CN104946729A - PCR (Polymerase Chain Reaction) specific primers and detection method for authenticating tortoise shell - Google Patents

Abstract

The invention belongs to the technical fields of traditional Chinese medicines and authentication of traditional Chinese medicinal materials and particularly relates to PCR (Polymerase Chain Reaction) specific primers and a detection method capable of specifically authenticating a tortoise shell. By authenticating the authenticity of the tortoise shell by using the method, the authentication on the authenticity of samples can be completed by only simple DNA (Deoxyribonucleic Acid) extraction, PCR specific amplification and fluorescence detection. The PCR specific primers and the detection method have the advantages of simplicity in operation, high specificity, good repeatability and the like and are mainly applied to the rapid authentication on tortoise shell medicinal materials, tortoise shell-containing preparations and tortoise shell in-vivo materials.

Description

The PCR special primer of qualification tortoise plastron and detection method thereof

Technical field

The present invention relates to Chinese medicine and Materia Medica Identification technical field, particularly a kind of can the PCR special primer of unique identification tortoise plastron and detection method thereof.Be mainly used in the Rapid identification of tortoise plastron medicinal material, preparation containing tortoise plastron and tortoise plastron living materials.

Background technology

Chinese medicinal materials tortoise plastron is carapace and the plastron of Testudinidae animal tortoise Chinemysreevesii (Gray), has nourishing and suppressing Yang, the strong bone of kidney-nourishing, bushing of nourishing blood, Gu the effect through only collapsing.Testudinidae animal species is numerous, in recent years due to demand rapid growth, market has occurred a large amount of tortoise plastron adulterants, has had a strong impact on the drug safety of tortoise plastron.Tradition differentiates that the method for tortoise plastron comprises character identification, microscopical identification, tlc discriminating, spectrography discriminating, ultraviolet spectroscopy discriminating etc.But traditional detection method depends on the chemical composition in medicinal material, and chemical composition is subject to the impact in kind and the place of production.Molecular marking technique is used successfully to identification and assessment of Chinese medicines, the Cyt b of Mitochondrial DNA (study by Chinese medicinal materials tortoise plastron cytochrome b specificity identification, Chinese Pharmaceutical Journal, 2012,4 (3): 182-185) and cytochrome C oxidase subunit base I (Cytochrome c oxidase subunitI, CoI), tortoise plastron (Cui Lina can be differentiated as DNA marker, Du He, Sun Jiaming etc., based on the tortoise plastron of COI bar code sequence and DNA molecular qualification [J] of mixed adulterant thereof. Jilin Chinese materia medica, 2012,2).Liu Zhongquan etc. utilize mtDNA 12S rRNA gene fragment order to establish the high specific PCR authentication method (Liu Zhongquan of tortoise plastron and former animal, Wang Yiquan, Zhou Kaiya etc., the high specific PCR identification research of Chinese medicinal materials tortoise plastron and former animal, Acta Pharmaceutica Sinica, 1999,12:941-945), but there is some new exotic speciess in the market.Li Mingcheng etc. disclose a kind of tortoise plastron DNA detection test kit and authentication method, and (application number: 201310178856.3), it only comprises 5 kinds of adulterants.

The present invention mainly in conjunction with Standard PCR and fluorescence detection method, within a short period of time Rapid identification tortoise plastron and common adulterant thereof, cost is low, suitability good.Before the present invention comes forth, not yet have any open or reported PCR primer mentioned in present patent application and method.

Summary of the invention

The invention provides a kind of PCR Auele Specific Primer and detection method thereof of tortoise plastron, the method is simple to operate, sample size is few, can identify tortoise plastron medicinal material, powder formulation and living materials fast and accurately, is specially adapted to qualification causes the Chinese medicine true and false that DNA content is few, form is broken qualification because of processing.

Identify special primer F1, F2 of tortoise plastron, its sequence is:

F1：5′-TTTGGAAACTGACTTGTACCTTTAACG-3′

F2：5′-GAGTAGTAATAGGACGGCTGTAATAAGTAGA-3′。

A PCR authentication method for tortoise plastron, is characterized in that, comprise the following steps:

A) from described material, extraction obtains DNA sample;

B) carry out PCR reaction with primer according to claim 1, PCR reaction parameter is after 95 DEG C of denaturation 5min, and carry out 30 circulations, loop parameter is 95 DEG C of 30s, 55-65 DEG C of 45s, rear extension 72 DEG C of 5min, obtains amplified production;

C) amplified production described in electrophoresis, if the unique DNA band that there is molecular weight 360bp, then determines that institute's sample material is tortoise plastron;

D) directly in described amplified production, add 2 μ L20 × SYBR Green I dyestuffs, vibration 30s fully mixes, under 365nm ultraviolet lamp, detect PCR primer; If reaction product sends intense green fluorescence, then determine that institute's sample material is tortoise plastron.

The present invention adopts fluorescence detection method to detect pcr amplification product, does not need electrophoresis that detection time is shortened greatly, and only needs simple and easy ultraviolet lamp to get final product complete operation, does not need to use electrophoresis equipment and gel imaging system.

In sum, the invention provides the PCR Auele Specific Primer and detection method of quick and precisely identifying tortoise plastron, with tortoise plastron DNA for template, can by it from other the Chelonian extremely near with its sibship, identify out efficiently and accurately, for tortoise plastron qualification provides a kind of technology of practicality.

Accompanying drawing explanation

The positive adulterant identification result of Fig. 1 tortoise plastron.M:100bp marker; P: positive control; N: negative control; B: blank; 1-12: tortoise plastron; 13: Malaysia tortoise; 14: intend crocodile tortoise; 15: Trachemys scripta; 16: Burma Testudo elongata; 17: Platysternon megacephalum Gray.

Fig. 2 tortoise plastron PCR primer Fluorescence Identification result.1: positive control; 2: Chinese grass tortoise; 3: Malaysia tortoise; 4: intend crocodile tortoise; 5: Trachemys scripta; 6: negative control

Embodiment

1. the design of high specific primer

The CO I sequence of certified products tortoise and Trachemys scripta, Mauremys mutica, plan crocodile tortoise, Annan tortoise, Burma's Testudo elongata adulterant is searched from GenBank.Carry out gene comparision with BioEdit software, find differential fragment, design diagnostic primers 5 '-TTTGGAAACTGACTTGTACCTTTAACG-3 ' and 5 '-GAGTAGTAATAGGACGGCTGTAATAAGTAGA-3 '.

2. the foundation of high specific PCR PCR method

2.1 template DNAs extract

Get it filled material (referring to table 1) about 50mg, puts in 2.0mL centrifuge tube, be ground into powder.Get powder 20mg, join in 2mL centrifuge tube respectively; Use pillar bone DNA extraction kit to extract DNA: the Extraction buffer adding 1000 μ L65 DEG C preheatings, 30s fully mixes; 65 DEG C of water-bath 30min; Add 300 μ L damping fluids and 200 μ L chloroforms; Abundant mixing, centrifugal 5min under 12000rpm; Get the 1.5mL centrifuge tube that 500 μ L supernatants to are new, add 500 μ L column-loading buffers, fully mix; Add in DNA purification column, centrifugal 1min under 12000rpm; Abandon and penetrate liquid; Add elutriant 500 μ L, centrifugal 1min under 12000rpm; Abandon and penetrate liquid, add elutriant 500 μ L, centrifugal 1min again under 12000rpm; Abandon and penetrate liquid, centrifugal DNA purification column 2min; Add 50 μ L aseptic double-distilled waters, room temperature to place after 2min centrifugal 1min under 12000rpm, gets to penetrate liquid and dilute 10 times and react for PCR.Separately get tortoise plastron control medicinal material and be about 50mg, be made in the same way of control medicinal material template D NA solution.

Table 1 sample table

The foundation of 2.2PCR reaction system

PCR reaction is carried out in 200 μ L centrifuge tubes.Reaction cumulative volume is 25 μ L, comprise 10 × PCR damping fluid 2.5 μ L, dNTP (2.5mmolL-1) 1 μ L, diagnostic primers (10 μm of olL-1) each 0.2 μ L, Taq archaeal dna polymerase (5UL-1) 0.2 μ L and template 1 μ L, supply reaction volume with aseptic double-distilled water.Centrifuge tube is put PCR instrument, PCR reaction parameter: 95 DEG C of denaturation 5min, circulating reaction 35 times (95 DEG C of 30s, 58 DEG C of 45s), 72 DEG C extend 5min, and 4 DEG C of insulations terminate reaction.Get 5 μ L reaction solutions through 1.5% agarose gel electrophoresis, EB dyes, and switches on power and carries out electrophoresis 20min by 5Vcm-1 constant voltage, after electrophoresis terminates, be placed in by gel on gel imaging instrument or imaging on ultraviolet transilluminator (Fig. 1).

2.3PCR product detects and positive adulterant interpretation

Get PCR reaction product, add 2 μ L20 × SYBR Green I dyestuffs, under 365nm ultraviolet lamp, detect PCR primer, compared with blank, send intense green fluorescence for positive (Fig. 2).

Claims (2)

1. identify special primer F1, F2 of tortoise plastron, its sequence is:

F1：5′-TTTGGAAACTGACTTGTACCTTTAACG-3′

F2：5′-GAGTAGTAATAGGACGGCTGTAATAAGTAGA-3′。

2. a PCR authentication method for tortoise plastron, is characterized in that, comprise the following steps:

A) from described material, extraction obtains DNA sample;

B) carry out PCR reaction with primer according to claim 1, PCR reaction parameter is after 95 DEG C of denaturation 5min, and carry out 30 circulations, loop parameter is 95 DEG C of 30s, 55-65 DEG C of 45s, rear extension 72 DEG C of 5min, obtains amplified production;

C) amplified production described in electrophoresis, if the unique DNA band that there is molecular weight 360bp, then determines that institute's sample material is tortoise plastron;

D) directly in described amplified production, add 2 μ L20 × SYBR Green I dyestuffs, vibration 30s fully mixes, under 365nm ultraviolet lamp, detect PCR primer; If reaction product sends intense green fluorescence, then determine that institute's sample material is tortoise plastron.