CN1661113A - A set of oligonucleotide probe for detecting intestinal hemorrhage type colibacillus and vibrio cholerae - Google Patents
A set of oligonucleotide probe for detecting intestinal hemorrhage type colibacillus and vibrio cholerae Download PDFInfo
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Abstract
A set of oligonucleotide probe for detecting the enterohemorrhagic E. coli O157:H7 and cholera vibrio O139 is designed on the Shiga-like toxin generating gene stx1 or stx2 and the beta-glucuronidase gene uidA of said enterohemorrhagic E coli O157:H7 and the enterotoxin A subset (ctxA), toxicity coordinate pilus A subset (tcpA) and glucosyltransferase (LPS gt) genes of cholera vibrio O139, and has high sensibility and specificity. It can be used to detect the enterohemorrhagic E. coli O157:H7, cholera vibrio O139 and other pathogenic genes from the specimen.
Description
Technical field
The invention belongs to the nucleic acid detection technique category of microorganism detection technology, especially pathogenic bacterium.The present invention relates to a cover and can detect the specific gene of enterorrhagia Bacillus coil 0157: H7 and cholera vibrio O 139 and the oligonucleotide probe of pathogenic toxicity plasmid, the sequence and the purposes of this cover probe.
Background technology
Enterorrhagia Bacillus coil 0157: H7 and cholera vibrio O 139 are two kinds of main pathogen enterobacterias, can cause with diarrhoea to be the transmissible disease of cardinal symptom.Can cause breaking out on a large scale by food and drinking-water [Nathalie.Analytical Biochemistry.2001,289:281-288].Pathogenic and a plurality of genes of O157:H7 are relevant, comprising shiga-like toxin producing gene stx1, stx2, and β-glucuronidase gene uidA [Law D.J Appl Microbiol, 2000,88:729-45.] such as (gusA).Remove O157:H7, non-H7 type and some other pathogenic colibacillus also may contain the stx gene among the O157 family member, therefore it is also unreliable only to identify at the detected result of stx gene, also must just can identify in conjunction with the detection of O157:H7 specific gene.In the uidA gene of O157:H7+and 93 bit bases are specific to sport G[Ken J.Molecular and CellularProbes by T, and 2003,17:275-2801 5], thereby O157:H7 and non-H7 type are differentiated.
The cholera vibrio O 139 type is by O1 form variation [Annick Robert-Pilot a.FEMS MicrobiologyEcology, 2002,40:39-46].O139 and O1 type vibrio cholerae all contain two kinds of main virulence gene ctxA and tcpA, but their glycosyltransferase gene (LPSgt) composition there are differences [R.M.Carter.Journal of ImmunologicalMethods, 1995,187:121-125].Distinguish cholerae strain and Non-cholera vibrio by identification ctxA and tcpA, distinguish O1 type and O139 bacterial strain according to the different dna sequence dnas of LPSgt gene then.
For a long time, the laboratory diagnosis of these two kinds of pathogenic bacterias mainly relies on traditional bacteriological detection method, complex steps and need a couple of days just can obtain the result.Immunological method also exists specificity and sensitive question.In recent years, the application of PCR and nucleic acid hybridization technique has promoted the development of Bacteria Detection technology, but has certain limitation.At present, detect the pathogenic agent that could identify simultaneously for polygene, biochip technology has been applied to the detection of pathogenic agent with its distinctive advantage.
Oligonucleotide gene chip (Oligochip) is the sophisticated a kind of high-throughput technique of gene detection of development in recent years (HaciaJ.Nature genetics (Supl), 1999,21 (1): 42-47).A main application of oligonucleotide gene chip is the parallel detection of polygene, is that a kind of polygene of good development prospect detects the true tumor technology on a large scale.The key foundation that biochip technology detects is to design, screen the coupling probe of a series of high specifics, highly sensitive, high stability, be fixed on the solid phase carriers such as sheet glass, utilize the hybridization of probe and sample, once experiment can be determined relevant information such as bacterial species and toxicity in the sample, has greatly improved detection usefulness.
Summary of the invention
The invention provides a cover and be used to detect the oligonucleotide probe of enterorrhagia Bacillus coil 0157: H7 and cholera vibrio O 139, comprise and followingly respectively organize the whole of probe or be the combination of part probe, the probe length of each several part is between the 19-60 base.
A. the shiga-like toxin producing gene stx1 detection probes of enterorrhagia Bacillus coil 0157: H7
B. the shiga-like toxin producing gene stx2 detection probes of enterorrhagia Bacillus coil 0157: H7
C. the β of enterorrhagia Bacillus coil 0157: H7-glucuronidase gene uidA detection probes
D. the ctxA of the enterotoxin A subunit detection probes of cholera vibrio O 139
E. the virulence of cholera vibrio O 139 is coordinated pili A subunit gene tcpA detection probes
F. the glycosyltransferase LPSgt gene test probe of cholera vibrio O 139
The present invention also provides the nucleotide sequence of each several part probe, sees table 1-1 for details.Wherein a probe is selected among P1～P10 1 or several; The b probe is selected among P11～P20 1 or several; The c probe is selected among P21～P30 1 or several; The d probe is selected among P31～P40 1 or several; The e probe is selected among P41～P50 1 or several; The f probe is selected among P51～P60 1 or several.
The present invention also provides a cover can detect the oligonucleotide probe of enterorrhagia Bacillus coil 0157: H7 and cholera vibrio O 139 simultaneously, comprises six parts of above-mentioned a～f.
The present invention further provides a cover specificity good, highly sensitive probe by preferred, and wherein a probe is P7, and the b probe is P20, and the c probe is P29, and the d probe is P36, and the e probe is P49, and the f probe is P60.
In addition, the present invention also provides the purposes of above-mentioned oligonucleotide probe, utilize a cover probe to detect the enterorrhagia Bacillus coil 0157 in the sample: H7 and cholera vibrio O 139, be particularly useful for detection based on biochip technology by nucleic acid hybridization reaction.Utilize probe can further detect enterorrhagia Bacillus coil 0157: H7 and whether carry shiga-like toxin producing gene stx1, stx2; Whether cholera vibrio O 139 carries enterotoxin A subunit (ctxA), virulence is coordinated pili A subunit pathogenic toxicity plasmids such as (tcpA).
Specifically, content of the present invention is achieved in that
Shiga-like toxin producing gene stx1, stx2 and β-glucuronidase gene (uidA) according to enterohemorrhagic Escherichia coli O 157: H7; The enterotoxin A subunit (ctxA) of cholera vibrio O 139, virulence are coordinated pili A subunit (tcpA), 60 oligonucleotide probes of glycosyltransferase (LPSgt) gene design, and probe length is between the 19-60 base.The base sequence consistent (probe sequence sees Table 1-1) of sequence oligonucleotide probe and said gene given zone domain dna normal chain.
Utilize an above-mentioned cover probe, can detect corresponding bacterium in the sample and toxicity plasmid, be particularly useful for detection, have higher sensitivity and specificity based on the gene chip principle by hybridization.When the detection of carrying out based on the gene chip principle, need probe all or wherein a part be fixed on carrier (as sheet glass, silicon substrate, polypropylene screen, nitrocellulose filter, nylon membrane etc.) with appropriate means, become the probe array that M * N arranges, be based on chip.Utilize the bacterium in the corresponding PCR primer amplification sample of optimizing to be checked then, selecting primer requirement is the fragment that comprises the probe region that can amplify all bacteriums.In amplification, introduce fluorescent mark, biotin labeling or other suitable markers.Amplified production is incubated 30 minutes to a few hours with chip under certain condition altogether as the target molecule of probe in detecting, utilizes fluorescent scanning instrument or other relevant detection equipment promptly can detect hybridization signal.The position that signal occurs according to probe can determine the kind of bacterium.
Except detection based on the gene chip principle, this cover probe also can be used for detection based on anti-phase dot hybridization: at first with probe stationary in suitable carriers, extract bacterial nucleic acid to be checked, and the nucleic acid of bacterium carried out mark, then, nucleic acid and the probe on the carrier that mark is good carry out hybridization, and be last, provides suitable plant and instrument to detect.
Probe provided by the invention can detect kind of the bacterium in the sample and entrained pathogenic toxicity plasmid thereof etc. fast, accurately and efficiently.If be prepared into the reagent corresponding box, can be widely used in the aspect such as diagnosis, antibiotic screening, Environmental Monitoring and Assessment, sanitary inspection and quarantine, bacteriology classification, epidemiology survey of clinical bacteria infectious diseases.
Description of drawings
Fig. 1 is target gene substance and triple PCR product electrophoresis detection result among O157:H7 and the O139.1:stx1,2:stx2,3:uidA, 4:O157:H7 triple PCR amplified production, 5:DL2000 Marker (bands visible is respectively 250bp and 500bp), 6:O139 triple PCR amplified production, 7:LPSgt, 8:ctxA, 9:tcpA
Fig. 2 is alternative oligonucleotide probe microarray synoptic diagram.It is the microarray of 10 row * 6 row that probe P1-P60 is arranged in order from left to right, from top to bottom.First walks to the alternative probe that the third line is respectively stx1, stx2 and uidA gene, detects Escherichia coli O 157: H7.Fourth line to the six row are respectively the alternative probe of ctxA, TcpA, LPSgt gene, detect cholera vibrio O 139.
Fig. 3 is alternative oligonucleotide probe screening hybridization scanner uni quantitative result (pcr template is 104 copy DNA).It is the microarray of 10 row * 6 row that probe P1-P60 is arranged in order from left to right, from top to bottom.First drains into the probe that the 3rd row is respectively stx1, stx2 and uidA gene, detects Escherichia coli O 157: H7.The 4th drains into the probe that the 6th row is respectively ctxA, tcpA, LPSgt gene, detects cholera vibrio O 139.
Fig. 4 is that enterorrhagia Bacillus coil 0157: H7 and cholera vibrio O 139 detect the gene chip probes arrangement position.
Fig. 5 be gene chip respectively with the fluorescent mark triple PCR product results of hybridization of enterorrhagia Bacillus coil 0157: H7 and cholera vibrio O 139.
Fig. 6 is enterorrhagia Bacillus coil 0157: H7 and cholera vibrio O 139 oligonucleotide chip sensitivity detected result.A: triple PCR product agarose gel electrophoresis detected result, when the PCR reaction template was 1000 copies, the triple PCR product is visible faint band on agarose gel electrophoresis.B: triple PCR product gene chip detected result, when the PCR reaction template is 80 copies, the visible fluorescent signals of triple PCR product and gene chip hybridization.
Embodiment:
In order to further specify embodiment and the purposes that a cover is used for detecting simultaneously the oligonucleotide probe of enterohemorrhagic Escherichia coli O 157: H7 and cholera vibrio O 139, describe with reference to following examples.Embodiment is in order to explain rather than to limit the scope of the invention by any way, and some change that those skilled in the art are made within the scope of the claims and adjusting also should be thought and belongs to scope of the present invention.
1. the design of the oligonucleotide probe of detection enterorrhagia Bacillus coil 0157: H7 and cholera vibrio O 139
Shiga-like toxin producing gene stx1, stx2 and β-glucuronidase gene (uidA) according to enterorrhagia Bacillus coil 0157: H7; Pili A subunit (tcpA), 60 oligonucleotide probes of glycosyltransferase (LPSgt) gene design are coordinated by the enterotoxin A subunit (ctxA) of cholera vibrio O 139, virulence, the base sequence consistent (probe sequence sees Table 1-1) of sequence oligonucleotide probe and said gene given zone domain dna normal chain.Probe length is between the 19-60 base.Amido modified at its 3 ' end when probe is synthetic, the active aldehyde radical that is used for probe and slide surface reacts and is fixed to carrier surface.
Table 1-1: the oligonucleotide probe alternative sequence that detects enterorrhagia Bacillus coil 0157: H7 and cholera vibrio O 139
Probes probes sequence probe is long
Sequence number degree (BP)
P1 CGTATGTAGA?TTCGCTGAAT?GTCATTCGCT?CTGCAATAGG?T 41
P2 CTGAATGTCA?TTCGCTCTGC?AATAGGTACT?CCATTACAGA?CTATTTCATC?AGG 53
P3 TGAATGTCAT?TCGCTCTGCA?ATAGGTACTC?CATTA 35
P4 GTCATTCGCT?CTGCAATAGG?TACTCCATTA?CAGACTATTT?CATCAGG 47
P5 ATCAGGAGGT?ACGTCTTTAC?TGATGATTGA?TAGTGGCACA?GGGGATAATT?TGTTTG 56
P6 GGAGGTACGT?CTTTACTGAT?GATTGATAGT?GGCACAGGGG?A 41
P7 GTACGTCTTT?ACTGATGATT?GATAGTGGCA?CAGGG 35
P8 TACTGATGAT?TGATAGTGGC?ACAGGGGATA?ATTTGTTTGC?AGTTGATG 48
P9 GTTTGCAGTT?GATGTCAGAG?GGATAGATCC?AGAGGAAGGG?CG 42
P10 GTCAGAGGGA?TAGATCCAGA?GGAAGGGCGG?TTTAA 35
P11 CGTAAATAGT?ATACGGACAG?AGATATCGAC?CCCTCTTGAA?CATATATCTC?AGG 53
P12 GTTAAATAGT?ATACGGACAG?AGATATCGAC?CCCTCTTGAA 40
P13 TAAATAGTAT?ACGGACAGAG?ATATCGACCC?CTCTT 35
P14 ATATCTCAGG?GGACCACATC?GGTGTCTGTT?ATTAACCACA 40
P15 TCTCAGGGGA?CCACATCGGT?GTCTGTTATT?AACCA 35
P16 ACCACATCGG?TGTCTGTTAT?TAACCACACC?CCACC 35
P17 CCACCGGGCA?GTTATTTTGC?TGTGGATATA?CGAGGGCTTG?ATGTCTATCA 50
P18 GGCAGTTATT?TTGCTGTGGA?TATACGAGGG?CTTGATGTCT 40
P19 GGCAGTTATT?TTGCTGTGGA?TATACGAGGG?CTTGATGTCT?ATCA 44
P20 AGTTATTTTG?CTGTGGATAT?ACGAGGGCTT?GATGT 35
P21 AAAACTCGAC?GGCCTGTGGG?CATTCAGTCT?GGATCGCGAA?AACTGTGGAA 50
P22 CTCGACGGCC?TGTGGGCATT?CAGTCTGGAT?CGCGAAAACT?GTGGA 45
P23 TGTGGGCATT?CAGTCTGGAT?CGCGAAAACT?GTGGAATTGA?GCAGCGTTGG 50
P24 TGTGGGCATT?CAGTCTGGAT?CGCGAAAACT?GTGGAATTGA?GCAGCGTTGG?TGGGAAAGCG 60
P25 CATTCAGTCT?GGATCGCGAA?AACTGTGGAA?TTGAGCAGCG?TTGGT 45
P26 CATTCAGTCT?GGATCGCGAA?AACTGTGGAA?TTGAGCAGCG?TTGGTGGGAA?AGCGC 55
P27 GGATCGCGAA?AACTGTGGAA?TTGAGCAGCG?TTGGTGGGAA?AG 42
P28 AAAACTGTGG?AATTGAGCAG?CGTTGGTGGG?AAAGCG 36
P29 TGGAATTGAG?CAGCGTTGG 19
P30 TGGAATTGAT?CAGCGTTGG 19
P31 ATGTTTCCAC?CTCAATTAGT?TTGAGAAGTG?CCCACTTAGT 40
P32 TATGTTATAG?CCACTGCACC?CAACATGT 28
P33 GCATACCGTC?CTCATCCAGA?TGAACAAGAA?GTTTCTGGCT?TAGGTGGGAT?TCCAT 55
P34 CATACAGTCC?TCATCCAGAT?GAACAAGAAG?TTTCTGCTTT?AGGTG 45
P35 ATGAACAAGA?AGTTTCTGCT?TTAGGTGGGA?TTCCA 35
P36 CATACAGTCC?TCATCCAGAT?GAACAAGAAG?TTTCTGCTTT?AGGTG 45
P37 AAGAAGTTTC?TGCTTTAGGT?GGGATTCCAT?ACTCCCAAAT 40
P38 ATATGGATGG?TATCGAGTTC?ATTTTGGGGT?GCTTG 35
P39 TGGATGGTAT?CGAGTTCATT?TTGGGGTGCT?TGATGAACAA?TTACA 45
P40 AACAAGAAGT?TTCTGCTTTA?GGTGGGATTC?CATACTCCCA?AATAT 45
P41 TACAAGCGTA?GGGGATATGT?TTCCATTTAT?CAACGTGAAA?GAAGG 45
P42 TACAAGCGTA?GGGGATATGT?TTCCATTTAT?CAACGTGAAA?GAAGGTGCTT 50
P43 TTCGAAACGA?GTGTCGCAGA?TGCTGCTACT?GGCGCTGGCG?TAATT 45
P44 CGAAACGAGT?GTCGCAGATG?CTGCTACTGG?CG 32
P45 AGTCCATTGC?ACCAGGAAGT?GCCAACTTAA?ACCTA 35
P46 AGTCCATTGC?ACCAGGAAGT?GCCAACTTAA?ACCTAACTAA?TATCACGCAT?GTTGAGAAGC 60
P47 CATTGCACCA?GGAAGTGCCA?ACTTAAACCT?AACTAATATC?ACGCATGTTG?AGAAG 55
P48 GCACCAGGAA?GTGCCAACTT?AAACCTAACT?AATATCACGC?ATGTTGAGAA 50
P49 CAGGAAGTGC?CAACTTAAAC?CTAACTAATA?TCACGCATGT?TGAGA 45
P50 GGAAGTGCCA?ACTTAAACCT?AACTAATATC?ACGCATGTTG?AG 42
P51 AATCATTTCA?TTCTTTCACT?TAATGAGCGC?ATTATTAACA?ATGTAACAGA 50
P52 TAATGAGCGC?ATTATTAACA?ATGTAACAGA?TTGTGATATG 40
P53 GAGCGCATTA?TTAACAATGT?AACAGATTGT?GATATGATAA?GAGCGCATCT 50
P54 AGCGCATTAT?TAACAATGTA?ACAGATTGTG?ATATGATAAG?AGCGCATCTT?TTAAA 55
P55 ATTAACAATG?TAACAGATTG?TGATATGATA?AGAGCGCATC 40
P56 CAATGTAACA?GATTGTGATA?TGATAAGAGC?GCATC 35
P57 AGTAGTTCTC?AAATTGAAAG?TAGCCAATTT?GATTCTTCTG?CTATAGAAAG 50
P58 ATTGAAAGTA?GCCAATTTGA?TTCTTCTGCT?ATAGAAAGGC?TTATG 45
P59 GCCAATTTGA?TTCTTCTGCT?ATAGAAAGGC?TTATG 35
P60 TCGATAAGAA?GAGATAAAGA?TCTGAGTTAT?CTAAAGATAT?TTG 43
2. amplification contains pulsating PCR primer design of target gene and optimization
According to virulence gene stx1, the stx2 of O157:H7 with contain ctxA, tcpA, glycosyltransferase gene (LPSgt) the gene order design primer of specific mutant locus gene uidA and 0139, primer sequence sees Table 1-2.Utilize the probe sequence that comprises in the PCR product sequence of primer amplification on each target fragment.All reverse primer sequence 5 ' ends are with fluorescein Cy3 mark, and to guarantee the PCR product behind hybridization, the reverse complemental chain that has fluorescence combines the generation detectable signal with nucleotide probe.Respectively O157:H7 and O139 are carried out the optimization (the PCR reaction electrophoresis detection of optimization the results are shown in accompanying drawing 1) of triple PCR amplification condition.
Table 1-2: be used to increase and contain the primer sequence of probe sequence target fragment
The gene primer sequence (5 '---3 ') product length remarks
Title (bp)
Stx1 GAATTTACCT TAGACTTCTC GAC 250 forward primers
TCCTGTTAAC AAATCCTGTC AC reverse primer
Stx2 TACGATAGAC TTTTCGACTC AAC 207 forward primers
TCAATAATCA GACGAAGATG GTC reverse primer
UidA TAATGAGGAG TCCCTTATGT TAC 179 forward primers
ACTGATCGTT AAAACTGCCT GG reverse primer
CtxA ACTCAGACGG GATTTGTTAG GC 304 forward primers
ATCTATCTCT GTAGCCCCTA TTAC reverse primer
TcpA TTGACCCAAG CACAATGTAA GAC 241 forward primers
CTACTGTGAA TGGAGCAGTT CC reverse primer
LPSgt ACATCTGTAG GGATTGTATT GAC 340 forward primers
ATAACAACTG AGATATCAAG CGTC reverse primer
3. the screening and optimizing of the preparation of oligonucleotide gene chip and probe
The probe purifying, quantitatively after, put on the aldehyde radical activatory slide glass with the gene chip sample applying instrument, make the alternative oligonucleotide probe microarray of O157:H7 and O139 (gene chip, accompanying drawing 2 is seen in the probe arrangement).With fluorescently-labeled pcr amplification product and gene chip hybridization, the crossbreeding effect that obtains each probe as shown in Figure 3.At six gene fragments of O157:H7 and two kinds of pathogenic agent of O139, select six probes that specificity is good, highly sensitive according to the chip hybridization result.The probe sequence that screening obtains sees Table 1-3.
Table 1-3: the oligonucleotide probe majorizing sequence that detects enterorrhagia Bacillus coil 0157: H7 and cholera vibrio O 139
Probes probes sequence probe length
Sequence number (BP)
P7 GTACGTCTTT?ACTGATGATT?GATAGTGGCA?CAGGG 35
P20 AGTTATTTTG?CTGTGGATAT?ACGAGGGCTT?GATGT 35
P29 TGGAATTGAG?CAGCGTTGG 20
P36 CATACAGTCC?TCATCCAGAT?GAACAAGAAG?TTTCTGCTTT?AGGTG 45
P49 CAGGAAGTGC?CAACTTAAAC?CTAACTAATA?TCACGCATGT?TGAGA 45
P60 TCGATAAGAA?GAGATAAAGA?TCTGAGTTAT?CTAAAGATAT?TTG 43
The specificity of embodiment 2. oligonucleotide probes and sensitivity evaluation
1. (6 * SSC 0.1%SDS) is diluted to final concentration 50 μ mol/L, gets 5ul and is transferred to 384 orifice plates with sampling liquid with oligonucleotide probe in the gene chip preparation.With Cartesian chip preparing instrument with probe points (probe arrange see accompanying drawing 4, chip is seen accompanying drawing 5 with the fluorescent mark triple PCR product results of hybridization of enterorrhagia Bacillus coil 0157: H7 and cholera vibrio O 139 respectively) to the aldehyde radical sheet.In this probe microarray,, contrast as negative probe as positive control with the reverse complementary sequence of fluorescent primer with irrelevant stochastic sequence, with the blank sampling liquid that do not contain probe as negative control.Probe sequence sees Table 2-1.
Table 2-1 enterorrhagia Bacillus coil 0157: H7 and cholera vibrio O 139 detect the gene chip probes sequence
Probe title probe sequence probe length
(BP)
stx1 GTACGTCTTT?ACTGATGATT?GATAGTGGCA?CAGGG 35
stx2 AGTTATTTTG?CTGTGGATAT?ACGAGGGCTT?GATGT 35
uidA TGGAATTGAG?CAGCGTTGG 20
ctxA CATACAGTCC?TCATCCAGAT?GAACAAGAAG?TTTCTGCTTT?AGGTG 45
tcpA CAGGAAGTGC?CAACTTAAAC?CTAACTAATA?TCACGCATGT?TGAGA 45
LPSgt TCGATAAGAA?GAGATAAAGA?TCTGAGTTAT?CTAAAGATAT?TTG 43
Negative probe contrast TCTTCGCCAG AGGCCTGCTA GCCTGGTTCA AGATACTACC 40
Positive probe contrast fluorescent primer reverse complementary sequence 20
2. standard plasmid makes up with the DNA that extracts in Escherichia coli O 157: H7 and the cholera vibrio O 139 reference culture as template, carries out conventional pcr amplification with the primer of unmarked fluorescence.Be connected with the pGEM-T carrier respectively after amplified production is purified and transformed into escherichia coli DH5 α, 37 ℃ are shaken bacterium and spend the night.Extract plasmid with the Promega plasmid extraction kit, order-checking is identified consistent with standard sequence.Calculate the copy number of the plasmid that extracts and carry out 10 times of serial dilutions ,-20 ℃ of preservations are as the template of pcr amplification.
3. enterorrhagia Bacillus coil 0157: H7 and the sensitivity of cholera vibrio O 139 oligonucleotide chip detect the 10 times of serial dilutions of positive plasmid that will make up, and carry out the triple PCR amplification with the template of different copy numbers.Amplified production and chip are hybridized, and result and agarose gel electrophoresis detection method compare.When the PCR reaction template was for 1000 whens copy, the triple PCR reaction product, the agarose gel electrophoresis test strip as seen, the chip hybridization signal is positive.When template concentrations was 500 copies, triple PCR amplified production electrophoresis detection was negative, and the chip hybridization reaction is positive.Template is carried out 2-10 more doubly dilute, wherein amplified production hybridization still has weak positive signal after 6 times of dilutions, continues to dilute then hybridization feminine gender (detected result is seen accompanying drawing 6).The result shows: reach the minimum sample size of chip detection, the dna profiling amount of multiplex PCR amplification should be greater than 80 copies.
4. enterorrhagia Bacillus coil 0157: H7 and cholera vibrio O 139 oligonucleotide chip specific detection
Optimizing under good the triple PCR amplification and hybridization condition, detecting the entero-bacte of 146 strain different serotypes.Comprising Escherichia coli O 157: H7 W933,882364 type strains, the local strain of 5 strains such as 97-O157:H7, the non-H7 type of 3 strain O157; Cholera vibrio O 139 M045,1837 type strains and 72 strain Local Isolates, O1 type vibrio cholerae 569B and Ogawa; Common entero-bacte: totally 146 strains such as enteroinvasive E 48017, STM408,191,197,247, AT colibacillus, golden Portugal bacterium 6538, Salmonellas, Yersinia, Bacillus cereus, Shigellae, listeria bacteria, pseudomonas, sex change bacillus.With the DNA of bacteria is that template is carried out two cover triple PCR amplifications, product and chip hybridization.The result is except that positive control, wherein 8 strain Escherichia coli O 157: H7 produce positive signal at stx1, stx2, uidA probe place, the non-H7 type of two strain O157 produces positive signal at stx1 and stx2 probe place, the intestinal bacteria of the not clear serotype of non-H7 type of other three strain O157 and 191,197 and one strain produce signal at stx1 probe place, but the non-H7 type of O157 is in all no signal generations of uidA probe.The O157:H7 that this explanation is detected contains stx1, stx2 virulence gene, but not H7 type and some entero-bacte also contain stx1 or stx2.The uidA probe can be used for detecting the mutational site of O157:H7, differentiates with non-H7 type.74 strain cholera vibrio O 139s all produce positive signal at ctxA, tcpA, LPSgt probe place.569B, Ogawa vibrio cholerae produce positive signal at ctxA, tcpA probe place, show that the O1 type vibrio cholerae that is detected contains ctxA, tcpA virulence gene.This experiment LPSgt probe then only with O139 type vibrio cholerae specific combination.And other entero-bacte and detection probe, all no signal produces.Selected being used to of this explanation differentiates that the probe of O157:H7 and O139 is special, illustrates that also this experiment is based upon on the two cover multiplex PCR bases, and the gene chip of qualitative detection O157:H7 and two kinds of pathogen enterobacterias of O139 is reliable simultaneously.This chip also can be used for the auxiliary O1 of discriminating type vibrio cholerae.
Table 2-2: applying gene chip detection 146 strain entero-bacte results
Bacterial strain quantity stx1 stx2 uidA ctxA tcpA LPSgt
O157:H7 8 + + + - - -
The non-H7 2 of O157++----
The non-H7 3 of O157+-----
191、197 2 + - - - - -
O139 74 - - - + + +
O1 2 - - - + + -
Other entero-bacte 55------
Embodiment 3. detects the application of enterorrhagia Bacillus coil 0157: H7 and cholera vibrio O 139 oligonucleotide chip
1. clinical sample is collected the anus swab of getting clinical patient morbidity and treatment different times with DNA extraction.Every increment originally is divided into two parts, and portion carries out conventional bacteriological detection with traditional bacterial culture, and detecting 89 increments altogether originally is 0139 type vibrio cholerae (the results are shown in Table 3-1); Another part increases bacterium 2-3h in basic peptone water, boiling lysis 10min then, and the centrifugal 2min of 12000rpm gets in the supernatant DNA as the multiplex PCR amplification template.
2. oligonucleotide chip detects and uses Escherichia coli O 157: H7 and cholera vibrio O 139 oligonucleotide chip to detect 342 parts of clinical samples altogether, with fluorescent primer clinical sample is carried out multiple asymmetric PCR amplification, and product mixes back and chip hybridization.The result has 137 parts positive signal to occur at chip ctxA, tcpA, LPSgt probe place, is accredited as O139 type vibrio cholerae, all the other 205 parts except that positive control, do not see hybridization signal (the results are shown in Table 3-1).The result shows the detection Escherichia coli O 157 that this research is set up: the gene chip positive rate of H7 and cholera vibrio O 139 is than conventional bacteriological detection method susceptibility height.
Table 3-1 clinical samples detected result
Detection method detection target detects positive sample and counts positive rate (%)
Microbial culture physiology, biochemical character 89 26.0
Gene chip ctxA, tcpA, LPSgt 137 40.0
The technical essential of above embodiment is as follows:
1. oligonucleotide synthetic oligonucleotide (primer or probe) adopts standard phosphoramidite chemical process upward synthetic at automatic DNA synthesizer DNA (ABi 8909).All fluorescent primers carry out mark with the phosphorus acylated reagent of Cy3 at 5 ' end in synthetic.All oligonucleotide probes 3 ' end carries out amido modifiedly, link to each other with spacerarm (polyoxyethylene glycol phosphorus esterification reagent) between amino and the probe sequence.Synthetic finishing with 55 ℃ of effects of strong aqua deprotection/cutting in 15 hours, OPC column purification.Ultraviolet is quantitative, and is frozen standby.
2. in the conventional PCR reaction system of multiplex PCR amplification 20ul, forward and reverse primer concentration is 1 μ mol/L, 100 μ mol/LdNTP, 1U TaqDNA polysaccharase, 1 * PCR reaction buffer, 50-100ng template DNA; Amplification condition is: and pre-sex change (94 ℃, 5min); 35 circulations: sex change (94 ℃, 30sec), annealing (55 ℃, 30sec) extend (72 ℃, 30sec/); Extend: (72 ℃, 5min/).
In the reaction of multiple asymmetric PCR, the change of concentration has tangible influence to the amplification efficiency of goal gene between the three pairs of primers, the ratio of the forward of each gene and reverse fluorescent primer and hybridization signal strong and weak closely related.Through optimum experimental repeatedly, make in the reaction tubes three products all can reach close and amplification efficiency and hybridization signal preferably.The result who optimizes is: the forward primer concentration of stx1, stx2 and uidA gene is respectively 0.15 μ mol/L, 0.1 μ mol/L, 0.1 μ mol/L in the O157:H7 multi-PRC reaction, and oppositely fluorescent dye primer concentration is respectively 0.75 μ mol/L, 0.5 μ mol/L, 0.5 μ mol/L; The forward primer concentration of ctxA, tcpA, LPSgt gene is respectively 0.15 μ mol/L, 0.15 μ mol/L, 0.2 μ mol/L in the O139 multi-PRC reaction, and oppositely fluorescent dye primer concentration is respectively 0.75 μ mol/L, 0.75 μ mol/L, 1.0 μ mol/L.In the 20ul reaction system, dNTP is 200 μ mol/L, MgCl
2Be 3mmol, 2U TaqDNA polysaccharase, 1 * PCR reaction buffer; Amplification condition is: and pre-sex change (94 ℃, 5min); 40 circulations: sex change (94 ℃, 30sec), annealing (56 ℃, 30sec) extend (72 ℃, 30sec/); Extend: (72 ℃, 5min/).
3. standard plasmid makes up and extracts DNA as template from standard O157:H7 W933 and cholera vibrio O 139 M045 bacterial strain, carries out conventional pcr amplification with every pair of primer (unmarked fluorescence).Be connected conversion after amplified production is purified respectively with the pGEM-T carrier and advance in the bacillus coli DH 5 alpha, 37 ℃ are shaken bacterium and spend the night usefulness Promega plasmid extraction kit extraction plasmid.Positive colony is identified in the order-checking of purified back, calculates the copy number of the plasmid that extracts and carries out 10 times of serial dilutions, and-20 ℃ of preservations are as the template of pcr amplification.
4. enterorrhagia Bacillus coil 0157: H7 and cholera vibrio O 139 oligonucleotide chip prepare oligonucleotide probe (6 * SSC 0.1%SDS) are diluted to final concentration 50 μ mol/L, gets 5ul and is transferred to 384 orifice plates with sampling liquid.With Cartesian chip preparing instrument probe points is arrived on the aldehyde radical sheet (Telechem), keeping temperature in the point sample instrument is 23 ℃, and relative humidity is greater than 85%.After the oligonucleotide chip preparation finishes, place the interior room temperature of Glass carrier box to place standby.Chip behind the point sample is placed 24h in room temperature before use at least.
5. hybridization of enterorrhagia Bacillus coil 0157: H7 and cholera vibrio O 139 oligonucleotide chip and detection
5.1 oligonucleotide gene chip pre-treatment oligonucleotide chip cleans 2 times with 0.2%SDS, then cleans 2 times with water, is used for hybridization after drying.
5.2 hybridization and the sex change of the fluorescently-labeled PCR product of hybridization aftertreatment (98 ℃, 5min, ice bath immediately) back and hybridization solution (5 * SSC, 5 * Denhardt, 10 μ g/ml salmon sperm DNAs, 0.3%SDS) mixing, get 10 μ l and be transferred to the chip reaction zone, chip places in the hybridizing box, immerses in 42 ℃ of water-baths together with hybridizing box and reacts 60 minutes, and the chip after the hybridization is successively at washing lotion A (1 * SSC, 0.2%SDS), washing lotion B (0.2 * SSC) and washing lotion C (respectively wash 1min in room temperature in 0.1 * SSC).Putting room temperature dries.
5.3 scanning and result judge that chip scans with chip scanner GenePix 4000B, with GenePix Pro 4.0 software analysis results.
Sequence table
＜110〉Institute of Radiation Medicine, Academy of Military Medical Sciences, PLA
＜120〉cover detects the oligonucleotide probe of enterohemorrhagic Escherichia coli and vibrio cholerae
<160>60
<210>1
<211>41
<212>DNA
＜213〉artificial sequence
<400>1
CGTATGTAGA?TTCGCTGAAT?GTCATTCGCT?CTGCAATAGG?T?41
<210>2
<211>53
<212>DNA
＜213〉artificial sequence
<400>2
CTGAATGTCA?TTCGCTCTGC?AATAGGTACT?CCATTACAGA?CTATTTCATC?AGG?53
<210>3
<211>35
<212>DNA
＜213〉artificial sequence
<400>3
TGAATGTCAT?TCGCTCTGCA?ATAGGTACTC?CATTA?35
<210>4
<211>47
<212>DNA
＜213〉artificial sequence
<400>4
GTCATTCGCT?CTGCAATAGG?TACTCCATTA?CAGACTATTT?CATCAGG?47
<210>5
<211>56
<212>DNA
＜213〉artificial sequence
<400>5
ATCAGGAGGT?ACGTCTTTAC?TGATGATTGA?TAGTGGCACA?GGGGATAATT?TGTTTG?56
<210>6
<211>41
<212>DNA
＜213〉artificial sequence
<400>6
GGAGGTACGT?CTTTACTGAT?GATTGATAGT?GGCACAGGGG?A?41
<210>7
<211>35
<212>DNA
＜213〉artificial sequence
<400>7
GTACGTCTTT?ACTGATGATT?GATAGTGGCA?CAGGG?35
<210>8
<211>48
<212>DNA
＜213〉artificial sequence
<400>8
TACTGATGAT?TGATAGTGGC?ACAGGGGATA?ATTTGTTTGC?AGTTGATG?48
<210>9
<211>42
<212>DNA
＜213〉artificial sequence
<400>9
GTTTGCAGTT?GATGTCAGAG?GGATAGATCC?AGAGGAAGGG?CG?42
<210>10
<211>35
<212>DNA
＜213〉artificial sequence
<400>10
GTCAGAGGGA?TAGATCCAGA?GGAAGGGCGG?TTTAA?35
<210>11
<211>53
<212>DNA
＜213〉artificial sequence
<400>11
CGTAAATAGT?ATACGGACAG?AGATATCGAC?CCCTCTTGAA?CATATATCTC?AGG?53
<210>12
<211>40
<212>DNA
＜213〉artificial sequence
<400>12
GTTAAATAGT?ATACGGACAG?AGATATCGAC?CCCTCTTGAA?40
<210>13
<211>35
<212>DNA
＜213〉artificial sequence
<400>13
TAAATAGTAT?ACGGACAGAG?ATATCGACCC?CTCTT?35
<210>14
<211>40
<212>DNA
＜213〉artificial sequence
<400>14
ATATCTCAGG?GGACCACATC?GGTGTCTGTT?ATTAACCACA?40
<210>15
<211>35
<212>DNA
＜213〉artificial sequence
<400>15
TCTCAGGGGA?CCACATCGGT?GTCTGTTATT?AACCA?35
<210>16
<211>35
<212>DNA
＜213〉artificial sequence
<400>16
ACCACATCGG?TGTCTGTTAT?TAACCACACC?CCACC?35
<210>17
<211>50
<212>DNA
＜213〉artificial sequence
<400>17
CCACCGGGCA?GTTATTTTGC?TGTGGATATA?CGAGGGCTTG?ATGTCTATCA?50
<210>18
<211>40
<212>DNA
＜213〉artificial sequence
<400>18
GGCAGTTATT?TTGCTGTGGA?TATACGAGGG?CTTGATGTCT?40
<210>19
<211>44
<212>DNA
＜213〉artificial sequence
<400>19
GGCAGTTATT?TTGCTGTGGA?TATACGAGGG?CTTGATGTCT?ATCA?44
<210>20
<211>35
<212>DNA
＜213〉artificial sequence
<400>20
AGTTATTTTG?CTGTGGATAT?ACGAGGGCTT?GATGT?35
<210>21
<211>50
<212>DNA
＜213〉artificial sequence
<400>21
AAAACTCGAC?GGCCTGTGGG?CATTCAGTCT?GGATCGCGAA?AACTGTGGAA?50
<210>22
<211>45
<212>DNA
＜213〉artificial sequence
<400>22
CTCGACGGCC?TGTGGGCATT?CAGTCTGGAT?CGCGAAAACT?GTGGA?45
<210>23
<211>50
<212>DNA
＜213〉artificial sequence
<400>23
TGTGGGCATT?CAGTCTGGAT?CGCGAAAACT?GTGGAATTGA?GCAGCGTTGG?50
<210>24
<211>60
<212>DNA
＜213〉artificial sequence
<400>24
TGTGGGCATT?CAGTCTGGAT?CGCGAAAACT?GTGGAATTGA?GCAGCGTTGG?TGGGAAAGCG?60
<210>25
<211>45
<212>DNA
＜213〉artificial sequence
<400>25
CATTCAGTCT?GGATCGCGAA?AACTGTGGAA?TTGAGCAGCG?TTGGT?45
<210>26
<211>55
<212>DNA
＜213〉artificial sequence
<400>26
CATTCAGTCT?GGATCGCGAA?AACTGTGGAA?TTGAGCAGCG?TTGGTGGGAA?AGCGC?55
<210>27
<211>42
<212>DNA
＜213〉artificial sequence
<400>27
GGATCGCGAA?AACTGTGGAA?TTGAGCAGCG?TTGGTGGGAA?AG?42
<210>28
<211>36
<212>DNA
＜213〉artificial sequence
<400>28
AAAACTGTGG?AATTGAGCAG?CGTTGGTGGG?AAAGCG?36
<210>29
<211>19
<212>DNA
＜213〉artificial sequence
<400>29
TGGAATTGAG?CAGCGTTGG?19
<210>30
<211>19
<212>DNA
＜213〉artificial sequence
<400>30
TGGAATTGAT?CAGCGTTGG?19
<210>31
<211>40
<212>DNA
＜213〉artificial sequence
<400>31
ATGTTTCCAC?CTCAATTAGT?TTGAGAAGTG?CCCACTTAGT?40
<210>32
<211>28
<212>DNA
＜213〉artificial sequence
<400>32
TATGTTATAG?CCACTGCACC?CAACATGT?28
<210>33
<211>55
<212>DNA
＜213〉artificial sequence
<400>33
GCATACCGTC?CTCATCCAGA?TGAACAAGAA?GTTTCTGGCT?TAGGTGGGAT?TCCAT?55
<210>34
<211>45
<212>DNA
＜213〉artificial sequence
<400>34
CATACAGTCC?TCATCCAGAT?GAACAAGAAG?TTTCTGCTTT?AGGTG?45
<210>35
<211>35
<212>DNA
＜213〉artificial sequence
<400>35
ATGAACAAGA?AGTTTCTGCT?TTAGGTGGGA?TTCCA?35
<210>36
<211>45
<212>DNA
＜213〉artificial sequence
<400>36
CATACAGTCC?TCATCCAGAT?GAACAAGAAG?TTTCTGCTTT?AGGTG?45
<210>37
<211>40
<212>DNA
＜213〉artificial sequence
<400>37
AAGAAGTTTC?TGCTTTAGGT?GGGATTCCAT?ACTCCCAAAT?40
<210>38
<211>35
<212>DNA
＜213〉artificial sequence
<400>38
ATATGGATGG?TATCGAGTTC?ATTTTGGGGT?GCTTG?35
<210>39
<211>45
<212>DNA
＜213〉artificial sequence
<400>39
TGGATGGTAT?CGAGTTCATT?TTGGGGTGCT?TGATGAACAA?TTACA?45
<210>40
<211>45
<212>DNA
＜213〉artificial sequence
<400>40
AACAAGAAGT?TTCTGCTTTA?GGTGGGATTC?CATACTCCCA?AATAT?45
<210>41
<211>45
<212>DNA
＜213〉artificial sequence
<400>41
TACAAGCGTA?GGGGATATGT?TTCCATTTAT?CAACGTGAAA?GAAGG?45
<210>42
<211>50
<212>DNA
＜213〉artificial sequence
<400>42
TACAAGCGTA?GGGGATATGT?TTCCATTTAT?CAACGTGAAA?GAAGGTGCTT?50
<210>43
<211>45
<212>DNA
＜213〉artificial sequence
<400>43
TTCGAAACGA?GTGTCGCAGA?TGCTGCTACT?GGCGCTGGCG?TAATT?45
<210>44
<211>32
<212>DNA
＜213〉artificial sequence
<400>44
CGAAACGAGT?GTCGCAGATG?CTGCTACTGG?CG?32
<210>45
<211>35
<212>DNA
＜213〉artificial sequence
<400>45
AGTCCATTGC?ACCAGGAAGT?GCCAACTTAA?ACCTA?35
<210>46
<211>60
<212>DNA
＜213〉artificial sequence
<400>46
AGTCCATTGC?ACCAGGAAGT?GCCAACTTAA?ACCTAACTAA?TATCACGCAT?GTTGAGAAGC?60
<210>47
<211>55
<212>DNA
＜213〉artificial sequence
<400>47
CATTGCACCA?GGAAGTGCCA?ACTTAAACCT?AACTAATATC?ACGCATGTTG?AGAAG?55
<210>48
<211>50
<212>DNA
＜213〉artificial sequence
<400>48
GCACCAGGAA?GTGCCAACTT?AAACCTAACT?AATATCACGC?ATGTTGAGAA?50
<210>49
<211>45
<212>DNA
＜213〉artificial sequence
<400>49
CAGGAAGTGC?CAACTTAAAC?CTAACTAATA?TCACGCATGT?TGAGA?45
<210>50
<211>42
<212>DNA
＜213〉artificial sequence
<400>50
GGAAGTGCCA?ACTTAAACCT?AACTAATATC?ACGCATGTTG?AG?42
<210>51
<211>50
<212>DNA
＜213〉artificial sequence
<400>51
AATCATTTCA?TTCTTTCACT?TAATGAGCGC?ATTATTAACA?ATGTAACAGA?50
<210>52
<211>40
<212>DNA
＜213〉artificial sequence
<400>52
TAATGAGCGC?ATTATTAACA?ATGTAACAGA?TTGTGATATG?40
<210>53
<211>50
<212>DNA
＜213〉artificial sequence
<400>53
GAGCGCATTA?TTAACAATGT?AACAGATTGT?GATATGATAA?GAGCGCATCT?50
<210>54
<211>55
<212>DNA
＜213〉artificial sequence
<400>54
AGCGCATTAT?TAACAATGTA?ACAGATTGTG?ATATGATAAG?AGCGCATCTT?TTAAA?55
<210>55
<211>40
<212>DNA
＜213〉artificial sequence
<400>55
ATTAACAATG?TAACAGATTG?TGATATGATA?AGAGCGCATC?40
<210>56
<211>35
<212>DNA
＜213〉artificial sequence
<400>56
CAATGTAACA?GATTGTGATA?TGATAAGAGC?GCATC?35
<210>57
<211>50
<212>DNA
＜213〉artificial sequence
<400>57
AGTAGTTCTC?AAATTGAAAG?TAGCCAATTT?GATTCTTCTG?CTATAGAAAG?50
<210>58
<211>45
<212>DNA
＜213〉artificial sequence
<400>58
ATTGAAAGTA?GCCAATTTGA?TTCTTCTGCT?ATAGAAAGGC?TTATG?45
<210>59
<211>35
<212>DNA
＜213〉artificial sequence
<400>59
GCCAATTTGA?TTCTTCTGCT?ATAGAAAGGC?TTATG?35
<210>60
<211>43
<212>DNA
＜213〉artificial sequence
<400>60
TCGATAAGAA?GAGATAAAGA?TCTGAGTTAT?CTAAAGATAT?TTG?43
Claims (7)
1. a cover is used to detect the oligonucleotide probe of enterorrhagia Bacillus coil 0157: H7 and cholera vibrio O 139, it is characterized in that comprising the following combination of respectively organizing all or part of probe of probe, and the probe length of each several part is between the 19-60 base.
A. the shiga-like toxin producing gene stx1 detection probes of enterorrhagia Bacillus coil 0157: H7
B. the shiga-like toxin producing gene stx2 detection probes of enterorrhagia Bacillus coil 0157: H7
C. the β of enterorrhagia Bacillus coil 0157: H7-glucuronidase gene uidA detection probes
D. the ctxA of the enterotoxin A subunit detection probes of cholera vibrio O 139
E. the virulence of cholera vibrio O 139 is coordinated pili A subunit gene tcpA detection probes
F. the glycosyltransferase LPSgt gene test probe of cholera vibrio O 139
2. a cover oligonucleotide probe as claimed in claim 1, wherein
The a probe is selected from the following oligonucleotide sequence 1 or several
P1 CGTATGTAGA?TTCGCTGAAT?GTCATTCGCT?CTGCAATAGG?T
P2 CTGAATGTCA?TTCGCTCTGC?AATAGGTACT?CCATTACAGA?CTATTTCATC?AGG
P3 TGAATGTCAT?TCGCTCTGCA?ATAGGTACTC?CATTA
P4 GTCATTCGCT?CTGCAATAGG?TACTCCATTA?CAGACTATTT?CATCAGG
P5 ATCAGGAGGT?ACGTCTTTAC?TGATGATTGA?TAGTGGCACA?GGGGATAATT?TGTTTG
P6 GGAGGTACGT?CTTTACTGAT?GATTGATAGT?GGCACAGGGG?A
P7 GTACGTCTTT?ACTGATGATT?GATAGTGGCA?CAGGG
P8 TACTGATGAT?TGATAGTGGC?ACAGGGGATA?ATTTGTTTGC?AGTTGATG
P9 GTTTGCAGTT?GATGTCAGAG?GGATAGATCC?AGAGGAAGGG?CG
P10 GTCAGAGGGA?TAGATCCAGA?GGAAGGGCGG?TTTAA
The b probe is selected from the following oligonucleotide sequence 1 or several
P11 CGTAAATAGT?ATACGGACAG?AGATATCGAC?CCCTCTTGAA?CATATATCTC?AGG
P12 GTTAAATAGT?ATACGGACAG?AGATATCGAC?CCCTCTTGAA
P13 TAAATAGTAT?ACGGACAGAG?ATATCGACCC?CTCTT
P14 ATATCTCAGG?GGACCACATC?GGTGTCTGTT?ATTAACCACA
P15 TCTCAGGGGA?CCACATCGGT?GTCTGTTATT?AACCA
P16 ACCACATCGG?TGTCTGTTAT?TAACCACACC?CCACC
P17 CCACCGGGCA?GTTATTTTGC?TGTGGATATA?CGAGGGCTTG?ATGTCTATCA
P18 GGCAGTTATT?TTGCTGTGGA?TATACGAGGG?CTTGATGTCT
P19 GGCAGTTATT?TTGCTGTGGA?TATACGAGGG?CTTGATGTCT?ATCA
P20 AGTTATTTTG?CTGTGGATAT?ACGAGGGCTT?GATGT
The c probe is selected from the following oligonucleotide sequence 1 or several
P21 AAAACTCGAC?GGCCTGTGGG?CATTCAGTCT?GGATCGCGAA?AACTGTGGAA
P22 CTCGACGGCC?TGTGGGCATT?CAGTCTGGAT?CGCGAAAACT?GTGGA
P23 TGTGGGCATT?CAGTCTGGAT?CGCGAAAACT?GTGGAATTGA?GCAGCGTTGG
P24 TGTGGGCATT?CAGTCTGGAT?CGCGAAAACT?GTGGAATTGA?GCAGCGTTGG?TGGGAAAGCG
P25 CATTCAGTCT?GGATCGCGAA?AACTGTGGAA?TTGAGCAGCG?TTGGT
P26 CATTCAGTCT?GGATCGCGAA?AACTGTGGAA?TTGAGCAGCG?TTGGTGGGAA?AGCGC
P27 GGATCGCGAA?AACTGTGGAA?TTGAGCAGCG?TTGGTGGGAA?AG
P28 AAAACTGTGG?AATTGAGCAG?CGTTGGTGGG?AAAGCG
P29 TGGAATTGAG?CAGCGTTGG
P30 TGGAATTGAT?CAGCGTTGG
The d probe is selected from the following oligonucleotide sequence 1 or several
P31 ATGTTTCCAC?CTCAATTAGT?TTGAGAAGTG?CCCACTTAGT
P32 TATGTTATAG?CCACTGCACC?CAACATGT
P33 GCATACCGTC?CTCATCCAGA?TGAACAAGAA?GTTTCTGGCT?TAGGTGGGAT?TCCAT
P34 CATACAGTCC?TCATCCAGAT?GAACAAGAAG?TTTCTGCTTT?AGGTG
P35 ATGAACAAGA?AGTTTCTGCT?TTAGGTGGGA?TTCCA
P36 CATACAGTCC?TCATCCAGAT?GAACAAGAAG?TTTCTGCTTT?AGGTG
P37 AAGAAGTTTC?TGCTTTAGGT?GGGATTCCAT?ACTCCCAAAT
P38 ATATGGATGG?TATCGAGTTC?ATTTTGGGGT?GCTTG
P39 TGGATGGTAT?CGAGTTCATT?TTGGGGTGCT?TGATGAACAA?TTACA
P40 AACAAGAAGT?TTCTGCTTTA?GGTGGGATTC?CATACTCCCA?AATAT
The e probe is selected from the following oligonucleotide sequence 1 or several
P41 TACAAGCGTA?GGGGATATGT?TTCCATTTAT?CAACGTGAAA?GAAGG
P42 TACAAGCGTA?GGGGATATGT?TTCCATTTAT?CAACGTGAAA?GAAGGTGCTT
P43 TTCGAAACGA?GTGTCGCAGA?TGCTGCTACT?GGCGCTGGCG?TAATT
P44 CGAAACGAGT?GTCGCAGATG?CTGCTACTGG?CG
P45 AGTCCATTGC?ACCAGGAAGT?GCCAACTTAA?ACCTA
P46 AGTCCATTGC?ACCAGGAAGT?GCCAACTTAA?ACCTAACTAA?TATCACGCAT?GTTGAGAAGC
P47 CATTGCACCA?GGAAGTGCCA?ACTTAAACCT?AACTAATATC?ACGCATGTTG?AGAAG
P48 GCACCAGGAA?GTGCCAACTT?AAACCTAACT?AATATCACGC?ATGTTGAGAA
P49 CAGGAAGTGC?CAACTTAAAC?CTAACTAATA?TCACGCATGT?TGAGA
P50 GGAAGTGCCA?ACTTAAACCT?AACTAATATC?ACGCATGTTG?AG
The f probe is selected from the following oligonucleotide sequence 1 or several
P51 AATCATTTCA?TTCTTTCACT?TAATGAGCGC?ATTATTAACA?ATGTAACAGA
P52 TAATGAGCGC?ATTATTAACA?ATGTAACAGA?TTGTGATATG
P53 GAGCGCATTA?TTAACAATGT?AACAGATTGT?GATATGATAA?GAGCGCATCT
P54 AGCGCATTAT?TAACAATGTA?ACAGATTGTG?ATATGATAAG?AGCGCATCTT?TTAAA
P55 ATTAACAATG?TAACAGATTG?TGATATGATA?AGAGCGCATC
P56 CAATGTAACA?GATTGTGATA?TGATAAGAGC?GCATC
P57 AGTAGTTCTC?AAATTGAAAG?TAGCCAATTT?GATTCTTCTG?CTATAGAAAG
P58 ATTGAAAGTA?GCCAATTTGA?TTCTTCTGCT?ATAGAAAGGC?TTATG
P59 GCCAATTTGA?TTCTTCTGCT?ATAGAAAGGC?TTATG
P60 TCGATAAGAA?GAGATAAAGA?TCTGAGTTAT?CTAAAGATAT?TTG
3. a cover oligonucleotide probe as claimed in claim 1 is characterized in that comprising simultaneously six groups of probes of a～f.
4. a cover oligonucleotide probe as claimed in claim 3, wherein a probe is selected from P7, and the b probe is selected from P20, and the c probe is selected from P29, and the d probe is selected from P36, and the e probe is selected from P49, and the f probe is selected from P60.
5. the purposes of the described arbitrary oligonucleotide probe of claim 1～4, it is characterized in that utilizing probe can the corresponding gene of the enterorrhagia Bacillus coil 0157 in the sample: H7 and cholera vibrio O 139 be detected, be particularly useful for detection based on biochip technology.
6. the purposes of the described oligonucleotide probe of claim 5 is characterized in that utilizing probe can detect enterorrhagia Bacillus coil 0157: H7 and whether carries shiga-like toxin producing gene stx1, stx2; Whether cholera vibrio O 139 carries the ctxA of enterotoxin A subunit, virulence is coordinated pathogenic toxin genes such as the pili A tcpA of subunit.
7. the purposes of the described oligonucleotide probe of claim 5 is characterized in that this detection can be used for clinical disease diagnosis, antibiotic screening, Environmental Monitoring and Assessment, sanitary inspection and quarantine, bacteriology classification and epidemiology survey etc.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2005100025141A CN1661113A (en) | 2005-01-21 | 2005-01-21 | A set of oligonucleotide probe for detecting intestinal hemorrhage type colibacillus and vibrio cholerae |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2005100025141A CN1661113A (en) | 2005-01-21 | 2005-01-21 | A set of oligonucleotide probe for detecting intestinal hemorrhage type colibacillus and vibrio cholerae |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1661113A true CN1661113A (en) | 2005-08-31 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2005100025141A Pending CN1661113A (en) | 2005-01-21 | 2005-01-21 | A set of oligonucleotide probe for detecting intestinal hemorrhage type colibacillus and vibrio cholerae |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100386443C (en) * | 2005-12-15 | 2008-05-07 | 深圳太太基因工程有限公司 | Primer and probe sequence for detecting nucleotide fragment of 0139 group choleraic vibrio |
| CN102260743A (en) * | 2011-07-13 | 2011-11-30 | 浙江省疾病预防控制中心 | Multiplex fluorescence polymerase chain reaction (PCR) detection kit and method for enterohemorrhagic Escherichia coli O104:H4 |
| CN101760514B (en) * | 2008-12-25 | 2012-06-27 | 天津生物芯片技术有限责任公司 | Gene chip and kit for detecting diarrheagenic escherichia coli in food and clinical samples |
| CN103409517A (en) * | 2013-07-24 | 2013-11-27 | 福建省亚明食品有限公司 | Biosensing chip capable of quickly detecting EHEC (Enterohemorrhagic Escherichia coli) O157:H7 |
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| CN103866028B (en) * | 2014-03-25 | 2016-06-15 | 广东出入境检验检疫局检验检疫技术中心 | A kind of detect alive can not the fluorescence PCR primer group of cultivation conditions Escherichia coli O 157 and detection method thereof |
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