and specific amplification bands, while other perciformes fish mainly show universal amplification bands. This technology demonstrates the feasibility of using "universal amplification signal + species-specific amplification signal" for rapid fish identification. However, this method targets spotted sea bass and perciformes fish, primarily targeting the transcriptional spacer region between the 5.8S rRNA and 28S rRNA genes, and relies on specific universal and specific primers and identification standard electrophoresis bands. It cannot directly provide stable and usable species-differentiating sites and specific primer combinations between *Sinocyclocheilus scutellatus* and *Sinocyclocheilus spinosatus*.

[0004] Chinese patent application CN116121354A discloses a method for rapid identification of the genetic sex of small sturgeon. This method uses female-specific primers and male-female shared primers in the same PCR detection system, determining genetic sex through a combination of a 172 bp specific amplification fragment and a 440 bp shared amplification fragment. This approach utilizes the shared amplification signal to assist in verifying the specific amplification results, reducing the risk of misjudgment when relying solely on specific bands. However, it addresses the problem of genetic sex identification within the same species. The specific markers, shared markers, and discrimination objects used are different from those used in identifying closely related fish species, and it does not address the molecular differences between *Sinocyclocheilus spp.* and *Sinocyclocheilus spinosa*.

[0005] In addition, Dong et al. published "Development of Species-Specific PCR Primers for the Rapid and Simultaneous Identification of the Six Species of Genus" in Developmental & Reproduction, Vol. 23, No. 4, 2019, pp. 367-375. Takifugu In this study, multiple species-specific primers were designed based on mitochondrial COI sequences, combined with control primers, to rapidly identify closely related fish species of the genus *Tetraodon* using multiplex PCR and amplification fragments of different sizes detected by agarose gel electrophoresis. This research indicates that screening for species differences in mitochondrial sequences and constructing multiplex PCR based on these differences is a feasible technical approach for molecular identification of fish. However, the stable differential sites for primer design, primer specificity, and matching relationships between multiple primers in the same tube for different species all require re-screening and validation for specific species and cannot be directly replaced by detection systems for other fish genera.

[0006] Therefore, existing technologies for fish DNA identification include DNA barcoding, specific PCR, multiplex PCR, and setting shared amplification signals for verification. However, for the closely related and historically confused species of the genus *Ctenopharynx tiaoxiensis* and *Ctenopharynx spinosaurus*, a dedicated molecular detection system targeting stable differential sites between the two species is still lacking. This system would allow for obtaining both a shared internal reference signal to confirm amplification validity and a specific signal to differentiate *Ctenopharynx spinosaurus* in a single routine PCR reaction, with direct interpretation based on different length electrophoretic bands. Especially in scenarios requiring batch testing, such as aquaculture seedlings, germplasm resource surveys, and verification of stock enhancement releases, further improvements in the specificity, intuitiveness of results, and process quality control are still necessary. Summary of the Invention

[0007] The purpose of this invention is to provide a molecular identification primer set, kit and identification method for *Sinocyclocheilus spicata* and *Sinocyclocheilus spinosa*, to solve the identification difficulty problem that exists when relying solely on morphological characteristics, and to reduce the risk of misjudgment due to DNA extraction or PCR amplification failure by setting a common internal reference for *Sinocyclocheilus spicata* and *Sinocyclocheilus spinosa*.

[0008] To achieve the above objectives, the specific solution of the present invention is as follows: A molecular identification primer set for distinguishing between *Sinocyclocheilus scutellatus* and *Sinocyclocheilus spinosatus* includes a *Sinocyclocheilus spinosatus*-specific primer pair and a common internal reference primer pair for both species. The *Sinocyclocheilus spinosatus*-specific primer pair includes a forward primer JF and a reverse primer JR. The nucleotide sequence of the forward primer JF is shown in SEQ ID NO: 1, and the nucleotide sequence of the reverse primer JR is shown in SEQ ID NO: 2. The shared internal reference primer pair includes a forward primer CF and a reverse primer CR. The nucleotide sequence of the forward primer CF is shown in SEQ ID NO: 3, and the nucleotide sequence of the reverse primer CR is shown in SEQ ID NO: 4.

[0009] SEQ ID NO: 1 CACTCGGACCCTAACATTGGC SEQ ID NO: 2 CAGAGGGTAGCCAAGGATGC SEQ ID NO: 3 CCAATGACTTGAAGAACCACCG SEQ ID NO: 4 GAATGATGCTCCGTTGGCGTG Preferably, the forward primer JF corresponds to positions 12313-12333 of the mitochondrial genome of *Spicata chinensis*, and the reverse primer JR corresponds to positions 12678-12697 of the mitochondrial genome of *Spicata chinensis*. The amplification product of the *Spicata chinensis*-specific primer pair is 385 bp in length, and its nucleotide sequence is shown in SEQ ID NO: 5.

[0010] SEQ ID NO: 5 CACTCGGACCCTAACATTGGCCGTTTCTTTAAATATCTACTCTTATTTCTGGTTGCTATGATTACCTTGGTTACAGCCAACAACATATTCCAGTTATTTATTGGCTGAGAAGGGTTGGCATTATGTCCTTCCTACTAATTGGATGATGATACGGCCGAGCGGACGCCAATACAGCAGCCCTTCAGGCTGTT ATTTATAACCGGGTTGGAGATATTGGATTAATTTTAAGCATGGCCTGATTTGCAATAAACCTTAACTCTTGAGAGATTCAGCAAATCTTCATTCTGTCGAAAGACTTTGACATAACAGTCCCCTAATAGGACTCATCCTCGCAGCCACTGGAAAATCAGCCCAATTTGGGCTGCATCCTTGGCTACCCTCTG Preferably, the forward primer CF corresponds to positions 14354-14375 of the mitochondrial genome of *Sinocyclocheilus scutellatus* and *Sinocyclocheilus spinosatus*, and the reverse primer CR corresponds to positions 14647-14667 of the mitochondrial genome of *Sinocyclocheilus scutellatus* and *Sinocyclocheilus spinosatus*. The amplification product of the common internal reference primer pair is 314 bp in length, and its nucleotide sequence is shown in SEQ ID NO: 6.

[0011] SEQ ID NO: 6 CCAATGACTTGAAGAACCACCGTTGTAGTTCAACTACAAGAACAATAATGGCAAGCCTGCGCAAAACCCATCCACTAATAAAAATCGCCAACGACGCACTAGTAGATCTCCCGACTCCCTCCAACATTTCCGTGTGATGAAACTTTGGATCCTTACT GGGACTCTGTCTAATTACTCAAATCCTCACCGGACTATTCCTAGCTATACACTACACCTCTGATATTTCCACTGCGTTTTCATCAGTAGTACATATTTGCCGAGACGTAAATTATGGATGACTTATTCGAAATCTGCACGCCAACGGAGCATCATTC The present invention also provides a detection kit for distinguishing between the tiaoxi tuna and the spiny-cheeked tuna, the detection kit comprising the above-mentioned molecular identification primer set.

[0012] Preferably, the test kit also includes a reaction buffer for PCR amplification, deoxyribonucleoside triphosphate, DNA polymerase, and water.

[0013] This invention also provides a molecular identification method for *Sinocyclocheilus scutellarioides* and *Sinocyclocheilus spinosa*, the molecular identification method comprising the following steps: S1. Extract DNA from the sample to be tested; S2. The DNA of the sample to be tested is amplified by double PCR using a molecular identification primer set, so that the specific fragment of the spiny-cheeked trevally and the internal reference fragment shared by the tiaoxi trevally and the spiny-cheeked trevally are amplified in the same detection system; S3. Perform electrophoresis detection on the PCR amplification products; when a 314bp internal reference band is detected and no 385bp *Scutellaria baicalensis*-specific amplification band is detected, the sample to be tested is determined to be *Scutellaria baicalensis*; when both a 314bp internal reference band and a 385bp *Scutellaria baicalensis*-specific amplification band are detected, the sample to be tested is determined to be *Scutellaria baicalensis*.

[0014] Preferably, in step S1, the sample to be tested is a fin sample from fish tissue.

[0015] Preferably, in step S2, the dual PCR amplification involves simultaneously adding the *Echinochloa crus-galli*-specific primer pair and the common internal reference primer pair to the same PCR reaction system.

[0016] Preferably, in step S3, the amplification product of the *Sinocyclocheilus spinosa*-specific primer pair is the 385 bp nucleotide sequence shown in SEQ ID NO: 5, and the amplification product of the common internal reference primer pair is the 314 bp nucleotide sequence shown in SEQ ID NO: 6.

[0017] This invention also provides applications of molecular identification primer sets and detection kits, specifically including the following (1), (2) and (3): (1) Species identification of Tiaoxi River Croaker and Spiny-cheeked Croaker; (2) Germplasm resource identification; (3) Verification of the germplasm for propagation and release.

[0018] Compared with methods that rely solely on morphological identification, the present invention has at least the following advantages: First, *Spodoptera litura*-specific amplification was achieved using differentially expressed mitochondrial genome sites between the two species, with clearly defined primer targets. Second, a shared internal reference fragment was introduced for simultaneous verification of the DNA template and amplification process within the same detection system. Third, intuitive judgment rules were established using amplification bands of two different lengths, 385 bp and 314 bp, eliminating the need for complex data analysis. Fourth, this method can be used for species verification in aquaculture seedlings, germplasm resource surveys, and pre-release stock enhancement, providing a molecular detection method for the resource management of *Spodoptera litura* and *Spodoptera litura*. Actual electrophoresis results further demonstrate that *Spodoptera litura* and *Spodoptera litura* samples can form stable and clearly differentiated band combinations, facilitating direct interpretation. Attached Figure Description

[0019] Figure 1 The image shows the agarose gel electrophoresis results obtained by using the dual PCR identification method of this invention to detect samples of *Sinocyclocheilus spp.* and *Sinocyclocheilus spinosa*. Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the following embodiments are used to illustrate the technical concept of the present invention and are not intended to limit the scope of protection of the present invention.

[0021] Example 1: Screening for Differential Loci Fin samples were collected from 12 individuals each of the spiny-cheeked trevally and the Tiaoxi trevally. Whole-genome resequencing was performed on these samples, and the mitochondrial genomes of both species were assembled using software such as mitofish. The 24 obtained mitochondrial genome sequences were then compared.

[0022] The comparison results showed that there were sequence differences between the spiny-cheeked wrasse and the Tiaoxi wrasse at positions 12331-12334 of the mitochondrial genome, with the spiny-cheeked wrasse's corresponding sequence being TGGC and the Tiaoxi wrasse's corresponding sequence being GAAT. This differential region can serve as a molecular target for constructing a spiny-cheeked wrasse-specific PCR identification system.

[0023] Example 2: Design of specific primers for the spiny scaly croaker Based on the differential sites obtained in Example 1, specific primer pairs for *Spiritobacter spinosum* were designed. The forward primer JF sequence is 5′-CACTCGGACCCTAACATTGGC-3′, corresponding to positions 12313-12333 in the *Spiritobacter spinosum* mitochondrial genome; the reverse primer JR sequence is 5′-CAGAGGGTAGCCAAGGATGC-3′, corresponding to positions 12678-12697 in the *Spiritobacter spinosum* mitochondrial genome.

[0024] When using *Spodoptera exigua* DNA as a template, the JF / JR primer pair can amplify a target fragment of 385 bp, the nucleotide sequence of which is shown in SEQ ID NO: 5. This primer pair generates a specific amplification signal for *Spodoptera exigua* based on the aforementioned mitochondrial differential region.

[0025] Example 3: Design of shared internal reference primers To simultaneously verify the DNA extraction and PCR amplification processes, the shared sequence from position 14354 to 14667 of the mitochondrial genome of *Sinoceras scoparia* and *Sinoceras taurensis* was selected as the internal reference region, and a shared internal reference primer pair was designed.

[0026] The forward primer (CF) for the internal control is 5′-CCAATGACTTGAAGAACCACCG-3′, corresponding to positions 14354-14375 in the mitochondrial genomes of both species; the reverse primer (CR) for the internal control is 5′-GAATGATGCTCCGTTGGCGTG-3′, corresponding to positions 14647-14667 in the mitochondrial genomes of both species. The CF / CR primer pair amplified the internal control fragment to a length of 314 bp, the nucleotide sequence of which is shown in SEQ ID NO: 6.

[0027] Example 4: Duplex PCR Molecular Identification DNA was extracted from the fish samples to be tested. The samples could be fin tissue or other tissue materials from which DNA could be obtained. The specific primer pair JF / JR for *Spicata chinensis* and the common internal control primer pair CF / CR were added to the same PCR reaction system to perform double PCR amplification of the DNA from the sample.

[0028] The reaction mixture consisted of 20 μL of DNA template (50 ng / μL), 1 μL of Premix Taq, 10 μL of primer JF (10 μM), 0.5 μL of primer JR (10 μM), 0.5 μL of primer CF (10 μM), 0.5 μL of primer CR (10 μM), and 7 μL of ultrapure water.

[0029] Amplification program settings: 94℃ for 3 min; 94℃ for 30 sec, 64℃ for 30 sec, 72℃ for 30 sec, 35 cycles; 72℃ for 10 min; 4℃ ∞.

[0030] After PCR amplification, the amplification products were analyzed by electrophoresis. Species identification was based on band positions: if the sample showed a 314 bp internal control amplification band but not a 385 bp *Scutellaria spicata*-specific amplification band, it was identified as *Scutellaria spicata*; if the sample showed both a 314 bp internal control amplification band and a 385 bp *Scutellaria spicata*-specific amplification band, it was identified as *Scutellaria spicata*. The actual double PCR electrophoresis results of the samples are shown below. Figure 1 As shown.

[0031] like Figure 1 As shown, samples numbered 1-24 were set up for testing for *Sinocyclocheilus scutellarioides* and *Sinocyclocheilus spinosa*, with samples numbered 1-12 in the top row and samples numbered 13-24 in the bottom row; the ladder-shaped band lanes between the two groups of samples are DNA molecular weight standards, used to refer to the size of the amplification products.

[0032] Depend on Figure 1 As can be seen, samples of *Sinocyclocheilus tauren* numbered 1-24 all showed a single main band corresponding to the 314 bp shared internal reference amplification fragment, and no specific band corresponding to the 385 bp *Sinocyclocheilus spinosa*-specific amplification fragment; samples of *Sinocyclocheilus spinosa* numbered 1-24 all showed two adjacent and distinguishable main amplification bands, the band patterns of which corresponded to the 314 bp shared internal reference amplification fragment and the 385 bp *Sinocyclocheilus spinosa*-specific amplification fragment, respectively. These detection results are consistent with the species determination rules established in this invention.

[0033] The above results demonstrate that, based on the normal amplification of samples from both species of wrasse by the shared internal reference primers CF / CR, the specific primers JF / JR can generate a recognizable 385 bp specific amplification signal in *Sinoceras scoparia* samples, while failing to produce a corresponding specific band in *Sinoceras taurensis* samples. This allows for direct molecular identification of the two species using the difference in single / double band patterns. The consistent banding patterns obtained from multiple samples also provide experimental support for the application of this identification method in batch sample detection.

[0034] The shared internal control band is used to characterize whether the DNA to be tested and the PCR amplification process meet the conditions for forming a detectable amplification product. Therefore, in actual testing, the presence of the 314 bp internal control band can be used to control the effectiveness of the detection.

[0035] Conclusion: The primer set, kit, and identification method of this invention can be used for species verification of fry or parents in the process of raising stream fish, as well as for field resource surveys, germplasm bank sample verification, and germplasm identification before stock enhancement and release. By extracting DNA from the samples, performing double PCR amplification, and electrophoresis, molecular differentiation between *Sinocyclocheilus spiculatus* and *Sinocyclocheilus spinosa* can be achieved without relying on individual external morphological differences.

Claims

1. A molecular identification primer set for distinguishing between *Sinocyclocheilus scutellarioides* and *Sinocyclocheilus spinosa*, characterized in that, Including specific primer pairs for the spiny-cheeked trevally and common internal reference primer pairs for both the tamarisk and spiny-cheeked trevally; The specific primer pair for the spiny stag beetle includes a forward primer JF and a reverse primer JR. The nucleotide sequence of the forward primer JF is shown in SEQ ID NO: 1, and the nucleotide sequence of the reverse primer JR is shown in SEQ ID NO:

2. The shared internal reference primer pair includes a forward primer CF and a reverse primer CR. The nucleotide sequence of the forward primer CF is shown in SEQ ID NO: 3, and the nucleotide sequence of the reverse primer CR is shown in SEQ ID NO:

4.

2. The molecular identification primer set according to claim 1, characterized in that, The forward primer JF corresponds to positions 12313-12333 of the mitochondrial genome of *Spiritobacter chinensis*, and the reverse primer JR corresponds to positions 12678-12697 of the mitochondrial genome of *Spiritobacter chinensis*. The amplification product of the *Spiritobacter chinensis*-specific primer pair is 385 bp in length, and its nucleotide sequence is shown in SEQ ID NO:

5.

3. The molecular identification primer set according to claim 1 or 2, characterized in that, The forward primer CF corresponds to positions 14354-14375 of the mitochondrial genome of *Sinocyclocheilus scutellatus* and *Sinocyclocheilus spinosatus*, and the reverse primer CR corresponds to positions 14647-14667 of the mitochondrial genome of *Sinocyclocheilus scutellatus* and *Sinocyclocheilus spinosatus*. The amplification product of the common internal reference primer pair is 314 bp in length, and its nucleotide sequence is shown in SEQ ID NO:

6.

4. A detection kit for distinguishing between the tamarisk and the spiny-cheeked tamarisk, characterized in that, Includes the molecular identification primer set as described in any one of claims 1-3.

5. The detection kit according to claim 4, characterized in that, It also includes one or more of the following: reaction buffer for PCR amplification, deoxyribonucleoside triphosphate, DNA polymerase, and water.

6. A molecular identification method for *Sinocyclocheilus scutellarioides* and *Sinocyclocheilus spinosa*, characterized in that, Includes the following steps: S1. Extract DNA from the sample to be tested; S2. The DNA of the sample to be tested is amplified by double PCR using the molecular identification primer set described in any one of claims 1-3, so that the specific fragment of *Sinocyclocheilus spinosa* and the internal reference fragment shared by *Sinocyclocheilus spicata* and *Sinocyclocheilus spinosa* are amplified in the same detection system. S3. Perform electrophoresis detection on the PCR amplification products; when a 314bp internal reference band is detected and no 385bp *Scutellaria baicalensis*-specific amplification band is detected, the sample to be tested is determined to be *Scutellaria baicalensis*; when both a 314bp internal reference band and a 385bp *Scutellaria baicalensis*-specific amplification band are detected, the sample to be tested is determined to be *Scutellaria baicalensis*.

7. The molecular identification method according to claim 6, characterized in that, In step S1, the sample to be tested is a fin sample from fish tissue.

8. The molecular identification method according to claim 6 or 7, characterized in that, In step S2, the dual PCR amplification simultaneously adds the *Echinochloa crus-galli*-specific primer pair and the common internal reference primer pair to the same PCR reaction system.

9. The molecular identification method according to any one of claims 6-8, characterized in that, In step S3, the amplification product of the *Spicata chinensis*-specific primer pair is the 385 bp nucleotide sequence shown in SEQ ID NO: 5, and the amplification product of the common internal reference primer pair is the 314 bp nucleotide sequence shown in SEQ ID NO:

6.

10. The application of the molecular identification primer set according to any one of claims 1-3 or the detection kit according to claim 4 or 5, comprising the following (1), (2) and (3): (1) Species identification of Tiaoxi River Croaker and Spiny-cheeked Croaker; (2) Germplasm resource identification; (3) Verification of the germplasm for propagation and release.

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