CN107022645A - Detect the molecular beacon probe and kit of urological genital tract causal agent gonococcus - Google Patents
Detect the molecular beacon probe and kit of urological genital tract causal agent gonococcus Download PDFInfo
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- CN107022645A CN107022645A CN201710449471.4A CN201710449471A CN107022645A CN 107022645 A CN107022645 A CN 107022645A CN 201710449471 A CN201710449471 A CN 201710449471A CN 107022645 A CN107022645 A CN 107022645A
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- molecular beacon
- gonococcus
- probe
- beacon probe
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
Abstract
The invention discloses a kind of molecular beacon probe for detecting urological genital tract causal agent gonococcus, the base sequence Probe GN of the molecular beacon probe are：5'CCCTACCCCATAAGTAGGTAGGGTAACCGCAAGGGTAGGG 3', the 5' ends of the molecular beacon probe are marked with fluorophor, and 3' ends are marked with quenching group.The molecular beacon probe of the present invention can urological genital tract causal agent gonococcus, and accuracy in detection is high, and sensitivity is high.
Description
Technical field
The present invention relates to a kind of molecular beacon probe, and in particular to a kind of point of detection urological genital tract causal agent gonococcus
Sub- beacon probe and kit.
Background technology
The most common pathogen of inflammation of genitourinary tracts is caused to include gonococcus, chlamydia trachomatis and Ureaplasma urealyticum.Mesh
The preceding diagnosis to gonococcus mainly has direct microscopy and cell culture.Although direct microscopy speed is fast but accuracy rate is relatively low, commonly use
Cell culture method accuracy although increase but time loss long, and detection process is more complicated.Therefore provide it is a kind of accurate,
Urological genital tract causal agent gonococcus detection method simple and in higher sensitivity has significant clinical meaning.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of molecular beacon for detecting urological genital tract causal agent gonococcus
Probe and kit.
In order to solve the above technical problems, the technical solution adopted by the present invention is：
A kind of molecular beacon probe for detecting urological genital tract causal agent gonococcus, the base sequence of the molecular beacon probe
It is classified as：
Probe GN：5'-CCCTACCCCATAAGTAGGTAGGGTAACCGCAAGGGTAGGG-3', the molecular beacon
The 5' ends of probe are marked with fluorophor, and 3' ends are marked with quenching group.
It is preferred that, the fluorophor be selected from FAM, Fluorescein, JOE, TET, HEX, Cyanine 3, ROX,
Texas Red, Cyanine 5 or Cyanine 5.5, the quenching group are selected from BHQ-1, BHQ-2, BHQ-2 or DABCYL.
Present invention also offers a kind of kit, it includes above-mentioned molecular beacon probe.
It is preferred that, mentioned reagent box also includes hybridization solution, and hybridization solution includes following components described in per 100ml：25mL 50*
Denhardtt's liquid, 10mL 50*SSC, 5mL 50*SSC, 100ug/mL denaturation salmon sperm DNA, 10g dextran sulfate.
It is preferred that, the use of the concentration of molecular beacon probe described above is 5pmol/ul.
Present invention also offers the method detected to urological genital tract causal agent gonococcus, this method includes following step
Suddenly：
(1) according to gonococcus (n.gonorrhoeae) 16S rna gene sequences Design fluorescence beacon probes, wherein drenching ball
Bacterium (n.gonorrhoeae) 16S rna gene sequences are：
TTGTACACACCGCCCGTCACACCATGGGAGTGGGGGATACCATAAGTAGGTAGGGTAACCGCAAGGAGC
CCGCTTACCACGGTATGCTTCATGACTGGGGTGAAGTCGTAACAAGGTAGCCGTAGGGGAACCTGCGGCTGGATCAC
CTCCTTTCTAGAGAGAGAAGGGCTTTAGGCATTCACACTTATCGGTAAACTGAAAAGATGCGGA
The molecular beacon probe Probe GN of designed synthesis base sequence is：
5'-CCCTACCCCATAAGTAGGTAGGGTAACCGCAAGGGTAGGG-3', its 5' end is marked with fluorophor,
3' ends are marked with quenching group；
(2) using urogenital system secretion as detection sample, smear is simultaneously fixed with fixer；
(3) molecular beacon probe is diluted with above-mentioned hybridization solution, then hybridized with detection sample；
(4) in fluorescence microscopy Microscopic observation result.
The principle that the present invention is detected is：Molecular beacon probe with unique " hair clip " space structure not with target sequence knot
During conjunction, molecular beacon is in " hair clip " structure, there is a ring sequence and a stem sequence, and its middle ring sequence is the base complementary with target site
Sequence, and stem sequence is the complementary series unrelated with target site；Fluorophor is marked with respectively at the two ends of probe and fluorescence is sudden
Go out group, and when probe is in hairpin structure, fluorophor and quencher are adjacent, produce resonance energy transfer effect so that
Fluorophor is quenched, it is impossible to produce fluorescence signal, and when probe is combined with target site, hairpin structure is opened, fluorescent base
Group and quencher are separated, and produce fluorescence signal, the fluorescence signal can be detected by fluorescence microscope.
The molecular beacon probe of the present invention can urological genital tract causal agent gonococcus, and accuracy in detection is high, sensitivity
It is high.
Embodiment
With reference to specific embodiment, the invention will be further described.Following examples are only used for clearly illustrating
Technical scheme, and can not be limited the scope of the invention with this.
According to gonococcus (n.gonorrhoeae) 16S rna gene sequences Design molecular beacon probes, the molecular beacon is visited
Pin base sequence is：
5'FAM-CCCTACCCCATAAGTAGGTAGGGTAACCGCAAGGGTAGGG-BHQ1 3'.Wherein 5' ends are marked
It is FAM, 3' ends mark is BHQ1, and the excitation wavelength of fluorophor is 494nm, and launch wavelength is 515nm.
According to gonococcus (n.gonorrhoeae) 16S rna gene sequences Design fluorescence labeling oligonucleotide probes Probe
GNc：5'-FAM-CATAAGTAGGTAGGGTAACCGCAA-3'.
Embodiment 1
Detection target is used as using the gonococcus (n.gonorrhoeae) of culture.Collect 2 female genitourinary system secretions
Thing is sample, proportionally adds the gonococcus of culture, and ratio is respectively the simple female urogenital of the simple gonococcus of A., B.
Be sample, C. in 10000 bacteriums/ml ratio add gonococcus.Tri- samples of the above A, B, C are diluted with 1xPBS respectively,
5000rpm is centrifuged, and collects sediment, adds 10% formalin (i.e. 1 part formalin adds 9 parts of water to be formulated) 250ul solid
Determine liquid and fix 5 minutes, washed 2 times with 1xPBS500ul, last 3000rpm centrifuges 5 points, supernatant is discarded, by the molecule through thermal denaturation
Beacon probe is diluted to 5pmol/ul with 1xDNA hybridization solutions, plus 200ul makes precipitation suspend, and 42 DEG C are incubated 15 minutes.It is added dropwise and is carrying
Slide, directly in fluorescence microscopy Microscopic observation and takes a picture, and calculates fluorescence developing bacterium.As a result it is not added with the sample unstressed configuration of gonococcus
Dyeing is feminine gender, and the whole bacteriums of simple gonococcus sample have the fluorescent staining i.e. positive, add in 10000 bacteriums/ml ratio
Enter the sample of gonococcus, it is observed that part bacterium has fluorescent staining that is, the positive.
Include following components per the above-mentioned DNA hybridization liquid of 100ml：25mL 50*denhardtt's liquid, 10mL 50*SSC,
5mL 50*SSC, 100ug/mL denaturation salmon sperm DNA, 10g dextran sulfate.
Embodiment 2
Detected using molecular beacon probe Probe GN：With known 50 gonococcal infection persons, 50 Healthy Peoples
Urethral secretions sample be used as detection target.It is applied to sample is light and slow in clean glass slide, after fully drying, uses acetone
10 minutes are fixed, is dried naturally.Molecular beacon probe through thermal denaturation is diluted to 5pmol/ul with 1xDNA hybridization solutions, plus
200ul makes precipitation suspend, and 42 DEG C are incubated 15 minutes.It is added dropwise in slide, diagosis is directly carried out under fluorescence microscope.
Detected using above-mentioned plain edition fluorescence labeling oligonucleotide probe Probe GNc：With known 50 pouring balls
Bacterium the infected, the urethral secretions sample of 50 Healthy Peoples are used as detection target.It is applied to sample is light and slow in clean glass slide,
After fully drying, 10 minutes are fixed using acetone, is dried naturally.It is subsequently added into 75 DEG C of denaturation, 5 minutes 5pmol/ul fluorescence mark
Remember oligonucleotide probe Probe GNc probes (probe with hybridization buffer (20mmol/L Tris, 0.9mmol/L NaCl,
20%formamide, 0.1%SDS, pH7.2) dilution), then it is incubated 45~60 minutes at 50 DEG C；By slide successively with washing
Liquid A (2xSSC, 50% formamide), washing lotion B (10mM PBS pH8.0,0.1%Nonidet P-40) respectively cleaning 2 times.In fluorescence
Diagosis is carried out under microscope.
Molecular beacon is identical with the criterion of common fluorescent labeled oligonucleotide probe experimental result to be：In 60 times of oil
Many visual field observations are carried out to sample under leaching object lens and the cell that fluorescence is sent in every visual field is counted and added up, by sentencing as follows
Disconnected standard：
Sample feminine gender (-)：The different visuals field of Continuous Observation 100, find no the cell for sending fluorescence；The sample positive (+)：
The different visuals field of Continuous Observation 100, the cell number for sending fluorescence is more than 1/100 visuals field, carries out experimental result judgement.
Judged by above-mentioned criterion, experimental result such as following table：
| Key sample (goldstandard) | Molecular beacon probe | Fluorogenic oligonucleotide probe | Sample number |
| + | + | + | 46 |
| + | + | - | 4 |
| - | - | - | 47 |
| - | - | + | 3 |
Note：In above-mentioned list, it is known that sample (goldstandard)：Known 50 Healthy People urethral secretions samples are used as negative gold
Standard, it is known that 50 gonococcal infection person's urethral secretions samples are used as positive goldstandard；“-”：Represent that feminine gender has not been detected
Gonococcal infection；“+”：Represent that the positive is that detection has gonococcal infection.
Using known 50 Healthy Peoples, 50 gonococcal infection person's urethral secretions samples as goldstandard, molecular beacon
Positive rate is (46+4)/(46+4) X100%=100%, and the positive rate of fluorescence labeling oligonucleotide probe is 46/
(46+4) X100%=92%；The false positive recall rate of molecular beacon is 0/ (47+3) X100%=0%, fluorescence labeling few nucleosides
The false positive recall rate of acid probe is 3/ (47+3) X100%=6%.From experimental result, molecular beacon probe is better than fluorescence
Oligonucleotide probe.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, some improvement and deformation can also be made, these improve and deformed
Also it should be regarded as protection scope of the present invention.
Claims (5)
1. a kind of molecular beacon probe for detecting urological genital tract causal agent gonococcus, it is characterised in that the molecular beacon is visited
The base sequence of pin is：
Probe GN：5'-CCCTACCCCATAAGTAGGTAGGGTAACCGCAAGGGTAGGG-3', the molecular beacon probe
5' ends be marked with fluorophor, 3' ends are marked with quenching group.
2. a kind of molecular beacon probe for detecting urological genital tract causal agent gonococcus according to claim 1, its feature
Be, the fluorophor be selected from FAM, Fluorescein, JOE, TET, HEX, Cyanine 3, ROX, Texas Red,
Cyanine 5 or Cyanine 5.5, the quenching group is selected from BHQ-1, BHQ-2, BHQ-2 or DABCYL.
3. a kind of kit, it is characterised in that including the molecular beacon probe described in claim 1 or 2.
4. a kind of kit according to claim 3, it is characterised in that also including hybridization solution, hybridization solution described in per 100ml
Including following components：25mL 50*denhardtt's liquid, 10mL 50*SSC, 5mL 50*SSC, 100ug/mL denaturation salmon
Smart DNA, 10g dextran sulfate.
5. a kind of kit according to claim 4, the concentration of the molecular beacon probe is 5pmol/ul.
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| CN201710449471.4A CN107022645A (en) | 2017-06-14 | 2017-06-14 | Detect the molecular beacon probe and kit of urological genital tract causal agent gonococcus |
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| EP1862557B1 (en) * | 2006-05-29 | 2011-05-18 | Bio-Rad Pasteur | Real-time multiplex detection of three bacterial species responsible for sexually-transmitted diseases |
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| EP3063292A2 (en) * | 2013-10-30 | 2016-09-07 | Mast Group Limited | Nucleic acid probe with single fluorophore label bound to internal cytosine for use in loop mediated isothermal amplification |
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Patent Citations (6)
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| EP1862557B1 (en) * | 2006-05-29 | 2011-05-18 | Bio-Rad Pasteur | Real-time multiplex detection of three bacterial species responsible for sexually-transmitted diseases |
| CN101333565A (en) * | 2007-06-27 | 2008-12-31 | 上海仁度生物科技有限公司 | Constant temperature synchronous amplification detecting process for nucleic acid and use thereof |
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