CN106868168A - Primer, probe and its application method for aquatic livestock derived bacterium sulfa drugs drug resistant gene triple fluorescent quantitative PCR detections - Google Patents
Primer, probe and its application method for aquatic livestock derived bacterium sulfa drugs drug resistant gene triple fluorescent quantitative PCR detections Download PDFInfo
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Abstract
The invention discloses the primer and TaqMan probe that are detected for aquatic livestock derived bacterium sulfa drugs drug resistant gene triple fluorescent quantitative PCR, three groups of primers and probe including being directed to aquatic livestock derived bacterium sulfa drugs drug resistant gene Su11, Su12 and Su13 respectively, three groups of primers and probe have the base sequence of sequence table SEQ .ID.No.1 to SEQ.ID.No.9 respectively.Accordingly, inventor is optimized as template with recombinant plasmid standard items to reaction condition, establishing a kind of while detect the triple fluorescent quantitative PCR method of above-mentioned 3 kinds of drug resistant genes, and can carry out the detection experiment of sensitiveness, specificity, repeatability and clinical strains to the method；Result shows, the characteristics of the method has quick, easy, accurate, can be used for the sulfa drugs drug resistant gene of clinical detection bacterium, for the investigation of the quick detection and drug resistant gene of sulfa drugs drug resistance provides a kind of brand-new technological means.
Description
Technical field
The invention belongs to sulfa drugs drug resistant gene detection technique field, more particularly, to aquatic livestock derived bacterium sulphur
Primer, probe and its application method that amine drug drug resistant gene triple fluorescent quantitative PCR is detected.
Background technology
Bacteriosis is the Major Diseases of Chinese Fishery cultivated animals, and it causes the loss of aquiculture animal to account for whole
More than the 50% of disease loss.In aquaculture process, it is using various Antibiotics Drug inhibitions or killing pathogenic bacteria
The preventing and treating maximally effective means of aquiculture animal bacteriosis.Sulfa drugs has antibacterial as main antibacterial agent use for fishery
Spectrum is wide, drug effect is notable, price is low, be the indispensable medicine of current each aquatic farm the advantages of easily store.Domestic numerous studies knot
Fruit shows that aquatic livestock derived bacterium has generated extensive drug resistance to sulfa drugs.Clinically bacterium is to sulfonamides
The resistance of thing is mainly (i.e. resistant by new dihydrofolate synthetase lower to sulfa drugs affinity on plasmid
DHPS) mediated, and R-plasmid can be shifted by modes such as conversion, transposition, engagements between bacterium, make R-Bacterium from
R+This resistance is obtained in bacterium.The DHPS resistant to sulfa drugs has 3 kinds, its corresponding encoding gene respectively by
Sul1, Sul2 and Sul3 are named as, the detection to this 3 genes can reflect resistance situation of the bacterium to sulfa drugs.
The Chinese detection at present clinically to bacterium sulfa drugs drug resistance mainly uses traditional susceptibility test methods,
Time-consuming for these methods, accuracy is poor, sensitivity is low, stability is not strong, clinically quick, accurate screening can not have been met sensitive
The need for medicine.In consideration of it, round pcr to be applied to domestic existing scholar the quick detection and resistance of sulfa drugs drug resistance
The Molecule Epidemiology Investigation of gene.Quantitative fluorescent PCR only have Standard PCR technology amplification efficiency it is high the characteristics of, also with spy
The features such as high degree of specificity of pin, the hypersensitivity of spectral technique and accuracy, and can be quantitative, pollution is not easily susceptible to, it is extensive
Apply to medical treatment, drug research and detection of pathogens.Multiple PCR technique can simultaneously detect multiple mesh in a PCR system
Mark gene, be widely used the features such as its is quick, efficient and inexpensive prospect.At present, relevant sulfa drugs is resistance to
The report of medicine gene by fluorescence quantitative PCR detection method is still few.
The content of the invention
The technical problem to be solved in the present invention is to provide triple for aquatic livestock derived bacterium sulfa drugs drug resistant gene
The primer of fluorescence quantitative PCR detection, probe and its application method, to realize that quick, easy, to carry out sulfa drugs exactly resistance to
The investigation of the quick detection and drug resistant gene of the property of medicine provides technical support.
In order to solve the above technical problems, the present invention uses following technical scheme：
For the primer and probe of aquatic livestock derived bacterium sulfa drugs drug resistant gene triple fluorescent quantitative PCR detections,
Three groups of primers and probe including being directed to aquatic livestock derived bacterium sulfa drugs drug resistant gene Sul1, Sul2 and Sul3 respectively,
Three groups of primers and probe have following base sequence respectively：
Sul1-F CTCGGTGTCGCGGAAATC,
Sul1-R CGCTGGACCCAGATCCTTT,
Sul1-P CGCCACCGTTGGCCTTCCTG；
Sul2-F TGGTGTGGCCTATCTCAATGAT,
Sul2-R GGCAGATGATTTCGCCAATT,
Sul2-P TTCGCGGTTTTCCAGACGCTGC；
Sul3-F CAACGCCCACTTCAGTTGTATC,
Sul3-R TCTGCATTTGGTTGAAGATGGA,
Sul3-P AAGCGGCTCCCAAATCAATCACATCTG。
5 ' the ends of probe Sul1-P, Sul2-P, Sul3-P are marked with fluorescent reporter group FAM, HEX, CY5,3 ' ends respectively
Mark has BHQ1.
The mol ratio of three groups of primers and probe is Sul1-F: Sul1-R: Sul1-P: Sul2-F: Sul2-R: Sul2-P:
Sul3-F: Sul3-R: Sul3-P=6: 6: 3: 4: 4: 2: 4: 4: 2.
Above-mentioned primer and spy for aquatic livestock derived bacterium sulfa drugs drug resistant gene triple fluorescent quantitative PCR detections
The application method of pin, expands aquatic livestock derived bacterium sulfa drugs drug resistant gene Sul1, Sul2 in triple fluorescent quantitative PCR
And Sul3.
The reaction system and response procedures of triple fluorescent quantitative PCR be：
Reaction system is：18 μ L, ROX Reference Dye II of Premix Ex Taq (2 ×) 0.5 μ L, 20 μm of ol/L
Sul1 upstream and downstream primer (Sul1-F/Sul1-R) are 0.9 μ L, and 10 μm of ol/L Sul1 probe (Sul1-P) consumptions are 0.9 μ L,
The upstream and downstream primer (Sul2-F/Sul2-R, Sul3-F/Sul3-R) of 20 μm of ol/L Sul2 and Sul3 is 0.6 μ L, 10 μ
Mol/L Sul2 and Sul3 probe (Sul2-P, Sul3-P) consumption is 0.6 μ L, the μ L of template 3, with sterilizing ddH2O complements to 30 μ
L；
Reaction condition is：95 DEG C of predegeneration 30s；95 DEG C of denaturation 5s, 60 DEG C of 45s that anneal and extend, expand 40 circulations；
Fluorescence signal is collected at the end of each 60 DEG C of circulation.
Lack the problem of effective detection method, inventor according to existing aquatic livestock derived bacterium sulfa drugs drug resistant gene
Conserved sequence with reference to multiple PCR technique for sulfa drugs 3 drug resistant genes Sul1, Sul2 and Sul3 in GenBank sets
Count and synthesized for aquatic livestock derived bacterium sulfa drugs drug resistant gene triple fluorescent quantitative PCR detection primer and
TaqMan probe, including respectively for three groups of aquatic livestock derived bacterium sulfa drugs drug resistant gene Sul1, Sul2 and Sul3
Primer and probe, three groups of primers and probe have the base sequence of sequence table SEQ .ID.No.1 to SEQ.ID.No.9 respectively.According to
This, inventor is optimized as template with recombinant plasmid standard items to reaction condition, and establishing one kind can be while detects above-mentioned 3 kinds
The triple fluorescent quantitative PCR method of drug resistant gene, and the inspection of sensitiveness, specificity, repeatability and clinical strains is carried out to the method
Test is tested.Result shows that the method quantification range is 1 × 101~1 × 108During copy/reaction, its standard curve is in good line
Sexual intercourse；The method sensitivity (the minimum plasmids detections limit of 3 genes can reach 10 copies/reaction) high, high specificity (with
Other pathogens and viral no cross reaction), reproducible (coefficient of variation is respectively less than 2%)；To the testing result of clinical strains
With the coincidence rate of drug sensitive test result up to 94.2%, and whole detection process can be completed in 2h.It can be seen that, the method has fast
Fast, easy, accurate the characteristics of, can be used for the sulfa drugs drug resistant gene of clinical detection bacterium, be sulfa drugs drug resistance
Quick detection and drug resistant gene investigation provide a kind of brand-new technological means.
Brief description of the drawings
Fig. 1 is the PCR amplification figures of Sul1, Sul2 and Sul3 gene.
In figure：M is the DNA Marker of DL 1000, and 1 is Sul1 genes, and 2 is Sul2 genes, and 3 is Sul3 genes, and NC is cloudy
Property control.
Fig. 2 is the triple fluorescent quantitative PCR amplification curves of Sul1, Sul2 and Sul3 gene
Fig. 3 is the standard curve that triple fluorescent quantitative PCR detects Sul1, Sul2 and Sul3 gene
Fig. 4 is the susceptibility results figure that triple fluorescent quantitative PCR detects Sul1 genes.
Fig. 5 is the susceptibility results figure that triple fluorescent quantitative PCR detects Sul2 genes.
Fig. 6 is the susceptibility results figure that triple fluorescent quantitative PCR detects Sul3 genes.
Fig. 7 is the specific detection result figure of triple fluorescent quantitative PCR.
Specific embodiment
1 materials and methods
1.1 materials and reagent
72 plants of bacterial strain to be checked, is 2013-2015 isolated from Guangxi aquatic livestock or aquatic products, specific such as table 1.
The bacterium source of table 1
Sul1, Sul2 and Sul3 gene-positive strains are Klebsiella Pneumoniae GX120222 separation strains, are identified through PCR and carried
Sul1, Sul2 and Sul3 gene.
GCRV (GCRV), white spot syndrome virus (WSSV), infectious subcutaneous and hematopoietic tissue necrosis disease
Malicious (IHHNV) is by Guangxi fishery disease control environmental monitoring and the identification of quality inspection center and preservation.
During sulphadiazine (Sulfadiazine, SD) and sulfamethoxazole (Sulfamethoxazole, SMZ) are purchased from
Veterinary drug biological products supervision institute of state, content is up to more than 99.5%；Nutrient agar, nutrient broth medium are purchased from Hangzhou shore and micro-
Biological reagent Co., Ltd；2 × Taq PCR MasterMix (archaeal dna polymerase containing Taq, dNTPs, MgCl2, reaction it is slow
Fliud flushing), Ago-Gel QIAquick Gel Extraction Kit, small amount plasmid extraction kit be purchased from Beijing Ai Delai bio tech ltd；
DNA Marker, pMD18-T, E.coli DH5 α competent cells of DL 1000, Premix Ex Taq (Probe qPCR) are
Purchased from precious bioengineering (Dalian) Co., Ltd；Ampicillin is purchased from Promega companies；It is biological that agarose creates Tianjin purchased from English Wei
Science and Technology Ltd.；It is pure that other reagents are domestic analysis.
1.2 methods
1.2.1 sensitivity testing of the aquatic livestock source clinical strains to sulfa drugs
72 plants of aquatic livestock source clinical strains are determined respectively using agar plate disk diffusion method (K-B methods) to SD and SMZ
Antibacterial circle diameter.The preparation of drug sensitive test paper is referring to document《Wear from English practicalities antibacterials [M] Shanghai：Shanghai science skill
Art publishing house, 1998.》, drug sensitivity tests interpretation standard is shown in document《Clinical and Laboratory Standards
Institute(CLSI).Performance standards for antimicrobial susceptibility
testing；Twentith informational supplement [S] .M100-S25, USA, 2015.》.
1.2.2 primer and probe are designed
Sul1, Sul2 and Sul3 gene of many kind bacteriums are compared with DNAStar softwares, using Primer
Express 3.0 and the softwares of Oligo 6.0, in conserved sequence region according to multiple fluorescence quantitative PCR design of primers principle, set altogether
Meter synthesis 3 sets of primers and probe, its middle probe 5 ' hold mark fluorescent reporter group FAM, HEX and CY5 successively, and 3 ' ends mark glimmering
Optical quenching group BHQ1, primer and probe synthesize by upper sea base health bio tech ltd.Each primer and probe sequence are shown in
Table 2.
The primer of table 2 and detecting probe information
1.2.3 the extraction of DNA of bacteria and the preparation of recombinant plasmid standard items
The single bacterium colony of test strain is inoculated in nutrient broth respectively, 36 DEG C of culture 18h；2mL bacteria suspensions are taken, using boiling
Boiling method extracts bacteria total DNA, is saved backup in -20 DEG C.
DNA with bacterial strain GX120222 expands each specificity purpose as template with Sul1, Sul2 and Sul3 gene primer
Fragment, the corresponding purpose fragment of recovery is purified with DNA QIAquick Gel Extraction Kits by specification respectively, the purpose fragment that then will be reclaimed with
PMD18-T carriers are connected.Connection product distinguishes Transformed E .coli DH5 α competent cells, and positive gram is screened by bacterium colony PCR
It is grand, and sequencing identification.The correct positive colony bacterium solution 5mL of sequencing result is taken, is carried according to small amount plasmid extraction kit specification
Plasmid is taken, and the plasmid of extraction is respectively designated as pMD18-T-Sul1, pMD18-T-Sul2 and pMD18-T-Sul3.With
GeneQuant nucleic acid-proteins analyzer determines the concentration of recombinant plasmid respectively, is converted to concentration according to A Fujiadeluo constants
Copy number, dilution DNA is 1.0 × 101~1.0 × 108Copy/μ L, with 8 gradient DNAs standard items, -20 DEG C
Preserve, it is standby.
1.2.4 the optimization of triple fluorescent quantitative PCR reaction conditions
With 1.0 × 106The standard items DNA of copy/μ L as template, by 0.1~0.8 μm of ol/L of primer final concentration and probe
0.1~0.5 μm of various combination of ol/L (being combined using matrix method) of final concentration carries out quantitative fluorescent PCR, obtains minimum facing
Primer and concentration and probe concentration when boundary's period (Ct) and relative intensity of fluorescence value added (Δ Rn) higher, as the single target base of optimization
The detection architecture of cause.On this basis, with 1.0 × 1063 kinds of standard items equal proportion mixed liquors of copy/μ L carry out three for template
Weight quantitative fluorescent PCR, is altered in steps each primer (the single optimal primer concentration ± 0.2 μm ol/L of target gene) and probe (single target
Optimal concentration and probe concentration ± 0.2 μm the ol/L of gene) concentration reaching optimal expanding effect.
The optimization of annealing temperature：With reference to the Tm values of each primer, 58~62 DEG C with gradient 1 DEG C carry out multiple fluorescence quantitative
PCR is expanded, and optimum annealing temperature is determined according to Ct values and amplification curve shape.
The optimization of Premix Ex Taq Mix：Make in reaction system Premix Ex Taq Mix additions for 0.8 ×,
0.9 ×, 1 ×, 1.1 ×, 1.2 ×, 1.3 ×, 1.4 ×, 1.5 ×, optimal Premix is determined according to Ct values and amplification curve shape
Ex Taq Mix reaction densities.
1.2.5 the foundation of standard curve and sensitiveness, specificity, replica test
With 10 times of gradient dilutions (1.0 × 101~1.0 × 108Copy/μ L) Sul1, Sul2 and Sul3 gene standard items etc.
Ratio hybrid dna is template, is expanded with the multiple fluorescence quantitative PCR method after optimization, and foundation is copied on template starting
The normal equation of shellfish number logarithm and Ct values, and the sensitiveness of the method is judged according to Ct values.
With positive strain and part sulfa drugs sensitive bacteria, GCRV, WSSV and IHHNV nucleic acid samples as template, use
Triple fluorescent quantitative PCR method after optimization detected, observes result.
Take 108、107、106Each standard items DNA of 3 gradients of copy/μ L carries out triple fluorescent quantitative PCR reactions, each
3 repetitions of concentration, calculate the coefficient of variation of Ct values.
The clinical practice of 1.3 triple fluorescent quantitative PCR methods
72 plants of Sul1, Sul2 and Sul3 genes of clinical strains are detected with triple fluorescent quantitative PCR method, amplification is will appear from
The gene of curve is judged to the positive, and amplification is compared with drug sensitive test result.
2 results and analysis
2.1 drug sensitive test results
Drug sensitive test result shows that 72 plants of aquatic livestock source clinical strains are respectively 68.1% to the resistant rate of SD and SMZ
(49/72) it is and 62.5% (45/72), 80.8% (42/72) to 2 kinds of crossing drug resistant rates of sulfa drugs.In 72 plants of bacteriums
There are 16 plants to quick in SD or SMZ, only 4 plants sensitive to SD or SMZ, and this shows resistance of the aquatic livestock derived bacterium to sulfa drugs
Phenomenon is very universal.
The preparation result of 2.2 recombinant plasmid standard items
Sul1, Sul2 and Sul3 gene amplification product are through agarose gel electrophoresis (Fig. 1), the stripe size difference for amplifying
For 67,76,83bp, it is in the same size with expection.Purpose fragment recovery purifying rear clone enters pMD18-T carriers, recombinant plasmid warp
PCR sequencing identifications, bacterium sulfa drugs drug resistant gene Sul1, Sul2 and Sul3 of the same race that gained sequence is announced with GenBank
Homology be 100%.Take positive colony bacterium solution and extract plasmid, determine its concentration and be converted to copy number, pMD18- can be obtained
The concentration of T-Sul1, pMD18-T-Sul2, pMD18-T-Sul3 is respectively 3.72 × 109、4.56×109、6.32×109Copy/
μ L, can meet the requirement of standard items.
The optimum results of 2.3 triple fluorescent quantitative PCR reaction conditions
It is final to determine that reaction system is after being optimized to each conditions of triple fluorescent quantitative PCR：Premix Ex Taq(2
×) μ L, Sul1 upstream and downstream primer (20 μm of ol/L) of 18 μ L, ROX Reference Dye II 0.5 are 0.9 μ L, Sul1 is visited
Pin consumption (10 μm of ol/L) is 0.6 μ L, Sul2 and Sul3 for the upstream and downstream primer (20 μm of ol/L) of 0.9 μ L, Sul2 and Sul3
Probe consumption (10 μm of ol/L) is 0.6 μ L, the μ L of template 3, with sterilizing ddH2O complements to 30 μ L.Reaction condition：95 DEG C of predegenerations
30s；95 DEG C of denaturation 5s, 60 DEG C of 45s that anneal and extend, expand 40 circulations；Fluorescence letter is collected at the end of each 60 DEG C of circulation
Number.
The foundation of 2.4 standard curves
With 10 times of gradient dilutions (1 × 101~1 × 108Copy/μ L) each standard items equal proportion hybrid dna be template, make
Expanded with the triple fluorescent quantitative PCR after optimization, amplification curve is in obvious " S " type, and negative control and blank pair
According to not occurring any amplification (Fig. 2).The DAS carried using instrument is analyzed, and abscissa (X) is represented
The logarithm of beginning template copy numbers, ordinate (Y) represents Ct values, obtains 3 standard curves (Fig. 3).
The calibration curve equation of Sul1 is Y=-3.572logX+42.12, and amplification efficiency (Eff.) and coefficient correlation side are worth
(RSq) it is respectively 90.5% and 0.996；The calibration curve equation of Sul2 is Y=-3.551logX+41.49, Eff. and RSq point
Wei 91.3% and 0.993；The calibration curve equation of Sul3 is respectively for Y=-3.712logX+3.46, Eff. and RSq
86.0% and 0.994.From above calibration curve equation, the logarithm of initial template copy number and Ct values are in good linear
Relation, can exactly reflect the amplification of purpose product.
The Sensitivity and Specificity and repeatability of 2.5 triple fluorescent quantitative PCR
2.5.1 sensitiveness
After testing, fluorescence signal value, 3 still be can detect when recombinant plasmid pMD18-T-Sul1 templates are 10 copies/reaction
The Ct values of individual repetition are respectively 37.66,37.80,38.22；When recombinant plasmid pMD18-T-Sul2 templates are 10 copy numbers/reaction
Also fluorescence signal value is can detect, 3 Ct values of repetition are respectively 37.34,37.54,38.06；Recombinant plasmid pMD18-T-
Sul3 templates also can detect fluorescence signal value when being 10 copy numbers/reaction, 3 Ct values of repetition are respectively 36.32,36.33
With 36.38.Result above shows that the triple fluorescent quantitative PCR set up in this research detects the spirit of Sul1, Sul2 and Sul3 gene
Sensitivity can reach 10 copies/reaction, with Standard PCR detection sensitivity 104Copy/reaction is compared, and sensitiveness significantly improves (figure
4~6).
2.5.2 it is specific
With the triple fluorescent quantitative PCR for setting up to positive strain and sulfa drugs sensitive bacteria, GCRV, WSSV and IHHNV
The result (Fig. 7) that is detected of nucleic acid show that only amplification curve occurs in positive strain, other bacterial strains and virus do not occur
Amplification curve, is feminine gender, shows inspections of the triple fluorescent quantitative PCR set up in this research to Sul1, Sul2 and Sul3 gene
Measuring tool has good specificity.
2.5.3 it is repeated
108、107、106The coefficient of variation of each standard items DNA triple fluorescent quantitatives PCR replica test Ct values of copy/μ L
(CV) 3 are shown in Table.Result shows that the coefficient of variation of Ct values is 0.30%~1.01%, illustrates the triple fluorescent quantitative PCR tools set up
There is good repeatability.
The triple fluorescent quantitative PCR of table 3 repeats result of the test
Testing results of the 2.6 triple fluorescent quantitative PCR to clinical strains
72 plants of clinical strains are detected with the triple fluorescent quantitative PCR method set up, is as a result shown：52 plants of resistance to SD
Or there are 49 plants to detected Sul1 or Sul2 or Sul3 genes, compared with drug sensitive test result, drug resistant gene in the bacterial strain of SMZ
Coincidence rate is 94.2%, wherein, there are 43 plants containing Sul1 genes, there are 26 plants of genes containing Sul2, there are 4 plants containing Sul3 genes, have
23 plants contain 2 kinds or gene of more than two kinds；There are 13 plants of detection drug resistant genes in 16 plants of medium sensitivity bacterium, wherein, there are 11 plants to contain
Sul1 genes, there is 7 plants of genes containing Sul2, have 1 plant containing Sul3 genes, have 6 plants to contain 2 kinds or gene of more than two kinds；4 plants quick
Any drug resistant gene is not detected in sense bacterium, coincidence rate is 100% compared with drug sensitive test result.Detect the bacterium of drug resistant gene
Strain, its drug resistant gene concentration is 109~106Copy/mL bacterium solutions.
3 conclusions and discussion
Survey using Method of paper diffusion to 72 plants of aquatic livestock source clinical strains to sulfa drugs sensitiveness
Determine result to show, 72 plants of bacterial strains are respectively 68.1% and 62.5% to the resistant rate of SD and SMZ, to 2 kinds of friendships of sulfa drugs
Fork resistant rate is 80.8%, and only 16 plants bacteriums have 4 plants to SD or SMZ sensitivities to quick in SD or SMZ, and its resistant rate compares Guangxi
Rofe source of fish pathogenic bacteria are high to the resistant rate (53.3%) of sulfa drugs, illustrate Guangxi aquatic livestock source bacterial strain to sulfonamides
The resistance phenomenon of thing is very serious, and has the trend of continuous exacerbation.Resistant rate and other livestock and poultry animal source bacterial strains in this experiment
Resistant rate to sulfa drugs is close, illustrates that sulfa drugs easily produces drug resistance in use, and this is with medication frequently
Rate is relevant with medication intensity, also produces the molecular mechanism of drug resistance closely related sulfa drugs with bacterium, because bacterium pair
The drug resistant gene of sulfonamides is predominantly located on plasmid or transposons, can easily in same kind or the bacterium of different genera
Between be transferred on another plasmid or chromosome from a plasmid.There are some researches show pathogen is to the easy generation of sulfa drugs
Cross resistance, this result of the test has also confirmed this conclusion.
Conserved sequence in this research according to Sul1, Sul2 and Sul3 gene in GenBank designs specific primer and phase
TaqMan probe is answered, and primed probe concentration proportioning, reaction system and reaction condition etc. are optimized, establishing can be while examines
Survey the triple fluorescent quantitative PCR method of above-mentioned 3 kinds of drug resistant genes.Sensitiveness, specificity and reperformance test result of the test show,
The method quantification range is wide, and detection template scope is 1 × 101~1 × 108During copy/reaction, its standard curve is in good line
Sexual intercourse；The method sensitivity is high, and the minimum plasmids detection limit of Sul1, Sul2 and Sul3 can reach 10 copies/reaction；The party
Method high specificity, with other pathogens and viral no cross reaction；The method is reproducible, and the coefficient of variation is respectively less than 2%；Entirely
Detection process can be completed in 2h, meet the demand that sulfa drugs drug resistance quick detection is clinically carried out to bacterial strain, be had
Application value higher.
The resistance in 72 plants of aquatic livestock source clinical strains is detected with the triple fluorescent quantitative PCR method set up in this research
Gene Sul1, Sul2 and Sul3, as a result show, have in 52 plants of bacterial strains of resistance to SD or SMZ 49 plants detected Sul1 or Sul2 or
Sul3 genes, the coincidence rate with drug sensitive test result is 94.2%, with accuracy higher；Wherein, 45 plants (account for
86.5%) detect Sul1 or (and) Sul2 genes, only 4 plants (accounting for 7.7%) detects Sul3 genes, illustrate aquatic products move
Material resource bacterial strain (91.8%) based on Sul1 and Sul2 genes, with livestock and poultry animal source bacterial strain (2/3 Sul1 and Sul2 genes, 1/3
Sul3 genes) differ larger.The bacterial strain of drug resistant gene is detected in this research, the copy number of its drug resistant gene is all higher, is
109~106Copy/mL bacterium solutions (about 108Individual bacterium), illustrate that drug resistant gene can singly copy/multicopy presence in single bacterium,
But drug resistant gene copy number might not be proportionate with drug-resistant intensity, this is probably because the drug resistant gene becomes XOR and lacks in itself
Caused by mistake causes miopragia or function to be lost, it is also possible to which the drug resistant gene is suppressed without obtaining table in bacterial strain
Up to caused.It can be seen that, bacterial resistance gene expression is obtained with the relation value of drug-resistant phenotype and drug-resistant intensity further to be studied.Additionally, this
The method set up in research can be detected including salmonella, Shigella, Aeromonas hydrophila, tarda and kerekou pneumonia
The sulfonamides drug resistant gene of many kind bacteriums such as primary bacterium, illustrate the method can extensive use, while illustrate Sul1, Sul2 and
Sul3 genes are guarded very much in the bacterium of different genera.
In a word, the triple fluorescent quantitative PCR method of Sul1, Sul2 and Sul3 gene is detected while foundation in this research not
Only have the advantages that quick, easy, sensitive, accurate, quantitative, reproducible, moreover it is possible to distinguish different drug resistant genes, can be clinic
The quick detection of bacterium sulfa drugs drug resistant gene and epidemiology survey provide fast and convenient, precise and high efficiency detection hand
Section.
SEQUENCE LISTING
<110>Guangxi Zhuang Autonomous Region aquatic science research institute
<120>Primer, spy for aquatic livestock derived bacterium sulfa drugs drug resistant gene triple fluorescent quantitative PCR detections
Pin and its application method
<130>Primer, spy for aquatic livestock derived bacterium sulfa drugs drug resistant gene triple fluorescent quantitative PCR detections
Pin and its application method
<160> 9
<170> PatentIn version 3.3
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Claims (5)
1. the primer and probe of aquatic livestock derived bacterium sulfa drugs drug resistant gene triple fluorescent quantitative PCR detections are used for, its
It is characterised by including three groups of primers respectively for aquatic livestock derived bacterium sulfa drugs drug resistant gene Sul1, Sul2 and Sul3
And probe, three groups of primers and probe have following base sequence respectively：
2. according to claim 1 for aquatic livestock derived bacterium sulfa drugs drug resistant gene triple fluorescent quantitative PCR
The primer and probe of detection, it is characterised in that：5 ' the ends of described probe Sul1-P, Sul2-P, Sul3-P are marked with fluorescence respectively
Reporter group FAM, HEX, CY5,3 ' ends mark have BHQ1.
3. according to claim 1 for aquatic livestock derived bacterium sulfa drugs drug resistant gene triple fluorescent quantitative PCR
The primer and probe of detection, it is characterised in that the mol ratio of three groups of primers and probe is Sul1-F: Sul1-R: Sul1-P:
Sul2-F: Sul2-R: Sul2-P: Sul3-F: Sul3-R: Sul3-P=6: 6: 3: 4: 4: 2: 4: 4: 2.
4. aquatic livestock derived bacterium sulfa drugs drug resistant gene triple fluorescent quantitative PCR detections are used for described in claim 1
The application method of primer and probe, it is characterised in that aquatic livestock derived bacterium sulfonamides is expanded in triple fluorescent quantitative PCR
Thing drug resistant gene Sul1, Sul2 and Sul3.
5. examined for aquatic livestock derived bacterium sulfa drugs drug resistant gene triple fluorescent quantitative PCR according to claim 4
Survey the application method of primer and probe, it is characterised in that the reaction system and response procedures of the triple fluorescent quantitative PCR be：Instead
The system is answered to be：Premix Ex Taq (2 ×) 18 μ L, ROX Reference Dye II0.5 μ L, 20 μm of ol/L Sul1 is upper and lower
Trip primer (Sul1-F/Sul1-R) is 0.9 μ L, and 10 μm of ol/LSul1 probes (Sul1-P) consumptions are 0.9 μ L, 20 μm of ol/
The upstream and downstream primer (Sul2-F/Sul2-R, Sul3-F/Sul3-R) of LSul2 and Sul3 is 0.6 μ L, 10 μm of ol/LSul2
0.6 μ L, the μ L of template 3 are with Sul3 probes (Sul2-P, Sul3-P) consumption, with sterilizing ddH2O complements to 30 μ L；
Reaction condition is：95 DEG C of predegeneration 30s；95 DEG C of denaturation 5s, 60 DEG C of 45s that anneal and extend, expand 40 circulations；At each
Fluorescence signal is collected at the end of 60 DEG C of circulation.
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| CN110184364A (en) * | 2019-05-14 | 2019-08-30 | 天津科技大学 | A kind of multi-PCR detection method and application for detecting the legionella pneumophilia of carrying Sulfonamides-resistant genes |
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| CN108300769A (en) * | 2018-01-10 | 2018-07-20 | 南方医科大学南方医院 | The quadruple fluorescence quantifying PCR method of germ oligotrophy unit cell and sul1, sul2, sul3 drug resistant gene is detected simultaneously |
| CN109825557A (en) * | 2019-04-11 | 2019-05-31 | 河北工程大学 | A kind of method of Sulfonamides-resistant genes sulI in detection atmospheric environment |
| CN110184364A (en) * | 2019-05-14 | 2019-08-30 | 天津科技大学 | A kind of multi-PCR detection method and application for detecting the legionella pneumophilia of carrying Sulfonamides-resistant genes |
| CN112779326A (en) * | 2021-02-04 | 2021-05-11 | 广西壮族自治区兽医研究所 | GeXP multiplex PCR detection kit for simultaneously identifying drug-resistant genes of sulfonamides antibiotics and primer group thereof |
| CN112779326B (en) * | 2021-02-04 | 2022-12-06 | 广西壮族自治区兽医研究所 | GeXP multiplex PCR detection kit for simultaneously identifying sulfonamide antibiotic drug resistance genes and primer group thereof |
| CN116769869A (en) * | 2023-08-18 | 2023-09-19 | 哈尔滨工业大学(深圳)(哈尔滨工业大学深圳科技创新研究院) | Method for detecting sulfamethoxazole environmental concentration and environmental risk |
| CN116769869B (en) * | 2023-08-18 | 2023-11-10 | 哈尔滨工业大学(深圳)(哈尔滨工业大学深圳科技创新研究院) | Method for detecting sulfamethoxazole environmental concentration and environmental risk |
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