CN103409535B - Detect the primer pair of Cronobacter sakazakii, probe and method - Google Patents
Detect the primer pair of Cronobacter sakazakii, probe and method Download PDFInfo
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Abstract
The present invention relates to for rapid detection Cronobacter sakazakii Oligonucleolide primers to and probe and comprise the test kit of described primer pair and probe.The present invention relates to the real time fluorescent PCR method utilizing described primer pair and probe or test kit rapid detection Cronobacter sakazakii.The invention still further relates to described primer pair and probe or test kit and detect the application in Cronobacter sakazakii.Utilize the present invention can high special, highly sensitive rapid detection Cronobacter sakazakii.
Description
Technical field
The invention belongs to biological technical field.Specifically, the Oligonucleolide primers that the present invention relates to special for height, highly sensitive rapid detection Cronobacter sakazakii to probe and the test kit comprising described primer pair and probe, the real time fluorescent PCR method of high special, highly sensitive rapid detection Cronobacter sakazakii, and described primer pair and probe or the application of test kit in rapid detection Cronobacter sakazakii.
Background technology
Cronobacter Pseudomonas (Cronobacter spp.) bacterium was once called as the enterobacter cloacae producing yellow pigment always, within 1980, was defined as Enterobacter sakazakii.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 advise this bacterium to be defined as a new genus, namely Cronobacter sakazakii belongs to.This genus comprises 5 novel species and 3 new subspecieses and 1 Cronobacter sakazakii gene kind.At present, the rugged Cronobacter sakazakii of slope (Cronobacter sakazakii) studies more one during Cronobacter sakazakii belongs to, 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%.It is all pathogenic that FAO and WHO thinks that Cronobacter sakazakii belongs to all kinds.At present, investigators it be unclear that the pollution source of Cronobacter sakazakii, but majority of cases report shows that Infant Formula Enterprises is the main channel that Cronobacter sakazakii is polluted.
The detection method of traditional Cronobacter sakazakii depends on biochemistry and Morphological Characteristics, and usual " in infant formula powder, the separation of Enterobacter sakazakii counts " of promulgating using U.S. FDA is as classical way in the world.Traditional detection method complex operation, longer and accuracy consuming time 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 Cronobacter sakazakii of slope, investigators have also carried out the PCR in real time detect delay of Cronobacter sakazakii, but fluorescence quantitative PCR detection is main still for the detection of the rugged Cronobacter sakazakii of slope.Rugged for slope Cronobacter sakazakii is classified as the essential items for inspection importing and exporting milk and milk products by China in October, 2005.Along with the change of Enterobacter sakazakii taxonomy, be the health of Protection of consumer and the safety of milk powder, the detection method setting up quick, sensitive and special Cronobacter sakazakii seems particularly necessary.
Polymerase chain reaction (PCR) is the DNA by the testing sample that increases, and can increase to be enough to reach the enormous quantity detecting limitation from initial minor amount.PCR method is in diversified gene test.Real-time fluorescence PCR adds fluorescent reporter group in PCR reaction system, utilizes fluorescent signal to carry out the whole PCR process of Real-Time Monitoring, carry out quantitative analysis, make round pcr develop into quantitative level from qualitative level finally by typical curve to unknown template concentrations.In recent years, in molecular biology research, utilize quantitative PCR to detect gene expression results, obtain application.Owing to it greatly improves the sensitivity of detection, specificity and accuracy and can effectively reducing in experimentation the danger producing pollution, be widely used in every field at present.
At present, domestic and international rare report can the real time fluorescent PCR method of Cronobacter sakazakii in simultaneously quick, special, sensitive detection food.
Therefore, this area is needed badly and is set up a kind of fast and easy, Cronobacter sakazakii detection method that specificity is good, highly sensitive, for the qualitative of Cronobacter sakazakii and detection by quantitative.
Summary of the invention
On the one hand, the invention provides Oligonucleolide primers and the probe for being detected Cronobacter sakazakii by real time fluorescent PCR method.
The present invention is based on Cronobacter sakazakii DNA, the region strong according to conservative property in slope rugged Cronobacter sakazakii alpha-glucosidase gene (EF635114.1), design the Auele Specific Primer that belongs to all kinds of bacteriums for Cronobacter sakazakii to and probe, utilize real time fluorescent PCR method rapid detection Cronobacter sakazakii.
The present invention can be used for detecting the Luo Nuo Bacillus bacteria in several samples (such as food).
In one embodiment, the invention provides Oligonucleolide primers for being detected Cronobacter sakazakii by real time fluorescent PCR method to and probe, the region that described primer pair and probe are strong according to conservative property in slope rugged Cronobacter sakazakii alpha-glucosidase gene (EF635114.1) is designed.
In one embodiment, the primer pair sequence used 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 Taqman probe.
In one embodiment, be connected with fluorescent quenching group at 3 ' end of probe, 5 ' end is connected with fluorescent reporter 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 fluorescent reporter group can be the normally used fluorescent reporter 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 the test kit for being detected Cronobacter sakazakii by real time fluorescent PCR method, wherein, described test kit comprises primer pair of the present invention and probe.
In one embodiment, described test kit also comprises the reagent for extracting Cronobacter sakazakii DNA, reagent, positive control, negative control, blank and the working instructions for real-time fluorescence PCR.
On the other hand, the invention provides and detect the method for Cronobacter sakazakii by real time fluorescent PCR method, described method comprises and 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 DEG C of 10min; 95 DEG C of 5s, 58 DEG C of 20s, 40 circulations.
In one embodiment, described detection method comprises step:
A () extracts DNA sample from product to be measured;
B condition that () provides real-time fluorescence PCR to react;
C () uses 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, along with the accumulation of PCR primer, the fluorescent signal that probe or dyestuff send strengthens, and fluorimetric detector can receive fluorescent signal, namely often produce a DNA chain, just have a fluorescence molecule to be formed, the accumulation and the PCR primer that achieve fluorescent signal form Complete Synchronization.Therefore can monitor whole PCR reaction process in real time, and finally detect the initial copy number of testing sample, thus can Cronobacter sakazakii belongs in rapid detection sample bacterium.
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 detecting Cronobacter sakazakii.
Real-time fluorescence PCR detection method of the present invention adopts complete stopped pipe to detect, and without the need to PCR aftertreatment, avoids crossed contamination and false positive.Utilize the real time fluorescent PCR method in the present invention can detect Cronobacter sakazakii belong in the bacterium of all kinds, the method in the present invention is simple and quick, high specificity and the highly sensitive Quantitative detection being suitable for Cronobacter sakazakii.The examination and controlling of Cronobacter sakazakii in sample such as food is had great importance.
Accompanying drawing explanation
Fig. 1 is the result of display real-time fluorescence PCR specific amplification Cronobacter sakazakii, wherein 1-24 is for comprising 5 Luo Nuo bacillus novel species: the rugged Cronobacter sakazakii of slope (Cronobacter sakazakii), the positive Cronobacter sakazakii (Cronobacter malonaticus) of malonate, Zurich Cronobacter bar (Cronobacterturicensis), Mu Tingsi Cronobacter sakazakii (Cronobacter muytjensii) and Dublin Cronobacter sakazakii (Cronobacter dublinensis) and 3 new subspecieses: Cronobacter sakazakii Dublin, Dublin subspecies (Cronobacter dublinensis subsp.Dublinensis), (Cronobacter genomospecies is in 24 interior strain Cronobacter sakazakii for Cronobacter sakazakii Lausanne, Dublin subspecies (Cronobacter dublinensis subsp.Lausannensis) and Dublin Cronobacter sakazakii milk powder subspecies (Cronobacter dublinensis subsp.Lactaridi) and 1 Cronobacter sakazakii 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 (Vibrioparahaemolyticus), streptococcus aureus (Staphyloccocu saureus), salmonella (Salmonella) and blank.
Fig. 2 is for the rugged Cronobacter sakazakii of slope, build plasmid standard, detect gram Luo Nuo Bacillus bacteria Auele Specific Primer of the present invention to the sensitivity results with probe combinations, wherein 1-9 is ten times of serial dilutions (8.26 × 10 of the plasmid standard carrying out real-time 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 real time PCR amplification obtains.
Fig. 4 is the detected result to delicatessen food sample.
Embodiment
By the mode of embodiment, the present invention is further illustrated, but the present invention is not limited only to following examples.
Embodiment 1
The present embodiment has carried out specificity verification by following experiment to the primer of Cronobacter sakazakii and probe.
By comprising 5 Cronobacter sakazakii novel species to contriver's Laboratories Accession: the rugged Cronobacter sakazakii of slope, the positive Cronobacter sakazakii of malonate, Zurich Cronobacter bar, Mu Tingsi Cronobacter sakazakii and Dublin Cronobacter sakazakii and 3 new subspecieses: Cronobacter sakazakii Dublin, Dublin subspecies, Dublin Cronobacter sakazakii Lausanne subspecies and Dublin Cronobacter sakazakii milk powder subspecies and 1 Cronobacter sakazakii gene kind are 24 interior strain Cronobacter sakazakii and enterobacter cloacaes, enteroaerogen, Listeria monocytogenes, Yersinia enterocolitica, escherichia coli O157:H7, shigella sonnei, Vibrio parahaemolyticus, streptococcus aureus, salmonella increases the partial sequence in its alpha-glucosidase gene, verify Auele Specific Primer and the probe of Cronobacter sakazakii of the present invention.
The main detecting instrument used:
Micropipet (10 μ L, 100 μ L, 1000 μ L, Eppendorf), whizzer (5804R, Eppendorf company of Germany), nucleic acid-protein analyser (DU640, German Beckman company), fluorescent PCR amplification instrument (Bio-Rad iQ5, Eppendorf company of Germany), electrophoresis apparatus (DYY-6C, Liuyi Instruments Plant, Beijing), gel imaging instrument (German Gene Genius company) etc.
Detection main agents:
Bacterial genomes DNA extraction kit is purchased from Tian Gen biotech firm.Real-time PCR reactions reagent FastStar Universal Probe Master (Rox) is purchased from Roche Holding Ag; Brain heart infusion agar (Brain heartinfusion, 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 24 strain Cronobacter sakazakii of above-mentioned 5 novel species and 3 new subspecieses and 1 Cronobacter sakazakii gene kind and enterobacter cloacae, enteroaerogen, Listeria monocytogenes, Yersinia enterocolitica, escherichia coli O157:H7, shigella sonnei, Vibrio parahaemolyticus, streptococcus aureus, salmonella inoculation be comprised in 5mL BHI substratum, 36 DEG C of overnight incubation.Get 1mL inoculum, extract genomic dna by bacterial genomes DNA extraction kit, concrete operations by specification carries out.Survey purity and the concentration of DNA with protein nucleic acid analyser, this is the DNA profiling detected.
2) primer pair of real-time PCR detection and probe
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:
Note: 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 Cronobacter sakazakii comprising 5 novel species and 3 new subspecieses and 1 Cronobacter sakazakii 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, absolutely prove that primer pair and the probe of this experimental design show good specificity to Cronobacter sakazakii.
Embodiment 2
The present embodiment for detecting basis, has carried out sensitivity technique by testing as follows to the primer pair of Cronobacter sakazakii and probe with the DNA of the rugged Cronobacter sakazakii of slope (ATCC 29544).
By detecting alpha-glucosidase gene sequence, the detection sensitivity of the primer pair that Cronobacter sakazakii is special and probe can be determined.
The Auele Specific Primer used to and probe sequence identical with embodiment 1.
The detection key instrument used:
Micropipet (10 μ L, 100 μ L, 1000 μ L Eppendorf), whizzer (5804R, Eppendorf company of Germany), nucleic acid-protein analyser (DU640, Beckman company of Germany), 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 kit, gel reclaim test kit purchased from Tian Gen biotech firm; Mini-scale plasmid extracts test kit, Nucleic acid purification kits, pMD19-T Vector, DH5 α competent cell purchased from the precious biotechnology company limited in Dalian; Real-time PCR reactions reagent Fast Star Universal ProbeMaster (Rox) is purchased from Roche Holding Ag; Brain heart infusion agar, enterobacteria enrichment broth are purchased from Beijing overpass company; For the PCR kit of DNA cloning purchased from the precious biotechnology company limited in Dalian.
Detect key step:
1) amplification of target sequence and purifying
With the genomic dna of the rugged Cronobacter sakazakii of slope (ATCC29544) for template, carry out Standard PCR reaction.Reaction system cumulative volume is 50 μ L, wherein template 2 μ L, PCR damping fluid (10 ×) 5 μ L, dNTP2.5 μ L, Ex Taq0.5U, upstream and downstream primer SEQ ID Nos.1 and 2 (10 μm of ol/L) each 0.5 μ L, DDW polishing 50 μ L.Reaction conditions: 95 DEG C of 10min; 95 DEG C of 15s, 58 DEG C of 30s, 72 DEG C of 30s, 35 circulations; 72 DEG C extend 7min.Product gel reclaims test kit to carry out cutting glue recovery, and concrete operations are carried out to specifications.
2) screening of recombinant plasmid
PCR primer after purifying is connected to pMD19-T carrier, imports DH5 α competent cell, carry out blue hickie screening.Random picking 5 positive colonies carry out PCR qualification, and positive colony is carried out sequencing analysis.Sequencing result and target gene sequence completely the same, be illustrated as correct positive colony.
3) preparation of plasmid standard
Inoculation has the positive colony bacterium of correct sequence in 5mL BHI substratum, 36 DEG C of overnight incubation.Plasmid extraction is carried out in the explanation of extracting test kit according to mini-scale plasmid, and measuring the plasmid concentration extracted is 25 μ g/ml.According to the molecular weight of plasmid, be copy number concentration by Plasmid samples concentration conversion.The calculating of plasmid copy number is according to formula: copy number concentration (copy/ml)=(quality/molecular weight) × 6.02 × 1023, molecular weight=660 × (2692+64)=31818960 of plasmid, wherein 660 is the molecular-weight average of base, and 2692 is the base number of carrier T.Be 8.26 × 10 by calculating the copy number concentration of obtained plasmid
12copy/mL.
4) preparation of typical curve and sensitivity technique
Carry out 10 times of serial dilutions, make plasmid concentration reach 8.26 × 10
11copy/ml to 8.26 × 10
2copy/mL, utilizes the real-time fluorescence PCR reaction system of embodiment 1 and condition to carry out 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 Cronobacter sakazakii of slope.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 Cronobacter sakazakii of slope, then corresponding Ct value display Monitoring lower-cut is 8.26 × 10
3copy/mL.As shown in Figure 2, the template amount added in each PCR reaction is 2 μ L, copy number then in each reaction tubes is 16.52, point out in each reaction tubes during the goal gene having 17 to copy and can be detected, show that the content that the real-time fluorescence PCR detection method set up can detect Cronobacter sakazakii is 17 copies/reaction system.The typical curve drawn by the CT value of plasmid concentration logarithmic value and correspondence thereof as shown in Figure 3.
Embodiment 3
The present inventor has carried out detection validation by following test to Cronobacter sakazakii in delicatessen food sample.
Choose 30 parts of dairy food sample and carried out real-time PCR detection, to verify set up real time fluorescent PCR method.According in the method that provides take 25g milk powder, put into the aseptic homogenizing bag filling 225mL physiological saline, 8000r/min ~ 10000r/min homogeneous 1min ~ 2min, make the even liquid of 1:10 sample; Cultivate 12h for 37 DEG C; Getting 10mL supernatant adds in 90mL enterobacteria enrichment broth, 1mL is taken out, the centrifugal 5min of 12000rpm, supernatant discarded after 37 DEG C of cultivation 12h, add the physiological saline of 1mL sterilizing, the centrifugal 5min of 12000rpm, supernatant discarded, adds 200 μ L sterile salines, boiling water bath 10min, the centrifugal 5min of 12000rpm, gets the template that supernatant reacts as PCR, carries out real-time fluorescence detection according to the step in example 1.Verify according to the bacterial strain of strains separation method of counting color developing culture medium to wherein test positive simultaneously.The each sample of real time fluorescent PCR method repeats 3 times.As shown in Figure 4, detect positive signal in two parts of dairy food sample, all the other milk powder samples and blank do not detect, and color developing culture medium checking also demonstrate that this detected result.Show, the method can detect Cronobacter sakazakii effectively.
Claims (9)
1. for by real time fluorescent PCR method detect Cronobacter sakazakii Oligonucleolide primers to and probe, the sequence of wherein said primer pair is SEQ ID No.s 1 and 2, and the sequence of described probe is SEQ ID No.3.
2. primer pair according to claim 1 and probe, wherein, described probe is Taqman probe.
3. primer pair according to claim 2 and probe, wherein, be connected with fluorescent quenching group at 3 ' end of probe, 5 ' end is connected with fluorescent reporter group.
4. primer pair according to claim 3 and probe, wherein, described fluorescent quenching group is BHQ, BHQ1, BHQ2 or TAMRA, and described fluorescent reporter group is FAM, CY3, CY5, HEX or TET.
5., for detecting the test kit of Cronobacter sakazakii by real time fluorescent PCR method, wherein said test kit comprises primer pair described in any one of claim 1-4 and probe.
6. test kit according to claim 5, it also comprises the reagent for extracting Cronobacter sakazakii DNA, reagent, positive control, negative control, blank and the working instructions for real-time fluorescence PCR.
7. the method for Cronobacter sakazakii is detected by real time fluorescent PCR method, described method comprises and uses primer pair described in any one of claim 1-4 and probe or the test kit according to any one of claim 5-6, and described method is not used in medical diagnosis on disease and therapeutic purpose.
8. method according to claim 7, comprises step:
A () extracts DNA sample from product to be measured;
B condition that () provides real-time fluorescence PCR to react;
C () uses primer pair described in any one of claim 1-4 and probe or the test kit according to any one of claim 5-6, carry out nucleic acid amplification reaction and detect amplified production by real time fluorescent PCR method.
9. the primer described in any one of claim 1-4 and probe or the test kit according to any one of claim 5-6 are in the application detecting Cronobacter sakazakii, and described application is not used in medical diagnosis on disease and therapeutic purpose.
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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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