CN110295241A - For detecting the primer sets of urinary tract infections and comprising the kit of the primer sets - Google Patents
For detecting the primer sets of urinary tract infections and comprising the kit of the primer sets Download PDFInfo
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- CN110295241A CN110295241A CN201910627996.1A CN201910627996A CN110295241A CN 110295241 A CN110295241 A CN 110295241A CN 201910627996 A CN201910627996 A CN 201910627996A CN 110295241 A CN110295241 A CN 110295241A
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Abstract
The invention discloses a kind of for detecting the primer sets of urinary tract infections encountered pathogenic bacteria, the primer sets comprising 5 groups of SEQ ID NO.1-SEQ ID NO.10.The invention also discloses a kind of for detecting the kit of urinary tract infections.Number of base avoids primer from forming dimer, improves reaction efficiency, enhance the specificity of amplification by modification in primer sets of the present invention.Primer sets of the present invention, being capable of quick, sensitive, specifically urinary tract infections bacterium in test sample in the kit of urinary tract infections.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to one group is used to detect the primer of urinary tract infections and draws comprising this
The kit of object group
Background technique
Urinary tract infections is also known as urinary system infection contamination, is inflammatory reaction caused by urothelium invades bacterium.Urinary tract infections
95% or more is as caused by single bacterium.Wherein 50% or so patient, pathogen are Escherichia coli.Except large intestine angstrom
Outside uncommon bacterium, enterococcus, Pseudomonas aeruginosa, proteus mirabilis, the bacteriums such as klebsiella spp be common in infect again, indwelling catheter
Manage, have the urinary tract infections person of complication.Clinical data shows to be more than 95% urinary tract infections to be by Escherichia coli, intestines ball
Bacterium, Klebsiella Pneumoniae, pseudomonas aeruginosa and proteus mirabilis cause.
Clinically the detection common method of urinary tract infections bacterium has bacterial cultivation at present.Bacterial cultivation is detection urinary tract
The goldstandard of bacterial infection.But this method needs professional technician's sterile working to site requirements height.And this method detects
At high cost, time-consuming.It is generally necessary to which 3-5 days can just see final testing result.The clinical diagnosis of urinary tract infections microorganism and
Identification is badly in need of simply, quickly, detection method at low cost.Isothermal duplication can be with fast-amplifying nucleic acid sequence, and does not need essence
The instrument of close temperature control has very big potentiality in field of fast detection.Isothermal duplication does not need high temperature unwinding, reaction speed and spy
The opposite sex is highly dependent on the design and experiment screening of primer.The general length for increasing primer sequence can accelerate reaction speed, but
The primer increased simultaneously is easy itself to combine generation dimer, reduces specificity and sensitivity, limits isothermal amplification technique
Application.
Summary of the invention
For overcome the deficiencies in the prior art, goal of the invention of the invention be to provide one group it is normal for detecting urinary tract infections
The primer for seeing pathogen, the RPA primer by modification are most common for specifically quick 5 kinds for detecting urinary tract infections bacterium
Bacterium.
Another object of the present invention is to provide a kind of for detecting the kit of urinary tract infections, which includes to pass through
The RPA primer sets of modification overcome the problems of urinary tract infections bacterium Present clinical detection, are the diagnosis and essence of urinary tract infections
Quasi- treatment provides new detection device.
To achieve the goals above, the technical solution adopted in the present invention content is specific as follows:
For detecting the primer sets of urinary tract infections, include
Primer sets 1: upstream primer: ATTTCATAACCCCCTGTTTATTGTGTTA*A*T*G*G (SEQ ID NO.1)
Downstream primer: ATTTTGTTAAACTCAAACGTGATAATGA*G*A*A*T (SEQ ID NO.2)
Primer sets 2:
Upstream primer: AACTCTCTACGTCGTATTTTATTGTCTTC*T*T*T*A (SEQ ID NO.3)
Downstream primer: AAGGGTTTTCTCTTCTTCAGCATATAATT*T*C*A*A (SEQ ID NO.4)
Primer sets 3:
Upstream primer: ACACTTTTCTCAATAACACCGAGCAGGAG*G*T*T*C (SEQ ID NO.5)
Downstream primer: TAAAAAGGGGCGCTACTATAGCACAAAGAT*A*A*A*C (SEQ ID NO.6)
Primer sets 4:
Upstream primer: TATTCCTAAATATAGTCAAGTTTCTTGTG*A*T*G*T (SEQ ID NO.7)
Downstream primer: ATTAAAACGAAAAATATTAAGAATATCGA*C*C*C*C (SEQ ID NO.8)
Primer sets 5: upstream primer: CCTGTTGTTCGTTGCGATCATGGGTG*T*T*T*C (SEQ ID NO.9)
Downstream primer: CCAGTTCCACTTTGTCATTCTCGGTC*T*T*G*C (SEQ ID NO.10)
Wherein, A*:2- aminopurine nucleoside；T*:2- sulphur thymidine；C*:N- ethylcytosine；G*: hypoxanthine.
As further embodiment, in primer sets of the present invention, A*, T*, G*, C* can be with corresponding T, A, C, G shapes
At stable hydrogen bond, to be matched according to Watson-Crick principle.But A*, T*, G*, C* and T*, A*, C*, G* cannot
Stable hydrogen bond is formed, can not be matched according to Watson-Crick principle.Therefore it can show:
A* can be matched with T, but A* cannot be matched with T*；
T* can be matched with A, but T* cannot be matched with A*；
G* can be matched with C, but G* cannot be matched with C*；
C* can be matched with G, but C* cannot be matched with G*.
It is a kind of for detecting the kit of urinary tract infections, which includes reaction solution, reaction enzyme system and primer sets, described
Primer sets are
Primer sets 1, upstream primer: ATTTCATAACCCCCTGTTTATTGTGTTA*A*T*G*G (SEQ ID NO.1),
Downstream primer: ATTTTGTTAAACTCAAACGTGATAATGA*G*A*A*T (SEQ ID NO.2)；
Primer sets 2, upstream primer: AACTCTCTACGTCGTATTTTATTGTCTTC*T*T*T*A (SEQ ID NO.3),
Downstream primer: AAGGGTTTTCTCTTCTTCAGCATATAATT*T*C*A*A (SEQ ID NO.4)；
Primer sets 3: upstream primer:
ACACTTTTCTCAATAACACCGAGCAGGAG*G*T*T*C (SEQ ID NO.5),
Downstream primer: TAAAAAGGGGCGCTACTATAGCACAAAGAT*A*A*A*C (SEQ ID NO.6)；
Primer sets 4: upstream primer: TATTCCTAAATATAGTCAAGTTTCTTGTG*A*T*G*T (SEQ ID NO.7),
Downstream primer: ATTAAAACGAAAAATATTAAGAATATCGA*C*C*C*C (SEQ ID NO.8)；
Primer sets 5: upstream primer: CCTGTTGTTCGTTGCGATCATGGGTG*T*T*T*C (SEQ ID NO.9),
Downstream primer: CCAGTTCCACTTTGTCATTCTCGGTC*T*T*G*C (SEQ ID NO.10)；
Wherein, A*:2- aminopurine nucleoside；T*:2- sulphur thymidine；C*:N- ethylcytosine；G*: hypoxanthine.
As further preferred scheme, reaction solution of the present invention includes Tris-HCl, KCl, dithiothreitol (DTT), sweet tea
Dish alkali, trehalose, PEG, BSA.
As further preferred scheme, the pH of Tris-HCl of the present invention is 7~9
As further preferred scheme, reaction enzyme system of the present invention include dNTPs, polymerase Bsu, albumen because
Son, recombinase.
As further preferred scheme, protein factor of the present invention is albumen RecF, RecO, RecR, single stranded DNA
One of binding protein SSB or a variety of.
As further preferred scheme, recombinase of the present invention is cre bacterium recombinase RecA, Archimycetes recombinase
One of RadA, bacteriophage recombinase UvsX or eucaryote recombinase Rad51 or a variety of.
As further preferred scheme, kit of the present invention further includes starting liquid, and the starting liquid is chlorination
Magnesium.
As further preferred scheme, the concentration of magnesium chloride of the present invention is 5mM~20mM.
Compared with prior art, the beneficial effects of the present invention are:
1. number of base avoids primer from forming dimer, improves reaction by modification in primer sets of the present invention
Efficiency enhances the specificity of amplification.
2. primer sets of the present invention are for can quick, sensitive, specifically detect in the kit of urinary tract infections
Urinary tract infections bacterium in sample.
The above description is only an overview of the technical scheme of the present invention, in order to better understand the technical means of the present invention,
And it can be implemented in accordance with the contents of the specification, and in order to allow above and other objects, features and advantages of the invention can
It is clearer and more comprehensible, it is special below to lift preferred embodiment, and cooperate attached drawing, detailed description are as follows.
Detailed description of the invention
Fig. 1 is the primer sets amplification comparing result figure after unmodified primer sets and modification, wherein
A group indicates to carry out isothermal amplification using unmodified primer sets, generates primer dimer, reduce amplification
The specificity and sensibility of reaction；A:DNA ladder in A group, b: Escherichia coli, chuA gene (AF280396), c: intestines ball
Bacterium, rpoA gene, NC_004668, d: Klebsiella Pneumoniae, khe gene, AF293352, e: unusual deformed rod ureR gene,
Z18752, f: pseudomonas aeruginosa, lasB gene, JN118955；
B group indicates the primer sequence using the present invention after modifying wherein 4 bases；G in B group: Escherichia coli, chuA
Gene (AF280396), h: enterococcus, rpoA gene, NC_004668, i: Klebsiella Pneumoniae, khe gene, AF293352, j:
Unusual deformed rod ureR gene, Z18752, k: pseudomonas aeruginosa, lasB gene, JN118955.
Specific embodiment
It is of the invention to reach the technical means and efficacy that predetermined goal of the invention is taken further to illustrate, below in conjunction with
Attached drawing and preferred embodiment, to specific embodiment, structure, feature and its effect according to the present invention, detailed description are as follows:
The present invention is used to detect the primer sets of urinary tract infections, includes
Primer sets 1:
Target gene: Escherichia coli, chuA gene (AF280396)
Upstream primer: ATTTCATAACCCCCTGTTTATTGTGTTA*A*T*G*G
Downstream primer: ATTTTGTTAAACTCAAACGTGATAATGA*G*A*A*T
Primer sets 2:
Target gene: enterococcus, rpoA gene, NC_004668
Upstream primer: AACTCTCTACGTCGTATTTTATTGTCTTC*T*T*T*A
Downstream primer: AAGGGTTTTCTCTTCTTCAGCATATAATT*T*C*A*A
Primer sets 3:
Target gene: Klebsiella Pneumoniae, khe gene, AF293352
Upstream primer: ACACTTTTCTCAATAACACCGAGCAGGAG*G*T*T*C
Downstream primer: TAAAAAGGGGCGCTACTATAGCACAAAGAT*A*A*A*C
Primer sets 4:
Target gene: unusual deformed rod ureR gene, Z18752
Upstream primer: TATTCCTAAATATAGTCAAGTTTCTTGTG*A*T*G*T
Downstream primer: ATTAAAACGAAAAATATTAAGAATATCGA*C*C*C*C
Primer sets 5:
Target gene: pseudomonas aeruginosa, lasB gene, JN118955
Upstream primer: CCTGTTGTTCGTTGCGATCATGGGTG*T*T*T*C
Downstream primer: CCAGTTCCACTTTGTCATTCTCGGTC*T*T*G*C
Wherein, A*:2- aminopurine nucleoside；T*:2- sulphur thymidine；C*:N- ethylcytosine；G*: hypoxanthine.
In the present invention, in 5 groups of primer sets, with A*:2- aminopurine nucleoside, T*:2- sulphur thymidine, C*:N- ethyl
Cytimidine, G*: hypoxanthine is modified, and in the primer sets after modification, A* can be matched with T, but A cannot match with T*
It is right；T* can be matched with A, but T* cannot be matched with A*；G* can be matched with C, but G* cannot be matched with C*；C* can be with
G pairing, but C* cannot be matched with G*, it is possible to prevente effectively from primer forms dimer, improved reaction efficiency, enhanced amplification
Specificity.
It is a kind of for detecting the kit of urinary tract infections, which includes reaction solution, reaction enzyme system and primer sets, described
Primer sets are
Primer sets 1, upstream primer: ATTTCATAACCCCCTGTTTATTGTGTTA*A*T*G*G,
Downstream primer: ATTTTGTTAAACTCAAACGTGATAATGA*G*A*A*T；
Primer sets 2, upstream primer: AACTCTCTACGTCGTATTTTATTGTCTTC*T*T*T*,
Downstream primer: AAGGGTTTTCTCTTCTTCAGCATATAATT*T*C*A*A；
Primer sets 3: upstream primer:
ACACTTTTCTCAATAACACCGAGCAGGAG*G*T*T*C, downstream primer:
TAAAAAGGGGCGCTACTATAGCACAAAGAT*A*A*A*C；
Primer sets 4: upstream primer: TATTCCTAAATATAGTCAAGTTTCTTGTG*A*T*G*T,
Downstream primer: ATTAAAACGAAAAATATTAAGAATATCGA*C*C*C*C；
Primer sets 5: upstream primer: CCTGTTGTTCGTTGCGATCATGGGTG*T*T*T*C,
Downstream primer: CCAGTTCCACTTTGTCATTCTCGGTC*T*T*G*C；
Wherein, A*:2- aminopurine nucleoside；T*:2- sulphur thymidine；C*:N- ethylcytosine；G*: hypoxanthine.
As further preferred scheme, reaction solution of the present invention includes Tris-HCl, KCl, dithiothreitol (DTT), sweet tea
Dish alkali, trehalose, PEG, BSA.
As further preferred scheme, the pH of Tris-HCl of the present invention is 7~9, it is preferred that pH 8.3.
As further preferred scheme, reaction enzyme system of the present invention include dNTPs, polymerase Bsu, albumen because
Son, recombinase.
As further preferred scheme, protein factor of the present invention is albumen RecF, RecO, RecR, single stranded DNA
One of binding protein SSB or a variety of.
As further preferred scheme, recombinase of the present invention is cre bacterium recombinase RecA, Archimycetes recombinase
One of RadA, bacteriophage recombinase UvsX or eucaryote recombinase Rad51 or a variety of.
As further preferred scheme, kit of the present invention further includes starting liquid, and the starting liquid is chlorination
Magnesium.
As further preferred scheme, the concentration of magnesium chloride of the present invention is 5mM~20mM.
It is specific embodiment of the present invention below.
Embodiment 1
It is a kind of for detecting the kit of urinary tract infections, including reaction solution, reaction enzyme system, primer sets and starting liquid, institute
Reaction solution is stated by comprising Tris-HCl (pH8.3) 50mM, KCl 60mM, dithiothreitol (DTT) 2mM, glycine betaine 1M, trehalose
5.5%, PEG 5.5%, BSA 0.1mg/mL；The reaction enzyme system includes dNTPs200 μM, polymerase Bsu 50U, single-stranded knot
Hop protein 262ng/ μ l, E.coli RecA protein 36 0ng/ μ l；The primer sets are
ATTTCATAACCCCCTGTTTATTGTGTTA*A*T*G*G (upstream primer) 200nM,
ATTTTGTTAAACTCAAACGTGATAATGA*G*A*A*T (downstream primer) 200nM；The starting liquid is magnesium chloride, concentration
5mM。
It draws 1ml sample to be transferred in 1.5mlEP pipe, 12,000g centrifugations 5 minutes.Supernatant is abandoned, 100 microlitres of cracking are added
Liquid mixes.Heating boils 100 DEG C, 5 minutes, exposes the DNA of bacterium.It takes 10 microlitres to be added in above-mentioned reaction solution after cooling, incubates
It educates 10 minutes, amplification is observed with agarose electrophoresis.The target gene of detection is Escherichia coli, chuA gene
(AF280396)。
Comparative example 1
According to 1 operation of embodiment, difference is the primer sets that use for unmodified primer sets, i.e. upstream primer:
ATTTCATAACCCCCTGTTTATTGTGTTAATGG(SEQ ID NO.11)；Downstream primer: ATTTTGTTAAACTCAAACGT
GATAATGAGAAT(SEQ ID NO.12)。
Embodiment 2
It is a kind of for detecting the kit of urinary tract infections, including reaction solution, reaction enzyme system, primer sets and starting liquid, institute
Reaction solution is stated by comprising Tris-HCl (pH8.3) 50mM, KCl 60mM, dithiothreitol (DTT) 2mM, glycine betaine 1M, trehalose
5.5%, PEG 5.5%, BSA 0.1mg/mL；The reaction enzyme system includes dNTPs200 μM, polymerase Bsu 50U, single-stranded knot
Hop protein 262ng/ μ l, E.coli RecA protein 36 0ng/ μ l；The primer sets are AACTCTCTACGTCGTATTTTATTGT
CTTC*T*T*T*A (upstream primer) 200nM, AAGGGTTTTCTCTTCTTCAGCATATAA TT*T*C*A*A (downstream primer)
200nM；The starting liquid is magnesium chloride, concentration 8mM.
It draws 1ml sample to be transferred in 1.5mlEP pipe, 12,000g centrifugations 5 minutes.Supernatant is abandoned, 100 microlitres of cracking are added
Liquid mixes.Heating boils 100 DEG C, 5 minutes, exposes the DNA of bacterium.It takes 10 microlitres to be added to after cooling and draws 200 μ l samples,
The DNA for boiling exposed bacterium is added in above-mentioned reaction solution, is incubated for 15 minutes, observes amplification with agarose electrophoresis.Inspection
The target gene of survey is enterococcus, rpoA gene, NC_004668.
Comparative example 2
According to 2 operation of embodiment, difference is the primer sets that use for unmodified primer sets, i.e. upstream primer:
AACTCTCTACGTCGTATTTTATTGTCTTCTTTA(SEQ ID NO.13)；Downstream primer: AAGGGTTTTCTCTTCTTCA
GCATATAATTTCAA(SEQ ID NO.14)。
Embodiment 3
It is a kind of for detecting the kit of urinary tract infections, including reaction solution, reaction enzyme system, primer sets and starting liquid, institute
Reaction solution is stated by comprising Tris-HCl (pH8.3) 50mM, KCl 60mM, dithiothreitol (DTT) 2mM, glycine betaine 1M, trehalose
5.5%, PEG 5.5%, BSA 0.1mg/mL；The reaction enzyme system includes dNTPs200 μM, polymerase Bsu 50U, single-stranded knot
Hop protein 262ng/ μ l, E.coli RecA protein 36 0ng/ μ l；The primer sets are ACACTTTTCTCAATAACACCGAGC
AGGAG*G*T*T*C (upstream primer) 200nM, TAAAAAGGGGCGCTACTATAGCACA AAGAT*A*A*A*C (draw in downstream
Object) 200nM；The starting liquid is magnesium chloride, concentration 10mM.
It draws 1ml sample to be transferred in 1.5mlEP pipe, 12,000g centrifugations 5 minutes.Supernatant is abandoned, 100 microlitres of cracking are added
Liquid mixes.Heating boils 100 DEG C, 5 minutes, exposes the DNA of bacterium.It takes 10 microlitres to be added in above-mentioned reaction solution after cooling, incubates
It educates 10 minutes, amplification is observed with agarose electrophoresis.Testing goal gene be Klebsiella Pneumoniae, khe gene,
AF293352。
Comparative example 3
According to 5 operation of embodiment, difference is the primer sets that use for unmodified primer sets, i.e. upstream primer:
ACACTTTTCTCAATAACACCGAGCAGGAGGTTC(SEQ ID NO.15)；Downstream primer: TAAAAAGGGGCGCTACTAT
AGCACAAAGATAAAC(SEQ ID NO.16)。
Embodiment 4
It is a kind of for detecting the kit of urinary tract infections, including reaction solution, reaction enzyme system, primer sets and starting liquid, institute
Reaction solution is stated by comprising Tris-HCl (pH8.3) 50mM, KCl 60mM, dithiothreitol (DTT) 2mM, glycine betaine 1M, trehalose
5.5%, PEG 5.5%, BSA 0.1mg/mL；The reaction enzyme system includes dNTPs200 μM, polymerase Bsu 50U, single-stranded knot
Hop protein 262ng/ μ l, E.coli RecA protein 36 0ng/ μ l；The primer sets are TATTCCTAAATATAGTCAAGTTTCTT
GTG*A*T*G*T (upstream primer) 200nM, ATTAAAACGAAAAATATTAAGAATA TCGA*C*C*C*C (downstream primer)
200nM；The starting liquid is magnesium chloride, concentration 15mM.
It draws 1ml sample to be transferred in 1.5mlEP pipe, 12,000g centrifugations 5 minutes.Supernatant is abandoned, 100 microlitres of cracking are added
Liquid mixes.Heating boils 100 DEG C, 5 minutes, exposes the DNA of bacterium.It takes 10 microlitres to be added in above-mentioned reaction solution after cooling, incubates
It educates 15 minutes, amplification is observed with agarose electrophoresis.Testing goal gene is unusual deformed rod ureR gene, Z18752.
Comparative example 4
According to 4 operation of embodiment, difference is the primer sets that use for unmodified primer sets, i.e. upstream primer:
TATTCCTAAATATAGTCAAGTTTCTTGTGATGT(SEQ ID NO.17)；Downstream primer: ATTAAAACGAAAAATATTA
AGAATATCGACCCC(SEQ ID NO.18)。
Embodiment 5
It is a kind of for detecting the kit of urinary tract infections, including reaction solution, reaction enzyme system, primer sets and starting liquid, institute
Reaction solution is stated by comprising Tris-HCl (pH8.3) 50mM, KCl 60mM, dithiothreitol (DTT) 2mM, glycine betaine 1M, trehalose
5.5%, PEG 5.5%, BSA 0.1mg/mL；The reaction enzyme system includes dNTPs200 μM, polymerase Bsu 50U, single-stranded knot
Hop protein 262ng/ μ l, E.coli RecA protein 36 0ng/ μ l；The primer sets are CCTGTTGTTCGTTGCGATC
ATGGGTG*T*T*T*C (upstream primer) 200nM, CCAGTTCCACTTTGTCATTC TCGGTC*T*T*G*C (downstream primer)
200nM；The starting liquid is magnesium chloride, concentration 20mM.
It draws 1ml sample to be transferred in 1.5mlEP pipe, 12,000g centrifugations 5 minutes.Supernatant is abandoned, 100 microlitres of cracking are added
Liquid mixes.Heating boils 100 DEG C, 5 minutes, exposes the DNA of bacterium.It takes 10 microlitres to be added in above-mentioned reaction solution after cooling, incubates
It educates 10 minutes, amplification is observed with agarose electrophoresis.The target gene of detection be pseudomonas aeruginosa, lasB gene,
JN118955。
Comparative example 5
According to 5 operation of embodiment, difference is the primer sets that use for unmodified primer sets, i.e. upstream primer:
CCTGTTGTTCGTTGCGATCATGGGTGTTTC (SEQ ID NO.19) downstream primer:
CCAGTTCCACTTTGTCATTCTCGGTCTTGC(SEQ ID NO.20)。
The electrophoresis result of embodiment 1- embodiment 5 and documents 1- documents 5 is referring to Fig. 1.The result of Fig. 1 is aobvious
Show, unmodified primer pair used by comparative example 1- comparative example 5 can in test generate primer dimer, reduce expansion
Increase the specificity and sensibility of reaction；And embodiment 1- implements 5 using the primer pair after modification, effectively avoids generating and draw
Object dimer enhances the specificity and sensibility of amplified reaction.
Application Example
100 parts of urine specimens are collected, the sensibility and specificity of urinary tract infections detection reagent is tested.
1. urine culture.1 microlitre is taken to be seeded to blood agar plate (5% sheep blood) in super-clean bench.37 DEG C, aerobic environment culture
12 hours, 24 hours, 48 hours.10 clones are occurred more than on single plate, show 10 > in urine4CFU·mL-1, explanation
It is positive sample.It otherwise is negative sample.As a result referring to table 1.
2. taking 1 milliliter of urine specimen, it is transferred in 1.5mlEP pipe, 12000g is centrifuged 5 minutes.Supernatant is abandoned, it is micro- to be added 100
Lysate is risen, is mixed.Heating boils 100 DEG C, 5 minutes.10 microlitres are taken to be added to urinary tract infections inspection of the present invention after cooling
It in test agent box reaction solution, is incubated for 10 minutes, agarose observes result.There is specific band, then explanation is positive sample.It is no
It is then negative sample.As a result referring to table 1.
Table 1
Wherein Positive UTI indicates urinary tract infections positive sample example, and Negative UTI indicates urinary tract infections negative sample
Example.Table 1 the result shows that: the primer sets and kit comprising the primer sets that we design can rapidly and sensitively detect urine
Road feel dye.
The above embodiment is only the preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto,
The variation and replacement for any unsubstantiality that those skilled in the art is done on the basis of the present invention belong to institute of the present invention
Claimed range.
Sequence table
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Claims (10)
1. the primer sets for detecting urinary tract infections, which is characterized in that include
Primer sets 1,
Upstream primer: ATTTCATAACCCCCTGTTTATTGTGTTA*A*T*G*G,
Downstream primer: ATTTTGTTAAACTCAAACGTGATAATGA*G*A*A*T；
Primer sets 2,
Upstream primer: AACTCTCTACGTCGTATTTTATTGTCTTC*T*T*T*A,
Downstream primer: AAGGGTTTTCTCTTCTTCAGCATATAATT*T*C*A*A；
Primer sets 3,
Upstream primer: ACACTTTTCTCAATAACACCGAGCAGGAG*G*T*T*C,
Downstream primer: TAAAAAGGGGCGCTACTATAGCACAAAGAT*A*A*A*C；
Primer sets 4:
Upstream primer: TATTCCTAAATATAGTCAAGTTTCTTGTG*A*T*G*T,
Downstream primer: ATTAAAACGAAAAATATTAAGAATATCGA*C*C*C*C；
Primer sets 5:
Upstream primer: CCTGTTGTTCGTTGCGATCATGGGTG*T*T*T*C,
Downstream primer: CCAGTTCCACTTTGTCATTCTCGGTC*T*T*G*C；
Wherein, A*:2- aminopurine nucleoside；T*:2- sulphur thymidine；C*:N- ethylcytosine；G*: hypoxanthine.
2. primer sets according to claim 1, which is characterized in that
A* can be matched with T, but A* cannot be matched with T*；
T* can be matched with A, but T* cannot be matched with A*；
G* can be matched with C, but G* cannot be matched with C*；
C* can be matched with G, but C* cannot be matched with G*.
3. a kind of for detecting the kit of urinary tract infections, which is characterized in that the kit includes reaction solution, reaction enzyme system and draws
Object group, the primer sets are
Primer sets 1, upstream primer: ATTTCATAACCCCCTGTTTATTGTGTTA*A*T*G*G,
Downstream primer: ATTTTGTTAAACTCAAACGTGATAATGA*G*A*A*T；
Primer sets 2, upstream primer: AACTCTCTACGTCGTATTTTATTGTCTTC*T*T*T*A, downstream primer:
AAGGGTTTTCTCTTCTTCAGCATATAATT*T*C*A*A；
Primer sets 3: upstream primer:
ACACTTTTCTCAATAACACCGAGCAGGAG*G*T*T*C,
Downstream primer: TAAAAAGGGGCGCTACTATAGCACAAAGAT*A*A*A*C；
Primer sets 4: upstream primer: TATTCCTAAATATAGTCAAGTTTCTTGTG*A*T*G*T, downstream primer:
ATTAAAACGAAAAATATTAAGAATATCGA*C*C*C*C；
Primer sets 5: upstream primer: CCTGTTGTTCGTTGCGATCATGGGTG*T*T*T*C, downstream primer:
CCAGTTCCACTTTGTCATTCTCGGTC*T*T*G*C；
Wherein, A*:2- aminopurine nucleoside；T*:2- sulphur thymidine；C*:N- ethylcytosine；G*: hypoxanthine.
4. kit according to claim 3, which is characterized in that the reaction solution includes Tris-HCl, KCl, two sulphur Soviet Union
Sugar alcohol, glycine betaine, trehalose, PEG, BSA.
5. kit according to claim 4, which is characterized in that the pH of the Tris-HCl is 7~9.
6. kit according to claim 3, which is characterized in that the reaction enzyme system includes dNTPs, polymerase Bsu, egg
Bai Yinzi, recombinase.
7. kit according to claim 6, which is characterized in that the protein factor be albumen RecF, RecO, RecR,
One of single-stranded DNA binding protein SSB or a variety of.
8. kit according to claim 5, which is characterized in that the recombinase is cre bacterium recombinase RecA, Archimycetes
One of recombinase RadA, bacteriophage recombinase UvsX or eucaryote recombinase Rad51 or a variety of.
9. according to the described in any item kits of claim 3-6, which is characterized in that further include starting liquid, the starting liquid is
Magnesium chloride.
10. kit according to claim 9, which is characterized in that the concentration of the magnesium chloride is 5mM~20mM.
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