CN103409535A - Primer pair, probe and method for detecting Cronobacter sakazakii - Google Patents
Primer pair, probe and method for detecting Cronobacter sakazakii Download PDFInfo
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Abstract
The invention relates to an oligonucleotide primer pair for quickly detecting Cronobacter sakazakii as well as a probe and a kit including the primer pair and the probe. The invention relates to a real-time fluorescence PCR (Polymerase Chain Reaction) method for quickly detecting the Cronobacter sakazakii by utilizing the primer pair and the probe or the kit. The invention further relates to an application of the primer pair and the probe or the kit to detecting the Cronobacter sakazakii. The Cronobacter sakazakii can be quickly detected with high specificity and high sensitivity by utilizing the primer pair, the probe and the method for detecting Cronobacter sakazakii disclosed by the invention.
Description
Technical field
The invention belongs to biological technical field.Particularly, the present invention relates to for the Oligonucleolide primers of special, the highly sensitive rapid detection of height Crow promise Bacillus bacteria to probe and the test kit that comprises described primer pair and probe, the real time fluorescent PCR method of high special, highly sensitive rapid detection Crow promise Bacillus bacteria, and described primer pair and probe or the application of test kit in rapid detection Crow promise Bacillus bacteria.
Background technology
Crow promise Pseudomonas (Cronobacter spp.) bacterium once was called as the enterobacter cloacae that produces yellow pigment always, was defined as Enterobacter sakazakii in 1980.Iversen in 2008 etc. have carried out new systematics classification according to 16S rRNA gene order, DNA-DNA cross experiment, amplified fluorescence fragment length polymorphism etc. to Enterobacter sakazakii, and suggestion is defined as a new genus by this bacterium, i.e. Crow promise Bacillaceae.This genus comprises 5 novel species and 3 new subspecieses and 1 Crow promise bacillus gene kind.At present, the rugged Crow promise of slope bacillus (Cronobacter sakazakii) is that in Crow promise Bacillaceae, research is more a kind of, it is a kind of conditioned pathogen, extensively be present in nature, can cause serious neonatal meningitis, necrotic colitis and microbemia, mortality ratio is up to more than 50%.FAO and WHO think that the Crow all kinds of promise Bacillaceae all cause a disease.At present, investigators it be unclear that the pollution source of Crow promise bacillus, are the main channels that promise bacillus in Crow pollutes but the majority of cases report shows Infant Formula Enterprises.
The detection method of traditional Crow promise bacillus mainly depends on biochemistry and Morphological Characteristics, and " separation of Enterobacter sakazakii counting in infant formula powder " that the U.S. FDA of usually usining is in the world promulgated is as classical way.Traditional detection method complex operation, length consuming time and accuracy and sensitivity are lower.Along with the development of molecular biotechnology, the widespread use in the detection of food-borne pathogens of the technology such as PCR, fluorescent PCR.For the different target genes of the rugged Crow promise of slope bacillus, the PCR in real time that investigators have also carried out Crow promise bacillus detects research, but fluorescence quantitative PCR detection is main or for the detection of the rugged Crow promise of slope bacillus.China classifies the rugged Crow promise of slope bacillus as the essential items for inspection of importing and exporting milk and milk products in October, 2005.Along with the variation of Enterobacter sakazakii taxonomy, be the health of Protection of consumer and the safety of milk powder, the detection method of setting up quick, sensitive and special Crow promise Bacillus bacteria seems particularly necessary.
Polymerase chain reaction (PCR) is by the DNA of amplification testing sample, can increase to from initial minor amount and be enough to reach the enormous quantity that detection is limited the quantity of.PCR method is for diversified gene test.Real-time fluorescence PCR is in the PCR reaction system, to add the fluorescence report group, utilizes fluorescent signal to carry out the whole PCR process of Real-Time Monitoring, finally by typical curve, unknown template concentrations is carried out to quantitative analysis, makes round pcr from qualitative levels to quantitative level.In recent years, in molecular biology research, utilize quantitative PCR to detect gene expression results, obtained application.Because it has greatly improved sensitivity, specificity and the accuracy detected and can effectively reduce in experimentation, produce the danger of polluting, be widely used at present every field.
At present, domestic and international rare report can simultaneously quick, special, sensitive detection food in the real time fluorescent PCR method of Crow promise Bacillus bacteria.
Therefore, this area is needed badly and is set up a kind of fast and easy, Crow promise Bacillus bacteria detection method that specificity is good, highly sensitive, for the qualitative and detection by quantitative of Crow promise bacillus.
Summary of the invention
On the one hand, the invention provides for by real time fluorescent PCR method, detecting Oligonucleolide primers and the probe of Crow promise Bacillus bacteria.
The present invention be take Crow promise bacillus DNA and is basis, according to the strong zone of conservative property in the rugged Crow promise of slope bacillus alpha-glucosidase gene (EF635114.1), design for the Auele Specific Primers of all kinds of bacteriums of Crow promise Bacillaceae to and probe, utilize real time fluorescent PCR method rapid detection Crow promise Bacillus bacteria.
The present invention can be used for detecting for example, in several samples (food) Luo Nuo Bacillus bacteria.
In one embodiment, the invention provides for the Oligonucleolide primers that detects promise Bacillus bacteria in Crow by real time fluorescent PCR method to and probe, described primer pair and probe are to design according to the strong zone of conservative property in the rugged Crow promise of slope bacillus alpha-glucosidase gene (EF635114.1).
In one embodiment, the primer pair sequence of using is:
CroF：5’-TGTGGACGACATCTGCCG-3’(SEQ ID NO.1)；
CroR：5’-TTCTCCCCCACCCACTCTT-3’(SEQ ID NO.2)；
Probe sequence is:
CroP：5’-GTCTACGACATGATGCGCTG-3’(SEQ ID NO.3)。
In one embodiment, described probe is the Taqman probe.
In one embodiment, be connected with the fluorescent quenching group at 3 ' end of probe, 5 ' end is connected with the fluorescence report group.
In the present invention, described fluorescent quenching group can be the normally used fluorescent quenching group in this area, such as BHQ, BHQ1, BHQ2 or TAMRA etc.; Described fluorescence report group can be the normally used fluorescence report group in this area, such as FAM, CY3, CY5, HEX or TET etc.
In one embodiment, be connected with FAM at 5 ' end of probe, 3 ' end is connected with BHQ.
On the one hand, the invention provides for by real time fluorescent PCR method, detecting the test kit of Crow promise Bacillus bacteria, wherein, described test kit comprises primer pair of the present invention and probe.
In one embodiment, described test kit also comprises be used to the reagent that extracts Crow promise Bacillus bacteria DNA, reagent, positive control, negative control, blank and the working instructions for real-time fluorescence PCR.
On the other hand, the invention provides the method that detects promise Bacillus bacteria in Crow by real time fluorescent PCR method, described method comprises uses primer pair of the present invention and probe or test kit of the present invention.
In one embodiment, described real-time fluorescence PCR reaction conditions is: 95 ℃ of 10min; 95 ℃ of 5s, 58 ℃ of 20s, 40 circulations.
In one embodiment, described detection method comprises step:
(a) from product to be measured, extracting the DNA sample;
(b) provide the condition of real-time fluorescence PCR reaction;
(c) use primer pair of the present invention and probe or test kit, carry out nucleic acid amplification reaction and detect amplified production by real time fluorescent PCR method.
Real-time fluorescence PCR is namely on the basis of conventional PCR method, add fluorescently-labeled probe or fluorescence dye, accumulation along with the PCR product, the fluorescent signal that probe or dyestuff send strengthens, and fluorimetric detector can receive fluorescent signal, be DNA chain of every generation, just have a fluorescence molecule to form, realized that the accumulation of fluorescent signal and PCR product form Complete Synchronization.Therefore can monitor in real time whole PCR reaction process, and finally detect the initial copy number of testing sample, thus the bacterium of Crow promise Bacillaceae in can the rapid detection sample.
Again on the one hand, the invention provides primer pair of the present invention and probe or test kit of the present invention in the application that detects Crow promise Bacillus bacteria.
Real-time fluorescence PCR detection method of the present invention adopts complete stopped pipe to detect, and without the PCR aftertreatment, has avoided crossed contamination and false positive.Utilize real time fluorescent PCR method in the present invention can detect the bacterium of all kinds in Crow promise Bacillaceae, the method Simple fast in the present invention, high specificity and the highly sensitive Quantitative detection that is suitable for Crow promise Bacillus bacteria.To sample for example in food the examination and controlling of Crow promise bacillus have great importance.
The accompanying drawing explanation
Fig. 1 is the result that shows real-time fluorescence PCR specific amplification Crow promise Bacillus bacteria, wherein 1-24 is for comprising 5 Luo Nuo bacillus novel species: the rugged Crow promise of slope bacillus (Cronobacter sakazakii), the positive Crow promise bacillus (Cronobacter malonaticus) of malonate, Crow, Zurich promise bar (Cronobacter turicensis), Mu Tingsi Crow promise bacillus (Cronobacter muytjensii) and Crow, Dublin promise bacillus (Cronobacter dublinensis) and 3 new subspecieses: Crow, Dublin promise bacillus Dublin subspecies (Cronobacter dublinensis subsp.Dublinensis), (Cronobacter genomospecies is at 24 interior strain Crow promise Bacillus bacterias for Crow, Dublin promise bacillus Lausanne subspecies (Cronobacter dublinensis subsp.Lausannensis) and Crow, Dublin promise bacillus milk powder subspecies (Cronobacter dublinensis subsp.Lactaridi) and 1 Crow promise bacillus gene kind, 25-34 is followed successively by enterobacter cloacae (Enterobacter cloacae), enteroaerogen (Enterobacter aerogenes), Listeria monocytogenes (Listeria monocytogenes), Yersinia enterocolitica (Yersinia enterocolitica), escherichia coli (E.coli) O157:H7, shigella sonnei (Shigella sonnei), Vibrio parahaemolyticus (Vibrio parahaemolyticus), streptococcus aureus (Staphyloccocu saureus), salmonella (Salmonella) and blank.
Fig. 2 take the rugged Crow promise of slope bacillus to be example, build plasmid standard, detect gram Luo Nuo Bacillus bacteria of the present invention Auele Specific Primer to the sensitivity result with probe combinations, wherein 1-9 is ten times of serial dilution concentration (8.26 * 10 of carrying out the plasmid standard of real-time fluorescence PCR detection
11Copy/ml to 8.26 * 10
2Copy/mL, 10 is blank, repeats 3 times.
Fig. 3 carries out according to the plasmid DNA of 10 times of serial dilutions the typical curve that the PCR in real time amplification obtains.
Fig. 4 is the detected result to the delicatessen food sample.
Embodiment
The present invention is further illustrated for mode by embodiment, but the present invention is not limited only to following examples.
Embodiment 1
The present embodiment has carried out the specificity checking by following experiment to primer and the probe of Crow promise Bacillus bacteria.
By 5 the Crow promise bacillus novel species that comprise to the preservation of contriver laboratory: the rugged Crow promise of slope bacillus, the positive Crow promise bacillus of malonate, Crow, Zurich promise bar, Mu Tingsi Crow promise bacillus and Crow, Dublin promise bacillus and 3 new subspecieses: Crow, Dublin promise bacillus Dublin subspecies, Crow, Dublin promise bacillus Lausanne subspecies and Crow, Dublin promise bacillus milk powder subspecies and 1 Crow promise bacillus gene kind are 24 interior strain Crow promise Bacillus bacteria and enterobacter cloacaes, enteroaerogen, Listeria monocytogenes, Yersinia enterocolitica, escherichia coli O157:H7, shigella sonnei, Vibrio parahaemolyticus, streptococcus aureus, the salmonella partial sequence in its alpha-glucosidase gene that increases, verify Auele Specific Primer and the probe of promise Bacillus bacteria in Crow of the present invention.
The main detecting instrument used:
Micropipet (10 μ L, 100 μ L, 1000 μ L, Eppendorf), whizzer (5804R, Germany Eppendorf company), nucleic acid-protein analyser (DU640, German Beckman company), fluorescent PCR amplification instrument (Bio-Rad iQ5, Germany Eppendorf company), electrophoresis apparatus (DYY-6C, Liuyi Instruments Plant, Beijing), gel imaging instrument (German Gene Genius company) etc.
Detect and use main agents:
Bacterial genomes DNA extraction test kit is purchased from sky root biotech firm.Real-time PCR reactions reagent Fast Star Universal Probe Master (Rox) is purchased from Roche Holding Ag; Brain heart infusion agar (Brain heart infusion, BHI), enterobacteria enrichment broth (Enterobacteria enrichment broth mossel, EE meat soup) are purchased from Beijing overpass company.
Detect key step:
1) preparation of strain culturing and template DNA
To comprise that 24 strain Crow promise Bacillus bacterias of above-mentioned 5 novel species and 3 new subspecieses and 1 Crow promise bacillus gene kind and enterobacter cloacae, enteroaerogen, Listeria monocytogenes, Yersinia enterocolitica, escherichia coli O157:H7, shigella sonnei, Vibrio parahaemolyticus, streptococcus aureus, salmonella inoculation are in 5mL BHI substratum, 36 ℃ of overnight incubation.Get the 1mL inoculum, extract genomic dna with bacterial genomes DNA extraction test kit, the concrete operations by specification carries out.With the protein nucleic acid analyser, survey purity and the concentration of DNA, this is the DNA profiling that detects use.
2) real-time fluorescence PCR detects primer pair and the probe of use
The primer pair sequence is SEQ ID Nos.1 and 2; Probe sequence is SEQ ID No.3, and is connected with FAM at probe 5 ' end, and 3 ' end is connected with BHQ.
3) real-time fluorescence PCR reaction system 25 μ L:
Annotate: each PCR detects and all sets up corresponding blank (replacing DNA profiling with the ultrapure water of preparation reaction system), and whether detection reagent is polluted).
4) real-time fluorescence PCR reaction parameter:
95℃ 10min；
95℃ 5s
58℃ 20s
40 circulations.
As shown in Figure 1, during with real time fluorescent PCR method amplification alpha-glucosidase gene, the 24 strain Crow promise Bacillus bacterias that comprise 5 novel species and 3 new subspecieses and 1 Crow promise bacillus gene kind all produce typical amplification curve, and enterobacter cloacae, enteroaerogen, Listeria monocytogenes, Yersinia enterocolitica, escherichia coli O157:H7, shigella sonnei, Vibrio parahaemolyticus, streptococcus aureus, salmonella and blank do not have amplification curve to produce, the primer pair and the probe that absolutely prove this experimental design show specificity preferably to Crow promise Bacillus bacteria.
Embodiment 2
The present embodiment be take the DNA of the rugged Crow promise of slope bacillus (ATCC 29544) and be to be detected basis, by following experiment, primer pair and the probe of Crow promise Bacillus bacteria has been carried out to the sensitivity detection.
By detecting the alpha-glucosidase gene sequence, can determine primer pair that Crow promise Bacillus bacteria is special and the detection sensitivity of probe.
The Auele Specific Primer used is identical with embodiment 1 to reaching probe sequence.
The detection key instrument used:
Micropipet (10 μ L, 100 μ L, 1000 μ L Eppendorf), whizzer (5804R, Germany Eppendorf company), nucleic acid-protein analyser (DU640, Germany Beckman company), fluorescent PCR amplification instrument (Bio-Rad iQ5, German Eppendorf company), electrophoresis apparatus (DYY-6C, Liuyi Instruments Plant, Beijing), gel imaging instrument (German Gene Genius company) etc.
Detect main agents:
Bacterial genomes DNA extraction test kit, gel reclaim test kit purchased from sky root biotech firm; Plasmid extraction kit, nucleic acid purification test kit, pMD19-T Vector, DH5 α competent cell are purchased from Dalian precious biotechnology company limited in a small amount; Real-time PCR reactions reagent Fast Star Universal Probe Master (Rox) is purchased from Roche Holding Ag; Brain heart infusion agar, enterobacteria enrichment broth are purchased from Beijing overpass company; Be used for the PCR test kit of DNA cloning purchased from Dalian precious biotechnology company limited.
Detect key step:
1) amplification of target sequence and purifying
The genomic dna of the rugged Crow promise of the slope of take bacillus (ATCC29544) is template, carries out conventional PCR reaction.The reaction system cumulative volume is 50 μ L, template 2 μ L wherein, PCR damping fluid (10 *) 5 μ L, dNTP2.5 μ L, Ex Taq0.5U, each 0.5 μ L of upstream and downstream primer SEQ ID Nos.1 and 2 (10 μ mol/L), DDW polishing 50 μ L.Reaction conditions: 95 ℃ of 10min; 95 ℃ of 15s, 58 ℃ of 30s, 72 ℃ of 30s, 35 circulations; 72 ℃ are extended 7min.Product reclaims test kit with gel and cuts the glue recovery, and concrete operations are carried out to specifications.
2) screening of recombinant plasmid
PCR product after purifying is connected to the pMD19-T carrier, imports DH5 α competent cell, carry out blue hickie screening.Random 5 positive colonies of picking carry out the PCR evaluation, and positive colony is carried out to sequencing analysis.Sequencing result and target gene sequence are in full accord, are illustrated as correct positive colony.
3) preparation of plasmid standard
The inoculation have correct sequence the positive colony bacterium in 5mL BHI substratum, 36 ℃ of overnight incubation.Plasmid extraction is carried out in explanation according to a small amount of plasmid extraction kit, and measuring the plasmid concentration extracted is 25 μ g/ml.According to the molecular weight of plasmid, the plasmid sample concentration is scaled to copy number concentration.The calculating of plasmid copy number is according to formula: copy number concentration (copy/ml)=(quality/molecular weight) * 6.02 * 1023, the molecular weight of plasmid=660 * (2692+64)=31818960, wherein 660 is the molecular-weight average of base, and 2692 is the base number of T carrier.By the copy number concentration that calculates the plasmid obtained, be 8.26 * 10
12Copy/mL.
4) preparation of typical curve and sensitivity detect
Carry out 10 times of serial dilutions, make plasmid concentration reach 8.26 * 10
11Copy/ml to 8.26 * 10
2Copy/mL, utilize the real-time fluorescence PCR reaction system of embodiment 1 and condition to carry out the PCR reaction.When plasmid concentration is 8.26 * 10
2During copy/mL, revision test, fluorescent signal is still lower than threshold value, and judgement can not detect the rugged Crow promise of slope bacillus.When plasmid concentration is 8.26 * 10
3During copy/mL, revision test, fluorescent signal is higher than detection threshold, and judgement can detect the rugged Crow promise of slope bacillus, and corresponding Ct value demonstration is limited to 8.26 * 10 under detecting
3Copy/mL.As shown in Figure 2, the template amount added in each PCR reaction is 2 μ L, the copy number in each reaction tubes is 16.52, while pointing out the goal gene that 17 copies are arranged in each reaction tubes, can be detected, the content that the real-time fluorescence PCR detection method that shows foundation can detect Crow promise Bacillus bacteria is 17 copy/reaction systems.By the typical curve of plasmid concentration logarithmic value and corresponding CT value drafting thereof as shown in Figure 3.
Embodiment 3
The present inventor has carried out detection validation by following test to promise Bacillus bacteria in Crow in the delicatessen food sample.
Chosen 30 parts of dairy products samples and carried out the real-time fluorescence PCR detection, to verify the real time fluorescent PCR method of being set up.According in the method that provides take 25g milk powder, put into the aseptic homogenizing bag that fills 225mL physiological saline, 8000r/min～10000r/min homogeneous 1min～2min, make the even liquid of 1:10 sample; Cultivate 12h for 37 ℃; Getting the 10mL supernatant adds in the 90mL enterobacteria enrichment broth, after 37 ℃ of cultivation 12h, take out 1mL, the centrifugal 5min of 12000rpm, supernatant discarded, add the sterilizing physiological saline of 1mL, the centrifugal 5min of 12000rpm, supernatant discarded, add 200 μ L sterile salines, boiling water bath 10min, the centrifugal 5min of 12000rpm, get the template of supernatant as the PCR reaction, carries out the real-time fluorescence detection according to the step in example 1.According to the strains separation method of counting, with color developing culture medium, the bacterial strain of test positive is wherein verified simultaneously.Each sample of real time fluorescent PCR method repeats 3 times.As shown in Figure 4, detect positive signal in two parts of dairy products samples, all the other milk powder samples and blank do not detect, and the color developing culture medium checking has also confirmed this detected result.Show, the method can detect Crow promise Bacillus bacteria effectively.
Claims (9)
- For the Oligonucleolide primers that detects promise Bacillus bacteria in Crow by real time fluorescent PCR method to and probe, the sequence of wherein said primer pair is SEQ ID No.s1 and 2, the sequence of described probe is SEQ ID No.3.
- 2. primer pair claimed in claim 1 and probe, wherein, described probe is the Taqman probe.
- 3. primer pair claimed in claim 2 and probe, wherein, be connected with the fluorescent quenching group at 3 ' end of probe, and 5 ' end is connected with the fluorescence report group.
- 4. primer pair claimed in claim 3 and probe, wherein, described fluorescent quenching group is BHQ, BHQ1, BHQ2 or TAMRA, described fluorescence report group is FAM, CY3, CY5, HEX or TET.
- 5. for by real time fluorescent PCR method, detecting the test kit of Crow promise Bacillus bacteria, wherein said test kit comprises the described primer pair of claim 1-4 any one and probe.
- 6. test kit claimed in claim 5, it also comprises be used to the reagent that extracts Crow promise Bacillus bacteria DNA, reagent, positive control, negative control, blank and working instructions for real-time fluorescence PCR.
- 7. by real time fluorescent PCR method, detect the method for Crow promise Bacillus bacteria, described method comprises that right to use requires the described test kit of any one in the described primer pair of 1-4 any one and probe or claim 5-6.
- 8. method claimed in claim 7 comprises step:(a) from product to be measured, extracting the DNA sample;(b) provide the condition of real-time fluorescence PCR reaction;(c) right to use requires the described test kit of any one in the described primer pair of 1-4 any one and probe or claim 5-6, carries out nucleic acid amplification reaction and detects amplified production by real time fluorescent PCR method.
- In the described primer of claim 1-4 any one and probe or claim 5-6 the described test kit of any one in the application that detects Crow promise Bacillus bacteria.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104593487A (en) * | 2014-12-15 | 2015-05-06 | 合肥工业大学 | PCR-RFLP molecular subtyping method for distinguishing different types of cronobacters |
| CN110699474A (en) * | 2019-12-09 | 2020-01-17 | 合肥工业大学 | Molecular typing method for distinguishing different species of Cronobacter on basis of RsaI single enzyme digestion gluA gene |
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| CN102978291A (en) * | 2012-12-25 | 2013-03-20 | 光明乳业股份有限公司 | Cronobacter sakazakii strain detecting method, kit and primer |
| CN103243171A (en) * | 2013-05-29 | 2013-08-14 | 光明乳业股份有限公司 | Method for detecting cronobacter sakazakii as well as kit and primer thereof |
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| CN102978291A (en) * | 2012-12-25 | 2013-03-20 | 光明乳业股份有限公司 | Cronobacter sakazakii strain detecting method, kit and primer |
| CN103243171A (en) * | 2013-05-29 | 2013-08-14 | 光明乳业股份有限公司 | Method for detecting cronobacter sakazakii as well as kit and primer thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104593487A (en) * | 2014-12-15 | 2015-05-06 | 合肥工业大学 | PCR-RFLP molecular subtyping method for distinguishing different types of cronobacters |
| CN110699474A (en) * | 2019-12-09 | 2020-01-17 | 合肥工业大学 | Molecular typing method for distinguishing different species of Cronobacter on basis of RsaI single enzyme digestion gluA gene |
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