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KR-20260065088-A - A genetic analysis method for identification of shrimp species

KR20260065088AKR 20260065088 AKR20260065088 AKR 20260065088AKR-20260065088-A

Abstract

The present invention relates to a genetic analysis method for identifying shrimp species comprising: (a) a step of extracting DNA from shrimp; (b) amplifying and detecting the extracted DNA using a PCR-based method with a primer pair specific to one or more molecular markers selected from CoI, 16S rRNA and Cytb; and (c) a step of selecting the shrimp species through the amplified and detected molecular marker; a method for monitoring shrimp products comprising the genetic analysis method; and a kit for identifying shrimp species comprising a primer pair specific to the molecular marker extracted from shrimp.

Inventors

  • 강태선
  • 김건희
  • 이지영

Assignees

  • 서울여자대학교 산학협력단

Dates

Publication Date
20260508
Application Date
20241031

Claims (13)

  1. (a) A step of extracting DNA from shrimp; (b) amplifying and detecting in extracted DNA using a PCR-based method with primer pairs specific to any one or more molecular markers selected from CoI, 16S rRNA and Cytb; and (c) A genetic analysis method for determining shrimp species, comprising the step of selecting shrimp species through the amplified and detected molecular markers.
  2. A genetic analysis method for identifying shrimp species according to claim 1, wherein the shrimp species is one or more selected from the group consisting of Metapenaeopsis dalei , Palaemon graviera , Euphausia pacific , Leptochela gracilis , Penaeus vannamei, Pandalus borealis , Penaeus monodon , and Pleoticus muelleri .
  3. A genetic analysis method for determining shrimp species according to claim 1, wherein the shrimp of (a) is fresh shrimp or a processed product.
  4. A genetic analysis method for identifying shrimp species according to paragraph 3, wherein the processed product is washed with distilled water and preserved in 99% ethanol, with the addition of a pretreatment step.
  5. A genetic analysis method for determining shrimp species, wherein, in paragraph 3, the processed product is one or more selected from the group consisting of frozen, dried, roasted fillets, soup, or blanched shells.
  6. A gene analysis method for determining shrimp species according to claim 1, wherein the primer pair of (b) targets a short sequence within 16S rRNA, CoI, or cytochrome b genes.
  7. A genetic analysis method for determining shrimp species according to claim 1, wherein the primer pair of (b) is one or more selected from the group consisting of i) to viii) below: i) a primer pair represented by SEQ ID NO. 1 and SEQ ID NO. 2 specific to the CoI marker of * Penaeus monodon *; ii) a primer pair indicated by SEQ ID NO. 3 and SEQ ID NO. 4 specific to the CoI marker of the northern pink shrimp ( Pandalus borealis ); iii) a primer pair indicated by SEQ ID NO. 5 and SEQ ID NO. 6 specific to the CoI marker of * Pleoticus muelleri *; iv) a primer pair indicated by sequence number 7 and sequence number 8 specific to the CoI marker of * Palememon graviera *; v) A primer pair represented by SEQ ID NO. 9 and SEQ ID NO. 10 specific to the CytB marker of the whiteleg shrimp ( Penaeus vannamei ); vi) a primer pair represented by SEQ ID NO. 11 and SEQ ID NO. 12 specific to the 16S rRNA marker of Metapenaeopsis dalei ; vii) a primer pair represented by SEQ ID NO. 13 and SEQ ID NO. 14 specific to the 16S rRNA marker of Euphausia pacific ; and viii) A primer pair represented by SEQ ID NO. 15 and SEQ ID NO. 16, specific to the 16S rRNA marker of * Leptochela gracilis *.
  8. A gene analysis method for shrimp species identification according to claim 1, wherein the PCR-based method of (b) is cPCR, RT-PCR, or multiplexed ultra-fast RT-PCR.
  9. A gene analysis method for identifying shrimp species according to claim 8, characterized in that the method using multiplex ultrafast RT-PCR reduces the gene analysis time to within 30 minutes.
  10. A shrimp product monitoring method comprising the method of any one of claims 1 to 9.
  11. A kit for identifying shrimp species comprising primer pairs specific to any one or more molecular markers selected from CoI, 16S rRNA, and Cytb extracted from shrimp.
  12. A kit for identifying shrimp species according to claim 11, wherein the shrimp species is one or more selected from the group consisting of Metapenaeopsis dalei , Palaemon graviera , Euphausia pacific , Leptochela gracilis , Penaeus vannamei , Pandalus borealis , Penaeus monodon , and Pleoticus muelleri .
  13. A shrimp species identification kit according to claim 11, wherein the primer pair is one or more selected from the group consisting of i) to viii) below: i) a primer pair represented by SEQ ID NO. 1 and SEQ ID NO. 2 specific to the CoI marker of * Penaeus monodon *; ii) a primer pair indicated by SEQ ID NO. 3 and SEQ ID NO. 4 specific to the CoI marker of the northern pink shrimp ( Pandalus borealis ); iii) a primer pair indicated by SEQ ID NO. 5 and SEQ ID NO. 6 specific to the CoI marker of * Pleoticus muelleri *; iv) a primer pair indicated by sequence number 7 and sequence number 8 specific to the CoI marker of * Palememon graviera *; v) A primer pair represented by SEQ ID NO. 9 and SEQ ID NO. 10 specific to the CytB marker of the whiteleg shrimp ( Penaeus vannamei ); vi) a primer pair represented by SEQ ID NO. 11 and SEQ ID NO. 12 specific to the 16S rRNA marker of Metapenaeopsis dalei ; vii) a primer pair represented by SEQ ID NO. 13 and SEQ ID NO. 14 specific to the 16S rRNA marker of Euphausia pacific ; and viii) A primer pair represented by SEQ ID NO. 15 and SEQ ID NO. 16, specific to the 16S rRNA marker of * Leptochela gracilis *.

Description

A genetic analysis method for identification of shrimp species The present invention relates to a genetic analysis method for identifying shrimp species, comprising: (a) a step of extracting DNA from shrimp; (b) amplifying and detecting one or more molecular markers selected from CoI, 16S rRNA and Cytb in the extracted DNA using a PCR-based method with a primer pair specific to them; and (c) a step of selecting shrimp species through the amplified and detected molecular markers; a method for monitoring shrimp products comprising the genetic analysis method; and a kit for identifying shrimp species comprising molecular markers extracted from shrimp. Food fraud refers to the act of intentionally deceiving consumers about food by falsely representing the product itself or related documents for economic gain (Lawrence, Elliott, Huisman, Dean, & van Ruth, 2022). While food fraud takes various forms, the most frequent are species mixing and species substitution, and reports of food fraud are on the rise globally. This increase is attributed to the growing proportion of processed products in the global food trade, where low-value species are often misclassified as high-value species for financial gain. Although it is extremely difficult to identify species substitution, particularly in highly processed products, due to similarities in morphological characteristics, taste, and texture, food fraud causes various social problems, including health, allergy, ethical issues, economic deception, and a decline in consumer trust. Meanwhile, seafood is highly popular due to its availability and price advantages, as well as its health benefits, which include providing high levels of protein, omega-3 fatty acids, and essential minerals compared to meat products. Global seafood consumption reached 20.2 kg per person in 2020 and is estimated to reach 21.4 kg per person by 2030 (FAO, 2022). Despite this tremendous popularity and consumption, seafood still carries a high risk of food fraud. DNA barcoding has emerged as a leading technology for identifying the species of such animal-derived products (Bartlett & Davidson, 1991; Hebert, Cywinska, Ball, & DeWaard, 2003), but it has inherent limitations such as requiring DNA sequencing and BLAST analysis, and being unable to identify or quantify foods containing multiple species. Against this backdrop, the inventors have made diligent efforts to develop a genetic analysis method for rapidly and efficiently identifying various shrimp species, and as a result, have completed a method that can efficiently identify shrimp species using various PCR-based methods with species-specific primers. Figure 1 shows the phylogenetic tree of reference specimens (R1-R8), commercial shrimp products (S1-S40), and 19 additional CoI sequences downloaded from the NCBI GenBank database, constructed using the neighbor-joining method. Figures 2 B to D show a set of 16S rRNA gene-based species-specific primers for identifying shrimp species Metapenaeopsis dalei, Eup. pacifica, and Lep. Gracilis. Figure 3A is a schematic diagram of a specially designed polymer chip for multiple ultrafast RT-PCR analysis, Figure 3B shows an amplification curve generated by the multiple ultrafast RT-PCR method, and Figure 3C shows a melting peak generated by the multiple ultrafast RT-PCR method. Figure 4 shows the results of PACs to verify the integrity of DNA extracted from 18 reference samples and 40 commercial products. Figure 5 shows the results of the sensitivity of cPCR analysis using species-specific primers for 8 shrimp species. Figure 6 shows the standard curve and melting peak of RT-PCR analysis using species-specific primers for 8 shrimp species. Figure 7 shows the results of verifying the SS primer specificity of 8 dominant shrimp species under optimal RT-PCR conditions. Figure 8 shows the arrangement of 16S rRNA gene sequences for six krill species. The present application will be explained in more detail below through examples. However, the following examples are merely preferred embodiments for illustrating the present application and are therefore not intended to limit the scope of the rights of the present application. Meanwhile, technical matters not described in this specification can be fully understood and easily implemented by a person skilled in the art who is proficient in the technical field of the present application or a similar technical field. Experimental Example 1: Sample Collection From January to June 2022, 40 commercially available shrimp products were purchased from local seafood markets and the internet. Muscle tissues of eight reference shrimp species identified morphologically and molecularly as Palaemongravieri , Alpheus japonicus , Trachypenaeus curvirostris , Leptochela gracilis , Metapenaeopsis dalei , Penaeus monodon , Penaeus vannamei , and Metanephrops thomsoni were obtained from the National Institute of Fisheries Science (Busan, Korea) and the National Marine Biological Resources In