CN104031129A - Peptide nucleic acid probe set and kit used for detecting staphylococcus aureus and/or staphylococcus epidermidis, use method of kit and application of kit - Google Patents
Peptide nucleic acid probe set and kit used for detecting staphylococcus aureus and/or staphylococcus epidermidis, use method of kit and application of kit Download PDFInfo
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- CN104031129A CN104031129A CN201410320018.XA CN201410320018A CN104031129A CN 104031129 A CN104031129 A CN 104031129A CN 201410320018 A CN201410320018 A CN 201410320018A CN 104031129 A CN104031129 A CN 104031129A
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- staphylococcus epidermidis
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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
Abstract
The invention relates to peptide nucleic acid probes used for rapidly detecting staphylococcus aureus and/or staphylococcus epidermidis through fluorescence in situ hybridization. The DNA sequences of the peptide nucleic acid probes are shown in SEQ NO: 1 and SEQ NO: 2. The invention further discloses a kit for detecting staphylococcus aureus and/or staphylococcus epidermidis and drug susceptibility of staphylococcus aureus and/or staphylococcus epidermidis in samples through the probes, and use steps of the kit. The probes and the kit can be used for detecting staphylococcus aureus and/or staphylococcus epidermidis and drug susceptibility of staphylococcus aureus and/or staphylococcus epidermidis. The DNA extraction step is omitted, and the whole identification process generally consumes at most two hours; meanwhile, the false positive is low, a few steps are needed, and efficiency is high. The staphylococcus aureus and staphylococcus epidermidis can be rapidly and accurately detected, and the peptide nucleic acid probes and the kit are of great importance in ensuring food safety, ensuring environmental health and preventing spreading of diseases.
Description
Technical field
The present invention relates to the detection field of Clinical Correlation microorganism, be specifically related to detect the peptide nucleic acid probe group of streptococcus aureus and/or staphylococcus epidermidis.
Background technology
Staphylococcus comprises that most is non-pathogenic bacteria, minority can cause disease, and streptococcus aureus (Staphylococcus aureus) and staphylococcus epidermidis (Staphylococcus epidermidis) are two kinds of Main Pathogenic Bacterias of Staphylococcus.The streptococcus aureus (Staphylococcus aureus) of 60%-70% can be by corresponding phage splitting, and (Staphylococcus epidermidis) is insensitive for staphylococcus epidermidis.Staphylococcus (Staphylococcus) is a group gram-positive cocci, because normal heap is polymerized to thyrsiform, therefore named.But in pus, milk, liquid nutrient medium, being two balls or short chain arranges.
Streptococcus aureus (Staphylococcus aureus) is a kind of important pathogen of the mankind, its infection causing is only second to intestinal bacteria and accounts for second, can cause localization pyogenic infection, also can cause pneumonia, pseudomembranous enteritis, pericarditis etc., the even systemic infection such as septicemia, Sepsis.Streptococcus aureus (Staphylococcus aureus) virulence power depends primarily on toxin and the aggressive enzyme of its generation.(Staphylococcus aureus) is ubiquitous at occurring in nature for streptococcus aureus, in the movement of air, water, dust and humans and animals, can find, and food is very easily subject to its pollution.Streptococcus aureus (Staphylococcus aureus) enterotoxin is a worldwide health difficult problem, the food poisoning causing at U.S. streptococcus aureus (Staphylococcus aureus) enterotoxin, account for 33% of whole bacterial food poisoning event, Canada is more, account for 45%, the food poisoning that Chinese streptococcus aureus (Staphylococcus aureus) causes also happens occasionally.Microbiotic is widely used hospital, makes the resistance of streptococcus aureus (Staphylococcus aureus) more and more stronger, and nearly all conventional antibiotics resistance bacterial strain can be separated to.Particularly resistance streptococcus aureus (Staphylococcus aureus), can resist all penicillin, comprises the penicillin of X-1497 and other anti-beta lactamases, only to vancomycin and teicoplanin sensitivity.
Staphylococcus epidermidis (Staphylococcus epidermidis) is the conditioned pathogen that is extensively present in skin surface, along with antibiotic widespread use, the progress of medical skill, this bacterium has become immune deficiency person's nosocomial infection common pathogen of (comprising that wound infection and intubate infect), and its trend that causes septicemia, chronic prostatitis all increased with day.The resistance phenomenon of staphylococcus epidermidis is very serious, and its penicillin to current widespread use of bibliographical information, Ampicillin Trihydrate, Oxazacillin, erythromycin etc. have very high resistance.
Peptide nucleic acid(PNA) (peptide nucleic acids, PNA) be a kind of brand-new DNA analog taking neutral amido linkage as skeleton by people's designs such as Denmark scientist Nielsen in 1991, 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, compared 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 staphylococcus 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 one and do not need to extract DNA, do not need pcr amplification can detect the peptide nucleic acid probe group of streptococcus aureus and/or staphylococcus epidermidis and test kit thereof, method, efficient quick, false positive is low.
For achieving the above object, technical scheme of the present invention is: for the peptide nucleic acid probe group of fluorescence in situ hybridization detection streptococcus aureus and/or staphylococcus epidermidis, 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 '-GCT TCT CGT CCG TTC-3 ', for detection of streptococcus aureus;
Wherein, described SEQ NO:2 sequence is: 5 '-AAT ATA TTA TCC GGT-3 ', for detection of staphylococcus epidermidis;
Wherein, described SEQ NO:1 sequence or SEQ NO:2 sequence can be used respectively, are used for detecting respectively streptococcus aureus or staphylococcus epidermidis; Also can use simultaneously, be used for detecting streptococcus aureus and staphylococcus epidermidis simultaneously.
Described peptide nucleic acid probe group can detect target sequence in the rRNA sequence of the rRNA of streptococcus aureus and/or staphylococcus epidermidis, rDNA or complementation.
The probe that detects streptococcus aureus 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; The probe that detects staphylococcus epidermidis in described peptide nucleic acid probe group 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 streptococcus aureus and/or staphylococcus epidermidis bacterial strain and drug susceptibility, the probe that described test kit comprises any one in claim 1～6.
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.
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 the using method of the test kit that detects streptococcus aureus and/or staphylococcus epidermidis bacterial strain 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 with the washing soln of Tris alkali, NaCl and Triton composition, the sample after hybridizing to be cleaned.
Described sample to be checked comes from blood, air, water, food, examination of living tissue and other medical inspection.
Described results of hybridization presents by fluorescence.
Fixed solution in the present invention is owing to using 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 the peptide nucleic acid probe group of fluorescence in situ hybridization detection streptococcus aureus and/or staphylococcus epidermidis, and described peptide nucleic acid probe group is for the detection of Gold Samples staphylococcus aureus and/or staphylococcus epidermidis.
The present invention is also provided for the purposes of the test kit that detects streptococcus aureus and/or staphylococcus epidermidis bacterial strain and drug susceptibility, and described test kit is for biological sample streptococcus aureus and/or the qualification of staphylococcus epidermidis and the detection of drug susceptibility thereof.
The present invention is also provided for the purposes of the using method of the test kit that detects streptococcus aureus and/or staphylococcus epidermidis bacterial strain and drug susceptibility, and described using method is for Gold Samples staphylococcus aureus and/or the qualification of staphylococcus epidermidis and the detection of 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 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 |
| Streptococcus aureus | SEQ?NO：1 | 5’-GCT?TCT?CGT?CCG?TTC-3’ |
| Staphylococcus epidermidis | SEQ?NO：2 | 5’-AAT?ATA?TTA?TCC?GGT-3’ |
First carry out the exploitation of PNA-FISH probe, owing to not understanding the suitable base quantity of probe, therefore need first to test the base number desirable for probe application.A large amount of Nucleotide within probe allows the high probe affinity of corresponding target spot, and at unusual hot operation, 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.Prediction is fixing/and mode that infiltrationization can be identical with previous description carries out, that is, 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 optimize these parameters in the situation that not 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 DEG C 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 stable probe/target mixture that is applicable to detect/qualification 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,, the test kit of streptococcus aureus (Staphylococcus aureus) and/or staphylococcus epidermidis (Staphylococcus epidermidis) and drug susceptibility thereof among detection or characterization of biological sample.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 that streptococcus aureus (Staphylococcus aureus) and/or staphylococcus epidermidis (Staphylococcus epidermidis) and drug susceptibility thereof detect or the test kit of qualification, comprise fixing, hybridization, washing soln 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 that produces thus specific binding nucleic acid with artificial molecule that uses the people of the technology relevant to nucleic acid conventionally to know;
(b) term " nucleotide sequence " contains subunit, the polymkeric substance of the compound of Nucleotide in this case with denotion;
(c) nucleotide sequence that is intended to detect in test streptococcus aureus (Staphylococcus aureus) and/or staphylococcus epidermidis (Staphylococcus epidermidis) 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 " censure can 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 streptococcus aureus (Staphylococcus aureus) and/or staphylococcus epidermidis (Staphylococcus epidermidis).
(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 streptococcus aureus (Staphylococcus aureus) and/or staphylococcus epidermidis (Staphylococcus epidermidis) 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 staphylococcus of each database (Staphylococcus) 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 streptococcus aureus in biological sample (Staphylococcus aureus) and/or staphylococcus epidermidis (Staphylococcus epidermidis) with and drug susceptibility, they needn't be only specific for this situation.
5’-GCT?TCT?CGT?CCG?TTC-3’(SEQ?NO：1)
5’-AAT?ATA?TTA?TCC?GGT-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 Gold Samples staphylococcus aureus (Staphylococcus aureus) and/or staphylococcus epidermidis (Staphylococcus epidermidis) with and the method for drug susceptibility.The PNA probe using comprises the nucleotide sequence that is similar to SEQ NO:1 and 2 at least 86% structure.
Method can contain to be made to have between the sample of one or more PNA probes of describing in this document and bacterium target sequence in hybridization conditions fully or under in situ hybridization condition, is contacting (as seen in embodiment) fully.Fluorescence in situ hybridization (FISH or PNA-FISH) or PCR in real time are the test forms for streptococcus aureus (Staphylococcus aureus) and/or staphylococcus epidermidis (Staphylococcus epidermidis) 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 (or other chemical denaturants) of use, 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 with the nonspecific hybridization of avoiding 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 streptococcus aureus (Staphylococcus aureus) and/or staphylococcus epidermidis (Staphylococcus epidermidis) detection case, it is the form with suspension.In water and air sample, sample is filtered to then liquid culture by black polycarbonate film or suitable body; 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 Gold Samples staphylococcus aureus (Staphylococcus aureus) and/or staphylococcus epidermidis (Staphylococcus epidermidis) and drug susceptibility thereof.Herein, mention 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 in a kind of at least 86% structure, and selects other reagent or composition for testing.
PNA probe of the present invention, their feature, method and test kit is 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 the analysis from 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 DEG C 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 each 10 minutes.
Hybridization
In this step, comprise 10% (wt/vol) T 500 by one, 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 of 15mM NaCl and 1% (vol/vol) Triton X-100 (pH10) composition.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 DEG C 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 obtain, green fluorescence represents streptococcus aureus, and red fluorescence represents staphylococcus epidermidis; 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, probe is verified with ProbeCheck and ncbi database.The result of ProbeCheck shows: probe SEQ NO:1 can detect 469 in 483 target streptococcus aureuses all in database, has 14 may hunt leak because of mononucleotide difference.And the non-target bacteria that other detect is all become estranged with streptococcus aureus relation, be not common pathogenic bacterium (table 2) yet.Subsequently PNA probe and large subunit (23S/28S) database matching are detected, find that probe SEQ NO:1 does not exist mispairing with 23S rRNA; Probe SEQ NO:2 can detect 240 in 245 target staphylococcus epidermidiss all in database, has 5 may hunt leak because of mononucleotide difference.And the non-target bacteria that other detect is all become estranged with staphylococcus epidermidis relation, be not common pathogenic bacterium (table 2) yet.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 detect various samples such as blood, air, water, food, living tissues.Therefore the present invention can save the plenty of time, saves and extracts the loaded down with trivial details step such as 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 of the present invention judges that susceptibility is high, high specificity, greatly reduces compared with traditional method false positives such as PCR methods based on fluoroscopic examination and form detection two portions.The present invention can detect streptococcus aureus and staphylococcus epidermidis quickly and accurately, for ensuring food safety, ensure 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-GCT?TCT?CGT?CCG?TTC(SEQ?NO：1)
Alexa546-O-AAT?ATA?TTA?TCC?GGT(SEQ?NO：2)
Streptococcus aureus to different subspecies and staphylococcus epidermidis carry out 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 DEG C 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 each 10 minutes.
Hybridization
Comprise 10% (wt/vol) T 500 by one, 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) TritonX-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.
Washing
After hybridization, remove cover glass, and by slide glass immerse pre-heating by 5mM Tris alkali, the washing soln of 15mM NaCl and 1% (vol/vol) Triton X-100 (pH10) composition.Washing step carries out 30 minutes at identical hybridization temperature.
Medicament sensitivity test
Bacterium in logarithmic phase is adjusted to concentration to 5 × 10
5cfu/mL cultivates 2 hours in 35 DEG C 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 obtain, green fluorescence represents streptococcus aureus, and red fluorescence represents staphylococcus epidermidis; 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 test (comprising following instance), and positive control test, dyes and substitute other probes with DAPI, and in negative control test, with blank alternative other probes.Result demonstration, all streptococcus aureuses are combined with probe SEQ NO:1; All staphylococcus epidermidiss 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 streptococcus aureus PNA probe
| Species | Numbering | Results of hybridization |
[0127]?
| ? | ? | DAPI | SEQ?NO：1 | Negative group |
| Staphylococcus?aureus | ATCC25923 | + | + | - |
| Staphylococcus?aureus | ATCC29213 | + | + | - |
| Staphylococcus?aureus | A4 | + | + | - |
| Staphylococcus?aureus | —— | + | + | - |
| Staphylococcus?aureus | —— | + | + | - |
| Staphylococcus?aureus | —— | + | + | - |
| Staphylococcus?aureus | —— | + | + | - |
| Staphylococcus?aureus | —— | + | + | - |
| Staphylococcus?aureus | —— | + | + | - |
| Staphylococcus?aureus | —— | + | + | - |
| Staphylococcus?aureus | —— | + | + | - |
Note:---for hospital distributing from, describe without strain number.
The sensitiveness test of table 4 staphylococcus epidermidis PNA probe
Note:---for hospital distributing from, describe without strain number.
Embodiment 2 PNA-FISH specificity checkings
Choose streptococcus aureus, staphylococcus epidermidis and other pathogenic bacterium, comprise the common pathogens such as Pseudomonas aeruginosa, faecalis, 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 |
| Staphylococcus?aureus | ATCC25923 | + | + | - |
| Staphylococcus?aureus | ATCC29213 | + | + | - |
| Staphylococcus?epidermidis | ATCC12228 | + | - | + |
| Staphylococcus?epidermidis | —— | + | - | + |
| Pseudomonas?aeruginosa | —— | + | - | - |
| Escherichia?coli | —— | + | - | - |
| Enterococcus?Faecium | —— | + | - | - |
| Enterococcus?faecalis | —— | + | - | - |
| Klebsiella?pneumoniae | —— | + | - | - |
| Acinerobacter?baumannii | —— | + | - | - |
| Streptococcus?pneumoniae | —— | + | - | - |
| Streptococcus?pyogenes | —— | + | - | - |
| Enterobacter?cloacae | —— | + | - | - |
| Streptococcus?viridans | —— | + | - | - |
Note:---for hospital distributing from, describe without strain number.
Embodiment 3 medicament sensitivity tests
Quality Control bacterial strain and the hospital distributing of choosing streptococcus aureus and staphylococcus epidermidis carry out medicament sensitivity test from the different pharmaceutical susceptibility streptococcus aureus obtaining and staphylococcus epidermidis bacterial strain, as described in Example 1, each test is simultaneously using K-B method as positive control for testing sequence and method.The results are shown in Table 6,7.
Result shows that the present invention can detect the drug susceptibility of aimed strain preferably.
Table 6 streptococcus aureus medicament sensitivity test
Note: A: fluorescence ratio method; B:K-B method; S: sensitivity; I: intermediary; R: resistance
Table 7 staphylococcus epidermidis medicament sensitivity test
Note: A: fluorescence ratio method; B:K-B method; S: sensitivity; I: intermediary; R: resistance
The 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 detect various samples such as blood, air, water, food, living tissues.Therefore the present invention can save the plenty of time, saves and extracts the loaded down with trivial details step such as DNA, if disregard bacterial growth repoductive time, general is consuming timely no more than 2 hours.Result of the present invention judges based on fluoroscopic examination and form detection two portions, greatly reduces compared with traditional method false positives such as PCR methods.The present invention can detect streptococcus aureus and staphylococcus epidermidis quickly and accurately, for ensuring food safety, ensure that environmental health, preventing disease propagate significant.
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 the peptide nucleic acid probe group of fluorescence in situ hybridization detection streptococcus aureus and/or staphylococcus epidermidis, 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 '-GCT TCT CGT CCG TTC-3 ', for detection of streptococcus aureus;
Wherein, described SEQ NO:2 sequence is: 5 '-AAT ATA TTA TCC GGT-3 ', for detection of staphylococcus epidermidis;
Wherein, described SEQ NO:1 sequence or SEQ NO:2 sequence can be used respectively, are used for detecting respectively streptococcus aureus or staphylococcus epidermidis; Also can use simultaneously, be used for detecting streptococcus aureus and staphylococcus epidermidis simultaneously.
2. the peptide nucleic acid probe group for fluorescence in situ hybridization detection streptococcus aureus and/or staphylococcus epidermidis as claimed in claim 1, it is characterized in that, described peptide nucleic acid probe group can detect target sequence in the rRNA sequence of the rRNA of streptococcus aureus and/or staphylococcus epidermidis, rDNA or complementation.
3. the peptide nucleic acid probe group for fluorescence in situ hybridization detection streptococcus aureus and/or staphylococcus epidermidis as claimed in claim 1, it is characterized in that, the probe that detects streptococcus aureus 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; The probe that detects staphylococcus epidermidis in described peptide nucleic acid probe group should at least comprise the DNA sequence dna that is similar to SEQ NO:2 in 86% structure.
4. the peptide nucleic acid probe group for fluorescence in situ hybridization detection streptococcus aureus and/or staphylococcus epidermidis as described in as arbitrary in claim 1～3, is characterized in that, described peptide nucleic acid probe group is at least connected to a kind of detectable fraction.
5. the peptide nucleic acid probe group for fluorescence in situ hybridization detection streptococcus aureus and/or staphylococcus epidermidis as claimed in claim 4, it is characterized in that, the type of described detectable fraction is selected from: conjugate, chromophore, fluorophore, radio isotope, enzyme, haptens or luminophor.
6. the peptide nucleic acid probe group for fluorescence in situ hybridization detection streptococcus aureus and/or staphylococcus epidermidis as claimed in claim 5, 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 streptococcus aureus and/or staphylococcus epidermidis bacterial strain 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 streptococcus aureus and/or staphylococcus epidermidis bacterial strain 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 10, is characterized in that, its results of hybridization presents by fluorescence.
The purposes of 13. peptide nucleic acid probe groups for fluorescence in situ hybridization detection streptococcus aureus and/or staphylococcus epidermidis as described in as arbitrary in claim 1～6, it is characterized in that, described peptide nucleic acid probe group is for the detection of Gold Samples staphylococcus aureus and/or staphylococcus epidermidis.
The purposes of 14. test kits for detection of streptococcus aureus and/or staphylococcus epidermidis bacterial strain and drug susceptibility as described in as arbitrary in claim 7～9, it is characterized in that, described test kit is applied to streptococcus aureus and/or the qualification of staphylococcus epidermidis and the detection of drug susceptibility thereof in biological sample.
The purposes of 15. using method as described in as arbitrary in claim 10～12, is characterized in that, described using method is for Gold Samples staphylococcus aureus and/or the qualification of staphylococcus epidermidis and the detection of drug susceptibility thereof.
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| CN111491667A (en) * | 2017-12-18 | 2020-08-04 | 文塔纳医疗系统公司 | Peptide nucleic acid conjugates |
| US11597925B2 (en) | 2016-12-19 | 2023-03-07 | Ventana Medical Systems, Inc. | Peptide nucleic acid conjugates |
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