CN110283920A - Detect the peptide nucleic acid Fluorescence in situ hybridization identification method and peptide nucleic acid probe of Shigella - Google Patents
Detect the peptide nucleic acid Fluorescence in situ hybridization identification method and peptide nucleic acid probe of Shigella Download PDFInfo
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- CN110283920A CN110283920A CN201910628531.8A CN201910628531A CN110283920A CN 110283920 A CN110283920 A CN 110283920A CN 201910628531 A CN201910628531 A CN 201910628531A CN 110283920 A CN110283920 A CN 110283920A
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- shigella
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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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Abstract
The invention discloses the peptide nucleic acid Fluorescence in situ hybridization identification methods and peptide nucleic acid probe of detection Shigella.It is related to the design for identifying Shigella in different types of sample (Shigella Castellani) peptide nucleic acid (peptide nucleic acid, PNA) probe, the DNA sequence dna of the PNA probe is as shown in SEQ ID:1.These probes and fluorescence in situ hybridization (fluorescence in situ hybridization, FISH) technology are combined for detecting Shigella.PNA of the invention has the high stability specifically bound with DNA and RNA, hybridizes rapidity and its good Cell permeable, the combination of PNA and FISH technology, there is detection method of the invention quickly, accurate and sensitive characteristic improves the accuracy rate of identification.
Description
Technical field
The present invention relates to the method for detecting microorganism associated with foodsafety more particularly to a kind of detection shigas
The peptide nucleic acid Fluorescence in situ hybridization identification method and peptide nucleic acid probe of Pseudomonas
Background technique
Food-borne pathogens are to cause one of principal element of food origin disease, by pass in world's field of food safety
Note.WHO report shows global 40~6,000,000,000 food origin diseases of generation every year, wherein about 70% is eaten because biogenic pollutes
Caused by product.There are about 1,800,000 populations to die of food origin disease every year for developing country, even also having every year in developed country
10% or more population infection food origin disease.The important pathogenic bacteria recognized in the world that can cause food origin disease, it is such as husky
Door Salmonella, staphylococcus aureus, Listeria monocytogenes, Escherichia coli O 157, Shigella etc. are widely present in dairy produce, vegetable
It is an important factor for causing food pollution in a variety of foods such as dish, aquatic products, meat products.Therefore, food-borne pathogens is effective
Detection is very important monitoring food safety.
Shigella (Shigella Castellani) is a kind of gram-Negative bacillus, is human bacterial's property dysentery
Most commonly seen pathogen is generally called shigella dysenteriae.Shigella by food infect the mankind, dairy produce, aquatic products, chicken,
It is often detected in the food such as fruit, bread.It, can threat to life if being given treatment to not in time when morbidity.Shigella is since by people
Since it was found that, it is quite mature identification and general survey technology have been separately cultured.However, with the development of science and technology, passing
Bacterium separation identification, culture and the identification of biochemical reaction of system can far from support diagnosis and stream to pathogenic microorganism
The research that row disease is learned, it is also much fast not as good as present pathogenic microorganisms detection speed, conveniently, specificity is high, the high and low consumption of sensibility
Energy.
The conventionally used cultural method of the detection of Shigella (Shigella Castellani) carries out.But these
Method is time-consuming and laborious, and therefore, many molecular detecting methods are developed, including polymerase chain reaction (PCR), genetic chip
Technology etc..The immunological test method of inspection, such as enzyme linked immunosorbent assay, SPA coagglutination method, immunoblotting, ring mediate
Isothermal amplification technique (LAMP) etc., the relevant detection method in relation to Shigella also report very much, such as Publication No.
CN107904320A patent is Shigella to be detected using method of loop-mediated isothermal amplification, but this method needs to extract bacterium to be measured
Genomic DNA, and the design of primer is complicated, causes the detection method time-consuming.Publication No. CN108660228A patent is benefit
Shigella is detected with fluorescent quantitative PCR technique, there are false positives, and its disclosed design of primers is complicated.Publication No.
The method of CN102424839A patent disclosure multiplex PCR detection Shigella, this method needs to increase bacterium 8-24 hour, and needs
Extract the DNA of bacterium to be measured, time and effort consuming.Publication No. CN108950037A patent discloses dual LAMP detection Shigella
Method, method is cumbersome, time-consuming.The detection method that standard SN/T 2754.3-2011 is announced is loop-mediated isothermal (LAMP) amplification inspection
Survey method, design of primers is complicated, needs to extract bacterial genomes DNA, time-consuming.
Skin nucleic acid is year designed by Danish scientist Nielsen et al. it is a kind of using neutral phthalein amine key as the completely new of skeleton
Analogies, skeleton structure unit are glycine, and base portion is connected in the amino N with main framing by methylene carboxyl, can
With sequence specific in conjunction with DNA, RNA.Its skeleton is electroneutral, compared with DNA-DNA or RNA-DNA complementary strand, PNA-DNA
Electrostatic repulsion is not present with PNA-RNA complementation, therefore there is very high DNA or RNA compatibility, no mispairing the case where
Lower its, which combines, has high stability, and hybridization speed is fast, has good Cell permeable, is the good selection of nucleic acid probe.
PNA probe combination Fluorescence in situ hybridization technology (FISH) simultaneously, compared with conventional biochemical identification, PNA-FISH method can be saved largely
Time.PNA-FISH result judgment method based on fluorescence detection and Morphology observation, improves the accuracy of detection, avoids vacation
Feminine gender, the appearance of false positive.
Summary of the invention
The first purpose of this invention is the peptide nucleic acid devised for detecting main food-borne pathogens Shigella
Fluorescence in situ identification PNA probe, the probe have Shigella specificity.
Another object of the present invention is to provide a kind of peptide nucleic acid fluorescence in situ identification method of Shigella, this method
In conjunction with FISH detection technique, carry out the quick detection to Shigella.
In order to realize above-mentioned first purpose, the invention adopts the following technical scheme:
For detecting the peptide nucleic acid probe of quantitative Shigella, the DNA sequence dna of the PNA probe is as follows: 5 '-
GATAATCTACGGCATATGGC-3'.The PNA probe can detect Shigella (Shigella Castellani)
The target sequence of the mutual bowl spares of the sequence of rRNA, rDNA or rRNA.Its N-terminal of the PNA probe is connected at least one type
Mark substance.The N-terminal mark substance is selected from one of following: fluorescein, biotin, digoxin.
In order to realize above-mentioned second purpose, the invention adopts the following technical scheme:
(1) 1-5 grams of measuring samples are taken, and sample are placed in the test tube containing 5-8ml sterilizing LB culture solution, 37 DEG C of oscillations
Culture 1 hour.
(2) thalline were collected by centrifugation, is gently cleaned with sterile water primary, then with sterile aqueous suspension, bacteria suspension is made.
(3) it takes the bacteria suspension point of 8-10 μ l step (2) on the glass slide sterilized, 5-10 μ l fixer is added and smoothens.
(4) step (3) glass slide is put into baking 20-30 minutes of 80 DEG C of baking oven.
(5) hybridization solution of the PNA probe containing 25ng/ml of 15-20 μ l is taken to be added drop-wise on glass slide, 55 DEG C hybridization 20-30 points
Clock.
(6) it is washed 2-3 times after hybridizing with cleaning solution, 5 minutes every time.
(7) its fluorescent brightness of micro- sem observation and ne ar after air-drying.
The fixer includes the paraformaldehyde of 0.04-0.06g/mL and the sodium hydroxide of 0.08-0.1g/mL.It is described miscellaneous
The formamide that friendship liquid is 2%-3% comprising percentage by volume, the dextran sulfate of 0.05-0.1g/mL, 10mM sodium chloride,
0.001-0.002g/mL ficoll, 5mM ethylenediamine tetra-acetic acid.The cleaning solution includes 10mM maleic acid, and percentage by volume is
The polysorbas20 of 0.3-0.5%.
The beneficial effects of the present invention are: hybridization is quick since PNA has the high stability specifically bound with DNA and RNA
Property and good Cell permeable, make detection method of the invention have quickly, accurately, sensitive characteristic.In combination with glimmering
Light hybridization in situ technique improves the identification accuracy rate of the program.
Specific embodiment
The design of embodiment 1.PNA probe
(http://www.ncbi.hlm.nih.gov/BLAST) has chosen several Shigella on the website of NCBI
16s rRNA sequence, be ranked up with the ClustalV algorithm of MegAlign software, the variable region after sequence devises
Shigella specific probe SC-16S-F.Probe sequence is as follows: 5 '-GATAATCTACGGCATATGGC-3 '
In order to verify the sensitivity and specificity of probe, with BLAST (http://www.ncbi.hlm.nih.gov/
BLAST) probe is verified.Blast search discovery, designed probe have very high specificity.Again by PNA probe
It is detected with large subunit (23S/28S) database matching, it is found that no sequence is matching, will not obscure.By BLAST points
The evaluation of analysis and software Pro beCheck, designed probe SC-16S-F theoretically have very high sensitivity and specificity.
It is used for subsequent detection together with positive control probe BacUin.Positive control probe BacUin sequence is as follows: 5 '-
CTGCCTCCCGTAGGA-3’
Then, the sequence of selection is synthesized, and accesses fluorescein FAM in its N-terminal.
The verifying of embodiment 2PNA-FISH susceptibility
It has chosen 11 plants of Shigella reference cultures and isolated strains has carried out sensitivity verifying, test method institute as above
It states.Test is all synchronous every time carries out positive control and negative control experiments, and positive control test substitutes other with probe BacUin
Probe, and in negative probes, other probes are substituted with blank.The results show that the Shigella bacterial strain of all experiments all with the positive
Control probe and probe SC-16S-F are positive (being shown in Table 1) in hybridization.The above results are consistent with default result, probe SC-16S-F energy
The aimed strain for detecting oneself has higher susceptibility.
Table 1PNA probe sensitivity verifying
Embodiment 3PNA-FISH specificity verification
It chooses 10 plants of representative gramnegative bacteriums and positive-bacterial strain carries out PNA-FISH specificity and tests
Card.Selection bacterial strain is Bacterium enteritidis, staphylococcus aureus, E.sakazakii, colon bacillus 0157: H7, single increasing Li Si
Special bacterium, enterococcus faecalis, vibrio parahemolyticus, yersinia enterocolitica, pseudomonas aeruginosa, campylobacter jejuni.Test side
Method is as described above.Only have positive control probe BacUin that can combine with all bacteriums as the result is shown, and probe SC-16S-F is not
It can illustrate that SC-16S-F probe has specificity well in conjunction with these non-targeted bacterial strains (table 2), can effectively avoid false positive
Appearance.
Table 2PNA probe specific verification
The PNA-FISH method and API method of 4. food sources Shigella isolated strains of embodiment, which are identified, to be compared
Simultaneously using PNA-FISH method and API method of the invention (the API LISTERIA strep of French Mei Liai company,
10300), to several food samples carry out Shigella detection, by sample collection, increase bacterium, separation and etc. after, sent out with this
Bright PNA-FISH is identified, while carrying out identification comparison referring to kit specification using API method.(table 3) as the result is shown,
In institute's inspection sample, probe SC-16S-F of the invention detects 16 parts of positive samples altogether, and is obtained with API approach identification
17 parts of positive samples (table 3), the results showed that have good inspection to Shigella with the PNA-FISH method that SC-16S-F probe hybridizes
Extracting rate, compared with traditional classical method, testing result is coincide substantially, can be used for the inspection of main food-borne pathogens Shigella
It surveys.
The testing result of table 3PNA-FISH method and API method in actual sample compares
Sequence table
<110>China's metering university
<120>the peptide nucleic acid Fluorescence in situ hybridization identification method and peptide nucleic acid probe of Shigella are detected
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gataatctac ggcatatggc 20
Claims (8)
1. the peptide nucleic acid probe for detecting Shigella, it is characterised in that: the DNA sequence dna of the peptide nucleic acid probe such as SEQ
Shown in ID NO:1.
2. peptide nucleic acid probe according to claim 1, which is characterized in that it can detect Shigella (Shigella
Castellani) the target sequence of the mutual bowl spares of the sequence of rRNA, rDNA or rRNA.
3. peptide nucleic acid according to claim 1 or 2, which is characterized in that its N-terminal is connected to the label of at least one type
Substance.
4. peptide nucleic acid according to claim 3, which is characterized in that N-terminal mark substance is selected from one of following: fluorescein, life
Object element, digoxin.
5. the method for detecting Shigella (Shigella Castellani), which is characterized in that it uses claim 1-
4 described in any item peptide nucleic acid probes, and itself the following steps are included:
(1) 1-5 grams of measuring samples are taken, and sample are placed in the test tube containing 5-8ml sterilizing LB culture solution, 37 DEG C of shaken cultivations
1 hour；
(2) thalline were collected by centrifugation, primary with sterile water wash, then with sterile aqueous suspension, bacteria suspension is made；
(3) it takes the bacteria suspension point of 8-10 μ l step (2) on the glass slide sterilized, 5-10 μ l fixer is added and smoothens；
(4) step (3) glass slide is put into baking 20-30 minutes of 80 DEG C of baking oven；
(5) hybridization solution of any one of the 1-4 of claim containing 25ng/ml of the 15-20 μ l peptide nucleic acid probe is taken to be added drop-wise to load glass
On piece, 55 DEG C hybridization 20-30 minutes；
(6) it is washed 2-3 times after hybridizing with cleaning solution, 5 minutes every time；
(7) its fluorescent brightness of micro- sem observation and ne ar after air-drying.
6. the method for detection Shigella (Shigella Castellani) according to claim 5, which is characterized in that
The fixer includes the paraformaldehyde of 0.04-0.06g/mL and the sodium hydroxide of 0.08-0.1g/mL.
7. the method for detection Shigella (Shigella Castellani) according to claim 5, which is characterized in that
The hybridization solution includes the formamide that percentage by volume is 2%-3%, the dextran sulfate of 0.05-0.1g/mL, 10mM chlorination
Sodium, 0.001-0.002g/mL ficoll, 5mM ethylenediamine tetra-acetic acid.
8. the method for detection Shigella (Shigella Castellani) according to claim 5, which is characterized in that
The cleaning solution includes 10mM maleic acid, and percentage by volume is the polysorbas20 of 0.3-0.5%.
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| CN201910628531.8A CN110283920A (en) | 2019-07-12 | 2019-07-12 | Detect the peptide nucleic acid Fluorescence in situ hybridization identification method and peptide nucleic acid probe of Shigella |
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| CN201910628531.8A CN110283920A (en) | 2019-07-12 | 2019-07-12 | Detect the peptide nucleic acid Fluorescence in situ hybridization identification method and peptide nucleic acid probe of Shigella |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586466A (en) * | 2012-03-23 | 2012-07-18 | 浙江省检验检疫科学技术研究院 | Method and PNA (peptide nucleic acid) probe for assaying salmonella by utilizing peptide nucleic acid fluorescent in-situ hybridization technique |
| US9663827B2 (en) * | 2011-04-01 | 2017-05-30 | Advandx, Inc. | Molecular gram stain |
-
2019
- 2019-07-12 CN CN201910628531.8A patent/CN110283920A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9663827B2 (en) * | 2011-04-01 | 2017-05-30 | Advandx, Inc. | Molecular gram stain |
| CN102586466A (en) * | 2012-03-23 | 2012-07-18 | 浙江省检验检疫科学技术研究院 | Method and PNA (peptide nucleic acid) probe for assaying salmonella by utilizing peptide nucleic acid fluorescent in-situ hybridization technique |
Non-Patent Citations (2)
| Title |
|---|
| ESIOBU, N: "The application of peptide nucleic acid probes for rapid detection and enumeration of eubacteria, Staphylococcus aureus and Pseudomonas aeruginosa in recreational beaches of S. Florida", 《JOURNAL OF MICROBIOLOGICAL METHODS》 * |
| STENDER, H: "Rapid detection, identification, and enumeration of Escherichia coli cells in municipal water by chemiluminescent in situ hybridization", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
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