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CN-116287360-B - Primer composition for identification or auxiliary identification of wild jujube seed and false product thereof and multiplex PCR detection method

CN116287360BCN 116287360 BCN116287360 BCN 116287360BCN-116287360-B

Abstract

The invention discloses a primer composition and a kit for identifying or assisting in identifying wild jujube seeds and counterfeit products thereof and a multiplex PCR detection method. The primer composition includes primers SF1, SR4, HAF1, HAR6, ZMF5 and ZMR5. The primer composition can specifically amplify DNA fragments of the wild jujube seed, the hovenia dulcis thunb and the spine date seed.

Inventors

  • PEI XIANGPING
  • LI KEFAN
  • Suo Xiaoxiong
  • DU CHENHUI
  • LI XIAOLAN
  • DING WEN

Assignees

  • 山西中医药大学

Dates

Publication Date
20260508
Application Date
20220309

Claims (8)

  1. 1. A primer composition for identifying or assisting in identifying wild jujube seed and its counterfeit products is characterized by comprising primers SF1, SR4, HAF1, HAR6, ZMF5 and ZMR5, The primer SF1 is a single-stranded DNA molecule shown in a sequence 1; the primer SR4 is a single-stranded DNA molecule shown in a sequence 2; the primer HAF1 is a single-stranded DNA molecule shown in a sequence 3; the primer HAR6 is a single-stranded DNA molecule shown in a sequence 4; The primer ZMF5 is a single-stranded DNA molecule shown in a sequence 5; The primer ZMR5 is a single-stranded DNA molecule shown in a sequence 6.
  2. 2. The primer composition of claim 1, wherein the primer SF1, the primer SR4, the primer ZMF5, the primer ZMR5, the primer HAF1 and the primer HAR6 are in a molar ratio of 4:4:4:4:5:5.
  3. 3. A reagent for identifying or assisting in identifying wild jujube seed and its counterfeit products, which is characterized by comprising the primer composition of claim 1.
  4. 4. A kit for identifying or assisting in identifying wild jujube seeds and counterfeit products thereof is characterized by comprising the primer composition of claim 1.
  5. 5. The kit of claim 4, further comprising a polymerase.
  6. 6. A multiplex PCR detection method for identifying or assisting in identifying wild jujube and false products thereof is characterized by comprising the steps of carrying out multiplex PCR on genomic DNA of a sample to be detected by adopting the primers SF1, SR4, HAF1, HAR6, ZMF5 and ZMR5 according to the claim 1, and identifying or assisting in identifying the authenticity of the wild jujube according to the result of the multiplex PCR; identifying whether the wild jujube seed is true or false as an amplification product of the multiplex PCR contains a DNA fragment A according to the multiplex PCR result, if so, the sample to be detected contains or is candidate to contain the wild jujube seed, if not, the sample to be detected does not contain or is candidate to contain the wild jujube seed, if so, the amplification product of the multiplex PCR contains or is candidate to contain the DNA fragment B, if so, the sample to be detected contains or is not candidate to contain the semen Hoveniae, if not, the sample to be detected does not contain or is candidate to contain the semen Hoveniae, if so, the amplification product of the multiplex PCR contains a DNA fragment C, if so, the sample to be detected contains or is candidate to contain the semen Ziziphi Spinosae, and if not, the sample to be detected does not contain or is candidate to not contain the semen Ziziphi Spinosae; the size of the DNA fragment A is 549 bp, the size of the DNA fragment B is 169 bp, and the size of the DNA fragment C is 389 bp.
  7. 7. The multiplex PCR detection method as defined in claim 6 wherein: The annealing temperature of the multiplex PCR is 62-65 ℃; the cycle number of the multiplex PCR is 23-24; The genome DNA consumption of the sample to be detected is 1.2-30ng; the molar ratio of the primer SF1, the primer SR4, the primer ZMF5, the primer ZMR5, the primer HAF1 and the primer HAR6 is 4:4:4:5:5; the concentrations of the primers SF1, SR4, ZMF5 and ZMR5 in the multiplex PCR system are all 0.08 mu mol.L -1 , and the concentrations of the primers HAF1 and HAR6 in the multiplex PCR system are all 0.1 mu mol.L -1 .
  8. 8. Use of the primer composition of claim 1 or 2, the reagent of claim 3 or the kit of claim 4 or 5 in any of the following: (1) Identifying or assisting in identifying the wild jujube seed; (2) Identifying or assisting in identifying semen Ziziphi Spinosae, semen Hoveniae and semen Ziziphi Spinosae; (3) Identifying or assisting in identifying whether the sample to be detected is semen Ziziphi Spinosae or semen Hoveniae or semen Ziziphi Spinosae; (4) Identifying or assisting in identifying whether the sample to be tested contains the wild jujube seeds, the hovenia dulcis thunb or the wild jujube seeds; (5) Preparing a kit for identifying or assisting in identifying the wild jujube seed; (6) Preparing a kit for identifying or assisting in identifying the wild jujube seeds, the hovenia dulcis thunb and the spine date seeds; (7) Preparing a kit for identifying or assisting in identifying whether the sample to be tested is the wild jujube seed or the hovenia dulcis thunb or the spine date seed; (8) Preparing a kit for identifying or assisting in identifying whether the sample to be tested contains the wild jujube seeds, the hovenia dulcis thunb or the wild jujube seeds.

Description

Primer composition for identification or auxiliary identification of wild jujube seed and false product thereof and multiplex PCR detection method Technical Field The invention belongs to the technical field of biology, and particularly relates to a primer composition for identifying or assisting identification of wild jujube seeds and pseudo products (semen zizyphi spinosae and semen hoveniae) thereof and a multiplex PCR detection method. Background The wild jujube seed is a dried mature seed of wild jujube Ziziphus jujuba Mill. Var. Spinosa (Bunge) Huex H.F.Chou, has flat nature, sweet and sour taste, has the effects of calming and hypnotizing, resisting anxiety, resisting depression, resisting oxidization and the like, is used as one of the first-choice traditional Chinese medicines for clinical sedation and tranquilization, and is prepared into various preparations for clinical application. Along with the continuous increase of the clinical demand of the wild jujube seeds and the limitation of the supply of wild resources, the price of the wild jujube seed medicinal material is increased, so that the wild jujube seeds are similar to the counterfeit products with different degrees of doping characteristics. The main mixed products appearing in the current medicinal material market are semen hoveniae Hovenia acerba Lindl, seed of the same genus plant, namely, fructus acanthopanacis glabrae Ziziphus mauritiana lam, and the like. At present, the authentication methods of basic source authentication, character authentication, microscopic authentication, physicochemical authentication, biological authentication and the like in the authentication of the wild jujube seed, and the authentication research of doping powders with different proportions based on a hydrogen nuclear magnetic technology are also available, and the reported biological authentication method is mostly a single site specific PCR technology, so that the authentication can only be carried out. Disclosure of Invention Aiming at the current application situation of the wild jujube, one of the purposes of the method is to adopt a molecular biological means to screen specific primers of the wild jujube, the hovenia dulcis thunb and the wild jujube based on SNP locus difference, construct a multiple locus specificity detection technology, detect different counterfeit products by using a multiple locus specificity method, not only can identify the authenticity of the wild jujube, but also can accurately determine specific adulterated varieties, and provide a reference for scientifically and comprehensively evaluating the quality of the wild jujube. The invention provides a primer composition for identifying or assisting in identifying wild jujube seeds and pseudo products thereof (semen zizyphi spinosae and semen hoveniae), which comprises primers SF1, SR4, HAF1, HAR6, ZMF5 and ZMR5, The primer SF1 is 1) or 2) as follows: 1) A single-stranded DNA molecule represented by sequence 1; 2) Single stranded DNA molecules resulting from deletion and/or addition and/or alteration of one or several nucleotides for sequence 1; the primer SR4 is 3) or 4) as follows: 3) A single-stranded DNA molecule represented by sequence 2; 4) Single stranded DNA molecules resulting from deletion and/or addition and/or alteration of one or several nucleotides for sequence 2; The primer HAF1 is 5) or 6) as follows: 5) A single-stranded DNA molecule represented by sequence 3; 6) Single stranded DNA molecules resulting from deletion and/or addition and/or alteration of one or several nucleotides for sequence 3; The primer HAR6 is 7) or 8) as follows: 7) A single-stranded DNA molecule represented by sequence 4; 8) Single stranded DNA molecules resulting from the deletion and/or addition and/or alteration of one or several nucleotides for sequence 4; the primer ZMF5 is 9) or 10) as follows: 9) A single-stranded DNA molecule represented by sequence 5; 10 A single-stranded DNA molecule obtained by deleting and/or adding and/or changing one or several nucleotides for sequence 5; The primer ZMR5 is 11) or 12) as follows: 11 A single-stranded DNA molecule represented by sequence 6; 12 A single stranded DNA molecule obtained by deleting and/or adding and/or changing one or several nucleotides to sequence 6. Optionally, in the primer composition, the semen zizyphi spinosae specific primer pair consists of a primer SF1 and a primer SR4, the semen hoveniae specific primer pair consists of a primer NAF1 and a primer HAR6, and the semen zizyphi spinosae specific primer pair consists of a primer ZMF5 and a primer ZMR 5. Optionally, in the primer composition, the molar ratio of the primer SF1, the primer SR4, the primer ZMF5, the primer ZMR5, the primer HAF1 and the primer HAR6 is 4:4:4:5:5. The invention also provides a reagent for identifying or assisting in identifying the wild jujube and the counterfeit products thereof, which comprises the primer composition. The invention also provides