CN106884037A - A kind of gene chip kit of detection bacterium drug resistant gene - Google Patents
A kind of gene chip kit of detection bacterium drug resistant gene Download PDFInfo
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Abstract
The invention discloses a kind of gene chip kit of detection bacterium drug resistant gene.Containing the primer pair group for detection bacterium drug resistant gene in kit provided by the present invention, it is made up of 13 primer pairs, its sequence is sequence 1-26 in sequence table；Also contain in the kit simultaneously and be fixed with 13 hybridization hybrid chips of ssDNA probe shown in sequence 27-39.The kit that the present invention is provided supports high flux, the quickly and accurately multiple bacterial resistance genes of detection, and examination is carried out to Chinese population, can effectively act as the effects such as Accurate Diagnosis, resistance are traced to the source, resistance is controlled, so that antibiotic usage amount, reduces resistance and produce.
Description
Technical field
The invention belongs to nucleic acid amplification technologies field, it is related to a kind of gene chip kit of detection bacterium drug resistant gene.
Background technology
Bacterial resistance gene detection has important clinical significance.The consumption that there is antibiotic in clinical diagnosis is big, high starting point,
The more random defect of species, so as to cause generation and the prevalence of resistance strain.Currently used for drug resistant gene detection still compared with
It is tradition, PCR (PCR) is subject to after substantially having physiological and biochemical test, serological test, drug sensitive test etc.
The method for expanding corresponding gene, and above-mentioned detection method needs the time long, method operation is loaded down with trivial details, report result is slow, logical
Amount is low, and often first identifies strain, could accordingly carry out the follow-up tests such as susceptibility, thus be difficult in adapt to clinic to control
The need for treatment.Therefore the molecular diagnostic techniques detection of exploitation fast high-flux, can assist quick diagnosis, instruct timely
Accurate medication.
Biochip technology is to be developed rapidly in life science in recent years and a ripe new and high technology, mainly
It is the miniature biochemical analysis system by micro-processing technology and microelectric technique in solid phase chip surface construction, can be real
Now to accurate, quick, bulk information the detection of cell, protein, nucleic acid and other various biotic components.It is raw
Thing chip be mainly characterized by high flux, miniaturization and automate.
The content of the invention
First purpose of the invention is to provide a kind of primer pair group for detection bacterium drug resistant gene.
Primer pair group for detection bacterium drug resistant gene provided by the present invention, is made up of following 13 primer pairs：
1) primer pair shown in following (a1) or (a2), is designated as primer pair 1：
(a1) primer pair that two single strand dnas shown in sequence in sequence table 1 and sequence 2 are constituted；
(a2) by by sequence in sequence table 1 and sequence 2 by the substitution of one or several nucleotides and/or missing and/
Or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (a1)
Identical primer pair；
2) primer pair shown in following (b1) or (b2), is designated as primer pair 2：
(b1) primer pair that two single strand dnas shown in sequence in sequence table 3 and sequence 4 are constituted；
(b2) by by sequence in sequence table 3 and sequence 4 by the substitution of one or several nucleotides and/or missing and/
Or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (b1)
Identical primer pair；
3) primer pair shown in following (c1) or (c2), is designated as primer pair 3：
(c1) primer pair that two single strand dnas shown in sequence in sequence table 5 and sequence 6 are constituted；
(c2) by by sequence in sequence table 5 and sequence 6 by the substitution of one or several nucleotides and/or missing and/
Or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (c1)
Identical primer pair；
4) primer pair shown in following (d1) or (d2), is designated as primer pair 4：
(d1) primer pair that two single strand dnas shown in sequence in sequence table 7 and sequence 8 are constituted；
(d2) by by sequence in sequence table 7 and sequence 8 by the substitution of one or several nucleotides and/or missing and/
Or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (d1)
Identical primer pair；
5) primer pair shown in following (e1) or (e2), is designated as primer pair 5：
(e1) primer pair that two single strand dnas shown in sequence in sequence table 9 and sequence 10 are constituted；
(e2) by by sequence in sequence table 9 and sequence 10 by the substitution of one or several nucleotides and/or missing and/
Or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (e1)
Identical primer pair；
6) primer pair shown in following (f1) or (f2), is designated as primer pair 6：
(f1) primer pair that two single strand dnas shown in sequence in sequence table 11 and sequence 12 are constituted；
(f2) by by sequence in sequence table 11 and sequence 12 by the substitution of one or several nucleotides and/or missing and
/ or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (f1)
Identical primer pair；
7) primer pair shown in following (g1) or (g2), is designated as primer pair 7：
(g1) primer pair that two single strand dnas shown in sequence in sequence table 13 and sequence 14 are constituted；
(g2) by by sequence in sequence table 13 and sequence 14 by the substitution of one or several nucleotides and/or missing and
/ or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (g1)
Identical primer pair；
8) primer pair shown in following (h1) or (h2), is designated as primer pair 8：
(h1) primer pair that two single strand dnas shown in sequence in sequence table 15 and sequence 16 are constituted；
(h2) by by sequence in sequence table 15 and sequence 16 by the substitution of one or several nucleotides and/or missing and
/ or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (h1)
Identical primer pair；
9) primer pair shown in following (i1) or (i2), is designated as primer pair 9：
(i1) primer pair that two single strand dnas shown in sequence in sequence table 17 and sequence 18 are constituted；
(i2) by by sequence in sequence table 17 and sequence 18 by the substitution of one or several nucleotides and/or missing and
/ or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (i1)
Identical primer pair；
10) primer pair shown in following (j1) or (j2), is designated as primer pair 10：
(j1) primer pair that two single strand dnas shown in sequence in sequence table 19 and sequence 20 are constituted；
(j2) by by sequence in sequence table 19 and sequence 20 by the substitution of one or several nucleotides and/or missing and
/ or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (j1)
Identical primer pair；
11) primer pair shown in following (k1) or (k2), is designated as primer pair 11：
(k1) primer pair that two single strand dnas shown in sequence in sequence table 21 and sequence 22 are constituted；
(k2) by by sequence in sequence table 21 and sequence 22 by the substitution of one or several nucleotides and/or missing and
/ or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (k1)
Identical primer pair；
12) primer pair shown in following (l1) or (l2), is designated as primer pair 12：
(l1) primer pair that two single strand dnas shown in sequence in sequence table 23 and sequence 24 are constituted；
(l2) by by sequence in sequence table 23 and sequence 24 by the substitution of one or several nucleotides and/or missing and
/ or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (l1)
Identical primer pair；
13) primer pair shown in following (m1) or (m2), is designated as primer pair 13：
(m1) primer pair that two single strand dnas shown in sequence in sequence table 25 and sequence 26 are constituted；
(m2) by the substitution by sequence in sequence table 25 and sequence 26 by one or several nucleotides and/or missing
And/or addition after gained sequence shown in two single strand dnas composition, and with primer pair work(described in (m1)
Can identical primer pair.
Wherein, the primer pair 1 is used to expand mecA genes；The primer pair 2 is used to expand vanA genes；Institute
Primer pair 3 is stated for expanding vanB genes；The primer pair 4 is used to expand blaDHA-1Gene；The primer pair 5
For expanding blaOXA-23Gene；The primer pair 6 is used to expand blaOXA-24Gene；The primer pair 7 is used to expand
Increase blaOXA-58Gene；The primer pair 8 is used to expand blaKPC-1Gene；The primer pair 9 is used to expand blaIMP-4
Gene；The primer pair 10 is used to expand blaVIM-8Gene；The primer pair 11 is used to expand blaNDM-1Gene；
The primer pair 12 is used to expand blaCTX-M-1Gene；The primer pair 13 is used to expand blaCTX-M-9Gene.
Second object of the present invention is to provide a kind of complete single stranded DNA for detection bacterium drug resistant gene.
Complete single stranded DNA for detection bacterium drug resistant gene provided by the present invention, specifically by probe groups and described draws
Thing is to a group composition；The probe groups are made up of following 13 ssDNA probes：In sequence table shown in sequence 27
SsDNA probe 1；SsDNA probe 2 in sequence table shown in sequence 28；In sequence table shown in sequence 29
SsDNA probe 3；SsDNA probe 4 in sequence table shown in sequence 30；The institute of sequence 31 in sequence table
The ssDNA probe 5 shown；SsDNA probe 6 in sequence table shown in sequence 32；Sequence 33 in sequence table
Shown ssDNA probe 7；SsDNA probe 8 in sequence table shown in sequence 34；Sequence in sequence table
SsDNA probe 9 shown in 35；SsDNA probe 10 in sequence table shown in sequence 36；Sequence in sequence table
SsDNA probe 11 shown in row 37；SsDNA probe 12 in sequence table shown in sequence 38；Sequence table
SsDNA probe 13 shown in middle sequence 39.
Wherein, the ssDNA probe 1 is used to detect the amplification of the primer pair 1；The single stranded DNA
Probe 2 is used to detect the amplification of the primer pair 2；The ssDNA probe 3 is used to detect the primer
To 3 amplification；The ssDNA probe 4 is used to detect the amplification of the primer pair 4；The list
Ssdna probe 5 is used to detect the amplification of the primer pair 5；The ssDNA probe 6 is used to detect institute
State the amplification of primer pair 6；The ssDNA probe 7 is used to detect the amplification of the primer pair 7；
The ssDNA probe 8 is used to detect the amplification of the primer pair 8；The ssDNA probe 9 is used for
Detect the amplification of the primer pair 9；The ssDNA probe 10 is used to detect the expansion of the primer pair 10
Increase result；The ssDNA probe 11 is used to detect the amplification of the primer pair 11；The single stranded DNA
Probe 12 is used to detect the amplification of the primer pair 12；The ssDNA probe 13 is used to detect described drawing
Thing to 13 amplification.
Third object of the present invention is to provide a kind of kit for detection bacterium drug resistant gene.
Kit for detection bacterium drug resistant gene provided by the present invention, contains the primer pair group, and hybridization
Chip；The hybridization hybrid chip is prepared according to the method for comprising the following steps：It is " NH by formula2-(T)n- miscellaneous
13 kinds of single-stranded detection probes of friendship probe sequence " are fixed to consolidating for aldehyde group modifiedization by amino with the reaction of aldehyde radical respectively
On phase carrier (such as slide), the hybridization hybrid chip is obtained；(T) in the formulanRepresent n continuous T, n
It is the integer more than or equal to 5, and less than or equal to 30；Corresponding to 13 kinds of single-stranded detection probes in the formula
13 kinds of hybridization probe sequences are respectively as shown in sequence 27-39 in sequence table.
As needed, can also contain through the random primer of fluorescence labeling in the kit；The sequence of the random primer
It is 5 '-NX- 3 ', N represents that any one in A, G, C and T, 6≤X≤15, and X are integer (such as X=9),
NXRepresent X continuous deoxyribonucleotide.
The above is for the amplified production to the primer pair in the primer pair group through the random primer of fluorescence labeling
Carry out fluorescence labeling.As needed, it is also possible to do not use the random primer through fluorescence labeling to enter amplified production
Row fluorescence labeling, but use other method.As will be described in primer pair group a primer of each primer pair 5 ' end
End carries out fluorescence labeling (such as TAMRA), and the primer being fluorescently labeled is that be present in amplified production can be by correspondent probe
Primer on the single stranded DNA of hybridization.
Hybridization solution can also be contained in the kit；Contain the unrelated single stranded DNA through fluorescence labeling in the hybridization solution
Molecule；The unrelated single strand dna is the non-single strand dna from the bacterial resistance gene.
The preparation method of the hybridization hybrid chip is also included surface chemistry Quality Control probe QC, and/or hybridization Quality Control probe
PC, and/or negative control probe BC is fixed to the solid phase carrier of aldehyde group modifiedization by amino with the reaction of aldehyde radical
Step on (such as slide)；
The surface chemistry Quality Control probe QC, the hybridization Quality Control probe PC and the negative control probe are single-stranded
Probe；Through amido modified, the other end has fluorescence labeling (such as Hex) for one end of the surface chemistry Quality Control probe QC；
One end of the hybridization Quality Control probe PC, and can be with the described unrelated single stranded DNA in the hybridization solution through amido modified
Molecule hybridizes；One end of the negative control probe BC through amido modified, and with from the bacterial resistance gene
Any single strand dna can not hybridize.
Further, in the present invention, structure compositions of the surface chemistry Quality Control probe QC from 5 ' ends to 3 ' ends is
“NH2-TCACTTGCTTCCGTTGAGG-Hex”；Knots of the hybridization Quality Control probe PC from 5 ' ends to 3 ' ends
Structure composition is " NH2-TTTTTTTTTTTTCCTCAACGGAAGCAAGTGAT”；The negative control is visited
Pin BC is held to the 3 ' structure compositions held from 5 '
“NH2-TTTTTTTTTTTTGTTGCTTCTGGAATGAGTTTGCT”。
The application of the primer pair group or the complete single stranded DNA or the kit in following (a) or (b)
Fall within protection scope of the present invention：
A () is detected or auxiliary detection bacterium drug resistant gene (non-diagnostic purpose), or prepare for detecting or aiding in detection
The product of bacterial resistance gene；
B () is detected or auxiliary bacterium (non-diagnostic purpose) of the detection containing bacterial resistance gene, or prepare for detecting
Or the product of auxiliary bacterium of the detection containing bacterial resistance gene.
In the present invention, at least one during the bacterial resistance gene is concretely following：MecA genes, vanA
Gene, vanB genes, blaDHA-1Gene, blaOXA-23Gene, blaOXA-24Gene, blaOXA-58Gene, blaKPC-1
Gene, blaIMP-4Gene, blaVIM-8Gene, blaNDM-1Gene, blaCTX-M-1Gene and blaCTX-M-9Gene.
The GenBank accession number of the mecA genes is AB221119.1 (update：2006-5-19)；The vanA
The GenBank accession number of gene is M97297.1 (update：2002-6-20)；The GenBank of the vanB genes
Accession number is AY655711.1 (update：2005-11-7)；The blaDHA-1The GenBank accession number of gene is
EF406115.1(update：2007-8-17)；The blaOXA-23The GenBank accession number of gene is JN665073.1
(update：2011-11-7)；The blaOXA-24The GenBank accession number of gene is JN207494.1 (update：
2011-11-27)；The blaOXA-58The GenBank accession number of gene is EU107372.1 (update：2007-9-11)；
The blaKPC-1The GenBank accession number of gene is AF297554.1 (update：2001-3-26)；The blaIMP-4
The GenBank accession number of gene is AF244145.1 (update：2001-2-27)；The blaVIM-8Gene
GenBank accession number is AY524987.1 (update：2004-11-5)；The blaNDM-1The GenBank of gene
Accession number is JF503991.1 (update：2012-12-11)；The blaCTX-M-1The GenBank accession number of gene is
AJ416342.1(update：2008-10-23)；The blaCTX-M-9The GenBank accession number of gene is AJ416345.1
(update：2005-4-15).
Accordingly, bacterium containing the mecA genes concretely staphylococcus aureus；Contain the vanA bases
The bacterium of cause concretely enterococcus faecalis or VREF；Bacterium containing the vanB genes concretely enterococcus faecalis
Or VREF；Contain the blaDHA-1The bacterium of gene concretely Klebsiella Pneumoniae；Contain the blaOXA-23
The bacterium of gene concretely Acinetobacter bauamnnii；Contain the blaOXA-24Concretely Bao Man is motionless for the bacterium of gene
Bacillus；Contain the blaOXA-58The bacterium of gene concretely Acinetobacter bauamnnii；Contain the blaKPC-1Gene
Bacterium concretely Klebsiella Pneumoniae or pseudomonas aeruginosa；Contain the blaIMP-4The bacterium of gene concretely Bao
Graceful acinetobacter calcoaceticus；Contain the blaVIM-8The bacterium of gene concretely Klebsiella Pneumoniae or Acinetobacter bauamnnii；Contain
There is the blaNDM-1The bacterium of gene concretely ETEC；Contain the blaCTX-M-1The bacterium tool of gene
Body can be proteus mirabilis；Contain the blaCTX-M-9The bacterium of gene concretely ETEC.
The application of the primer pair group or the complete single stranded DNA in the kit is prepared falls within of the invention
Protection domain.
The present invention is studied the bacterial resistance gene of very big sample size.The kit that the present invention is provided supports high pass
Amount, quickly and accurately detects multiple drug resistant genes, and examination is carried out to Chinese population, can effectively act as Accurate Diagnosis,
The effect such as resistance is traced to the source, resistance is controlled, so that antibiotic usage amount, reduces resistance and produce.
Brief description of the drawings
Fig. 1 is the dot matrix arrangement schematic diagram (i.e. chip probe schematic layout pattern) of hybridization hybrid chip.
Fig. 2 is the specific analysis result of the kit for detection bacterium drug resistant gene.The corresponding chip probe of the figure
Schematic layout pattern is as shown in Figure 1.Wherein, the corresponding reference material DNA plasmid of chip detection result shown in A is
ZL-CTX-M-1 (correspondence blaCTX-M-1Gene)；The corresponding reference material DNA plasmid of chip detection result shown in B is
ZL-CTX-M-9 (correspondence blaCTX-M-9Gene)；The corresponding reference material DNA plasmid of chip detection result shown in C is
ZL-DHA-1 (correspondence blaDHA-1Gene)；The corresponding reference material DNA plasmid of chip detection result shown in D is
ZL-IMP-4 (correspondence blaIMP-4Gene)；The corresponding reference material DNA plasmid of chip detection result shown in E is
ZL-KPC-1 (correspondence blaKPC-1Gene)；The corresponding reference material DNA plasmid of chip detection result shown in F is
ZL-NDM-1 (correspondence blaNDM-1Gene)；The corresponding reference material DNA plasmid of chip detection result shown in G is
ZL-OXA-23 (correspondence blaOXA-23Gene)；The corresponding reference material DNA plasmid of chip detection result shown in H is
ZL-OXA-24 (correspondence blaOXA-24Gene)；The corresponding reference material DNA plasmid of chip detection result shown in I is
ZL-OXA-58 (correspondence blaOXA-58Gene)；The corresponding reference material DNA plasmid of chip detection result shown in J is
ZL-vanA (correspondence vanA genes)；The corresponding reference material DNA plasmid of chip detection result shown in K is ZL-vanB
(correspondence vanB genes)；The corresponding reference material DNA plasmid of chip detection result shown in L is ZL-VIM-8 (correspondences
blaVIM-8Gene)；The corresponding reference material DNA plasmid of chip detection result shown in M is ZL-mecA (correspondence mecA
Gene).
Fig. 3 is three parts of chip detection result figures of clinical sample.Wherein, A is No. 1 clinical sample；B faces for No. 2
Bed sample；C is No. 3 clinical samples.The corresponding chip probe schematic layout pattern of the figure is as shown in Figure 1.
Specific embodiment
Experimental technique used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.
Embodiment 1, for detection bacterium drug resistant gene kit preparation and its use
First, for detection bacterium drug resistant gene kit assembling with prepare
1st, for 13 primers and hybridization probe of bacterial resistance gene design
The primer designed for 13 bacterial resistance genes and single-stranded hybridization probe particular sequence are as shown in Table 1 and Table 2.
Table 1 is for 13 primers of bacterial resistance gene design
Table 2 is for 13 single-stranded hybridization probes of bacterial resistance gene design
2nd, hybridization probe is fixed on hybridization hybrid chip
Hybridization hybrid chip is to be respectively fixed with 13 kinds of substrates of single-stranded detection probe.Every kind of detection probe is all that one section of amino is repaiied
The oligonucleotide probe of decorations, 13 kinds of formulas of single-stranded detection probe are " NH2- TTTTTTTTTTTTTTT- hybridization is visited
Pin sequence ", wherein " hybridization probe sequence " is 13 kinds of single-stranded hybridization probes shown in sequence 27-39 in table 2 ".
With gene sampling liquid, (Capitalbio Corporation Co., Ltd.'s product, its catalog number is each detection probe respectively
CP.440010) dissolve, final concentration of 10 μM, in triplicate slide (rich biological collection difficult to understand of the point system to aldehyde radicalization modification
Group's Co., Ltd product, its catalog number is CP.420022) on, so that by the reaction of amino and aldehyde radical by 13
Probe is planted to be fixed on hybridization hybrid chip.In addition, again by amino and aldehyde radical reaction by surface chemistry Quality Control probe QC,
Hybridization Quality Control probe PC and negative control probe BC is again secured on hybridization hybrid chip.QC is that one end marks with Hex,
The other end has amido modified single strand oligonucleotide probes, and for observing chip point sample and fixed efficiency, it is from 5 '
The structure composition held to 3 ' ends is NH2-TCACTTGCTTCCGTTGAGG-Hex.PC is one section amido modified
Single strand oligonucleotide probes, can with hybridization solution add the unrelated single strand dna through fluorescence labeling
(C-PC) hybridize, for the Quality Control of crossover process, its structure composition from 5 ' ends to 3 ' ends is
NH2-TTTTTTTTTTTTCCTCAACGGAAGCAAGTGAT.BC is one section of amido modified few nucleosides
Sequence all to be detected in acid probe, with hybridization system will not hybridize, for observing whether there is Non-specific hybridization, its
It is to the 3 ' structure compositions held from 5 ' ends
NH2-TTTTTTTTTTTTGTTGCTTCTGGAATGAGTTTGCT.Fig. 1 is arranged for the dot matrix of hybridization hybrid chip
Cloth schematic diagram.
3rd, for detection bacterium drug resistant gene kit composition
It is provided by the present invention for detection bacterium drug resistant gene (13 kinds of bacterial resistance bases being related in Tables 1 and 2
Cause) kit contain：
(1) 13 primer pairs in step 1 shown in table 1；
(2) 13 kinds of detection probes and surface chemistry Quality Control probe QC, hybridization Quality Control probe are fixed with step 2
The hybridization hybrid chip of PC and negative control probe BC；
(3) through the random primer of fluorescence labeling；The sequence of the random primer is 5 '-N9- 3 ', N represent A, G,
Any one in C and T, N9Represent 9 continuous deoxyribonucleotides.
(4) hybridization solution；Containing the unrelated single strand dna (C-PC) through fluorescence labeling in the hybridization solution, its
Nucleotides sequence is classified as 5 '-ATCACTTGCTTCCGTTGAGG-3 '.
2nd, for detection bacterium drug resistant gene kit application method
1st, sample PCR (PCR) amplification
1 μ L DNA sample solution to be measured is taken, with it as template, 13 primer pairs shown in table 1 is respectively adopted
Enter performing PCR amplification.20 μ L PCR amplification systems are as follows：10 × PCR buffer (contain Mg2+)2μL；2.5mM
dNTP 1.6μL；10 μM of μ L of sense primer 0.5；10 μM of μ L of anti-sense primer 0.5；The μ L of template 1；5U/μL rTaq
0.2μL；Moisturizing is to 20 μ L.PCR amplification programs are as follows：94℃5min；(94℃30s；55℃30s；72
DEG C 1min) 30 circulations；72℃10min.PCR reactions are obtained 13 parts of pcr amplification products after terminating.
2nd, the fluorescence labeling of pcr amplification product
Every kind of pcr amplification product takes 5 μ L, is added in different sample cells, and 3 μ L concentration are added in each sample cell
For 100 μM through TAMRA fluorescence labelings 9N random primers (nucleotides sequence is classified as 5 '-N9-3 ', N represent A,
Any one in G, C and T, N9 represents 9 continuous deoxyribonucleotides, work of specifically making a living bioengineering (on
Sea) limited company's product), moisturizing to 19 μ L, concussion is mixed, brief centrifugation；It is placed on ice after 95 DEG C of denaturation,
Add 6 μ L fluorescence labeling reaction systems mix (compositions：10×Klenow Buffer 2.5μL；5U/μL Klenow
The μ L of enzyme 1；2.5mM dNTP 2.5μL).Follow procedure carries out fluorescence labeling, and program is as follows：37℃90min；70
℃10min.13 parts of TAMRA fluorescent mark products are obtained.
3rd, the purifying of TAMRA fluorescent mark products
The Nucleo Spin Gel and PCR Clean-up kit (products produced using Macherey-Nagel companies
Article No.：REF740609*250), the TAMRA fluorescent mark products that step 2 is obtained are purified, concrete operations
Carried out according to kit specification.
4th, hybridize
The unrelated single strand dna through TAMRA fluorescence labelings will be contained
(5 '-ATCACTTGCTTCCGTTGAGG-3 ') hybridization buffer (Capitalbio Corporation Co., Ltd.'s product,
Its catalog number is CP.440030) in 50 DEG C of thawings, (wherein step 3 is after purification to prepare 13 parts of hybrid mixed liquid
The μ L of TAMRA fluorescent mark products solution 15, hybridization buffer 5 μ L), the 13 parts of hybrid mixed liquid that will be prepared
It is added on the hybridization hybrid chip in step one 2, every part of hybrid mixed liquid one intact hybridization chip of correspondence, 95 DEG C of denaturation
3min, places on ice immediately, and 50 DEG C of water-bath hybridization 2h.
5th, clean
With two kinds of different washing lotion (washing lotion I：2 × SSC, 0.2%SDS, are preheated to 50 DEG C；Washing lotion II：0.2 × SSC,
Be preheated to 50 DEG C) be respectively washed 4min according to the order of washing lotion II after first washing lotion I after, chip is placed on
In SlideWasher_8 chip cleaning devices, centrifuging process is selected, (or centrifuge 1000rpm centrifugations 2min) is centrifuged,
Dry.
6th, scanner uni result judgement
Scanning (selection green channel, setting are completed using rich Austria crystalline substance core LuxScan 10K micro-array chip scanners
The parameter area of " Power " is 50-90 and the parameter area of " PMT " is 500-900), and pressed according to scanning result
Determine whether contain 13 kinds of bacterial resistance genes being related in table 1, and tool in DNA sample to be measured according to following method
Body contains 13 kinds of any or which kinds of bacterial resistance gene：For detecting the probe of certain bacterial resistance gene in chip
If on fixed position detect fluorescence signal, judge in corresponding DNA sample to be measured containing corresponding thin
Bacterium drug resistant gene；Corresponding bacterial resistance gene is not contained then otherwise.In addition, software can be according to detection in scanning process
The fluorescence signal intensity for arriving assigns different colours, and color is gradually strengthened by blueness to white, signal, and then can tentatively be sentenced
The height of target bacteria drug resistant gene content in DNA sample to be measured of breaking.
Embodiment 2, for detection bacterium drug resistant gene kit specificity and sensitivity determination
First, the preparation of reference material DNA plasmid
The structure of the plasmid the 1st, containing mecA gene target fragments
By mecA genes (GenBank：AB221119.1, update：1001-1926 institute 2006-5-19)
Show that DNA fragmentation is connected on pGEM-T Easy Vector carriers (promega Products), obtain recombinating matter
Grain ZL-mecA.And it is correct through sequence verification.
The structure of the plasmid the 2nd, containing vanA gene target fragments
By vanA genes (GenBank：M97297.1, update：Shown in 7000-7988 2002-6-20)
DNA fragmentation is connected on pGEM-T Easy Vector carriers, obtains recombinant plasmid ZL-vanA.And through sequence verification
Correctly.
The structure of the plasmid the 3rd, containing vanB gene target fragments
By vanB genes (GenBank：AY655711.1, update：Shown in 14-1029 2005-11-7)
DNA fragmentation is connected on pGEM-T Easy Vector carriers, obtains recombinant plasmid ZL-vanB.And through sequence verification
Correctly.
4th, bla is containedDHA-1The structure of the plasmid of gene target fragment
By blaDHA-1Gene (GenBank：EF406115.1, update：Shown in 1-1113 2007-8-17)
DNA fragmentation is connected on pGEM-T Easy Vector carriers, obtains recombinant plasmid ZL-DHA-1.And tested through sequencing
Card is correct.
5th, bla is containedOXA-23The structure of the plasmid of gene target fragment
By blaOXA-23Gene (GenBank：JN665073.1, update：97-899 institute 2011-11-7)
Show that DNA fragmentation is connected on pGEM-T Easy Vector carriers, obtain recombinant plasmid ZL-OXA-23.And through surveying
Sequence checking is correct.
6th, bla is containedOXA-24The structure of the plasmid of gene target fragment
By blaOXA-24Gene (GenBank：JN207494.1, update：4168-4882 2011-11-27)
DNA fragmentation shown in position is connected on pGEM-T Easy Vector carriers, obtains recombinant plasmid ZL-OXA-24.And
It is correct through sequence verification.
7th, bla is containedOXA-58The structure of the plasmid of gene target fragment
By blaOXA-58Gene (GenBank：EU107372.1, update：42-835 institute 2007-9-11)
Show that DNA fragmentation is connected on pGEM-T Easy Vector carriers, obtain recombinant plasmid ZL-OXA-58.And through surveying
Sequence checking is correct.
8th, bla is containedKPC-1The structure of the plasmid of gene target fragment
By blaKPC-1Gene (GenBank：AF297554.1, update：154-1003 institute 2001-3-26)
Show that DNA fragmentation is connected on pGEM-T Easy Vector carriers, obtain recombinant plasmid ZL-KPC-1.And through sequencing
Checking is correct.
9th, bla is containedIMP-4The structure of the plasmid of gene target fragment
By blaIMP-4Gene (GenBank：AF244145.1, update：Shown in 1-470 2001-2-27)
DNA fragmentation is connected on pGEM-T Easy Vector carriers, obtains recombinant plasmid ZL-IMP-4.And tested through sequencing
Card is correct.
10th, bla is containedVIM-8The structure of the plasmid of gene target fragment
By blaVIM-8Gene (GenBank：AY524987.1, update：Shown in 89-798 2004-11-5)
DNA fragmentation is connected on pGEM-T Easy Vector carriers, obtains recombinant plasmid ZL-VIM-8.And tested through sequencing
Card is correct.
11st, bla is containedNDM-1The structure of the plasmid of gene target fragment
By blaNDM-1Gene (GenBank：JF503991.1, update：118437-119168 2012-12-11)
DNA fragmentation shown in position is connected on pGEM-T Easy Vector carriers, obtains recombinant plasmid ZL-NDM-1.And
It is correct through sequence verification.
12nd, bla is containedCTX-M-1The structure of the plasmid of gene target fragment
By blaCTX-M-1Gene (GenBank：AJ416342.1, update：567-1373 2008-10-23)
DNA fragmentation shown in position is connected on pGEM-T Easy Vector carriers, obtains recombinant plasmid ZL-CTX-M-1.And
It is correct through sequence verification.
13rd, bla is containedCTX-M-9The structure of the plasmid of gene target fragment
By blaCTX-M-9Gene (GenBank：AJ416345.1, update：2005-4-15) 218-958
Shown DNA fragmentation is connected on pGEM-T Easy Vector carriers, obtains recombinant plasmid ZL-CTX-M-9.And pass through
Sequence verification is correct.
2nd, the specificity for the kit of detection bacterium drug resistant gene is analyzed
It is 10 that 13 kinds of reference material DNA plasmids that step one builds are diluted into concentration4Copy/μ L, respectively with dilution
13 kinds of reference material DNA plasmids afterwards are DNA sample to be measured, are then operated according to the step 2 of embodiment 1,
Detect that whether specific decision method is referring to the step of embodiment 1 containing purposeful bacterial resistance gene in DNA sample to be measured
26.
Result is as shown in Fig. 2 be only to secure phase as seen from the figure for each reference material DNA plasmid
The point of detection probe is answered to be able to detect that obvious fluorescence signal, other points do not detect fluorescence signal.The result table
The bright kit for detection bacterium drug resistant gene provided by the present invention has stronger specificity.
3rd, for detection bacterium drug resistant gene kit sensitivity analysis
13 kinds of reference material DNA plasmids that above-mentioned steps one build are carried out into gradient dilution respectively, concentration is obtained and is followed successively by
104Copy/μ L, 103Copy/μ L, 102Copy/μ L, 101The dilution of copy/μ L.Respectively with different dilution factors
13 kinds of reference material DNA plasmids are DNA sample to be measured, are then operated according to the step 2 of embodiment 1, are detected
Whether contain purposeful bacterial resistance gene in DNA sample to be measured, specific decision method is referring to the step 26 of embodiment 1.
Result shows：For each reference material DNA plasmid, concentration is 104Copy/μ L, 103Copy/μ L,
102The point of corresponding detection probe can detect obvious fluorescence signal during copy/μ L；Only concentration is 101Copy/μ L's
Reference material DNA plasmid does not detect fluorescence signal.The result shows provided by the present invention resistance to for detection bacterium
The kit of medicine gene has sensitivity higher.
The detection of embodiment 3, actual clinical sample
First, clinical sample type
The present embodiment using clinical sample come from The People's Hospital of Peking University collection the sticky fester in people wound (this
The voluntary principle of this picker), totally three parts.
2nd, in clinical sample DNA sample extraction
1st, the clinical sample 1-3mL of step one is taken；
2nd, 4 times of NaOH of volume 4% (4g/100mL) are added, is shaken up, room temperature places 30min liquefaction；
3rd, the NaOH of fester and 0.5mL 4% (4g/100mL) after 0.5mL liquefies, room temperature, 10min are taken；
4th, 12 000rpm centrifugations 15min；
5th, supernatant, plus SPSS 1mL are abandoned, is mixed, 12 000rpm centrifugations 5min；
6th, supernatant is abandoned, is precipitated and is extracted for DNA；
7th, (Capitalbio Corporation Co., Ltd.'s product, its catalog number is to add 50 μ L nucleic acid extractions liquid
CP.360090), fully shaking is mixed, and precipitation is suspended completely；
8th, suspension is transferred in nucleic acid extraction pipe, screws lid, be put into instrument for extracting nucleic acid or vortex concussion instrument most
Big rotating speed concussion 5min；
9th, 95 DEG C of metal bath heating 5min；
10th, 5000rpm centrifugations 1min, supernatant is transferred in 1.5mL centrifuge tubes, and -20 DEG C save backup.
3rd, the detection of actual clinical sample
Three parts of clinical sample DNA that step 2 is obtained are DNA sample to be measured, then according to the step 2 of embodiment 1
Operated, whether purposeful bacterial resistance gene is contained in detection DNA sample to be measured, specific decision method is referring to reality
Apply the step 26 of example 1.
Result is as shown in figure 3, three parts of sample hybridization signals are clear and single as seen from the figure.Compared through with probe location,
Detection obtains signal for drug resistant gene bla in No. 1 clinical sampleCTX-M-9And blaKPC-1, detected in No. 2 clinical samples
It is drug resistant gene bla to signalOXA-23、blaOXA-58And blaVIM-8, it is resistance to that detection obtains signal in No. 3 clinical samples
Medicine gene blaCTX-M-1And blaDHA-1。
In order to further determine that the accuracy of above testing result of the present invention, the present inventor is by three parts of clinical samples
Pcr amplification product carried out agarose gel electrophoresis and sequence verification, as a result confirm：No. 1 clinical sample only with
The amplified production of primer pair CTX-M9-F/CTX-M9-R and KPC-F/KPC-R has obvious electrophoretic band, and
The amplified production of other primer pairs does not detect obvious electrophoretic band, further to primer pair
The amplified production sequencing of CTX-M9-F/CTX-M9-R and KPC-F/KPC-R finds that its sequence respectively just is
blaCTX-M-9Gene (GenBank：AJ416345.1, update：218-958 2005-4-15) and blaKPC-1
Gene (GenBank：AF297554.1, update：2001-3-26) 154-1003；No. 2 clinical samples
Only with primer pair OXA23-F/OXA23-R, OXA58-F/OXA58-R and the amplified production of VIM-F/VIM-R
With obvious electrophoretic band, and the amplified production of other primer pairs does not detect obvious electrophoretic band, enters one
Walk and the amplified production of primer pair OXA23-F/OXA23-R, OXA58-F/OXA58-R and VIM-F/VIM-R is surveyed
Sequence finds that its sequence respectively just is blaOXA-23Gene (GenBank：JN665073.1, update：2011-11-7)
97-899, blaOXA-58Gene (GenBank：EU107372.1, update：42-835 2007-9-11)
Position and blaVIM-8Gene (GenBank：AY524987.1, update：2004-11-5) 89-798；No. 3
Clinical sample has obvious only with the amplified production of primer pair CTX-M1-F/CTX-M1-R and DHA-F/DHA-R
Electrophoretic band, and the amplified production of other primer pairs does not detect obvious electrophoretic band, further to primer
Amplified production sequencing to CTX-M1-F/CTX-M1-R and DHA-F/DHA-R finds that its sequence respectively just is
blaCTX-M-1Gene (GenBank：AJ416342.1, update：567-1373 2008-10-23) and blaDHA-1
Gene (GenBank：EF406115.1, update：2007-8-17) 1-1113.The result proves to utilize
The testing result of kit of the present invention is accurately and reliably.
Claims (10)
1. it is used for the primer pair group of detection bacterium drug resistant gene, is made up of following 13 primer pairs：
1) primer pair shown in following (a1) or (a2), is designated as primer pair 1：
(a1) primer pair that two single strand dnas shown in sequence in sequence table 1 and sequence 2 are constituted；
(a2) by by sequence in sequence table 1 and sequence 2 by the substitution of one or several nucleotides and/or missing and/
Or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (a1)
Identical primer pair；
2) primer pair shown in following (b1) or (b2), is designated as primer pair 2：
(b1) primer pair that two single strand dnas shown in sequence in sequence table 3 and sequence 4 are constituted；
(b2) by by sequence in sequence table 3 and sequence 4 by the substitution of one or several nucleotides and/or missing and/
Or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (b1)
Identical primer pair；
3) primer pair shown in following (c1) or (c2), is designated as primer pair 3：
(c1) primer pair that two single strand dnas shown in sequence in sequence table 5 and sequence 6 are constituted；
(c2) by by sequence in sequence table 5 and sequence 6 by the substitution of one or several nucleotides and/or missing and/
Or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (c1)
Identical primer pair；
4) primer pair shown in following (d1) or (d2), is designated as primer pair 4：
(d1) primer pair that two single strand dnas shown in sequence in sequence table 7 and sequence 8 are constituted；
(d2) by by sequence in sequence table 7 and sequence 8 by the substitution of one or several nucleotides and/or missing and/
Or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (d1)
Identical primer pair；
5) primer pair shown in following (e1) or (e2), is designated as primer pair 5：
(e1) primer pair that two single strand dnas shown in sequence in sequence table 9 and sequence 10 are constituted；
(e2) by by sequence in sequence table 9 and sequence 10 by the substitution of one or several nucleotides and/or missing and/
Or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (e1)
Identical primer pair；
6) primer pair shown in following (f1) or (f2), is designated as primer pair 6：
(f1) primer pair that two single strand dnas shown in sequence in sequence table 11 and sequence 12 are constituted；
(f2) by by sequence in sequence table 11 and sequence 12 by the substitution of one or several nucleotides and/or missing and
/ or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (f1)
Identical primer pair；
7) primer pair shown in following (g1) or (g2), is designated as primer pair 7：
(g1) primer pair that two single strand dnas shown in sequence in sequence table 13 and sequence 14 are constituted；
(g2) by by sequence in sequence table 13 and sequence 14 by the substitution of one or several nucleotides and/or missing and
/ or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (g1)
Identical primer pair；
8) primer pair shown in following (h1) or (h2), is designated as primer pair 8：
(h1) primer pair that two single strand dnas shown in sequence in sequence table 15 and sequence 16 are constituted；
(h2) by by sequence in sequence table 15 and sequence 16 by the substitution of one or several nucleotides and/or missing and
/ or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (h1)
Identical primer pair；
9) primer pair shown in following (i1) or (i2), is designated as primer pair 9：
(i1) primer pair that two single strand dnas shown in sequence in sequence table 17 and sequence 18 are constituted；
(i2) by by sequence in sequence table 17 and sequence 18 by the substitution of one or several nucleotides and/or missing and
/ or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (i1)
Identical primer pair；
10) primer pair shown in following (j1) or (j2), is designated as primer pair 10：
(j1) primer pair that two single strand dnas shown in sequence in sequence table 19 and sequence 20 are constituted；
(j2) by by sequence in sequence table 19 and sequence 20 by the substitution of one or several nucleotides and/or missing and
/ or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (j1)
Identical primer pair；
11) primer pair shown in following (k1) or (k2), is designated as primer pair 11：
(k1) primer pair that two single strand dnas shown in sequence in sequence table 21 and sequence 22 are constituted；
(k2) by by sequence in sequence table 21 and sequence 22 by the substitution of one or several nucleotides and/or missing and
/ or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (k1)
Identical primer pair；
12) primer pair shown in following (l1) or (l2), is designated as primer pair 12：
(l1) primer pair that two single strand dnas shown in sequence in sequence table 23 and sequence 24 are constituted；
(l2) by by sequence in sequence table 23 and sequence 24 by the substitution of one or several nucleotides and/or missing and
/ or addition after gained sequence shown in two single strand dnas composition, and with primer pair function described in (l1)
Identical primer pair；
13) primer pair shown in following (m1) or (m2), is designated as primer pair 13：
(m1) primer pair that two single strand dnas shown in sequence in sequence table 25 and sequence 26 are constituted；
(m2) by the substitution by sequence in sequence table 25 and sequence 26 by one or several nucleotides and/or missing
And/or addition after gained sequence shown in two single strand dnas composition, and with primer pair work(described in (m1)
Can identical primer pair.
2. the complete single stranded DNA of detection bacterium drug resistant gene, the primer as described in probe groups and claim 1 are used for
To a group composition；
The probe groups are made up of following 13 ssDNA probes：Single stranded DNA in sequence table shown in sequence 27
Probe 1；SsDNA probe 2 in sequence table shown in sequence 28；Single stranded DNA in sequence table shown in sequence 29
Probe 3；SsDNA probe 4 in sequence table shown in sequence 30；Single stranded DNA in sequence table shown in sequence 31
Probe 5；SsDNA probe 6 in sequence table shown in sequence 32；Single stranded DNA in sequence table shown in sequence 33
Probe 7；SsDNA probe 8 in sequence table shown in sequence 34；Single stranded DNA in sequence table shown in sequence 35
Probe 9；SsDNA probe 10 in sequence table shown in sequence 36；Single stranded DNA in sequence table shown in sequence 37
Probe 11；SsDNA probe 12 in sequence table shown in sequence 38；It is single-stranded shown in sequence 39 in sequence table
DNA probe 13.
3. the kit of detection bacterium drug resistant gene is used for, it is containing the primer pair group described in claim 1 and miscellaneous
Hand over chip；
The hybridization hybrid chip is prepared according to the method for comprising the following steps：It is " NH by formula2-(T)n- hybridization
13 kinds of single-stranded detection probes of probe sequence " are fixed to the solid phase of aldehyde group modifiedization by amino with the reaction of aldehyde radical respectively
On carrier, the hybridization hybrid chip is obtained；(T) in the formulanRepresent the continuous T of n, n be more than or equal to 5,
And the integer less than or equal to 30；Corresponding to 13 kinds of hybridization probe sequences of 13 kinds of single-stranded detection probes in the formula
Row are respectively as shown in sequence 27-39 in sequence table.
4. kit according to claim 3, it is characterised in that：Also contain through fluorescence mark in the kit
The random primer of note；The sequence of the random primer is 5 '-NX- 3 ', N represents any one in A, G, C and T,
6≤X≤15, and X is integer, NXRepresent X continuous deoxyribonucleotide.
5. the kit according to claim 3 or 4, it is characterised in that：Also contain hybridization in the kit
Liquid；Contain the unrelated single strand dna through fluorescence labeling in the hybridization solution；The unrelated single strand dna is
The non-single strand dna from the bacterial resistance gene.
6. kit according to claim 5, it is characterised in that：The preparation method of the hybridization hybrid chip is also wrapped
Include and surface chemistry Quality Control probe QC, and/or hybridization Quality Control probe PC, and/or negative control probe BC are passed through into amino
The step on the solid phase carrier of aldehyde group modifiedization is fixed to the reaction of aldehyde radical；
The surface chemistry Quality Control probe QC, the hybridization Quality Control probe PC and the negative control probe are single-stranded
Probe；
Through amido modified, the other end has fluorescence labeling for one end of the surface chemistry Quality Control probe QC；
One end of the hybridization Quality Control probe PC through amido modified, and can with it is described unrelated single-stranded in the hybridization solution
DNA molecular hybridizes；
One end of the negative control probe BC through amido modified, and with from any of the bacterial resistance gene
Single strand dna can not hybridize.
7. kit according to claim 6, it is characterised in that：The surface chemistry Quality Control probe QC is from 5 '
The structure composition held to 3 ' ends is " NH2-TCACTTGCTTCCGTTGAGG-Hex”；
The hybridization Quality Control probe PC is held to the 3 ' structure compositions held from 5 '
“NH2-TTTTTTTTTTTTCCTCAACGGAAGCAAGTGAT”；
The negative control probe BC is held to the 3 ' structure compositions held from 5 '
“NH2-TTTTTTTTTTTTGTTGCTTCTGGAATGAGTTTGCT”。
8. the complete single stranded DNA or claim 3-7 described in the primer pair group or claim 2 described in claim 1
In application of any described kit in following (a) or (b)：
A () is detected or auxiliary detection bacterium drug resistant gene, or prepare for detecting or aiding in detection bacterium drug resistant gene
Product；
B () is detected or auxiliary bacterium of the detection containing bacterial resistance gene, or prepare for detecting or aiding in detection to contain
There is the product of the bacterium of bacterial resistance gene.
9. according to any primer pair group in claim 1-8 or complete single stranded DNA or kit or application, its
It is characterised by：The bacterial resistance gene be it is following at least one：MecA genes, vanA genes, vanB bases
Cause, blaDHA-1Gene, blaOXA-23Gene, blaOXA-24Gene, blaOXA-58Gene, blaKPC-1Gene, blaIMP-4
Gene, blaVIM-8Gene, blaNDM-1Gene, blaCTX-M-1Gene and blaCTX-M-9Gene.
10. the complete single stranded DNA described in the primer pair group or claim 2 described in claim 1 is preparing right
It is required that the application in 3-7 in any described kit.
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| CN111876508A (en) * | 2020-09-01 | 2020-11-03 | 上海市动物疫病预防控制中心(上海市兽药饲料检测所、上海市畜牧技术推广中心) | Fluorescent quantitative PCR kit for detecting Escherichia coli CTX-M gene |
| CN112309499A (en) * | 2020-11-09 | 2021-02-02 | 浙江大学 | Method and device for quickly annotating bacterial pdif |
| CN113249507A (en) * | 2021-07-05 | 2021-08-13 | 广州赛哲生物科技股份有限公司 | Co-detection method for existence and expression condition of pathogen drug resistance gene |
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