CN105483251A - Probe, reagent kit and method for detecting bacteria in sputum - Google Patents
Probe, reagent kit and method for detecting bacteria in sputum Download PDFInfo
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- CN105483251A CN105483251A CN201511026690.9A CN201511026690A CN105483251A CN 105483251 A CN105483251 A CN 105483251A CN 201511026690 A CN201511026690 A CN 201511026690A CN 105483251 A CN105483251 A CN 105483251A
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- probe
- sputum
- bacterium
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- fluorophor
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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
- C12Q1/6841—In situ hybridisation
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- 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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Abstract
The invention provides a probe for detection and particularly relates to a probe, reagent kit and method for detecting bacteria in a sputum sample. The reagent kit comprises a liquefaction solution, a lysis solution, a probe solution, a stop solution, an immobilization solution, slide glass and cover glass. According to the probe, reagent kit and method for detecting bacteria in the sputum sample, after the sputum sample is liquefied, cell lysis, probe hybridization, fluorescence immobilization and the like are performed, and bacteria are observed by naked eyes, counted, analyzed and the like through a fluorescence microscope. The reagent kit is simple in operation method, brief in program and easy and convenient to operate, the detection result is strong in specificity, high in sensitivity, clear and high in credibility, and the probe, the reagent kit and the method can be used for directly, easily, conveniently and effectively detecting bacteria in the sputum sample.
Description
Technical field
The present invention relates to a kind of probe detected, being specifically related to a kind of probe, test kit and method for detecting bacterium in sputum sample.
Background technology
Microbial culture and plate coating checking are one of etiological diagnosis technology be most widely used, and wherein sputum is cultivated and accounted for more than 50%.Sputum sample inspection has important clinical meaning for the diagnosis of respiratory tract disease and discriminating.Normal people's lower respiratory tract is aseptic, and when lung or the segmental bronchus generation bacterial infection of human body, sputum secretory volume can obviously increase.Through sputum culture, isolate pathogenic bacteria, contribute to the Diagnosis and Treat to Lower respiratory tract infection.Cultivate through sputum and find common pathogenic bacterium:
Gram positive organism: streptococcus pneumoniae, streptococcus aureus, mycobacterium tuberculosis, actinomycetes, slave block bacterium, anaerobic cocci, diphtheria corynebacterium etc.;
Gram-negative bacteria: catarrh Blanc Chinese bacterium, Neisseria meningitidis, hemophilus influenzae, Klebsiella Pneumoniae, enterobacteria, pseudomonas, legionella etc.
In the related sanitary standard of China, measure the method for bacterial number, be carry out under the cultural method and culture condition of strict regulation, make each living bacterial cells adapting to these conditions can generate a macroscopic bacterium colony, the total number of bacterial colony generated is the total plate count of detection.
Its clinical coincidence rate of the sputum sample bacteriological analysis of traditional habit program is not high, and its reason is many-sided.Except sputum specimen gathers the cultivation results brought of the defective sample caused lack of standardization and not to conform to patient infection, also because the dependency of microorganism and human body inherently has complicacy: the dialectical property being derived from the pathogenic of bacterium and opportunistic, as pathogenic bacterium may for normally to carry, and bacterium the abnormal patient of pulmonary ventilation function lower respiratory tract can there is simple field planting or infection, prove that the dependency of cultivation results and infection is the difficult problem that medical microbiology faces, this also makes all the time very slow at the Standardization for Sputum culturing.
Sputum culture positive rate is general lower, the clinical key factor meeting rate variance is that sputum specimen collection is lack of standardization, the chance directly causing pathogenic bacteria separated reduces by defective sputum specimen, a large amount of normal microflora also can suppress the growth of pathogenic bacteria or disturb detecting of pathogenic bacteria, reduces clinical coincidence rate; Having is exactly the lack of standardization of schedule of operation again, thinks little of direct smear, the imprecision that checkout procedure controls; And the kind of bacterium is a lot of in sputum, their physiological property and required culture condition are not quite similar.If adopt method bacterial detection kind and the quantity of cultivation, must adopt different substratum and culture condition, its workload is very large, is more than the major reason causing clinical coincidence rate low.
Therefore, under current sputum method of detecting bacterium can not meet the condition that sputum bacteria sample is easy, rapid detection requires, a kind of instrument application extensively cheap, easy and simple to handle, result judges that directly perceived, highly sensitive detection method is ready to appear.
Fluorescence in situ hybridization (FluorescenceInSituHybridization, FISH) is a kind of molecular and cytogenetic techniques, is a kind of nonradioactivein situhybridization technology grown up phase late 1980s.The ultimate principle of FISH is probe with fluorescently-labeled single-chain nucleic acid, carries out specific binding, form the heteroduplex nucleic acid that can be detected with single-chain nucleic acid unknown in material to be checked.The probe of using fluorescence mark directly and karyomit(e) carry out hybridizing thus chromosomal localization carried out to gene.This technology has been widely used in many fields such as the structural research of animal-plant gene group, chromosomal structural variation analysis, viral infection assays, mankind's antenatal diagnosis, cancer genetics and genome evolution research at present.
Molecular beacon (molecularbeacon) is a kind of a kind of novel nucleic acid fluorescent probe based on FRET (fluorescence resonance energy transfer) principle design, it is combined the change of meeting recurring structure thus affects fluorescent signal with target sequence, because its detection has high sensitivity, high specific, and can be used for live body and detect in real time, be thus widely used in the numerous areas such as biology.
In pathogenic bacteria genetic information, the ribosomal RNA gene being representative with 16SrRNA gene is encoding ribosomal RNA(rRNA on pathogenic bacteria) corresponding DNA sequence dna, be present in the genome of all pathogenic bacterias.This gene have height conservative property and specificity with, the ribosome-RNA(rRNA) detection technique being representative with 16SrRNA gene has been become to a kind of powerful tool of detection of pathogens and qualification.
The advantage of the present invention effective matching fluorescence in situ hybridization technique, molecular beacons technology, fluorescence microscopy, to the bacterium in sputum sample carry out direct, easy, effectively detect.
Summary of the invention
The object of invention is: provide a kind of efficiently, detect the probe of bacterium in sputum sample, method and test kit rapidly.
Technical scheme of the present invention is: provide a kind of probe, method and test kit for detecting bacterium in sputum sample.Described probe is molecular beacon structure, namely probe sequence is made up of loop-stem structure two portions, total length 25 ~ 50 bases, wherein in the middle of ring Sequence and bacterium in rrna RNA(16srRNA, 23srRNA etc.) sequence matches, two ends stem is divided into 5 ~ 10 complementary DNA sequence dnas, the 5'-end fluorophor of probe marks, 3'-end fluorescent quenching group mark, at normal temperature, under unbound state, probe can be annealed formation hairpin structure automatically, fluorophor and fluorescent quenching group do not send fluorescent signal due to close together, only have after probe and target sequence successful cross, fluorophor and fluorescent quenching group separated from one another, and then send fluorescent signal, described probe is the bacterium detected by the principle of nucleic acid hybridization in sputum, the described probe for detecting bacterium in sputum, it is characterized in that, the fluorophor that described probe 5'-holds is the one in FAM, JOE, HEX, VIC, ROX, CY3, CY5, and the fluorophor that described probe 3'-holds is the one in DABCYL, TAMRA, BHQ.
The invention provides a kind of test kit for detecting bacterium in sputum sample, described test kit comprises:
(1) probe described in claim 1 or 2;
(2) liquefier;
(3) lysate;
(4) stop buffer;
(5) stationary liquid;
Described liquefier comprises: 1% (w/v) PEG, 0.6mMDTT, 13.4mMNaCl, 0.3mMKCl, 1mMNaH
2pO
4, 0.1mMKH
2pO
4, 50mMEDTA, 0.1% (v/v) TritonX-100,50mMTrisHClpH8.0;
Described lysate comprises: 5M Guanidinium hydrochloride, 0.2mMDTT, 50mMEDTA, 0.5% (w/v) ammonium persulphate, 0.1% (v/v) TritonX-100,10mMTrisHClpH7.0;
Described stop buffer comprises 50mMTrisHClpH10.0,0.5MNaCl, 0.1% (v/v) NP-40,0.5% (v/v) TritonX-100;
Described stationary liquid comprises: 5% (w/v) glycerine, 0.2mMDTT, 25mMNaCl, 0.3mMKCl, 0.6mMNaH
2pO
4, 0.6mMKH
2pO
4, 50mMEDTA, 10mMTrisHClpH7.0.
The invention provides a kind of method for detecting bacterium in sputum sample, described method adopts mentioned reagent box to detect, and said method comprising the steps of:
A () gets sputum sample to be checked 100 ~ 500ul, join in 1 ~ 3ml liquefier, and the volume ratio of sputum sample to be checked and liquefier is 1:1 ~ 1:10, concussion mixing;
B () is got the liquid 3 ~ 20ul obtained by step (a) operation and is dropped on slide glass, dry 3 ~ 20min for 45 ~ 65 DEG C;
C slide glass that () will obtain by step (b) operation immerses 3 ~ 20min in ethanol, dries 3 ~ 20min for 45 ~ 65 DEG C;
D () adds people's probe 3 ~ 20ul in the slide glass well obtained by step (c) operation, be placed in hot-plate 45 ~ 65 DEG C of lucifuges and dry 3 ~ 20min;
E slide glass that () will obtain by step (d) operation immerses 1 ~ 60sec in stop buffer, is placed in hot-plate 45 ~ 65 DEG C and dries 3 ~ 20min;
F () adds stationary liquid 3 ~ 20ul in the slide glass well obtained by step (e) operation, add a cover cover glass, observes with fluorescence microscopy, observes counting with 10 × object lens, observes ne ar with 40 × or 100 × object lens.
The technical essential of test kit of the present invention and detection method or principle: liquefier of the present invention carries out liquefaction processing to sputum sample, strengthen its homogeneity, mobility, is easily combined with slide glass well.Fluorescence in situ hybridization (fluorescenceinsituhybridization, FISH) is a kind of application molecular probe technology, is detected the method for bacterial genomes specific DNA or RNA in sputum by the method for fluorescence in situ hybridization; Molecular beacon is a kind of a kind of novel nucleic acid fluorescent probe based on FRET (fluorescence resonance energy transfer) principle design, and it is combined the change of meeting recurring structure thus affects fluorescent signal with target sequence, this signal detects by fluorescent microscope.
The present invention uses the multiple FISH technology of enhancing, in existing FISH technical foundation, use the probe that fluorescently-labeled molecular beacon detects as pathogenic agent microorganism conserved nucleic acid sequence (ribosome rRNA), it is the improvement to traditional F ISH technology, and significantly decrease experimental period: utilize the molecular beacons technology of nucleic acid high specific and susceptibility to carry out Rapid nucleic acid hybridization check to pathogenic micro-organism conserved nucleic acid sequence (ribosome rRNA), by fluorescent microscope, macroscopic direct result judgement is carried out to the fluorescent signal that molecular beacon fluorophor sends.The samples sources of this technology for detection is in clinical patient respiratory tract juice---sputum, without the need to through any cultivation before detecting, microorganism pathogenic bacteria common clinically can be detected, whole testing process only needs fluorescent microscope and hot-plate two instrument, easy handling; Have and do not need nucleic acid extraction, short, pollution-free, the advantage such as effective directly perceived easy and simple to handle, consuming time.
Accompanying drawing explanation
Fig. 1 is the fluorescence microscopy design sketch of a kind of embodiment being detected bacterium in sputum sample by detection method of the present invention.
Embodiment
Embodiment 1 is for detecting the Design and synthesis of colibacillary specific molecular beacon probe in sputum
By the sequential analysis comparison to intestinal bacteria rrna 16srRNA in sputum, filter out and there is the specific probe sequence of bacillus coli gene, 5 '-GGCGGTTTGTTAAGTCAGATGTGAAA-3 ', this section of sequence is the loop section of molecular beacon probe, on this basis, add 6 complementary bases respectively at these sequence two ends, form the stem portion of probe, and connect fluorophor and quenching group at sequence two ends, just constitute complete molecular beacon probe:
5’-FAM-CACGCAGGCGGTTTGTTAAGTCAGATGTGAAATGCGTG-DABCYL-3’，
This molecular beacon 5 '-hold with FAM mark, 3 '-holding with DABCYL mark, fluorophor FAM requires excitation wavelength 492nm, determined wavelength 517nm.
Embodiment 2 uses the intestinal bacteria in the molecular beacon probe detection sputum of design and synthesis in embodiment 1
Detection method:
A () gets sputum sample 500ul to be checked, join in 1ml liquefier, concussion mixing;
B () is got the liquid 10ul obtained by step (a) operation and is dropped on slide glass, be placed in hot-plate 52 DEG C and dry 5min;
C slide glass that () will obtain by step (b) operation immerses 5min in ethanol, is placed in hot-plate 52 DEG C and dries 5min;
D () adds people's probe 10ul in the slide glass well obtained by step (c) operation, be placed in hot-plate 52 DEG C of lucifuges and dry 5min;
E slide glass that () will obtain by step (d) operation immerses 40sec in stop buffer, is placed in hot-plate 52 DEG C and dries 5min;
F () adds stationary liquid 15ul in the slide glass well obtained by step (e) operation, add a cover cover glass, observes with fluorescence microscopy, observes counting with 10 × object lens, observes ne ar with 100 × object lens.In dark background, intestinal bacteria send green fluorescence, as shown in Figure 1.
Nucleotides sequence list
SEQUENCELISTING
<110> Shanghai Xingyao Medical Technology Development Co., Ltd.
Shanghai Fosun Pharmaceutical (Group) Co., Ltd.
Late, sharp greatly
Wu, controls greatly
Summer, virtuous
<120> mono-kind is for detecting the probe of bacterium in sputum, test kit and method
<130>bacteriadetectionfromsputum
<140>cn
<141>2015-11-05
<160>1
<170>PatentInversion3.3
<210>1
<211>40
<212>DNA
<213>E.coli
<220>
<221>SEQIDNo.1
<222>(2)..(39)
<223>b=FAM;d=DABCYL
<400>1
bcacgcaggcggtttgttaagtcagatgtgaaatgcgtgd
Claims (5)
1. one kind for detecting the probe of bacterium in sputum, it is characterized in that: described probe is molecular beacon probe, this molecular beacon probe is made up of loop-stem structure two portions, total length 25 ~ 50 bases, wherein in the middle of ring Sequence and bacterium in rrna RNA(16srRNA, 23srRNA etc.) sequence matches, two ends stem Sequence is the DNA sequence dna of 5 ~ 10 base complementrities, the 5'-end fluorophor of molecular beacon probe marks, 3'-end fluorescent quenching group mark, at normal temperature, under unbound state, molecular beacon probe can be annealed formation hairpin structure automatically, fluorophor and fluorescent quenching group do not send fluorescent signal due to close together, only have after ring Sequence in molecular beacon probe and target sequence specific binding, fluorophor and fluorescent quenching group separated from one another, and then send fluorescent signal, described probe is the bacterium detected by the principle of nucleic acid hybridization in sputum.
2. the probe for detecting bacterium in sputum according to claim 1, it is characterized in that, the fluorophor that described probe 5'-holds is the one in FAM, JOE, HEX, VIC, ROX, CY3, CY5, and the fluorophor that described probe 3'-holds is the one in DABCYL, TAMRA, BHQ.
3. for detecting a test kit for bacterium in sputum, it is characterized in that, described test kit comprises:
(1) probe described in claim 1 or 2;
(2) liquefier;
(3) lysate;
(4) stop buffer;
(5) stationary liquid.
4. the test kit for detecting bacterium in sputum according to claim 3, is characterized in that, described liquefier comprises: 1% (w/v) PEG, 0.6mMDTT, 13.4mMNaCl, 0.3mMKCl, 1mMNaH
2pO
4, 0.1mMKH
2pO
4, 50mMEDTA, 0.1% (v/v) TritonX-100,50mMTrisHClpH8.0;
Described lysate comprises: 5M Guanidinium hydrochloride, 0.2mMDTT, 50mMEDTA, 0.5% (w/v) ammonium persulphate, 0.1% (v/v) TritonX-100,10mMTrisHClpH7.0;
Described stop buffer comprises 50mMTrisHClpH10.0,0.5MNaCl, 0.1% (v/v) NP-40,0.5% (v/v) TritonX-100;
Described stationary liquid comprises: 5% (w/v) glycerine, 0.2mMDTT, 25mMNaCl, 0.3mMKCl, 0.6mMNaH
2pO
4, 0.6mMKH
2pO
4, 50mMEDTA, 10mMTrisHClpH7.0.
5. adopt as claim 3,4 arbitrary as described in the method for detecting bacterium in sputum sample of test kit, it is characterized in that, said method comprising the steps of:
A () gets sputum sample to be checked, join in liquefier, concussion mixing;
B () is got the liquid 3 ~ 20ul obtained by step (a) operation and is dropped on slide glass, dry 3 ~ 20min for 45 ~ 65 DEG C;
C slide glass that () will obtain by step (b) operation immerses 3 ~ 20min in ethanol, dries 3 ~ 20min for 45 ~ 65 DEG C;
D () adds probe 3 ~ 20ul in the slide glass well obtained by step (c) operation, be placed in hot-plate 45 ~ 65 DEG C of lucifuges and dry 3 ~ 20min;
E slide glass that () will obtain by step (d) operation immerses 1 ~ 60sec in stop buffer, is placed in hot-plate 45 ~ 65 DEG C and dries 3 ~ 20min;
F () adds stationary liquid 3 ~ 20ul in the slide glass well obtained by step (e) operation, add a cover cover glass, observes with fluorescence microscopy, observes counting with 10 × object lens, observes ne ar with 40 × or 100 × object lens.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201511026690.9A CN105483251A (en) | 2015-12-31 | 2015-12-31 | Probe, reagent kit and method for detecting bacteria in sputum |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201511026690.9A CN105483251A (en) | 2015-12-31 | 2015-12-31 | Probe, reagent kit and method for detecting bacteria in sputum |
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| Publication Number | Publication Date |
|---|---|
| CN105483251A true CN105483251A (en) | 2016-04-13 |
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| CN201511026690.9A Pending CN105483251A (en) | 2015-12-31 | 2015-12-31 | Probe, reagent kit and method for detecting bacteria in sputum |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018196691A1 (en) * | 2017-04-24 | 2018-11-01 | 倪燕翔 | Precise recognition method for nucleic acid |
| EP3625358A4 (en) * | 2017-05-17 | 2021-02-24 | Microbio Pty Ltd | Biomarkers and uses thereof |
-
2015
- 2015-12-31 CN CN201511026690.9A patent/CN105483251A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018196691A1 (en) * | 2017-04-24 | 2018-11-01 | 倪燕翔 | Precise recognition method for nucleic acid |
| EP3625358A4 (en) * | 2017-05-17 | 2021-02-24 | Microbio Pty Ltd | Biomarkers and uses thereof |
| US11739389B2 (en) | 2017-05-17 | 2023-08-29 | Microbio Pty Ltd | Biomarkers and uses thereof |
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Application publication date: 20160413 |