CN105420394A - Primer pair, probe and kit for detecting bacterium MCR-1 gene - Google Patents
Primer pair, probe and kit for detecting bacterium MCR-1 gene Download PDFInfo
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- CN105420394A CN105420394A CN201511026072.4A CN201511026072A CN105420394A CN 105420394 A CN105420394 A CN 105420394A CN 201511026072 A CN201511026072 A CN 201511026072A CN 105420394 A CN105420394 A CN 105420394A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Abstract
The invention relates to a primer pair, a probe and a kit for detecting the bacterium MCR-1 drug-resistant gene. The primer pair comprises an MCR-1 gene detecting upstream primer including the sequence shown by SEQ ID NO.1 and an MCR-1 gene detecting downstream primer including the sequence shown by SEQ ID NO.2. The probe for the MCR-1 gene comprises a sequence shown by SEQ ID NO.3, a fluorescence group is integrated at the 5' end, and a quenching group is integrated at the 3' end of the probe for the MCR-1 gene. The kit for detecting the bacterium MCR-1 drug-resistant gene comprises the primer pair and the probe. The primer pair, the probe and the kit for detecting the bacterium MCR-1 drug-resistant gene have the advantages of being capable of detecting the bacterium MCR-1 drug-resistant gene, good in sensitivity, high in specificity, accurate in rationing and the like. The primer pair, the probe and the kit are good for clinical detection and monitoring of the bacterium MCR-1 drug-resistant gene and guiding antibiotic therapy of patients.
Description
Technical field
The invention belongs to antibiotic resistance genes detection technique field, be specifically related to for the primer pair of bacterial detection MCR-1 drug resistant gene, probe and test kit.
Background technology
From the twenties in 20th century, a large amount of natural antibiont comprises penicillin, Streptomycin sulphate etc. and is in succession found, and the mankind open the microbiotic epoch thus, with the antagonism of bacterium in have overwhelming superiority.But along with depending on unduly antibiotic usage, the anti-medicine problem of bacterium highlights day by day.Screened and the enrichments etc. of the drug tolerant bacteria of antibiotic abuse to random variation, make antibiotic medicine power generation upon generation of weaken, even lose efficacy.The war of the mankind and bacterium proceeds to the situation of " protract war " gradually around " resistance " problem.Polymyxin (Polymyxins) is the one group of polypeptide antibiotics raised in poly-viscosity bacillus nutrient solution, there are 5 kinds of composition (A, B, C, D, E). clinical conventional be PXB (PolymyxinB) and Polymyxin E (colislin, colistin).Polymyxin be one comprise medicinal Totazina and in livestock industry widely used microbiotic.Polymyxin is considered to last line of defense that the mankind resist bacterium.
At present, researchist finds that the intestinal bacteria sample memory gathered in some animals and raw meat is at a kind of novel antibiotic resistance genes MCR-1 gene; In addition, in the intestinal bacteria and klebsiella pneumoniae sample of inpatient, have also discovered MCR-1 gene.MCR-1 gene is that carrier is present in bacterium with plasmid, can carry out the exchange of genetic material in Horizontal Gene Transfer mode between different strains.This transfer mode breaks the boundary of sibship, can propagate, and speed is fast between different bacterium.This characteristic is similar with the drug resistance gene NDM-1 situation found in India several years ago, and the bacterium carrying NDM-1 almost can resist all microbiotic, comprises " trump card " carbapenems.The discovery of MCR-1 gene, imply that microbiotic last line of defense---polymyxin is threatened already.
Therefore, the detection for MCR-1 gene breaks out for monitoring drug-resistant bacteria, instructs patient infection's medication to have vital role.
But not yet there is the primer, probe and the test kit that are applicable to MCR-1 gene test at present.
Summary of the invention
In view of the problem existing for prior art, the invention provides a kind of primer pair for bacterial detection MCR-1 gene, probe, test kit and its using method, MCR-1 drug resistant gene can be detected, and realize drug resistant gene quantitative assay, there is high specificity, sensitivity advantages of higher.
The technical scheme that the present invention solves the problems of the technologies described above is as follows:
The invention provides a kind of primer pair for bacterial detection MCR-1 gene, comprise following primer pair:
Detect the upstream primer of MCR-1 gene, comprise the sequence shown in SEQIDNO.1, the sequence shown in SEQIDNO.1 is 5 '-TTGCTCTACTGGCATTTATTTGG-3 ';
Detect the downstream primer of MCR-1 gene, comprise the sequence shown in SEQIDNO.2, the sequence shown in SEQIDNO.2 is 5 '-ACCATGGTTCAGCATCAGACAA-3 '.
Preferably, the upstream primer detecting MCR-1 gene is the sequence shown in SEQIDNO.1, and the downstream primer detecting MCR-1 gene is the sequence shown in SEQIDNO.2.
By the size of the PCR primer of above-mentioned primer pair amplifies all in the scope of 40bp to 200bp, this can make it possible to specific gene at short notice.The present invention is chosen by above-mentioned primer, can fast, the detection MCR-1 gene of convenience, highly sensitive, high specificity.And for the qualitative and quantitative analysis of MCR-1 gene.
The present invention also provides a kind of probe for bacterial detection MCR-1 gene, comprise with above-mentioned detection MCR-1 gene primer the MCR-1 probe mated, MCR-1 probe comprises the sequence shown in SEQIDNO.3, and the sequence shown in SEQIDNO.3 is 5 '-ATGAGAATTTTGAGGGAGAATCAATAAACTATT-3 '.
Further, 5 ' end of described MCR-1 probe is combined with fluorescent marker, and 3 ' end of described MCR-1 probe is combined with quencher.
Preferably, MCR-1 probe is the sequence shown in SEQIDNO.3, and has following structural: 5 '-fluorophor/GTTTGCCAAATTCACGCCAGTGTGTG/ quenching group-3 '.
Above-mentioned probe can select following arbitrary fluorophor at its 5 ' end mark, such as: FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670, NED; Select following arbitrary quenching group at its 3 ' end mark, such as: the non-prominent light essence of 6-TAMRA, BHQ-1 ~ 3 and binding molecule ditch is gone out agent (MinorGrooveBindernonfluorescentquencher, MGBNFQ).
Preferably, the 5 ' end of described SEQIDNO.3 indicates fluorophor FAM, and the 3 ' end of SEQIDNO.3 indicates quenching group BHQ-1.
The invention provides a kind of test kit for bacterial detection MCR-1 gene, comprise above-mentioned primer pair and above-mentioned MCR-1 probe.
Further, also comprise interior label primer to internal control probe;
Described interior label primer is to being the primer pair for the E.coli standard substance that increase;
Described internal control probe is the E.coli probe mated with the primer pair for the E.coli standard substance that increase;
Described E.coli standard substance sequence is for shown in SEQIDNO.8, and the nucleotide sequence shown in SEQIDNO.8 is specifically:
TGTTATTGCCGGGAAAAGTGTACGTATCACTGTTTGTGTGAACAACGAACTGAACTGGCAGACTATCCCGCCGGGAATGGTGATTACCGACGAAAACGGCAAGAAAAAGCAGTCTTACTTTCATGATTTCTTTAACTACGCCGGGATCCATCGCAGCGTAATGCTCTACACCACGCCGAACACCTGGGTGGACGATAT；
The upstream primer of amplification E.coli standard substance comprises the sequence shown in SEQIDNO.4, sequence shown in SEQIDNO.4 is 5 '-TCTTACCCTAGTACATGGAGTAATGTTG-3 ', preferably, the upstream primer of amplification E.coli standard substance is the sequence shown in SEQIDNO.4;
The downstream primer of amplification E.coli standard substance comprises the sequence shown in SEQIDNO.5, sequence shown in SEQIDNO.5 is 5 '-GCATGTTCTGGTACCAAATAATCCGTGA-3 ', preferably, the downstream primer of amplification E.coli standard substance is the sequence shown in SEQIDNO.5;
E.coli probe comprises the sequence shown in SEQIDNO.6, preferably, E.coli probe is the sequence shown in SEQIDNO.6, sequence shown in SEQIDNO.6 is 5 '-CGGGAATGGTGATTACCGACGAAAACG-3 ', be combined with fluorophor at 5 ' end of this sequence, be combined with quenching group at 3 ' end of this sequence.
By the size of the PCR primer of above-mentioned primer pair amplifies all in the scope of 40bp to 200bp, this can make it possible to specific gene at short notice.Above-mentioned probe can select following arbitrary fluorophor at its 5 ' end mark, such as: FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670, NED; Select following arbitrary quenching group at its 3 ' end mark, such as: the non-prominent light essence of 6-TAMRA, BHQ-1 ~ 3 and binding molecule ditch is gone out agent (MinorGrooveBindernonfluorescentquencher, MGBNFQ).
Preferably, the 5 ' end of described SEQIDNO.6 indicates fluorophor HEX, and the 3 ' end of SEQIDNO.6 indicates quenching group BHQ-1.
Further, PCR reaction mixture and Taq DNA polymerase is also comprised; Described PCR reaction mixture comprises following component: the upstream primer 0.6 μM of Buffer10 × damping fluid 8 μ L, the upstream primer 0.6 μM detecting MCR-1 gene, the downstream primer 0.6 μM detecting MCR-1 gene, MCR-1 probe 0.3 μM, amplification E.coli standard substance, downstream primer 0.6 μM, internal control probe 0.3 μM, each component 0.8mM of dNTP of amplification E.coli standard substance.
Further, positive reference substance and negative controls is also comprised;
Described positive reference substance is comprise the plasmid that MCR-1 is detected fragment, and concentration is 10
3copy/μ L;
The physiological saline of described negative controls to be concentration be 0.9% (per-cent of the quality of sodium-chlor and the volume of physiological saline).
Further, described MCR-1 is detected fragment and comprises the nucleotide sequence shown in SEQIDNO.7, and concrete sequence is:
ATGACTTTGTCGCTGCCAATAACGGCAAAGATATGCTGATCATGCTGCACCAAATG GGCAATCACGGGCCTGCGTATTTTAAGCGATATGATGAAAAGTTTGCCAAATTCAC GCCAGTGTGTGAAGGTAATGAGCTTGCCAAGTGCGAACATCAGTCCTTGATCAATG CTTATGACAATGCCTTGCTTGCCACCGATGATTTCATCGCTCAAAGTATCCAGTGG CTGCAGACGCACAGCAATGCCTATGATGTCTCAATGCTGTATGTCAGCGATCAT, described plasmid is pUC57 plasmid.
Above-mentioned primer pair, probe and test kit have good application in the diagnosis or complementary diagnosis of bacterium MCR-1 gene.
For sample, it can be the samples sources that ight soil, urine, sweat, fester, hydrocephalus, blood, sputum, Nasopharyngeal swabs etc. may contain MCR-1 drug resistant gene; Described sample to be tested is the DNA extracted from sample.
In the present invention, MCR-1 bacterium is specifically as follows the microorganism that intestinal bacteria, Klebsiella pneumonia, clostridium difficile, streptococcus aureus, gonococcus etc. may carry MCR-1 gene.
For a detection method for the test kit of bacterial detection MCR-1 gene, comprise the following steps:
5 μ L samples to be tested, 0.6 μ LTaqDNA polysaccharase are mixed with 34.4 μ LPCR reaction mixtures, described PCR reaction mixture comprises following component: the upstream primer 0.6 μM of Buffer10 × damping fluid 8 μ L, the upstream primer 0.6 μM detecting MCR-1 gene, the downstream primer 0.6 μM detecting MCR-1 gene, MCR-1 probe 0.3 μM, amplification E.coli standard substance, downstream primer 0.6 μM, internal control probe 0.3 μM, each component 0.8mM of dNTP of amplification E.coli standard substance;
Increase according to condition below:
The i.e. first stage: 94 DEG C 5 minutes; Subordinate phase: 94 DEG C 20 seconds, 58 DEG C 30 seconds, 10 circulations; Phase III: 94 DEG C 20 seconds, 58 DEG C 30 seconds, 35 circulations, collect FAM/HEX fluorescent signal.
Detection kit for bacterium MCR-1 drug resistant gene provided by the invention, can convenient, bacterial detection MCR-1 drug resistant gene fast and accurately.For practical application, detected result can be obtained in 1.5 hours, select significant to quick ancillary drug treatment guidance and medication.Meanwhile, can be used for epidemiological survey and epidemic situation monitoring with research or understand bacterium MCR-1 drug resistant gene in China and even global popularity.
Accompanying drawing explanation
Fig. 1 is recombinant plasmid pUC57-MCR-13 × 10 containing MCR-1 gene
4copy/μ L, 3 × 10
3copy/μ L, 3 × 10
2copy/μ L, 3 × 10
1copy/μ L, 3 × 10
0the different concns gradient dilution liquid augmentation detection results such as copy/μ L;
Fig. 2 is sample amplification figure in the embodiment of the present invention 4.
Embodiment
Be described principle of the present invention and feature below in conjunction with accompanying drawing, example, only for explaining the present invention, is not intended to limit scope of the present invention.
The experimental technique used in following embodiment if no special instructions, is the ordinary method of this area.
The material used in following embodiment, reagent etc., if no special instructions, be routine business and obtain
Embodiment 1 is for the test kit and preparation method thereof of bacterial detection MCR-1 drug resistant gene
Test kit for bacterial detection MCR-1 drug resistant gene provided by the present invention is composed as follows:
PCR reaction mixture
The each component of PCR reaction mixture and concentration composed as follows: Buffer10 × damping fluid 8 μ L, the upstream primer 0.6 μM detecting MCR-1 gene, the downstream primer 0.6 μM detecting MCR-1 gene, MCR-1 probe 0.3 μM, the upstream primer 0.6 μM of the E.coli standard substance that increase, the downstream primer 0.6 μM of the E.coli standard substance that increase, internal control probe 0.3 μM, each component 0.8mM of dNTP.
Wherein, in specific embodiment, Buffer10 × damping fluid is 10 × ExTaqBuffer (Mg
2+plus), 10 × ExTaqBuffer (Mg
2+plus) damping fluid is purchased from precious biotechnology (Dalian) company limited of TAKARA, model 9152A.
Detect the upstream primer sequence of MCR-1 gene as shown in SEQIDNO.1, detect the downstream primer sequence of MCR-1 gene as shown in SEQIDNO.2; The sequence of MCR-1 probe, as shown in SEQIDNO.3, is 5 '-GTTTGCCAAATTCACGCCAGTGTGTG-3 ', and 5 ' end of this sequence indicates fluorophor FAM, and 3 ' end of this sequence indicates quenching group BHQ-1; Above-mentioned primer and probe all synthesize in Nanjing Jin Sirui Bioisystech Co., Ltd (hereinafter referred to as " Jin Sirui ").
The sequence of the upstream primer of amplification E.coli standard substance is as shown in SEQIDNO.4, and the sequence of the downstream primer of amplification E.coli standard substance is as shown in SEQIDNO.5; The sequence of internal control probe, as shown in SEQIDNO.6, is 5 '-CGGGAATGGTGATTACCGACGAAAACG-3 ', and its 5 ' end indicates fluorophor HEX, and 3 ' end indicates quenching group BHQ-1; Above-mentioned primer and probe all synthesize at Jin Sirui.
The each component of dNTP purchased from precious biotechnology (Dalian) company limited of TAKARA, model 430Q.
Taq enzyme: i.e. Taq DNA polymerase, purchased from the HSTaq enzyme of TAKARA.
Positive reference substance: 10
3the MCR-1 that comprises of copy/μ L is detected fragment of plasmid
It is SEQIDNO.7 that described MCR-1 is detected fragment, and concrete sequence is as follows:
ATGACTTTGTCGCTGCCAATAACGGCAAAGATATGCTGATCATGCTGCACCAAATGGGCAATCACGGGCCTGCGTATTTTAAGCGATATGATGAAAAGTTTGCCAAATTCACGCCAGTGTGTGAAGGTAATGAGCTTGCCAAGTGCGAACATCAGTCCTTGATCAATGCTTATGACAATGCCTTGCTTGCCACCGATGATTTCATCGCTCAAAGTATCCAGTGGCTGCAGACGCACAGCAATGCCTATGATGTCTCAATGCTGTATGTCAGCGATCAT；
Synthesize SEQIDNO.7 sequence at Jin Sirui, pUC57 plasmid (being provided by Jin Sirui) is provided, identify correct through double digestion and order-checking.Measure plasmid concentration, and be 10 by plasmid dilution
3copy/μ L is as positive reference substance.
Negative controls: the physiological saline of preparation 0.9%, wherein the per-cent of the quality of sodium-chlor and the volume of physiological saline is 0.9%.
Embodiment 2 is for the using method of the test kit of bacterial detection MCR-1 drug resistant gene
1, sample DNA extracts:
Test kit used is that OMEGA bacterial genomes extracts test kit, and concrete steps are see specification sheets, and description is as follows:
(1) bacteria culture fluid or the centrifugal 1min of bacterium 1mL, 10000 × g is got.
(2) remove supernatant, add 250 μ L solution I (containing RNaseA), the concussion of vortex vibrator suspends completely to thalline.
(3) add 250 μ L solution II, gentleness puts upside down centrifuge tube 4-6 time, obtains the lysate of clarification.Best incubated at room 2min, violent mixing can make shearing chromosomal DNA, reduces plasmid purity.(storage solutions II should tighten bottle cap).
(4) add 350 μ L solution III, gentleness puts upside down mixing for several times, to occurring white flock precipitate, and the centrifugal 10min of room temperature 10000 × g.
(5) SC draws supernatant, moves to clean assembling in the absorption column of volume 2mL centrifuge tube.Ensure not suck precipitation and cell debris.The centrifugal 1min of room temperature 10000 × g, passes through absorption column completely to lysate.
(6) abandon filtered solution, add 500 μ LBufferHB, the centrifugal 1min of 10000 × g, cleaning absorption column, removing residual protein ensures the purity of DNA.If following step is not high to plasmid purity requirement, as other screening methods such as enzyme digestions, this step can be omitted.
(7) filtered solution is abandoned, absorption column is cleaned again with 750 μ LWashBuffer of 100% alcohol dilution, 10000 × g is centrifugal, and 1min notes: WashBuffer concentrated solution with before must dilute with straight alcohol, if method is shown in that label is through freezing, with before must recover room temperature.
(8) this step is optional: add 750 μ LWashBuffer again and clean absorption column.
(9) centrifugal for absorption column 10000 × g 1min must be guaranteed that ethanol is removed, ethanol can affect step below.
(10) absorption column is put into clean 1.5mL centrifuge tube, add 50-100 μ L (depending on the final concentration of needs) aseptic deionized water or TE damping fluid on filter membrane, the centrifugal 5min of 10000 × g, once can adhere to DNA (wash-out again can be carried out, but DNA concentration can be reduced) by wash-out 75-80%.
The reagent such as the solution I in above-mentioned, solution II, solution III, BufferHB, 100% ethanol, WashBuffer, TE damping fluid are OMEGA bacterial genomes and extract test kit self-contained reagent.
2, reaction system is prepared
| Component | Volume |
| PCR reaction mixture | 34.4μL |
| Taq enzyme | 0.6μL |
| Sample DNA | 5μL |
| Cumulative volume | 40μL |
Get the PCR reaction solution of 34.4 μ L, 0.6 μ LTaq enzyme and 5 μ L sample DNAs and be mixed into 40 μ L reaction solns, centrifugal after vortex concussion evenly.
Positive controls and negative control group are set simultaneously;
Sample DNA in positive controls is the pUC57 plasmid containing SEQIDNO.7 of above-mentioned structure; In negative control group, " sample DNA " is alternative with the physiological saline of isopyknic 0.9%; Other parts are all identical with experimental group.
3, fluorescence real-time quantitative PCR reaction and detection
Quantitative fluorescent PCR reaction conditions is as shown above: the first stage: 94 DEG C of 5min; Subordinate phase: 94 DEG C of 20s, 58 DEG C of 30s, 10 circulations; Phase III: 94 DEG C of 20s, 58 DEG C of 30s, 35 circulations, collect FAM/HEX signal.
4, result judges
After reaction terminates, according to the basis for estimation whether amplification curve and the cycle number Ct of sample in each reaction chamber exist as MCR-1 gene.
If sample to be tested does not produce S type amplification curve, illustrate that this amplification is invalid.
If sample to be tested produces S type amplification curve, and Ct value is more than or equal to 40 and is less than 45, duplicate detection 1 time; If Ct value is less than 40, then there is MCR-1 drug resistant gene in described sample, otherwise do not exist in described sample; Ct value is more than or equal to 40, then there is not MCR-1 drug resistant gene in described sample.
Embodiment 3. is for the sensitivity analysis of bacterial detection MCR-1 drug resistant gene test kit
One, the preparation of reference material DNA nucleic acid
Structure comprises MCR-1 gene plasmid
The MCR-1 gene (SEQIDNO.7) as described in summary of the invention is synthesized by Jin Sirui.Synthetic gene is connected to pUC57 carrier (being provided by Jin Sirui), builds and comprise pUC57-MCR1 plasmid, and order-checking check and correction.
Two, for detecting sensitivity and the specificity analyses of the test kit of MCR-1 drug resistant gene
The reference material preparation of different concns
The MCR-1 gene plasmid that comprises that above-mentioned steps is synthesized is carried out quantitatively, and is quantitatively 3 × 10
10copy/μ L, and gradient dilution, obtain 3 × 10
5copy/μ L, 3 × 10
4copy/μ L, 3 × 10
3copy/μ L, 3 × 10
2copy/μ L, 3 × 10
1copy/μ L, 3 × 10
0the different concns gradient dilution liquid such as copy/μ L.
Reaction system is prepared
| Component | Volume |
| PCR reaction mixture | 34.4μL |
| Taq enzyme | 0.6μL |
| Sample DNA | 5μL |
| Cumulative volume | 40μL |
Get the PCR reaction solution of 34.4 μ L, 0.6 μ LTaq enzyme and 5 μ L sample DNAs and be mixed into 40 μ L reaction solns, centrifugal after vortex concussion evenly.
Fluorescence real-time quantitative PCR reaction and detection
Quantitative fluorescent PCR reaction conditions is as shown above: the first stage: 94 DEG C of 5min; Subordinate phase: 94 DEG C of 20s, 58 DEG C of 30s, 10 circulations; Phase III: 94 DEG C of 20s, 58 DEG C of 30s, 35 circulations, collect FAM/HEX signal.
Three, result
As shown in Figure 1, for recombinant plasmid, reaction system is minimum detects 3 × 10 to result
0the template of copy/μ L, amplification all has obvious S type amplification curve.These results suggest that this test kit has higher amplification sensitivity.
Embodiment 4: for the specificity analyses of bacterial detection MCR-1 drug resistant gene test kit
1. bacterial strain
Bacterial strain and numbering see the following form:
| Bacterial strain | Numbering | Bacterial strain | Numbering |
| Clostridium difficile 1 | ATCC9689 | Clostridium difficile 2 | ATCC BAA1805 7 --> |
| Colon bacillus | ATCC8739 | Subtilis | ATCC6633 |
| Shigella flexneri | CMCC44149 | Pathogenic colon bacillus | CMCC44149 |
| Enteroaerogen | ATCC13048 | Dysentery is congratulated Salmonella | CMCC51105 |
| Shigella dysenteriae | CMCC51105 | Enterobacter cloacae | CMCC45301 |
| Produce malicious intestinal bacteria | CMCC44814 | Salmonella typhimurium | CMCC50013 |
ATCC9689, ATCCBAA1805, CMCC44814, CMCC51105 are purchased from CGMCC, and all the other bacterial strains are all purchased from CCTCC.
2. bacterium is cultivated
All bacterial strains are in the medium in 37 DEG C of training 24h to 48h.
3. use sky root ight soil genome DNA extracting reagent kit to extract the bacterial genomes DNA of above-mentioned bacterium respectively, concrete operations are as follows:
1) get 200 μ L bacterium liquid in 2mL centrifuge tube, and pipe is placed on ice.
2) in sample, add 1.4mL buffer A SL, interrupted oscillation mixes to sample for 1 minute.
3) 5 minutes are hatched for 70 DEG C;
4) vortex 15 seconds, centrifugal 1 minute of 13000rpm, transfer supernatant liquor 1.2mL is to new 2mL centrifuge tube.
5) add an inhibitor suction sheet InhibitEX, vibrate and thoroughly open resuspended to suction sheet, incubated at room 1 minute, makes suction sheet fully act on.
6) centrifugal 3 minutes of 13000rpm.
7) previous step gained supernatant liquor is transferred to new 1.5mL centrifuge tube, repeating step 6).
8) shift gained supernatant liquor 200 μ L to new 1.5mL centrifuge tube, add 15 μ L Proteinase Ks.
9) 200 μ L buffer A L are added, vortex 15 seconds.
10) 10 minutes are hatched for 70 DEG C.
11) add 200 μ L dehydrated alcohols, vortex mixes.
12) join in an adsorption column (adsorption column puts into collection tube) by previous step gained solution, centrifugal 30 seconds of 12000rpm, outwells waste liquid, adsorption column is put into collection tube.
13) in adsorption column, add 500 μ L buffer A W1, centrifugal 30 seconds of 12000rpm (~ 13400 × g), outwells waste liquid, is put into by suction post and receives EP pipe.
14) in adsorption column, enter 700 μ L rinsing liquid AW2, centrifugal 30 seconds of 12000rpm (~ 13400 × g), outwells waste liquid, is put into by suction post and receives EP pipe.
15) suction post is put into receipts EP pipe, centrifugal 2 minutes of 12000rpm (~ 13400 × g), outwell waste liquid, adsorption column is placed in room temperature and places several minutes, is placed in room temperature and places several minutes, thoroughly to dry rinsing remaining in sorbing material.
16) adsorption column is proceeded in a clean centrifuge tube, the unsettled dropping 200 in middle part to adsorption film μ L elution buffer AE, room temperature places 2-5 minute, 12000rpm (~ 13400 × g) centrifugal 2 minutes, by solution collection in centrifuge tube.
4. reaction system preparation
| Component | Volume |
| PCR reaction mixture | 34.4μL |
| Taq enzyme | 0.6μL |
| Sample DNA | 5μL 8 --> |
| Cumulative volume | 40μL |
Get the PCR reaction solution of 34.4 μ L, 0.6 μ LTaq enzyme and 5 μ L sample DNAs and be mixed into 40 μ L reaction solns, centrifugal after vortex concussion evenly.Arrange negative control group and positive controls, concrete grammar is see embodiment 3 simultaneously.
Fluorescence real-time quantitative PCR reaction and detection
Quantitative fluorescent PCR reaction conditions is as shown above: the first stage: 94 DEG C of 5min; Subordinate phase: 94 DEG C of 20s, 58 DEG C of 30s, 10 circulations; Phase III: 94 DEG C of 20s, 58 DEG C of 30s, 35 circulations, collect FAM/HEX signal.
Fig. 2 is sample amplification figure in the embodiment of the present invention 4, and as can be seen from Figure 2, following 6 sample standard deviations can not expand.
Detected result sees the following form:
| Bacterial strain | Result | Bacterial strain | Result |
| Clostridium difficile 1 | - | Clostridium difficile 2 | - |
| Colon bacillus | - | Subtilis | - |
| Shigella flexneri | - | Pathogenic colon bacillus | - |
| Enteroaerogen | - | Dysentery is congratulated Salmonella | - |
| Shigella dysenteriae | - | Enterobacter cloacae | - |
| Produce malicious intestinal bacteria | - | Salmonella typhimurium | - |
Detected result is feminine gender, carries out sequence verification after amplified production being cut glue, and the result of sequence verification is consistent with the result of above-mentioned quantitative fluorescent PCR, illustrates that the specificity being used for bacterial detection MCR-1 drug resistant gene test kit is good.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. for the primer pair of bacterial detection MCR-1 gene, it is characterized in that, comprise following primer pair:
Detect the upstream primer of MCR-1 gene, comprise the sequence shown in SEQIDNO.1, the sequence shown in SEQIDNO.1 is 5 '-TTGCTCTACTGGCATTTATTTGG-3 ';
Detect the downstream primer of MCR-1 gene, comprise the sequence shown in SEQIDNO.2, the sequence shown in SEQIDNO.2 is 5 '-ACCATGGTTCAGCATCAGACAA-3 '.
2. for the probe of bacterial detection MCR-1 gene, it is characterized in that, comprise the MCR-1 probe mated with primer pair according to claim 1, MCR-1 probe comprises the sequence shown in SEQIDNO.3, and the sequence shown in SEQIDNO.3 is 5 '-ATGAGAATTTTGAGGGAGAATCAATAAACTATT-3 '.
3. according to claim 2 for the probe of bacterial detection MCR-1 gene, it is characterized in that, 5 ' end of described MCR-1 probe is combined with fluorophor, and 3 ' end of described MCR-1 probe is combined with quenching group.
4. for the test kit of bacterial detection MCR-1 gene, it is characterized in that, comprise primer pair according to claim 1 and MCR-1 probe according to claim 3.
5., according to claim 4 for the test kit of bacterial detection MCR-1 gene, it is characterized in that, also comprise interior label primer to internal control probe;
Described interior label primer is to being the primer pair for the E.coli standard substance that increase;
Described internal control probe is the E.coli probe mated with the primer pair for the E.coli standard substance that increase;
Described E.coli standard substance sequence is as shown in SEQIDNO.8;
The upstream primer of amplification E.coli standard substance comprises the sequence shown in SEQIDNO.4, and the sequence shown in SEQIDNO.4 is 5 '-TCTTACCCTAGTACATGGAGTAATGTTG-3 ';
The downstream primer of amplification E.coli standard substance comprises the sequence shown in SEQIDNO.5, and the sequence shown in SEQIDNO.5 is 5 '-GCATGTTCTGGTACCAAATAATCCGTGA-3 ';
E.coli probe comprises the sequence shown in SEQIDNO.6, and the sequence shown in SEQIDNO.6 is 5 '-CGGGAATGGTGATTACCGACGAAAACG-3 ', is combined with fluorophor at 5 ' end of this sequence, is combined with quenching group at 3 ' end of this sequence.
6. according to claim 5 for the test kit of bacterial detection MCR-1 gene, it is characterized in that, also comprise PCR reaction mixture and Taq DNA polymerase;
Described PCR reaction mixture comprises following component: the upstream primer 0.6 μM of Buffer10 × damping fluid 8 μ L, the upstream primer 0.6 μM detecting MCR-1 gene, the downstream primer 0.6 μM detecting MCR-1 gene, MCR-1 probe 0.3 μM, amplification E.coli standard substance, downstream primer 0.6 μM, internal control probe 0.3 μM, each component 0.8mM of dNTP of amplification E.coli standard substance.
7. according to any one of claim 4 to 6 for the test kit of bacterial detection MCR-1 gene, it is characterized in that, also comprise positive reference substance and negative controls;
Described positive reference substance is comprise the plasmid that MCR-1 is detected fragment; Described negative controls is physiological saline.
8. according to claim 7 for the test kit of bacterial detection MCR-1 gene, it is characterized in that, described MCR-1 is detected fragment and comprises the nucleotide sequence shown in SEQIDNO.7, and the concentration of described positive reference substance is 10
3copy/μ L, the concentration of described physiological saline is 0.9%.
9. diagnosing or the complementary application diagnosing bacterium MCR-1 gene for the test kit of bacterial detection MCR-1 gene described in the probe described in primer pair according to claim 1, Claims 2 or 3, any one of claim 4 to 8.
10. for a detection method for the test kit of bacterial detection MCR-1 gene, it is characterized in that, comprise the following steps:
Sample to be tested, Taq DNA polymerase are mixed with PCR reaction mixture, described PCR reaction mixture comprises following component: the upstream primer 0.6 μM of Buffer10 × damping fluid 8 μ L, the upstream primer 0.6 μM detecting MCR-1 gene, the downstream primer 0.6 μM detecting MCR-1 gene, MCR-1 probe 0.3 μM, amplification E.coli standard substance, downstream primer 0.6 μM, internal control probe 0.3 μM, each component 0.8mM of dNTP of amplification E.coli standard substance;
Increase according to condition below:
The i.e. first stage: 94 DEG C 5 minutes; Subordinate phase: 94 DEG C 20 seconds, 58 DEG C 30 seconds, 10 circulations; Phase III: 94 DEG C 20 seconds, 58 DEG C 30 seconds, 35 circulations, collect fluorescent signal.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106191043A (en) * | 2016-07-26 | 2016-12-07 | 吉林大学 | A kind of genetic fragment, carrier pPlasmid Clearance and application |
| CN106967812A (en) * | 2017-04-17 | 2017-07-21 | 张嵘 | A kind of screening technique of the positive bacterias of mcr 1 |
| CN107460242A (en) * | 2017-08-15 | 2017-12-12 | 北京紫萌医药科技有限公司 | A kind of detection kit that can detect a variety of high drug resistant genes simultaneously and its application |
| CN108504723A (en) * | 2018-06-01 | 2018-09-07 | 北京农学院 | A kind of Primer composition and application for detection bacterium MCR-1 genes |
| CN110643693A (en) * | 2019-10-10 | 2020-01-03 | 首都儿科研究所 | Primer, probe, kit and detection method for detecting Mcr by RAA fluorescence method |
| CN112522378A (en) * | 2020-11-18 | 2021-03-19 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | Kit for detecting MCR gene, detection method and application thereof |
| CN113817855A (en) * | 2021-10-22 | 2021-12-21 | 上海市计量测试技术研究院 | Digital PCR primer probe composition, kit and method for detecting polymyxin drug resistance gene |
| CN113943824A (en) * | 2021-10-22 | 2022-01-18 | 上海市计量测试技术研究院 | Digital PCR primer probe composition for detecting polymyxin drug resistance gene and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104928376A (en) * | 2015-06-05 | 2015-09-23 | 武汉大学 | Composition, kit and method for detecting high-virulence bacterial strains and/or toxin type of clostridium difficile |
-
2015
- 2015-12-31 CN CN201511026072.4A patent/CN105420394A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104928376A (en) * | 2015-06-05 | 2015-09-23 | 武汉大学 | Composition, kit and method for detecting high-virulence bacterial strains and/or toxin type of clostridium difficile |
Non-Patent Citations (2)
| Title |
|---|
| H HASMAN，ET AL.: "Detection of mcr-1 encoding plasmid-mediated colistin-resistant Escherichia coli isolates from human bloodstream infection and imported chicken meat, Denmark 2015", 《EURO SURVEILL》 * |
| YI-YUN LIU, ET AL.: "Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study", 《LANCET INFECT DIS》 * |
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| CN106967812A (en) * | 2017-04-17 | 2017-07-21 | 张嵘 | A kind of screening technique of the positive bacterias of mcr 1 |
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| CN107460242B (en) * | 2017-08-15 | 2022-06-10 | 北京紫萌医药科技有限公司 | Detection kit capable of simultaneously detecting multiple high-drug-resistance genes and application thereof |
| CN108504723A (en) * | 2018-06-01 | 2018-09-07 | 北京农学院 | A kind of Primer composition and application for detection bacterium MCR-1 genes |
| CN110643693A (en) * | 2019-10-10 | 2020-01-03 | 首都儿科研究所 | Primer, probe, kit and detection method for detecting Mcr by RAA fluorescence method |
| CN112522378A (en) * | 2020-11-18 | 2021-03-19 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | Kit for detecting MCR gene, detection method and application thereof |
| CN112522378B (en) * | 2020-11-18 | 2023-10-31 | 深圳市疾病预防控制中心（深圳市卫生检验中心、深圳市预防医学研究所） | Kit for detecting MCR gene, detection method and application thereof |
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