CN106754906A - LAMP primer composition and its application for detecting seven kinds of drug resistant genes of Carbapenem-resistant class antibiotic - Google Patents

LAMP primer composition and its application for detecting seven kinds of drug resistant genes of Carbapenem-resistant class antibiotic Download PDF

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CN106754906A
CN106754906A CN201710017455.8A CN201710017455A CN106754906A CN 106754906 A CN106754906 A CN 106754906A CN 201710017455 A CN201710017455 A CN 201710017455A CN 106754906 A CN106754906 A CN 106754906A
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sequence
primer
drug resistant
resistant gene
oxa
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张岩
刘莹莹
王颢婷
邢婉丽
程京
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CapitalBio Corp
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    • C12Q2600/16—Primer sets for multiplex assays

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Abstract

LAMP primer composition and its application the invention discloses seven kinds of drug resistant genes for detecting Carbapenem-resistant class antibiotic.Present invention firstly provides a kind of combination of primer, 42 kinds of DNA moleculars shown in sequence 1 to sequence 42 are constituted.Whether primer combination contains drug resistant gene OXA 23 and/or drug resistant gene OXA 24 and/or drug resistant gene OXA 58 and/or drug resistant gene OXA 66 and/or drug resistant gene KPC 2 and/or drug resistant gene IMP 4 and/or drug resistant gene VIM 2 in can be applied to detect bacterium to be measured or sample to be tested.The primer combination identification that the present invention is provided is used to detect seven kinds of drug resistant genes of Carbapenem-resistant class antibiotic, with high specific and high sensitivity, it is possible to achieve easy, quick, accurate detection.The present invention has great promotional value.

Description

LAMP primer group for detecting seven kinds of drug resistant genes of Carbapenem-resistant class antibiotic Close and its apply
Technical field
The invention belongs to biological technical field, and in particular to seven kinds of resistance bases for detecting Carbapenem-resistant class antibiotic The LAMP primer composition of cause and its application.
Background technology
China is one of abuse of antibiotics situation country the most serious, however as the increasing of antibiotic usage amount, carefully Bacterium resistance situation is also serious all the more.Antibiotics are various, and mechanism of action is various, and the carbon wherein in beta-lactam antibiotic is blue or green Mould alkenes can be by the height parent with macromolecule PBP (PBP) in gram-positive bacterium or negative bacteria And power, play bactericidal action by suppressing bacteria cell wall synthesis.Drug resistant gene (such as drug resistant gene in drug-resistant bacteria body OXA-23, drug resistant gene OXA-24, drug resistant gene OXA-58, drug resistant gene OXA-66, drug resistant gene KPC-2, drug resistant gene IMP-4 or drug resistant gene VIM-2) then can by produce inactivation carbapenem antibiotic beta-lactamase, OprD lack or There is efflux pump, PBP Target alterations cause the mechanism such as affinity decline to complete the effect for inactivating carbapenem antibiotic.
The reason for bacterium produces drug resistance is a lot, is broadly divided into generation inactivator, antibacterials target site and changes, changes Bacterial outer membrane permeability, influence are actively pumped out six aspects such as system, the formation of influence Bacterial biofilm and cross resistance.
Mainly there is following several method on the detection to bacterial drug resistance at present：Bacterial resistance phenotype detection (including Resistance screening test, the instrumentation of break sensitization test and drug sensitive test and automation etc.), beta-lactamase detection, it is special resistance to Medicine bacterium is detected and drug resistant gene is detected etc..Wherein the most intuitively detection method is, with the drug test of in vitro culture, but still to deposit In the defect such as incubation time is long.Research with resistance mechanism is progressively goed deep into, and molecular biology method detection bacterium resistance is gradually Received by clinic.The method of common detection bacterium drug resistant gene mainly has：PCR, PCR- Restriction Fragment Lengths Polymorphism analysis, PCR- single-strand conformation polymorphism analysis, biochip technology, automated DNA sequencing etc..Traditional identified for genes side Method is PCR methods and DNA sequencing method, wherein traditional PCR method detection cycles are more long, it usually needs 3-4 hour；Sanger DNA PCR sequencing PCR is higher to primer specificity requirement, and price is costly, and later data analysis is more complicated, and being not suitable for clinical expansion makes With.
Ring mediated isothermal amplification method (loop-mediated isothermal amplification, LAMP) is to utilize one The Bst archaeal dna polymerases with strand-displacement activity and waterfall type nucleic acid amplification function are planted, the change of nucleic acid is carried out under isothermal conditions The strand displacement nucleic acid amplification reaction of property and automatic cycle.LAMP designs 4 special primers for 6 regions of target sequence, utilizes The archaeal dna polymerase for possessing strand displacement function constantly replicates DNA amplification at a constant temperature.In order to improve reaction efficiency, can be anti- Two ring primers of addition in system are answered, is allowed to be combined with loop-stem structure respectively, start strand displacement synthesis, recursive copying.
LAMP has easy, quick, sensitive, special advantage, is particluarly suitable for the application of development at the basic level LAMP kit Promote.In LAMP technology, primer is the key factor for determining testing result sensitivity and specificity.
The content of the invention
The purpose of the present invention is to provide a kind of seven kinds of LAMP of drug resistant gene for detecting Carbapenem-resistant class antibiotic Primer is combined and its applied.
It is following (a1) or (a2) or (a3) present invention firstly provides a kind of combination of primer：
(a1) by primer sets I, primer sets II, primer sets III, primer sets IV, primer sets V, primer sets VI and primer sets VII Composition；
(a2) by the primer sets I, the primer sets II, the primer sets III, the primer sets IV, the primer sets Vth, in the primer sets VI and the primer sets VII any two, any three, any four, any five or any six Individual composition；
(a3) primer sets I, the primer sets II, the primer sets III, the primer sets IV, the primer sets V, The primer sets VI or the primer sets VII.
The primer sets I can by-the F3 of the primer I ,-B3 of the primer I ,-FIP of the primer I ,-BIP of the primer I ,-LF of primer I and primer I- LB is constituted；
- the F3 of the primer I can be following (b1) or (b2)；
(b1) single strand dna shown in the sequence 1 of sequence table；
(b2) sequence 1 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 1 The DNA molecular of identical function；
- the B3 of the primer I can be following (b3) or (b4)；
(b3) single strand dna shown in the sequence 2 of sequence table；
(b4) sequence 2 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 2 The DNA molecular of identical function；
- the FIP of the primer I can be following (b5) or (b6)；
(b5) single strand dna shown in the sequence 3 of sequence table；
(b6) sequence 3 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 3 The DNA molecular of identical function；
- the BIP of the primer I can be following (b7) or (b8)；
(b7) single strand dna shown in the sequence 4 of sequence table；
(b8) sequence 4 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 4 The DNA molecular of identical function；
- the LF of the primer I can be following (b9) or (b10)；
(b9) single strand dna shown in the sequence 5 of sequence table；
(b10) sequence 5 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 5 The DNA molecular of identical function；
- the LB of the primer I can be following (b11) or (b12)；
(b11) single strand dna shown in the sequence 6 of sequence table；
(b12) sequence 6 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 6 The DNA molecular of identical function.
The primer sets II can by-the F3 of primer II ,-the B3 of primer II ,-the FIP of primer II ,-the BIP of primer II ,-the LF of primer II and - the LB of primer II is constituted；
- the F3 of the primer II can be following (c1) or (c2)；
(c1) single strand dna shown in the sequence 7 of sequence table；
(c2) sequence 7 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 7 The DNA molecular of identical function；
- the B3 of the primer II can be following (c3) or (c4)；
(c3) single strand dna shown in the sequence 8 of sequence table；
(c4) sequence 8 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 8 The DNA molecular of identical function；
- the FIP of the primer II can be following (c5) or (c6)；
(c5) single strand dna shown in the sequence 9 of sequence table；
(c6) sequence 9 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 9 The DNA molecular of identical function；
- the BIP of the primer II can be following (c7) or (c8)；
(c7) single strand dna shown in the sequence 10 of sequence table；
(c8) sequence 10 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 10 There is the DNA molecular of identical function；
- the LF of the primer II can be following (c9) or (c10)；
(c9) single strand dna shown in the sequence 11 of sequence table；
(c10) sequence 11 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 11 There is the DNA molecular of identical function；
- the LB of the primer II can be following (c11) or (c12)；
(c11) single strand dna shown in the sequence 12 of sequence table；
(c12) sequence 12 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 12 There is the DNA molecular of identical function.
The primer sets III can by-the F3 of primer III ,-the B3 of primer III ,-the FIP of primer III ,-the BIP of primer III ,-the LF of primer III and - the LB of primer III is constituted；
- the F3 of the primer III can be following (d1) or (d2)；
(d1) single strand dna shown in the sequence 13 of sequence table；
(d2) sequence 13 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 13 There is the DNA molecular of identical function；
- the B3 of the primer III can be following (d3) or (d4)；
(d3) single strand dna shown in the sequence 14 of sequence table；
(d4) sequence 14 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 14 There is the DNA molecular of identical function；
- the FIP of the primer III can be following (d5) or (d6)；
(d5) single strand dna shown in the sequence 15 of sequence table；
(d6) sequence 15 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 15 There is the DNA molecular of identical function；
- the BIP of the primer III can be following (d7) or (d8)；
(d7) single strand dna shown in the sequence 16 of sequence table；
(d8) sequence 16 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 16 There is the DNA molecular of identical function；
- the LF of the primer III can be following (d9) or (d10)；
(d9) single strand dna shown in the sequence 17 of sequence table；
(d10) sequence 17 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 17 There is the DNA molecular of identical function；
- the LB of the primer III can be following (d11) or (d12)；
(d11) single strand dna shown in the sequence 18 of sequence table；
(d12) sequence 18 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 18 There is the DNA molecular of identical function.
The primer sets IV can by-the F3 of primer IV ,-the B3 of primer IV ,-the FIP of primer IV ,-the BIP of primer IV ,-the LF of primer IV and - the LB of primer IV is constituted；
- the F3 of the primer IV can be following (e1) or (e2)；
(e1) single strand dna shown in the sequence 19 of sequence table；
(e2) sequence 19 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 19 There is the DNA molecular of identical function；
- the B3 of the primer IV can be following (e3) or (e4)；
(e3) single strand dna shown in the sequence 20 of sequence table；
(e4) sequence 20 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 20 There is the DNA molecular of identical function；
- the FIP of the primer IV can be following (e5) or (e6)；
(e5) single strand dna shown in the sequence 21 of sequence table；
(e6) sequence 21 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 21 There is the DNA molecular of identical function；
- the BIP of the primer IV can be following (e7) or (e8)；
(e7) single strand dna shown in the sequence 22 of sequence table；
(e8) sequence 22 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 22 There is the DNA molecular of identical function；
- the LF of the primer IV can be following (e9) or (e10)；
(e9) single strand dna shown in the sequence 23 of sequence table；
(e10) sequence 23 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 23 There is the DNA molecular of identical function；
- the LB of the primer IV can be following (e11) or (e12)；
(e11) single strand dna shown in the sequence 24 of sequence table；
(e12) sequence 24 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 24 There is the DNA molecular of identical function.
The primer sets V can by-the F3 of primer V ,-the B3 of primer V ,-the FIP of primer V ,-the BIP of primer V ,-the LF of primer V and - the LB of primer V is constituted；
- the F3 of the primer V can be following (f1) or (f2)；
(f1) single strand dna shown in the sequence 25 of sequence table；
(f2) sequence 25 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 25 There is the DNA molecular of identical function；
- the B3 of the primer V can be following (f3) or (f4)；
(f3) single strand dna shown in the sequence 26 of sequence table；
(f4) sequence 26 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 26 There is the DNA molecular of identical function；
- the FIP of the primer V can be following (f5) or (f6)；
(f5) single strand dna shown in the sequence 27 of sequence table；
(f6) sequence 27 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 27 There is the DNA molecular of identical function；
- the BIP of the primer V can be following (f7) or (f8)；
(f7) single strand dna shown in the sequence 28 of sequence table；
(f8) sequence 28 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 28 There is the DNA molecular of identical function；
- the LF of the primer V can be following (f9) or (f10)；
(f9) single strand dna shown in the sequence 29 of sequence table；
(f10) sequence 29 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 29 There is the DNA molecular of identical function；
- the LB of the primer V can be following (f11) or (f12)；
(f11) single strand dna shown in the sequence 30 of sequence table；
(f12) sequence 30 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 30 There is the DNA molecular of identical function.
The primer sets VI can by-the F3 of primer VI ,-the B3 of primer VI ,-the FIP of primer VI ,-the BIP of primer VI ,-the LF of primer VI and - the LB of primer VI is constituted；
- the F3 of the primer VI can be following (g1) or (g2)；
(g1) single strand dna shown in the sequence 31 of sequence table；
(g2) sequence 31 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 31 There is the DNA molecular of identical function；
- the B3 of the primer VI can be following (g3) or (g4)；
(g3) single strand dna shown in the sequence 32 of sequence table；
(g4) sequence 32 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 32 There is the DNA molecular of identical function；
- the FIP of the primer VI can be following (g5) or (g6)；
(g5) single strand dna shown in the sequence 33 of sequence table；
(g6) sequence 33 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 33 There is the DNA molecular of identical function；
- the BIP of the primer VI can be following (g7) or (g8)；
(g7) single strand dna shown in the sequence 34 of sequence table；
(g8) sequence 34 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 34 There is the DNA molecular of identical function；
- the LF of the primer VI can be following (g9) or (g10)；
(g9) single strand dna shown in the sequence 35 of sequence table；
(g10) sequence 35 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 35 There is the DNA molecular of identical function；
- the LB of the primer VI can be following (g11) or (g12)；
(g11) single strand dna shown in the sequence 36 of sequence table；
(g12) sequence 36 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 36 There is the DNA molecular of identical function.
The primer sets VII can by-the F3 of primer VII ,-the B3 of primer VII ,-the FIP of primer VII ,-the BIP of primer VII ,-the LF of primer VII and - the LB of primer VII is constituted；
- the F3 of the primer VII can be following (h1) or (h2)；
(h1) single strand dna shown in the sequence 37 of sequence table；
(h2) sequence 37 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 37 There is the DNA molecular of identical function；
- the B3 of the primer VII can be following (h3) or (h4)；
(h3) single strand dna shown in the sequence 38 of sequence table；
(h4) sequence 38 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 38 There is the DNA molecular of identical function；
- the FIP of the primer VII can be following (h5) or (h6)；
(h5) single strand dna shown in the sequence 39 of sequence table；
(h6) sequence 39 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 39 There is the DNA molecular of identical function；
- the BIP of the primer VII can be following (h7) or (h8)；
(h7) single strand dna shown in the sequence 40 of sequence table；
(h8) sequence 40 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 40 There is the DNA molecular of identical function；
- the LF of the primer VII can be following (h9) or (h10)；
(h9) single strand dna shown in the sequence 41 of sequence table；
(h10) sequence 41 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 41 There is the DNA molecular of identical function；
- the LB of the primer VII can be following (h11) or (h12)；
(h11) single strand dna shown in the sequence 42 of sequence table；
(h12) sequence 42 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 42 There is the DNA molecular of identical function.
In the primer sets I ,-the F3 of the primer I ,-B3 of the primer I ,-FIP of the primer I ,-BIP of the primer I ,-LF of primer I and primer I- The mol ratio of LB concretely 0.5：0.5：2：2：1：1.
In the primer sets II ,-the F3 of primer II ,-the B3 of primer II ,-the FIP of primer II ,-the BIP of primer II ,-the LF of primer II and The mol ratio of-the LB of primer II concretely 0.5：0.5：2：2：1：1.
In the primer sets III ,-the F3 of primer III ,-the B3 of primer III ,-the FIP of primer III ,-the BIP of primer III ,-the LF of primer III and The mol ratio of-the LB of primer III concretely 0.5：0.5：2：2：1：1.
In the primer sets IV ,-the F3 of primer IV ,-the B3 of primer IV ,-the FIP of primer IV ,-the BIP of primer IV ,-the LF of primer IV and The mol ratio of-the LB of primer IV concretely 0.5：0.5：2：2：1：1.
In the primer sets V ,-the F3 of primer V ,-the B3 of primer V ,-the FIP of primer V ,-the BIP of primer V ,-the LF of primer V and The mol ratio of-the LB of primer V concretely 0.5：0.5：2：2：1：1.
In the primer sets VI ,-the F3 of primer VI ,-the B3 of primer VI ,-the FIP of primer VI ,-the BIP of primer VI ,-the LF of primer VI and The mol ratio of-the LB of primer VI concretely 0.5：0.5：2：2：1：1.
In the primer sets VII ,-the F3 of primer VII ,-the B3 of primer VII ,-the FIP of primer VII ,-the BIP of primer VII ,-the LF of primer VII and The mol ratio of-the LB of primer VII concretely 0.5：0.5：2：2：1：1.
The present invention also protects application of the primer combination in reagent preparation box；The purposes of the kit can be as follows Or (y2) or (y3) (y1)：
(y1) drug resistant gene OXA-23 and/or drug resistant gene OXA-24 and/or drug resistant gene OXA-58 and/or resistance are identified Gene OXA-66 and/or drug resistant gene KPC-2 and/or drug resistant gene IMP-4 and/or drug resistant gene VIM-2；
(y2) be used to detect in sample to be tested whether containing drug resistant gene OXA-23 and/or drug resistant gene OXA-24 and/or Drug resistant gene OXA-58 and/or drug resistant gene OXA-66 and/or drug resistant gene KPC-2 and/or drug resistant gene IMP-4 and/or resistance to Medicine gene VIM-2；
(y3) it is used to detect whether bacterium to be measured contains drug resistant gene OXA-23 and/or drug resistant gene OXA-24 and/or resistance Gene OXA-58 and/or drug resistant gene OXA-66 and/or drug resistant gene KPC-2 and/or drug resistant gene IMP-4 and/or resistance base Because of VIM-2.
The present invention also kit of the protection containing primer combination；The purposes of the kit can for following (y1) or Or (y3) (y2)：
(y1) drug resistant gene OXA-23 and/or drug resistant gene OXA-24 and/or drug resistant gene OXA-58 and/or resistance are identified Gene OXA-66 and/or drug resistant gene KPC-2 and/or drug resistant gene IMP-4 and/or drug resistant gene VIM-2；
(y2) be used to detect in sample to be tested whether containing drug resistant gene OXA-23 and/or drug resistant gene OXA-24 and/or Drug resistant gene OXA-58 and/or drug resistant gene OXA-66 and/or drug resistant gene KPC-2 and/or drug resistant gene IMP-4 and/or resistance to Medicine gene VIM-2；
(y3) it is used to detect whether bacterium to be measured contains drug resistant gene OXA-23 and/or drug resistant gene OXA-24 and/or resistance Gene OXA-58 and/or drug resistant gene OXA-66 and/or drug resistant gene KPC-2 and/or drug resistant gene IMP-4 and/or resistance base Because of VIM-2.
The present invention also protects the preparation method of the kit, including the step of each bar primer is individually packed.
The present invention also protects whether a kind of detection bacterium to be measured contains drug resistant gene OXA-23 and/or drug resistant gene OXA-24 And/or drug resistant gene OXA-58 and/or drug resistant gene OXA-66 and/or drug resistant gene KPC-2 and/or drug resistant gene IMP-4 and/ Or the method for drug resistant gene VIM-2, comprise the following steps：
(1) genomic DNA of bacterium to be measured is extracted；
(2) genomic DNA with step (1) extraction is respectively adopted each primer sets in the primer combination as template Ring mediated isothermal amplification is carried out, is then made the following judgment：
If using the primer sets I can realize the specific amplification with the genomic DNA as template, in bacterium to be measured Containing or doubtful contain drug resistant gene OXA-23；If using the primer sets I cannot realize with the genomic DNA as mould The specific amplification of plate, does not contain in bacterium to be measured or doubtful does not contain drug resistant gene OXA-23；
If using the primer sets II can realize the specific amplification with the genomic DNA as template, bacterium to be measured In contain or doubtful contain drug resistant gene OXA-24；If using the primer sets II cannot realize with the genomic DNA It is the specific amplification of template, is not contained in bacterium to be measured or doubtful do not contain drug resistant gene OXA-24；
If using the primer sets III can realize the specific amplification with the genomic DNA as template, bacterium to be measured In contain or doubtful contain drug resistant gene OXA-58；If using the primer sets III cannot realize with the genomic DNA It is the specific amplification of template, is not contained in bacterium to be measured or doubtful do not contain drug resistant gene OXA-58；
If using the primer sets IV can realize the specific amplification with the genomic DNA as template, bacterium to be measured In contain or doubtful contain drug resistant gene OXA-66；If using the primer sets IV cannot realize with the genomic DNA It is the specific amplification of template, is not contained in bacterium to be measured or doubtful do not contain drug resistant gene OXA-66；
If using the primer sets V can realize the specific amplification with the genomic DNA as template, bacterium to be measured In contain or doubtful contain drug resistant gene KPC-2；If use the primer sets V cannot realize be with the genomic DNA The specific amplification of template, does not contain in bacterium to be measured or doubtful does not contain drug resistant gene KPC-2；
If using the primer sets VI can realize the specific amplification with the genomic DNA as template, bacterium to be measured In contain or doubtful contain drug resistant gene IMP-4；If use the primer sets VI cannot realize be with the genomic DNA The specific amplification of template, does not contain in bacterium to be measured or doubtful does not contain drug resistant gene IMP-4；
If using the primer sets VII can realize the specific amplification with the genomic DNA as template, bacterium to be measured In contain or doubtful contain drug resistant gene VIM-2；If use the primer sets VII cannot realize be with the genomic DNA The specific amplification of template, does not contain in bacterium to be measured or doubtful does not contain drug resistant gene VIM-2.
Whether the present invention contains drug resistant gene OXA-23 and/or drug resistant gene OXA- in also protecting a kind of detection sample to be tested 24 and/or drug resistant gene OXA-58 and/or drug resistant gene OXA-66 and/or drug resistant gene KPC-2 and/or drug resistant gene IMP-4 And/or the method for drug resistant gene VIM-2, comprise the following steps：
(1) STb gene of sample to be tested is extracted；
(2) as template, each primer sets being respectively adopted in the primer combination are carried out the STb gene with step (1) extraction Ring mediated isothermal amplification, then makes the following judgment：
If using the primer sets I can realize the specific amplification with the STb gene as template, contain in sample to be tested Have or doubtful contain drug resistant gene OXA-23；If using the primer sets I cannot realize the spy with the STb gene as template Specific amplification, does not contain in sample to be tested or doubtful does not contain drug resistant gene OXA-23；
If using the primer sets II can realize the specific amplification with the STb gene as template, in sample to be tested Containing or doubtful contain drug resistant gene OXA-24；If using the primer sets II cannot realize with the STb gene as template Specific amplification, do not contained in sample to be tested or doubtful do not contain drug resistant gene OXA-24；
If using the primer sets III can realize the specific amplification with the STb gene as template, in sample to be tested Containing or doubtful contain drug resistant gene OXA-58；If using the primer sets III cannot realize with the STb gene as template Specific amplification, do not contained in sample to be tested or doubtful do not contain drug resistant gene OXA-58；
If using the primer sets IV can realize the specific amplification with the STb gene as template, in sample to be tested Containing or doubtful contain drug resistant gene OXA-66；If using the primer sets IV cannot realize with the STb gene as template Specific amplification, do not contained in sample to be tested or doubtful do not contain drug resistant gene OXA-66；
If using the primer sets V can realize the specific amplification with the STb gene as template, in sample to be tested Containing or doubtful contain drug resistant gene KPC-2；If using the primer sets V cannot realize with the STb gene as template Specific amplification, does not contain in sample to be tested or doubtful does not contain drug resistant gene KPC-2；
If using the primer sets VI can realize the specific amplification with the STb gene as template, in sample to be tested Containing or doubtful contain drug resistant gene IMP-4；If using the primer sets VI cannot realize with the STb gene as template Specific amplification, does not contain in sample to be tested or doubtful does not contain drug resistant gene IMP-4；
If using the primer sets VII can realize the specific amplification with the STb gene as template, in sample to be tested Containing or doubtful contain drug resistant gene VIM-2；If using the primer sets VII cannot realize with the STb gene as template Specific amplification, does not contain in sample to be tested or doubtful does not contain drug resistant gene VIM-2.
In any of the above methods described, during using the primer sets I, draw in the reaction system of the ring mediated isothermal amplification The molar concentration of-the F3 of the thing I ,-B3 of the primer I ,-FIP of the primer I ,-BIP of the primer I ,-LF of primer I and the-LB of primer I be followed successively by 0.5 μM, 0.5μM、2μM、2μM、1μM、1μM。
In any of the above methods described, during using the primer sets II, in the reaction system of the ring mediated isothermal amplification The molar concentration of-the F3 of the primer II ,-B3 of the primer II ,-FIP of the primer II ,-BIP of the primer II ,-LF of primer II and the-LB of primer II is successively It is 0.5 μM, 0.5 μM, 2 μM, 2 μM, 1 μM, 1 μM.
In any of the above methods described, during using the primer sets III, in the reaction system of the ring mediated isothermal amplification The molar concentration of-the F3 of the primer III ,-B3 of the primer III ,-FIP of the primer III ,-BIP of the primer III ,-LF of primer III and the-LB of primer III is successively It is 0.5 μM, 0.5 μM, 2 μM, 2 μM, 1 μM, 1 μM.
In any of the above methods described, during using the primer sets IV, in the reaction system of the ring mediated isothermal amplification The molar concentration of-the F3 of the primer IV ,-B3 of the primer IV ,-FIP of the primer IV ,-BIP of the primer IV ,-LF of primer IV and the-LB of primer IV is successively It is 0.5 μM, 0.5 μM, 2 μM, 2 μM, 1 μM, 1 μM.
In any of the above methods described, during using the primer sets V, in the reaction system of the ring mediated isothermal amplification The molar concentration of-the F3 of the primer V ,-B3 of the primer V ,-FIP of the primer V ,-BIP of the primer V ,-LF of primer V and the-LB of primer V is successively It is 0.5 μM, 0.5 μM, 2 μM, 2 μM, 1 μM, 1 μM.
In any of the above methods described, during using the primer sets VI, in the reaction system of the ring mediated isothermal amplification The molar concentration of-the F3 of the primer VI ,-B3 of the primer VI ,-FIP of the primer VI ,-BIP of the primer VI ,-LF of primer VI and the-LB of primer VI is successively It is 0.5 μM, 0.5 μM, 2 μM, 2 μM, 1 μM, 1 μM.
In any of the above methods described, during using the primer sets VII, in the reaction system of the ring mediated isothermal amplification The molar concentration of-the F3 of the primer VII ,-B3 of the primer VII ,-FIP of the primer VII ,-BIP of the primer VII ,-LF of primer VII and the-LB of primer VII is successively It is 0.5 μM, 0.5 μM, 2 μM, 2 μM, 1 μM, 1 μM.
In any of the above methods described, loop-mediated isothermal amplification condition is：65 DEG C of constant temperature 50min.
The present invention also protects the primer combination detecting whether bacterium to be measured contains drug resistant gene OXA-23 and/or resistance base Because of OXA-24 and/or drug resistant gene OXA-58 and/or drug resistant gene OXA-66 and/or drug resistant gene KPC-2 and/or drug resistant gene Application in IMP-4 and/or drug resistant gene VIM-2.
The present invention also protects whether primer combination contains drug resistant gene OXA-23 and/or resistance in sample to be tested is detected Medicine gene OXA-24 and/or drug resistant gene OXA-58 and/or drug resistant gene OXA-66 and/or drug resistant gene KPC-2 and/or resistance Application in gene IMP-4 and/or drug resistant gene VIM-2.
The present invention also protect primer combination in identification drug resistant gene OXA-23 and/or drug resistant gene OXA-24 and/or Drug resistant gene OXA-58 and/or drug resistant gene OXA-66 and/or drug resistant gene KPC-2 and/or drug resistant gene IMP-4 and/or resistance to Application in medicine gene VIM-2.
Sample to be tested described in any of the above can be the fester of people (Homo sapiens).
Concretely No. Genebank nucleotide sequence for KP203815.1 of drug resistant gene OXA-23 described in any of the above Shown DNA molecular.Concretely No. Genebank nucleosides for KR922888.1 of drug resistant gene OXA-24 described in any of the above DNA molecular shown in acid sequence.Drug resistant gene OXA-58 described in any of the above concretely No. Genebank be KF740447.1 Nucleotide sequence shown in DNA molecular.Drug resistant gene OXA-66 described in any of the above is for No. Genebank concretely DNA molecular shown in the nucleotide sequence of DQ923479.1.The concretely Genebank of drug resistant gene KPC-2 described in any of the above Number for KT001101.1 nucleotide sequence shown in DNA molecular.Drug resistant gene IMP-4 described in any of the above is concretely No. Genebank DNA molecular shown in the nucleotide sequence of AY795963.1.Drug resistant gene VIM-2 is specific described in any of the above Can be No. ID DNA molecular shown in 14678525 nucleotide sequence of NCBI Gene.
Loop-mediated isothermal amplification technique (loop-mediated isothermal amplification, LAMP) is in recent years Come a kind of sensitive, special, the simple, fast nucleic acid amplification technologies for developing, its principle is that have strand-displacement activity in one kind Archaeal dna polymerase in the presence of, recognize the 6-8 4-6 bar primer in region, under isothermal conditions quickly, specifically expand purpose Gene, can be applied to and fast and accurately detect common Carbapenem-resistant gene.LAMP method has sensitivity High, the specific good, reaction time is short, result of determination is convenient, do not need the advantages such as expensive instrument.
The primer combination identification that the present invention is provided is used to detect seven kinds of drug resistant genes of Carbapenem-resistant class antibiotic, has High specific and high sensitivity, it is possible to achieve easy, quick, accurate detection.The present invention has great promotional value.
Brief description of the drawings
Fig. 1 is the testing result in embodiment 2 using primer sets I.
Fig. 2 is the testing result in embodiment 2 using primer sets II.
Fig. 3 is the testing result in embodiment 2 using primer sets III.
Fig. 4 is the testing result in embodiment 2 using primer sets IV.
Fig. 5 is the testing result in embodiment 2 using primer sets V.
Fig. 6 is the testing result in embodiment 2 using primer sets VI.
Fig. 7 is the testing result in embodiment 2 using primer sets VII.
Fig. 8 is the testing result of sample one in embodiment 4.
Fig. 9 is the testing result of sample two in embodiment 4.
Figure 10 is the testing result of sample three in embodiment 4.
Specific embodiment
The present invention is further described in detail with reference to specific embodiment, the embodiment for being given is only for explaining The bright present invention, rather than in order to limit the scope of the present invention.Experimental technique in following embodiments, unless otherwise specified, is Conventional method.Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.Following reality The quantitative test in example is applied, three repetitions is respectively provided with and is tested, results averaged.
Reaction solution is the product of Capitalbio Corporation Co., Ltd., and catalog number is CP.440020.PUC57 plasmids are The product of Sangon Biotech (Shanghai) Co., Ltd..- T Easy plasmids are the product of Promega companies.
The computational methods of DNA copy number are as follows：
The 1 A260 absorbances=μ g/ml of ds DNA 50；
Nucleic acid concentration=(OD260) × (extension rate) × (50)=x ng/ μ l；
Mean molecule quantity (MW) representative gram/mol, unit dalton (dolton), i.e. 1dolton=1g/mol；
Mole=6.02 × 1023；
Mean molecule quantity (MW)：DsDNA=(base number) × (660 dalton/base)；
Copy number computing formula：
(6.02×1023Copies/ moles) × (x ng/ μ l × 10-9)/(DNA length × 660)=copies/ μ l.
The preparation of embodiment 1, kit
Kit is made up of seven LAMP primer groups, and each primer sets is used to detect a kind of Carbapenem-resistant class antibiotic Drug resistant gene.
Primer sets for detecting drug resistant gene OXA-23 are as follows：
Outer primer F3：5 '-GAATATGTGCCAGCCTCTA-3 ' (sequence 1 in sequence table)；
Outer primer B3：5 '-TCTTTTTGCATGAGATCAAGAC-3 ' (sequence 2 in sequence table)；
Inner primer FIP：5 '-CCTTTTCTCGCCCTTCCATTTAAATTTGAATGCCCTGATCGGA-3 ' are (in sequence table Sequence 3)；
Inner primer BIP：5 '-TACCGCTTGGGAAAAAGACATGGTCGCGCAAGTTCCTGA-3 ' (sequences in sequence table 4)；
Ring primer LF：5 '-CCGTTTTCTGGTTCTCCAA-3 ' (sequence 5 in sequence table)；
Ring primer LB：5 '-ACACTAGGAGAAGCCATGA-3 ' (sequence 6 in sequence table).
Primer sets for detecting drug resistant gene OXA-24 are as follows：
Outer primer F3：5 '-CTGTGTTCCAATATTTGTATTTCC-3 ' (sequence 7 in sequence table)；
Outer primer B3：5 '-GCAACAACAAATGAGATTTTCA-3 ' (sequence 8 in sequence table)；
Inner primer FIP：5 '-ATCAAGAGCTTGCAAGACGGACATTAACCCGCTTTACTTCTTTCTG-3 ' (sequence tables Middle sequence 9)；
Inner primer BIP：5 '-CTGGAACTGCTGACAATGCCATTGAATGGGATGGTAAAAAAAGAACTT-3 ' (sequences Sequence 10 in table)；
Ring primer LF：5 '-TGGCCTAGAGCTAATG-3 ' (sequence 11 in sequence table)；
Ring primer LB：5 '-CCTCACCTAAAGTCATATC-3 ' (sequence 12 in sequence table).
Primer sets for detecting drug resistant gene OXA-58 are as follows：
Outer primer F3：5 '-CCAATTAGTGCATTGGCAATT-3 ' (sequence 13 in sequence table)；
Outer primer B3：5 '-TTGGGGCTTGTGCTGAG-3 ' (sequence 14 in sequence table)；
Inner primer FIP：5 '-TGTGTTTGTCACATATGATGGTCAAAGATGCAGGAATATAAGCTGTT-3 ' (sequences Sequence 15 in table)；
Inner primer BIP：5 '-CGCTTGAACATTCTGATCGATGATTATGAGTCGAGCAAAAACAAGTACA-3 ' (sequences Sequence 16 in list)；
Ring primer LF：5 '-GCATTTAGACCGAGCA-3 ' (sequence 17 in sequence table)；
Ring primer LB：5 '-GAGTTATTCACTTGTGGAA-3 ' (sequence 18 in sequence table).
Primer sets for detecting drug resistant gene OXA-66 are as follows：
Outer primer F3：5 '-CTAATAAAACGCTTCCATTTAGC-3 ' (sequence 19 in sequence table)；
Outer primer B3：5 '-GAACAGAGCTAGGTATTCCTT-3 ' (sequence 20 in sequence table)；
Inner primer FIP：5 '-ACATCCCATCCCCAACCACTTTTTGTCCAAGATGAAGTGCAATCC-3 ' (sequence tables Middle sequence 21)；
Inner primer BIP：5 '-CCACAAGTAGGCTGGTTAACTGTCTAAGTTAAGGGAGAACGCTACA-3 ' (sequence tables Middle sequence 22)；
Ring primer LF：5 '-CCATTCTTTTCTTCTATGAATAGCA-3 ' (sequence 23 in sequence table)；
Ring primer LB：5 '-TTGTTCAGCCTCAAGGGAAT-3 ' (sequence 24 in sequence table).
Primer sets for detecting drug resistant gene KPC-2 are as follows：
Outer primer F3：5 '-CTGACGGCCTTCATGCG-3 ' (sequence 25 in sequence table)；
Outer primer B3：5 '-GCCAATCAACAAACTGCT-3 ' (sequence 26 in sequence table)；
Inner primer FIP：5 '-ATGGCGGAGTTCAGCTCCATCTATCGGCGATACCACGT-3 ' (sequences in sequence table 27)；
Inner primer BIP：5 '-AGGCGATGCGCGCGATACCCAGTGTCAGTTTTTGTAAGC-3 ' (sequences in sequence table 28)；
Ring primer LF：5 '-CCAGCGGTCCAGACG-3 ' (sequence 29 in sequence table)；
Ring primer LB：5 '-GCGCCGTGACGGAAA-3 ' (sequence 30 in sequence table).
Primer sets for detecting drug resistant gene IMP-4 are as follows：
Outer primer F3：5 '-TCTAATTGACACTCCATTTACG-3 ' (sequence 31 in sequence table)；
Outer primer B3：5 '-AGTTAACCCCGCCAAAT-3 ' (sequence 32 in sequence table)；
Inner primer FIP：5 '-ATGAAAATGAGAGGAAATACTGCCTATACTGAAAAGTTAGTCACTTGGT-3 ' (sequences Sequence 33 in list)；
Inner primer BIP：5 '-TGGCTTAATTCTCAATCCATCCCTTAGCTTGAACCTTACCGTCT-3 ' are (in sequence table Sequence 34)；
Ring primer LF：5 '-ATAGCCACGTTCCACAA-3 ' (sequence 35 in sequence table)；
Ring primer LB：5 '-CGTATGCGTCTGAATTAAC-3 ' (sequence 36 in sequence table).
Primer sets for detecting drug resistant gene VIM-2 are as follows：
Outer primer F3：5 '-GGCACTTCTCGCGGAGA-3 ' (sequence 37 in sequence table)；
Outer primer B3：5 '-GCTCGATGAGAGTCCTTCT-3 ' (sequence 38 in sequence table)；
Inner primer FIP：5 '-CGACGCGGTCGTCATGAAAGCAAATTGGACTTCCTGTAACG-3 ' (sequences in sequence table Row 39)；
Inner primer BIP：5 '-GGCTGGGGTGGCAACGTACAGTGCGTGGGAATCTCG-3 ' (sequence 40 in sequence table)；
Ring primer LF：5 '-GCGTGGAGACTGCAC-3 ' (sequence 41 in sequence table)；
Ring primer LB：5 '-GCATCACCGTCGACAC-3 ' (sequence 42 in sequence table).
Primer sets for detecting drug resistant gene OXA-23 are named as primer sets I.For detecting drug resistant gene OXA-24's Primer sets are named as primer sets II.Primer sets for detecting drug resistant gene OXA-58 are named as primer sets III.It is resistance to for detecting The primer sets of medicine gene OXA-66 are named as primer sets IV.Primer sets for detecting drug resistant gene KPC-2 are named as primer sets Ⅴ.Primer sets for detecting drug resistant gene IMP-4 are named as primer sets VI.Primer sets for detecting drug resistant gene VIM-2 It is named as primer sets VII.
Embodiment 2, specificity experiments
Sample to be tested 1：Plasmid containing drug resistant gene OXA-23.The preparation method of the plasmid containing drug resistant gene OXA-23 is： WillDNA fragmentation between the EcoRI and SpeI of-T Easy plasmids replaces with the nucleosides that No. Genebank is KP203815.1 DNA molecular shown in acid sequence, the recombinant plasmid for obtaining is the plasmid containing drug resistant gene OXA-23.
Sample to be tested 2：Plasmid containing drug resistant gene OXA-24.The preparation method of the plasmid containing drug resistant gene OXA-24 is： WillDNA fragmentation between the EcoRI and SpeI of-T Easy plasmids replaces with the nucleosides that No. Genebank is KR922888.1 DNA molecular shown in acid sequence, the recombinant plasmid for obtaining is the plasmid containing drug resistant gene OXA-24.
Sample to be tested 3：Plasmid containing drug resistant gene OXA-58.The preparation method of the plasmid containing drug resistant gene OXA-58 is： WillDNA fragmentation between the EcoRI and SpeI of-T Easy plasmids replaces with the nucleosides that No. Genebank is KF740447.1 DNA molecular shown in acid sequence, the recombinant plasmid for obtaining is the plasmid containing drug resistant gene OXA-58.
Sample to be tested 4：Plasmid containing drug resistant gene OXA-66.The preparation method of the plasmid containing drug resistant gene OXA-66 is： It is the DNA molecular shown in DQ923479.1 that the interval DNA fragmentations of the MCS of pUC57 plasmids are replaced with into No. Genebank, is obtained Recombinant plasmid is the plasmid containing drug resistant gene OXA-66.
Sample to be tested 5：Plasmid containing drug resistant gene KPC-2.The preparation method of the plasmid containing drug resistant gene KPC-2 is：WillDNA fragmentation between the EcoRI and SpeI of-T Easy plasmids replaces with the nucleotides that No. Genebank is KT001101.1 DNA molecular shown in sequence, the recombinant plasmid for obtaining is the plasmid containing drug resistant gene KPC-2.
Sample to be tested 6：Plasmid containing drug resistant gene IMP-4.The preparation method of the plasmid containing drug resistant gene IMP-4 is：WillDNA fragmentation between the EcoRI and SpeI of-T Easy plasmids replaces with the nucleotides that No. Genebank is AY795963.1 DNA molecular shown in sequence, the recombinant plasmid for obtaining is the plasmid containing drug resistant gene IMP-4.
Sample to be tested 7：Plasmid containing drug resistant gene VIM-2.The preparation method of the plasmid containing drug resistant gene VIM-2 is：WillDNA fragmentation between the EcoRI and SpeI of-T Easy plasmids replaces with the nucleosides that NCBI Gene ID are 14678525 DNA molecular shown in acid sequence, the recombinant plasmid for obtaining is the plasmid containing drug resistant gene VIM-2.
Each sample to be tested carries out following steps respectively：
1st, the DNA of sample to be tested is extracted.
2nd, the DNA extracted with step 1 is template, and each primer sets that the preparation of embodiment 1 is respectively adopted carry out ring Jie Lead isothermal duplication.
Reaction system (10 μ L)：7.0 μ L reaction solutions, 1 μ L primer mixtures, 1 μ L DNAs (5pg-50pg), moisturizing is extremely 10μL.Primer mixture is the mixture of each bar primer composition in primer sets.In reaction system, outer primer F3 and outer primer B3 Final concentration be 0.5 μM, the final concentration of inner primer FIP and inner primer BIP is 2 μM, the end of ring primer LF and ring primer LB Concentration is 1 μM.
Reaction condition：65 DEG C of constant temperature 50min.In course of reaction, fluorescence signal is detected using fluorescent PCR instrument.
According to the method described above, DNA is replaced with into sterilized water, other steps are constant, used as blank.
The result of primer sets I is used to see that (" S types " amplification curve is sample to be tested 1 to Fig. 1, and non-" S types " amplification curve is other Sample to be tested or blank).Sample to be tested shows positive amplification curve (i.e. when being the plasmid containing drug resistant gene OXA-23 Amplification curve is " S types " amplification curve).Sample to be tested is sample to be tested 2, sample to be tested 3, sample to be tested 4, sample to be tested 5, treats Positive amplification curve is not shown when test sample sheet 6 or sample to be tested 7.
The result of primer sets II is used to see that (" S types " amplification curve is sample to be tested 2 to Fig. 2, and non-" S types " amplification curve is it Its sample to be tested or blank).Sample to be tested shows positive amplification curve when being the plasmid containing drug resistant gene OXA-24 (i.e. amplification curve is " S types " amplification curve).Sample to be tested is sample to be tested 1, sample to be tested 3, sample to be tested 4, sample to be tested 5th, sample to be tested 6 or positive amplification curve is not shown when sample to be tested 7.
The result of primer sets III is used to see that (" S types " amplification curve is sample to be tested 3 to Fig. 3, and non-" S types " amplification curve is it Its sample to be tested or blank).Sample to be tested shows positive amplification curve when being the plasmid containing drug resistant gene OXA-58 (i.e. amplification curve is " S types " amplification curve).Sample to be tested is sample to be tested 1, sample to be tested 2, sample to be tested 4, sample to be tested 5th, sample to be tested 6 or positive amplification curve is not shown when sample to be tested 7.
The result of primer sets IV is used to see that (" S types " amplification curve is sample to be tested 4 to Fig. 4, and non-" S types " amplification curve is it Its sample to be tested or blank).Sample to be tested shows positive amplification curve when being the plasmid containing drug resistant gene OXA-66 (i.e. amplification curve is " S types " amplification curve).Sample to be tested is sample to be tested 1, sample to be tested 2, sample to be tested 3, sample to be tested 5th, sample to be tested 6 or positive amplification curve is not shown when sample to be tested 7.
The result of primer sets V is used to see that (" S types " amplification curve is sample to be tested 5 to Fig. 5, and non-" S types " amplification curve is it Its sample to be tested or blank).Sample to be tested shows positive amplification curve when being the plasmid containing drug resistant gene KPC-2 (i.e. amplification curve is " S types " amplification curve).Sample to be tested is sample to be tested 1, sample to be tested 2, sample to be tested 3, sample to be tested 4th, sample to be tested 6 or positive amplification curve is not shown when sample to be tested 7.
The result of primer sets VI is used to see that (" S types " amplification curve is sample to be tested 6 to Fig. 6, and non-" S types " amplification curve is it Its sample to be tested or blank).Sample to be tested shows positive amplification curve when being the plasmid containing drug resistant gene IMP-4 (i.e. amplification curve is " S types " amplification curve).Sample to be tested is sample to be tested 1, sample to be tested 2, sample to be tested 3, sample to be tested 4th, sample to be tested 5 or positive amplification curve is not shown when sample to be tested 7.
The result of primer sets VII is used to see that (" S types " amplification curve is sample to be tested 7 to Fig. 7, and non-" S types " amplification curve is it Its sample to be tested or blank).Sample to be tested shows positive amplification curve when being the plasmid containing drug resistant gene VIM-2 (i.e. amplification curve is " S types " amplification curve).Sample to be tested is sample to be tested 1, sample to be tested 2, sample to be tested 3, sample to be tested 4th, sample to be tested 5 or positive amplification curve is not shown when sample to be tested 6.
Result above shows that seven primer sets that the present invention is provided have specificity very high to its target gene respectively.
Embodiment 3, sensitivity experiment
Sample to be tested 1：The plasmid containing drug resistant gene OXA-23 prepared in embodiment 2.
Sample to be tested 2：The plasmid containing drug resistant gene OXA-24 prepared in embodiment 2.
Sample to be tested 3：The plasmid containing drug resistant gene OXA-58 prepared in embodiment 2.
Sample to be tested 4：The plasmid containing drug resistant gene OXA-66 prepared in embodiment 2.
Sample to be tested 5：The plasmid containing drug resistant gene KPC-2 prepared in embodiment 2.
Sample to be tested 6：The plasmid containing drug resistant gene IMP-4 prepared in embodiment 2.
Sample to be tested 7：The plasmid containing drug resistant gene VIM-2 prepared in embodiment 2.
1st, the DNA of sample to be tested is extracted, gradient dilution is carried out with sterilized water, obtain each dilution.
2nd, the dilution obtained with step 1 is template, and the primer sets that the preparation of embodiment 1 is respectively adopted carry out ring mediated isothermal Amplification.
When sample to be tested is sample to be tested 1, ring mediated isothermal amplification is carried out using primer sets I.Sample to be tested is to treat test sample During sheet 2, ring mediated isothermal amplification is carried out using primer sets II.When sample to be tested is sample to be tested 3, ring is carried out using primer sets III Mediated isothermality amplification.When sample to be tested is sample to be tested 4, ring mediated isothermal amplification is carried out using primer sets IV.Sample to be tested is During sample to be tested 5, ring mediated isothermal amplification is carried out using primer sets V.When sample to be tested is sample to be tested 6, using primer sets VI Carry out ring mediated isothermal amplification.When sample to be tested is sample to be tested 7, ring mediated isothermal amplification is carried out using primer sets VII.
Reaction system (10 μ L)：7.0 μ L reaction solutions, 1 μ L primer mixtures, 1 μ L dilutions (contain in 1 μ L dilutions Genome copy numbers are respectively 103、5×102、102Or 101), moisturizing to 10 μ L.Primer mixture is that each bar in primer sets draws The mixture of thing composition.In reaction system, the final concentration of outer primer F3 and outer primer B3 is 0.5 μM, inner primer FIP and interior draws The final concentration of thing BIP is 2 μM, and the final concentration of ring primer LF and ring primer LB is 1 μM.
Reaction condition：65 DEG C of constant temperature 50min.In course of reaction, fluorescence signal is detected using fluorescent PCR instrument.
If occurring positive amplification curve (i.e. amplification curve is " S types " amplification curve) in 50min, show reaction system In corresponding gene group content can be detected.If not occurring positive amplification curve in 50min, show reactant Corresponding gene group content in system can not be detected.
The sensitivity of the detection target gene of primer sets I is 5 × 102Individual copy number/reaction system, the detection target of primer sets II The sensitivity of gene is 5 × 102Individual copy number/reaction system, the sensitivity of the detection target gene of primer sets III is 5 × 102It is individual to copy Shellfish number/reaction system, the sensitivity of the detection target gene of primer sets IV is 5 × 102Individual copy number/reaction system, primer sets V The sensitivity for detecting target gene is 5 × 102Individual copy number/reaction system, the sensitivity of the detection target gene of primer sets VI is 5 ×102Individual copy number/reaction system, the sensitivity of the detection target gene of primer sets VII is 5 × 102Individual copy number/reaction system.
Embodiment 4, application
Sample to be tested is following sample one, sample two or sample three：
Sample one：Identified by susceptibility and PCR sequencing identifications confirm the fester containing drug resistant gene OXA-23；
Sample two：Identified by susceptibility and PCR sequencing identifications confirm the fester containing drug resistant gene KPC-2；
Sample three：Identified by susceptibility and PCR sequencing identifications confirm the fester containing drug resistant gene IMP-4.
1st, the STb gene of sample to be tested is extracted.
2nd, the STb gene of the sample to be tested extracted with step 1 is template, and each primer sets of the preparation of embodiment 1 are respectively adopted Carry out ring mediated isothermal amplification.
Reaction system is with reaction condition with embodiment 2.In course of reaction, fluorescence signal is detected using fluorescent PCR instrument.
According to the method described above, the STb gene of sample to be tested is replaced with into sterilized water, other steps are constant, used as blank pair According to.
The result of sample one is shown in that (" S types " amplification curve is primer sets I to Fig. 8, and non-" S types " amplification curve is other primer sets Or blank).Positive amplification curve is shown when using primer sets I (i.e. amplification curve is " S types " amplification curve).Using Positive amplification curve is not shown when other six primer sets or blank beyond primer sets I, it is complete with actual conditions It is complete consistent.
The result of sample two is shown in that (" S types " amplification curve is primer sets V to Fig. 9, and non-" S types " amplification curve is other primer sets Or blank).Positive amplification curve is shown when using primer sets V (i.e. amplification curve is " S types " amplification curve).Adopt Positive amplification curve is not shown when with other six primer sets or blank beyond primer sets V, with actual conditions It is completely the same.
The result of sample three is shown in that (" S types " amplification curve is primer sets VI to Figure 10, and non-" S types " amplification curve is other primers Group or blank).Positive amplification curve is shown when using primer sets VI (i.e. amplification curve is " S types " amplification curve). Positive amplification curve is not shown when using other six primer sets or blank beyond primer sets VI, with actual feelings Condition is completely the same.
Result above shows that the kit provided using the present invention can be to seven kinds of resistances of Carbapenem-resistant class antibiotic Gene detected, as a result accurately and reliably.
<110>Capitalbio Corporation Co., Ltd.
<120>LAMP primer composition and its application for detecting seven kinds of drug resistant genes of Carbapenem-resistant class antibiotic
<160> 42
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
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<223>
<400> 1
gaatatgtgc cagcctcta 19
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 2
tctttttgca tgagatcaag ac 22
<210> 3
<211> 43
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 3
ccttttctcg cccttccatt taaatttgaa tgccctgatc gga 43
<210> 4
<211> 39
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 4
taccgcttgg gaaaaagaca tggtcgcgca agttcctga 39
<210> 5
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 5
ccgttttctg gttctccaa 19
<210> 6
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 6
acactaggag aagccatga 19
<210> 7
<211> 24
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 7
ctgtgttcca atatttgtat ttcc 24
<210> 8
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 8
gcaacaacaa atgagatttt ca 22
<210> 9
<211> 46
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 9
atcaagagct tgcaagacgg acattaaccc gctttacttc tttctg 46
<210> 10
<211> 48
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 10
ctggaactgc tgacaatgcc attgaatggg atggtaaaaa aagaactt 48
<210> 11
<211> 16
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 11
tggcctagag ctaatg 16
<210> 12
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 12
cctcacctaa agtcatatc 19
<210> 13
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 13
ccaattagtg cattggcaat t 21
<210> 14
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 14
ttggggcttg tgctgag 17
<210> 15
<211> 47
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 15
tgtgtttgtc acatatgatg gtcaaagatg caggaatata agctgtt 47
<210> 16
<211> 49
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 16
cgcttgaaca ttctgatcga tgattatgag tcgagcaaaa acaagtaca 49
<210> 17
<211> 16
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 17
gcatttagac cgagca 16
<210> 18
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 18
gagttattca cttgtggaa 19
<210> 19
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 19
ctaataaaac gcttccattt agc 23
<210> 20
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 20
gaacagagct aggtattcct t 21
<210> 21
<211> 45
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 21
acatcccatc cccaaccact ttttgtccaa gatgaagtgc aatcc 45
<210> 22
<211> 46
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 22
ccacaagtag gctggttaac tgtctaagtt aagggagaac gctaca 46
<210> 23
<211> 25
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 23
ccattctttt cttctatgaa tagca 25
<210> 24
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 24
ttgttcagcc tcaagggaat 20
<210> 25
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 25
ctgacggcct tcatgcg 17
<210> 26
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 26
gccaatcaac aaactgct 18
<210> 27
<211> 38
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 27
atggcggagt tcagctccat ctatcggcga taccacgt 38
<210> 28
<211> 39
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 28
aggcgatgcg cgcgataccc agtgtcagtt tttgtaagc 39
<210> 29
<211> 15
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 29
ccagcggtcc agacg 15
<210> 30
<211> 15
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 30
gcgccgtgac ggaaa 15
<210> 31
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 31
tctaattgac actccattta cg 22
<210> 32
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 32
agttaacccc gccaaat 17
<210> 33
<211> 49
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 33
atgaaaatga gaggaaatac tgcctatact gaaaagttag tcacttggt 49
<210> 34
<211> 44
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 34
tggcttaatt ctcaatccat cccttagctt gaaccttacc gtct 44
<210> 35
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 35
atagccacgt tccacaa 17
<210> 36
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 36
cgtatgcgtc tgaattaac 19
<210> 37
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 37
ggcacttctc gcggaga 17
<210> 38
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 38
gctcgatgag agtccttct 19
<210> 39
<211> 41
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 39
cgacgcggtc gtcatgaaag caaattggac ttcctgtaac g 41
<210> 40
<211> 36
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 40
ggctggggtg gcaacgtaca gtgcgtggga atctcg 36
<210> 41
<211> 15
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 41
gcgtggagac tgcac 15
<210> 42
<211> 16
<212> DNA
<213>Artificial sequence
<220>
<223>
<400> 42
gcatcaccgt cgacac 16

Claims (9)

1. primer combination, is following (a1) or (a2) or (a3)：
(a1) it is made up of primer sets I, primer sets II, primer sets III, primer sets IV, primer sets V, primer sets VI and primer sets VII；
(a2) by the primer sets I, the primer sets II, the primer sets III, the primer sets IV, the primer sets V, institute State any two in primer sets VI and the primer sets VII, any three, any four, any five or any six groups Into；
(a3) the primer sets I, primer sets II, the primer sets III, the primer sets IV, the primer sets V, described Primer sets VI or the primer sets VII；
The primer sets I are by-the F3 of the primer I ,-B3 of the primer I ,-FIP of the primer I ,-BIP of the primer I ,-LF of primer I and the-LB groups of primer I Into；
- the F3 of the primer I is following (b1) or (b2)；
(b1) single strand dna shown in the sequence 1 of sequence table；
(b2) sequence 1 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 1 identical The DNA molecular of function；
- the B3 of the primer I is following (b3) or (b4)；
(b3) single strand dna shown in the sequence 2 of sequence table；
(b4) sequence 2 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 2 identical The DNA molecular of function；
- the FIP of the primer I is following (b5) or (b6)；
(b5) single strand dna shown in the sequence 3 of sequence table；
(b6) sequence 3 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 3 identical The DNA molecular of function；
- the BIP of the primer I is following (b7) or (b8)；
(b7) single strand dna shown in the sequence 4 of sequence table；
(b8) sequence 4 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 4 identical The DNA molecular of function；
- the LF of the primer I is following (b9) or (b10)；
(b9) single strand dna shown in the sequence 5 of sequence table；
(b10) sequence 5 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 5 identical The DNA molecular of function；
- the LB of the primer I is following (b11) or (b12)；
(b11) single strand dna shown in the sequence 6 of sequence table；
(b12) sequence 6 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 6 identical The DNA molecular of function；
The primer sets II are by-the F3 of the primer II ,-B3 of the primer II ,-FIP of the primer II ,-BIP of the primer II ,-LF of primer II and primer II-LB is constituted；
- the F3 of the primer II is following (c1) or (c2)；
(c1) single strand dna shown in the sequence 7 of sequence table；
(c2) sequence 7 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 7 identical The DNA molecular of function；
- the B3 of the primer II is following (c3) or (c4)；
(c3) single strand dna shown in the sequence 8 of sequence table；
(c4) sequence 8 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 8 identical The DNA molecular of function；
- the FIP of the primer II is following (c5) or (c6)；
(c5) single strand dna shown in the sequence 9 of sequence table；
(c6) sequence 9 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 9 identical The DNA molecular of function；
- the BIP of the primer II is following (c7) or (c8)；
(c7) single strand dna shown in the sequence 10 of sequence table；
(c8) sequence 10 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 10 The DNA molecular of congenerous；
- the LF of the primer II is following (c9) or (c 10)；
(c9) single strand dna shown in the sequence 11 of sequence table；
(c10) sequence 11 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 11 The DNA molecular of congenerous；
- the LB of the primer II is following (c11) or (c12)；
(c11) single strand dna shown in the sequence 12 of sequence table；
(c12) sequence 12 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 12 The DNA molecular of congenerous；
The primer sets III are by-the F3 of the primer III ,-B3 of the primer III ,-FIP of the primer III ,-BIP of the primer III ,-LF of primer III and primer III-LB is constituted；
- the F3 of the primer III is following (d1) or (d2)；
(d1) single strand dna shown in the sequence 13 of sequence table；
(d2) sequence 13 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 13 The DNA molecular of congenerous；
- the B3 of the primer III is following (d3) or (d4)；
(d3) single strand dna shown in the sequence 14 of sequence table；
(d4) sequence 14 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 14 The DNA molecular of congenerous；
- the FIP of the primer III is following (d5) or (d6)；
(d5) single strand dna shown in the sequence 15 of sequence table；
(d6) sequence 15 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 15 The DNA molecular of congenerous；
- the BIP of the primer III is following (d7) or (d8)；
(d7) single strand dna shown in the sequence 16 of sequence table；
(d8) sequence 16 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 16 The DNA molecular of congenerous；
- the LF of the primer III is following (d9) or (d10)；
(d9) single strand dna shown in the sequence 17 of sequence table；
(d10) sequence 17 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 17 The DNA molecular of congenerous；
- the LB of the primer III is following (d11) or (d12)；
(d11) single strand dna shown in the sequence 18 of sequence table；
(d12) sequence 18 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 18 The DNA molecular of congenerous；
The primer sets IV are by-the F3 of the primer IV ,-B3 of the primer IV ,-FIP of the primer IV ,-BIP of the primer IV ,-LF of primer IV and primer IV-LB is constituted；
- the F3 of the primer IV is following (e1) or (e2)；
(e1) single strand dna shown in the sequence 19 of sequence table；
(e2) sequence 19 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 19 The DNA molecular of congenerous；
- the B3 of the primer IV is following (e3) or (e4)；
(e3) single strand dna shown in the sequence 20 of sequence table；
(e4) sequence 20 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 20 The DNA molecular of congenerous；
- the FIP of the primer IV is following (e5) or (e6)；
(e5) single strand dna shown in the sequence 21 of sequence table；
(e6) sequence 21 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 21 The DNA molecular of congenerous；
- the BIP of the primer IV is following (e7) or (e8)；
(e7) single strand dna shown in the sequence 22 of sequence table；
(e8) sequence 22 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 22 The DNA molecular of congenerous；
- the LF of the primer IV is following (e9) or (e10)；
(e9) single strand dna shown in the sequence 23 of sequence table；
(e10) sequence 23 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 23 The DNA molecular of congenerous；
- the LB of the primer IV is following (e11) or (e12)；
(e11) single strand dna shown in the sequence 24 of sequence table；
(e12) sequence 24 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 24 The DNA molecular of congenerous；
The primer sets V are by-the F3 of the primer V ,-B3 of the primer V ,-FIP of the primer V ,-BIP of the primer V ,-LF of primer V and primer V-LB is constituted；
- the F3 of the primer V is following (f1) or (f2)；
(f1) single strand dna shown in the sequence 25 of sequence table；
(f2) sequence 25 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 25 The DNA molecular of congenerous；
- the B3 of the primer V is following (f3) or (f4)；
(f3) single strand dna shown in the sequence 26 of sequence table；
(f4) sequence 26 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 26 The DNA molecular of congenerous；
- the FIP of the primer V is following (f5) or (f6)；
(f5) single strand dna shown in the sequence 27 of sequence table；
(f6) sequence 27 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 27 The DNA molecular of congenerous；
- the BIP of the primer V is following (f7) or (f8)；
(f7) single strand dna shown in the sequence 28 of sequence table；
(f8) sequence 28 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 28 The DNA molecular of congenerous；
- the LF of the primer V is following (f9) or (f10)；
(f9) single strand dna shown in the sequence 29 of sequence table；
(f10) sequence 29 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 29 The DNA molecular of congenerous；
- the LB of the primer V is following (f11) or (f12)；
(f11) single strand dna shown in the sequence 30 of sequence table；
(f12) sequence 30 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 30 The DNA molecular of congenerous；
The primer sets VI are by-the F3 of the primer VI ,-B3 of the primer VI ,-FIP of the primer VI ,-BIP of the primer VI ,-LF of primer VI and primer VI-LB is constituted；
- the F3 of the primer VI is following (g1) or (g2)；
(g1) single strand dna shown in the sequence 31 of sequence table；
(g2) sequence 31 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 31 The DNA molecular of congenerous；
- the B3 of the primer VI is following (g3) or (g4)；
(g3) single strand dna shown in the sequence 32 of sequence table；
(g4) sequence 32 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 32 The DNA molecular of congenerous；
- the FIP of the primer VI is following (g5) or (g6)；
(g5) single strand dna shown in the sequence 33 of sequence table；
(g6) sequence 33 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 33 The DNA molecular of congenerous；
- the BIP of the primer VI is following (g7) or (g8)；
(g7) single strand dna shown in the sequence 34 of sequence table；
(g8) sequence 34 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 34 The DNA molecular of congenerous；
- the LF of the primer VI is following (g9) or (g10)；
(g9) single strand dna shown in the sequence 35 of sequence table；
(g10) sequence 35 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 35 The DNA molecular of congenerous；
- the LB of the primer VI is following (g11) or (g12)；
(g11) single strand dna shown in the sequence 36 of sequence table；
(g12) sequence 36 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 36 The DNA molecular of congenerous；
The primer sets VII are by-the F3 of the primer VII ,-B3 of the primer VII ,-FIP of the primer VII ,-BIP of the primer VII ,-LF of primer VII and primer VII-LB is constituted；
- the F3 of the primer VII is following (h1) or (h2)；
(h1) single strand dna shown in the sequence 37 of sequence table；
(h2) sequence 37 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 37 The DNA molecular of congenerous；
- the B3 of the primer VII is following (h3) or (h4)；
(h3) single strand dna shown in the sequence 38 of sequence table；
(h4) sequence 38 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 38 The DNA molecular of congenerous；
- the FIP of the primer VII is following (h5) or (h6)；
(h5) single strand dna shown in the sequence 39 of sequence table；
(h6) sequence 39 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 39 The DNA molecular of congenerous；
- the BIP of the primer VII is following (h7) or (h8)；
(h7) single strand dna shown in the sequence 40 of sequence table；
(h8) sequence 40 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 40 The DNA molecular of congenerous；
- the LF of the primer VII is following (h9) or (h10)；
(h9) single strand dna shown in the sequence 41 of sequence table；
(h10) sequence 41 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 41 The DNA molecular of congenerous；
- the LB of the primer VII is following (h11) or (h12)；
(h11) single strand dna shown in the sequence 42 of sequence table；
(h12) sequence 42 had into phase by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 42 The DNA molecular of congenerous.
2. application of the primer combination in reagent preparation box described in claim 1；The purposes of the kit be following (y1) or Or (y3) (y2)：
(y1) drug resistant gene OXA-23 and/or drug resistant gene OXA-24 and/or drug resistant gene OXA-58 and/or drug resistant gene are identified OXA-66 and/or drug resistant gene KPC-2 and/or drug resistant gene IMP-4 and/or drug resistant gene VIM-2；
(y2) whether it is used to detect in sample to be tested contain drug resistant gene OXA-23 and/or drug resistant gene OXA-24 and/or resistance Gene OXA-58 and/or drug resistant gene OXA-66 and/or drug resistant gene KPC-2 and/or drug resistant gene IMP-4 and/or resistance base Because of VIM-2；
(y3) it is used to detect whether bacterium to be measured contains drug resistant gene OXA-23 and/or drug resistant gene OXA-24 and/or drug resistant gene OXA-58 and/or drug resistant gene OXA-66 and/or drug resistant gene KPC-2 and/or drug resistant gene IMP-4 and/or drug resistant gene VIM-2。
3. the kit of primer combination described in claim 1 is contained；The purposes of the kit be following (y1) or (y2) or (y3)：
(y1) drug resistant gene OXA-23 and/or drug resistant gene OXA-24 and/or drug resistant gene OXA-58 and/or drug resistant gene are identified OXA-66 and/or drug resistant gene KPC-2 and/or drug resistant gene IMP-4 and/or drug resistant gene VIM-2；
(y2) whether it is used to detect in sample to be tested contain drug resistant gene OXA-23 and/or drug resistant gene OXA-24 and/or resistance Gene OXA-58 and/or drug resistant gene OXA-66 and/or drug resistant gene KPC-2 and/or drug resistant gene IMP-4 and/or resistance base Because of VIM-2；
(y3) it is used to detect whether bacterium to be measured contains drug resistant gene OXA-23 and/or drug resistant gene OXA-24 and/or drug resistant gene OXA-58 and/or drug resistant gene OXA-66 and/or drug resistant gene KPC-2 and/or drug resistant gene IMP-4 and/or drug resistant gene VIM-2。
4. the preparation method of kit described in claim 3, including the step of each bar primer is individually packed.
5. whether a kind of detection bacterium to be measured contains drug resistant gene OXA-23 and/or drug resistant gene OXA-24 and/or drug resistant gene OXA-58 and/or drug resistant gene OXA-66 and/or drug resistant gene KPC-2 and/or drug resistant gene IMP-4 and/or drug resistant gene The method of VIM-2, comprises the following steps：
(1) genomic DNA of bacterium to be measured is extracted；
(2) genomic DNA with step (1) extraction as template, draw by each being respectively adopted during primer described in claim 1 is combined Thing group carries out ring mediated isothermal amplification, then makes the following judgment：
If using the primer sets I can realize the specific amplification with the genomic DNA as template, contain in bacterium to be measured Or doubtful contain drug resistant gene OXA-23；If using the primer sets I cannot realize with the genomic DNA as template Specific amplification, does not contain in bacterium to be measured or doubtful does not contain drug resistant gene OXA-23；
If using the primer sets II can realize the specific amplification with the genomic DNA as template, contain in bacterium to be measured Have or doubtful contain drug resistant gene OXA-24；If using the primer sets II cannot realize with the genomic DNA as mould The specific amplification of plate, does not contain in bacterium to be measured or doubtful does not contain drug resistant gene OXA-24；
If using the primer sets III can realize the specific amplification with the genomic DNA as template, contain in bacterium to be measured Have or doubtful contain drug resistant gene OXA-58；If using the primer sets III cannot realize with the genomic DNA as mould The specific amplification of plate, does not contain in bacterium to be measured or doubtful does not contain drug resistant gene OXA-58；
If using the primer sets IV can realize the specific amplification with the genomic DNA as template, contain in bacterium to be measured Have or doubtful contain drug resistant gene OXA-66；If using the primer sets IV cannot realize with the genomic DNA as mould The specific amplification of plate, does not contain in bacterium to be measured or doubtful does not contain drug resistant gene OXA-66；
If using the primer sets V can realize the specific amplification with the genomic DNA as template, contain in bacterium to be measured Have or doubtful contain drug resistant gene KPC-2；If using the primer sets V cannot realize with the genomic DNA as template Specific amplification, do not contained in bacterium to be measured or doubtful do not contain drug resistant gene KPC-2；
If using the primer sets VI can realize the specific amplification with the genomic DNA as template, contain in bacterium to be measured Have or doubtful contain drug resistant gene IMP-4；If using the primer sets VI cannot realize with the genomic DNA as template Specific amplification, do not contained in bacterium to be measured or doubtful do not contain drug resistant gene IMP-4；
If using the primer sets VII can realize the specific amplification with the genomic DNA as template, contain in bacterium to be measured Have or doubtful contain drug resistant gene VIM-2；If using the primer sets VII cannot realize with the genomic DNA as template Specific amplification, do not contained in bacterium to be measured or doubtful do not contain drug resistant gene VIM-2.
6. whether drug resistant gene OXA-23 and/or drug resistant gene OXA-24 and/or resistance base are contained in a kind of detection sample to be tested Because of OXA-58 and/or drug resistant gene OXA-66 and/or drug resistant gene KPC-2 and/or drug resistant gene IMP-4 and/or drug resistant gene The method of VIM-2, comprises the following steps：
(1) STb gene of sample to be tested is extracted；
(2) STb gene with step (1) extraction is respectively adopted each primer sets in primer combination described in claim 1 as template Ring mediated isothermal amplification is carried out, is then made the following judgment：
If using the primer sets I can realize the specific amplification with the STb gene as template, in sample to be tested contain or It is doubtful to contain drug resistant gene OXA-23；If using the primer sets I cannot realize the specificity with the STb gene as template Amplification, does not contain in sample to be tested or doubtful does not contain drug resistant gene OXA-23；
If using the primer sets II can realize the specific amplification with the STb gene as template, contain in sample to be tested Or doubtful contain drug resistant gene OXA-24；If using the primer sets II cannot realize the spy with the STb gene as template Specific amplification, does not contain in sample to be tested or doubtful does not contain drug resistant gene OXA-24；
If using the primer sets III can realize the specific amplification with the STb gene as template, contain in sample to be tested Or doubtful contain drug resistant gene OXA-58；If using the primer sets III cannot realize the spy with the STb gene as template Specific amplification, does not contain in sample to be tested or doubtful does not contain drug resistant gene OXA-58；
If using the primer sets IV can realize the specific amplification with the STb gene as template, contain in sample to be tested Or doubtful contain drug resistant gene OXA-66；If using the primer sets IV cannot realize the spy with the STb gene as template Specific amplification, does not contain in sample to be tested or doubtful does not contain drug resistant gene OXA-66；
If using the primer sets V can realize the specific amplification with the STb gene as template, contain in sample to be tested Or doubtful contain drug resistant gene KPC-2；If it is special with the STb gene as template to use the primer sets V to realize Property amplification, do not contained in sample to be tested or doubtful do not contain drug resistant gene KPC-2；
If using the primer sets VI can realize the specific amplification with the STb gene as template, contain in sample to be tested Or doubtful contain drug resistant gene IMP-4；If it is special with the STb gene as template to use the primer sets VI to realize Property amplification, do not contained in sample to be tested or doubtful do not contain drug resistant gene IMP-4；
If using the primer sets VII can realize the specific amplification with the STb gene as template, contain in sample to be tested Or doubtful contain drug resistant gene VIM-2；If it is special with the STb gene as template to use the primer sets VII to realize Property amplification, do not contained in sample to be tested or doubtful do not contain drug resistant gene VIM-2.
7. primer combination described in claim 1 is detecting whether bacterium to be measured contains drug resistant gene OXA-23 and/or drug resistant gene OXA-24 and/or drug resistant gene OXA-58 and/or drug resistant gene OXA-66 and/or drug resistant gene KPC-2 and/or drug resistant gene Application in IMP-4 and/or drug resistant gene VIM-2.
8. whether primer combination contains drug resistant gene OXA-23 and/or resistance base in sample to be tested is detected described in claim 1 Because of OXA-24 and/or drug resistant gene OXA-58 and/or drug resistant gene OXA-66 and/or drug resistant gene KPC-2 and/or drug resistant gene Application in IMP-4 and/or drug resistant gene VIM-2.
9. primer combination described in claim 1 is in identification drug resistant gene OXA-23 and/or drug resistant gene OXA-24 and/or resistance base Because of OXA-58 and/or drug resistant gene OXA-66 and/or drug resistant gene KPC-2 and/or drug resistant gene IMP-4 and/or drug resistant gene Application in VIM-2.
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