CN108048583A - A kind of probe, genetic chip and kit for pseudomonas aeruginosa detection of nucleic acids - Google Patents
A kind of probe, genetic chip and kit for pseudomonas aeruginosa detection of nucleic acids Download PDFInfo
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- CN108048583A CN108048583A CN201711271879.3A CN201711271879A CN108048583A CN 108048583 A CN108048583 A CN 108048583A CN 201711271879 A CN201711271879 A CN 201711271879A CN 108048583 A CN108048583 A CN 108048583A
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- 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
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- C40B40/04—Libraries containing only organic compounds
- C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
- C40B40/08—Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
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Abstract
A kind of probe, genetic chip and kit for pseudomonas aeruginosa detection of nucleic acids.The invention discloses one group of probes for pseudomonas aeruginosa detection, and including the bacterium PAK gene probes 1 and 2, sequence is respectively SEQ ID NO.1~2 and the bacterium Pf4 gene probes 1 and 2, and sequence is respectively SEQ ID NO.3~4.The invention also discloses the kits of the genetic chip containing above-mentioned probe and the genetic chip：Hybridization signal is amplified by visible light optical, signal resolution is high, can direct interpretation.The present invention is easy to operate, cheap, result is readable, meets the field quick detection demand of hospital clinical, as a result accurately and reliably.
Description
Technical field
The invention belongs to biology fields, are related to a kind of probe, gene for pseudomonas aeruginosa detection of nucleic acids
Chip and kit.
Background technology
Pseudomonas aeruginosa（Pseudomonas aeruginosa）Also known as Pseudomonas aeruginosa, the bacterium are conditioned pathogen, are
One of the main pathogenic fungi of inside-hospital infection.Often cause postoperative wound infection, can also cause bedsore, abscess, suppurative middle ear
Inflammation etc..Charrin disease can betide many regions of anatomy, including skin, subcutaneous tissue, bone, ear, eye, urinary tract and the heart
Dirty valve.Infection site is related with the entrance of bacterium and the neurological susceptibility of patient.During burn, region can become a large amount of bacteriums under eschar
The place of infringement, and then become and cause bacteremic lesion, and bacteremia is often the lethal complication of burn.P. aeruginosa
Microbial many infection are happened at weak or immunocompromised host inpatient, it is that the second that intensive care unit is infected is most normal
The pathogen seen is the common cause of Ventilator Associated Pneumonia.In addition to being infected in hospital, HIV infection person is easy to
Community obtains the infection of the bacterium, and once often may occur in which the sign of late stage HIV infection by charrin disease.And
The germ also has the characteristics that multidrug resistant, and has congenital and acquired drug resistance, and metainfective clinical treatment is very
It is difficult.Therefore, it is particularly important to the detection of pseudomonas aeruginosa.
At present, the detection technique in pseudomonas aeruginosa includes biochemical identification and PCR is detected, and is still continued to use in national and foreign standards
Traditional Physico-chemical tests method, including rubbing method, more test tube MPN methods, filter membrane method etc., it is necessary to which pure culture and biochemical identification etc. are more
A step, entire detection cycle 3-5 days, sensitivity is low, and biochemical identification normal nothing when result is not certain in practical operation
Method accurate judgement.And PCR identifications are needed to be sequenced and compared, and it is time-consuming longer, generally take 5 days or so.Two kinds of detection method detections
Cycle is longer, it is impossible to meet the needs of field quick detection.
Biochip technology is that substantial amounts of probe molecule is fixed on solid support simultaneously, miscellaneous by nucleic acid molecules
The characteristic of mating pair to the sequence information of DNA samples efficiently understand and analyze.It is detected using genetic chip,
It is big to detect flux, can a kind of multiple genes of pathogen are detected or disposably be completed several simultaneously on a chip
Kind pathogen detection, carries out directly against pathogen gene, as a result more accurate, high, specific good with detection sensitivity, required
The advantages that sample size is few.
The detection of traditional biochip technology is detected based on the fluorescence radiation of fluorescence labeling probe.Its
Signal is weak, it is necessary to by excitation, could shine, it is necessary to the Laser Scanning Equipment of costliness.Genetic chip of the present invention is sent out using chemistry
Light detects hybridization signal, and crossover process can be completed at normal temperatures, large-scale instrument and equipment is not required, reduce the investment of detection into
Sheet and application cost.
The content of the invention
It is an object of the invention to provide a kind of quick detection, specific good, the accurate pseudomonas aeruginosas of visual result
Detection of nucleic acids probe, genetic chip and kit to simplify detecting step, improve detection speed, reduce testing cost and application
Cost, and detection sensitivity is improved, meet the needs of field quick detection.
The present invention realizes that the technical solution of its purpose is as follows：
For the probe of pseudomonas aeruginosa detection of nucleic acids, the PAK gene probes 1 including the bacterium, 2, Pf4 gene probes 1,2；
The probe sequence is as follows：
PAK gene probes 1,2 sequences are SEQ ID NO.1~2；
Pf4 gene probes 1,2 sequences are SEQ ID NO.3~4.
Further, present invention further includes a kind of genetic chip for pseudomonas aeruginosa detection of nucleic acids, including
Solid phase and foregoing probes.
Further, present disclosure further includes a kind of genetic chip reagent for pseudomonas aeruginosa detection of nucleic acids
Box, including forementioned gene chip.
Further, the gene chip kit of pseudomonas aeruginosa detection of nucleic acids detection, further includes following draw
Object pair：PAK gene primers pair and Pf4 gene primers pair, the sequence of the primer pair are as follows：
PAK gene primers pair, sequence are SEQ ID NO.5~6；
Pf4 gene primers pair, sequence are SEQ ID NO.7~8.
Further, biotin labeling is contained on the primer.
Further, it is right to further include multi-PRC reaction system, feminine gender for the pseudomonas aeruginosa kit for detecting nucleic acid
According to product, positive reference substance, hybridization buffer, Avidin（Horseradish peroxidase-labeled）, cleaning solution, ECL developing solutions.
Further, the negative controls are physiological saline, and the positive reference substance is the PAK containing target gene
The mixed liquor of gene plasmid, Pf4 gene plasmids, the objective gene sequence are respectively SEQ ID NO.9~10.
Further, the hybridization buffer for Quick Reaction Buffer (Chongqing prestige this rise biological Co., Ltd
There is provided), the cleaning formula of liquid is to distill water dissolution 8g NaCl, 0.2g KCl and 3g Tris-base with 800ml.With HCl tune
To ph7.4, with distilled water constant volume to 1L.
Further, the Avidin（Horseradish peroxidase-labeled）It needs to be diluted to conjunction with hybridization buffer during use
Suitable concentration.
Further, the multiplex PCR system is：1 μ L, Premix Taq of amplification template, 10 μ of 50~150ng/ μ L
L, concentration are 8~12 μM of each 0 .6 μ L of different upstream and downstream primers.
The beneficial effects of the invention are as follows：By the selection of specific primer and probe, multiple PCR technique and gene make use of
The method that chip technology is combined, using chemiluminescence detection gene chip hybridization signal, the Physico-chemical tests method of beyond tradition,
Large-scale instrument and equipment is not required, not only reduced inspection step, shortens round of visits, reduce the cost of investment of detection and be applied to
This, and detection sensitivity and specificity are improved, meet the demand of the Site Detection of hospital etc., there is wide clinical practice city
Field potentiality.
Description of the drawings
Attached drawing 1 is product gel electrophoretogram of two genes of pseudomonas aeruginosa after multiplexed PCR amplification（M：DL500
DNA Marker；CK-：Negative control sample amplification；CK+：Positive control amplification；PA：Pseudomonas aeruginosa
Sample amplification）.
A is the genechip detection pseudomonas aeruginosa gene group amplified hybridization result of the detection present invention in attached drawing 2；B is
Positive control amplified hybridization result；C is negative control sample amplified hybridization result.
Specific embodiment
Below in conjunction with attached drawing, the preferred embodiment of the present invention is described in detail.
【Embodiment 1】Target fragment, the design of primer and probe
The gene order of pseudomonas aeruginosa is searched by ncbi database, the conserved sequence of PAK genes, Pf4 genes is selected to make
For target（ID is respectively CP020659 and KM975472）, according to primer and probe design principle, design each pathogen gene
Amplimer and probe, in order to can be directly by naked eyes interpretation, in each pathogen target base after chip results is made to be developed the color with developing solution
5 ' the end of upstream and downstream primer of cause is marked with biotin, and particular sequence is as follows：
PAK genes
PAK gene target sequence amplification primers
F primers (PAK-F)：Sequence is SEQ ID NO.5；
R primers (PAK-R)：Sequence is SEQ ID NO.6；
PAK probes 1：Sequence is SEQ ID NO.1；
PAK probes 2：Sequence is SEQ ID NO.2；
Pf4 genes
F primers (Pf4-F)：Sequence is SEQ ID NO.7；
R primers (Pf4-R)：Sequence is SEQ ID NO.8；
Pf4 probes 1：Sequence is SEQ ID NO.3；
Pf4 probes 2：Sequence is SEQ ID NO.4.
【Embodiment 2】The preparation of chip
By amido modified probe and reference material probe, arranged according to table 1 respectively by probe point sample to NC, every group sets two
Parallel point of sample, UV crosslinking 20min fix probe.
Reference material ProbeT probe sequences are SEQ ID NO.11.
1 pseudomonas aeruginosa chip matrix of table
【Embodiment 3】Detection method
1st, genome extracts：This kit does not provide bacterial genomes DNA extracts reagents, limited using precious bioengineering (Dalian)
The DNA of bacteria extracts kit of company, article No. 9763.
2nd, amplification system, component are shown in Table 2.
2 PAK, Pf4 gene multiplexed PCR amplification system of table
PCR response procedures are：95℃ 5min；95 DEG C of 10s, 58 DEG C of 30s, 72 DEG C of 30s, 32 Xun Huans；72℃ 10min.
3rd, hybridize
Material：Negative controls are distilled water；Positive control is that target sequence and 19T using PAK, Pf4 gene as template amplification carry
The plasmid of body structure；Hybridization buffer is Quick Reaction Buffer (Chongqing prestige this rise biological Co., Ltd provide), clearly
Lotion prescription is to distill water dissolution 8g NaCl, 0.2g KCl and 3g Tris-base with 800ml.PH7.4 is adjusted to HCl, with steaming
Distilled water constant volume adds 1ml polysorbas20s to 1L；Avidin（Horseradish peroxidase-labeled）It needs to be diluted with hybridization buffer during use
To suitable concentration；ECL developing solutions are purchased in Thermo Fischer Scient Inc., article No. 32106；Gene core is made according to embodiment 2
Piece.
Hybridization step is as follows：
A. by the heating 3 minutes of 95 DEG C of PCR amplified productions, it is immediately placed on ice；
B. 10 μ l PCR products is taken to be mixed with 2mL hybridization buffers, are put into togerther with chip in hybridizing box, at normal temperatures, Yu Shui
60min is incubated on yawing bed.
C. cleaned 2 times using hybridization buffer, each 5min.
D. after cleaning, by Avidin（Horseradish peroxidase-labeled）It is added in hybridizing box, is incubated 10min.
E. cleaned 3 times with cleaning solution, each 5min
F. the point sample ECL developing solutions on the genetic chip dried, the Tanon produced using Shanghai Tian Neng Science and Technology Ltd.s
4600 Full-automatic chemiluminescence image analysis systems develop the color.
The present invention make use of multiple PCR technique and biochip technology phase by the selection of the primer and probe of specificity
With reference to method, not only increase accuracy and the specificity of detection, and at normal temperatures can be complete
Into crossover process, using chemiluminescence detection gene chip hybridization signal, large-scale instrument and equipment is not required, reduces detection
Cost of investment and application cost, meet the demand of the Site Detection of hospital etc..
Sequence table
<110>Chongqing prestige this rise biological medicine science and technology limited Company
<120>A kind of probe, genetic chip and kit for pseudomonas aeruginosa detection of nucleic acids
<130> 2017
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 50
<212> DNA
<213>Artificial sequence (unknown)
<400> 1
ccgcgaccag ggctcgcagg tgatgcccct gccctggctc aaggcgttgc 50
<210> 2
<211> 50
<212> DNA
<213>Artificial sequence (unknown)
<400> 2
accccaaggc gcccgcggaa ggcctgccgg tgggcttcac cgtagccggc 50
<210> 3
<211> 50
<212> DNA
<213>Artificial sequence (unknown)
<400> 3
ggcagcttct ccgccccgcg ctcccgagcc ctcggcggca agagcgggat 50
<210> 4
<211> 50
<212> DNA
<213>Artificial sequence (unknown)
<400> 4
gtggccgatc cggtcaaggg ccgcgctccc ggctcgtcgg aacagcctca 50
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence (unknown)
<400> 5
gcaaggaatg gaccgaaagc 20
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence (unknown)
<400> 6
aacaggtcat gccgaccatc 20
<210> 7
<211> 23
<212> DNA
<213>Artificial sequence (unknown)
<400> 7
atgcttgcta agaccctgaa agc 23
<210> 8
<211> 21
<212> DNA
<213>Artificial sequence (unknown)
<400> 8
ctagcgaacc aagccaacca c 21
<210> 9
<211> 234
<212> DNA
<213> PAK(PAK)
<400> 9
gcaaggaatg gaccgaaagc caccgccagg acttctacag ccgcgaccag ggctcgcagg 60
tgatgcccct gccctggctc aaggcgttgc gacagccgga tggaacgcct ttcctcgccg 120
acagcctggc ccgctacggc tatttgccca accccaaggc gcccgcggaa ggcctgccgg 180
tgggcttcac cgtagccggc acgggcgccc ggcagatggt cggcatgacc tgtt 234
<210> 10
<211> 378
<212> DNA
<213> Pf4(Pf4)
<400> 10
atgcttgcta agaccctgaa agcgctgctc ctgctctgcc tgatccaggc cgcccgcacc 60
gtggccgatc cggtcaaggg ccgcgctccc ggctcgtcgg aacagcctca ccgttccggc 120
gaacggaagc acgggcggag cgcacccttg aacgcctccc ccctgaaaca gcctccgctg 180
gggagtgtgg ggcagcttct ccgccccgcg ctcccgagcc ctcggcggca agagcgggat 240
gacaagggca gagcccttgg tgttgctctg cgggttccaa ggggaagcgt tccccttggc 300
cgtcggagac gacgttgcga tagggatcgt tccccgaatg ggccgagacg aacacccgtg 360
gttggcttgg ttcgctag 378
<210> 11
<211> 28
<212> DNA
<213>Artificial sequence (unknown)
<400> 11
tttttttttt tttttttttt tttttttt 28
Claims (10)
1. a kind of probe for pseudomonas aeruginosa, it is characterised in that：PAK gene probes 1,2, Pf4 bases including the bacterium
Because of probe 1,2, the PAK gene probes 1,2 sequences are respectively shown in SEQ ID NO.1~2；The Pf4 gene probes 1,2 sequences
Row are respectively SEQ ID NO.3~4.
2. a kind of genetic chip for pseudomonas aeruginosa detection of nucleic acids, it is characterised in that：Including solid phase and claim
1 probe groups.
3. a kind of gene chip kit for pseudomonas aeruginosa detection of nucleic acids, it is characterised in that：Including claim 2
The genetic chip.
4. according to the gene chip kit of the pseudomonas aeruginosa detection of nucleic acids described in claim 3, it is characterised in that：Also wrap
PAK gene primers pair and Pf4 gene primers pair, the PAK gene primers pair are included, sequence is respectively SEQ ID NO.5~6；Pf4
Gene primer pair, sequence are respectively SEQ ID NO.7~8.
5. according to the pseudomonas aeruginosa kit for detecting nucleic acid described in claim 4, it is characterised in that：Above and below the primer pair
Contain biotin labeling in 5 ' ends of trip primer.
6. according to the pseudomonas aeruginosa kit for detecting nucleic acid described in claim 4 and 5, it is characterised in that：Further include multiplex PCR
Reaction system, negative controls, positive reference substance, hybridization buffer, Avidin（Horseradish peroxidase-labeled）, cleaning solution,
ECL developing solutions.
7. according to the gene chip kit of the pseudomonas aeruginosa detection of nucleic acids described in claim 6, it is characterised in that：Institute
Negative controls are stated as physiological saline, positive reference substance is the PAK gene plasmids containing target gene, Pf4 gene plasmids it is mixed
Liquid is closed, the objective gene sequence is respectively SEQ ID NO.9~10.
8. according to the gene chip kit of the pseudomonas aeruginosa detection of nucleic acids described in claim 6, it is characterised in that：It is described
Hybridization buffer is Quick Reaction Buffer (Chongqing prestige this rise biological medicine science and technology limited Company provide), institute
It is that formula is to distill water dissolution 8g NaCl, 0.2g KCl and 3g Tris-base with 800ml to state cleaning solution, is adjusted to HCl
PH7.4 with distilled water constant volume to 1L, adds 1ml polysorbas20s.
9. according to the pseudomonas aeruginosa kit for detecting nucleic acid described in claim 6, it is characterised in that：The Avidin（Horseradish
Peroxidase labelling）It needs to be diluted to suitable concentration with hybridization buffer during use.
10. according to the pseudomonas aeruginosa kit for detecting nucleic acid described in claim 6, it is characterised in that：The multiplex PCR system
For：1 μ L, Premix Taq of amplification template, the 10 μ L of 50~150ng/ μ L, concentration are that 8 ~ 12 μM of different upstream and downstream primers are each
0 .6μL。
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| CN201711271879.3A CN108048583A (en) | 2017-12-06 | 2017-12-06 | A kind of probe, genetic chip and kit for pseudomonas aeruginosa detection of nucleic acids |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111394488A (en) * | 2020-05-06 | 2020-07-10 | 南开大学 | Liquid phase chip for detecting 19 serotypes of pseudomonas aeruginosa and application |
Citations (3)
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|---|---|---|---|---|
| CN103911459A (en) * | 2014-04-29 | 2014-07-09 | 西南大学 | LAMP rapid detection kit for toxin genes of pseudomonas aeruginosa type III secretion system and application and method thereof |
| WO2016014575A1 (en) * | 2014-07-22 | 2016-01-28 | Board Of Trustees Of The Leland Stanford Junior University | Organization of polymers by inovirus bacteriophage |
| CN106520984A (en) * | 2016-12-06 | 2017-03-22 | 湖南圣湘生物科技有限公司 | Pseudomonas aeruginosa nucleic acid fluorescent PCR (polymerase chain reaction) detection kit and detection method |
-
2017
- 2017-12-06 CN CN201711271879.3A patent/CN108048583A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103911459A (en) * | 2014-04-29 | 2014-07-09 | 西南大学 | LAMP rapid detection kit for toxin genes of pseudomonas aeruginosa type III secretion system and application and method thereof |
| WO2016014575A1 (en) * | 2014-07-22 | 2016-01-28 | Board Of Trustees Of The Leland Stanford Junior University | Organization of polymers by inovirus bacteriophage |
| CN106520984A (en) * | 2016-12-06 | 2017-03-22 | 湖南圣湘生物科技有限公司 | Pseudomonas aeruginosa nucleic acid fluorescent PCR (polymerase chain reaction) detection kit and detection method |
Non-Patent Citations (2)
| Title |
|---|
| PETAR KNEZEVIC等: "Prevalence of Pf1-like(pro)phage genetic elements among Pseudomonas aeruginosa isolates", 《VIROLOGY》 * |
| 石慧等: "铜绿假单胞菌重要外毒素基因多重PCR 检测方法的建立", 《中国食品学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111394488A (en) * | 2020-05-06 | 2020-07-10 | 南开大学 | Liquid phase chip for detecting 19 serotypes of pseudomonas aeruginosa and application |
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Application publication date: 20180518 |