CN101240335A - Gene chip and kit for detecting common pathogen in dairy products - Google Patents
Gene chip and kit for detecting common pathogen in dairy products Download PDFInfo
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- CN101240335A CN101240335A CNA2007101665303A CN200710166530A CN101240335A CN 101240335 A CN101240335 A CN 101240335A CN A2007101665303 A CNA2007101665303 A CN A2007101665303A CN 200710166530 A CN200710166530 A CN 200710166530A CN 101240335 A CN101240335 A CN 101240335A
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Abstract
The present invention provides a gene chip for detecting common pathogen in dairy, which comprises a solid-phase vector and an oligonucleotide probe fixed on the solid-phase vector, wherein the oligonucleotide probe includes DNA fragment or its complementary DNA or RNA sequence selected from E. sakazakii, streptococcus pyogenes, staphylococcus aureus, Klebsiella pneumoniae, Klebsiella oxytoca, Listeria monocytogenes, salmonella and 16S-23S rDNA intergenic spacer region of Bacillus cereus, as well as ipaH gene of Shigella or gyrB gene of citrobacter freundii. The invention further provides a reagent box which uses the gene chip to detect pathogen in dairy. The gene chip and the reagent box of the invention for detecting pathogen in dairy are easy to operate, highly precise and greatly repeatable.
Description
Technical field
The present invention relates to a kind of gene chip and use test kit, relate in particular to the gene chip and the test kit that detect common pathogen in the milk preparation with detecting
Background technology
Since in April, 2004, " milk powder inferior " incident took place in the Fuyang, the milk powder safety problem became the focus of various circles of society's extensive concern.The main consumer group of milk powder is the lower crowds of immunizing power such as infant, children, old man or patient, especially the infant is the important consumer group of milk powder, therefore, the security of milk powder is particularly important, is subjected to the great attention of State Council, functional department and governments at all levels always." 15 " country great science and technology milk industry special project had been assigned by the Department of Science and Technology in 2002, and several frequently seen pathogenic bacterium in the milk-product have been carried out the research of method for quick.This shows that the method that research and foundation quick and precisely detect the pathogenic bacterium in milk powder and the milk preparation is very necessary with the safety that ensures milk preparation.
According to the relevant regulations of Chinese national standard and State General Administration for Quality Supervision's rower, inspection and quarantine department to the pathogenic bacterium project that milk powder detects is at present: Salmonellas, Shigellae, streptococcus aureus, micrococcus scarlatinae and Enterobacter sakazakii.The present invention has increased Klebsiella pneumonia, acid-producing Klebsiella bacterium, Listeria monocytogenes, citrobacter freundii and the Bacillus cereus that causes concern in recent years again in addition.Therefore ten kinds of common pathogenic bacterias are in the milk powder of the present invention's detection and the milk preparation: Salmonellas, Shigellae, streptococcus aureus, micrococcus scarlatinae, Enterobacter sakazakii, Klebsiella pneumonia, acid-producing Klebsiella bacterium, Listeria monocytogenes, citrobacter freundii and Bacillus cereus.
Mostly the method that detects pathogenic bacterium in the GB is to adopt the traditional separation and Culture and the method for biochemical identification, the report assay roughly needs 5～7 days, in addition the method for inspection loaded down with trivial details, waste time and energy, not only become a heavy burden of inspection department, and more and more can not satisfy the needs of food safety and growing international trade.In quality testing department inside, adopt traditional technique in measuring Salmonellas, Shigellae, streptococcus aureus and micrococcus scarlatinae, the method of employing industry standard usefulness PCR detects the Enterobacter sakazakii in the milk powder, but does not all detect the method for Klebsiella pneumonia in GB and the rower.
In July, 1997, Affymetrics, Inc. (Santa Clara, CA) the 6th, 228, No. 575 U.S. Patent Publications of the people such as Thomas R. of company invention the method for microorganism being carried out deciding kind and phenotype analytical with biochip technology.Since nineteen nineties, biochip technology is set up as a kind of brand-new foranalysis of nucleic acids detection technique, and the progressively development along with carrying out of the Human Genome Project.Technology such as molecular biology, unicircuit manufacturing, computer, semi-conductor, confocal laser scanning, fluorescent mark have been used in this technological synthesis, make testing process have characteristics such as susceptibility height, high specificity, large scale integration, automatization, simple and efficient to handle, efficient height, be widely used at aspects such as genetic expression correlative study and bio-pharmaceuticals researchs.Therefore,, not only can simplify detection means greatly, improve detection speed, and have high accuracy, highly sensitive, high-throughput, repeatable plurality of advantages such as strong if biochip technology is used for the evaluation of bacterium.
The target molecule of the microorganism identification of the most normal use at present is 16S rRNA and 23S rRNA, existing both at home and abroad a lot of bibliographical informations.Utilize 16S rRNA and 23S rRNA bacterial micro-organism can be identified to planting or belonging to, but for the nearer bacterium kind of sibship or the evaluation very difficult (BodrossyL, Sessitsch be microarrays in microbial diagnostics.Current Opinion in Microbiology 7:245-25 A.2004.Oligonucleotide) of genus.The evaluation that utilizes between 16S-23S rRNA the district to carry out bacterium as target molecule has at present become focus (the Nubel U.SchmidtPM of research gradually, Rei β E, et a1.2004.Oligonucleotide microarray for identification ofBacillus anthracis based on intergenic transcribed spacers in ribosomal DNA.FEMS Microbiology Letters 240:215-223.), its variation speed is equivalent to 16SrRNA or 23S rRNA ten times, therefore has higher resolving power, even bacterium can be distinguished type, have 16S rRNA and the incomparable advantage of 23S rRNA for the differentiation of the nearer kind of sibship.Two ends are 16S rRNA gene and 23S rRNA gene regions of guarding simultaneously, can can avoid the problem of many primer dimers that primer is brought at the conserved regions design universal primer at two ends, length is between 200bp-1000bp, the size easy handling, thereby the amplification of target sequence and labeling process are simplified more, also easier control, the actual requirement that match operation is simple and efficient.
Summary of the invention
An object of the present invention is to provide a kind of gene chip that detects pathogenic bacterium in the milk preparation, to remedy the defective of the time-consuming consumption power that the common pathogen detection technique exists in traditional detection milk powder and the milk preparation, expansion pathogenic bacteria sensing range, improve detection sensitivity and specificity, reduce labour intensity, shorten sense cycle.
The gene chip of pathogenic bacterium comprises solid phase carrier and the oligonucleotide probe that is fixed on this solid phase carrier in the detection milk preparation of the present invention, and wherein said this oligonucleotide probe comprises one or more sequences of choosing from following sequence:
(1) dna sequence dna of between the 16S-23S rDNA of Enterobacter sakazakii, micrococcus scarlatinae, streptococcus aureus, Klebsiella pneumonia, acid-producing Klebsiella bacterium, Listeria monocytogenes, Salmonellas and Bacillus cereus, choosing the gyrB gene of ipaH (invasion plasmid antigengene) gene of district and Shigellae or citrobacter freundii;
(2) complementary dna sequence of the dna sequence dna of choosing in above-mentioned (1);
(3) the complementary RNA sequence of the dna sequence dna of choosing in above-mentioned (1) or (2).
Wherein, the above-mentioned dna fragmentation of choosing gyrB (the DNA gyrase subunit B) gene of the ipaH gene of district and Shigellae or citrobacter freundii between the 16S-23S rDNA of Enterobacter sakazakii, micrococcus scarlatinae, streptococcus aureus, Klebsiella pneumonia, acid-producing Klebsiella bacterium, Listeria monocytogenes, Salmonellas and Bacillus cereus has one or more dna sequence dnas in the dna sequence dna shown in the SEQ IDNO:2-SEQ ID NO:28; The dna fragmentation of choosing from bacterium 16s rDNA conserved regions has the dna sequence dna shown in the SEQ ID NO:1.
Wherein, above-mentioned oligonucleotide probe also comprises positive control probe, negative control probe and fluorescent probe.Above-mentioned positive control probe is preferably dna fragmentation or its complementary DNA or the RNA sequence of choosing from bacterium 16s rDNA conserved regions, above-mentioned positive control probe has the dna sequence dna shown in the SEQ ID NO:1 in the preferred embodiment of the present invention.
Another object of the present invention provides the application of above-mentioned gene chip, and it can be used at least a detection of Enterobacter sakazakii, micrococcus scarlatinae, streptococcus aureus, Klebsiella pneumonia, acid-producing Klebsiella bacterium, Listeria monocytogenes, Salmonellas, Shigellae, citrobacter freundii, Bacillus cereus.
Wherein, applied detection primer comprises at least a in the dna sequence dna shown in the SEQ ID NO:29-SEQ ID NO:34.
A further object of the present invention provides a kind of test kit that utilizes pathogenic bacterium in the said gene chip detection milk preparation, this test kit comprises the above-mentioned gene chip of the present invention, and this test kit also comprises at least a of the dna sequence dna shown in the detection primer SEQ ID NO:29-SEQ ID NO:34 or its complementary dna sequence.
Test kit of the present invention also comprises interpretation software and the specification sheets that hybridizing box, hybridization solution and Analysis and Identification result use.
A further object of the present invention provides the application of above-mentioned test kit, and it can be used at least a detection of Enterobacter sakazakii, micrococcus scarlatinae, streptococcus aureus, Klebsiella pneumonia, acid-producing Klebsiella bacterium, Listeria monocytogenes, Salmonellas, Shigellae, citrobacter freundii, Bacillus cereus.
As seen from the above technical solutions, the present invention introduces biochip technology common pathogen detection range in milk powder and the milk preparation first, set up a kind of quick, sensitive, the accuracy height, common pathogen detects gene chip and detection method thereof in brand-new milk powder that repeatability is strong and the milk preparation, utilize gene chip of the present invention can reach the purpose that detects pathogenic bacterium common in milk powder and the milk preparation, because it is easy and simple to handle, the accuracy height, repeatability is strong, for as inspection and quarantining for import/export department the inspection and quarantine of milk powder and milk preparation is still all had important use value to the milk powder and the milk preparation safety verification of milk preparation processing enterprise and large supermarket.
For above and other objects of the present invention, feature and advantage can be become apparent, below especially exemplified by preferred embodiment, and cooperate Figure of description, be described in detail below.
Description of drawings
Fig. 1 is the profile synoptic diagram of an embodiment of gene chip of the present invention.
Fig. 2 is the rule synoptic diagram of arranging of single dot matrix probe on the embodiment of gene chip of the present invention.
Fig. 3 A is the results of hybridization when utilizing gene chip of the present invention to detect Enterobacter sakazakii in milk powder and the milk preparation.
Fig. 3 B is the results of hybridization when utilizing gene chip of the present invention to detect micrococcus scarlatinae in milk powder and the milk preparation.
Fig. 3 C is the results of hybridization when utilizing gene chip of the present invention to detect Shigellae in milk powder and the milk preparation.
Fig. 3 D is the results of hybridization when utilizing gene chip of the present invention to detect streptococcus aureus in milk powder and the milk preparation.
Fig. 3 E is the results of hybridization when utilizing gene chip of the present invention to detect acid-producing Klebsiella bacterium in milk powder and the milk preparation.
Fig. 3 F is the results of hybridization when utilizing gene chip of the present invention to detect Salmonellas in milk powder and the milk preparation.
Fig. 3 G is the results of hybridization when utilizing gene chip of the present invention to detect Listeria monocytogenes in milk powder and the milk preparation.
Fig. 3 H is the results of hybridization when utilizing gene chip of the present invention to detect Bacillus cereus in milk powder and the milk preparation.
Fig. 3 I is the results of hybridization when utilizing gene chip of the present invention to detect Klebsiella pneumonia in milk powder and the milk preparation.
Fig. 3 J is the results of hybridization when utilizing gene chip of the present invention to detect citrobacter freundii in milk powder and the milk preparation.
Embodiment
The design and the preparation of embodiment 1 probe
1. sequence obtains:
(1) acquisition of bacterium 16s rDNA sequence: download the whole 16s rDNA sequences that obtain ten kinds of bacterium from the GenBank public database.
(2) acquisition of region sequence between 16S-23S rDNA: download region sequence between all 16S-23S rDNA that obtain Salmonellas, streptococcus aureus, micrococcus scarlatinae, Enterobacter sakazakii and their nearly edge bacterium from the GenBank public database.
Region sequence between 21 of 9 strain bacterium is only arranged in the region sequence public database between Klebsiella pneumonia, and region sequence has only 1 sequence of 1 strain bacterium between acid-producing Klebsiella bacterium, does not satisfy the demand of screening specific probe.This laboratory is to 22 Klebsiella pneumoniaes, 3 strain ozena klebsiellas, 2 strain noses scleromas klebsiella, 4 strain acid-producing Klebsiella bacteriums, autochthonal klebsiella of 1 strain and 1 strain plant living klebsiella and 2 strains separate the ornithine klebsiella carried out between the order-checking in district.Distinguish between the primer amplification of 16s rDNA and 23s rDNA sequences Design with above-mentioned, be connected to behind the PCR product purification on the T carrier, the back electricity changes to advance in the DH5 α competence, and picking contains the plasmid order-checking of 500bp-1kbp, sequenator ABI 3700.The sequence that records is with the splicing of Staden Package software, thus obtain Klebsiella pneumonia and acid-producing Klebsiella bacterium and nearly edge bacterium thereof between region sequence.
(3) acquisition of ipaH gene order: download the whole ipaH gene orders that obtain four kinds of Shigellae from the GenBank public database.
(4) acquisition of gyrB gene order: we have downloaded three incomplete sequences of citrobacter freundii in the GenBank public database.In addition, University of Washington and Sanger carry out genome sequencing to Ke Shi citric acid bacillus and grinding tooth citric acid bacillus respectively, and the applicant obtains its gyrB gene order through bioinformatic analysis.The gyrB gene that while applicant's laboratory does not have propanedioic acid citric acid bacillus, 1 strain grinding tooth citric acid bacillus to 9 strain citrobacter freundiis, 1 strain checks order.
2. probe design:
(1) general probe of bacterium: 16s rDNA sequence that ten kinds of bacterium are whole and the 16srDNA sequence of other bacterium import in the Glustal X software, choose near between the district one section 16s rDNA conserved sequence as probe, satisfy length 27bp ± 2bp, 68 ℃ ± 3 ℃ of Tm values.
(2) region probe between: region sequence between all 16S-23S rDNA of Enterobacter sakazakii, micrococcus scarlatinae, streptococcus aureus, Klebsiella pneumonia, acid-producing Klebsiella bacterium, Listeria monocytogenes, Salmonellas and Bacillus cereus is imported in the Glustal X software respectively, thereby learn this bacterium between region sequence several types is arranged, choose one for every type and represent sequence to do the blastn comparison in common data NCBI, determining could be as the position of special target spot and special target spot.Contrast Glustal X comparison result is chosen and is satisfied the character that this section is not all guarded between homophyletic, while length 27bp ± 2bp, 68 ℃ ± 3 ℃ of Tm values.
(3) ipaH gene probe: the above-mentioned ipaH gene order of downloading four kinds of Shigellae that obtain from the GenBank public database is compared with sequence alignment software Glustal X, find this gene conservative section, should guard section and import in the OligoArray2.0 software, parameter setting is as follows :-n20;-1 30;-L 40;-D 3000;-t 79;-T 90; 65 ℃ of-s; 65 ℃ of-x;-N 2;-p 33, and-P 65;-m GGGGG CCCCC TTTTT AAAAA;-g 15.The online designing probe of working procedure.From the output result, select length, the probe that the Tm value is 68 ℃ ± 3 ℃ at 27bp ± 2bp.
(4) gyrB gene probe: in the gyrB sequence importing Glustal X software of whole citrobacter freundiis that above-mentioned download and own order-checking are obtained, find this gene conservative section, should guard section and import in the OligoArray2.0 software, parameter setting is as follows :-n 20;-l 30;-L 40:-D 3000;-t 79;-T 90; 65 ℃ of-s; 65 ℃ of-x;-N 2;-p 33, and-P 65;-m GGGGGCCCCC TTTTT AAAAA;-g 15.The online designing probe of working procedure.From the output result, select length, the probe that the Tm value is 68 ℃ ± 3 ℃ at 27bp ± 2bp.
3. probe is synthetic: entrust probe Synesis Company (Beijing AudioCodes company) synthetic, standby after 5 ' of the probe sequence in the following table 1 is held prolongation 10 T (10 T that do not comprise prolongation in the fluorescent probe sequence shown in the table 1) and amination.
4. probe screening: will synthesize the good also an amount of dilution of probe dissolving back and on glass chip, make gene chip with gene chip sample applying instrument point, carry out the probe screening by hybrid experiment, finally obtain being used to prepare gene chip of the present invention required special, sensitive probe.
In a preferred embodiment of the invention, selected 31 length, and carried out the probe screening, finally obtained probe as shown in table 1 by 269 hybrid experiments at 35bp ± 2bp, Tm75 ℃ ± 2 ℃ probe.Wherein, the probe sequence that is numbered NO.1 (SEQ ID NO:1) is selected from the 16s rDNA of all bacteriums, be used for detecting as positive control whether bacterium is arranged, the probe that is numbered NO.2 is as fluorescent probe, the probe that is numbered NO.3 is a poly T fragment, as negative control, the probe that is numbered NO.4 is 50% DMSO, as blank, 21 probe sequences (SEQ ID NO:2-SEQ ID NO:22) of numbering NO.5-NO.25 are selected from common pathogen (Enterobacter sakazakii, micrococcus scarlatinae, streptococcus aureus, Klebsiella pneumonia, acid-producing Klebsiella bacterium, Listeria monocytogenes, Salmonellas and Bacillus cereus) 16S-23SrDNA between distinguish, 4 probe sequences of numbering NO.26-NO.29 (SEQ ID NO:23-SEQ ID NO:26) are selected from the ipaH gene of Shigellae, and 2 probe sequences of numbering NO.30-NO.31 (SEQ IDNO:27-SEQ ID NO:28) are selected from the gurB gene of citrobacter freundii.
Table 1: sequence oligonucleotide probe of selecting for use on the gene chip of the present invention and detectable pathogenic bacterium
| The probe numbering | SEQ ID | Probe sequence (5 '-3 ') | Detectable pathogenic bacterium |
| NO. 1 | NO：1 | TTGTACACACCGCCCGTCACACCAT | Bacterium is over against photograph |
| NO. 2 | Cy3_TTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTTT | Fluorescent probe | |
| NO. 3 | TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTT | Negative control | |
| NO. 4 | 50％DMSO | Blank | |
| NO. 5 | NO：2 | GTCAGAGTCTCTCAAACTCGCAGCACG | Enterobacter sakazakii |
| NO. 6 | NO：3 | CCACCATCACTTCAGAGTGTACTCAGT GAG | Enterobacter sakazakii |
| NO. 7 | NO：4 | ACAGACACGCTGCTGTATTTCTCCGTA AT | Enterobacter sakazakii |
| NO. 8 | NO：5 | GGCTCCATCAGGATACAATCCTACTAA ACTT | Micrococcus scarlatinae |
| NO. 9 | NO：6 | CACATGGTCAGATTCCTAATTTTCTAC AGA | Micrococcus scarlatinae |
| NO. 10 | NO：7 | GCTAAAGCGAGCGTTGCTTAGTATCCT A | Micrococcus scarlatinae |
| NO. 11 | NO：8 | GCTTATGCGAGCGCTTGACAATCTATT CT | Streptococcus aureus |
| NO. 12 | NO：9 | TAAAGCAGTATGCGAGCGCTTGACTA AA | Streptococcus aureus |
| NO. | NO：10 | ATGTGAACGTTTGACTTATAAAAATGG | Golden yellow grape |
| 13 | TGG | Coccus | |
| NO. 14 | NO：11 | CTGATAGATGTAAAGAAGCAAGACGG C | Acid-producing Klebsiella bacterium |
| NO. 15 | NO：12 | ACGGCTGCGAAGTCGCGACACCT | Acid-producing Klebsiella bacterium |
| NO. 16 | NO：13 | TCTAGCGGTTAGGACTCCGCCCT | Acid-producing Klebsiella bacterium |
| NO. 17 | NO：14 | TGAAAGGCACAACCAACCGATATCT | Acid-producing Klebsiella bacterium |
| NO. 18 | NO：15 | ATTTGAAGAGGTTGCAAACGATGGG | Klebsiella pneumonia |
| NO. 19 | NO：16 | GGCCTACCAAATTTGCGAAGCAA | Klebsiella pneumonia |
| NO. 20 | NO：17 | GAGGTTCTGACTACACGATGGGGCTAT | Salmonellas |
| NO. 21 | NO：18 | AGGCACTATGCTTGAAGCATCGCGC | Listeria monocytogenes |
| NO. 22 | NO：19 | AAGAAATACAAATAATCATACCCTTTT ATG | Listeria monocytogenes |
| NO. 23 | NO：20 | TTTCTTTCTGACACAAGAAATACAAAT AATCATA | Listeria monocytogenes |
| NO. 24 | NO：21 | ATCAATATAAGTTTCCGTGTTTCGTTTT CG | Bacillus cereus |
| NO. 25 | NO：22 | TTCTTTGAAAACTAGATAACAGTGTAG CTCAT | Bacillus cereus |
| NO. 26 | NO：23 | GATAATGATACCGGCGCTCTGCTCTCC | Shigellae |
| NO. 27 | NO：24 | AGATAGAAGTCTACCTGGCCTTCCAGA CCA | Shigellae |
| NO. 28 | NO：25 | AGGAAATGCGTTTCTATGGCGTGTCG | Shigellae |
| NO. 29 | NO：26 | ACCATGGCATGCTGTACTGAAGCGTAC | Shigellae |
| NO. 30 | NO：27 | CGTAAAGACGCTGAACTGAACCTGT | Citrobacter freundii |
| NO. 31 | NO：28 | CTGATGAAGCGAAAGTCACTGCCTG | Citrobacter freundii |
Embodiment 2 primer design and preparation
1. sequence obtains: the sequence of the same designing probe.
2. design primer:
(1) region sequence primer design between the amplification: after the 16S rDNA of 10 kinds of bacteriums that download obtains from public database NCBI compares with sequence alignment software Glustal X, choose near between the district one section 16s rDNA conserved sequence as upstream primer, length meets 50 ℃ ± 5 ℃ of Tm values, length 17bp ± 2bp, Hairpin:NONE, Dimer:NONE, False Priming:NONE, CrossDimer:NONE, and comprises bacterium general probe sequence interior.
(2) amplification ipaH gene order primer design: the above-mentioned ipaH gene order of downloading four kinds of Shigellae that obtain from the GenBank public database is compared with sequence alignment software Glustal X, find this gene conservative section, should guard section imports in primer-design software PrimerPremier 5.0 softwares, relevant parameter is set as follows: Search For:PCR Primers, Searchtypes:Both.Search Ranges:Sense Primer 1 to 672, Anti-sense Primer 1 to 672, PCR Product Size:100bp to 1000bp.Primer Length:20bp ± 2bp.Search Mode:Automatic.From the output result, choose 50 ℃ ± 5 ℃ of Tm values, length 17bp ± 2bp, Hairpin:NONE, Dimer:NONE, False Priming:NONE, Cross Dimer:NONE and comprise probe sequence at interior primer.
(3) amplification gyrB gene order primer design: all citrobacter freundii gyrB gene orders that above-mentioned download obtained and oneself checks order are compared with sequence alignment software Glustal X, find this gene conservative section, should guard section imports in primer-design software Primer Premier 5.0 softwares, relevant parameter is set as follows: Search For:PCR Primers, Search types:Both.Search Ranges:Sense Primer 1 to 2415, Anti-sense Primer 1 to 2415, PCRProduct Size:100bp to 1000bp.Primer Length:20bp ± 2bp.Search Mode:Automatic.From the output result, choose 50 ℃ ± 5 ℃ of Tm values, length 17bp ± 2bp, Hairpin:NONE, Dimer:NONE, False Priming:NONE, Cross Dimer:NONE and comprise probe sequence at interior primer.
3. primer is synthetic: entrust primer Synesis Company (Beijing AudioCodes) synthetic, standby primer sequence in the following table 2.
4. primer screening: will synthesize good primer dissolving and an amount of dilution, on the one hand detect the amplification of primer by increase the respectively gyrB gene order of district, Shigellae ipaH gene and citrobacter freundii between 16S-23S rDNA of PCR reaction, another fermentation, three pairs of primers carry out the consistency of three pairs of primers of augmentation detection simultaneously to the different strains of 10 kinds of bacterium, finally obtain being used to prepare gene chip of the present invention required special, sensitive primer.
In a preferred embodiment of the invention, 6 of primers that not only comprise probe but also be fit to use simultaneously common pathogen (Salmonellas, Shigellae, streptococcus aureus, micrococcus scarlatinae, Enterobacter sakazakii, Klebsiella pneumonia, acid-producing Klebsiella bacterium, Listeria monocytogenes, citrobacter freundii and Bacillus cereus) DNA in 10 kinds of milk powder of 3 pairs of primer amplifications and the milk preparation have been chosen, for adapting to the PCR of three pairs of primers simultaneously, through the information biology primary dcreening operation and by a large amount of PCR experiment screenings, filter out suitable primer as shown in table 2.
Table 2 is used for the drawing of pcr amplification that 10 kinds of common pathogens of milk powder and milk preparation detect DNA
The thing sequence
| The primer numbering | SEQ ID | Primer sequence (5 '-3 ') | Amplification effect |
| P-1 | NO：29 | TGTACACACCGCCCGTC | Distinguish upstream primer between 16S-23S rDNA |
| P-2 | NO：30 | GGTACTTAGATGTTTCAGTTC | Distinguish downstream primer between 16S-23S rDNA |
| P-3 | NO：31 | TGACCGCCTTTCCGATA | Shigellae ipaH upstream region of gene primer |
| P-4 | NO：32 | GCCAGTACCTCGTCAGTCA | Shigellae ipaH upstream region of gene primer |
| P-5 | NO：33 | GCGTTGTCCGAACTGTACCTTGTGG | Citrobacter freundii gyrB upstream region of gene primer |
| P-6 | NO：34 | GCCCATCAGCGTGGTGAACAGC | Citrobacter freundii gyrB gene downstream primer |
Embodiment 3 gene chips preparation---chip point sample
1. dissolving probe: synthetic probe among the embodiment 1 is dissolved in respectively in the 50%DMSO solution, and dilution makes the final concentration of probe reach 1 μ g/ μ l.
2. splice: will dissolve the corresponding position that good probe adds 384 orifice plates, every hole 10 μ l.
3. point sample: aldehyde radical slide (the CEL Associates of the cleaning of 57.5mm * 25.5mm * 1mm (length * wide * height) that will be as shown in Figure 1, Inc.) be put on the Stage microscope of chip point sample instrument (Spotarray 72), use the control software (Tele chem smp3 stealty pin) of SpotArray, working procedure, in the point sample district by arrangement mode point 4.5mm * 4.5mm on the slide of aldehyde radicalization shown in Figure 2, the array arrangement rule is identical in the low density DNA micromatrix in the formation, six dot matrix areas on the slide.Dot spacing 250 μ m in the dot matrix area domain sizes 3mm * 2.25mm, this dot matrix, matrix: 12 * 9,12 * 250 μ m=3mm, 9 * 250 μ m=2.25mm, standard film base size: 75.5mm * 25.5mm * 1mm.
4. dry: dried overnight under the chip room temperature that will put, then 45 ℃ of oven dryings 2 hours.
5. crosslinked: with crosslinked 2 times of crosslinked instrument (uvpcl-2000M ultraciolet Crosslinker) 600J.Crosslinked good chip is put back in the clean chip cartridges, standby.
As seen from Figure 2, in each point sample district be the individual probe points in 12 (OK) * 9 (row).The position of NO.1 frame district signal be bacterial detection over against according to probe, the position of NO.2 frame district signal is a fluorescent probe, the position of NO.3 frame district signal is negative contrast probe, other be the specific probe (numbering corresponding to the correspondent probe in the table 1) of each pathogenic bacterium.
Embodiment 4 utilizes common pathogen in gene chip rapid detection milk powder and the milk preparation
1. sample preparation: 25g milk powder and milk preparation add in the 225ml 2YT substratum for preparing in advance, and 37 ℃, the 200rpm overnight shaking is cultivated.
2. extraction genome: the pathogenic bacterium thalline that centrifugal 5 minutes precipitations of the sample 8000rpm of 1ml incubated overnight may exist, abandon supernatant (empty as far as possible doing).It is resuspended to add the 100ul deionized water in precipitation, and 8000rpm centrifugal 5 minutes, removes supernatant.The lysate (it is as follows to fill a prescription) that adds 100ul, 100 ℃ of boiling water baths 15 minutes, centrifugal 3 minutes of 12000rpm, supernatant is the dna profiling of slightly carrying.
Attached: the lysate prescription:
1 * PCR damping fluid (contains Mg
+)
0.5% NP 40
0.5% Tween 20
3. amplified target sequence: get the 3ul middle layer supernatant that said gene group extracting method extracts and add in the PCR reaction mixture as template, PCR reaction mixture prescription is as shown in table 3 below.(annotate: the PCR damping fluid in the following table 3-table 4, MgCl
2, the dNTP mixture, the Taq enzyme is all available from Sangon company)
Table 3 Multiplex PCR reaction mixture prescription
| Composition | Concentration | Application of sample amount (μ l) |
| ddH 2O 10 * PCR damping fluid MgCl 2DNTP mixture P-1 to P-2 P-3 and P-4 P-5 and P-6 | - 10× 25mM 10mM 10μM 10μM 10μM | 36 55 0.5 each 1 each 0.3 each 1.2 |
| The Taq enzyme | 5U/μl | 0.5 |
Annotate: P-1 to P-2 and P-3 and P-4 are primer listed in the table 2 in the table.
Reaction tubes is put into PCR instrument (Biometra), and the loop parameter of setting is as follows:
94 ℃ 5 minutes
94 ℃ 30 seconds
50 ℃ 30 seconds
Got back to second step, totally 35 circulations in 1 minute for 72 ℃
72 ℃ 5 minutes
4 ℃ 20 minutes
3. purifying: with purification column (MILIPORE company) purifying, concrete steps are as follows with the pcr amplification product of above-mentioned acquisition:
(1) the PCR product is transferred in the purification column, adds water and complement to 400 μ l.
Centrifugal 15 minutes of (2) 25 ℃, 6000rpm abandon collection tube.
(3) purification column is transferred in the centrifuge tube of new 1.5ml, added the ultrapure water (MilliQ) of 25 μ l, placed 5 minutes for 37 ℃.
(4) the purification column inversion is placed on the centrifuge tube of 1.5ml, centrifugal 2 minutes of 6000rpm collects product.
4. labels targets sequence: get 12 μ l purified products, add in the mark mixed solution, the labeled reactant mixture formula is as shown in table 4 below.
Table 4 mark mixture formula
| Composition | Concentration | Application of sample amount (μ l) |
| ddH 2O 10 * PCR damping fluid MgCl 2DNTP mixture P-2, P-4 and | - 10× 25mM 10mM 10μM | 9.3 33 0.3 each 0.6 |
| P-6 Cy3-dUTP Taq enzyme | 25nM 5U/μl | 0.3 0.3 |
Annotate: P-2 and P-4 are primer listed in the table 2 in the table.
Reaction tubes is put into PCR instrument (Biometra), and the loop parameter of setting is as follows:
94 ℃ 5 minutes
94 ℃ 30 seconds
50 ℃ 30 seconds
Got back to second step, totally 35 circulations in 1 minute for 72 ℃
72 ℃ 5 minutes
4 ℃ 20 minutes
5. oven dry: marked product is put 65 ℃ of oven for drying.
6. hybridization: the pre-70 μ l ddH that add in hybridizing box (Bo Ao company)
2O is to keep humidity.12 μ l hybridization solutions (it is as follows to fill a prescription) return molten oven dry product and be added in the milk powder of preparation among the embodiment three and milk preparation in common pathogen detect the probe array zone of gene chip, cover the cover plate (Bo Ao company) (noting between cover plate and the slide glass bubble being arranged) of customization, cover tight hybridizing box, hybridization is 16 hours in 40 ℃ of water-baths.
7. washing: when hybridizing to, take out hybridizing box, remove cover plate, gene chip was washed 3 minutes in washing lotion A successively, washing is 3 minutes among the washing lotion B, and washing is 90 seconds among the washing lotion C, and is air-dry in the air.
Hybridization solution prescription: 10% T 500 (dextran Sulfate); 25% methane amide (formamide); 0.1%SDS (sodium lauryl sulphate); 6 * SSPE
Washing lotion A:1 * SSC (sodium-chlor-sodium citrate solution); 0.1%SDS
Washing lotion B:0.05 * SSC
Washing lotion C:95% ethanol
8. scanning: with GenePix personal 4100A biochip scanner (AXONinstrument) scanning, used parameter is as follows:
Software and version: GenePix Pro 6.0
official name：575DF35
PMT Gain：550
Scanning resolution: 10 μ m
Scanning result saves as JPG, TIF, GPR form
Hybridization scanning result when detecting pathogenic bacterium in common milk powder and the milk preparation (Salmonellas, Shigellae, streptococcus aureus, micrococcus scarlatinae, Enterobacter sakazakii, Klebsiella pneumonia, acid-producing Klebsiella bacterium, Listeria monocytogenes, citrobacter freundii and Bacillus cereus) respectively with gene chip of the present invention is shown in Fig. 3 A-3J.
9. analysis interpretation: because milk and milk preparation have sterilization steps in process of manufacture, so other food pathogenic are less relatively relatively, therefore present quality testing department includes only five kinds of bacterium (Salmonellas, Shigellae, streptococcus aureus, micrococcus scarlatinae, Enterobacter sakazakii) to the pathogenic bacterium detection of milk powder and milk preparation, and the present invention is directed to the actual situation that detects has increased Klebsiella pneumonia, acid-producing Klebsiella bacterium, Listeria monocytogenes, citrobacter freundii and 10 kinds of pathogenic bacterium of Bacillus cereus total again.Number of bacteria is less, and probe points is also less, and detected result can be judged by naked eyes.According to the hybridization image that scans, with over against according to the position of probe as image coordinate, judge the position of the specific probe that fluorescent signal occurs, contrast dot matrix layout viewing is judged pathogenic bacterium.If only, then do not have this ten kinds of pathogenic bacterium over against signal being arranged according to probe.
5 pairs of gene chips of embodiment carry out the specificity evaluation and sensitivity detects
Common pathogen in the milk powder of preparation among the embodiment 3 and the milk preparation being detected the specificity of gene chip identifies as follows:
Amount to the type strains of 205 strain pathogenic bacterium and detect strain and their nearly edge bacterial strain is identified the specificity of common pathogen detection gene chip in the milk powder of preparation among the embodiment three and the milk preparation.In this specificity qualification test, all bacterial strain situations of use see Table 5.Utilize gene chip of the present invention and above-mentioned detection method to hybridize detection, all shown correct results of hybridization, this illustrates that gene chip of the present invention has good specificity.
Table 5: the bacterial strain that the specificity test is used
| Bacteria name | Reference culture (strain) | Clinical strains number (strain) |
| The slow staphylococcus of salmonella Shigella staphylococcus aureus micrococcus scarlatinae Enterobacter sakazakii Friedlanders bacillus acid-producing Klebsiella bacterium Listeria monocytogenes citrobacter freundii Bacillus cereus Escherichia coli enterobacter cloacae clostridium perfringen reunion enterobacteria day ditch dimension enterobacteria Wo Shi staphylococcus MRSH imitation staphylococcus staphylococcus saprophyticus MRSE Staphylococcus sciuri | 18 12 3 3 3 22 5 5 9 5 5 4 3 1 1 1 1 1 1 1 1 1 | 10 8 20 2 8 17 6 3 |
| Calf staphylococcus Staphylococcus caprae head staphylococcal pneumonia streptococcus streptococcus salivarius streptococcus lactis Streptococcusagalactiae streptococcus faecalis streptococcus fecalis Streptococcus suis bargen's streptococcus Klebsiella ozaenae nose scleroma Klebsiella Klebsiella terrigena is planted the harmless listeria spp Wei Shi of living Klebsiella solution ornithine Klebsiella vibrio parahaemolytious Liszt Bacillus subtillis grinding tooth citric acid bacillus is not had the malonic acid citric acid bacillus | 1 1 1 2 1 1 1 1 1 1 1 3 2 1 1 2 1 1 1 1 1 1 | 1 |
The sensitivity that common pathogen in the milk powder of preparation among the embodiment 3 and the milk preparation is detected gene chip detects as follows:
The detection sensitivity of this gene chip is through the checking of 107 hybrid experiments, 0.1ng micro-genomic dna or every 25g (ml) milk powder and milk preparation in have (1-5) cfu just can guarantee that above-mentioned 10 kinds of pathogenic bacterium have stable, good results of hybridization, this illustrates that gene chip of the present invention has very high detection sensitivity.
Description according to technical scheme of the present invention and preferred embodiment thereof; any those skilled in the art; without departing from the spirit and scope of the present invention; can make various possible being equal to and change or replacement, and all these changes or replacement all should belong to the protection domain of claim of the present invention.
Sequence table
＜110〉Tianjin Biochip Technology Co., Ltd
Tianjin Exit-Entrance Check and Guarantine Bureau
＜120〉gene chip and the test kit of common pathogen in the detection milk preparation
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cgtaaagacg ctgaactgaa cctgt 25
<210>28
<211>25
<212>DNA
＜213〉based on the gyrB gene design of citrobacter freundii and the probe sequence of synthetic
<400>28
ctgatgaagc gaaagtcact gcctg 25
<210>29
<211>17
<212>DNA
＜213〉design the also general detection upstream primer sequence of synthetic based on district between Enterobacter sakazakii, micrococcus scarlatinae, streptococcus aureus, acid-producing Klebsiella bacterium, Klebsiella pneumonia Listeria monocytogenes, bacillus cereus 16S-23S rDNA
<400>29
tgtacacacc gcccgtc 17
<210>30
<211>21
<212>DNA
＜213〉design the also general detection downstream primer sequence of synthetic based on district between Enterobacter sakazakii, micrococcus scarlatinae, streptococcus aureus, acid-producing Klebsiella bacterium, Klebsiella pneumonia Listeria monocytogenes, bacillus cereus 16S-23S rDNA
<400>30
ggtacttaga tgtttcagtt c 21
<210>31
<211>17
<212>DNA
＜213〉based on the detection of Shigellae ipaH gene design and synthetic upstream primer sequence
<400>31
tgaccgcctt tccgata 17
<210>32
<211>19
<212>DNA
＜213〉based on the detection of Shigellae ipaH gene design and synthetic downstream primer sequence
<400>32
gccagtacct cgtcagtca 19
<210>33
<211>25
<212>DNA
＜213〉based on the detection of citrobacter freundii gyrB gene design and synthetic upstream primer sequence
<400>33
gcgttgtccg aactgtacct tgtgg 25
<210>34
<211>22
<212>DNA
＜213〉based on the detection of citrobacter freundii gyrB gene design and synthetic downstream primer sequence
<400>34
gcccatcagc gtggtgaaca gc 22
Claims (11)
1. a gene chip that detects pathogenic bacterium in the milk preparation comprises solid phase carrier and the oligonucleotide probe that is fixed on this solid phase carrier, it is characterized in that described this oligonucleotide probe comprises one or more sequences of choosing from following sequence:
(1) dna sequence dna of between the 16S-23S rDNA of Enterobacter sakazakii, micrococcus scarlatinae, streptococcus aureus, Klebsiella pneumonia, acid-producing Klebsiella bacterium, Listeria monocytogenes, Salmonellas and Bacillus cereus, choosing the gyrB gene of ipaH gene of district and Shigellae or citrobacter freundii;
(2) complementary dna sequence of the dna sequence dna of choosing in described (1);
(3) the complementary RNA sequence of the dna sequence dna of choosing in described (1) or (2).
2. gene chip according to claim 1 is characterized in that the described dna fragmentation of choosing the gyrB gene of district and the ipaH gene of Shigellae or citrobacter freundii has one or more dna sequence dnas in the dna sequence dna shown in the SEQ ID NO:2-SEQ ID NO:28 between the 16S-23S rDNA of Enterobacter sakazakii, micrococcus scarlatinae, streptococcus aureus, Klebsiella pneumonia, acid-producing Klebsiella bacterium, Listeria monocytogenes, Salmonellas and Bacillus cereus.
3. gene chip according to claim 1 is characterized in that described oligonucleotide probe also comprises positive control probe, negative control probe and fluorescent probe.
4. gene chip according to claim 3 is characterized in that described positive control probe is selected from dna fragmentation or its complementary DNA or the RNA sequence of choosing in the bacterium 16s rDNA conserved regions.
5. gene chip according to claim 4 is characterized in that described positive control probe has the dna sequence dna shown in the SEQ ID NO:1.
6. the application of each described gene chip of claim 1-5 is characterized in that this gene chip is used at least a detection of Enterobacter sakazakii, micrococcus scarlatinae, streptococcus aureus, Klebsiella pneumonia, acid-producing Klebsiella bacterium, Listeria monocytogenes, Salmonellas, Shigellae, citrobacter freundii, Bacillus cereus.
7. the application of gene chip according to claim 6 is characterized in that applied detection primer comprises at least a in the dna sequence dna shown in the SEQ ID NO:29-SEQ ID NO:34.
8. a test kit that detects pathogenic bacterium in the milk preparation is characterized in that described test kit comprises each described gene chip of claim 1-5.
9. test kit according to claim 8 is characterized in that described test kit also comprises at least a of the dna sequence dna shown in the detection primer SEQ ID NO:29-SEQ ID NO:34 or its complementary dna sequence.
10. test kit according to claim 8 is characterized in that described test kit also comprises interpretation software and specification sheets that hybridizing box, hybridization solution and Analysis and Identification result use.
11. the application of the described test kit of claim 8 is characterized in that described test kit is used at least a detection of Enterobacter sakazakii, micrococcus scarlatinae, streptococcus aureus, Klebsiella pneumonia, acid-producing Klebsiella bacterium, Listeria monocytogenes, Salmonellas, Shigellae, citrobacter freundii, Bacillus cereus.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2007101665303A CN101240335B (en) | 2007-02-09 | 2007-11-05 | Gene chip and kit for detecting common pathogen in dairy products |
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| CN200710004918.3 | 2007-02-09 | ||
| CN200710004918 | 2007-02-09 | ||
| CN2007101665303A CN101240335B (en) | 2007-02-09 | 2007-11-05 | Gene chip and kit for detecting common pathogen in dairy products |
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| CN101240335A true CN101240335A (en) | 2008-08-13 |
| CN101240335B CN101240335B (en) | 2011-02-02 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN2007101665303A Expired - Fee Related CN101240335B (en) | 2007-02-09 | 2007-11-05 | Gene chip and kit for detecting common pathogen in dairy products |
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