CN104032033A - Peptide nucleic acid probe set for fluorescence in-situ hybridization detection of enterococcus faecalis and/or other enterococcocci, reagent kit, using method and applications - Google Patents
Peptide nucleic acid probe set for fluorescence in-situ hybridization detection of enterococcus faecalis and/or other enterococcocci, reagent kit, using method and applications Download PDFInfo
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Abstract
The invention relates to a peptide nucleic acid probe for rapid fluorescence in-situ hybridization detection of enterococcus faecalis and/or other enterococcocci. The DNA sequence of the probe is shown as the SEQ NO:1 or the SEQ NO:2. The invention further discloses a reagent kit for appraising the enterococcus faecalis and/or other enterococcocci in samples and the drug susceptibility of the enterococcus faecalis and/or other enterococcocci through the probe and provides a using method of the reagent kit. The probe and the reagent kit can be used for detecting the enterococcus faecalis and/or other enterococcocci in the samples and the drug susceptibility of the enterococcus faecalis and/or other enterococcocci. The DNA extraction step is avoided, and the time for a whole appraising process is generally less than two hours; besides, the false positive rate is low, the number of steps is small, efficiency is high, sensitivity is high, and specificity is high. The enterococcus faecalis and other enterococcocci can be rapidly and accurately detected, and important significance to food safety guaranteeing, environmental sanitation guaranteeing and pathophoresis preventing is achieved.
Description
Technical field
The present invention relates to the detection field of Clinical Correlation microorganism, be specifically related to detect enterococcus faecalis and/or other enterococcal peptide nucleic acid probe groups.
Background technology
Faecalis (Enterococcus) is gram-positive, becomes coccus, the oval of two or short catenation, and without brood cell, without pod membrane, part faecalis has sparse flagellum.Nutritional requirement is high, aerobic and facultative anaerobe, 35 ℃ of optimum growth temperatures.
Commonly enterococcus faecalis (Enterococcus faecalis) and faecium (Enterococcus faecium) clinically, be one of most important pathogenic bacteria infecting in Hospitals at Present, minority is separable to enterococcus avium (Enterococcus avium), steadfast and persevering faecalis (Enterococcus durans), E. casselflavus (Enterococcus casseliflavus) etc.
Enterococcus spp is mankind's normal intestinal flora, in external environment, also exists.Such bacterium can cause ward infection, also can cause community infection, and especially application, indwelling catheter, blood and the peritoneal dialysis etc. of cephalosporins etc. are the Hazard Factor that cause faecalis ward infection.Urinary tract infections is the most common in the infection that causes of faecalis, shows as upper and lower urinary tract infections more, and minority is prostatitis and kidney periphery abscess; What during endocarditis infects, faecalis caused accounts for 5～15%; Faecalis can infect abdominal cavity, pelvic cavity jointly with intestinal bacteria and bacillus fragilis, and rare occasion infects separately abdominal cavity and pelvic cavity; Under other a few cases, faecalis can cause wound infection, phlegmon, meningitis, seldom causes respiratory tract infection.After the endocarditis that faecalis causes, urinary tract, abdominal cavity, pelvic infection, burn, wound infection etc. all can become septicemia.Septicemia is intensive care unit common bacterial grave infection of hospital, and lethality rate is high.
Enterococcal resistance is divided into natural drug resistance and acquired resistance.For general dosage or middle dosage aminoglycoside resistant be often natural resistance to the low resistance of vancomycin, drug resistant gene is present in karyomit(e).Acquired resistance strain is in recent years on the increase, and shows as to aminoglycoside high level resistance with to vancomycin, teicoplanin height resistance.At present, enterococcal resistance problem comprises: the faecalis of (1) penicillin resistant and Ampicillin Trihydrate.The susceptibility of Ampicillin Trihydrate and penicillin can be used to the susceptibility of prediction to amoxycilline Trihydrate bp, ampicillin/sulbactam, amoxicillin/clavulanate, piperacillin and piperacillin/Tazobactam Sodium.(2) faecalis of aminoglycoside high level resistance (HLAR).Heavy dose of gentamicin and Streptomycin sulphate screening are generally applied in Clinical microorganism laboratory, other aminoglycosides do not need to test, because they are not better than gentamicin and Streptomycin sulphate to enterococcal activity, responsive result indication Ampicillin Trihydrate, penicillin or vancomycin and this aminoglycoside antibiotics have synergy, and resistance result (HLAR) indicates and between them, do not have synergy.(3) faecalis of vancomycin resistance (VRE).1988 there is VRE in reported first, and at present domestic three grades have first-classly accounted for separated enterococcal 1%～5% with the VRE that goes to the hospital.Faecalis can be divided into low-level resistance (MIC is 8～32mg/L) and high-level resistance (MIC >=64mg/L) to the resistance of vancomycin.
Peptide nucleic acid(PNA) (peptide nucleic acids, PNA) be that the people such as 1991 Nian You Denmark scientist Nielsen design a kind of be take the brand-new DNA analog that neutral amido linkage is skeleton, its skeleton structure unit is (2-aminoethyl) glycine, base portion is connected in by methylene radical carbonyl in the amino N of main framing, can be combined with DNA, RNA sequence specific.Its skeleton is electric neutrality, compare with DNA-DNA or RNA-DNA complementary strand, there is not electrostatic repulsion in PNA-DNA and PNA-RNA complementation, therefore there is very high DNA or RNA affinity, without mispairing in the situation that, it is in conjunction with having high stability, and hybridization speed is fast, there is good cell-penetrating, be the good selection of nucleic acid probe.
Existing faecalis detection method all needs DNA of bacteria to extract, and then carries out pcr amplification, or bacterium liquid is increased, then detects with amplified production, time-consuming taking a lot of work, and complex steps, and false positive is high, easily causes misjudgement.
Summary of the invention
The object of this invention is to provide a kind of DNA that do not need to extract, do not need pcr amplification can detect enterococcus faecalis and/or other enterococcal peptide nucleic acid probe groups and test kit thereof, method, efficient quick, false positive is low.
For achieving the above object, technical scheme of the present invention is: for fluorescence in situ hybridization detection enterococcus faecalis and/or other enterococcal peptide nucleic acid probe groups, it is characterized in that: the DNA sequence dna of this peptide nucleic acid probe group is as shown in SEQ NO:1, SEQ NO:2;
Wherein, described SEQ NO:1 sequence is: 5 '-TTA TCC CCC TCT GAT GGG-3 ', for detection of enterococcus faecalis;
Wherein, described SEQ NO:2 sequence is: 5 '-TAC TGA TCG GCA GCT-3 ', for detection of other faecalis;
Wherein, described SEQ NO:1 sequence and SEQ NO:2 sequence can be used respectively, are used for detecting respectively enterococcus faecalis or other faecalis, also can use simultaneously, are used for detecting enterococcus faecalis and other faecalis simultaneously.
Described peptide nucleic acid probe group can detect target sequence in the rRNA sequence of enterococcus faecalis and/or other enterococcal rRNA, rDNA or complementation.
The probe that detects enterococcus faecalis in described peptide nucleic acid probe group should at least comprise the DNA sequence dna that is similar to SEQ NO:1 in 86% structure; In described peptide nucleic acid probe group, detect other enterococcal probes and should at least comprise the DNA sequence dna that is similar to SEQ NO:2 in 86% structure.
Described peptide nucleic acid probe group is at least connected to a kind of detectable fraction.
The type of described detectable fraction is selected from: conjugate, chromophore, fluorophore, radio isotope, enzyme, haptens or luminophor.
Described fluorophore group is at least one in following: fluorophore Alexa series, Alexa Fluor series, cyanin, 5-(and-6) carboxyl-2 ', 7 '-dichlorofluorescein, 5-ROX (5-carboxyl-X-rhodamine, triethyl ammonium salt).
The present invention is also provided for detecting the test kit of enterococcus faecalis and/or other faecalis bacterial strains and drug susceptibility, the probe that described test kit comprises any one in claim 1～6.。
Described test kit comprises following solution: fixed solution, hybridization solution and drug susceptibility detect solution;
Wherein, fixed solution comprises paraformaldehyde and ethanol;
Wherein, hybridization solution comprises Triton and methane amide;
Wherein, drug susceptibility detection solution is M-H liquid.
The ethanol of the paraformaldehyde that described fixed solution comprises 2～8% (wt/vol) and 25～75% (vol/vol); Composition and the composition of described hybridization solution are: 10% (wt/vol) T 500,10mM NaCl, 50% (v/v) methane amide, 0.1% (wt/vol) trisodium phosphate, 0.2% (wt/vol) polyvinyl pyrroline, 0.2% (wt/vol) FICOLL (ficoll), 5mM EDETATE SODIUM, 0.1% (vol/vol) Triton X-100 (Triton), 50mM Tris-HCl.
The present invention is also provided for detecting the using method of the test kit of enterococcus faecalis and/or other faecalis bacterial strains and drug susceptibility, and described using method comprises the following steps:
(a) fixing: sample to be checked to be put into fixed solution and be fixed;
(b) hybridization: peptide nucleic acid probe is contacted in hybridization solution, peptide nucleic acid probe and the target sequence hybridization that is present in the microorganism in sample to be checked with sample to be checked;
(c) washing: the sample after hybridization is cleaned;
(d) strain identification result viewing;
(e) cell of logarithmic phase growth shaking culture in the M-H liquid of setting antibacterials concentration;
(f) sampling is fixed, hybridizes, is washed, observation counting fluorescent value;
(g) counting fluorescence ratio, determines drug susceptibility.
Wherein, washing step is that the washing soln forming with Tris alkali, NaCl and Triton cleans the sample after hybridizing.
Described sample to be checked comes from blood, air, water, food, examination of living tissue and other medical inspection.
Wherein, described results of hybridization presents by fluorescence.
Fixed solution in the present invention is owing to being used paraformaldehyde and ethanol fixed cell, and fixed effect is better.
In hybridization solution of the present invention, add a certain proportion of methane amide, can make the sensitivity of hybridization higher, Triton can increase the penetrance of cytolemma.
The present invention is also provided for the purposes of fluorescence in situ hybridization detection enterococcus faecalis and/or other enterococcal peptide nucleic acid probe groups, and described peptide nucleic acid probe group is for sample enterococcus faecalis and/or other enterococcal detections.
The present invention is also provided for detecting the purposes of the test kit of enterococcus faecalis and/or other faecalis bacterial strains and drug susceptibility, and described test kit is for the detection of biological sample enterococcus faecalis and/or other enterococcal evaluations and drug susceptibility thereof.
The present invention is also provided for detecting the purposes of using method of the test kit of enterococcus faecalis and/or other faecalis bacterial strains and drug susceptibility, and described using method is for the detection of sample enterococcus faecalis and/or other enterococcal evaluations and drug susceptibility thereof.
PNA probe of the present invention has good specificity, and susceptibility is very high, and 1 or 2 relevant nucleotide sequences of different IPs thuja acid can distinguish well.
The PNA probe of describing in the present invention can detect rRNA, corresponding to the genome sequence (rDNA) of rRNA, or its complementary sequence, the specific detection of permission target species.
The nucleotide sequence of the PNA probe of describing is in the present invention selected from and at least 86% structure, is similar to sequence in table 1:
Table 1 probe sequence
| Species | Title | Characteristic sequence |
| Enterococcus faecalis | SEQ?NO：1 | 5’-TTA?TCC?CCC?TCT?GAT?GGG-3’ |
| Other faecalis | SEQ?NO：2 | 5’-TAC?TGA?TCG?GCA?GCT-3’ |
First carry out the exploitation of PNA-FISH probe, owing to not understanding the suitable base quantity of probe, therefore need first test for the desirable base number of probe application.A large amount of Nucleotide within probe allows the high probe affinity of corresponding target spot, under high temperature, working very much, and oligonucleotide number hint occurs not enough in conjunction with free energy for hybridization.In the case, find 12～18 Nucleotide can reach optimum.
For each particular case, the best experiment of research FISH (fluorescent in situ hybridization) condition, because probe hybridization successfully depends on hybridization conditions, and depends on fixing/infiltrationization and washing step.First the condition for detection of bacterium of mentioning in disclosed documents and materials before considering.Fix/infiltrationization of prediction can the mode identical with previous description be carried out, that is, and and with 4% paraformaldehyde and 50% ethanol.Therefore, the parameter of main research is temperature, methane amide concentration and hybridization and washing time, and due to from these conditions of probe before, characteristic can not be extrapolated to this new probe.
Because must not optimize these parameters in the situation that knowing which factor affects PNA-FISH method negatively simultaneously, this process is complicated and consuming time.In the disclosed document of other investigators, mention, probe is up to now all because of inefficiency, and makes them be difficult to be applied to PNA-FISH method.
In this case, Best Times, temperature and methane amide concentration are accredited as the washing step in 55 ℃ and the hybridization of lower 60 minutes of 30% methane amide and 30 minutes.
Good successfully hybridization can detect specified microorganisms and quantity thereof by fluorescent microscope afterwards.
To this, consider that the PNA probe fraction of the applicable stable probe/target mixture that detects/identify is also important.The detectable fraction of probe is selected from one of following group: conjugate, chromophore, fluorophore, radio isotope, enzyme, haptens or luminophor.
The method of describing in the present invention comprises contacting of sample and PNA probe.According to method, about the hybridization of PNA sequence and target sequence under the suitable hybridization conditions of being associated in of they detect or characterization of biological sample among microorganism.Thereby the uniqueness of analyzing the judgement based on fluorescence, form and quantity is tested.On the contrary, current many phenotypic characteristics based on relating to several tests for the traditional method of analyzing microorganism and drug susceptibility thereof.
Theme of the present invention also comprises and is applicable to carrying out for measuring, that is, detect or characterization of biological sample among the test kit of enterococcus faecalis (Enterococcus faecalis) and/or other faecalis (Enterococcus) and drug susceptibility thereof.Test kit comprises two kinds of PNA probes, carries out reagent or the compound of in situ hybridization and needed other selections of drug susceptibility test.
In more preferred implementation method, be applicable to carrying out the test kit that enterococcus faecalis (Enterococcus faecalis) and/or other faecalis (Enterococcus) and drug susceptibility thereof detect or identify, comprise fixed solution, hybridization solution and drug susceptibility and detect solution.
Probe application of the present invention did not relate to reagent or the enzyme for membrane permeability before hybridization, and PNA probe can directly apply in slide specimen.
The term definition using in the present invention:
(a) as used herein, term " Nucleotide " comprises the natural compound of specific binding nucleic acid that produces thus with artificial molecule that uses that the people of the technology relevant to nucleic acid knows conventionally;
(b) term " nucleotide sequence " contains subunit with denotion, in this case the polymkeric substance of the compound of Nucleotide;
(c) nucleotide sequence that is intended to detect in test enterococcus faecalis (Enterococcus faecalis) and/or other faecalis (Enterococcus) censured in term " target sequence ", and wherein the part of nucleotide probe is designed to hybridization;
(d) polymkeric substance that has nucleotide sequence and be specific to the subunit of the PNA of hybridizing with the target sequence of object microorganism censured in term " PNA probe ".Pna molecule is the DNA analog that the achirality that formed by the N-by repeating (2-amino-ethyl) glycine unit of electronegative sugar-phosphate backbone structure and electric neutrality replace;
(e) term " detectable fraction " refers to be connected in probe and causes thus probe to pass through instrument or the detectable molecule of method;
(f) term " sample " refer to contain for detection of microorganism or any biological sample of target sequence, comprise blood, air, water, food, examination of living tissue and other medical inspection.
(g) term " drug susceptibility " is censured the minimum working concentration of certain antibiotics medicine and can be killed enterococcus faecalis (Enterococcus faecalis) and/or other faecalis (Enterococcus).
(h) conjugate: be different from that outside chromophore, fluorophore, radio isotope, enzyme, haptens or luminophor, other can be connected in probe and cause thus probe to pass through instrument or the detectable molecule of method.
PNA probe concept of the present invention
PNA probe groups of the present invention is selected as the target sequence with enterococcus faecalis (Enterococcus faecalis) and/or other faecalis (Enterococcus) kind feature in conserved regions responsible and that microbial species symbolic animal of the birth year is closed.Thus, the 16S rRNA sequence of the enterococcus spp of each database (Enterococcus) is compared, the variation zone design after sequence streptococcus aureus and staphylococcus epidermidis specific probe, probe sequence is in Table 1.
PNA probe of the present invention comprises 15 nucleotide sequences.According to these standards, select to be similar at least 86% structure the sequence of SEQ NO:1, SEQ NO:2, although this probe be described for detection of enterococcus faecalis in biological sample (Enterococcus faecalis) and/or other faecalis (Enterococcus) with and drug susceptibility, they must be only not specific for this situation.
5’-TTA?TCC?CCC?TCT?GAT?GGG-3’(SEQ?NO：1)
5’-TAC?TGA?TCG?GCA?GCT-3’(SEQ?NO：2)
Alternatively, the variation in probe nucleotide sequence is also contained in the present invention.This variation can especially comprise disappearance, insert.Thereby, as described in, probe nucleotide sequence should at least 86% with coming from above-mentioned sequence.
The detectable fraction of PNA probe
Be not limited to the following example, the detectable fraction of PNA probe can comprise various types of molecules, especially such as the dextran of puting together, chromophore, fluorophore, radio isotope, enzyme, haptens, chemiluminescence compound.
As an example, among fluorophore class, can preferably use (but being not limited to): Alexa Fluor series, cyanin, 5-(and-6) carboxyl-2 ', 7 '-dichlorofluorescein, 5-ROX (5-carboxyl-X-rhodamine, triethyl ammonium salt).
Method
The invention discloses for detection of enterococcus faecalis in sample (Enterococcus faecalis) and/or other faecalis (Enterococcus) with and the method for drug susceptibility.The PNA probe using comprises the nucleotide sequence that is similar to SEQ NO:1 and/or SEQ NO:2 at least 86% structure.
Method can contain between the sample that makes to have one or more PNA probes of describing in this document and bacterium target sequence in hybridization conditions fully or under in situ hybridization condition, contact (as seen in embodiment) fully.Fluorescence in situ hybridization (FISH or PNA-FISH) or PCR in real time are the test forms for enterococcus faecalis (Enterococcus faecalis) and/or other faecalis (Enterococcus) and medicament sensitivity analysis thereof.
Thus, method can be divided into: sample preparation, and medicament sensitivity test, cell is fixed, hybridization, washing and result visualization.This method can be carried out in the cell adhering to or suspend.
Hybridization conditions
Several factors of controlling target sequence and PNA probe hybridization stringency, these factors comprise: the pH value of per-cent, salt concn and the ionic strength of the methane amide of use (or other chemical denaturants), hybridization temperature, detergent concentration, hybridization buffer and other.In order to measure hybridization top condition, the essential fixing different factors, and change individually each factor, until reach the degree of distinguishing of expectation.
Another non-target sequence in target sequence and sample is more approaching, and the various factors that limits impact hybridization needs strictly to control greatly.Non-target sequence can have only 1 different IPs thuja acid with target sequence in the present invention, and the difference of level is essential to avoid the nonspecific hybridization of PNA probe and non-target sequence like this.
Sample preparation
Sample to be analyzed can especially obtain from blood, air, water, food and medical inspection.In enterococcus faecalis (Enterococcus faecalis) and/or other faecalis (Enterococcus) detection case, it is the form with suspension.In water and air sample, sample is filtered by black polycarbonate film or suitable body, then liquid culture; Blood and medical inspection sample directly carry out liquid culture; For food samples, sample is immersed into water or in aseptic buffer solution after in bacterium paddle blender, smash to pieces, separation and Culture.Culture is made suspension and is directly used in crossover process.
Test kit
The present invention also relates to allow to detect the test kit of enterococcus faecalis (Enterococcus faecalis) in sample and/or other faecalis (Enterococcus) and drug susceptibility thereof.Herein, mentioned the PNA probe using in this test kit, its feature and the method relating to.
Test kit of the present invention comprises the nucleotide sequence that is similar to SEQ NO:1 and 2 at least 86% structure, and selects other reagent or composition for testing.
PNA probe of the present invention, their feature, method and test kit are suitable for analyzing deposits the nucleotide sequence being in or be not within object biomass cells in object biomass cells.The present invention can be used for biological analysis or from the analysis of nucleic acid object biological extraction or source, shows not limit target sequence source of the present invention.
Operation steps
Bacterial strain
Bacterial strain is the reference strain that clinical isolates strain and American type culture collection (ATCC) obtain.Each strain culture is added to slide glass, is placed in 55 ℃ and is dried.
Fixing
In order to prevent the loss during 16S rRNA hybridization, sample is immersed in to 4% paraformaldehyde (wt/vol) and 50% ethanol (vol/vol) solution respectively 10 minutes.
Hybridization
In this step, by one, comprise 10% (wt/vol) T 500,10mM NaCl, 50% (v/v) methane amide, 0.1% (wt/vol) trisodium phosphate, 0.2% (wt/vol) polyvinyl pyrroline, 0.2% (wt/vol) FICOLL, 5mM EDETATE SODIUM, 0.1% (vol/vol) Triton X-100, the hybridization solution of the PNA probe of 50mM Tris-HCl and 200nM adds sample.Sample covers to guarantee probe expansion with cover glass, and incubation 60 minutes.During this, probe can enter cytolemma, and is incorporated into 16S rRNA complementary sequence.The existence of cover glass and sample l Water Paper is around for preventing that hybridization solution evaporation from being necessary.
Washing
After hybridization, remove cover glass, and by slide glass immerse pre-heating by 5mM Tris alkali, the washing soln that 15mM NaCl and 1% (vol/vol) Triton X-100 (pH10) forms.Washing step carries out 30 minutes at identical hybridization temperature.
Medicament sensitivity test
In this test, the bacterium in logarithmic phase is adjusted to concentration to 5 * 10
5cfu/mL cultivates 2 hours in 35 ℃ of concussions in the M-H liquid of setting antibiolics substrate concentration, and sampling is fixing, hybridization, washing and fluorescence counting, calculates fluorescence ratio.
Result
Bacteria Identification result by the spectral filter the fluorochrome signal being applicable within probe is housed (, it is coupled to the emission wavelength of the fluorochrome of probe) fluorescent microscope in observe to obtain, green fluorescence represents enterococcus faecalis, and red fluorescence represents other faecalis; Bacterium drug susceptibility is determined by the scope of fluorescence ratio, sensitivity: fluorescence ratio≤0.75, intermediary: 0.75 < fluorescence ratio < 1.4, resistance: fluorescence ratio >=1.4.
The susceptibility of probe and specificity theoretical validation
In order to verify susceptibility and the specificity of probe, with ProbeCheck and ncbi database, probe is verified.The result of ProbeCheck shows: probe SEQ NO:1 can detect 358 in 360 target enterococcus faecalis all in database, has 2 because of mononucleotide difference, may hunt leak.And the non-target bacteria that other detect is all become estranged with enterococcus faecalis relation, also and non-pathogenic bacteria (table 2).Subsequently PNA probe and large subunit (23S/28S) database matching are detected, find that probe SEQ NO:1 does not exist mispairing with 23SrRNA; Probe SEQ NO:2 can detect 721 in 796 other faecalis of target all in database, has 75 because of single or multiple nucleotide differences, may hunt leak.And the non-target bacteria that other detect is all become estranged with faecalis relation, morphological differences is large, also and non-pathogenic bacteria (table 2).Subsequently PNA probe and large subunit (23S/28S) database matching are detected, find that probe SEQ NO:2 does not exist mispairing with 23S rRNA.
Through ProbeCheck checking, probe of the present invention has very high sensitivity and specificity.
Table 2 ProbeCheck detects susceptibility and the specificity of PNA probe
All non-object bacteria bacterial strain numbers in the non-object bacteria bacterial strain number/database of specificity=do not detect;
All object bacteria bacterial strain numbers in the object bacteria bacterial strain number/database of susceptibility=detect.
The present invention adopts PNA probe in conjunction with Fluorescence in situ hybridization (FISH) technology, with traditional biochemical identification comparison, the present invention does not need bacterium to carry out DNA extraction, does not need membrane permeability, can directly to various samples such as blood, air, water, food, living tissues, detect.Therefore the present invention can save the plenty of time, saves loaded down with trivial details steps such as extracting DNA, and the used time is few, and efficiency is high, if disregard bacterial growth repoductive time, general is consuming timely no more than 2 hours.And result judgement of the present invention detects two portions based on fluoroscopic examination and form, susceptibility is high, high specificity, compared with traditional method false positives such as PCR methods, greatly reduces.The present invention can detect enterococcus faecalis and other faecalis quickly and accurately, for ensuring food safety, guarantee that environmental health, preventing disease propagate significant.
Embodiment
Below in conjunction with embodiment, the present invention is further detailed explanation.
Embodiment 1, the checking of PNA-FISH susceptibility
PNA probe:
Alexa488-O-TTA?TCC?CCC?TCT?GAT?GGG(SEQ?NO：1)
Alexa546-O-TAC?TGA?TCG?GCA?GCT(SEQ?NO：2)
The enterococcus faecalis of different subspecies and other faecalis are carried out to susceptibility checking.
Experimental procedure is as described below.
Bacterial strain
Bacterial strain is the reference strain that clinical isolates strain and American type culture collection (ATCC) obtain.Each strain culture is added to slide glass, is placed in 55 ℃ and is dried.
Fixing
In order to prevent the loss during 16S rRNA hybridization, sample is immersed in to 4% paraformaldehyde (wt/vol) and 50% ethanol (vol/vol) solution respectively 10 minutes.
Hybridization
In this step, by one, comprise 10% (wt/vol) T 500,10mM NaCl, 50% (v/v) methane amide, 0.1% (wt/vol) trisodium phosphate, 0.2% (wt/vol) polyvinyl pyrroline, 0.2% (wt/vol) FICOLL, 5mM EDETATE SODIUM, 0.1% (vol/vol) Triton X-100, the hybridization solution of the PNA probe of 50mM Tris-HCl and 200nM adds sample.Sample covers to guarantee probe expansion with cover glass, and incubation 60 minutes.During this, probe can enter cytolemma, and is incorporated into 16S rRNA complementary sequence.The existence of cover glass and sample l Water Paper is around for preventing that hybridization solution evaporation from being necessary.
Washing
After hybridization, remove cover glass, and by slide glass immerse pre-heating by 5mM Tris alkali, the washing soln that 15mM NaCl and 1% (vol/vol) Triton X-100 (pH10) forms.Washing step carries out 30 minutes at identical hybridization temperature.
Medicament sensitivity test
In this test, the bacterium in logarithmic phase is adjusted to concentration to 5 * 10
5cfu/mL cultivates 2 hours in 35 ℃ of concussions in the M-H liquid of setting antibiolics substrate concentration, and sampling is fixing, hybridization, washing and fluorescence counting, calculates fluorescence ratio.
Result
Bacteria Identification result by the spectral filter the fluorochrome signal being applicable within probe is housed (, it is coupled to the emission wavelength of the fluorochrome of probe) fluorescent microscope in observe to obtain, green fluorescence represents Pseudomonas aeruginosa, red fluorescence represents Klebsiella pneumonia, and blue-fluorescence represents Acinetobacter bauamnnii; Bacterium drug susceptibility is determined by the scope of fluorescence ratio, sensitivity: fluorescence ratio≤0.75, intermediary: 0.75 < fluorescence ratio < 1.4, resistance: fluorescence ratio >=1.4.
Positive control and negative control test are all synchronously carried out in each experiment (comprising following instance), and positive control test substitutes other probes with DAPI dyeing, and in negative control test, with blank other probes that substitute.Result demonstration, all enterococcus faecalis are combined with probe SEQ NO:1; Other all faecalis are combined (table 3,4) with probe SEQ NO:2.Result is consistent with expection, and SEQ NO:1 and 2 has good susceptibility.
The sensitiveness test of table 3 enterococcus faecalis PNA probe
Note:---for separated in hospital, food, environment, without strain number, describe.
Other faecalis PNA probe sensitiveness tests of table 4
Note:---for separated in hospital, food, environment, without strain number, describe.
The checking of embodiment 2PNA-FISH specificity
Choose enterococcus faecalis, faecium and other pathogenic bacterium, comprise the common pathogens such as Pseudomonas aeruginosa, streptococcus aureus, staphylococcus epidermidis, primary gram of bacterium of kerekou pneumonia and Acinetobacter bauamnnii, testing sequence and method are with described in embodiment 1.
Result shows to only have positive control (DAPI) and all bacteriums can be in conjunction with, and probe SEQ NO:1 and 2 only can be combined with aimed strain (table 5).Consistent with expected results, SEQ NO:1 and 2 has good specificity.
The checking of table 5 PNA probe specificity
| Species | Numbering | DAPI | SEQ?NO：1 | SEQ?NO：2 |
| Enterococcus?faecalis | ATCC29212 | + | + | - |
| Enterococcus?faecium | ATCC35667 | + | - | + |
| Enterococcus?avium | —— | + | - | + |
| Enterococcus?durans | —— | + | - | + |
| Pseudomonas?aeruginosa | —— | + | - | - |
| Escherichia?coli | —— | + | - | - |
| Staphylococcus?aureus | ATCC29213 | + | - | - |
| Staphylococcus?epidermidis | ATCC12228 | + | - | - |
| Klebsiella?pneumoniae | —— | + | - | - |
| Acinetobacter?baumannii | —— | + | - | - |
| Streptococcus?pneumoniae | —— | + | - | - |
| Streptococcus?pyogenes | —— | + | - | - |
| Enterobacter?cloacae | —— | + | - | - |
| Streptococcus?viridans | —— | + | - | - |
Note:---for separated in hospital, food, environment, without strain number, describe.
Embodiment 3 medicament sensitivity tests
Choose the Quality Control bacterial strain of faecalis number and carry out medicament sensitivity test with the separated different pharmaceutical susceptibility enterococcus faecalis obtaining and other faecalis bacterial strains in hospital, food, environment, testing sequence and method are with described in embodiment 1, and each test is usingd K-B method as positive control simultaneously.The results are shown in Table 6.
Result shows that the present invention can detect the drug susceptibility of aimed strain preferably.
1table 6 faecalis medicament sensitivity test
Note: A: fluorescence ratio method; B:K-B method; S: sensitivity; I: intermediary; R: resistance
2the present invention adopts PNA probe in conjunction with Fluorescence in situ hybridization (FISH) technology, with traditional biochemical identification comparison, do not need bacterium to carry out DNA extraction, do not need membrane permeability, can directly to various samples such as blood, air, water, food, living tissues, detect.Therefore the present invention can save the plenty of time, saves loaded down with trivial details steps such as extracting DNA, if disregard bacterial growth repoductive time, general is consuming timely no more than 2 hours.Result judgement of the present invention detects two portions based on fluoroscopic examination and form, compared with traditional method false positives such as PCR methods, greatly reduces.
Above-described is only the preferred embodiment of the present invention, it should be pointed out that for the person of ordinary skill of the art, without departing from the concept of the premise of the invention, can also make some distortion and improvement, and these all belong to protection scope of the present invention.
Claims (15)
1. for fluorescence in situ hybridization detection enterococcus faecalis and/or other enterococcal peptide nucleic acid probe groups, it is characterized in that: the DNA sequence dna of described peptide nucleic acid probe group is as shown in SEQ NO:1, SEQ NO:2;
Wherein, described SEQ NO:1 sequence is: 5 '-TTA TCC CCC TCT GAT GGG-3 ', for detection of enterococcus faecalis;
Wherein, described SEQ NO:2 sequence is: 5 '-TAC TGA TCG GCA GCT-3 ', for detection of other faecalis;
Wherein, described SEQ NO:1 sequence or SEQ NO:2 sequence can be used respectively, are used for detecting respectively enterococcus faecalis or other faecalis; Also can use simultaneously, be used for detecting enterococcus faecalis and other faecalis simultaneously.
2. as claimed in claim 1 for fluorescence in situ hybridization detection enterococcus faecalis and/or other enterococcal peptide nucleic acid probe groups, it is characterized in that, described peptide nucleic acid probe group can detect target sequence in the rRNA sequence of enterococcus faecalis and/or other enterococcal rRNA, rDNA or complementation.
3. as claimed in claim 1 for fluorescence in situ hybridization detection enterococcus faecalis and/or other enterococcal peptide nucleic acid probe groups, it is characterized in that, the probe that detects enterococcus faecalis in described peptide nucleic acid probe group should at least comprise the DNA sequence dna that is similar to SEQ NO:1 in 86% structure; In described peptide nucleic acid probe group, detect other enterococcal probes and should at least comprise the DNA sequence dna that is similar to SEQ NO:2 in 86% structure.
4. as described in as arbitrary in claim 1～3 for fluorescence in situ hybridization detection enterococcus faecalis and/or other enterococcal peptide nucleic acid probe groups, it is characterized in that, described peptide nucleic acid probe group is at least connected to a kind of detectable fraction.
5. as claimed in claim 4 for fluorescence in situ hybridization detection enterococcus faecalis and/or other enterococcal peptide nucleic acid probe groups, it is characterized in that, the type of described detectable fraction is selected from: conjugate, chromophore, fluorophore, radio isotope, enzyme, haptens or luminophor.
6. as claimed in claim 5 for fluorescence in situ hybridization detection enterococcus faecalis and/or other enterococcal peptide nucleic acid probe groups, it is characterized in that, described fluorophore group is at least one in following: fluorophore Alexa series, Alexa Fluor series, cyanin, 5-(and-6) carboxyl-2 ', 7 '-dichlorofluorescein, 5-ROX.
7. for detection of the test kit of enterococcus faecalis and/or other faecalis bacterial strains and drug susceptibility, it is characterized in that the probe that described test kit comprises any one in claim 1～6.
8. test kit as claimed in claim 7, is characterized in that, described test kit also comprises following solution: fixed solution, hybridization solution and drug susceptibility detect solution;
Wherein, fixed solution comprises paraformaldehyde and ethanol;
Wherein, hybridization solution comprises Triton and methane amide;
Wherein, drug susceptibility detection solution is M-H liquid.
9. test kit as claimed in claim 8, is characterized in that, described fixed solution comprises 2～8% paraformaldehyde and 25～75% ethanol; Composition and the composition of described hybridization solution are: 10% T 500,10mM NaCl, 50% methane amide, 0.1% trisodium phosphate, 0.2% polyvinyl pyrroline, 0.2%FICOLL, 5mM EDETATE SODIUM, 0.1%Triton X-100,50mM Tris-HCl.
10. for detection of the using method of the test kit of enterococcus faecalis and/or other faecalis bacterial strains and drug susceptibility, it is characterized in that, described using method comprises the following steps:
(a) fixing: sample to be checked to be put into fixed solution and be fixed;
(b) hybridization: peptide nucleic acid probe is contacted in hybridization solution, peptide nucleic acid probe and the target sequence hybridization that is present in the microorganism in sample to be checked with sample to be checked;
(c) washing: the sample after hybridization is cleaned;
(d) strain identification result viewing;
(e) cell of logarithmic phase growth shaking culture in the M-H liquid of setting antibacterials concentration;
(f) sampling is fixed, hybridizes, is washed, observation counting fluorescent value;
(g) counting fluorescence ratio, determines drug susceptibility.
11. using method as claimed in claim 10, is characterized in that, described sample to be checked comes from blood, air, water, food, examination of living tissue and other medical inspection.
12. using method as claimed in claim 11, is characterized in that, its results of hybridization presents by fluorescence.
13. purposes for fluorescence in situ hybridization detection enterococcus faecalis and/or other enterococcal peptide nucleic acid probe groups as described in as arbitrary in claim 1～6, is characterized in that, described peptide nucleic acid probe group is for sample enterococcus faecalis and/or other enterococcal detections.
The purposes of 14. test kits for detection of enterococcus faecalis and/or other faecalis bacterial strains and drug susceptibility as described in as arbitrary in claim 7～9, it is characterized in that, described test kit is for the detection of biological sample enterococcus faecalis and/or other enterococcal evaluations and drug susceptibility thereof.
The purposes of 15. using method as described in as arbitrary in claim 10～12, is characterized in that, described using method is for the detection of sample enterococcus faecalis and/or other enterococcal evaluations and drug susceptibility thereof.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105063188A (en) * | 2015-07-29 | 2015-11-18 | 广州华银医学检验中心有限公司 | Fluorescence in situ hybridization detection kit for detecting mycobacterium tuberculosis infection |
| CN113025737A (en) * | 2021-05-10 | 2021-06-25 | 临沂大学 | Microdroplet digital PCR detection method for enterococcus faecalis in medical food |
| US11834703B2 (en) | 2008-05-27 | 2023-12-05 | Agilent Technologies, Inc. | Hybridization compositions and methods |
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| NL2022147B1 (en) * | 2018-12-06 | 2020-07-02 | Biotrack Holding B V | Methods for detecting cell wall-deficient bacteria (CWDB) in clinical samples. |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP0957175A1 (en) * | 1998-04-20 | 1999-11-17 | Academisch Ziekenhuis Groningen | Method for the rapid determination of bacteria |
-
2014
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| EP0957175A1 (en) * | 1998-04-20 | 1999-11-17 | Academisch Ziekenhuis Groningen | Method for the rapid determination of bacteria |
Non-Patent Citations (1)
| Title |
|---|
| G. J. JANSEN ET AL.: "Rapid identification of bacteria in blood cultures by using fluorescently labeled oligonucleotide probes", 《JOUNAL OF CLINICAL MICROBIOLOGY》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11834703B2 (en) | 2008-05-27 | 2023-12-05 | Agilent Technologies, Inc. | Hybridization compositions and methods |
| CN105063188A (en) * | 2015-07-29 | 2015-11-18 | 广州华银医学检验中心有限公司 | Fluorescence in situ hybridization detection kit for detecting mycobacterium tuberculosis infection |
| CN105063188B (en) * | 2015-07-29 | 2016-08-24 | 广州华银医学检验中心有限公司 | The fluorescence in situ hybridization detection test kit of mycobacterium tuberculosis infection detection |
| CN113025737A (en) * | 2021-05-10 | 2021-06-25 | 临沂大学 | Microdroplet digital PCR detection method for enterococcus faecalis in medical food |
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