CN108998544A - A kind of identification American cockroach primer pair, kit and its application - Google Patents
A kind of identification American cockroach primer pair, kit and its application Download PDFInfo
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- CN108998544A CN108998544A CN201811019760.1A CN201811019760A CN108998544A CN 108998544 A CN108998544 A CN 108998544A CN 201811019760 A CN201811019760 A CN 201811019760A CN 108998544 A CN108998544 A CN 108998544A
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- 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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Abstract
The present invention provides a kind of identification American cockroach primer pair, kit and its applications, are related to Med Mat Appreciation technical field.The nucleotide sequence of primer pair of the present invention is as shown in SEQ ID NO.2 and SEQ ID NO.3.Easy to operate using primer pair of the invention or kit identification American cockroach, sample is restricted low, detects specificity height, stability is good, and practicability is wide.For the American cockroach sample of separate sources, either fresh goods or dry product, insect medicinal material processing product or powdered samples, full worm or part polypide, larva or adult all has 98% or more accuracy and precision.
Description
Technical field
The invention belongs to Med Mat Appreciation fields, and in particular to a kind of identification American cockroach primer pair, kit and its answer
With.
Background technique
American cockroach (Periplaneta americana Linn.) is prolonged application in civil animal drugs medicinal material, often
It is used as medicine with dry or fresh adult, cures mainly infantile malnutrition, tonsillitis, body mass, ulcerative carbuncle swelling and pain and centipede and bite.In recent years
Studies have shown that American cockroach, as insect drug, clinical application record shows it with antitumor, treatment cardiovascular disease, rush
Into tissue repair, antibacterial, antiviral, anti-oxidant, enhancing is immune etc., and pharmacological actions, the application of pharmaceutical preparation clinically are also got over
Come more extensive, and is the basis for being used as medicine and carrying out pharmaceutical research to the authenticity of American cockroach medicinal material.At present in international model
Enclosing the interior Identification of Species to insect is mainly the morphological feature according to adult, and the medicinal material market in China, American cockroach medicine
Material is usually used as medicine in the form of powder or processed product etc..Lack accurate internal anatomy feature either since traditional character identifies
Form is seriously damaged during storage, Processing methods, therefore the taxonomic description stability based on morphosis nuance
It is poor, with a low credibility, cause the identification of medicinal American cockroach to there is very big difficulty.
Since traditional character discrimination method can not accurately and rapidly identify the American cockroach medicinal material that medicinal material market is sold,
Cause to have part adulterant (even such as drug effect not as good as the insect that the Australian cockroach of American cockroach, Groton bug, other sections belong to) to exist
It circulates in market, market is more chaotic.Therefore, the quality of the Med Mat Appreciation of the insect drug and bulk drug medicine materical crude slice control etc. urgently
It need to seek a kind of new method, to make up the defect and deficiency of traditional taxonomy method.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of identification American cockroach primer pair, kit and its application,
It realizes Rapid identification of the American cockroach as insect raw medicinal material, qualification time can be shortened, improve the accuracy of qualification result.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of identification American cockroach primer pair, the nucleotide sequence of the primer pair such as SEQ ID
Shown in NO.2 and SEQ ID NO.3.
The present invention provides a kind of identification American cockroach kits, include above-mentioned primer pair, further include PCR Buffer.
Preferably, the PCR Buffer includes: dNTP, 20mmol/L of the Taq polymerase of 0.1U, 500 μm of ol/L
The MgCl of KCl and 3 μm of ol/L of Tris-HCl, 100mmol/L2。
The present invention also provides the application of above-mentioned primer pair or mentioned reagent box in identification American cockroach.
Preferably, it is described identification the following steps are included: 1) extract sample gene group DNA, obtain template DNA；2) by primer pair,
PCR amplification is carried out after the template DNA mixing that PCRBuffer and step 1) obtain, obtains amplified production；3) step 2) is obtained
The amplified production through agarose gel electrophoresis, when the size of the amplified production is 705bp, the sample is that America is big
Lian.
Preferably, the sample includes fresh goods or dry product.
Preferably, the sample includes the full worm or part polypide of larva or adult.
Preferably, the reaction system of PCR amplification described in every 50 μ L includes: 19 μ L, PCR Buffer of distilled water, 25 μ L, is drawn
2 μ L of each 2 μ L of object and template DNA.
Preferably, the response procedures of the PCR amplification include: 94 DEG C of initial denaturation 3.5min；94 DEG C of 35s, 49 DEG C of 35s, 72
DEG C 45s, 33 circulations；72 DEG C extend 5 minutes.
The present invention provides a kind of identification American cockroach primer pair, kit and its application, it can be achieved that American cockroach makees
For the Rapid identification of insect raw medicinal material, shortens qualification time, improve the accuracy of qualification result.In embodiments of the present invention,
Identify accuracy up to 98% or more.
Detailed description of the invention
Fig. 1 is the agarose gel electrophoresis figure of the embodiment of the present invention 3；
Fig. 2 is the agarose gel electrophoresis figure of the embodiment of the present invention 4.
Fig. 3 is the agarose gel electrophoresis figure of the embodiment of the present invention 5；
Fig. 4 is the agarose gel electrophoresis figure of the embodiment of the present invention 6.
Specific embodiment
The present invention provides a kind of identification American cockroach primer pair, the nucleotide sequence of the primer pair such as SEQ ID
Shown in NO.2:GGTCAACAAATCATAAAGATATTGG and SEQ ID NO.3: TAAACTTCAGGGTGACCAAAAAATCA.
Primer pair of the present invention is preferably designed according to the DNA bar code gene of American cockroach, the DNA bar code gene
Preferably COI gene.The nucleotide sequence of COI gene of the present invention is as shown in SEQ ID NO.1:
GGTCAACAAATCATAAAGATATTGGAACATTATATTTTATATTCGGTGCCTGATCAGGTATAGTGGGAACATCACTA
AGAATATTAATTCGTGCTGAGCTCGGGCAACCAGGTTCACTAATTGGAGATGATCAAATTTATAATGTAATCGTTAC
TGCCCATGCCTTCATTATAATTTTCTTTATAGTAATACCAATCATAATTGGGGGATTTGGTAATTGATTAGTACCAC
TAATATTAGGAGCCCCAGATATAGCCTTCCCACGAATAAATAATATAAGATTCTGATTATTACCACCTTCATTAACT
TTATTACTAGCTAGTAGTATAGTAGAAAGAGGTGCCGGAACAGGATGAACAGTATACCCACCACTAGCAAGAGGCAT
TGCTCATGCCGGAGCATCTGTTGATCTAGCAATTTTTTCATTACATCTAGCAGGTGTATCCTCAATTCTAGGAGCTG
TAAATTTTATCTCCACAACAATTAATATAAAACCTATTAATATAAAACCAGAACGAATTCCCCTTTTCGTATGATCA
GTAGCTATTACAGCATTATTATTATTATTATCTCTACCAGTGCTTGCTGGAGCAATTACTATATTATTAACTGACCG
AAATCTAAATACATCCTTTTTTGATCCAGCAGGAGGGGGTGACCCAATTTTATATCAACACTTATTCTGATTCTTTG
GTCATCCAGAAG。
The present invention provides a kind of identification American cockroach kits, include above-mentioned primer pair, further include PCR Buffer.
PCR Buffer of the present invention is preferably included: dNTP, 20mmol/L of the Taq polymerase of 0.1U, 500 μm of ol/L
Tris-HCl, 100mmol/L KCl and 3 μm of ol/L MgCl2。
The present invention also provides the application of above-mentioned primer pair or mentioned reagent box in identification American cockroach.
Identification of the present invention preferably includes following steps: 1) extracting sample gene group DNA, obtain template DNA；2) by primer
PCR amplification is carried out after the template DNA mixing obtained to, PCRBuffer and step 1), obtains amplified production；3) by step 2)
The obtained amplified production is through agarose gel electrophoresis, and when the size of the amplified production is 705bp, the sample is America
Big Lian.
The present invention extracts sample gene group DNA, obtains template DNA when identifying the American cockroach.The present invention is to the sample
There is no particular determinations for the type of product or source, preferably include fresh goods or dry product, also may include larva or adult full worm or
Part polypide.There is no particular determinations for extracting method of the present invention to the genomic DNA, preferably include phenol chloroform method.This
The concentration for inventing the template DNA is preferably 20~50ng/ μ L.
After obtaining template DNA, the present invention will carry out PCR amplification after the mixing of primer pair, PCR Buffer and template DNA, must expand
Increase production object.When PCR amplification system of the present invention is 50 μ L system, 19 μ L, PCR Buffer of distilled water, 25 μ is preferably included
2 μ L of L, each 2 μ L of primer and template DNA.The concentration of primer of the present invention is preferably 5~15 μm of ol/L, more preferably 8~12 μ
Mol/L, most preferably 10 μm of ol/L.The response procedures of PCR amplification of the present invention preferably include: 94 DEG C of initial denaturation 3.5min；
94 DEG C of 35s, 49 DEG C of 35s, 72 DEG C of 45s, 33 circulations；72 DEG C extend 5 minutes.The present invention is when carrying out the PCR amplification, preferably
Positive control is set.The template DNA of positive control of the present invention is nucleotide sequence gene as shown in SEQ ID NO.1.
After amplified production, the present invention by the amplified production through agarose gel electrophoresis, it is big when the amplified production
Small is 705bp, and the sample is American cockroach.The present invention to the method for the agarose gel electrophoresis there is no particular determination,
Utilize the conventional method of this field.Whether the present invention is when identifying sample is American cockroach, preferably by the template DNA
Amplified production and band of the amplified production on Ago-Gel of positive control compare, the two in same position or
The size for being amplified production is 705bp, it can be determined that the sample is American cockroach.
Identification American cockroach primer pair provided by the invention, kit and its application are carried out in detail below with reference to embodiment
Thin explanation, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
The musculature of American cockroach is impregnated 3 hours in 75% alcohol, is then immersed in pure water and impregnates 3
Hour, it is rinsed well later with pure water.Sample is cut into small pieces with sterilized operating scissors later, shreds to be placed in rapidly and grind
In alms bowl, liquid nitrogen is added, rapid grind into powder is respectively charged into centrifuge tube, and 50mg sample is added in general each centrifuge tube.
SDS- Proteinase K is added in each centrifuge tube, is placed in 56 DEG C of water-baths and digests 1~3 hour, until mixed liquor digests
Until very limpid.
Isometric balance phenol is added in mixed liquor, it is slowly ten times reverse up and down, it is sure not acutely to vibrate, in desk type high speed
6000r/min is centrifuged 10 minutes on centrifuge, takes supernatant.It repeats the above steps until the interface of water phase and phenol phase can't see
Until the protein layer of white, supernatant is taken.
Isometric chloroform: isoamyl alcohol (24:1) is added in supernatant, slowly reverse ten times up and down, desk type high speed from
8000r/min is centrifuged 10 minutes in scheming, takes supernatant.
The cold dehydrated alcohol (being set in -20 DEG C of refrigerators in advance) that 2 × volume is added in supernatant precipitates DNA, puts in refrigerator
It sets 10 minutes, 12000r/min is centrifuged 10 minutes, abandons supernatant.
1 milliliter 70% of cold ethanol washing DNA is added in the centrifuge tube there are precipitating, on table model high speed centrifuge
10000r/min is centrifuged 5 minutes, and incline supernatant.
By the corresponding 10 microlitres of TE buffer solution DNA of 10 milligrams of samples, template DNA is done.
Embodiment 2
It is anti-that PCR is carried out after taking 19 μ L of sterile purified water, 25 μ L of PCR Buffer, each 2 μ L primer and 2 μ L template DNAs to mix
It answers, 94 DEG C of initial denaturation 3.5min is set；94 DEG C of 35s, 49 DEG C of 35s, 72 DEG C of 45s, 33 circulations；72 DEG C extend 5 minutes.
The pcr amplification product for taking 5 μ L, with 1.2% agarose gel electrophoresis (90V voltage, electrophoresis 40 minutes), ethidium bromide
Dyeing.Gel imaging system detection, the pcr amplification product commission Hua Da gene to pillar location in 500~750bp are surveyed
Sequence.Sequencing result is as follows:
GGTCAACAAATCATAAAGATATTGGAACATTATATTTTATATTCGGTGCCTGATCAGGTATAGTGGGAACATCACTA
AGAATATTAATTCGTGCTGAGCTCGGGCAACCAGGTTCACTAATTGGAGATGATCAAATTTATAATGTAATCGTTAC
TGCCCATGCCTTCATTATAATTTTCTTTATAGTAATACCAATCATAATTGGGGGATTTGGTAATTGATTAGTACCAC
TAATATTAGGAGCCCCAGATATAGCCTTCCCACGAATAAATAATATAAGATTCTGATTATTACCACCTTCATTAACT
TTATTACTAGCTAGTAGTATAGTAGAAAGAGGTGCCGGAACAGGATGAACAGTATACCCACCACTAGCAAGAGGCAT
TGCTCATGCCGGAGCATCTGTTGATCTAGCAATTTTTTCATTACATCTAGCAGGTGTATCCTCAATTCTAGGAGCTG
TAAATTTTATCTCCACAACAATTAATATAAAACCTATTAATATAAAACCAGAACGAATTCCCCTTTTCGTATGATCA
GTAGCTATTACAGCATTATTATTATTATTATCTCTACCAGTGCTTGCTGGAGCAATTACTATATTATTAACTGACCG
AAATCTAAATACATCCTTTTTTGATCCAGCAGGAGGGGGTGACCCAATTTTATATCAACACTTATTCTGATTCTTTG
GTCATCCAGAAG；
Obtained sequence will be sequenced and carry out BLAST inspection in NCBI, what determination obtained is COI genetic fragment.
Embodiment 3
Fetch bit is in the extraordinary pharmaceutical insects exploitation country local joint project research center of Dali Bai Autonomous prefecture, Yunnan
The American cockroach that city's base batch raising is made in the pharmaceutical insects standardization cultivation of subordinate is sample to be tested, comprising: just deviates from ootheca
1 day old larva, larva, adult, and the adult processed product through baking oven flash baking, and buy and be accredited as with medicinal material market
The adult of American cockroach is positive control sample.The part musculature and sample to be tested for taking positive control sample are numbered simultaneously respectively
It is stored in dehydrated alcohol.
DNA extraction method leads to embodiment 1.
PCR amplification and agarose gel electrophoresis method for detecting are arranged negative control and (are free of in reaction system with embodiment 2
Template, water is instead of template).Gel imaging system detection, electrophorogram is as shown in Figure 1, wherein M3 is 1 age in days for deviating from ootheca
The amplified band of larva, M2 are the amplified band of larva, the amplified band that M4 is adult, and M5 is the expansion of the drying processed product of adult
Increase band, M1 is positive control, MA DNAMarker.
Obtained pcr amplification product is sequenced, sequencing result is shown: just the sequence of 1 day old larva of de- sheath with such as
The homology of COI gene order shown in SEQ ID NO.1 be 98%, adult, adult processed product and positive control sample with such as
The homology of COI gene order shown in SEQ ID NO.1 is 99%.
Embodiment 4
The Blattaria Blattidae Periplaneta insect Australian cockroach Periplaneta belonged to American cockroach is taken respectively
The not equal Germany of australasiae Fabr. (picking up from Yunnan Province Yuxi Salvador in Yuanjiang County of Yunnan Province), same to mesh (Blattaria Blattodea)
Small Lian Blattlla germanica Linn. (picking up from Dali Bai Autonomous prefecture, Yunnan Dali), ground bettle (are purchased from Zhejiang
Pan'an Chinese Medicinal Materials Markets) and the American cockroach (fresh goods) of Honghe state, Yunnan Province wild stocks is picked up from as sample to be tested.It takes to test sample
The part musculature of product is numbered respectively and is stored in -20 DEG C of refrigerator-freezers.
DNA extraction method leads to embodiment 1.
PCR amplification and agarose gel electrophoresis method for detecting are arranged negative control and (are free of in reaction system with embodiment 2
Template DNA, water is instead of template) and positive control (the COI gene order SEQ ID NO.1 of adult is template DNA).Gel at
As system detection, for electrophorogram as shown in Fig. 2, wherein W6 and W7 is the amplified band of Australian cockroach, W8 is the expansion of Groton bug
Increase band, W9 is the amplified band of ground bettle, and W10 is the amplified band of wild stocks American cockroach fresh goods.
Pcr amplification product is sequenced, sequencing result and DNA bar code standard gene pair shown in SEQ ID NO.1
Than: ground bettle can not obtain amplified band, and the sequence of Australian cockroach and the homology of SEQ ID NO.1 are 87%, Groton bug
Homology with SEQ ID NO.1 is 77%, the sequence and SEQ ID of the American cockroach from Honghe state, Yunnan Province wild stocks
The homology of NO.1 is 99%.
Embodiment 5
Take respectively and pick up from the wild American cockroach of Wenshan Prefecture of Yunnan Province (fresh goods), America that Zhejiang Chinese Medicinal Materials Markets are bought it is big
The America that American cockroach (dry product) that Lian (dry product), Hui nationality's Chinese Medicinal Materials Markets are bought, Hebei Anguo Chinese Medicinal Materials Markets are bought
Big Lian (processed product) is sample to be tested, takes local joint project research center (Yunnan Province) subordinate of extraordinary pharmaceutical insects exploitation country
Pharmaceutical insects standardization cultivation make city base raising adult bombay canary (fresh goods) be positive control.Sample to be tested is distinguished
It numbers and is stored in -20 DEG C of refrigerator-freezers.
DNA extraction method leads to embodiment 1.
PCR amplification and agarose gel electrophoresis method for detecting are the same as embodiment 2.Gel imaging system detection, electrophorogram such as Fig. 3 institute
Show, wherein Y1 is the amplified band of positive control, and Y2 is the American cockroach processed product amplified band of Hebei Anguo Chinese Medicinal Materials Markets,
Y3 is the American cockroach dry product amplified band in Hui nationality's Chinese medicine city, and Y4 is the wild American cockroach fresh goods of Wenshan Prefecture of Yunnan Province
Amplified band, Y5 are the American cockroach dry product amplified band of Zhejiang Chinese Medicinal Materials Markets.
Pcr amplification product is sequenced, the DNA bar shaped as shown in SEQ ID NO.1 of sequencing result and nucleotide sequence
Code standard gene comparison, the sequence and SEQ ID NO.1 of sample to be tested Y2 (Hebei strain), Y3 (Anhui plant) and Y5 (Zhejiang strain)
Homology is all 98%；The homology of the sequence and SEQ ID NO.1 of sample to be tested Y1 (positive control) and Y4 (Yunnan strain) is all
It is 99%.
Embodiment 6
The American cockroach incompleteness polypide for intending abandoning after medicinal material finished product is screened (to sample local from extraordinary pharmaceutical insects exploitation country
Joint project research center subordinate's pharmaceutical insects standardization cultivation makees city base raising American cockroach), Yunnan Province's medicinal material market
(sampling is supported from the county Dali Prefecture, Yunnan Province Wei Shan American cockroach profession for the adult bombay canary (dry product) of purchase, American cockroach larva
Grow family breeding room), powdered American cockroach (sample from the good doctor in Sichuan climb Western medicine industry limited liability company production American cockroach
Fine powder) it is sample to be tested.Take sampling from extraordinary pharmaceutical insects exploitation country local joint project research center subordinate's pharmaceutical insects
The adult bombay canary (fresh goods) that the raising of city base is made in standardization cultivation is positive control.Sample to be tested and positive control are distinguished
It numbers and is stored in -20 DEG C of refrigerator-freezers.
DNA extraction method leads to embodiment 1.
PCR amplification and agarose gel electrophoresis method for detecting are the same as embodiment 2.Gel imaging system detection, electrophorogram such as Fig. 4 institute
Show, wherein B1 is the amplified band of positive control, and B3 is the amplified band of American cockroach dry product, and B2 is American cockroach incompleteness polypide
Amplified band, B4 be American cockroach larva amplified band, B5 be powdered American cockroach amplified band.
Pcr amplification product is sequenced, the DNA bar shaped as shown in SEQ ID NO.1 of sequencing result and nucleotide sequence
Code standard gene comparison, the sequence of sample to be tested larva (B4) and the homology of SEQ ID NO.1 are 98%, and sample to be tested is positive
Compare the sequence and SEQ ID of (B1), American cockroach incompleteness polypide (B2), American cockroach dry product (B3) and powdered samples (B5)
The homology of NO.1 is all 99%.
Collected remaining larva is placed in suitable environment and is raised, after about 45 days, to grow up to the polypide of adult into
The confirmation of row Morphological Identification is American cockroach.
Shown in sum up, the present invention provides a kind of identification American cockroach primer pair, kit and its applications, identify America
Big Lian is easy to operate, and sample is restricted low, detects specificity height, stability is good, and practicability is wide.It is big for the America of separate sources
Lian sample, either fresh goods or dry product, insect medicinal material processing product or powdered samples, full worm or part polypide, larva or adult
All there is 98% or more accuracy and precision.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Dali university
<120>a kind of identification American cockroach primer pair, kit and its application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 705
<212> DNA
<213> Periplaneta americana
<400> 1
ggtcaacaaa tcataaagat attggaacat tatattttat attcggtgcc tgatcaggta 60
tagtgggaac atcactaaga atattaattc gtgctgagct cgggcaacca ggttcactaa 120
ttggagatga tcaaatttat aatgtaatcg ttactgccca tgccttcatt ataattttct 180
ttatagtaat accaatcata attgggggat ttggtaattg attagtacca ctaatattag 240
gagccccaga tatagccttc ccacgaataa ataatataag attctgatta ttaccacctt 300
cattaacttt attactagct agtagtatag tagaaagagg tgccggaaca ggatgaacag 360
tatacccacc actagcaaga ggcattgctc atgccggagc atctgttgat ctagcaattt 420
tttcattaca tctagcaggt gtatcctcaa ttctaggagc tgtaaatttt atctccacaa 480
caattaatat aaaacctatt aatataaaac cagaacgaat tccccttttc gtatgatcag 540
tagctattac agcattatta ttattattat ctctaccagt gcttgctgga gcaattacta 600
tattattaac tgaccgaaat ctaaatacat ccttttttga tccagcagga gggggtgacc 660
caattttata tcaacactta ttctgattct ttggtcatcc agaag 705
<210> 2
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ggtcaacaaa tcataaagat attgg 25
<210> 3
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
taaacttcag ggtgaccaaa aaatca 26
Claims (9)
1. a kind of identification American cockroach primer pair, which is characterized in that the nucleotide sequence of the primer pair such as SEQ ID NO.2
With shown in SEQ ID NO.3.
2. a kind of identification American cockroach kit further includes PCR Buffer comprising primer pair described in claim 1.
3. kit according to claim 2, which is characterized in that the PCR Buffer include: 0.1U Taq polymerase,
The MgCl of KCl and 3 μm of ol/L of Tris-HCl, 100mmol/L of dNTP, 20mmol/L of 500 μm of ol/L2。
4. any one of primer pair described in claim 1 or claim 2~3 kit answering in identification American cockroach
With.
5. application according to claim 4, which is characterized in that 1) identification is the following steps are included: extract sample gene
Group DNA, obtains template DNA；2) PCR expansion is carried out after the template DNA for obtaining primer pair, PCRBuffer and step 1) mixes
Increase, obtains amplified production；3) amplified production for obtaining step 2) is through agarose gel electrophoresis, big when the amplified production
Small is 705bp, and the sample is American cockroach.
6. application according to claim 5, which is characterized in that the sample includes fresh goods or dry product.
7. application according to claim 5 or 6, which is characterized in that the sample includes full worm or the portion of larva or adult
Divide polypide.
8. application according to claim 5, which is characterized in that the reaction system of PCR amplification described in every 50 μ L includes: distillation
19 μ L, PCR Buffer of water 25 μ L, 2 μ L of each 2 μ L of primer and template DNA.
9. the application according to claim 5 or 8, which is characterized in that the response procedures of the PCR amplification include: 94 DEG C pre-
It is denaturalized 3.5min；94 DEG C of 35s, 49 DEG C of 35s, 72 DEG C of 45s, 33 circulations；72 DEG C extend 5 minutes.
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| CN201811019760.1A CN108998544A (en) | 2018-09-03 | 2018-09-03 | A kind of identification American cockroach primer pair, kit and its application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111434781A (en) * | 2019-01-14 | 2020-07-21 | 四川好医生攀西药业有限责任公司 | PCR identification method of novel rehabilitation liquid |
| CN111635949A (en) * | 2020-07-02 | 2020-09-08 | 大理大学 | DNA barcode standard detection fragment for identifying wasp with yellow veins and application thereof |
| CN112458178A (en) * | 2019-09-06 | 2021-03-09 | 内蒙古京新药业有限公司 | Periplaneta americana PCR identification primer and identification method |
| CN114150069A (en) * | 2020-09-08 | 2022-03-08 | 四川好医生攀西药业有限责任公司 | Periplaneta americana medicinal material specificity detection method based on TaqMan probe qPCR technology |
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| CN114150069A (en) * | 2020-09-08 | 2022-03-08 | 四川好医生攀西药业有限责任公司 | Periplaneta americana medicinal material specificity detection method based on TaqMan probe qPCR technology |
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