CN110184364A - A kind of multi-PCR detection method and application for detecting the legionella pneumophilia of carrying Sulfonamides-resistant genes - Google Patents
A kind of multi-PCR detection method and application for detecting the legionella pneumophilia of carrying Sulfonamides-resistant genes Download PDFInfo
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Abstract
The present invention relates to a kind of for detecting the multi-PCR detection method for carrying the legionella pneumophilia of Sulfonamides-resistant genes, and the method uses following two pairs of specific primers: F1:SEQ NO.1, i.e. 5 '-TCCGCCTGGACTAATCCTAAAG-3 ' when detecting；R1:SEQ NO.2, i.e. 5 '-CACCAACCATCCCTATCTCATTAC-3 '；F2:SEQ NO.3, i.e. 5 '-CGTATTGCGCCGCTCTTAG-3 '；R2:SEQ NO.4, i.e. 5 '-ATCTAACCCTCGGTCTCTGGC-3 '.Nucleic acid sequence of the present invention can be used for the drug resistance situation of the clinical quickly sulfa antibiotics of checkout and diagnosis patients with pneumonia pathogenic bacteria, provide reference frame for clinical treatment.
Description
Technical field
The invention belongs to technical field of microbial detection, are related to a kind of using molecular biology method detection microorganism drug resistance
The method of gene, it is especially a kind of for detecting the multi-PCR detection method for carrying the legionella pneumophilia of Sulfonamides-resistant genes
And application.
Background technique
Légionaires' disease is the acute infections of respiratory tract disease with environmental correclation, with human diseases relationship it is the closest be
Legionella pneumophilia (Legionella pneumophila, Lp).Therefore, legionella pneumophilia is the important disease for causing Legionnaires Pneumonia
Opportunistic pathogen.Since Legionella is parasitized breeding in the cell, antibiotic need to reach could preferably be killed in alveolar cell it is thin
Bacterium.Thoroughly treatment légionaires' disease need to select fat-soluble strong antibiotic just effective.Although the treatment common antibiotic of Legionella is
Macrolides or fluoroquinolone antibiotics, but fortimicin, rifampin, sulfonamides and fluorine quinoline promise class drug are also optional
With.
Sulfa drugs is artificial synthesized antimicrobial, and for nearly 50 years clinical, it has antimicrobial spectrum compared with wide, property is steady
It is fixed, price is low, using easy, production when the advantages that not consuming grain, therefore be widely used.Although a large amount of antibiosis Plain Questions
Generation, but sulfa drugs is still important chemotherapeutic agent.As sulfa drugs uses on a large scale, clinical various bacteriums
Separation strains its resistant rate is constantly risen.There is also apparent differences for resistance mechanism of all kinds of bacteriums to sulfa drugs.(1)
Wherein the sulfanilamide (SN) resistance mechanism of staphylococcus aureus is persister excess generation aminobenzoic acid (PABA), and confrontation sulfanilamide (SN) is competing
It strives.(2) the sulfanilamide (SN) resistance mechanism of streptococcus pneumonia is that the dihydropteroic acid synthetase-coding gene of persister is mutated.(3) gram
The sulfanilamide (SN) resistance mechanism of negative bacterium is different from them, and sulfanilamide (SN) resistance mechanism is divided into two kinds, and one is that bacterium obtains sul gene
(coding dihydropteroic acid synzyme), so that bacterium dihydropteroic acid synzyme intracellular increases, sulfanilamide (SN) and PABA do not constitute competitive relation.
Secondly being obtained dfr gene (coding dihyrofolate reductase) for bacterium, antagonism trimethoprim, so that folic acid synthesis is not by shadow
It rings.Therefore Gram-negative bacteria expresses sul gene and dfr gene, and folic acid can be synthesized normally to which bacterium is not by sulphur in bacterium
The influence of amine drug.Research shows that: sul gene and dfr gene can be by the removable heredity such as plasmid, integron, transposons
Element mediates, therefore easily propagates in bacterium of the same race and not of the same race.
The detection to drug resistant gene is carried out from molecular level both at home and abroad, clinical sample Resistant genetype has been examined at present
The method of survey has very much, wherein the Molecular tools inspection based on DNA probe, polymerase chain reaction (PCR), quantitative fluorescent PCR etc.
Drug resistant gene is surveyed to have made great progress.When carrying out detection drug resistant gene using these Molecular tools, gene detected is past
Past is the gene order being widely present, and has ignored some genes with specific gene sequences.
By comparing discovery in the gene order library of NCBI, Sulfonamides-resistant genes sul1 is for example big in Gram-negative bacteria
Enterobacteria, pseudomonas aeruginosa, salmonella, Klebsiella pneumoniae, enterobacter cloacae, shigella dysenteriae, Freund citric acid bar
Conserved sequence homology in the various bacterias such as bacterium, corynebacterium, Aeromonas hydrophila, shigella flexneri is up to 100%, and
In Gram-negative bacteria legionella pneumophilia there is specificity in the sequence of sul1 gene, only with the homologys of other Gram-negative bacterias
It is 14.3%.Therefore in the method for most of detection Sulfonamides-resistant genes sul1, this specific sequence is all had ignored.It leads
It causes existing to cover the detection method of Sulfonamides-resistant genes sul1 well all Sulfonamides-resistant genes sul1.
Comprehensive inspection not can be carried out for the drug resistance situation of the sulfa antibiotics of clinical quick checkout and diagnosis patients with pneumonia pathogenic bacteria
It surveys.
By retrieval, such as next chapter patent publication us relevant to present patent application is found:
A kind of animal derived bacterium sulfa drugs drug resistant gene multiplex PCR detection technique (CN1944670), this method include
The three pairs of PCR primers and pcr template reagent preparation and bacterial resistance gene multiplexed PCR amplification of amplification tested bacteria drug resistant gene
Reagent can expand the drug resistant gene of strain to be tested sulfonamides using the multiplex PCR detection technique.It has high sensitivity, spy
Anisotropic height has a wide range of application, as a result accurate and reliable feature.
By comparison, there is essential difference in present patent application and above-mentioned patent publication us.
Summary of the invention
Place that the purpose of the present invention is to overcome the deficiency in the prior art provides a kind of for detecting carrying sulfamido drug resistance base
The multi-PCR detection method of the legionella pneumophilia of cause and application.Nucleic acid sequence of the present invention can be used for clinical quickly detection
The drug resistance situation of the sulfa antibiotics of diagnosis of pneumonia patient's pathogenic bacteria, provides reference frame for clinical treatment.
The technical solution adopted by the present invention to solve the technical problems is:
It is a kind of for detect carry Sulfonamides-resistant genes legionella pneumophilia multi-PCR detection method, the method
Following two pairs of specific primers are used when detecting:
F1:SEQ NO.1, i.e. 5 '-TCCGCCTGGACTAATCCTAAAG-3 '；
R1:SEQ NO.2, i.e. 5 '-CACCAACCATCCCTATCTCATTAC-3 '；
F2:SEQ NO.3, i.e. 5 '-CGTATTGCGCCGCTCTTAG-3 '；
R2:SEQ NO.4, i.e. 5 '-ATCTAACCCTCGGTCTCTGGC-3 '.
Moreover, the method is when detecting, and the target fragment length different sizes after amplification, legionella pneumophilia sul1 gene
Primer size is 1390bp after amplification, is lp-sul1 by this unnamed gene；Other gram-negatives in addition to legionella pneumophilia
Property bacterium Sulfonamides-resistant genes sul1 amplification after primer size be 643bp, by this unnamed gene be nor-sul1, pass through fine jade
Sepharose electrophoresis directly carries out separating discrimination.
Moreover, described other Gram-negative bacterias in addition to legionella pneumophilia are Escherichia coli, pseudomonas aeruginosa, sand
Door Salmonella, Klebsiella pneumoniae, enterobacter cloacae, shigella dysenteriae, citrobacter freundii, corynebacterium, thermophilic aqueous vapor list
Born of the same parents bacterium or shigella flexneri.
Moreover, steps are as follows:
(1) the preparation of DNA profiling: carry out culture carrying Sulfonamides-resistant genes sul1 in the medium respectively removes thermophilic lung
Other Gram-negative bacterias except Legionella and the culture carrying sulfamido drug resistance base in charcoal yeast extract culture medium
Because of the legionella pneumophilia of sul1, the extracting of DNA is carried out respectively, the DNA of extraction is saved -20, and the detection mould as PCR
Plate；
(2) multi-PRC reaction system: using the Plasmid DNA no more than 50ng or the genomic DNA no more than 200ng as mould
Plate, using two pairs of primers Fs 1 and R1, F2 and R2, final concentration be 0.2 μM, include dNTPs and 2mM Mg2+2 ×
12.5 μ L of PCRbuffer, 0.5 μ L of archaeal dna polymerase, finally uses ddH2O is mended to 25 μ L；
(3) multi-PRC reaction program: 94 DEG C of 2min of initial denaturation；98 DEG C of 10s are denaturalized, anneal 57 DEG C of 30s, extends 68 DEG C of 45s
Totally 30 circulations；Extend 68 DEG C of 2min eventually；Product is saved at 4 DEG C；
(4) 2-5 μ L multiple PCR products are taken, are mixed with 2 μ 6 × Loading of L buffer, multiple PCR products are through 1.0% fine jade
Sepharose carries out electrophoresis detection；
The electrophoretic band judging result presented according to gel imaging system: if only there is specific band at 1390bp,
Illustrate there is the legionella pneumophilia for carrying Sulfonamides-resistant genes sul1 in the DNA sample；If only there is specificity at 643bp
Band illustrates there are other bacterial strains for carrying Sulfonamides-resistant genes sul1 in the DNA sample；If in 643bp and 1390bp
There is specific band at place, illustrates that the bacterium is to carry the legionella pneumophilia of Sulfonamides-resistant genes sul1 and exist simultaneously carrying
Other bacterial strains of Sulfonamides-resistant genes sul1；No band is then feminine gender, illustrates the bacterium neither existing and carries sulfamido drug resistance
The legionella pneumophilia of gene sul1, nor there are other bacterial strains for carrying Sulfonamides-resistant genes sul1.
Multi-PCR detection method as described above for detecting the legionella pneumophilia of carrying Sulfonamides-resistant genes is fast
Application in terms of the drug resistance situation of the sulfa antibiotics of fast checkout and diagnosis pathogenic bacteria.
The advantages of present invention obtains and good effect are as follows:
1, there is the primer that the method for the present invention uses preferable specificity and conservative, the reaction can quickly detect sample
Middle hybrid dna sample whether there is target gene nor-sul1, and can detect simultaneously in sample with the presence or absence of carrying sulfamido
The legionella pneumophilia of drug resistant gene lp-sul1.Nucleic acid sequence of the present invention can be used for clinical quickly checkout and diagnosis pneumonia and suffer from
The drug resistance situation of the sulfa antibiotics of person's pathogenic bacteria, provides reference frame for clinical treatment.
2, the invention discloses the primers that the legionella pneumophilia multiplex PCR for carrying Sulfonamides-resistant genes quickly detects
And its application, this method can be detected in legionella pneumophilia simultaneously in Sulfonamides-resistant genes sul1 and other Gram-negative bacterias
Sul1 gene, the primer that the method for the present invention uses have good specificity, and this method can quickly detect in primary first-order equation
Whether legionella pneumophilia and Sulfonamides-resistant genes sul1 out are can detecte out in environmental samples DNA in the short time containing carrying
The legionella pneumophilia of Sulfonamides-resistant genes sul1, while whether can detect in the mixing sample containing carrying sulfamido drug resistance base
Because of other Gram-negative bacterias of sul1, such as Escherichia coli, pseudomonas aeruginosa, salmonella, Klebsiella pneumoniae, cloaca
In the various bacterias such as enterobacteria, shigella dysenteriae, citrobacter freundii, corynebacterium, Aeromonas hydrophila, shigella flexneri
Sulfonamides-resistant genes sul1.Without specific band, non-false positive, without expensive instrument, because the method is more accurate, just
It is prompt, efficient.The present invention can provide certain foundation for clinical treatment pneumonia.
Detailed description of the invention
Fig. 1 is electrophoresis detection result figure of the multiple PCR products through 1.0% Ago-Gel in the present invention；Wherein, swimming lane 1
For blank control, swimming lane 2 is nor-sul1 gene substance PCR, and swimming lane 3 is lp-sul1 gene substance PCR, and swimming lane 4 is nor-
Sul1 gene and lp-sul1 gene multiplex PCR；
Fig. 2 is the electrophoresis detection result figure of template minimum detection limit in multiplex PCR system in the present invention；Wherein, swimming lane 1-7
It is followed successively by using 1-7 in table 2 as the PCR result of template.
Specific embodiment
The embodiment of the present invention is described in detail below, it should be noted that the present embodiment is narrative, is not limited
, this does not limit the scope of protection of the present invention.
Raw material used in the present invention is unless otherwise specified conventional commercial product；Used in the present invention
Method is unless otherwise specified the conventional method of this field.
It is a kind of for detect carry Sulfonamides-resistant genes legionella pneumophilia multi-PCR detection method, the method
Following two pairs of specific primers are used when detecting:
F1:SEQ NO.1, i.e. 5 '-TCCGCCTGGACTAATCCTAAAG-3 '；
R1:SEQ NO.2, i.e. 5 '-CACCAACCATCCCTATCTCATTAC-3 '；
F2:SEQ NO.3, i.e. 5 '-CGTATTGCGCCGCTCTTAG-3 '；
R2:SEQ NO.4, i.e. 5 '-ATCTAACCCTCGGTCTCTGGC-3 '.
More preferably, the method when detecting, the target fragment length different sizes after amplification, legionella pneumophilia sul1 base
Primer size is 1390bp after gene-amplification, is lp-sul1 by this unnamed gene；Other grams in addition to legionella pneumophilia
Primer size is 643bp after the Sulfonamides-resistant genes sul1 amplification of negative bacterium, is nor-sul1 by this unnamed gene, passes through
Agarose gel electrophoresis directly carries out separating discrimination.
More preferably, described other Gram-negative bacterias in addition to legionella pneumophilia be Escherichia coli, pseudomonas aeruginosa,
Salmonella, Klebsiella pneumoniae, enterobacter cloacae, shigella dysenteriae, citrobacter freundii, corynebacterium, thermophilic aqueous vapor
Monad or shigella flexneri.
More preferably, steps are as follows:
(1) the preparation of DNA profiling: carry out culture carrying Sulfonamides-resistant genes sul1 in the medium respectively removes thermophilic lung
Other Gram-negative bacterias except Legionella and the culture carrying sulfamido drug resistance base in charcoal yeast extract culture medium
Because of the legionella pneumophilia of sul1, the extracting of DNA is carried out respectively, the DNA of extraction is saved -20, and the detection mould as PCR
Plate；
(2) multi-PRC reaction system: using the Plasmid DNA no more than 50ng or the genomic DNA no more than 200ng as mould
Plate, using two pairs of primers Fs 1 and R1, F2 and R2, final concentration be 0.2 μM, include dNTPs and 2mM Mg2+2 ×
12.5 μ L of PCRbuffer, 0.5 μ L of archaeal dna polymerase, finally uses ddH2O is mended to 25 μ L；
(3) multi-PRC reaction program: 94 DEG C of 2min of initial denaturation；98 DEG C of 10s are denaturalized, anneal 57 DEG C of 30s, extends 68 DEG C of 45s
Totally 30 circulations；Extend 68 DEG C of 2min eventually；Product is saved at 4 DEG C；
(4) 2-5 μ L multiple PCR products are taken, are mixed with 2 μ 6 × Loading of L buffer, multiple PCR products are through 1.0% fine jade
Sepharose carries out electrophoresis detection；
The electrophoretic band judging result presented according to gel imaging system: if only there is specific band at 1390bp,
Illustrate there is the legionella pneumophilia for carrying Sulfonamides-resistant genes sul1 in the DNA sample；If only there is specificity at 643bp
Band illustrates there are other bacterial strains for carrying Sulfonamides-resistant genes sul1 in the DNA sample；If in 643bp and 1390bp
There is specific band at place, illustrates that the bacterium is to carry the legionella pneumophilia of Sulfonamides-resistant genes sul1 and exist simultaneously carrying
Other bacterial strains of Sulfonamides-resistant genes sul1；No band is then feminine gender, illustrates the bacterium neither existing and carries sulfamido drug resistance
The legionella pneumophilia of gene sul1, nor there are other bacterial strains for carrying Sulfonamides-resistant genes sul1.
Multi-PCR detection method as described above for detecting the legionella pneumophilia of carrying Sulfonamides-resistant genes can
It applies in terms of the drug resistance situation of the sulfa antibiotics of quick checkout and diagnosis pathogenic bacteria.
Specifically, a kind of for detecting the multi-PCR detection method for carrying the legionella pneumophilia of Sulfonamides-resistant genes, institute
It states method and uses two pairs of specific primers such as table 1 when detecting:
The present invention is according to the Sulfonamides-resistant genes sul1 of legionella pneumophilia and the sulfamido drug resistance base of Gram-negative bacteria
Because the feature target gene and sequence design of sul1 go out two couples of specific primers F1/R1, F2/R2, it is shown in Table 1, wherein legionella pneumophilia
With primers F 1/R1, the size of product is 1390bp after amplification: other Gram-negative bacterias share pair of primers F2/R2, Qi Tage
Lan Shi negative bacterium is (for example, Escherichia coli, pseudomonas aeruginosa, salmonella, Klebsiella pneumoniae, enterobacter cloacae, dysentery
Bacillus, citrobacter freundii, corynebacterium, Aeromonas hydrophila or shigella flexneri etc.) size of product is after amplification
643bp.Each specific primer has the conservative of height, expands post-fragment different sizes, can directly be distinguished by electrophoresis.
1 multiplex PCR of table quickly detects specific primer sequence
More preferably, the step of specific detection method is as follows:
1, the preparation of DNA profiling: the Escherichia coli of culture carrying Sulfonamides-resistant genes (sul1) and work in LB respectively
Property charcoal yeast extract (BCYE) culture medium in culture carry Sulfonamides-resistant genes (sul1) legionella pneumophilia, according to difference
Bacterial genomes extracts kit method carries out the extracting of DNA, the DNA of extraction is saved -20, and the detection mould as PCR
Plate；
2, multi-PRC reaction system: using the Plasmid DNA no more than 50ng or the genomic DNA no more than 200ng as mould
Plate, two pairs of primers described in table 1, final concentration are 0.2 μM, and 2 × PCRbuffer, 12.5 μ L (includes dNTPs and 2mM
Mg2+), 0.5 μ L of archaeal dna polymerase finally uses ddH2O is mended to 25 μ L；
3, multi-PRC reaction program: 94 DEG C of 2min of initial denaturation；98 DEG C of 10s are denaturalized, anneal 57 DEG C of 30s, extends 68 DEG C of 45s
Totally 30 circulations；Extend 68 DEG C of 2min eventually；Product is saved at 4 DEG C；
4, PCR product is sequenced: DNA sequencing carried out to positive PCR product of the PCR containing drug resistant gene respectively, examining order by
Suzhou Jin Weizhi Biotechnology Co., Ltd completes；
5,2 μ L PCR products are taken, are mixed with 2 μ 6 × Loading of L buffer, multiple PCR products are through 1.0% agarose
Gel carries out electrophoresis detection.The electrophoretic band judging result presented according to gel imaging system.If only there is spy at 1390bp
Anisotropic band illustrates there is the legionella pneumophilia for carrying Sulfonamides-resistant genes sul1 in the DNA sample；If only in 643bp
There is specific band at place, illustrates there are the Escherichia coli for carrying Sulfonamides-resistant genes sul1 in the DNA sample；If
There is specific band at 643bp and 1390bp, illustrates that the bacterium is the legionella pneumophilia for carrying Sulfonamides-resistant genes sul1
And exist simultaneously the Escherichia coli for carrying Sulfonamides-resistant genes sul1；No band be then it is negative, illustrate the bacterium neither in the presence of
The legionella pneumophilia of Sulfonamides-resistant genes sul1 is carried, nor there is the large intestine bar for carrying Sulfonamides-resistant genes sul1
Bacterium.
By the observation to agarose gel electrophoresis results, each purpose band is all relatively clear, and without non-specificity
Band, this illustrates that the multiplex PCR system established in the present invention has specificity and sensitivity well, primarily determines that this is anti-
Answer system can sul1 drug resistant gene in effective test sample, and can specificity detect no to have that carry sulfamido resistance to
The legionella pneumophilia of medicine gene.
More specifically, it is a kind of for detecting the multi-PCR detection method for carrying the legionella pneumophilia of Sulfonamides-resistant genes,
Steps are as follows:
Material used is as follows:
(1) plasmid and bacterial strain
The plasmid of the lp-sul1 and nor-sul1 on pMD-19 are constructed, names pLP-T and pNOR-T respectively.Detection institute
Wild-type strain is commercial product.
(2) main agents
LB solid medium, LB liquid medium, (LB liquid medium has Amp resistance to Amp resistance, is in order to more preferable
The feasibility of ground detection primer)；Plasmid extraction kit is purchased from TIANGEN Biotech (Beijing) Co., Ltd.；KOD-Multi&Purchased from TOYOBO；Marker III is purchased from TIANGEN Biotech (Beijing) Co., Ltd.；TAE buffer, agarose, EB dye
Material.
(3) key instrument
37 DEG C of constant-temperature tables, 37 DEG C of constant incubators, PCR instrument, electrophoresis apparatus, gel imager.
Step are as follows:
1, for detecting the design of primers of sul1 gene and legionella pneumophilia sul1 gene in Gram-negative bacteria
According to the document delivered, due to Sulfonamides-resistant genes sul gene and dfr gene can by plasmid, integron,
The Mobile genetic elements such as transposons mediate, therefore easily propagate in Gram-negative bacteria bacterium of the same race and not of the same race.?
The sequence discovery of Sulfonamides-resistant genes (sul1) between different genera, the conserved sequence 840bp of sul1 gene are compared in NCBI
The homology of the Gram-negative bacteria of different genera has reached 100%, as Escherichia coli, pseudomonas aeruginosa, salmonella, gram
Thunder Bai Shi pulmonitis strain, enterobacter cloacae, shigella dysenteriae, citrobacter freundii, corynebacterium, Aeromonas hydrophila, Freund
Sulfonamides-resistant genes (sul1) in the various bacterias such as shigella dysenteriae, the present invention in by this section of sequence designations be nor-sul1.This
It is also the reason of only carrying out sul1 gene magnification with pair of primers in most of document.But further comparison discovery, Shi Fei legion
The homology of the sul1 gene of the sul1 gene and other bacteriums of bacterium is only 14.30%, and the present invention is a length of by legionella pneumophilia
The sul1 unnamed gene of 2307bp is lp-sul1.There is legionella pneumophilia Sulfonamides-resistant genes (sul1) inter-species of height to protect
Keeping property, consideration are since the length of this section of sequence is longer than the length of general drug resistant gene very much, and concrete reason also needs further
Experimental study proves.
Two to for expanding Gram-negative bacteria nor-sul1 gene and specific amplification legionella pneumophilia lp-sul1 draws
Object, the primer sequence of 2 drug resistant genes are as follows:
2, PCR primer specific detection
The legionella pneumophilia of Escherichia coli and sul1 gene to the gene containing sul1 carries out substance PCR respectively, then to institute
There is the combination of strain to carry out double PCR.
(1) PCR reaction system: KOD-Multi&2 × PCRbuffer, 12.5 μ L (includes dNTPs and 2mM Mg2 +), primer totally 2 μ L (two pairs of primers Fs 1 and R1, F2 and R2, final concentration be 0.2 μM), KOD-Multi&DNA is poly-
0.5 μ L of synthase, template totally 2 μ L, finally uses ddH2O is mended to 25 μ L
(2) multi-PRC reaction program: 94 DEG C of 2min of initial denaturation；98 DEG C of 10s are denaturalized, anneal 57 DEG C of 30s, extends 68 DEG C of 45s
Totally 30 circulations；Extend 68 DEG C of 2min eventually；Product is saved at 4 DEG C.
(3) 2 μ L PCR products are taken, are mixed with 2 μ 6 × Loading of L buffer, multiple PCR products are through 1.0% agarose
Gel carries out electrophoresis detection.
(4) results and discussion
As shown in Figure 1, according to gel imaging the results show that the substance PCR band of each gene is clear and single, instruction sheet
Preferably, amplification efficiency is high for the specificity of one primer.The multiplex PCR band of two genes is clear, without other non-specific amplifications, card
Bright two pairs of PCR primers in multiplex PCR system of the present invention, have reached higher amplification efficiency by optimization.It can be used for
The detection of late-run sample, to detect save the cost and time.
3, the analysis of multiplex PCR template minimum detection limit
(1) primer mixed liquor: by two pairs of primers, i.e. 4 nucleotide sequence (i.e. two pairs of specific primers: F1:SEQ
NO.1；R1:SEQ NO.2；F2:SEQ NO.3；R2:SEQ NO.4) give Suzhou Jin Weizhi Biotechnology Co., Ltd to synthesize,
Then it is mixed in a pipe, uses ddH2O dissolution, final concentration of 0.2 μM of every primer, -20 DEG C of preservations.
(2) prepared by template: carrying out 10 times of dilutions to two plasmids of pLP-T and pNOR-T, forms 109、108、107、106、
105、104、103Copies/ μ L concentration gradient.Template after dilution is mixed according to described in following table, hybrid template is named as
1-7 is saved -20.
The template of 2 minimal detectable concentration of table analysis
(3) PCR reaction system: KOD-Multi&2 × PCRbuffer, 12.5 μ L (includes dNTPs and 2mM Mg2 +), primer mixture 2 μ L, KOD-Multi&0.5 μ L of archaeal dna polymerase, 2 μ L of hybrid template, finally uses ddH2O is mended to 25 μ
L。
(4) multi-PRC reaction program: 94 DEG C of 2min of initial denaturation；98 DEG C of 10s are denaturalized, anneal 57 DEG C of 30s, extends 68 DEG C of 45s
Totally 30 circulations；Extend 68 DEG C of 2min eventually；Product is saved at 4 DEG C.
(5) 2 μ L PCR products are taken, are mixed with 2 μ 6 × Loading of L buffer, multiple PCR products are through 1.0% agarose
Gel carries out electrophoresis detection.
(6) results and discussion
As a result as shown in Fig. 2, as shown in Figure 2, using hybrid template 1-7 as template, there are two at 643bp and 1390bp
Specific band.The concentration of two plasmids gradually decreases in hybrid template 1-7, with the reduction of template concentrations,
Two electrophoretic band brightness at 643bp and 1390bp gradually weaken.When two templates are diluted to 103When Copies/ μ L,
Without electrophoretic band at 643bp and 1390bp, illustrate that multiplex PCR system of the invention reaches the minimum detection limit of two templates
To 104Copies/μL。
Although disclosing the embodiment of the present invention for the purpose of illustration, it will be appreciated by those skilled in the art that: not
Be detached from the present invention and spirit and scope of the appended claims in, various substitutions, changes and modifications be all it is possible, therefore, this
The range of invention is not limited to the embodiment and attached drawing disclosure of that.
Sequence table
<110>University Of Science and Technology Of Tianjin
<120>a kind of for detecting the multi-PCR detection method and the application that carry the legionella pneumophilia of Sulfonamides-resistant genes
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA/RNA
<213>primers F 1 (Unknown)
<400> 1
tccgcctgga ctaatcctaa ag 22
<210> 2
<211> 24
<212> DNA/RNA
<213>primer R1 (Unknown)
<400> 2
caccaaccat ccctatctca ttac 24
<210> 3
<211> 19
<212> DNA/RNA
<213>primers F 2 (Unknown)
<400> 3
cgtattgcgc cgctcttag 19
<210> 4
<211> 21
<212> DNA/RNA
<213>primer R2 (Unknown)
<400> 4
atctaaccct cggtctctgg c 21
Claims (5)
1. a kind of for detecting the multi-PCR detection method for carrying the legionella pneumophilia of Sulfonamides-resistant genes, it is characterised in that:
The method uses following two pairs of specific primers when detecting:
F1:SEQ NO.1；
R1:SEQ NO.2；
F2:SEQ NO.3；
R2:SEQ NO.4.
2. according to claim 1 for detecting the multiplex PCR detection for carrying the legionella pneumophilia of Sulfonamides-resistant genes
Method, it is characterised in that: the method when detecting, the target fragment length different sizes after amplification, legionella pneumophilia sul1
Primer size is 1390bp after gene magnification, is lp-sul1 by this unnamed gene；Other leather in addition to legionella pneumophilia are blue
Primer size is 643bp after the Sulfonamides-resistant genes sul1 amplification of family name's negative bacterium, is nor-sul1 by this unnamed gene, leads to
Agarose gel electrophoresis is crossed directly to carry out separating discrimination.
3. according to claim 1 for detecting the multiplex PCR detection for carrying the legionella pneumophilia of Sulfonamides-resistant genes
Method, it is characterised in that: described other Gram-negative bacterias in addition to legionella pneumophilia are Escherichia coli, P. aeruginosa
Bacterium, salmonella, Klebsiella pneumoniae, enterobacter cloacae, shigella dysenteriae, citrobacter freundii, corynebacterium, thermophilic water
Aeromonas or shigella flexneri.
4. according to any one of claims 1 to 3 for detecting the more of the legionella pneumophilia of carrying Sulfonamides-resistant genes
Weight PCR detection method, it is characterised in that: steps are as follows:
(1) the preparation of DNA profiling: carry out culture carrying Sulfonamides-resistant genes sul1 in the medium respectively removes Shi Fei legion
Other Gram-negative bacterias except bacterium and the culture carrying Sulfonamides-resistant genes in charcoal yeast extract culture medium
The legionella pneumophilia of sul1 carries out the extracting of DNA respectively, the DNA of extraction is saved -20, and the detection template as PCR；
(2) multi-PRC reaction system: using the Plasmid DNA no more than 50ng or the genomic DNA no more than 200ng as template, make
With two pairs of primers Fs 1 and R1, F2 and R2, final concentration is 0.2 μM, includes dNTPs and 2mM Mg2+2 × PCRbuffer
12.5 μ L, 0.5 μ L of archaeal dna polymerase, finally use ddH2O is mended to 25 μ L；
(3) multi-PRC reaction program: 94 DEG C of 2min of initial denaturation；98 DEG C of 10s are denaturalized, anneal 57 DEG C of 30s, extends 68 DEG C of 45s totally 30
A circulation；Extend 68 DEG C of 2min eventually；Product is saved at 4 DEG C；
(4) 2-5 μ L multiple PCR products are taken, are mixed with 2 μ 6 × Loading of L buffer, multiple PCR products are through 1.0% agarose
Gel carries out electrophoresis detection；
The electrophoretic band judging result presented according to gel imaging system: if only there is specific band at 1390bp, explanation
There is the legionella pneumophilia for carrying Sulfonamides-resistant genes sul1 in the DNA sample；If only there is specific item at 643bp
Band illustrates there are other bacterial strains for carrying Sulfonamides-resistant genes sul1 in the DNA sample；If at 643bp and 1390bp
There is specific band, illustrates that the bacterium is to carry the legionella pneumophilia of Sulfonamides-resistant genes sul1 and exist simultaneously carrying sulphur
Other bacterial strains of amine drug resistant gene sul1；No band is then feminine gender, illustrates the bacterium neither existing and carries sulfamido drug resistance base
Because of the legionella pneumophilia of sul1, nor there are other bacterial strains for carrying Sulfonamides-resistant genes sul1.
5. such as 1 to 4 described in any item multiplex PCR detection sides for being used to detect the legionella pneumophilia for carrying Sulfonamides-resistant genes
Method in terms of the drug resistance situation of the sulfa antibiotics of quick checkout and diagnosis pathogenic bacteria in application.
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