CN108559785A - Primer combination, probe, kit and Carbapenems Drug-resistant genes blaVIM detection method - Google Patents
Primer combination, probe, kit and Carbapenems Drug-resistant genes blaVIM detection method Download PDFInfo
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Abstract
The invention discloses a kind of combinations of primer, including forward primer and reverse primer, for expanding Carbapenems Drug-resistant genes blaVIMIn the sequence as shown in SEQ ID NO.1.The invention also discloses a kind of probes, the probe includes oligonucleotide sequence, the quenching group with the fluorophor of 5 ' end connections of the oligonucleotide sequence and with 3 ' end connections of the oligonucleotide sequence, and the oligonucleotide sequence can be matched with sequence shown in SEQ ID NO.1.The invention also discloses a kind of kits, including are used to detect Carbapenems Drug-resistant genes blaVIMThe detection reagent of sequence shown in middle SEQ ID NO.1.The invention also discloses a kind of Carbapenems Drug-resistant genes blaVIMDetection method.
Description
Technical field
The present invention relates to biotechnologies, more particularly to a kind of primer combination, probe, kit and carbapenem
Class Drug-resistant genes blaVIMDetection method.
Background technology
It is found in penicillin 90 years so far from nineteen twenty-eight, abuse of the antibacterials in human being's production, life and unreasonable
Using causing bacterial resistance problem to grow in intensity, more drug resistances, general drug resistance, full antibody-resistant bacterium are increasing, for global public health
Bring severe challenge.The genetic mechanism of bacterial resistance includes intrinsic resistance and acquisition drug resistance.Intrinsic resistance refers to bacterium
It is naturally insensitive to certain antibacterials；It is since the gene of bacterium changes so as to cause which give resistance to obtain drug resistance
Pharmacological property.Bacterium, which obtains drug resistance, usually following two approach：One is bacterial chromosomal genes to be mutated acquisition drug resistance；It is another
Kind is the removable drug resistant gene element obtained from environment or other bacteriums.At present no matter in people doctor, animal doctor, environment drug resistance
Property etc. fields, by drug resistant gene element send out caused by acquired resistance receive significant attention, bacterium can in several ways from
The external world, which obtains, moves drug resistant gene element, such as engages transfer, transposons by the transduction of bacteriophage mediation, R-plasmid and turns
Change etc..blaVIMIt is a kind of Carbapenems Drug-resistant genes being located on transferable plasmid, carries the bacterium of the gene
Production carbapenem enzyme ability is obtained to be resistant to such antibacterials.
The genotype detection of bacterial resistance gene element is a kind of method of bacterial drug resistance detection and monitoring.Due to drug resistance
There are a variety of hypotypes, current detection methods to be detected for a certain specific hypotype for the genotype of gene, lacks general
The method of adaptive is for detecting a variety of hypotypes.
Invention content
Based on this, it is necessary to it is anti-to provide a kind of primer combination, a kind of probe, a kind of kit and a kind of Carbapenems
Bacterium Drug-resistant gene blaVIMDetection method, realize to drug resistant gene blaVIMA variety of hypotypes detection.
A kind of primer combination, including forward primer and reverse primer, for expanding Carbapenems antibiotic resistance base
Because of blaVIMIn the sequence as shown in SEQ ID NO.1.
The sequence of the forward primer is as shown in SEQ ID NO.2 in one of the embodiments, the reverse primer
Sequence is as shown in SEQ ID NO.3.
The Carbapenems Drug-resistant genes bla in one of the embodiments,VIMIncluding blaVIM-1、
blaVIM-2、blaVIM-3、blaVIM-4、blaVIM-5、blaVIM-6、blaVIM-8、blaVIM-9、blaVIM-10、blaVIM-11、blaVIM-12、
blaVIM-14、blaVIM-15、blaVIM-16、blaVIM-17、blaVIM-18、blaVIM-19、blaVIM-23、blaVIM-24、blaVIM-25、
blaVIM-26、blaVIM-27、blaVIM-28、blaVIM-29、blaVIM-30、blaVIM-31、blaVIM-32、blaVIM-33、blaVIM-34、
blaVIM-35、blaVIM-36、blaVIM-37、blaVIM-38、blaVIM-39、blaVIM-42、blaVIM-43、blaVIM-44、blaVIM-45With
blaVIM-46In it is one or more.
A kind of probe, the probe include oligonucleotide sequence, the fluorescence with 5 ' end connections of the oligonucleotide sequence
Group and the quenching group for holding connection with the 3 ' of the oligonucleotide sequence, the oligonucleotide sequence can be with SEQ ID
Sequence shown in NO.1 is matched.
The oligonucleotide sequence is as shown in SEQ ID NO.4 in one of the embodiments,.
A kind of kit, including it is used to detect Carbapenems Drug-resistant genes blaVIMMiddle SEQ IDNO.1 institutes
Show the detection reagent of sequence.
The detection reagent includes the primer combination in one of the embodiments,.
The detection reagent further includes the probe in one of the embodiments,.
A kind of Carbapenems Drug-resistant genes blaVIMDetection method, include the following steps：
The sample to be tested of predetermined amount is taken, total DNA is extracted；And
Using the total DNA as template, PCR amplification is carried out with the primer combination；And
Measure the sequence length of amplified production.
A kind of Carbapenems Drug-resistant genes blaVIMDetection method, include the following steps：S10 provides institute
The primer combination stated and the probe are different dense to containing using the standard items containing sequence shown in SEQ ID NO.1 as template
Multiple standard solutions of the standard items of degree carry out real-time fluorescence quantitative PCR reaction, obtain real-time fluorescence quantitative PCR reaction
Standard curve；And
S20, using sample to be tested as template, combined using primer identical with the step S10, probe and reaction condition into
Row real-time fluorescence quantitative PCR reacts, and obtained Ct values are substituted into the standard curve, obtain containing SEQ in the sample to be tested
The starting copies concentration of the target gene of sequence shown in ID NO.1.
The SEQ ID NO.1 sequences are in the Carbapenems Drug-resistant genes blaVIMMost of hypotype
In be conservative, it has been found that Carbapenems Drug-resistant genes VIM groups 41 hypotypes in have 39 hypotypes
Including the SEQ ID NO.1 sequences, the present invention is described to draw for primer combination described in the SEQ ID NO.1 sequence designs
Object combination can be to the Carbapenems Drug-resistant genes blaVIMInclude SEQ ID NO.1 sequences hypotype into
Row amplification whether there is Carbapenems Drug-resistant genes bla so as to be used to detect in sampleVIM, or even can
For detecting also undiscovered Carbapenems Drug-resistant genes blaVIMHypotype.
Description of the drawings
Fig. 1 is that the real-time fluorescence quantitative PCR of one embodiment of the invention expands kinetic curve photo；
Fig. 2 is the real-time fluorescence quantitative PCR standard curve photo of one embodiment of the invention.
Specific implementation mode
In order to make the purpose , technical scheme and advantage of the present invention be clearer, by the following examples, it and combines attached
Figure, to primer combination, probe, kit and the Carbapenems Drug-resistant genes bla of the present inventionVIMDetection side
Method is further elaborated.It should be appreciated that described herein, specific examples are only used to explain the present invention, is not used to
Limit the present invention.
The embodiment of the present invention provides a kind of primer combination, including forward primer and reverse primer, the sequence of the forward primer
Row are as shown in SEQ ID NO.2, and the sequence of the reverse primer is as shown in SEQ ID NO.3.
The forward primer and the reverse primer and the Carbapenems Drug-resistant genes blaVIMIn spy
Determine conserved sequence to match, can be used in the amplification Carbapenems Drug-resistant genes bla of specificityVIM, according to can
Amplified production is obtained to judge to whether there is the Carbapenems Drug-resistant genes bla in sample to be testedVIM。
The embodiment of the present invention also provides a kind of primer combination, including forward primer and reverse primer, for expanding carbon mould
Carbapenem antibacterial Drug-resistant gene blaVIMIn the sequence as shown in SEQ ID NO.1.
The SEQ ID NO.1 sequences are in the Carbapenems Drug-resistant genes blaVIMMost of hypotype
In be conservative, it has been found that Carbapenems Drug-resistant genes VIM groups 41 hypotypes in have 39 hypotypes
Including the SEQ ID NO.1 sequences.The present invention is described to draw for primer combination described in the SEQ ID NO.1 sequence designs
Object combination can be to the Carbapenems Drug-resistant genes blaVIMInclude SEQ ID NO.1 sequences hypotype into
Row amplification whether there is Carbapenems Drug-resistant genes bla so as to be used to detect in sampleVIM, or even can
For detecting also undiscovered Carbapenems Drug-resistant genes blaVIMHypotype.
The amplimer is to including forward primer and reverse primer, the forward primer in one of the embodiments,
Sequence as shown in SEQ ID NO.2, the sequence of the reverse primer is as shown in SEQ ID NO.3.The forward direction of the length is drawn
Mismatch rate is lower when object and the reverse primer make the amplimer to being used to expand SEQ ID NO.1 sequences, expands effect
Rate higher.In practical applications, there may be the inclined of 1-3 base for the primer sequence of the forward primer and the reverse primer
Difference does not interfere with the result of amplification.
The Carbapenems Drug-resistant genes bla in one of the embodiments,VIMIncluding blaVIM-1(SEQ
ID NO.5)、blaVIM-2(SEQ ID NO.6)、blaVIM-3(SEQ ID NO.7)、blaVIM-4(SEQ ID NO.8)、blaVIM-5
(SEQ ID NO.9)、blaVIM-6(SEQ ID NO.10)、blaVIM-8(SEQ ID NO.11)、blaVIM-9(SEQ ID
NO.12)、blaVIM-10(SEQ ID NO.13)、blaVIM-11(SEQ ID NO.14)、blaVIM-12(SEQ ID NO.15)、
blaVIM-14(SEQ ID NO.16)、blaVIM-15(SEQ ID NO.17)、blaVIM-16(SEQ ID NO.18)、blaVIM-17(SEQ
ID NO.19)、blaVIM-18(SEQ ID NO.20)、blaVIM-19(SEQ ID NO.21)、blaVIM-23(SEQ ID NO.22)、
blaVIM-24(SEQ ID NO.23)、blaVIM-25(SEQ ID NO.24)、blaVIM-26(SEQ ID NO.25)、blaVIM-27(SEQ
ID NO.26)、blaVIM-28(SEQ ID NO.27)、blaVIM-29(SEQ ID NO.28)、blaVIM-30(SEQ ID NO.29)、
blaVIM-31(SEQ ID NO.30)、blaVIM-32(SEQ ID NO.31)、blaVIM-33(SEQ ID NO.32)、blaVIM-34(SEQ
ID NO.33)、blaVIM-35(SEQ ID NO.34)、blaVIM-36(SEQ ID NO.35)、blaVIM-37(SEQ ID
NO.36)、blaVIM-38(SEQ ID NO.37)、blaVIM-39(SEQ ID NO.38)、blaVIM-42(SEQ ID NO.39)、
blaVIM-43(SEQ IDNO.40)、blaVIM-44(SEQ ID NO.41)、blaVIM-45(SEQ ID NO.42) and blaVIM-46
It is one or more in (SEQID NO.43).The bla of 39 kinds of Carbapenems Drug-resistant genesVIMIn hypotype all
Including the SEQ ID NO.1 sequences, the SEQ ID NO.1 sequences are in 39 kinds of blaVIMThe 208- of hypotype drug resistant gene
The position of 269bp can expand 39 kinds of hypotypes by the primer pair, and it is independent need not to be directed to each hypotype
The universality of design primer, the primer combination is stronger.
The embodiment of the present invention also provides a kind of probe, and the probe can be used for hybridizing with SEQ ID NO.1 sequence specifics
Indicate the increase of SEQ ID NO.1 sequence amplification products, can be carried out using the probe real-time fluorescence quantitative PCR reaction from
And to the Carbapenems Drug-resistant genes bla of sample to be testedVIMCarry out quantitative analysis.It is carried out using the probe real
When quantitative fluorescent PCR reaction identification step of the method due to increasing probe and target sequence, specific higher, quantitative analysis knot
Fruit is more acurrate.
In one embodiment, the probe can be TaqMan probe, and TaqMan probe is a kind of oligonucleotide probe, glimmering
Light group is connected to 5 ' ends of probe, and quenching group is connected to 3 ' ends.Probe is matched with target sequence, when probe is complete,
Fluorophor transmitting fluorescence because with 3 ' hold quenching group close to due to be quenched.When carrying out extension, Taq DNA polymerizations
The 5 prime excision enzyme activity of the 5 ' of enzyme -3 ' cuts off probe so that fluorophor is detached with quenching group, to which fluorescence monitoring system can be with
Detect fluorescence.Often expanding a DNA chain, just there are one fluorescent moleculars to generate, and with the increase of amplification cycles number, releases
Fluorophor constantly accumulate, it is fully synchronized to realize that the accumulation of fluorescence signal and PCR product are formed.According to the accumulation of fluorescence signal
Amount can obtain the starting copies concentration of SEQ ID NO.1 sequences in sample to be tested, be reacted by the size of starting copies concentration
Wait for that side sample is strong and weak to the drug resistance of Carbapenems antibacterials.
In one embodiment, the probe may include oligonucleotide sequence, 5 ' the end companies with the oligonucleotide sequence
The fluorophor that connects and quenching group with 3 ' end connections of the oligonucleotide sequence, the oligonucleotide sequence can be with
The SEQ ID NO.1 sequences pairing.When real-time fluorescence quantitative PCR starts, if there are the SEQ in sample to be tested
ID NO.1 sequences, then the oligonucleotide sequence and the SEQ ID NO.1 sequences pairing in sample to be tested, with extension
It carries out, 5 ' -3 ' 5 prime excision enzyme activities of Taq archaeal dna polymerases cut off the probe, to make the fluorophor quench with described
The group that goes out detaches so that fluorescence monitoring system can detect fluorescence, and often one DNA chain of amplification will there are one fluorescent moleculars to produce
It is raw, it may determine that the SEQ ID NO.1 sequences in sample with the presence or absence of described by the intensity of the fluorescence signal monitored
SEQ ID NO.1 sequences and obtain the SEQ ID NO.1 sequences concentration size.
The length of the oligonucleotide sequence and position need forward primer, reverse primer with real-time fluorescence quantitative PCR
And target sequence SEQ ID NO.1 sequences match, the forward primer on the oligonucleotide sequence and the target sequence and
Sequence pairing between the reverse primer.In one embodiment, the oligonucleotide sequence is as shown in SEQ ID NO.4.
In practical application, the oligonucleotide sequence may exist the deviation of 1-3 base, not interfere with the result of amplification.
In one embodiment, the fluorophor includes Fluoresceincarboxylic acid, 2', 4', 5', 7', Isosorbide-5-Nitrae-chlordene fluorescein,
2', 7'- dimethoxy-4 ' ', one kind in bis- chloro- 6- Fluoresceincarboxylic acids of 5'- and 1,4- tetrachlorofluoresceins.The quenching group packet
Include one kind in Dabcyl (4- [4- (dimethylamino) benzeneazo] benzoic acid), BHQ and MGB.
Preferably, the quenching group is MGB groups.MGB groups are non-fluorescence quenching group, itself does not generate fluorescence,
The intensity of background signal can be substantially reduced.Melting temperature (Tm) can be improved about 3 by MGB and the connection of oligonucleotides one end
DEG C to 6 DEG C.In order to obtain the probe of same Tm values, the probe for connecting MGB can be than the oligonucleotides of general T aqMan probes
Sequence design obtains shorter, both reduces synthesis cost, but also the success rate of probe design greatly improves.
The embodiment of the present invention also provides a kind of kit, including is used to detect Carbapenems Drug-resistant genes
blaVIMThe detection reagent of sequence shown in middle SEQ ID NO.1.Using sample to be tested as template, PCR expansions are carried out using the kit
Increase, can be qualitatively judged in sample to be tested with the presence or absence of sequence shown in SEQ ID NO.1 according to amplified production can be obtained, from
And judge whether Carbapenems Drug-resistant genes blaVIM。
In one embodiment, the detection reagent includes the primer combination.If containing such as SEQ IDNO.1 in sample
Shown in sequence Carbapenems antibacterial drug resistance gene, then primer combination can carry out PCR amplification.Pass through inspection
The length of amplified band is surveyed, if do not contained and Carbapenems Drug-resistant genes bla in sampleVIM, then cannot obtain
The amplified band of specific length.It can be qualitatively judged by primer combination and whether there is Carbapenems antimicrobial in sample
Object drug resistant gene blaVIM。
In one embodiment, the detection reagent further includes the probe.The probe and primer combination are matched
It closes, can be reacted by real-time fluorescence quantitative PCR in the Carbapenems antibacterial drug resistance gene quantitatively detected in sample
SEQ ID NO.1 shown in sequence concentration, to realize Carbapenem-resistant gene bla in sampleVIMAbsolute quantitation.
In one embodiment, the detection reagent further includes real-time fluorescence quantitative PCR reaction reagent.The real-time fluorescence is fixed
It may include Taq archaeal dna polymerases, dNTP and buffer buffer solutions etc. to measure PCR reaction reagents.
The embodiment of the present invention also provides a kind of kit, including primer combination.The primer combination includes that forward direction is drawn
Object and reverse primer, the sequence of the forward primer is as shown in SEQ ID NO.2, the sequence such as SEQ ID of the reverse primer
Shown in NO.3.
Since the forward primer and the reverse primer are Carbapenems Drug-resistant genes blaVIMIn it is specific
The specificity amplification primer pair of gene carries out PCR using sample to be tested as template using the forward primer and the reverse primer
Can amplification can qualitatively judge anti-with the presence or absence of Carbapenems in sample to be tested according to the amplified production that obtain specific length
Bacterium Drug-resistant gene blaVIM。
In one embodiment, the kit further includes the probe.The probe include oligonucleotide sequence, with it is described
The fluorophor of 5 ' end connections of oligonucleotide sequence and the quenching group for holding connection with the 3 ' of the oligonucleotide sequence, institute
Oligonucleotide sequence is stated as shown in SEQ ID NO.4.The primer combination and the probe match carry out real time fluorescent quantitative
PCR reacts, and can quantitative determine the Carbapenems Drug-resistant genes bla with test sample sheetVIMThe starting of middle specific gene
Copy concentrations, so that it is determined that the power of the Carbapenems antibacterial drug resistance of sample to be tested.
The embodiment of the present invention also provides a kind of Carbapenems Drug-resistant genes blaVIMDetection method, including
Following steps：
The sample to be tested of predetermined amount is taken, total DNA is extracted；And
Using the total DNA as template, PCR amplification is carried out with the primer combination；And
Measure the sequence length of amplified production.
In the Carbapenems Drug-resistant genes blaVIMDetection method in, the forward primer and described
Reverse primer matches with sequence shown in SEQ ID NO.1, if including and sequence shown in SEQ IDNO.1 in sample to be tested
Target gene, then the primer combination can expand to obtain the target gene of SEQ ID NO.1.By detecting amplification purpose base
Because of the length of band, such as agarose electrophoresis, if not including the purpose with sequence shown in SEQ ID NO.1 in sample to be tested
Gene, then the primer combination can not expand to obtain the target gene band of 62bp length.According to can expand to obtain 62bp
Target gene band, can qualitatively judge in sample to be tested whether there is Carbapenems Drug-resistant genes blaVIM。
Further, according to the shade of the obtained target gene band can be rough the carbon qualitatively judged in sample to be tested it is green
Mould carbapenem antibacterial Drug-resistant gene blaVIMContent, the more deep then content of color is higher, then sample is to Carbapenems antimicrobial
The drug resistance of object is stronger.
The sample to be detected can have separate sources, including but not limited to saliva, blood, urine, excrement etc..
For further to the Carbapenems Drug-resistant genes bla in sample to be testedVIMIntensity judged, can
With to the Carbapenems Drug-resistant genes bla in sample to be testedVIMSEQ ID NO.1 shown in sequence concentration into
The quantitative detection of row.
In one embodiment, the Carbapenems Drug-resistant genes blaVIMDetection method include following step
Suddenly：
S10 provides the probe described in the primer combination and root, with the standard containing sequence shown in SEQ ID NO.1
Product are template, carry out real-time fluorescence quantitative PCR reaction to multiple standard solutions of the standard items containing various concentration, obtain
The standard curve reacted to real-time fluorescence quantitative PCR；And
S20, using sample to be tested as template, combined using primer identical with the step S10, probe and reaction condition into
Row real-time fluorescence quantitative PCR reacts, and obtained Ct values are substituted into the standard curve, obtain containing SEQ in the sample to be tested
The starting copies concentration of the target gene of sequence shown in ID NO.1.
In the step S10, the standard items can be that will contain the purpose base of sequence shown in SEQ ID NO.1
The recombinant plasmid that cause is connected with cloning vector.The target gene can be connected to the clone by digestion and connection
On carrier, structure forms the recombinant vector for including the target gene, including the recombinant vector of the target gene can conduct
Standard items are used for the foundation of real-time fluorescence quantitative PCR standard curve.Recombinant plasmid by the way that a certain concentration gradient is arranged is used as mark
Quasi- product, using the recombinant plasmid as template, with primer combination to for primer, being carried out using the probe as marker real-time
Quantitative fluorescent PCR reacts.Due to different starting copies concentration (C0) recombinant plasmid generate fluorescence signal accumulation not
Together, different kinetic curves is will present in fluoroscopic examination, the recombinant plasmid of each corresponding initial concentration can detect
Different Ct values.Ct values are that fluorescence signal reaches the recurring number undergone when given threshold.The Ct values and the template of each template
The logarithm of starting copy number C0 there are linear relationships, according to the correspondence of obtained corresponding Ct values and starting copies concentration C 0
Relationship can be obtained using log C0 as abscissa, using Ct as the standard curve of ordinate.
In the step S20, unknown sample to be tested is carried out by the identical reaction systems of the step S10 glimmering in real time
Fluorescent Quantitative PCR react, by fluoroscopic examination to kinetic curve obtain the Ct values of sample to be tested.The Ct values are updated to step
In the standard curve that rapid S10 is obtained, you can obtain rising for the target gene of sequence as shown in SEQ ID NO.1 in sample to be tested
Beginning copy concentrations.The starting copies concentration and Carbapenems antibacterial drug resistance of the target gene of sample to be tested are directly proportional.
Embodiment 1
(1) extraction standard product genomic DNA
Sample is selected from the room preservation of Inst of Infection Disease Prevention and Control, Chinese Diseases Prevention an's bacterial resistance
Klebsiella Pneumoniae (A2509) strain.The genomic DNA of sample is extracted using DNA extraction kit.
(2) PCR amplification
Using the reverse primer of such as forward primer of SEQ ID NO.2 sequences and such as SEQ ID NO.3 sequences, with (1)
The genomic DNA of step extraction is template, carries out Standard PCR, is expanded to the target gene of such as SEQ ID NO.1 sequences.
Pcr amplification product is detected using the agarose gel electrophoresis of mass fraction 1%, determines amplified production length
For 61bp, judgement amplified production is the target gene such as SEQ ID NO.1 sequences.
(3) construction recombination plasmid
The target gene obtained using plastic recovery kit (QIAGEN) recycling step (2) agarose gel electrophoresis.
Use pEASY-T1Simple Cloning Vector kits (Beijing Quanshijin Biotechnology Co., Ltd) structure
Recombinant plasmid is built, is connected by target gene and with T1Simple cloning vectors.
After PCR and sequence verification are converted successfully, recombinant plasmid is extracted.
(4) real-time fluorescence quantitative PCR standard curve is established
10 times of concentration ratio dilutions are carried out using the concentration of spectrophotometric determination recombinant plasmid, and to recombinant plasmid, respectively
Obtain 8 groups a concentration of 5 × 109、5×108、5×107、5×106、5×105、5×104、5×103、5×102Copies/ μ L's
Recombinant plasmid, as recombinant plasmid standard items template.
Respectively Taqman fluorescent quantitative PCRs, the spy are carried out by template of the recombinant plasmid standard items of various concentration
Needle is 5 ' FAM-TGATGAGTTGCTTTTGATTG-MGB 3 ', and PCR reaction systems are as shown in table 1, and PCR reaction conditions are：(1)
95 DEG C of pre-degeneration 3min；(2) 95 DEG C of denaturation 5s, 59 DEG C of annealing 20s, totally 40 recycle.
1 quantitative fluorescent PCR reaction system of table
Amplification kinetic curve and standard curve are obtained by quantitative fluorescent PCR.As shown in Figures 1 and 2, standard concentration
5×109~5 × 102It is in good linear relationship in copies/ μ L, the coefficient R 2 of standard curve is 0.997, and slope is
3.534, intercept 46.05, amplification efficiency 0.94, standard curve y=-3.534x+46.05, standard curve regression equation
Formula:Ct=-3.534 × log C0+46.05.Wherein, Ct is cycle threshold, and C0 is the mesh such as SEQ ID NO.1 sequences in sample
Gene starting copies concentration.
(5) pattern detection
Sample to be tested is taken, the fluorescence quantifying PCR method established according to step (4) is detected, and obtains the Ct for waiting for side sample
Value, according to standard regressive method formula, the Ct values of sample to be tested substitute into formula can calculate in sample to be tested such as SEQ ID
The starting copies concentration of the starting copies concentration of the target gene of NO.1 sequences, target gene is higher, illustrates carbon in sample to be tested
Penems Drug-resistant genes blaVIMConcentration it is higher, illustrate that sample to be tested drug resistance is stronger.
Each technical characteristic of embodiment described above can be combined arbitrarily, to keep description succinct, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, it is all considered to be the range of this specification record.
Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
Cannot the limitation to the scope of the claims of the present invention therefore be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect range.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Sequence table
<110>Inst of Infection Disease Prevention and Control, Chinese Diseases Prevention an
<120>Primer combination, probe, kit and Carbapenems Drug-resistant genes blaVIM detection method
<160> 43
<170> SIPOSequenceListing 1.0
<210> 1
<211> 62
<212> DNA
<213> Pseudomonas aeruginosa
<400> 1
aatggtctca ttgtccgtga tggtgatgag ttgcttttga ttgatacagc gtggggtgcg 60
aa 62
<210> 2
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 2
aatggtctca ttgtccgtga tg 22
<210> 3
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 3
ttcgcacccc acgctgta 18
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 4
tgatgagttg cttttgattg 20
<210> 5
<211> 801
<212> DNA
<213> Pseudomonas aeruginosa
<400> 5
atgttaaaag ttattagtag tttattggtc tacatgaccg cgtctgtcat ggctgtcgca 60
agtccgttag cccattccgg ggagccgagt ggtgagtatc cgacagtcaa cgaaattccg 120
gtcggagagg tccgacttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcggtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgaa 300
aagcaaattg gacttcccgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggcagaggg gaacgagatt cccacgcatt ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gagctcttct atcctggtgc tgcgcattcg 540
accgacaatc tggttgtata cgtcccgtca gcgaacgtgc tatacggtgg ttgtgccgtt 600
catgagttgt caagcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccgttg agcggattca aaaacactac ccggaagcag aggtcgtcat tcccgggcac 720
ggtctaccgg gcggtctaga cttgctccag cacacagcga acgttgtcaa agcacacaaa 780
aatcgctcag tcgccgagta g 801
<210> 6
<211> 801
<212> DNA
<213> Pseudomonas aeruginosa
<400> 6
atgttcaaac ttttgagtaa gttattggtc tatttgaccg cgtctatcat ggctattgcg 60
agtccgctcg ctttttccgt agattctagc ggtgagtatc cgacagtcag cgaaattccg 120
gtcggggagg tccggcttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcagtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgag 300
aagcaaattg gacttcctgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggtagaggg gaacgagatt cccacgcact ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gaactcttct atcctggtgc tgcgcattcg 540
accgacaact tagttgtgta cgtcccgtct gcgagtgtgc tctatggtgg ttgtgcgatt 600
tatgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccattg agcggattca acaacactac ccggaagcac agttcgtcat tccggggcac 720
ggcctgccgg gcggtctaga cttgctcaag cacacaacga atgttgtaaa agcgcacaca 780
aatcgctcag tcgttgagta g 801
<210> 7
<211> 801
<212> DNA
<213> Pseudomonas aeruginosa
<400> 7
atgttcaaac ttttgagtaa gttattggtc tatttgaccg cgtctatcat ggctattgcg 60
agtccgctcg ctttttccgt agattctagc ggtgagtatc cgacagtcag cgaaattccg 120
gtcggggagg tccggcttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgaag 180
tcgtttgatg gcgcagtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgag 300
aagcaaattg gacttcctgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggtagaggg gagcgagatt cccacgcact ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gaactcttct atcctggtgc tgcgcattcg 540
accgacaact tagttgtgta cgtcccgtct gcgagtgtgc tctatggtgg ttgtgcgatt 600
tatgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccattg agcggattca acaacactac ccggaagcac agttcgtcat tccggggcac 720
ggcctgccgg gcggtctaga cttgctcaag cacacaacga atgttgtaaa agcgcacaca 780
aatcgctcag tcgttgagta g 801
<210> 8
<211> 801
<212> DNA
<213> Pseudomonas aeruginosa
<400> 8
atgttaaaag ttattagtag tttattggtc tacatgaccg cgtctgtcat ggctgtcgca 60
agtccgttag cccattccgg ggagccgagt ggtgagtatc cgacagtcaa cgaaattccg 120
gtcggagagg tccgacttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcggtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgaa 300
aagcaaattg gacttcccgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggcagaggg gaacgagatt cccacgcatt ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gagctcttct atcctggtgc tgcgcattcg 540
accgacaatc tggttgtata cgtcccgtca gcgaacgtgc tatacggtgg ttgtgccgtt 600
catgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccgttg agcggattca aaaacactac ccggaagcag aggtcgtcat tcccgggcac 720
ggtctaccgg gcggtctaga cttgctccag cacacagcga acgttgtcaa agcacacaaa 780
aatcgctcag tcgccgagta g 801
<210> 9
<211> 801
<212> DNA
<213> Klebsiella pneumoniae
<400> 9
atgttaaaag ttattagtag tttattggtc tacatgaccg cgtctgtcat ggctgtagct 60
agtccgttag cccattccgg ggagccgagt ggtgagtatc cgacagtcaa cgaaattccg 120
gtcggagagg tccggcttta ccagattgct gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcggtcta cccatccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cccttctcgc ggagattgag 300
aagcaaattg gacttcccgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg gaaggctgga gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggcagaggg gaacgagatt cccacgcact ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gagctcttct atcccggtgc tgcgcattcg 540
accgacaatc tggttgtata cgtcccgtca gcgaacgtgc tatacggtgg ttgtgccgtt 600
cttgcgttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccgttg agcggattca aaaacactac ccggaagcag aggtcgtcat tcccgggcac 720
ggtctaccgg gcggtctaga cttgctccag cacacagcga acgttgtcac agcacacaaa 780
aatcgctcag tcgccgagta g 801
<210> 10
<211> 828
<212> DNA
<213> Pseudomonas putida
<400> 10
atgttcaaac ttttgagtaa gttattggtc tatttgaccg cgtctatcat ggctattgcg 60
agtccgctcg ctttttccgt agattctagc ggtgagtatc cgacagtcag cgaaattccg 120
gtcggggagg tccggcttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcgg 180
tcgtttgatg gcgcagtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgag 300
aagcaaattg gacttcctgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggtagaggg gagcgagatt cccacgcact ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gaactcttct atcctggtgc tgcgcattcg 540
accgacaact tagttgtgta cgtcccgtct gcgagtgtgc tctatggtgg ttgtgcgatt 600
tatgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccattg agcggattca acaacactac ccggaagcac agttcgtcat tccggggcac 720
ggcctgccgg gcggtctaga cttgctcaag cacacaacga atgttgtaaa agcgcacaca 780
aatcgctcag tcgttgagta gcaggcagat gcggcataac atgaagtt 828
<210> 11
<211> 801
<212> DNA
<213> Pseudomonas aeruginosa
<400> 11
atgttcaaac ttttgagtaa gttattggtc tatttgaccg cgtctatcat ggctattgcg 60
agtccgctcg ctttttccgt agattctagc ggtgagtatc cgacagtcag cgaaattccg 120
gtcggggagg tccggcttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcagtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgag 300
aagcaaattg gacttcctgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcggcacgc 420
cggctagccg aggtagaggg gaacgagatt cccacgcact ctctagaagg actctcatcg 480
agcggggacg cggtgcgctt cggtccagta gaactcttct atcctggtgc tgcgcattcg 540
accgacaact tagttgtgta cgtcccgtct gcgagtgtgc tctatggtgg ttgtgcgatt 600
tatgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccattg agcggattca acaacactac ccagaagcac agttcgtcat tccggggcac 720
ggcctgccgg gcggtctaga cttgctcaag cacacaacga atgttgtaaa agcgcacaca 780
aatcgctcag tcgttgagta g 801
<210> 12
<211> 801
<212> DNA
<213> Pseudomonas aeruginosa
<400> 12
atgttcaaac ttttgagtaa gttattggtc tatttgaccg cgtctatcat ggctattgcg 60
agtccgctcg ctttttccgt agattctagc ggtgagtatc cgacagtcag cgaaattccg 120
gtcggggagg tccggcttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcagtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgag 300
aagcaaattg gacttcctgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgatacgc 420
cggctagccg aggtagaggg gaacgagatt cccacgcact ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gaactcttct atcctggtgc tgcgcattcg 540
accgacaact tagttgtgta cgtcccgtct gcgagtgtgc tctatggtgg ttgtgcgatt 600
tatgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccattg agcggattca acaacactac ccggaagcac agttcgtcat tccggggcac 720
ggcctgccgg gcggtctaga cttgctcaag cacacaacga atgttgtaaa agcgcacaca 780
aatcgctcag tcgttgagta g 801
<210> 13
<211> 801
<212> DNA
<213> Pseudomonas aeruginosa
<400> 13
atgttcaaac ttttgagtaa gttattggtc tatttgaccg cgtctatcat ggctattgcg 60
agtccgctcg ctttttccgt agattctagc ggtgagtatc cgacagtcag cgaaattccg 120
gtcggggagg tccggcttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcagtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgag 300
aagcaaattg gacttcctgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggtagaggg gaacgagatt cccacgcact ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gaactcttct atcctggtgc tgcgcattcg 540
accgacaact tagttgtgta cgtcccgtct gcgagtgtgc tctatggtgg ttgtgcgatt 600
tatgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccattg agcggattca acaacactac ccggaagcac agtacgtcat tccggggcac 720
ggcctgccgg gcggtctaga cttgctcaag cacacaacga atgttgtaaa agcgcacaca 780
aatcgctcag tcgttgagta g 801
<210> 14
<211> 801
<212> DNA
<213> Pseudomonas aeruginosa
<400> 14
atgttcaaac ttttgagtaa gttattggtc tatttgaccg cgtctatcat ggctattgcg 60
agtccgctcg ctttttccgt agattctagc ggtgagtatc cgacagtcag cgaaattccg 120
gtcggggagg tccggcttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcagtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgag 300
aagcaaattg gacttcctgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggtagaggg gagcgagatt cccacgcact ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gaactcttct atcctggtgc tgcgcattcg 540
accgacaact tagttgtgta cgtcccgtct gcgagtgtgc tctatggtgg ttgtgcgatt 600
tatgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccattg agcggattca acaacactac ccggaagcac agttcgtcat tccggggcac 720
ggcctgccgg gcggtctaga cttgctcaag cacacaacga atgttgtaaa agcgcacaca 780
aatcgctcag tcgttgagta g 801
<210> 15
<211> 801
<212> DNA
<213> Klebsiella pneumoniae
<400> 15
atgttaaaag ttattagtag tttattggtc tacatgaccg cgtctgtcat ggctgtcgca 60
agtccgttag cccattccgg ggagccgagt ggtgagtatc cgacagtcaa cgaaattccg 120
gtcggagagg tccgacttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcggtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgaa 300
aagcaaattg gacttcccgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggcagaggg gaacgagatt cccacgcatt ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gagctcttct atcctggtgc tgcgcattcg 540
accgacaatc tggttgtata cgtcccgtca gcgaacgtgc tatacggtgg ttgtgccgtt 600
catgagttgt caagcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccattg agcggattca acaacactac ccggaagcac agttcgtcat tccggggcac 720
ggcctgccgg gcggtctaga cttgctcaag cacacaacga atgttgtaaa agcgcacaca 780
aatcgctcag tcgttgagta g 801
<210> 16
<211> 801
<212> DNA
<213> Pseudomonas aeruginosa
<400> 16
atgttaaaag ttattagtag tttattggtc tacatgaccg cgtctgtcat ggctgtcgca 60
agtccgttag cccattccgg ggagccgagt agtgagtatc cgacagtcaa cgaaattccg 120
gtcggagagg tccgacttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcggtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgaa 300
aagcaaattg gacttcccgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggcagaggg gaacgagatt cccacgcatt ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gagctcttct atcctggtgc tgcgcattcg 540
accgacaatc tggttgtata cgtcccgtca gcgaacgtgc tatacggtgg ttgtgccgtt 600
catgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccgttg agcggattca aaaacactac ccggaagcag aggtcgtcat tcccgggcac 720
ggtctaccgg gcggtctaga cttgctccag cacacagcga acgttgtcaa agcacacaaa 780
aatcgctcag tcgccgagta g 801
<210> 17
<211> 801
<212> DNA
<213> Pseudomonas aeruginosa
<400> 17
atgttcaaac ttttgagtaa gttattggtc tatttgaccg cgtctatcat ggctattgcg 60
agtccgctcg ctttttccgt agattctagc ggtgagtatc cgacagtcag cgaaattccg 120
gtcggggagg tccggcttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcagtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgag 300
aagcaaattg gacttcctgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggtagaggg gaacgagatt cccacgcact ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gaactcttct atcctggtgc tgcgcattcg 540
accgacaact tagttgtgta cgtcccgtct gcgagtgtgc tctttggtgg ttgtgcgatt 600
tatgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccattg agcggattca acaacactac ccggaagcac agttcgtcat tccggggcac 720
ggcctgccgg gcggtctaga cttgctcaag cacacaacga atgttgtaaa agcgcacaca 780
aatcgctcag tcgttgagta g 801
<210> 18
<211> 801
<212> DNA
<213> Pseudomonas aeruginosa
<400> 18
atgttcaaac ttttgagtaa gttattggtc tatttgaccg cgtctatcat ggctattgcg 60
agtccgctcg ctttttccgt agattctagc ggtgagtatc cgacagtcag cgaaattccg 120
gtcggggagg tccggcttta ccagattgcc gatggtgttt ggttgcatat cgcaacgcag 180
tcgtttgatg gcgcagtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgag 300
aagcaaattg gacttcctgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggtagaggg gaacgagatt cccacgcact ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gaactcttct atcctggtgc tgcgcattcg 540
accgacaact tagttgtgta cgtcccgtct gcgagtgtgc tctatggtgg ttgtgcgatt 600
tatgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccattg agcggattca acaacactac ccggaagcac agttcgtcat tccggggcac 720
ggcctgccgg gcggtctaga cttgctcaag cacacaacga atgttgtaaa agcgcacaca 780
aatcgctcag tcgttgagta g 801
<210> 19
<211> 801
<212> DNA
<213> Pseudomonas aeruginosa
<400> 19
atgttcaaac ttttgagtaa gttattggtc tatttgaccg cgtctatgat ggctattgcg 60
agtccgctcg ctttttccgt agattctagc ggtgagtatc cgacagtcag cgaaattccg 120
gtcggggagg tccggcttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcagtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgag 300
aagcaaattg gacttcctgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggtagaggg gaacgagatt cccacgcact ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gaactcttct atcctggtgc tgcgcattcg 540
accgacaact tagttgtgta cgtcccgtct gcgagtgtgc tctatggtgg ttgtgcgatt 600
tatgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccattg agcggattca acaacactac ccggaagcac agttcgtcat tccggggcac 720
ggcctgccgg gcggtctaga cttgctcaag cacacaacga atgttgtaaa agcgcacaca 780
aatcgctcag tcgttgagta g 801
<210> 20
<211> 789
<212> DNA
<213> Pseudomonas aeruginosa
<400> 20
atgttcaaac ttttgagtaa gttattggtc tatttgaccg cgtctatcat ggctattgcg 60
agtccgctcg ctttttccgt agattctagc ggtgagtatc cgacagtcag cgaaattccg 120
gtcggggagg tccggcttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcgg 180
tcgtttgatg gcgcagtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgag 300
aagcaaattg gacttcctgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagcga acgagattcc cacgcactct ctagaaggac tctcatcgag cggggacgca 480
gtgcgcttcg gtccagtaga actcttctat cctggtgctg cgcattcgac cgacaactta 540
gttgtgtacg tcccgtctgc gagtgtgctc tatggtggtt gtgcgattta tgagttgtca 600
cgcacgtctg cggggaacgt ggccgatgcc gatctggctg aatggcccac ctccattgag 660
cggattcaac aacactaccc ggaagcacag ttcgtcattc cggggcacgg cctgccgggc 720
ggtctagact tgctcaagca cacaacgaat gttgtaaaag cgcacacaaa tcgctcagtc 780
gttgagtag 789
<210> 21
<211> 801
<212> DNA
<213> Escherichia coli
<400> 21
atgttaaaag ttattagtag tttattggtc tacatgaccg cgtctgtcat ggctgtcgca 60
agtccgttag cccattccgg ggagccgagt ggtgagtatc cgacagtcaa cgaaattccg 120
gtcggagagg tccgacttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcggtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgaa 300
aagcaaattg gacttcccgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggcagaggg gaacgagatt cccacgcatt ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gagctcttct atcctggtgc tgcgcattcg 540
accgacaatc tggttgtata cgtcccgtca gcgaaagtgc tatacggtgg ttgtgccgtt 600
catgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccgttg agcggattca aaaacactac ccggaagcag aggtcgtcat tcccgggcac 720
ggtctaccgg gcggtctaga cttgctccag cacacagcga acgttgtcaa agcacacaaa 780
aatcgctcag tcgccgagta g 801
<210> 23
<211> 803
<212> DNA
<213> Enterobacter cloacae
<400> 23
atgttcaaac ttttgagtaa gttattggtc tatttgaccg cgtctatcat ggctattgcg 60
agtccgctcg ctttttccgt agattctagc ggtgagtatc cgacagtcag cgaaattccg 120
gtcggggagg tccggcttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcagtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgag 300
aagcaaattg gacttcctgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggtagaggg gaacgagatt cccacgcact ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gaactcttct atcctggtgc tgcgcattcg 540
accgacaact tagttgtgta cgtcccgtct gcgagtgtgc tctatggtgg ttgtgcgatt 600
tatgagttgt caagcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccattg agcggattca acaacactac ccggaagcac agttcgtcat tccggggcac 720
ggcctgccgg gcggtctaga cttgctcaag cacacaacga atgttgtaaa agcgcacaca 780
aatcgctcag tcgttgagta gca 803
<210> 23
<211> 801
<212> DNA
<213> Klebsiella pneumoniae
<400> 23
atgttcaaac ttttgagtaa gttattggtc tatttgaccg cgtctatcat ggctattgcg 60
agtccgctcg ctttttccgt agattctagc ggtgagtatc cgacagtcag cgaaattccg 120
gtcggggagg tccggcttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcagtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgag 300
aagcaaattg gacttcctgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggtagaggg gaacgagatt cccacgcact ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gaactcttct atcctggtgc tgcgcattcg 540
accgacaact tagttgtgta cgtcccgtct gcgagtgtgc tctatggtgg ttgtgcgatt 600
tatgagttgt cactcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccattg agcggattca acaacactac ccggaagcac agttcgtcat tccggggcac 720
ggcctgccgg gcggtctaga cttgctcaag cacacaacga atgttgtaaa agcgcacaca 780
aatcgctcag tcgttgagta g 801
<210> 24
<211> 801
<212> DNA
<213> Proteus mirabilis
<400> 24
atgttaaaag ttattagtag tttattggtc tacatgaccg cgtctgtcat ggctgtagct 60
agtccgttag cccattccgg ggagccgagt ggtgagtatc cgacagtcaa cgaaattccg 120
gtcggagagg tccggcttta ccagattgct gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcggtcta cccatccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cccttctcgc ggagattgag 300
aagcaaattg gacttcccgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg gaaggctgga gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggcagaggg gaacgagatt cccacgcact ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gagctcttct atcccggtgc tgcgcattcg 540
accgacaatc tggttgtata cgtcccgtca gcgaacgtgc tatacggtgg ttgtgccgtt 600
cttgcgttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccgttg agcggattca aaaacactac ccggaagcac agttcgtcat tccggggcac 720
ggcctgccgg gcggtctaga cttgctcaag cacacaacga atgttgtaaa agcgcacaca 780
aatcgctcag tcgttgagta g 801
<210> 25
<211> 801
<212> DNA
<213> Klebsiella pneumoniae
<400> 25
atgttaaaag ttattagtag tttattggtc tacatgaccg cgtctgtcat ggctgtcgca 60
agtccgttag cccattccgg ggagccgagt ggtgagtatc cgacagtcaa cgaaattccg 120
gtcggagagg tccgacttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcggtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgaa 300
aagcaaattg gacttcccgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggcagaggg gaacgagatt cccacgcatt ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gagctcttct atcctggtgc tgcgcattcg 540
accgacaatc tggttgtata cgtcccgtca gcgaacgtgc tatacggtgg ttgtgccgtt 600
cttgagttgt caagcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccgttg agcggattca aaaacactac ccggaagcag aggtcgtcat tcccgggcac 720
ggtctaccgg gcggtctaga cttgctccag cacacagcga acgttgtcaa agcacacaaa 780
aatcgctcag tcgccgagta g 801
<210> 26
<211> 801
<212> DNA
<213> Klebsiella pneumoniae
<400> 26
atgttaaaag ttattagtag tttattggtc tacatgaccg cgtctgtcat ggctgtcgca 60
agtccgttag cccattccgg ggagccgagt ggtgagtatc cgacagtcaa cgaaattccg 120
gtcggagagg tccgacttta ccagattgcc gatggtgttt ggtcgcatat ctcaacgcag 180
tcgtttgatg gcgcggtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgaa 300
aagcaaattg gacttcccgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggcagaggg gaacgagatt cccacgcatt ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gagctcttct atcctggtgc tgcgcattcg 540
accgacaatc tggttgtata cgtcccgtca gcgaacgtgc tatacggtgg ttgtgccgtt 600
catgagttgt caagcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccgttg agcggattca aaaacactac ccggaagcag aggtcgtcat tcccgggcac 720
ggtctaccgg gcggtctaga cttgctccag cacacagcga acgttgtcaa agcacacaaa 780
aatcgctcag tcgccgagta g 801
<210> 27
<211> 801
<212> DNA
<213> Pseudomonas aeruginosa
<400> 27
atgttaaaag ttattagtag tttattggtc tacatgaccg cgtctgtcat ggctgtcgca 60
agtccgttag cccattccgg ggagccgagt ggtgagtatc cgacagtcaa cgaaattccg 120
gtcggagagg tccgacttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcggtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgaa 300
aagcaaattg gacttcccgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggcagaggg gaacgagatt cccacgcatt ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gagctcttct atcctggtgc tgcgcattcg 540
accgacaatc tggttgtata cgtcccgtca gcgaacgtgc tatacggtgg ttgtgccgtt 600
cttgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccgttg agcggattca aaaacactac ccggaagcag aggtcgtcat tcccgggcac 720
ggtctaccgg gcggtctaga cttgctccag cacacagcga acgttgtcaa agcacacaaa 780
aatcgctcag tcgccgagta g 801
<210> 28
<211> 801
<212> DNA
<213> Escherichia coli
<400> 28
atgttaaaag ttattagtag tttattggtc tacatgaccg cgtctgtcat ggctgtcgca 60
agtccgttag cccattccgg ggagccgagt ggtgagtatc cgacagtcaa cgaaattccg 120
gtcggagagg tccgacttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcggtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgaa 300
aagcaaattg gacttcccgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggcagaggg gaacgagatt cccacgcatt ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gagctcttct atcctggtgc tgcgcattcg 540
accgacaatc tggttgtata cgtcccgtca gcgaaagtgc tatacggtgg ttgtgccgtt 600
catgagttgt caagcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccgttg agcggattca aaaacgctac ccggaagcag aggtcgtcat tcccgggcac 720
ggtctaccgg gcggtctaga cttgctccag cacacagcga acgttgtcaa agcacacaaa 780
aatcgctcag tcgccgagta g 801
<210> 29
<211> 801
<212> DNA
<213> Pseudomonas aeruginosa
<400> 29
atgttcaaac ttttgagtaa gttattggtc tatttgaccg cgtctatcat ggctattgcg 60
agtccgctcg ctttttccgt agattctagc ggtgagtatc cgacagtcaa cgaaattccg 120
gtcggggagg tccggcttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcagtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgag 300
aagcaaattg gacttcctgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggtagaggg gaacgagatt cccacgcact ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gaactcttct atcctggtgc tgcgcattcg 540
accgacaact tagttgtgta cgtcccgtct gcgagtgtgc tctatggtgg ttgtgcgatt 600
tatgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccattg agcggattca acaacactac ccggaagcac agttcgtcat tccggggcac 720
ggcctgccgg gcggtctaga cttgctcaag cacacaacga atgttgtaaa agcgcacaca 780
aatcgctcag tcgttgagta g 801
<210> 30
<211> 801
<212> DNA
<213> Enterobacter cloacae
<400> 30
atgttcaaac ttttgagtaa gttattggtc tatttgaccg cgtctatcat ggctattgcg 60
agtccgctcg ctttttccgt agattctagc ggtgagtatc cgacagtcag cgaaattccg 120
gtcggggagg tccggcttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcagtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgag 300
aagcaaattg gacttcctgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggtagaggg gaacgagatt cccacgcact ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gaactcttct atcctggtgc tgcgcattcg 540
accgacaact tagttgtgta cgtcccgtct gcgagtgtgc tctatggtgg ttgtgcgatt 600
catgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccattg agcggattca acaacgctac ccggaagcac agttcgtcat tccggggcac 720
ggcctgccgg gcggtctaga cttgctcaag cacacaacga atgttgtaaa agcgcacaca 780
aatcgctcag tcgttgagta g 801
<210> 31
<211> 801
<212> DNA
<213> Klebsiella oxytoca
<400> 31
atgttaaaag ttattagtag tttattggtc tacatgaccg cgtctgtcat ggctgtcgca 60
agtccgttag cccattccgg ggagccgagt ggtgagtatc cgacagtcaa cgaaattccg 120
gtcggagagg tccgacttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcggtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgaa 300
aagcaaattg gacttcccgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggcagcggg gaacgagatt cccacgcatt ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gagctcttct atcctggtgc tgcgcattcg 540
accgacaatc tggttgtata cgtcccgtca gcgaacgtgc tatacggtgg ttgtgccgtt 600
catgagttgt caagcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccgttg agcggattca aaaacactac ccggaagcag aggtcgtcat tcccgggcac 720
ggtctaccgg gcggtctaga cttgctccag cacacagcga acgttgtcaa agcacacaaa 780
aatcgctcag tcgccgagta g 801
<210> 32
<211> 801
<212> DNA
<213> Klebsiella pneumoniae
<400> 32
atgttaaaag ttattagtag tttattggtc tacatgaccg cgtctgtcat ggctgtcgca 60
agtccgttag cccattccgg ggagccgagt ggtgagtatc cgacagtcaa cgaaattccg 120
gtcggagagg tccgacttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcggtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgaa 300
aagcaaattg gacttcccgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggcagaggg gaacgagatt cccacgcatt ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gagctcttct atcctggtgc tgcgcattcg 540
accgacaatc tggttgtata cgtcccgtca gcgaacgtgc tattcggtgg ttgtgccgtt 600
catgagttgt caagcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccgttg agcggattca aaaacactac ccggaagcag aggtcgtcat tcccgggcac 720
ggtctaccgg gcggtctaga cttgctccag cacacagcga acgttgtcaa agcacacaaa 780
aatcgctcag tcgccgagta g 801
<210> 33
<211> 801
<212> DNA
<213> Klebsiella pneumoniae
<400> 33
atgttaaaag ttattagtag tttattggtc tacatgaccg cgtctgtcat ggctgtcgca 60
agtccgttag cccattccgg ggagccgagt ggtgagtatc cgacagtcaa cgaaattccg 120
gtcggagagg tccgacttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcggtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgaa 300
aagcaaattg gacttcccgt aacgcgtgca atctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggcagaggg gaacgagatt cccacgcatt ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gagctcttct atcctggtgc tgcgcattcg 540
accgacaatc tggttgtata cgtcccgtca gcgaacgtgc tatacggtgg ttgtgccgtt 600
catgagttgt caagcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccgttg agcggattca aaaacactac ccggaagcag aggtcgtcat tcccgggcac 720
ggtctaccgg gcggtctaga cttgctccag cacacagcga acgttgtcaa agcacacaaa 780
aatcgctcag tcgccgagta g 801
<210> 34
<211> 801
<212> DNA
<213> Klebsiella oxytoca
<400> 34
atgttaaaag ttattagtag tttattggtc tacatgaccg cgtctgtcat ggctgtcgca 60
agtccgttag cccattccgg ggagccgagt ggtgagtatc cgacagtcaa cgaaattccg 120
gtcggagagg tccgacttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcggtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgaa 300
aagcaaattg gacttcccgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggcagaggg gaacgagatt cccacgcatt ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gagctcttct atcctggtgc tgcgcattcg 540
accgacaatc tggttgtata cgtcccgtca gcgaacgtgc tatacggtgg ttgtgccgtt 600
catgagttgt caagcacgtc tgcggggaac gtgaccgatg ccgatctggc tgaatggccc 660
acctccgttg agcggattca aaaacactac ccggaagcag aggtcgtcat tcccgggcac 720
ggtctaccgg gcggtctaga cttgctccag cacacagcga acgttgtcaa agcacacaaa 780
aatcgctcag tcgccgagta g 801
<210> 35
<211> 801
<212> DNA
<213> Pseudomonas aeruginosa
<400> 35
atgttcaaac ttttgagtaa gttattggtc tatttgaccg cgtctatcat ggctattgcg 60
agtccgctcg ctttttccgt agattctagc ggtgagtatc cgacagtcag cgaaattccg 120
gtcggggagg tccggcttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcgg 180
tcgtttgatg gcgcagtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgag 300
aagcaaattg gacttcctgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggtagaggg gaacgagatt cccacgcact ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gaactcttct atcctggtgc tgcgcattcg 540
accgacaact tagttgtgta cgtcccgtct gcgagtgtgc tctatggtgg ttgtgcgatt 600
tatgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccattg agcggattca acaacactac ccggaagcac agttcgtcat tccggggcac 720
ggcctgccgg gcggtctaga cttgctcaag cacacaacga atgttgtaaa agcgcacaca 780
aatcgctcag tcgttgagta g 801
<210> 36
<211> 801
<212> DNA
<213> Pseudomonas aeruginosa
<400> 36
atgttaaaag ttattagtag tttattggtc tacatgaccg cgtctgtcat ggctgtcgca 60
agtccgttag cccattccgg ggagccgagt ggtgagtatc cgacagtcaa cgaaattccg 120
gtcggagagg tccgacttta ccagattgcc gatggtgttt ggtcgcatat ctcaacgcag 180
tcgtttgatg gcgcggtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgaa 300
aagcaaattg gacttcccgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggcagaggg gaacgagatt cccacgcatt ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gagctcttct atcctggtgc tgcgcattcg 540
accgacaatc tggttgtata cgtcccgtca gcgaacgtgc tatacggtgg ttgtgccgtt 600
catgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccgttg agcggattca aaaacactac ccggaagcag aggtcgtcat tcccgggcac 720
ggtctaccgg gcggtctaga cttgctccag cacacagcga acgttgtcaa agcacacaaa 780
aatcgctcag tcgccgagta g 801
<210> 37
<211> 801
<212> DNA
<213> Pseudomonas aeruginosa
<400> 37
atgttaaaag ttattagtag tttattggtc tatttgaccg cgtctgtcat ggctgtagct 60
agtccgttag cccattccgg ggagccgagt ggtgagtatc cgacagtcaa cgaaattccg 120
gtcggagagg tccggcttta ccagattgct gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcggtcta cccatccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cccttctcgc ggagattgag 300
aagcaaattg gacttcccgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg gaaggctgga gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggcagaggg gaacgagatt cccacgcact ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gagctcttct atcccggtgc tgcgcattcg 540
accgacaatc tggttgtata cgtcccgtca gcgaacgtgc tatacggtgg ttgtgccgtt 600
cttgcgttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccgttg agcggattca aaaacactac ccggaagcag aggtcgtcat tcccgggcac 720
ggtctaccgg gcggtctaga cttgctccag cacacagcga acgttgtcac agcacacaaa 780
aatcgctcag tcgttgagta g 801
<210> 38
<211> 801
<212> DNA
<213> Klebsiella pneumoniae
<400> 38
atgttaaaag ttattagtag tttattggtc tacatgaccg cgtctgtcat ggctgtcgca 60
agtccgttag cccattccgg ggagccgagt ggtgagtatc cggcagtcaa cgaaattccg 120
gtcggagagg tccgacttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcggtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgaa 300
aagcaaattg gacttcccgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggcagaggg gaacgagatt cccacgcatt ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gagctcttct atcctggtgc tgcgcattcg 540
accgacaatc tggttgtata cgtcccgtca gcgaacgtgc tatacggtgg ttgtgccgtt 600
cttgagttgt caagcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccgttg agcggattca aaaacactac ccggaagcag aggtcgtcat tcccgggcac 720
ggtctaccgg gcggtctaga cttgctccag cacacagcga acgttgtcaa agcacacaaa 780
aatcgctcag tcgccgagta g 801
<210> 39
<211> 801
<212> DNA
<213> Klebsiella pneumoniae
<400> 39
atgttaaaag ttattagtag tttattggtc tacatgaccg cgtctgtcat ggctgtcgca 60
agtccgttag cccattccgg ggagccgagt ggtgagtatc cgacagtcaa cgaaattccg 120
gtcggagagg tccgacttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcggtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgaa 300
aagcaaattg gacttcccgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggcagaggg gaacgagatt cccacgcatt ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gagctcttct atcctggtgc tgcgcattcg 540
accgacaatc tggttgtata cgtcccgtca gcgaacgtgc tatacggtgg ttgtgccgtt 600
catgagttgt caagcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccattg agcggattca aaaacactac ccggaagcag aggtcgtcat tcccgggcac 720
ggtctaccgg gcggtctaga cttgctccag cacacagcga acgttgtcaa agcacacaaa 780
aatcgctcag tcgccgagta g 801
<210> 40
<211> 801
<212> DNA
<213> Pseudomonas aeruginosa
<400> 40
atgttaaaag ttattagtag tttattggtc tacatgaccg cgtctgtcat ggctgtcgca 60
agtccgttag tccattccgg ggagccgagt ggtgagtatc cgacagtcaa cgaaattccg 120
gtcggagagg tccgacttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcggtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgaa 300
aagcaaattg gacttcccgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggcagaggg gaacgagatt cccacgcatt ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gagctcttct atcctggtgc tgcgcattcg 540
accgacaatc tggttgtata cgtcccgtca gcgaacgtgc tatacggtgg ttgtgccgtt 600
catgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccgttg agcggattca aaaacactac ccggaagcag aggtcgtcat tcccgggcac 720
ggtctaccgg gcggtctaga cttgctccag cacacagcga acgttgtcaa agcacacaaa 780
aatcgctcag tcgccgagta g 801
<210> 41
<211> 801
<212> DNA
<213> Pseudomonas aeruginosa
<400> 41
atgttcaaac ttttgagtaa gttattggtc tatttgaccg cgtctatcat ggctattgcg 60
agtccgctcg ctttttccgt agattctagc ggtgagtatc cgacagtcag cgaaattccg 120
gtcggggagg tccggcttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcagtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgag 300
aagcaaattg gacttcctgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcgcc gtcgacacgc 420
cggctagccg aggtagaggg gaacgagatt cccacgcact ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gaactcttct atcctggtgc tgcgcattcg 540
accgacaact tagttgtgta cgtcccgtct gcgagtgtgc tctatggtgg ttgtgcgatt 600
tatgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccattg agcggattca acaacactac ccggaagcac agttcgtcat tccggggcac 720
ggcctgccgg gcggtctaga cttgctcaag cacacaacga atgttgtaaa tgcgcacaca 780
aatcgctcag tcgttgagta g 801
<210> 42
<211> 801
<212> DNA
<213> Pseudomonas aeruginosa
<400> 42
atgttcaaac ttttgagtaa gttattggtc tatttgaccg cgtctatcat ggctattgcg 60
agtccgctcg ctttttccgt agattctagc ggtgagtatc cgatagtcag cgaaattccg 120
gtcggggagg tccggcttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcagtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgag 300
aagcaaattg gacttcctgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggtagaggg gaacgagatt cccacgcact ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gaactcttct atcctggtgc tgcgcattcg 540
accgacaact tagttgtgta cgtcccgtct gcgagtgtgc tctatggtgg ttgtgcgatt 600
tatgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccattg agcggattca acaacactac ccggaagcac agttcgtcat tccggggcac 720
ggcctgccgg gcggtctaga cttgctcaag cacacaacga atgttgtaaa agcgcacaca 780
aatcgctcag tcgttgagta g 801
<210> 43
<211> 801
<212> DNA
<213> Pseudomonas aeruginosa
<400> 43
atgttcaaat ttttgagtaa gttattggtc tatttgaccg cgtctatcat ggctattgcg 60
agtccgctcg ctttttccgt agattctagc ggtgagtatc cgacagccag cgaaattccg 120
gtcggggagg tccggcttta ccagattgcc gatggtgttt ggtcgcatat cgcaacgcag 180
tcgtttgatg gcgcagtcta cccgtccaat ggtctcattg tccgtgatgg tgatgagttg 240
cttttgattg atacagcgtg gggtgcgaaa aacacagcgg cacttctcgc ggagattgag 300
aagcaaattg gacttcctgt aacgcgtgca gtctccacgc actttcatga cgaccgcgtc 360
ggcggcgttg atgtccttcg ggcggctggg gtggcaacgt acgcatcacc gtcgacacgc 420
cggctagccg aggtagaggg gaacgagatt cccacgcact ctctagaagg actctcatcg 480
agcggggacg cagtgcgctt cggtccagta gaactcttct atcctggtgc tgcgcattcg 540
accgacaact tagttgtgta cgtcccgtct gcgagtgtgc tctatggtgg ttgtgcgatt 600
tatgagttgt cacgcacgtc tgcggggaac gtggccgatg ccgatctggc tgaatggccc 660
acctccattg agcggattca acaacactac ccggaagcac agttcgtcat tccggggcac 720
ggcctgccgg gcggtctaga cttgctcaag cacacaacga atgttgtaaa agcgcacaca 780
aatcgctcag tcgttgagta g 801
Claims (10)
1. a kind of primer combination, which is characterized in that including forward primer and reverse primer, for expanding Carbapenems antimicrobial
Object drug resistant gene blaVIMIn the sequence as shown in SEQ ID NO.1.
2. primer combination according to claim 1, which is characterized in that the sequence of the forward primer such as SEQ ID NO.2
Shown, the sequence of the reverse primer is as shown in SEQ ID NO.3.
3. primer combination according to claim 1, which is characterized in that the Carbapenems Drug-resistant genes
blaVIMIncluding blaVIM-1、blaVIM-2、blaVIM-3、blaVIM-4、blaVIM-5、blaVIM-6、blaVIM-8、blaVIM-9、blaVIM-10、
blaVIM-11、blaVIM-12、blaVIM-14、blaVIM-15、blaVIM-16、blaVIM-17、blaVIM-18、blaVIM-19、blaVIM-23、
blaVIM-24、blaVIM-25、blaVIM-26、blaVIM-27、blaVIM-28、blaVIM-29、blaVIM-30、blaVIM-31、blaVIM-32、
blaVIM-33、blaVIM-34、blaVIM-35、blaVIM-36、blaVIM-37、blaVIM-38、blaVIM-39、blaVIM-42、blaVIM-43、
blaVIM-44、blaVIM-45And blaVIM-46In it is one or more.
4. a kind of probe, which is characterized in that the probe includes oligonucleotide sequence, 5 ' the end companies with the oligonucleotide sequence
The fluorophor that connects and quenching group with 3 ' end connections of the oligonucleotide sequence, the oligonucleotide sequence can be with
Sequence shown in SEQ ID NO.1 is matched.
5. probe according to claim 4, which is characterized in that the oligonucleotide sequence is as shown in SEQ ID NO.4.
6. a kind of kit, which is characterized in that including being used to detect Carbapenems Drug-resistant genes blaVIMMiddle SEQ
The detection reagent of sequence shown in ID NO.1.
7. kit according to claim 6, which is characterized in that the detection reagent includes as claims 1 to 3 is any
Primer combination described in.
8. kit according to claim 6, which is characterized in that the detection reagent further includes such as claim 4 to 5 times
Probe described in one.
9. a kind of Carbapenems Drug-resistant genes blaVIMDetection method, include the following steps：
The sample to be tested of predetermined amount is taken, total DNA is extracted；And
Using the total DNA as template, is combined with primer according to any one of claims 1 to 3 and carry out PCR amplification；And
Measure the sequence length of amplified production.
10. a kind of Carbapenems Drug-resistant genes blaVIMDetection method, include the following steps：S10 provides root
According to the combination of claims 1 to 3 any one of them primer and according to claim 4 to 5 any one of them probe, to contain
The standard items of sequence shown in SEQ ID NO.1 are template, to multiple standard solutions of the standard items containing various concentration
Real-time fluorescence quantitative PCR reaction is carried out, the standard curve of real-time fluorescence quantitative PCR reaction is obtained；And
S20 is combined, probe and reaction condition carry out in fact using sample to be tested as template using primer identical with the step S10
When quantitative fluorescent PCR react, obtained Ct values are substituted into the standard curve, obtain containing SEQ ID in the sample to be tested
The starting copies concentration of the target gene of sequence shown in NO.1.
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