CN110484640A - The ARMS-PCR primer and its molecular detecting method of sulfanilamide (SN) drug resistance Eimeria Tenella - Google Patents
The ARMS-PCR primer and its molecular detecting method of sulfanilamide (SN) drug resistance Eimeria Tenella Download PDFInfo
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Abstract
The invention belongs to parasite molecules detection technique fields, are related to a kind of molecular detecting method of sulfanilamide (SN) drug resistance Eimeria Tenella, especially a kind of method using four primer amplifications retardance system-PCR detection sulfanilamide (SN) drug resistance Eimeria Tenella.The present invention designs two specific primers pair according to the dhps gene order of chicken Eimeria Tenella, the nucleotide sequence of the primer pair is respectively shown in SEQ ID NO:2 to SEQ ID NO:5, then sample total DNA is extracted, ARMS-PCR amplified reaction is carried out, whether is determined containing sulfanilamide (SN) drug-resistant worm plant finally by agarose gel electrophoresis in sample.High sensitivity of the present invention, high specificity, can be used for the quick identification of sulfanilamide (SN) drug resistance Eimeria Tenella.
Description
Technical field
The invention belongs to parasite molecules detection technique field, it is related to a kind of sulfanilamide (SN) drug resistance Eimeria Tenella
ARMS-PCR primer and its molecular detecting method.It is that a kind of four primer amplifications of utilization block system-PCR (amplification
Refractory mutation system-PCR) the technology method that quickly detects sulfanilamide (SN) drug resistance Eimeria Tenella.
Technical background
Chicken coccidiasis is to colonize in a kind of disease caused by enterocyte as a variety of coccidias of Eimeriidae Eimeria,
It is extremely serious to the harm of chicken.The chick disease incidence of 15-50 age in days is high, and the death rate is up to 80%.The whole world is used to prevent every year
The expense of globidiosis has been more than multi-billion dollar.Eimeria species are common to share 7 kinds, wherein being soft in the most commonly used coccidia of distribution in China
Tender eimeria tenella, while Eimeria Tenella is also one of stronger Eimeria species of pathogenicity.
Up to the present, the common control method of chicken coccidiasis is still relied on adds anticoccidial drug in feed.Anti- ball
Worm medicine mainly includes chemical synthesis anticoccidial drug and polyether ionophore antibiotic anticoccidial drug.Sulfa drugs is a kind of important
Common chemical synthetic drug, it mainly reaches Anti-human globulin test by inhibiting the growth and development of coccidia, and without direct
Anti-human globulin test.Due to the long-term a wide range of unreasonable coccidia point for using sulfa drug, making it possible to resist that sulfa drug acts on
Cloth is more and more extensive, brings bigger challenge to clinically control chicken coccidiasis.Therefore, urgent need, which develops one kind, can quickly detect sulphur
The molecular detecting method of amine drug resistance Eimeria Tenella.
Four primer amplifications block system-PCR (amplification refractory mutation system-PCR)
It is the instant proofreading activity for lacking 3 ' -5 ' archaeal dna polymerases at low concentrations using Taq DNA polymerase, to be reacted at one
In system, using a pair of primer reversed each other on particular bases mutational site, SNP parting is carried out to sample DNA.
It is not able to detect the molecular detecting method for the Eimeria Tenella of resistance to sulfa drug at present.The discovery such as Gong Zhenxing is soft
The mutation (Asn-Asp) in 403 site of the functional areas PPPK-DHPS of tender eimeria tenella and Eimeria Tenella sulfa drug are anti-
The generation of pharmacological property is related.By the base mutation in sequencing discovery site, being compared again after sequencing is still current monitoring SNP
Goldstandard, but this method spends higher, the required time is longer, and the present invention with ARMS-PCR be based on molecular water
The flat dhps gene to chicken Eimeria Tenella carries out specific amplification, and this method does not need special equipment, it is only necessary to
One PCR instrument and conventional PCR reagent, thus greatly reduce the expense of detection, can be applied more broadly in resistance to sulfa drug
The reality of the clinical diagnosis of chicken Eimeria Tenella and the Eimeria Tenella drug-resistant worm plant for the purpose of non-clinical diagnosis
Test room screening identification.
Summary of the invention
The object of the present invention is to provide the ARMS-PCR primers of one group of sulfanilamide (SN) drug resistance Eimeria Tenella, and utilizing should
Primer pair sulfanilamide (SN) drug resistance Eimeria Tenella carries out Molecular Detection.
Concrete scheme of the invention the following steps are included:
1) design of primers: according to reported chicken Eimeria Tenella dhps gene (2713bp, sequence table SEQ ID
Shown in NO:1).The gene is selected to design a group-specific primers, the primer pair nucleotide sequence is as follows:
Outer primer is to F-outer:5'-CGGTGTCAGACACGCTTAGATGGATTGA-3'；
Outer primer is to R-outer:5'-AGATATGCACTTCAGGCGTGGGACTGAC-3'；
Inner primer is to F-inner:5'-CGTTGATGTGGGAGGAGAGGCAAAAA-3'；
Inner primer is to R-inner:5'-CGACACTTGCTCTTGCACGAAAGGTTC-3'；
Above-mentioned nucleotides sequence, which is listed in sequence table, is successively named as SEQ ID NO:2 to SEQ ID NO:5；
2) sample total DNA is extracted, carries out ARMS-PCR amplified reaction using the primer of step 1)；
3) amplified production is detected using agarose gel electrophoresis method.
ARMS-PCR amplification system: 2.5ul 10 × PCR Buffer Mg2+Free, 1.5ul dNTP, 1.0ul 25mM
MgCl2, 14.1ul ddH2O, 0.4ul F-outer, 0.4ul R-outer, 2.0ul F-inner, 2.0ul R-inner, 1ul
DNA profiling, 0.1ul 5U/ul rTaq.
ARMS-PCR amplification reaction condition: 95 DEG C of initial denaturation 5min, later carry out 35 circulation 95 DEG C of 30s, 62 DEG C
30s, 72 DEG C of 60s finally carry out 72 DEG C of terminations and extend 5min.
Agarose gel electrophoresis: taking 5ul final reacting product, is added containing 0.5ug/mL ethidium bromide (EB) dyestuff
In 2% Ago-Gel, the electrophoresis 30min under 120V voltage, the imaging result on gel imaging system.
Imaging results determine: if there is the band of a 483bp and the band of a 216bp, showing in the sample only
The worm strain of Drug Sensitivity containing sulfanilamide (SN)；If there is the band of 483bp and the band of a 320bp, show in the sample containing only
The worm strain of sulfa drug drug resistance；If there is the band of the band of 483bp and 320bp and the band of a 216bp,
It proves in the sample not only containing the strain of sulfanilamide (SN) Drug Sensitivity worm but also containing the strain of drug resistance worm.
Quickly, specifically, high sensitivity has the advantage that method established by the present invention compared with prior art
1, low in cost: required reagent is all common PCR reagent, such as: Taq enzyme.
2, high sensitivity: the detection method high sensitivity, minimum detection limit have reached 40fg, can stablize screening clinic sample
Template type in product.
3, easy to operate: to can be completed with common PCR instrument in laboratory etc., do not need complicated instrumentation step.
4, accuracy is high: ARMS-PCR method testing result and sequencing result comparison result consistency are up to 100%.
5. high specificity: four primers ensure that the high degree of specificity of ARMS-PCR method amplification.
6. quickly time saving: the used time is short, and reaction is completed usually in 90min, can determine whether by observation bin number situation
Whether sample is the worm strain of sulfa drugs resistance, without amplified production is sequenced.
Detailed description of the invention
Fig. 1: the ARMS-PCR agarose gel electrophoresis results of sensitive insect strain, the strain of resistance worm and mixed infection worm plant.Figure
In: swimming lane M:DNA Marker 2K；1 is the strain of resistance worm；2 be sensitive insect strain；3 be the strain of mixed infection worm；4 be water.
Fig. 2: the gel electrophoresis spectrum of regular-PCR amplified production.In figure: swimming lane M:DNA Marker 2K；1 is resistance worm
Strain.
The sensitivity experiments result of Fig. 3: ARMS-PCR method.In figure: swimming lane M:DNA Marker；1-8 template is respectively
The DNA of 40ng/ul, 4ng/ul, 400pg/ul, 40pg/ul, 4pg/ul, 400fg/ul, 40fg/ul, 4fg/ul.
Fig. 4: the sensitivity experiments result of regular-PCR method.In figure: swimming lane M:DNA Marker；1-8 template is respectively
The DNA of 40ng/ul, 4ng/ul, 400pg/ul, 40pg/ul, 4pg/ul, 400fg/ul, 40fg/ul, 4fg/ul.
Specific embodiment
Below by specific embodiment, the present invention is described in detail.
Embodiment 1: the detection for the Eimeria Tenella of resistance to sulfa drug is carried out to chicken manure sample
1. the extraction of clinical chicken manure sample total DNA: the egg capsule suspension 500ul of Sporulated quickly being prepared with sample be
Egg capsule suspension, is placed on centrifuge at 10000r/min is centrifuged 1min later by system, and it is spare to retain precipitating；It is molten with that will precipitate
In 200ulGA, later plus 20ul protein kinase K digests 8h in 56 DEG C of water-baths；Reagent is extracted according to Tiangeng blood DNA below
Box (centrifugal column type is purchased from QIAGEN company, catalog number: No.51304) carries out the extraction of subsequent DNA.
2. the selection of gene
We select dihydrofolate synthetase gene and according to the tender Amy that worm strain dhps announced in Gene Bank
Gene (2713bp), then sequence alignment is carried out with Snapgene software, as a result, it has been found that Eimeria Tenella dhps gene
(2713bp) shows high specificity to protozoon and various hosts.Therefore, Eimeria Tenella dhps gene is selected
Candidate template as design of primers.
3. design of primers
It is set according to relevant primer design principle, and with reference to http://primer1.soton.ac.uk/primer1.htm1
Meter requires, and by Shanghai, Sheng Gong biotech company synthesizes following primer.
Outer primer is to F-outer:5'-CGGTGTCAGACACGCTTAGATGGATTGA-3'；
Outer primer is to R-outer:5'-AGATATGCACTTCAGGCGTGGGACTGAC-3'；
Inner primer is to F-inner:5'-CGTTGATGTGGGAGGAGAGGCAAAAA-3'；
Inner primer is to R-inner:5'-CGACACTTGCTCTTGCACGAAAGGTTC-3'；
3.ARMS-PCR amplification: it establishes four primer amplifications retardance mutation system (ARMS-PCR) reaction system: 1. will
2.5ul 10 × PCRBufferMg2+free, 1.5ul dNTP, 1.0ul 25mM Mgcl2,2.0ul F-inner, 2.0ul
R-inner, 0.4ul F-outer, 0.4ul R-outer, 0.1ul Taq archaeal dna polymerase, 1ulDNA template, sterilize distilled water
14.1ul mixing and slight oscillatory, are centrifuged the several seconds；2. above-mentioned 25 μ l overall reaction liquid is placed individually into PCR instrument, according to 95 DEG C
Initial denaturation 5min carries out 95 DEG C of 30s, 62 DEG C of 30s, the 72 DEG C of 60s of 35 circulations later, finally carries out 72 DEG C of terminations and extends
5min.3. agarose gel electrophoresis: taking 5ul final reacting product, be added and contain the 2% of 0.5ug/mL ethidium bromide (EB) dyestuff
Ago-Gel in, the electrophoresis 30min under 120V voltage, the imaging result on gel imaging system.
4.ARMS-PCR reacts amplified production analysis:
Imaging results determine: if there is the band of a 483bp and the band of a 216bp, showing in the sample only
The worm strain of Drug Sensitivity containing sulfanilamide (SN)；If there is the band of 483bp and the band of a 320bp, show in the sample containing only
The worm strain of sulfa drug drug resistance；If there is the band of the band of 483bp and 320bp and the band of a 216bp,
It proves in the sample not only containing the strain of sulfanilamide (SN) Drug Sensitivity worm but also containing the strain of drug resistance worm.
2 sulfanilamide (SN) drug resistance Eimeria Tenella detection method specific test of embodiment
1. being chosen respectively according to the method for embodiment 1 containing to sulfa drug drug resistance, sensitivity or mixed infection worm strain sample
Middle total DNA template.
2. using primer pair and method progress ARMS-PCR amplification and detection in embodiment 1.
As a result such as Fig. 1, it can be seen that it is clear to the purpose band of sulfa drug drug resistance, sensitivity or mixed infection worm strain, it has no
Miscellaneous band illustrates that this method has preferable specificity.
3. the sequencing of Eimeria Tenella dhps gene gDNA is identified:
Send the PCR product 20ul at the 483bp band of amplification to survey.Sequencing result is imported into ncbi database and carries out sequence
It compares, so that drug resistance, sensitivity or the mixed infection three types that gDNA is Eimeria Tenella are identified, these three types
DNA sequence dna difference is shown in Table 1.
The sequence difference of 1 three kinds of worm strains of table
| Worm strain type | Mutational site |
| Drug-resistant worm plant | It is G in No. 1129 sites of base |
| Sensitive insect strain | It is A in No. 1129 sites of base |
| Mixed infection worm strain | It is G/A in No. 1129 sites of base |
Embodiment 3: sulfanilamide (SN) drug resistance Eimeria Tenella detection method sensitivity tests
1. expanding target fragment with regular-PCR method: choosing the total DNA containing sulfa drug drug-resistant worm plant and expand tender Amy
Your coccidia includes the partial gene fragments of the dhps of mutation, and the nucleotide sequence of the primer pair is as follows.
Upstream primer F2:5'-GTTGCGGCATTCTGCATCAAGTTCAGGA-3'；
Downstream primer R2:5'-CTGCATGCGGCAACAACTTATAAGAGGAC-3'；
Reaction system are as follows: Phanta Max Super-Fidelity DNA Polymerase 1.0ul, 2 × Phanta
Max Buffer25ul, dNTP Mixture 1ul, total DNA template 2ul, ddH2O 17ul, each 2ul of upstream and downstream primer.Reaction
Total system is 50ul.Above-mentioned PCR final product 5ul is added to 2% agar for containing 0.5ug/mL ethidium bromide (EB) dyestuff
In sugared gel, the electrophoresis 30min under 120V voltage, in gel at the map of imaging amplified production on phase system.Amplification produces
It is Eimeria Tenella dhps partial gene fragments PCR product that object has single band at 800bp.As a result such as Fig. 2.
2. using ddH2It is dilute that gDNA regular-PCR product containing the strain of sulfa drug resistance worm in above-mentioned steps 1 is first carried out multiple proportions by O
It releases.Specific dilution process is as follows: 10 times of dilutions: taking 1ul regular-PCR product that 9ul ddH is added2In O, 1ul is taken after mixing well
Another pipe is added and has 9ul ddH2In the EP pipe of O, takes 1ul that next pipe is added after mixing again and have 9ul ddH2The EP of O is managed
In, and so on.
3.ARMS-PCR sensitivity experiments: 1. by 10 × PCRBufferMg of 2.5ul2+Free, 1.5uldNTP, 1.0ul
25mM MgCl2, 2.0ulF-inner, 2.0ulR-inner, 0.4ul F-outer, 0.4ul R-outer, 0.1ul Taq
Archaeal dna polymerase, 1ulDNA template (DNA after regular-PCR product doubling dilution), sterilizing distilled water 14.1ul are mixed and are slightly shaken
It swings, is centrifuged the several seconds；2. above-mentioned 25 μ l overall reaction liquid is placed individually into PCR instrument, according to 95 DEG C of initial denaturation 5min, carry out later
95 DEG C of 30s, 62 DEG C of 30s, the 72 DEG C of 60s of 35 circulations finally carry out 72 DEG C of terminations and extend 5min.3. agarose gel electrophoresis:
5ul final reacting product is taken, is added in 2% Ago-Gel containing 0.5ug/mL ethidium bromide (EB) dyestuff, in 120V
Electrophoresis 30min under voltage, the imaging result on gel imaging system.
Faint item still can be detected to 40fg containing sulfa drug resistance worm strain regular-PCR product dilution for ARMS-PCR method
Band, result such as Fig. 3；
And it can detect with the method for regular-PCR being diluted to 400fg containing the positive PCR product of sulfa drug resistance worm strain
Faint band simultaneously can be completed to be sequenced, result such as Fig. 4.
As can be seen from the above results, ARMS-PCR method is sensitiveer than regular-PCR method.
Sequence table
<110>Hua Zhong Agriculture University
<120>the ARMS-PCR primer and its molecular detecting method of sulfanilamide (SN) drug resistance Eimeria Tenella
<160> 5
<170> SIPOSequenceListing 1.0
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<212> DNA
<213>Eimeria Tenella (Eimeria tenella)
<400> 1
atgtttcatt taaccagtcc ccgcacatcg cttcaatggt tttacaaaag gtgtttctca 60
tttgtaaatg gccgcagcta cagcgtacgc cggtttgtac gttgcgtgac tactgcgtcc 120
tccggcgaat ccacacgacc caaacggcca gtggcatata tctccatggg gacgaatttg 180
ggtggtgaaa ggcggcttag tatactagaa agcgctcttt caagcattcg gagagaagtc 240
ggttcccttg acgcctgttc ctgcctgtac gaatcacttc ctggttatga cgtggatcat 300
cggagccgag atgaacacga catgtcgatt ccgcttcatc tgaacgcagt ggtgcgcgtc 360
gagacagatt caagtgatcc ccaagtaatc ttgaaaattt tgcagcgcat agaggcaaac 420
catggtagag accgatcggc cacagccggc agattgcgca gagcattaga ccttgacctt 480
ctgttattcg aaaatttaga ctcacacagc atgcaggtaa acaccaccaa cctcacgcta 540
cctcatcctc gactggctca gaggaatttc atcctcttcc cactgtgcga catcaagcca 600
gatttggtgc atcccacaga gagggaaaca atgaagaatt tgattttgaa gaacatgcga 660
cgcagagagg aggcgttgcg gcattctgca tcaagttcag gagaaacttc tgatgcagct 720
gtaaagttca accgcaaatc gccatacacc cttgacggca accttgcaat tccccgcagg 780
tgcttcgcag tcaataaccg ccagttatgg acagtgaagg gaggtacaga acttgcggtg 840
tcagacacgc ttagatggat tgaagaagtg ctgacaaggt atgggcttga ggacaactgt 900
gatcagcagg atccaaacct tcctgcgttt gctgggcccc tcctttccct caagcgtttt 960
cttttacggg agcgacgctc acccagattg atgggggtcc tcaatgttac tcccgactcc 1020
ttcagtgacg gccagaagta ctacgacaat gtggaagctg cattgaggca tgctcggcga 1080
atgagggacg acggcgctga catcgttgat gtgggaggag aggcaacaaa ccctttcgtg 1140
caagagcaag tgtcggtaca ctctgaaata cagcgcgtag tgccagtggt cgaggcagtg 1200
aggagagaga tgggaagaga tgttattatc tcagttgaca caaggcgaag gcccgtggcg 1260
gaggccgcga cagcggcagg tgctgacctg gtcagtccca cgcctgaagt gcatatctac 1320
ggccgttgtg agaaccagct gtaggagcgc ggtattttcc tgcatagcag tagaattagt 1380
tatcaacagc attaaatcgc aaaggatagt tcttgaactg tagtagtctc tgagtgacta 1440
accaggtcct cttataagtt gttgccgcat gcagatcaac gacgtgagcg caggcgaggt 1500
ggacccagac cttctcacgt ttgccgcatc cagaggcgtc ccgcttgttc tgatgcactc 1560
acgtgggcct ccgcagttaa tggacagtct ggtgcgtttg cacactgaag tactcagctg 1620
atactagcga tagtaagtgc atgctgtaga ccgtgaatgg cgctaacaaa gtacagccgc 1680
ctgtagagcg tgttcctgtt cggcccatag gcaatctacg acgatgttat tgctgaagtg 1740
gcgaagtact tgactgataa gactgagacg ctaattaaaa tggggctgcc gcggtaaaac 1800
tttcactgag ttcacttgca ctcaaggtga cgggtgcaga agcgcgcatt gcatatacgt 1860
aatccggcgt tagttttaag ctacactaca ttgctatcta gtacacctac cttggtgcag 1920
cagaaatttt tggggcacgt attacggaaa cagagatgta acatcctatt ccgctgagct 1980
gaaattaacc gcagcaaata gaaaacaaac tctattatgt ccctcttagg tggcgtctgg 2040
ttctggacgc gggtttaggc tttgcaaaga ctgccgatca ctcctttgag atactccgca 2100
gacttcgaga tcttcgaact aaactgccca gcggtctgcc gatgcttgtt ggtcattccc 2160
ggaaaaggta ggattcttcg tgtttgagct tcgcgtggct ccgcagccta gcttagctag 2220
tgtaacagta acaggtcttt cgtctgtaag tggacattta caggttcatt ggttcggcca 2280
tagcagctat gccgaacagt cacgcaaaac aggactcacc gggaaacaag gtaaatgatg 2340
gcgaaggaaa actgacagaa gaacctatgt caaaccgtga cgtcgcggga ttagctgtag 2400
catgttgggc tgcgaaggac aactgtgtcg atatcctcag agtacacgat gtcaaggtac 2460
gcactacaca caggcaagct ttcgcgtggc gcatattaag gagagtttcg aagccaagga 2520
ggggaaagta cgccacgtag tacccgtgcc agcgcttgcc ctatcaatat gctgttgcaa 2580
aggacatgct gtgcaatctt gcgttgtgca gcgaacagcc ctagtctact ccgtcatgaa 2640
tgagctgacg aaaaggtcaa agacagacac tgaaaccgac tttaaggcga tgtttcccgc 2700
ggaaagcagc tag 2713
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<211> 28
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<213>artificial sequence (Artificial sequence)
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cggtgtcaga cacgcttaga tggattga 28
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<213>artificial sequence (Artificial sequence)
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agatatgcac ttcaggcgtg ggactgac 28
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<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 4
cgttgatgtg ggaggagagg caaaaa 26
<210> 5
<211> 27
<212> DNA
<213>artificial sequence (Artificial sequence)
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cgacacttgc tcttgcacga aaggttc 27
Claims (6)
1. the ARMS-PCR primer of one group of sulfanilamide (SN) drug resistance Eimeria Tenella, it is characterised in that including following two primer pairs:
Outer primer is to F-outer:5'-CGGTGTCAGACACGCTTAGATGGATTGA-3'；
Outer primer is to R-outer:5'-AGATATGCACTTCAGGCGTGGGACTGAC-3'；
Inner primer is to F-inner:5'-CGTTGATGTGGGAGGAGAGGCAAAAA-3'；
Inner primer is to R-inner:5'-CGACACTTGCTCTTGCACGAAAGGTTC-3'.
2. ARMS-PCR primer described in claim 1 answering in the non-diagnostic purpose detection of sulfanilamide (SN) drug resistance Eimeria Tenella
With.
3. a kind of non-diagnostic molecules of interest detection method of sulfanilamide (SN) drug resistance Eimeria Tenella, feature the following steps are included:
1) group-specific primers, the sequence of the primer are designed according to Eimeria Tenella dihydrofolate synthetase gene order
Column are as follows:
Outer primer is to F-outer:5'-CGGTGTCAGACACGCTTAGATGGATTGA-3'；
Outer primer is to R-outer:5'-AGATATGCACTTCAGGCGTGGGACTGAC-3'；
Inner primer is to F-inner:5'-CGTTGATGTGGGAGGAGAGGCAAAAA-3'；
Inner primer is to R-inner:5'-CGACACTTGCTCTTGCACGAAAGGTTC-3'；
2) sample total DNA is extracted, carries out ARMS-PCR amplified reaction using the primer of step 1)；
3) amplified production is detected using agarose gel electrophoresis method.
4. the non-diagnostic molecules of interest detection method of sulfanilamide (SN) drug resistance Eimeria Tenella as claimed in claim 3, feature exist
In the system of the amplified reaction are as follows: 2.5ul 10 × PCR Buffer Mg2+Free, 1.5ul dNTP, 1.0ul 25mM
MgCl2, 14.1ul ddH2O, 0.4ul F-outer, 0.4ul R-outer, 2.0ul F-inner, 2.0ul R-inner, 1ul
DNA profiling, 0.1ul 5U/ul rTaq.
5. the non-diagnostic molecules of interest detection method of sulfanilamide (SN) drug resistance Eimeria Tenella as claimed in claim 3, feature exist
In the condition of the amplified reaction are as follows: 95 DEG C of initial denaturation 5min, later carry out 35 circulation 95 DEG C of 30s, 62 DEG C of 30s, 72 DEG C
60s finally carries out 72 DEG C of terminations and extends 5min.
6. the non-diagnostic molecules of interest detection method of sulfanilamide (SN) drug resistance Eimeria Tenella as claimed in claim 3, feature exist
In: the agarose gel electrophoresis, which sentences method for distinguishing, is: taking the ARMS-PCR amplified production of 5ul, is added and contains 0.5ug/mL bromine
In 2% Ago-Gel for changing second ingot dyestuff, the electrophoresis 30min under 120V voltage, the imaging on gel imaging system
As a result, showing in the sample if there is the band of a 483bp and the band of a 216bp containing only sulfanilamide (SN) Drug Sensitivity worm
Strain；If there is the band of a 483bp and the band of a 320bp, show in the sample containing only the worm strain of sulfa drug drug resistance；
If there is the band of the band of 483bp and 320bp and the band of a 216bp, prove both to contain in the sample
There is the strain of sulfanilamide (SN) Drug Sensitivity worm again containing the strain of drug resistance worm.
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