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CN-122012730-A - Multiplex PCR primer combination for identifying pangolin species and application thereof

CN122012730ACN 122012730 ACN122012730 ACN 122012730ACN-122012730-A

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a multiplex PCR primer combination for identifying pangolin species and application thereof. The invention designs three pairs of specific primers aiming at species-specific conserved regions in three pangolin mitochondrial genes, and the specific primers respectively target COI genes of Chinese pangolin, malaytea scurfpea and south African pangolin. By utilizing the primer group, synchronous identification of three pangolin species can be realized in the same PCR reaction system, and visual interpretation can be carried out according to the size of amplified fragments through agarose gel electrophoresis. The detection method has the advantages of rapidness, accuracy, strong specificity, simple and convenient operation and the like, is suitable for species identification of squama Manis, tissues, blood and products thereof, and provides a high-efficiency and reliable technical means for illegal trade tracing, species protection monitoring and identification of the squama Manis.

Inventors

  • SHEN YONGYI
  • XIANG CHENGWEI
  • HUA YAN
  • Zou Zanjian
  • CHEN FANGTING
  • WANG ZHIGUANG
  • HOU FANGHUI
  • SHEN XUEJUAN
  • CHEN WU
  • CHEN XIAOYUAN
  • Huang wanhe

Assignees

  • 华南农业大学

Dates

Publication Date
20260512
Application Date
20260213

Claims (8)

  1. 1. The multiplex PCR detection primer combination for synchronously identifying the pangolin scales, the malaytea scurfpea scales and the south Africa pangolin scales is characterized by comprising the following three pairs of specific primers, wherein the three pairs of primers can realize balanced amplification in the same PCR reaction system and generate products with different lengths which can be distinguished by electrophoresis: The first primer pair for identifying pangolin scales has nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2 respectively; The second primer pair for identifying the pangolin scales has nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4 respectively; the nucleotide sequences of the third primer pair for identifying the pangolins in south Africa are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6.
  2. 2. A multiplex PCR assay kit for identifying species of pangolins, comprising the primer combination of claim 1.
  3. 3. The kit of claim 2, further comprising DNA extraction reagents and PCR amplification reagents.
  4. 4. A method of identifying a species of pangolin, the method comprising the steps of: s1, extracting total DNA from a sample; s2, carrying out multiplex PCR amplification reaction on the sample by using the DNA extracted in the step S1 as a template and using the primer group of claim 1 to obtain an amplification product; And S3, performing agarose gel electrophoresis analysis on the PCR amplification product in the step S2, and observing the result under a gel imaging system to determine the species type.
  5. 5. The method according to claim 4, wherein the PCR amplification reaction system of step S2 comprises 2X SuperTaq PCR StarMix 12.5.5 uL,10uM ZH-COI-F/ZH-COI-R, 10. Mu.M ML-COI-F/ML-COI-R each 1.5. Mu.L, NF-COI-F/NF-COI-R template DNA 2. Mu.L, and deionized water to 25. Mu.L.
  6. 6. The method according to claim 4, wherein the reaction conditions of the multiplex PCR amplification reaction of step S2 are 95℃for 3min, 95℃for 15S,60℃for 15S,72℃for 15S, 25 cycles, and finally 72℃for 5min.
  7. 7. The method according to claim 4, wherein the determining the species type in step S3 is: when there is no amplification product, then there is no such species in the sample; when the amplified product is a fragment with the size of 372bp, the sample is Chinese pangolin; When the amplified product is a fragment with the size of 594bp, the sample is pangolin scales of south Africa; When the amplified product is two fragments with the sizes of 594bp and 1169bp, the sample is the pangolin scales.
  8. 8. Use of a primer combination according to claim 1 for the preparation of a reagent or kit for simultaneous identification of chinese pangolin scales, maleic pangolin scales and south african pangolin scales.

Description

Multiplex PCR primer combination for identifying pangolin species and application thereof Technical Field The invention belongs to the technical field of molecular biology, and particularly relates to a multiplex PCR primer combination for identifying pangolin species and application thereof. Background Pangolin scales are one of the most serious mammals for global illegal trade, and all species are listed in CITES appendix I, prohibiting international commercial trade. Chinese squama Manis, malaysia squama Manis and south Africa squama Manis are morphologically indistinguishable, especially in scales, tissues, blood or processed products, and conventional morphological identification methods have large limitations. At present, species identification is based on DNA bar code technology, such as sequencing mitochondrial gene fragments of COI, cyt b and the like, and the method has high accuracy, long time consumption and high cost, and is not suitable for large-scale screening or on-site rapid detection. The multiplex PCR technology can synchronously amplify a plurality of specific targets in a reaction system, realizes parallel detection through the difference of product length or marks, has the outstanding advantages of high flux, high speed, low cost, small sample demand and the like, and is particularly suitable for the fields of species identification, pathogen typing, genetic screening and the like. Therefore, there is a need for a multiplex PCR detection primer capable of rapidly and accurately distinguishing between Chinese, malaysia and south Africa pangolins. Disclosure of Invention The invention aims to provide a group of multiplex PCR detection primer combinations for synchronously identifying pangolin scales, malaytea scurfpea pangolin scales and south African pangolin scales, and the primer groups can be utilized to realize synchronous identification of three pangolin scales in the same PCR reaction system and carry out visual interpretation according to the size of amplified fragments through agarose gel electrophoresis. In order to achieve the above object, the present invention provides the following technical solutions: The invention provides a group of multiplex PCR detection primer combinations for synchronously identifying pangolin scales, malaytea scurfpea scales and pangolin scales in south Africa, which consists of the following three pairs of specific primers, wherein the three pairs of primers can realize balanced amplification in the same PCR reaction system and generate products with different lengths which can be distinguished by electrophoresis: The first primer pair for identifying pangolin scales has nucleotide sequences shown as SEQ ID NO.1 and SEQ ID NO.2 respectively; The second primer pair for identifying the pangolin scales has nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4 respectively; the nucleotide sequences of the third primer pair for identifying the pangolins in south Africa are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6. The invention also provides a multiplex PCR detection kit for identifying pangolin species, which comprises the primer combination. Preferably, the kit further comprises DNA extraction reagents and PCR amplification reagents. The invention also provides a method for identifying pangolin scales, which comprises the following steps: s1, extracting total DNA from a sample; s2, carrying out multiplex PCR amplification reaction on the sample by using the DNA extracted in the step S1 as a template and using the primer group of claim 1 to obtain an amplification product; And S3, performing agarose gel electrophoresis analysis on the PCR amplification product in the step S2, and observing the result under a gel imaging system to determine the species type. Preferably, the reaction system for PCR amplification in step S2 comprises 2X SuperTaq PCR StarMix 12.5.5 uL,10uM ZH-COI-F/ZH-COI-R, 10. Mu.M ML-COI-F/ML-COI-R each 1.5. Mu.L, and NF-COI-F/NF-COI-R template DNA 2. Mu.L, and deionized water to 25. Mu.L. Preferably, the reaction conditions of the multiplex PCR amplification reaction described in step S2 are 95℃for 3min, 95℃for 15S,60℃for 15S,72℃for 15S, 25 cycles, and finally 72℃for 5min. Preferably, the determining method for determining the species type in step S3 is as follows: when there is no amplification product, then there is no such species in the sample; when the amplified product is a fragment with the size of 372bp, the sample is Chinese pangolin; When the amplified product is a fragment with the size of 594bp, the sample is pangolin scales of south Africa; When the amplified product is two fragments with the sizes of 594bp and 1169bp, the sample is the pangolin scales. The invention also provides application of the primer combination in preparation of a reagent or a kit for synchronously identifying Chinese pangolin scales, maleic pangolin scales and south African pangolin scales. Compared with the prior art, the invention