CN105316398A - Amplification primer for detecting food-borne pathogenic microorganisms and liquid chip kit - Google Patents
Amplification primer for detecting food-borne pathogenic microorganisms and liquid chip kit Download PDFInfo
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Abstract
The invention discloses an amplification primer for detecting food-borne pathogenic microorganisms and a liquid chip kit. The kit comprises an amplification primer pair designed for microorganism genes to be detected; and a forward primer and a reverse primer of microorganisms to be detected comprise at least one pair of SEQ ID NO. 1 and SEQ ID NO. 2 for enterobacter sakazakii, SEQ ID NO. 3 and SEQ ID NO. 4 for monocyte listeria monocytogenes, SEQ ID NO. 5 and SEQ ID NO. 6 for escherichia coli O157, SEQ ID NO. 7 and SEQ ID NO. 8 for staphylococcus aureus, SEQ ID NO. 9 and SEQ ID NO. 10 for shigella, SEQ ID NO. 11 and SEQ ID NO. 12 for salmonella, and SEQ ID NO. 13 and SEQ ID NO. 14 for vibrio parahaemolyticus, and magnetic beads coated with Anti-tag sequences. The amplification primer designed by the invention has excellent specificity, and can accurately distinguish and specifically amplify gene segments corresponding to various target detection pathogenic microorganisms. In addition, the sensibility of the detecting kit designed by the invention is greatly improved. An experiment result shows that sensibility of detection of seven food-borne pathogenic microorganisms can reach 10 CFU/mL.
Description
Technical field
The invention belongs to biology field, particularly relate to amplimer and the liquid phase chip reagent box of food-borne pathogenic microorganism detection.
Background technology
It is one of main source producing food safety risk in food and beverage sevice link processing and making process that pathogenic microorganism pollutes, all likely bring pathogenic microorganism into pollute dining food from food raw material, the course of processing, each link of Dining tool, increase food safety risk.Common pathogenic bacterium have: Enterobacter sakazakii, Listeria monocytogenes, colon bacillus O157, streptococcus aureus, Shigellae, Salmonellas, Vibrio parahemolyticus etc.
The detection technique of food-borne pathogenic microorganism is the key technique of the Prevention and controls of food origin disease.At present, the method detecting food-borne pathogenic microorganism in food has traditional method (national standard GT4789), based on the method (real-time fluorescence PCR, regular-PCR method and loop-mediated isothermal amplification method) based on nucleic acid and immunological method (as enzyme-linked immunosorbent assay (ELISA), Colloidal Gold).Traditional Bacteria Identification mainly relies on the methods such as microbial culture, serology, biological chemistry and Gern morphology to carry out taxonomic identification, although traditional method is accurate, comprehensively, sense cycle is long, program is complicated, required reagent is various, and actual testing amount is huge; Immune colloid gold applies one of comparatively general method, but its specificity and sensitivity lower; Enzyme-linked immunosorbent assay (ELISA) technical requirements is low, easy to carry, often occurring and easily commercialization with the form of test kit, is be most widely used at present and proven technique, but the method needs high quality antibody, testing cost is large and operating process is loaded down with trivial details; Regular-PCR method and quantitative fluorescent PCR are owing to detecting the limitation of flux, and primary first-order equation, can not practical requirement only to detect an index.
In addition, also have prior art openly to detect with putting down formula PCR combined with fluorescent microballoon signal, the method greatly enhances accuracy and the sensitivity of detection.But the method is more complicated still, and the complicacy detected because of food-borne pathogenic microorganism, the sensitivity of detection method also needs to improve.
Summary of the invention
Based on this, an object of the present invention is to provide a kind of high specificity, highly sensitive and detection method simple food-borne pathogenic microorganism detection liquid phase chip reagent box, Enterobacter sakazakii, Listeria monocytogenes is detected for independent or arbitrary combination, colon bacillus O157, streptococcus aureus, Shigellae, Salmonellas, Vibrio parahemolyticus.
The technical scheme realizing above-mentioned purpose is as follows.
A kind of food-borne pathogenic microorganism detects liquid phase chip reagent box, includes:
(A) for the amplimer pair that microbial gene to be detected designs, often pair of amplimer includes amplimer F1 and amplimer R1, described amplimer F1 holds successively by tag sequence from 5 ' end to 3 ', spacerarm, the forward primer composition of often kind of corresponding microorganism to be detected, described amplimer R1 is the reverse primer that 5 ' end band has biotin labeled, corresponding microorganism to be detected, described tag sequence is selected from SEQIDNO.17-SEQIDNO.28, described spacerarm is selected from any one in C3Spacer, Spacer9, dSpacer, 3 ' C6Spacer, Spacer12, Spacer18 or PCSpacer, the forward primer of described microorganism to be detected and reverse primer include following at least one pair of: for SEQIDNO.1 and SEQIDNO.2 of Enterobacter sakazakii, for SEQIDNO.3 and SEQIDNO.4 of Listeria monocytogenes, for SEQIDNO.5 and SEQIDNO.6 of colon bacillus O157, for SEQIDNO.7 and SEQIDNO.8 of streptococcus aureus, for SEQIDNO.9 and SEQIDNO.10 of Shigellae, for Salmonellas SEQIDNO.11 and SEQIDNO.12, for SEQIDNO.13 and SEQIDNO.14 of Vibrio parahemolyticus,
(B) be coated with the magnetic bead of Anti-tag sequence, the tag complementary of described Anti-tag sequence and corresponding microorganism to be detected matches, and described Anti-tag sequence is selected from SEQIDNO.29-SEQIDNO.40; Magnetic bead for difference microorganism to be detected has different fluorescence-encoded.
Wherein in an embodiment, the forward primer of described microorganism to be detected and reverse primer also include SEQIDNO.15 and SEQIDNO.16 for internal control bacterium.
Wherein in an embodiment, described spacerarm is C3Spacer.
Wherein in an embodiment, described tag sequence is: the SEQIDNO.17 for Enterobacter sakazakii, the SEQIDNO.18 for Listeria monocytogenes, the SEQIDNO.19 for colon bacillus O157, the SEQIDNO.20 for streptococcus aureus, for the SEQIDNO.21 of Shigellae, for Salmonellas SEQIDNO.22, SEQIDNO.13 and SEQIDNO.23 for Vibrio parahemolyticus.
Wherein in an embodiment, the tag sequence for internal control bacterium is SEQIDNO.24.
Wherein in an embodiment, between described magnetic bead and Anti-tag sequence, be also provided with spacer sequence; Further, described spacer sequence is preferably 5-10 T.
Another object of the present invention is to provide the amplimer that a kind of food-borne pathogenic microorganism detects.
The technical scheme realizing above-mentioned purpose is as follows.
The amplimer that food-borne pathogenic microorganism detects, include following at least one pair of: SEQIDNO.1 and SEQIDNO.2 for Enterobacter sakazakii, SEQIDNO.3 and SEQIDNO.4 for Listeria monocytogenes, SEQIDNO.5 and SEQIDNO.6 for colon bacillus O157, SEQIDNO.7 and SEQIDNO.8 for streptococcus aureus, for SEQIDNO.9 and SEQIDNO.10 of Shigellae, for Salmonellas SEQIDNO.11 and SEQIDNO.12, SEQIDNO.13 and SEQIDNO.14 for Vibrio parahemolyticus.
Wherein in an embodiment, also include SEQIDNO.15 and SEQIDNO.16 of directed toward bacteria internal control sequence.
Major advantage of the present invention is:
1. seven kinds of food-borne pathogenic microorganisms designed by the present invention detect liquid-phase chip and have extraordinary signal-noise ratio, hybridization can be carried out under homogeneous reaction conditions, do not form hairpin structure, palindrome between designed amplimer and tag sequence, there is not specific binding, cross reaction is there is not between anti-tag sequence, tag sequence and amplified production, simultaneously, the present invention that combinationally uses of multiple sequence label and amplimer forms the intact system of a Detection results, thus realizes the parallel detection of multiple pathogenic microorganism target gene.The identical rate of detection method provided by the present invention and National Standard Method is up to 100%.
2. the amplimer of the present invention's design has extraordinary specificity, accurately can distinguish the corresponding gene fragment of various target detect pathogenic microorganisms with specific amplification, and pass through the specific binding of tag sequence and anti-tag sequence, realize the accurate differentiation of seven kinds of pathogenic microorganism amplified productions, and, the detection kit of the present invention's design, its sensitivity is greatly improved, from experimental result, the detection sensitivity of seven kinds of food-borne pathogenic microorganisms can reach 10CFU/mL.
3. detection method step of the present invention is simple, one step multiplex PCR can complete the amplification reaching genes involved target sequence in seven kinds of target microorganisms, avoid the many uncertain factors existed in the complex operations processes such as repeated multiple times PCR, thus greatly can improve Detection accuracy, embody accurate qualitative analysis feature.
4. the time required for detection method provided by the present invention is 4 ~ 5 hours, and well below the detection method that national standard is recommended, realistic especially detection needs.
Embodiment
For the ease of understanding the present invention, below by specific embodiment, the present invention is described more fully.The present invention can realize in many different forms, is not limited to embodiment described herein.On the contrary, provide the object of these embodiments be make the understanding of disclosure of the present invention more comprehensively thorough.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition, the people such as such as Sambrook, molecular cloning: laboratory manual (NewYork:ColdSpringHarborLaboratoryPress, 1989) condition described in, or according to the condition that manufacturer advises.Various conventional chemical reagent used in embodiment, is commercially available prod.
Unless otherwise defined, all technology used in the present invention and scientific terminology are identical with belonging to the implication that those skilled in the art of the present invention understand usually.The object of the term used in specification sheets of the present invention just in order to describe specific embodiment, is not used in restriction the present invention.Term "and/or" used in the present invention comprises arbitrary and all combinations of one or more relevant Listed Items.
Embodiment 1
Food-borne pathogenic microorganism described in the present embodiment detects liquid phase chip reagent box, mainly includes:
One, PCR primer
For the rpsU-dnaG gene of Enterobacter sakazakii, the prfA gene of Listeria monocytogenes, the flic gene of colon bacillus O157, the Sa442 gene of streptococcus aureus, the ipaH gene of Shigellae, the fim gene of Salmonellas, the toxR gene of Vibrio parahemolyticus, bacterium internal control sequence 16S-rRNA, respectively design of amplification primers pair.
Wherein, amplimer F1 is made up of three parts, and 5 ' end is held successively to 3 ': tag sequence, spacerarm, the forward amplimer of corresponding gene, simultaneously, amplimer R1 is the reverse primer of microorganism to be detected biotin labeled, corresponding in 5 ' end band, and primer sequence is as shown in the table:
Table 1 food-borne pathogenic microorganism gene and amplimer
Table 2tag sequence
| SEQ ID NO. | Tag sequence |
| 17 | CTTTTCATCAATAATCTTACCTTT |
| 18 | CTTCTCATTAACTTACTTCATAAT |
| 19 | CAATTAACTACATACAATACATAC |
| 20 | TCATTCATATACATACCAATTCAT |
| 21 | TCATTTCAATCAATCATCAACAAT |
| 22 | AAACAAACTTCACATCTCAATAAT |
| 23 | TCATTTACTCAACAATTACAAATC |
| 24 | CTTTTTCAATCACTTTCAATTCAT |
| 25 | TACACTTTATCAAATCTTACAATC |
| 26 | CAATTCATTTACCAATTTACCAAT |
| 27 | CTACTATACATCTTACTATACTTT |
| 28 | TCAATTACCTTTTCAATACAATAC |
About spacerarm:
Amplimer F1 of the present invention is made up of three parts, and 5 ' end is held successively to 3 ': tag sequence, spacerarm, the forward amplimer of corresponding gene.
In the present invention, a spacer segment arm is inserted between Tag sequence and forward amplimer, spacerarm can connect two sections of base sequences (tag sequence and forward amplimer), but can not match with deoxyribonucleotide bases, in pcr amplification process, this spacerarm can stop DNA polyreaction to be carried out forward, thus makes PCR primer without the anti-tag sequence of matching with tag complementary, greatly improves the intensity of hybridization efficiency and detection signal.
Described spacerarm can be selected from: the one in C3Spacer, Spacer9, dSpacer, 3 ' C6Spacer, Spacer12, Spacer18 or PCSpacer, the present embodiment spacerarm is preferably C3Spacer.
All PCR primer are synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every bar primer after synthesis is mixed with the stock solution of 50umol/mL respectively with 10mmol/LTrisBuffer.
Two, the magnetic bead of Anti-tag sequence bag quilt
According to designed amplimer fragment, select tag sequence, reduce the secondary structure that between forward amplimer and tag sequence, specific binding may be formed to greatest extent, and the specific binding between anti-tag sequence, tag sequence and forward amplified production, oppositely amplimer, the anti-tag sequence that the magnetic bead numbering of selection is corresponding on magnetic bead is as shown in table 3:
Table 3 magnetic bead numbering and Anti-tag sequence
The magnetic bead purchased from American Luminex company selected, is coated in anti-tag sequence on magnetic bead.Be connected with the spacer sequence of 5-10 T between anti-tag sequence and magnetic bead, before each anti-tag sequence, namely add the spacer sequence of the preceding paragraph 5-10 T, anti-tag sequence is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.By the anti-tag sequence sterilizing ddH of synthesis
2o is made into the stock solution of 100nmol/ml.Described spacer sequence is for by anti-tag and magnetic bead surfaces is spaced apart or anti-tag is placed in the sequence of hydrophilic environments.By arranging the spacer sequence of suitable length between anti-tag sequence and magnetic bead, can reduce sterically hindered, improving the efficiency of hybridization and the specificity of hybridization.Common spacer sequence comprises poly dT, i.e. poly (dT), and oligomerization four polyoxyethylene glycol and (CH2) n spacerarm (n >=3), as (CH2) 12, (CH2) 18 etc.In addition, if there is poly (dA) interference, poly (TTG) can also be used as spacerarm.The present embodiment spacer sequence is preferably 5-10 T, and the process of magnetic bead bag quilt is as follows:
Get 5 × 10 respectively
6the carboxylated magnetic bead (purchased from Luminex company) of individual above-mentioned numbering is suspended in (pH4.5) in the MES solution of 50ul0.1mol/L, adds the anti-tag molecule (100nmol/ml) of 10ul synthesis.EDC (N-(3-Dimethylaminopropyl-N-ethylcarbodiimide) (purchased from the PierceChemical company) working fluid of preparation 10ng/ml.The EDC working fluid of 2.5ul is added, constant-temperature incubation 30 minutes, then the EDC working fluid adding 2.5ul in suspension containing magnetic beads, then constant-temperature incubation 30 minutes.After reaction terminates, the Tween-20 washing with 0.02% once, then is washed once with the SDS liquid of 0.1%.The magnetic bead being coated with anti-tag sequence after washing is resuspended in the Tris-EDTA solution of 100ul, and [10mmol/LTris (pH8.0), in 1mmol/LEDTA, 2-8 DEG C keeps in Dark Place.
During test kit embody rule of the present invention, the tag sequence of amplimer centering and the Anti-tag sequence of magnetic bead bag quilt are specifically selected see embodiment 2 and embodiment 3.
The synchronous detection of seven kinds of food-borne pathogenic microorganism gene test liquid phase chip reagent boxes described in embodiment 2 Application Example 1
The formula of described various solution is as follows:
The MES damping fluid (pH5.0) formula (250mL) of 50mM:
2 × Tm hybridization buffer
4 DEG C are stored in after filtration.
One, experimental technique
Relevant molecular biology experiment operative technique, all with reference to " molecular cloning ".
Two, the pcr amplification of testing sample
Primer5.0 is utilized to design eight pairs of amplimers (bacterial 16 S-rRNA conservative region containing as internal control sequence), multiplex PCR one step amplifies seven food-borne pathogenic microorganism conserved sequences, product size is respectively 224bp, 156bp, 180bp, 102bp, 290bp, 153bp, 127bp, and internal control sequence 169bp, the composition of its forward primer and reverse primer to be shown in embodiment 1 shown in table 1.
First multiple PCR primer working fluid is prepared: amplimer stock solution (50uM) 100uL getting corresponding gene respectively, in 1.5ml Eppendorf tube, mixes and is multiple PCR primer working fluid.Multi-PRC reaction system is as follows:
Pcr amplification program is: 95 DEG C of 3min; 94 DEG C of 20s, 56 DEG C of 30s, 72 DEG C of 30s, 30 circulations; 72 DEG C of 10min; 4 DEG C save backup.
Three, hybridization
1. (magnetic bead concentration is 2.5 × 10 to select corresponding eight kinds of magnetic beads
5individual/ml).Often kind of magnetic bead is respectively with different colours coding, and often kind of magnetic bead surfaces is connected to the anti-tag sequence that one section of pin can match with tag complementary simultaneously; Specifically as shown in table 4.The amplimer of 8 kinds of microorganisms to be detected to be coated with the magnetic bead of Anti-tag sequence as described in embodiment 1 and table 4.
The composition of table 4 detection kit tag and anti-tag
2. get 1uL respectively and often plant the magnetic bead of numbering in the Eppendorf tube of 1.5mL;
3. magnetic bead is in >=centrifugal the 1-2min of 10000g;
4. supernatant discarded, magnetic bead is resuspended in 2 × Tm hybridization buffer of 100uL, and vortex mixes;
5. get the above-mentioned suspension containing magnetic beads of 25uL in the 96 corresponding holes of hole filter plate, control wells adds the ddH of 25uL
2o;
6. the amplified production getting 5-25uL, in corresponding hole, uses ddH
2o complements to 50uL;
7. encase 96 orifice plates with lucifuge with masking foil, 95 DEG C of 60s, 52 DEG C of 15min hatch hybridization;
8. the magnetic bead after hybridization is in >=centrifugal the 2-5min of 3000g;
9. remove supernatant, magnetic bead is resuspended in 1 × Tm hybridization buffer of 75uL;
10. magnetic bead is in >=centrifugal the 2-5min of 3000g;
Magnetic bead is resuspended in 1 × Tm hybridization buffer of 75uL by 11, adds streptavidin-phycoerythrin (SA-PE) that 15uL concentration is 10ug/mL;
Hatch 15min for 12.52 DEG C, detect on Luminex instrument.
Four, result detects and data analysis
Reaction after product is detected by Luminex serial analysis instrument.Carrier using magnetic bead as reaction, using fluorescence detector as detection platform, carries out high-throughout multi objective parallel detection to nucleic acid molecule.In the manufacturing processed of magnetic bead, mix ruddiness and the infrared light staining agent of different ratios, thus form the magnetic bead of as many as 100 kinds of different colours codings.Different magnetic bead covalent attachment is for the nucleic acid molecule of difference thing to be detected as probe molecule, and reporter molecules with biotin labeling, and uses high-sensitive fluorescent dyeing.These magnetic beads and determinand, reporter molecules, fluorescent marker form the reading of complete magnetic bead detection system for Luminex system.Luminex reading system respectively excitated red laser and green laser is used for the detection of magnetic bead system.
Following requirement is had to fluorescent value (MFI) and data processing:
1. bacterium internal control sequence MFI is greater than 300 and is greater than PCR negative control MFI;
2.NETMFI=sample MFI-PCR negative control MFI (NETMFI be less than 0 represent with 0);
3. meet the data of above two conditions, can carry out subsequent analysis, not be inconsistent if any a condition, then this experiment is invalid;
4. according to the MFI definite threshold (cut-off value) of 50 routine dummies to each gene, to divide yin and yang attribute scope.Arranging of threshold value (cut-off value) is as follows: the average of 50 routine dummies and 2 times of variance sums, namely
refer to table 5.
5. result judges: after meeting above condition, the NETMFI of each gene compares with corresponding Cut-off value, if NETMFI is greater than Cut-off value, then this gene is positive, represents that sample exists the pathogenic microorganism kind corresponding with this gene; If NETMFI is less than Cut-off value, then this gene is negative, represents that sample does not exist the pathogenic microorganism kind corresponding with this gene or this microorganism below the detectability of this test kit.
Table 5 threshold value (cut-off value)
| Gene | Cut-off value |
| rpsU-dnaG | 189 |
| prfA | 164 |
| flic | 201 |
| Sa442 | 115 |
| ipaH | 133 |
| fim | 175 |
| toxR | 160 |
| 16S-rRNA | 130 |
Use present method to detect 20 increments food-borne pathogenic microorganism gene originally, experimental data meets above-mentioned requirements, by comparing with cut-off value in above table, judges that it is negative or positive.Mark examination criteria described in " food microbiological analysis " pushing away GT4789 recommendation using food safety country and, as the contrast of the inventive method, calculate the identical rate of qualitative checking method detected result provided by the present invention.
Table 6. Luminex sample fluorescence value (MFI)
Table 7 Luminex sample yin and yang attribute result of determination [25mg (mL) sample size]
Table 8 National Standard Method pattern detection result [25mg (mL) sample size]
From above-mentioned detected result, present method detects 20 this food-borne pathogenic microorganism of increment gene test results and the National Standard Method rate of coincideing reaches 100%.Whether food-borne pathogenic microorganism gene test liquid-phase chip provided by the present invention accurately can exist corresponding pathogenic microorganism in qualitative analysis sample, and result is reliable and stable.
The liquid-phase chip of the amplimer of the different tag sequence of embodiment 3 is to the detection of target gene
One, the design (selection of Tag sequence and Anti-Tag sequence) prepared of liquid-phase chip
For the prfA gene test liquid-phase chip of the rpsU-dnaG gene of Enterobacter sakazakii, Listeria monocytogenes, respectively for rpsU-dnaG and prfA gene design amplimer, described amplimer is made up of three parts, 5 ' end is held successively to 3 ': the forward amplimer of tag sequence+spacerarm+corresponding gene, meanwhile, biotin labeling in 5 ' end band of the reverse amplimer of amplimer R1.Wherein, the tag sequence in described amplimer is selected from SEQIDNO.17-SEQIDNO.28 respectively, accordingly, is coated in and magnetic bead is selected from SEQIDNO.29-SEQIDNO.40 with the anti-tag sequence that corresponding tag complementary matches.Specific design is as shown in following table (table 9).The synthesis, detection method etc. of amplimer as described in embodiment 1 and embodiment 2, Tag sequence and anti-tag sequence bag as shown in table 9 by the selection of magnetic bead.
Wherein, all use internal control 16S-rRNA in 3 groups of experimental group, tag sequence uses SEQIDNO.24, anti-tag to be SEQIDNO.36, uses No. 54 encoded magnetic bead.The synthesis of amplimer, anti-tag sequence bag by magnetic bead, detection method etc. as described in embodiment 1 and embodiment 2.
Design prepared by table 9 liquid-phase chip
One, sample detection
Adopt liquid-phase chip prepared by above-mentioned design, by testing process described in embodiment 2 and method, sequence number 21-40 sample is detected, detected result is in table 10 and table 11, simultaneously, examination criteria is as the contrast method of the present embodiment to use food safety country mark to push away described in " food microbiological analysis " that GT4789 recommends, and detected result is in table 12:
Table 10 pattern detection results and analysis (MFI)
Table 11 pattern detection results and analysis
Table 12 pattern detection results and analysis
As can be seen from the present embodiment, for the liquid-phase chip of different target genes, amplimer uses Tag sequences different in embodiment 1 table 2, its result is still reliable and stable, but when amplimer selects that in embodiment 2, tag sequence and specific primer sequence are arranged in pairs or groups, effect better (signal to noise ratio is better), see the present embodiment test group 1.
Other different tag sequence and specific primer sequence are arranged in pairs or groups, and with coming to the same thing of embodiment 2 and the present embodiment, concrete data are omitted.
The liquid-phase chip of embodiment 4 different interval arm is to the detection of target gene
One, the design (selection of spacerarm) prepared of liquid-phase chip
Amplimer F1 of the present invention is made up of three parts, and 5 ' end is held successively to 3 ': tag sequence, spacerarm, the forward amplimer of corresponding gene.In the present invention, a spacer segment arm is inserted between Tag sequence and forward amplimer, spacerarm can connect two sections of base sequences (tag sequence and amplimer), but can not match with deoxyribonucleotide bases, in pcr amplification process, this spacerarm can stop DNA polyreaction to be carried out forward, thus makes PCR primer without the anti-tag sequence of matching with tag complementary, greatly improves the intensity of hybridization efficiency and detection signal.
The present embodiment, for the ipaH gene test liquid-phase chip of Shigellae, adopts spacerarm C3Spacer, Spacer9, dSpacer, Spacer12 respectively, designs corresponding amplimer, contrast its Detection results.Specific design is as shown in following table (table 13).The synthesis of amplimer, anti-tag sequence bag by magnetic bead, detection method etc. as described in embodiment 1 and embodiment 2.
The selection of table 13 spacerarm
| Spacerarm kind | Experimental group |
| C3Spacer | 1 |
| Spacer9 | 2 |
| dSpacer | 3 |
| Spacer12 | 4 |
Two, sample detection
Adopt liquid-phase chip prepared by above-mentioned design, by testing process described in embodiment 2 and method, sample 41-45 detected, detected result following (in table, data are for detecting fluorescent value):
Table 14 sample detection result (detecting fluorescent value <MFI value >)
| Catalogue number(Cat.No.) | Experimental group 1 | Experimental group 2 | Experimental group 3 | Experimental group 4 |
| N | 12 | 6 | 12 | 5 |
| 41 | 52 | 78 | 25 | 56 |
| 42 | 40 | 50 | 84 | 72 |
| 43 | 1557 | 1337 | 1288 | 1170 |
| 44 | 60 | 34 | 39 | 42 |
| 45 | 29 | 84 | 40 | 72 |
The detection fluorescent value that 4 groups are designed is carried out statistical analysis, proves that 4 groups of Detection results designed do not have difference.Therefore, the design of these 4 kinds of spacerarms is equivalent, but when spacerarm selects C3Spacer, effect better (signal to noise ratio is better).Other detects the liquid-phase chip of the tag sequence of the gene intervening sequence different between forward amplimer for different target, result is as this embodiment, when namely selecting C3Spacer, Spacer9, dSpacer, Spacer12, its result is still reliable and stable, but for design chips test kit of the present invention, when spacerarm selects C3Spacer, signal to noise ratio can be better.Because result is roughly the same, concrete data are omitted.
The performance verification of embodiment 5 food-borne pathogenic microorganism gene test liquid-phase chip
One, inside authentication
Adopting food-borne pathogenic microorganism gene test liquid-phase chip in above-described embodiment 1, is that sample detects with reference culture, carries out performance verification to 7 kinds of food-borne pathogenic microorganism liquid-phase chip detection methods of the present invention.The synthesis of amplimer, anti-tag sequence bag by magnetic bead, detection method etc. as described in embodiment 1 and embodiment 2.
1, specificity
Short run specific test adopts bacterial strain in table 15, and detected result is in table 16.
Table 15 short run specific test adopts bacterial strain
Note: wherein ATCC is the standard bacteria of AmericanTypeCultureCollection, CMCC is the standard bacteria of Chinese medicine bacterium preservation administrative center.NCTC is Britain's state-run type species collection shop standard bacteria.
Table 16 short run specific test detected result (Net-MFI value)
From the detected result of table 16, primer all good with the specificity of probe, without non-specific no cross reaction, non-false positive and false negative result, with a high credibility.
2, sensitivity/detect and track
1) inoculate seven kinds of bacterium to be respectively equipped with in the small test tube of respective standard enrichment liquid substratum in 5, grow to about 1 × 10
8cFU/mL (measures OD
600value about 0.5).
2) respectively get a certain amount of bacterium liquid respectively, add physiological saline with 10 times of dilutions, be diluted to 10 respectively
4, 10
3, 10
2, 10
1, 10
0cFU/mL.
3) get seven kinds of each 1mL of bacterium of each gradient respectively, get equal-volume physiological saline is blank simultaneously, and detect according to the inventive method, determine the detection limit of the inventive method, detected result is in table 17.
4) seven kinds of bacterium being carried out being mixed into total bacterium amount is 10
4cFU/mL, 10
3cFU/mL, 10
2cFU/mL, 10
1cFU/mL, 10
0cFU/mL, detected result is in table 18.
Use test kit of the present invention to detect above-mentioned bacterial classification, the synthesis of amplimer, anti-tag sequence bag by magnetic bead, detection method etc. as described in embodiment 1 and embodiment 2.
The single pathogenic microorganism sensitivity results of table 17 (Net-MFI value)
Table 18 pathogenic microorganism Mixed Sensitivity result (Net-MFI value)
As can be seen here, the sensitivity results of comprehensive single microbial and seven kinds of microorganism mixing is known, and the sensitivity of seven kinds of food-borne pathogenic microorganisms is 10CFU/mL.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (9)
1. food-borne pathogenic microorganism detects a liquid phase chip reagent box, it is characterized in that, includes:
(A) for the amplimer pair that microbial gene to be detected designs, often pair of amplimer includes amplimer F1 and amplimer R1, described amplimer F1 holds successively by tag sequence from 5 ' end to 3 ', spacerarm, the forward primer composition of often kind of corresponding microorganism to be detected, described amplimer R1 is the reverse primer that 5 ' end band has biotin labeled, corresponding microorganism to be detected, described tag sequence is selected from SEQIDNO.17-SEQIDNO.28, described spacerarm is selected from any one in C3Spacer, Spacer9, dSpacer, 3 ' C6Spacer, Spacer12, Spacer18 or PCSpacer, the forward primer of described microorganism to be detected and reverse primer include following at least one pair of: for SEQIDNO.1 and SEQIDNO.2 of Enterobacter sakazakii, for SEQIDNO.3 and SEQIDNO.4 of Listeria monocytogenes, for SEQIDNO.5 and SEQIDNO.6 of colon bacillus O157, for SEQIDNO.7 and SEQIDNO.8 of streptococcus aureus, for SEQIDNO.9 and SEQIDNO.10 of Shigellae, for Salmonellas SEQIDNO.11 and SEQIDNO.12, for SEQIDNO.13 and SEQIDNO.14 of Vibrio parahemolyticus,
(B) be coated with the magnetic bead of Anti-tag sequence, the tag complementary of described Anti-tag sequence and corresponding microorganism to be detected matches, and described Anti-tag sequence is selected from SEQIDNO.29-SEQIDNO.40; Magnetic bead for difference microorganism to be detected has different fluorescence-encoded.
2. food-borne pathogenic microorganism according to claim 1 detects liquid phase chip reagent box, and it is characterized in that, described spacerarm is C3Spacer.
3. food-borne pathogenic microorganism according to claim 1 detects liquid phase chip reagent box, it is characterized in that, described tag sequence is: the SEQIDNO.17 for Enterobacter sakazakii, the SEQIDNO.18 for Listeria monocytogenes, the SEQIDNO.19 for colon bacillus O157, the SEQIDNO.20 for streptococcus aureus, for the SEQIDNO.21 of Shigellae, for Salmonellas SEQIDNO.22, SEQIDNO.13 and SEQIDNO.23 for Vibrio parahemolyticus.
4. the food-borne pathogenic microorganism according to any one of claim 1-3 detects liquid phase chip reagent box, and it is characterized in that, the forward primer of described microorganism to be detected and reverse primer also include SEQIDNO.15 and SEQIDNO.16 for internal control bacterium.
5. food-borne pathogenic microorganism according to claim 4 detects liquid phase chip reagent box, and it is characterized in that, the tag sequence for internal control bacterium is SEQIDNO.24.
6. the food-borne pathogenic microorganism according to any one of claim 1-3 detects liquid phase chip reagent box, it is characterized in that, is also provided with spacer sequence between described magnetic bead and Anti-tag sequence.
7. food-borne pathogenic microorganism according to claim 6 detects liquid phase chip reagent box, and it is characterized in that, described spacer sequence is 5-10 T.
8. the amplimer of food-borne pathogenic microorganism detection, it is characterized in that, include following at least one pair of: for SEQIDNO.1 and SEQIDNO.2 of Enterobacter sakazakii, for SEQIDNO.3 and SEQIDNO.4 of Listeria monocytogenes, for SEQIDNO.5 and SEQIDNO.6 of colon bacillus O157, for SEQIDNO.7 and SEQIDNO.8 of streptococcus aureus, for SEQIDNO.9 and SEQIDNO.10 of Shigellae, for Salmonellas SEQIDNO.11 and SEQIDNO.12, for SEQIDNO.13 and SEQIDNO.14 of Vibrio parahemolyticus.
9. the amplimer of food-borne pathogenic microorganism detection according to claim 8, is characterized in that, also include SEQIDNO.15 and SEQIDNO.16 for internal control bacterium.
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