CN109439770A - A kind of loop-mediated isothermal amplification (LAMP) primer and the method for identifying the Chinese medicine zaocys dhumnade true and false - Google Patents
A kind of loop-mediated isothermal amplification (LAMP) primer and the method for identifying the Chinese medicine zaocys dhumnade true and false Download PDFInfo
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- CN109439770A CN109439770A CN201811524677.XA CN201811524677A CN109439770A CN 109439770 A CN109439770 A CN 109439770A CN 201811524677 A CN201811524677 A CN 201811524677A CN 109439770 A CN109439770 A CN 109439770A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention discloses a kind of loop-mediated isothermal amplification (LAMP) primer and the methods of the identification Chinese medicine zaocys dhumnade true and false, the primer includes cyt b-4-F3, cyt b-4-B3, cyt b-4-FIP and cyt b-4-BIP, and sequence is successively as shown in SEQ.ID.NO.1-SEQ.ID.NO.4；Identify the method for the Chinese medicine zaocys dhumnade true and false the following steps are included: extracting the genomic DNA of Chinese medicine as template, primer described in claim 1 is added, 65 DEG C of incubation 40min carry out LAMP reaction, after reaction, nucleic acid dye is added, the color change of amplified production is visually observed under fluorescent lamp and ultraviolet lamp；If expanding and successfully then showing green, amplification failure is then shown orange；Amplification is successfully zaocys dhumnade certified products.This method is easy to operate, is easy to grasp, accuracy height, can be in 1 hour or so completion discrimination process.
Description
Technical field
The invention belongs to the identifications of Chinese medicine to identify field, and in particular to Molecular Identification, in particular to a kind of ring mediation etc.
Warm amplimer and the method for identifying the Chinese medicine zaocys dhumnade true and false.
Background technique
Visited according to market and literature survey it is found that the adulterant of Chinese medicine zaocys dhumnade have Ptyas korras (Ptyas korros),
Dinodon rufozonatum snake (Dinodon rufozonatum), water snake (Hurria rhynchops), indian cobra (Naja naja), Meng Jia
It draws cobra (Naja kaouthia), dodge squama snake (Xenopeltis unicolor), Natrix piscater (Xenochrophis
Piscator), Bungarus multicinctus (Bungarus multicinctus), Malaysia krait (Bungarus candidus), long-nosed pit viper
(Agkistrodon acutus) etc..
In current authentication technique, " Chinese Pharmacopoeia " 2015 edition owner will be identified using properties and characteristics and microscopic features,
Foundation are as follows: surface dark brown or green black, Density Diamond scale: back squama line number is in pairs, carries on the back the strong barring of center 2-4 row scale,
It forms two and passes through all black lines.For cephalic disc in centre, oblate, eye lower recess greatly is glossy.8 pieces of upper labial, the 4th, 5
Piece enter socket of the eye, 1 piece of cheek squama, descends 1 piece of squama at the moment, smaller, 2 pieces of squama after eye.Spine is towering at ridge.Abdomen splits edge to curls inward
Song, ridge muscle is thick, yellow-white or light brown, it is seen that the rib cage of marshalling.Tail portion is tapered and grows, urostege duplicate rows.Skinner
Only let the hair grow the skin squama of tail, middle section is more smooth.Gas raw meat, it is lightly seasoned.Scutellum is close colourless or faint yellow, and surface has longitudinal stripe.Table
Gather brown or brown-black pigment particle are seen in epidermis face, and it is agglomerating to be often linked to be netted, branched or aggregation.Rhabdium is faint yellow
Or it is close colourless.There is light and dark fine and closely woven band.The nearly colourless or light gray of bone chip, is in irregular fragment, bone lacuna spindle shape,
Most equidirectional arrangement, bone canalicules are close and relatively thick.These identify main points and identification person are required to have identification of experience quite abundant, no
The identification between zaocys dhumnade and mixed adulterant is not can be effectively used to then.
At " Chinese Pharmaceutical Journal ", once report utilizes the 12srRNA gene order in mitochondria to Tang Xiaojing etc. within 2007, if
1 pair of primer (HWL-1 and HWH-1) is counted, for identifying zaocys dhumnade and other Its Common Confused Varieties.But when because of the operation of entire discrimination process
Between it is longer, to zaocys dhumnade it is quick identification have some limitations, so medicinal material identification in applicability be limited.
Summary of the invention
The primary purpose of the present invention is that being directed to the conservative gene cyt b of zaocys dhumnade, design provides one group of ring mediated isothermal
Expand (LAMP) primer.
Another object of the present invention is to provide a kind of methods (LAMP method) for quickly identifying the Chinese medicine zaocys dhumnade true and false.
The purpose of the invention is achieved by the following technical solution:
One group of loop-mediated isothermal amplification (LAMP) primer is cyt b-4-F3, cyt b-4-B3, cyt b-4-FIP and cyt b-4-
BIP, sequence is successively as shown in SEQ.ID.NO.1-SEQ.ID.NO.4.This group of primer is directed to the cyt b gene of zaocys dhumnade and sets
Meter, cyt b gene overall length are 307bp, and primer cyt b-4-F3 length is 18bp, positioned at 25~42 sites of the gene；Primer
Cyt b-4-B3 length is 23b, positioned at 194~216 sites of the gene；Primer cyt b-4-FIP length is 39b, and being located at should
83~103 and 43~60 sites of gene；Primer cyt b-4-BIP length is 43bp, positioned at the 118~142 of the gene and 176
~193 sites.
A kind of method of the quick identification Chinese medicine zaocys dhumnade true and false, comprising the following steps:
The genomic DNA of Chinese medicine is extracted as template, above-mentioned primer is added, it is anti-that 65 DEG C of incubation 40min carry out LAMP
It answers, after reaction, nucleic acid dye is added, the color change of amplified production is visually observed under fluorescent lamp and ultraviolet lamp；If expanding
Increase and successfully then show green, amplification failure is then shown orange；Amplification is successfully zaocys dhumnade certified products；
The Chinese medicine includes zaocys dhumnade certified products and its mixed adulterant；
The mixed adulterant is Ptyas korras, dinodon rufozonatum snake, water snake, indian cobra, Naja kaouthia, dodges squama snake, fishing trip
One or more of snake, Bungarus multicinctus, Malaysia krait or long-nosed pit viper；
The genomic DNA for extracting Chinese medicine, is using Animal genome DNA extraction kitDNA
Mini Kit (QIAGEN company, Germany) chooses without the medicinal material sample 30mg for going mouldy and damaging by worms, is cut with what is sterilized
Knife is cut into fragment, is placed in 2mL centrifuge tube；180 μ L Buffer ATL and 20 μ L proteinase K, oscillation treatment is added
20s, 56 DEG C of water-baths to tissue cracking completely；Of short duration centrifugation, to remove in centrifuge tube lid along liquid；200 μ L Buffer are added
AL vibrates 15s, 70 DEG C of water-bath 10min, of short duration centrifugation；200 μ L dehydrated alcohols are added, vibrate 15s, of short duration centrifugation；By the above institute
It obtains mixed liquor to be carefully added into centrifugal column, centrifugal column is put into 2mL collecting pipe, 1min is centrifuged with 6000 × g, by centrifugal column
It takes out, is put into a clean 2mL collecting pipe；500 μ L Buffer AW1 are added into centrifugal column, with 6000 × g centrifugation
1min takes out centrifuge tube, is put into a clean 2mL collecting pipe；500 μ L Buffer AW2 are added into centrifugal column, with
20000 × g is centrifuged 3min, and centrifuge tube is taken out, is put into a clean 2mL collecting pipe；50 μ L Buffer AE, room is added
Temperature is lower to stand 1min, and 6000 × g is centrifuged 1min, collecting pipe is covered, -20 DEG C spare；
The described LAMP reaction, reaction system are as follows: totally 25 μ L, archaeal dna polymerase containing Bst (800U) 1 μ L, 10 ×
TherrmoPol Reaction Buffer 2.5 μ L, MgSO4(100mM) 1.5 μ L, dNTPs (10mM) 3.5 μ L, outer primer FIP
(10 μM) 4 μ L, outer primer BIP (10 μM) 4 μ L, inner primer F3 (10 μM) 0.5 μ L of 0.5 μ L, inner primer B3 (10 μM), DNA profiling
(60~82ng/ μ L) 1 μ L, sterile water complement to 25 μ L.
The preferred SYBR Green I of the nucleic acid dye.
The present invention relative to existing polymerase chain reaction method (PCR) have following advantages and effects
(1) high specificity, accuracy rate are high.Round pcr needs 2 primers, and LAMP technology is directed to 6 areas of target gene
Domain, design obtain 4 primers, and specificity and accuracy increase.
(2) rapid reaction, efficiently.LAMP technology 15~40min it is amplifiable go out a large amount of purpose nucleic acid segment, 1 hour
Interior achievable entire qualification process.
(3) easy to operate, it is cheap.LAMP technology does not need PCR cycle amplification instrument, it is only necessary to use common water-bath or
Amplified reaction can be completed in heating device, and amplified production does not need to carry out gel electrophoresis, and laboratory operating procedures are reduced, experimental cost
Reduction.
Detailed description of the invention
Fig. 1 is zaocys dhumnade and mixed adulterant genome dna electrophoresis figure；
Fig. 2 is the color of the LAMP reaction product of zaocys dhumnade and other 10 kinds of mixed adulterants under fluorescent light；
Fig. 3 is the color of the LAMP reaction product of zaocys dhumnade and other 10 kinds of mixed adulterants in the UV lamp；
Wherein, 1 zaocys dhumnade, 2 indian cobras, 3 Malaysia kraits, 4 long-nosed pit vipers, 5 dinodon rufozonatum snakes, 6 Ptyas korras, 7 Bungarus multicinctus, 8 dodge
Squama snake, 9 Naja kaouthias, 10 Natrix piscaters, 11 water snakes, N negative control；
M:DNA ladder most bright band is 500bp, is followed successively by 400bp, 300bp, 200bp, 100bp downwards.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Embodiment
A kind of method of the quick identification Chinese medicine zaocys dhumnade true and false, comprising the following steps:
(1) it extracts genomic DNA: choosing without the medicinal material sample about 30mg for going mouldy and damaging by worms, be cut into the scissors sterilized
Fragment is placed in 2mL centrifuge tube.It is added 180 μ L Buffer ATL and 20 μ L proteinase K, oscillation treatment 20s, 56 DEG C
Water-bath cracks completely to tissue.Of short duration centrifugation, to remove in centrifuge tube lid along liquid.200 μ L Buffer AL are added, vibrate
15s, 70 DEG C of water-bath 10min, of short duration centrifugation.200 μ L dehydrated alcohols are added, vibrate 15s, of short duration centrifugation.By mixing obtained as above
Liquid is carefully added into centrifugal column, and centrifugal column is put into 2mL collecting pipe, is centrifuged 1min with 6000 × g, centrifugal column is taken out,
It is put into a clean 2mL collecting pipe.500 μ L Buffer AW1 are added into centrifugal column, 1min is centrifuged with 6000 × g, it will
Centrifuge tube takes out, and is put into a clean 2mL collecting pipe.500 μ L Buffer AW2 are added into centrifugal column, with 20000 ×
G is centrifuged 3min, and centrifuge tube is taken out, is put into a clean 2mL collecting pipe.50 μ L Buffer AE are added, it is quiet at room temperature
1min is set, 6000 × g is centrifuged 1min, collecting pipe is covered, -20 DEG C spare.
The medicinal material is 11 kinds of zaocys dhumnade and other class medicinal materials in the markets such as Guangzhou, Anhui, Jiangxi, Sichuan, totally 13
Item, respectively zaocys dhumnade (Zaocys dhumnades), Ptyas korras (Ptyas korros), dinodon rufozonatum snake (Dinodon
Rufozonatum), water snake (Hurria rhynchops), indian cobra (Naja naja), Naja kaouthia (Naja
Kaouthia), squama snake (Xenopeltis unicolor), Natrix piscater (Xenochrophis piscator), Bungarus multicinctus are dodged
(Bungarus multicinctus), Malaysia krait (Bungarus candidus), long-nosed pit viper (Agkistrodon acutus).
The equal Successful amplification of genomic DNA (Fig. 1) of zaocys dhumnade and other 10 kinds of mixed adulterants, ultramicron UV, visible light are divided light
The purity and concentration of degree meter 11 kinds of class medicinal material sample solutions of detection, A260/A280 value minimum 1.78, up to 1.92,
It can be used for subsequent gene magnification.
(2) LAMP reacts:
LAMP primer:
Cyt b-4-F3:CTAGCCATCCACTACACAG (SEQ.ID.NO.1)；
Cyt b-4-B3:GTCAGACATTTTTGTTTAGGTAAG (SEQ.ID.NO.2)；
Cyt b-4-FIP:
ATTCATCCATAGGGGACGTCTCCCAACATCAACCTTGCCTT(SEQ.ID.NO.3)；
Cyt b-4-BIP:
CATACAAAACCTTCATGCAATTGGATCCGTAGTATAGTCCGCG(SEQ.ID.NO.4)。
Reaction system: the 25 μ L large fragment of archaeal dna polymerase containing Bst (800U), 1 μ L, 10x TherrmoPol Reaction
Buffer 2.5 μ L, MgSO4 (100mM) 1.5 μ L, dNTPs (10mM) 3.5 μ L, outer primer FIP (10 μM) 4 μ L, outer primer BIP
(10 μM) 4 μ L, inner primer F3 (10 μM) 0.5 μ L, inner primer B3 (10 μM) 0.5 μ L, DNA profiling (60~82ng/ μ L) 1 μ L, nothing
Bacterium water complements to 25 μ L.
Reaction condition: reaction heat inactivation 10min at 65 DEG C of incubation 40min, 85 DEG C.After reaction, 0.5 μ L is added
SYBR Green I nucleic acid dye visually observes the color change of amplified production under fluorescent lamp and ultraviolet lamp.If expanding successfully
Then show green, amplification failure is then shown orange.
Using ring mediated isothermal amplification (LAMP) primer cyt b-4-F3, the cyt b-4- of zaocys dhumnade cyt b gene segment
B3, cyt b-4-FIP and cyt b-4-BIP are carried out amplification reaction, using Fluorometric assay amplified production (Fig. 2-Fig. 3), only
Positive reaction is presented in zaocys dhumnade sample, reacts and green is presented in EP pipe, and orange is presented in other 10 kinds mixed adulterant sample amplification failures
Color.Show that LAMP reaction can quickly identify zaocys dhumnade and other mixed adulterants in 1 hour, and there is simple, quick, Gao Teyi
Property and it is low in cost the advantages that.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Sequence table
<110>Ji'nan University
<120>a kind of method of loop-mediated isothermal amplification (LAMP) primer and the identification Chinese medicine zaocys dhumnade true and false
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223> cyt b-4-F3
<400> 1
ctagccatcc actacacag 19
<210> 2
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223> cyt b-4-B3
<400> 2
gtcagacatt tttgtttagg taag 24
<210> 3
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223> cyt b-4-FIP
<400> 3
attcatccat aggggacgtc tcccaacatc aaccttgcct t 41
<210> 4
<211> 43
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223> cyt b-4-BIP
<400> 4
catacaaaac cttcatgcaa ttggatccgt agtatagtcc gcg 43
Claims (8)
1. one group of loop-mediated isothermal amplification (LAMP) primer, it is characterised in that: be cyt b-4-F3, cyt b-4-B3, cyt b-4-FIP and
Cyt b-4-BIP, sequence is successively as shown in SEQ.ID.NO.1-SEQ.ID.NO.4.
2. a kind of method for quickly identifying the Chinese medicine zaocys dhumnade true and false, it is characterised in that the following steps are included:
The genomic DNA of Chinese medicine is extracted as template, primer described in claim 1 is added, 65 DEG C of incubation 40min are carried out
LAMP reaction is added nucleic acid dye, the color change of amplified production is visually observed under fluorescent lamp and ultraviolet lamp after reaction；
If expanding and successfully then showing green, amplification failure is then shown orange；Amplification is successfully zaocys dhumnade certified products.
3. according to the method described in claim 2, it is characterized by: the Chinese medicine includes zaocys dhumnade certified products and its mixed puppet
Product.
4. according to the method described in claim 3, it is characterized by: the mixed adulterant is Ptyas korras, dinodon rufozonatum snake, water snake, print
One or more of spend cobra, Naja kaouthia, dodge squama snake, Natrix piscater, Bungarus multicinctus, Malaysia krait or long-nosed pit viper.
5. according to the method described in claim 2, it is characterized by: the genomic DNA for extracting Chinese medicine, is using animal
Genome DNA extracting reagent kitDNA Mini Kit, chooses without the medicinal material sample 30mg for going mouldy and damaging by worms, with disappearing
The scissors that poison is crossed is cut into fragment, is placed in 2mL centrifuge tube；180 μ L Buffer ATL and 20 μ L proteinase K are added, shake
Swing processing 20s, 56 DEG C of water-baths to tissue cracking completely；Of short duration centrifugation, to remove in centrifuge tube lid along liquid；200 μ L are added
Buffer AL vibrates 15s, 70 DEG C of water-bath 10min, of short duration centrifugation；200 μ L dehydrated alcohols are added, vibrate 15s, of short duration centrifugation；
Mixed liquor obtained as above is carefully added into centrifugal column, centrifugal column is put into 2mL collecting pipe, 1min is centrifuged with 6000 × g,
Centrifugal column is taken out, is put into a clean 2mL collecting pipe；500 μ L Buffer AW1 are added into centrifugal column, with 6000
× g is centrifuged 1min, and centrifuge tube is taken out, is put into a clean 2mL collecting pipe；500 μ L Buffer AW2 are added to centrifugation
In column, 3min is centrifuged with 20000 × g, centrifuge tube is taken out, is put into a clean 2mL collecting pipe；50 μ L are added
Buffer AE, stands 1min at room temperature, and 6000 × g is centrifuged 1min, collecting pipe is covered, -20 DEG C spare.
6. according to the method described in claim 2, it is characterized by: the LAMP reacts, reaction system are as follows: 25 μ L contain Bst
1 μ L, 10 × TherrmoPol Reaction Buffer of archaeal dna polymerase 2.5 μ L, MgSO41.5 3.5 μ L of μ L, dNTPs, outside
Primers F IP 4 μ L, outer primer BIP 4 μ L, inner primer F3 0.5 μ L, inner primer B3 0.5 μ L, 1 μ L of DNA profiling, sterile water are supplied
To 25 μ L.
7. according to the method described in claim 6, it is characterized by: DNA concentration is 60~82ng/ μ in the DNA profiling
L。
8. according to the method described in claim 2, it is characterized by: the nucleic acid dye is SYBR Green I.
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| CN110951909A (en) * | 2019-12-27 | 2020-04-03 | 青岛大学附属医院 | Loop-mediated isothermal amplification primer group, method, kit and application for identifying traditional Chinese medicine fleece-flower root and adulterants thereof |
| CN112501320A (en) * | 2021-02-08 | 2021-03-16 | 中国农业大学 | Snake origin component rapid detection kit and application thereof |
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| CN110951909A (en) * | 2019-12-27 | 2020-04-03 | 青岛大学附属医院 | Loop-mediated isothermal amplification primer group, method, kit and application for identifying traditional Chinese medicine fleece-flower root and adulterants thereof |
| CN112501320A (en) * | 2021-02-08 | 2021-03-16 | 中国农业大学 | Snake origin component rapid detection kit and application thereof |
| CN112501320B (en) * | 2021-02-08 | 2021-04-27 | 中国农业大学 | Snake origin component rapid detection kit and application thereof |
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Application publication date: 20190308 |