CN1683565A - Oligonucleotide probe kit for detecting common intestine trac kpathogenic bacteria and its use - Google Patents
Oligonucleotide probe kit for detecting common intestine trac kpathogenic bacteria and its use Download PDFInfo
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Abstract
The present invention belongs to the field of microbe detecting technology. The oligonucleotide probe for detecting common intestinal tract pathogenic bacteria is designed on 16S rRNA and 23S rRNA of bacteria, ipaH of dysentery bacillus giant plasmid, VipR of Salmonella typhi and other gene sequence, has length of 25-50 bp, and relatively high sensitivity and specificity. The oligonucleotide probe is suitable for detection based on nucleic acid hybridization principle, especially detection based on gene chip principle. Under certain use condition, it can detect Listeria, parahemolutic vibrio, Campylobacter, etc. It may be used in many aspects, such as disease diagnosis, environment detection, food poisoning detection, etc.
Description
Technical field
The invention belongs to the nucleic acid detection technique category of microorganism detection technology, especially pathogen enterobacteria.The present invention relates to the oligonucleotide probe that a cover can detect Lee Bordetella, Vibrio parahemolyticus, Campylobacter, yersinia entero-colitica, Yersinia, streptococcus aureus, Clostridium botulinum, wax-like bacillus genus, Salmonellas, salmonella typhi, Shigellae, dysentery bacterium, clostridium perfringens, Bacillus proteus, brucella, enterorrhagia Bacillus coil 0157: H7 and vibrio cholerae, the sequence and the purposes of this cover probe.
Background technology
Pathogen enterobacteria is to cause that clinically with diarrhoea, vomiting be the main pathogens of symptom transmissible disease.Common pathogenic bacterium have Lee Bordetella, Vibrio parahemolyticus, Campylobacter, yersinia entero-colitica, Yersinia, streptococcus aureus, Clostridium botulinum, wax-like bacillus genus, Salmonellas, salmonella typhi, Shigellae, dysentery bacterium, clostridium perfringens, Bacillus proteus, brucella, enterorrhagia Bacillus coil 0157: 17 kinds such as H7 and vibrio cholerae, and wherein bacillus cereus, anthrax bacillus etc. detect as a bacterioid; Lee Bordetella is mainly common singly to increase Lee Salmonella; Yersinia is mainly based on Yersinia pestis and yersinia entero-colitica; The crooked bacterium of campylobacter jejuni and colon the most commonly in the Campylobacter.These bacteriums can cause breaking out on a large scale by approach such as food and drinking-water, the serious threat mankind's safe diet and orthobiosis, and brought heavy economical load to society.According to The World Health Organization's incomplete statistics report, the whole world had 2,100,000 people to die from diarrhoea in 2000, most of pollution owing to pathogenic bacterium in the food and drinking water; And the actual sickness rate of intestinal tract disease manyed 300-500 doubly than the cases reported number especially in recent years, and global trouble patient is several hundred million to reach, and annual statistical value all remains high.The disease that the U.S. has 7,600 ten thousand routine pathogen enterobacterias to cause every year approximately wherein has 32500 people's hospital cares, 5000 people's death; The intestinal tract disease that bacterial contamination causes (food poisoning) also is distinct issues in China's food safety and the clinical diagnosis.According to the health ministry statistics, received altogether that great food report reached 150, dead 135 people of 6237 people that poison in 2000; 2,001 185 of great food poisonings take place altogether, and 15715 people poison, 146 people's death.These are owing to all be that other reasons causes that disease can't be obtained more than wide, the critical health crowd of range of countries of intestinal tract diseases influence that various pathogens causes.
16S rRNA and 23S rRNA identify to learn to have important biological significance in the classification of bacterium, get slowly than other genetic evolution in the organic evolution process, and eubacterial 16S rRNA and 23S rRNA conservative property are strong, are referred to as " living fossil that bacterium is evolved ".But this conservative property also is relative, that is to say to exist every kind of specific fragment of bacterium on 16S rRNA and 23S rRNA sequence.Found that by what the 16S rDNA of various bacteriums and 23S rDNA sequence were compared sequence variable region and constant region on it are staggered.Therefore a pair of primer of choosing on 16S rDNA and 23S rDNA sequence respectively in this patent is chosen specific sequence as probe betwixt as universal primer, and the bacterium of above-mentioned 17 kinds is differentiated.
Bacillary dysentery is a kind of disease that is caused by dysentery bacterium.Case history is more clinically, and infectivity is strong.IpaH is the fragment gene of coding invasion and attack plasmid antigen H on the big plasmid, its specific being present in the dysentery bacterium, therefore the four kinds of pathogenic bacterium and the enteroinvasive E that comprise Shigella at ipaH design primer and probe, can be differentiated bacillary dysentery.
Salmonellas is a kind of common clinically pathogen enterobacteria, is divided into salmonella typhi and Salmonella enteritidis etc. according to clinical symptom and biochemical character.Wherein salmonella typhi is the most representative in this Pseudomonas, and it can cause typhoid, and symptom is not clearly clinically, and this just brings certain trouble to diagnosis; And it still is a big event in the biological anti-terrorism task.VipR is the gene of a kind of regulation and control Vi antigen presentation of encoding of being arranged in salmonella typhi karyomit(e) ViaB zone, and compares with sequence in other pathogen enterobacterias, has very high specificity.Therefore choose this gene as the purpose fragment that detects, design primer and probe being accredited as under the prerequisite of Salmonellas through the probe on the 16S rDNA sequence, are further screened salmonella typhi.
For a long time, laboratory gold standard identification of means for pathogen enterobacteria also rests on traditional bacteriological detection levels such as cultivation, serum and biochemical aspect, complex steps and need a couple of days just can obtain the result, on certain degree, limited Rapid identification, also brought inconvenience to clinical treatment to pathogen enterobacteria.The time of experimental implementation has been shortened in the application of immunological method, but also there are problems such as the singularity of its laboratory sample and result's false positive rate is higher in this method.In recent years, the application of PCR and nucleic acid hybridization technique simple to operate with it, fast and characteristics such as visual result greatly promoted the development of Bacteria Detection technology.For the requirement that the pathogen enterobacteria of majority amount is identified simultaneously, method commonly used at present all can't reach.Therefore the DNA chip technology has been applied in the detection evaluation of pathogen enterobacteria with its distinctive advantage.
DNA chip (DNA microarrays) is the sophisticated a kind of high-throughput technique of gene detection of development in recent years, is the another major technological breakthrough on the molecular biology development history, is characterized in integrated, microminiaturized, the automatization of experimental implementation and data processing.A main application of DNA chip is the parallel detection of polygene, is that the wide polygene of a kind of development prospect detects new technology on a large scale.The crucial part that the DNA chip 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 sample and probe, after scanning, analysis, can draw correct result.With regard to this experiment, just can determine the kind of pathogen enterobacteria in the sample greatly to have improved detection efficiency by experimental implementation, identifying for clinical diagnosis and food provides simple, fast technique means.
Summary of the invention
The invention provides a cover and be used to detect the oligonucleotide probe of common pathogen enterobacteria, 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 25-50 base.
A. the 16S rDNA gene test probe of streptococcus aureus
B. the 16S rDNA gene test probe that belongs to of listeria spp
C. the 23S rDNA gene test probe of enterorrhagia Bacillus coil 0157: H7
D. the 16S rDNA gene test probe of vibrio cholerae
E. the 16S rDNA gene test probe of Yersinia
F. the 16S rDNA gene test probe of yersinia entero-colitica
G. the 16S rDNA gene test probe of salmonella
H. salmonella typhi plays the gene VipR detection probes of positive regulating and controlling effect to Vi antigen presentation gene ViaB
I. the 16S rDNA gene test probe of Shigella
J. coding is attacked plasmid antigen H gene ipaH detection probes on the big plasmid of dysentery bacterium
K. the 16S rDNA gene test probe of Vibrio parahemolyticus
L. the 23S rDNA gene test probe of Vibrio parahemolyticus
M. the total 16S rDNA gene test probe of Shigella, salmonella and intestinal bacteria
N. the 16S rDNA gene test probe of clostridium perfringens
O. the 16S rDNA gene test probe of Campylobacter
P. the 16S rDNA gene test probe of Bacillus proteus
Q. the 16S rDNA gene test probe that belongs to of wax-like bacillus
R. the 16S rDNA gene test probe of Clostridium botulinum
S. the general 16S rDNA gene test probe of eubacterium
T. brucellar 16S rDNA gene test probe
The present invention also provides the nucleotide sequence of each several part probe, sees table 1-1 for details.Also provide a cover can detect the oligonucleotide probe of common pathogen enterobacteria simultaneously, comprised 20 parts of above-mentioned a～t.
The present invention further provides a cover specificity good, highly sensitive probe by preferred, wherein a probe is SA5, and the b probe is LI2 and LI3, and the c probe is O157:H7-1 and O157:H7-2, the d probe is VC1, and the e probe is YE1, and the f probe is ye-2e, the g probe is SHI1, the h probe is V2 and V3, and the i probe is SAL1, and the j probe is I-1 and I-2, the k probe is VP1,1 probe is VP23, and the m probe is SS5, and the n probe is CP-new-1, the o probe is Cam-5, the p probe is PR-2, and the q probe is B-6, and the r probe is CB-1, the s probe is common-2-1 and common-3-1, and the t probe is Bru-2.
In addition, the present invention also provides the purposes of above-mentioned oligonucleotide probe, utilizes a cover probe to detect the common pathogen enterobacteria in the sample by nucleic acid hybridization reaction, is particularly useful for the detection based on biochip technology.Whether utilize the probe can also be when identifying Salmonellas and Shigellae, further determining be salmonella typhi and dysentery bacterium.
Specifically, content of the present invention is achieved in that
(every kind of bacterium is wanted three kinds of different Genebank sequence numbers at least according to the complete sequence of 16S rDNA, the 23S rDNA of the nearly 30 kinds of pathogen enterobacterias chosen and relevant nearly source kind thereof, in order to avoid difference appears in institute's indivedual bases of favored area), 16S rDNA, the 23S rDNA gene order of nearly 30 kinds of bacteriums that use clustalx1.8.msw Alignment program will retrieve from the nucleic acid database of Genebank are compared, and obtain the result of base alignment figure and cluster analysis.Can mark off conserved regions in 16S rDNA, the 23S rDNA gene and the variation zone that differs greatly according to base alignment figure, and then use primer5.0 software to seek universal primer in conserved regions, seek specific probe in variation zone, make that the Tm value of each probe and the Tm value between the two pairs of primers are basic identical.Then respectively on the dysentery bacterium big plasmid of (comprising four kinds of pathogenic bacterium and enteroinvasive E in the Shigella) coding invasion and attack plasmid antigen H genes (ipaH) go up and the gene (VipR) that Vi antigen presentation gene ViaB is played positive regulating and controlling effect of salmonella typhi is gone up and designed primer and probe, design 91 oligonucleotide probes altogether, probe length is between the 25-50 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 the corresponding pathogenic bacteria in the sample, be particularly useful for detection, have higher sensitivity and specificity based on DNA chip principle by hybridization.When the detection of carrying out based on DNA 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 the DNA chip.Utilize the pathogenic 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 pathogenic bacterium in the sample.
Except detection based on DNA 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 the kind of the pathogenic bacterium in the sample 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 food quarantine, bacteriology classification, epidemiology survey of clinical bacteria infectious diseases.
Description of drawings
Fig. 1 is pathogenic bacterium 16S rDNA primer PCR product electrophoresis detection result.1:DL2000 Marker, 2: singly increase Lee Salmonella, 3: campylobacter jejuni, 4: yersinia entero-colitica, 5: vibrio cholerae,: Vibrio parahemolyticus, 7: Bacillus proteus, 8: streptococcus aureus, 9: Bacillus cereus, 10: clostridium perfringens, 11: Shigellae, 12 Salmonellass.
Fig. 2 is pathogenic bacterium 23S rDNA primer PCR product electrophoresis detection result.1:100bp DNA Ladder Marker, 2: singly increase Lee Salmonella, 3: campylobacter jejuni, 4: yersinia entero-colitica, 5: vibrio cholerae, 6: Vibrio parahemolyticus, 7: Bacillus proteus, 8: streptococcus aureus, 9: Bacillus cereus, 10: clostridium perfringens, 11: Shigellae, 12 Salmonellass.
Fig. 3 is 16S rDNA, ipaH and 23S rDNA, vipR two-fold PCR result.1: salmonella typhi 23S rDNA primer amplification result, 2: salmonella typhi vipR primer amplification result, 3: salmonella typhi 23S rDNA and vipR primer two-fold pcr amplification result, 4:DL2000 Marker, 5-9: dysentery bacterium (four kinds of bacteriums of Shigella and enteroinvasive E) 16S rDNA, ipaH two-fold pcr amplification result, 10: dysentery bacterium ipaH primer amplification result, 11: dysentery bacterium 16S rDNA primer amplification result.
Fig. 4 is that 17 kinds of pathogen enterobacterias detect the gene chip probes arrangement position.
Fig. 5 be gene chip respectively with the fluorescent mark PCR product results of hybridization of 17 kinds of pathogen enterobacterias and other negative bacterias.Wherein salmonella typhi and dysentery bacterium hybridization figure all are double pcr amplification product results of hybridization.
Embodiment:
In order to further specify embodiment and the purposes that a cover is used for detecting simultaneously above-mentioned 17 common pathogen enterobacteria oligonucleotide probes of kind, 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. detect the design of the oligonucleotide probe of 17 kind pathogen enterobacterias
Probe designs respectively on the gene orders such as invasion and attack plasmid antigen H (ipaH) and salmonella typhi regulation and control Vi antigen presentation of encoding on the big plasmid in bacterial 16 S rRNA and 23S rRNA and dysentery bacterium, have higher sensitivity and specificity, 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 25-50 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 common pathogen enterobacteria
Probes probes sequence probe is long
Title (5 '-3 ') degree (BP)
SA1????aac?gga?cga?gga?gct?tgc?ttc?tct?gat?gtt?agc????????????????33
SA2????caa?aag?tga?aag?acg?gtc?ttg?ctg?tca?ctt?ata?ga?????????????35
SA3????aaa?ctc?tgt?tat?tag?gga?aga?aca?tat?gtg?taa?gta????????????36
SA4????gtg?cac?atc?ttg?acg?gta?cct?aat?cag?aaa?gcc?acg????????????36
SA5??????aca?tat?gtg?taa?gta?act?gtg?cac?atc?ttg?acg?gta????????????36
SA-6?????tga?cta?ctt?gta?agc?aca?cgg?ttt?cag?gtt?cta?ttt????????????36
SA-7?????aat?att?ttg?aac?cgc?atg?gtt?caa?aag?tga?aag????????????????33
SA-8?????tac?gac?caa?ata?cta?aac????????????????????????????????????18
SA-9?????caa?taa?tca?tca?ctt?gag?gc?????????????????????????????????20
LI1??????aac?gga?gga?aga?gct?tgc?tct?tcc?aaa?gtt?agt????????????????33
LI2??????tgt?tgt?tag?aga?aga?aca?agg?ata?aga?gta?act?gct????????????36
LI3??????tag?aga?aga?aca?agg?ata?aga?gta?act?gct?tgt?ccc????????????36
Lis-23???gtg?gca?tgc?gcc?aca?ctt?tat?c??????????????????????????????22
VP1??????aaa?cga?gtt?atc?tga?acc?ttc?ggg?gaa?cga?taa?cgg????????????36
VP23?????ggt?acg?cag?tca?cag?gac?aaa?gcc????????????????????????????24
VC1??????cag?cac?aga?gga?act?tgt?tcc?ttg?ggt?ggc?gag????????????????33
YE1??????gcg?gca?gcg?gga?agt?agt?tta?cta?ctt?tgc?cgg????????????????33
ye-3?????agg?ggt?tga?gtt?taa?tac?gct?caa?tca?ttg????????????????????30
ye-2e????cat?aaa?ggt?taa?taa?cct?ttg?tga?ttg?acg?t??????????????????31
SHI1?????ggg?agt?aaa?gtt?aat?acc?ttt?gct?cat?tga????????????????????30
V-1??????ctg?gcc?tcc?gaa?tga?tat?cta?ttt????????????????????????????24
V-2??????ctt?caa?taa?tgc?cag?cag?ctc?caa?ccc?cga?aat?aga?ta?????????38
V-3??????aca?ggc?tgt?agc?gat?tta?ggt?tca?caa?aca?aat?aat?tt?????????38
V-4??????cgt?tat?cat?ttc?aag?ttc?gcg?act?a??????????????????????????25
V-5??????cgg?caa?cat?cag?tcc?agg?ata?tta?tca?gaa?atg????????????????33
SAL1?????ggt?gtt?gtg?gtt?aat?aac?cgc?agc?aat?tga????????????????????30
I-1??????gaa?ggc?ctt?ttc?gat?aat?gat?acc?ggc?gct?ctg?ctc?tcc?c??????40
I-2??????gga?ccg?tgt?cgc?gct?cac?atg?gaa?caa?tct?ccg?gaa?aac?cct????42
I-3??????agt?ctt?tcg?ctg?ttg?ctg?ctg?atg?cca?c??????????????????????28
I-4??????cct?ctg?cgg?agc?ttc?gac?agc?agt?ctt?tcg?ctg?tt?????????????35
I-5??????ata?ccg?tct?ctg?cac?gca?ata?cct?ccg?gat?tcc?gt?????????????35
I-6??????tcc?gga?ttc?cgt?gaa?cag?gtc?gct?gca?tgg?ct?????????????????32
H7-1?????gca?gtc?acc?cca?taa?aag?agg?ct?????????????????????????????23
H7-2?????tca?ccc?cat?aaa?aga?ggc?tcc?cac?tgc????????????????????????27
Cam-1????act?cct?gct?taa?cac?aag?ttg?agt?ag?????????????????????????26
Cam-2??????tcg?gtg?tag?gat?gag?act?ata?tag?????????????????????????????????????24
Cam-3??????act?cct?gct?taa?cac?aag?ttg?agt?agg?gaa?agt?ttt?tcg?g???????????????40
Cam-4??????ggt?ata?gtt?aat?ctg?ccc?tac?aca?aga?????????????????????????????????27
Cam-5??????ctc?ctt?ttc?tta?ggg?aag?aat?tct?gac?ggt?acc?taa?gga?at??????????????41
Cam-6??????act?cct?gct?taa?cac?aag?ttg?agt?agg?gaa?ag??????????????????????????32
Cam-7??????gat?ttg?cct?gta?taa?cct?cct?acg?acc?tta?gac?tag?????????????????????36
Cam-8??????cgg?agt?aaa?tcc?taa?tac?aaa?gct?aac?ca??????????????????????????????29
Cam-9??????ttc?tat?cca?tcc?gaa?gac?ttc?aaa?aag?cct?t???????????????????????????31
Cam-3-1????act?cct?gct?taa?cac?aag?ttg?agt?agg?gaa?agt?ttt?tcg?g???????????????40
B-1????????cat?ttt?gaa?ccg?cat?ggt?tcg?aaa?ttg?aaa?ggc?????????????????????????33
B-2????????gga?taa?cat?ttt?gaa?ccg?cat?ggt?tcg?aaa?????????????????????????????30
B-3????????ggc?tgt?cac?tta?tgg?atg?gac?ccg?cgt?c???????????????????????????????28
B-4????????tgc?tag?ttg?aat?aag?ctg?gca?cct?????????????????????????????????????24
B-5????????ggc?ttc?ggc?tgt?cac?tta?tgg?atg?gac?ccg?cgt?c???????????????????????34
B-6????????tgc?tag?ttg?aat?aag?ctg?gca?cct?tga?cg??????????????????????????????29
B-41???????tgc?gta?gcc?gac?ctg?aga?ggg?tgt?????????????????????????????????????24
CP-1???????cga?gct?caa?agt?atg?t???????????????????????????????????????????????16
CP-2???????gcc?gag?ctc?aaa?gta?tgt?????????????????????????????????????????????18
CP-3???????gcc?gag?ctc?aaa?gta?tgt?ggc?att?????????????????????????????????????24
CP-4???????act?act?cat?act?cga?cgc?tag?c???????????????????????????????????????22
CP-5???????gtg?ata?agg?ttc?ggt?aag?cgc?tat?gcc?????????????????????????????????27
CP-6???????ggc?ata?gcg?ctt?acc?gaa?cct?tat?cac?????????????????????????????????27
cp-16??????aag?atg?gca?tca?tca?ttc?aac?caa?agg?agc?aat?c???????????????????????34
CP-16-1????aag?atg?gca?tca?tca?ttc?aac?caa?agg?agc?aat?cgc?tat?g???????????????40
CP1????????gaa?aga?tgg?cat?cat?cat?tca?acc?aaa?gga?gca?atc?????????????????????36
CP-new-1???atg?gca?tca?tca?ttc?aac?caa?agg?agc?aat?ccg?cta?tga?gat?gga?ccc?????48
CP-new-2???atg?gca?tca?tca?ttc?aac?caa?agg?agc?aat?ccg?cta?tga?gat?gga?????????45
common-1???cca?gac?tcc?tac?ggg?agg?cag?cag?t???????????????????????????????????25
common-2???ctc?cta?cgg?gag?gca?gca?gta?ggg?aat?????????????????????????????????27
common-3???cac?act?gga?act?gag?aca?cgg?tc??????????????????????????????????????23
common21???act?gag?aca?cgg?tcc?aga?ctc?cta?cgg?gag?gca?gca?gta?ggg?aat?attg????49
Common31?????cac?act?gga?act?gag?aca?cgg?tcg?aga?ctc?cta?cgg?ga???????????????38
SS3??????????tgt?ctg?gga?aac?tgc?ctg?atg?gag?g????????????????????????????????25
SS4??????????cgt?cgc?aag?acc?aaa?gag?ggg?gac?ctt?cgg?g????????????????????????31
SS5??????????ggt?ttc?agg?ttc?ttt?ttc?act?ccc?ctc?gcc?g????????????????????????31
SS6??????????aac?ctg?ccc?atg?gct?aga?tca?ccg?ggt?ttc?ggg?tct?ata?ccc?t????????43
SS7??????????aat?ttt?tca?aca?tta?gtc?ggt?tcg?gtc?ctc?cag?tta?gtg??????????????39
SS8??????????cct?ctt?gcc?atc?gga?tgt?gcc?cag?atg??????????????????????????????27
CB-1?????????tat?aag?aga?atc?gca?tga?ttt?tct?tat?cca?aag?att?tat??????????????39
CB-2?????????atg?aag?ctt?cct?tcg?gga?agt?gga?tta?gcg?gc???????????????????????32
CB-3?????????ggg?ata?gcc?ttc?cga?aag?gaa?gat?taa?tac??????????????????????????30
SAL，SHI1????gaa?cgg?taa?cag?gaa?gca?gct?tgc?tgt?ttg?ctg??????????????????????33
SAL，SHI2????aac?gtc?gca?aga?cca?aag?agg??????????????????????????????????????21
Bru-1????????gtg?ccc?ttc?ggg?gga?aag?att?tat?cgg?caa?atg?atc?ggc?ccg?cgt?t????46
Bru-2????????cgt?acc?att?tgc?tac?gga?ata?act?cag?gga?aac?ttg?tg???????????????38
PR1??????????taa?cag?gag?aaa?gct?tgc?ttt?ctt?gct?gac??????????????????????????30
PR-2?????????cgt?cga?gtt?cac?aat?aac?agc?atc?ttc?aga??????????????????????????30
pr-2?????????cga?gcg?gta?aca?gga?gaa?agc?ttg?ctt?tct?tgc?tga??????????????????36
pr-3?????????cga?gcg?gta?aca?gga?gaa?agc?ttg?ctt?t????????????????????????????28
pr-4?????????gta?aca?gga?gaa?agc?ttg?ctt?tct?tgc?tga?cga?gc???????????????????35
pr-5?????????gtc?tac?gga?cca?aag?cag?ggg?ctc?ttc?gga?cct?tgc??????????????????36
pr-6?????????gtg?ata?aag?tta?ata?cct?tta?tca?att?g????????????????????????????28
pr-7?????????att?gcg?taa?cag?aga?gaa?agc?ttg?ctt?tct?tgc?tga??????????????????36
pr-8?????????att?gcg?taa?cag?aga?gaa?agc?ttg?ctt?t????????????????????????????28
pr-9?????????taa?cag?aga?gaa?agc?ttg?ctt?tct?tgc?tga?ctg?agc??????????????????36
pr-10????????gtg?ata?aag?tta?ata?cct?ttg?tca?att?g????????????????????????????28
2. amplification contains pulsating PCR primer design of target gene and optimization
According to coding invasion and attack plasmid antigen H (ipaH) on the big plasmid in bacterial 16 S rRNA and 23S rRNA and the dysentery bacterium and salmonella typhi regulation and control Vi antigen presentation (VipR) gene order design primer, 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 16S rDNA, ipaH and 23S rDNA, VipR primer are carried out the optimization (the PCR reaction electrophoresis detection of optimization the results are shown in accompanying drawing 3) of the double pcr amplification condition of two covers.
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)
16S rDNA cgc tgg cgg cag gcc taa cac atg c 500 forward primers
Cgc ggc tgc tgg cac gga gtt agc c reverse primer
23S rDNA tta aat gat ggc tgc ttc taa gcc 500 forward primers
Acc gat agt gaa cca gta ccg tga g reverse primer
IpaH gtt cct tga ccg cct ttc cga tac 330 forward primers
Gag agt tct gac ttt atc ccg reverse primer
VipR ggt ttc atc att tct ggc ctc cg 337 forward primers
Ctc tgc tcc gtc aag atc ttt tca cc reverse primer
3. the screening and optimizing of the preparation of oligonucleotide gene chip and probe
With alternative oligonucleotide probe purifying, quantitatively after, put on the aldehyde radical activatory slide glass with the gene chip sample applying instrument, make the probe microarray.With fluorescently-labeled pcr amplification product and gene chip hybridization, obtain the hybridization figure of each probe.At the gene fragment of various pathogenic bacteria, select good, the highly sensitive probe of specificity separately 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 common pathogen enterobacteria
Probes probes sequence probe length
Title (BP)
SA5??????aca?tat?gtg?taa?gta?act?gtg?cac?atc?ttg?acg?gta???????????36
LI2??????tgt?tgt?tag?aga?aga?aca?agg?ata?aga?gta?act?gct???????????36
LI3??????tag?aga?aga?aca?agg?ata?aga?gta?act?gct?tgt?ccc???????????36
VC1??????cag?cac?aga?gga?act?tgt?tcc?ttg?ggt?ggc?gag???????????????33
YE1??????gcg?gca?gcg?gga?agt?agt?tta?cta?ctt?tgc?cgg???????????????33
ye-2e????cat?aaa?ggt?taa?taa?cct?ttg?tga?ttg?acg?t?????????????????31
SHI1?????ggg?agt?aaa?gtt?aat?acc?ttt?gct?cat?tga???????????????????30
V-2??????ctt?caa?taa?tgc?cag?cag?ctc?caa?ccc?cga?aat?aga?ta????????38
V-3????????aca?ggc?tgt?agc?gat?tta?ggt?tca?caa?aca?aat?aat?tt??????????????38
SAL1???????ggt?gtt?gtg?gtt?aat?aac?cgc?agc?aat?tga?????????????????????????30
I-1????????gaa?ggc?ctt?ttc?gat?aat?gat?acc?ggc?gct?ctg?ctc?tcc?c???????????40
I-2????????gga?ccg?tgt?cgc?gct?cac?atg?gaa?caa?tct?ccg?gaa?aac?cct?????????42
VP1????????aaa?cga?gtt?atc?tga?acc?ttc?ggg?gaa?cga?taa?cgg?????????????????36
VP23???????ggt?acg?cag?tca?cag?gac?aaa?gcc?????????????????????????????????24
H7-1???????gca?gtc?acc?cca?taa?aag?agg?ct??????????????????????????????????23
H7-2???????tca?ccc?cat?aaa?aga?ggc?tcc?cac?tgc?????????????????????????????27
SS5????????ggt?ttc?agg?ttc?ttt?ttc?act?ccc?ctc?gcc?g???????????????????????31
CP-new-1???atg?gca?tca?tca?ttc?aac?caa?agg?agc?aat?ccg?cta?tga?gat?gga?????48
ccc
Cam-5??????ctc?ctt?ttc?tta?ggg?aag?aat?tct?gac?ggt?acc?taa?gga?at??????????41
pr-2???????cga?gcg?gta?aca?gga?gaa?agc?ttg?ctt?tct?tgc?tga?????????????????36
CB-1???????tat?aag?aga?atc?gca?tga?ttt?tct?tat?cca?aag?att?tat?????????????39
com21??????act?gag?aca?cgg?tcc?aga?ctc?cta?cgg?gag?gca?gca?gta?ggg?aat?????49
attg
com31??????cac?act?gga?act?gag?aca?cgg?tcg?aga?ctc?cta?cgg?ga??????????????38
Bru-2??????cgt?acc?att?tgc?tac?gga?ata?act?cag?gga?aac?ttg?tg??????????????38
B-6????????tgc?tag?ttg?aat?aag?ctg?gca?cct?tga?cg??????????????????????????29
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 PCR product results of hybridization of 17 kind pathogen enterobacterias 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.
The detection gene chip probes sequence of the common pathogen enterobacteria of table 2-1
Probe title probe sequence probe length
(BP)
SA5????????aca?tat?gtg?taa?gta?act?gtg?cac?atc?ttg?acg?gta????????????????36
LI2????????tgt?tgt?tag?aga?aga?aca?agg?ata?aga?gta?act?gct????????????????36
LI3???????????tag?aga?aga?aca?agg?ata?aga?gta?act?gct?tgt?ccc?????????????36
VC1???????????cag?cac?aga?gga?act?tgt?tcc?ttg?ggt?ggc?gag?????????????????33
YE1???????????gcg?gca?gcg?gga?agt?agt?tta?cta?ctt?tgc?cgg?????????????????33
ye-2e?????????cat?aaa?ggt?taa?taa?cct?ttg?tga?ttg?acg?t???????????????????31
SHI1??????????ggg?agt?aaa?gtt?aat?acc?ttt?gct?cat?tga?????????????????????30
V-2???????????ctt?caa?taa?tgc?cag?cag?ctc?caa?ccc?cga?aat?aga?ta??????????38
V-3???????????aca?ggc?tgt?agc?gat?tta?ggt?tca?caa?aca?aat?aat?tt??????????38
SAL1??????????ggt?gtt?gtg?gtt?aat?aac?cgc?agc?aat?tga?????????????????????30
I-1???????????gaa?ggc?ctt?ttc?gat?aat?gat?acc?ggc?gct?ctg?ctc?tcc?c???????40
I-2???????????gga?ccg?tgt?cgc?gct?cac?atg?gaa?caa?tct?ccg?gaa?aac?cct?????42
VP1???????????aaa?cga?gtt?atc?tga?acc?ttc?ggg?gaa?cga?taa?cgg?????????????36
VP23??????????ggt?acg?cag?tca?cag?gac?aaa?gcc?????????????????????????????24
H7-1??????????gca?gtc?acc?cca?taa?aag?agg?ct??????????????????????????????23
H7-2??????????tca?ccc?cat?aaa?aga?ggc?tcc?cac?tgc?????????????????????????27
SS5???????????ggt?ttc?agg?ttc?ttt?ttc?act?ccc?ctc?gcc?g???????????????????31
CP-ncw-1??????atg?gca?tca?tca?ttc?aac?caa?agg?agc?aat?ccg?cta?tga?gat?????48
gga?ccc
Cam-5?????????ctc?ctt?ttc?tta?ggg?aag?aat?tct?gac?ggt?acc?taa?gga?at??????41
pr-2??????????cga?gcg?gta?aca?gga?gaa?agc?ttg?ctt?tct?tgc?tga?????????????36
CB-1??????????tat?aag?aga?atc?gca?tga?ttt?tct?tat?cca?aag?att?tat?????????39
com21?????????act?gag?aca?cgg?tcc?aga?ctc?cta?cgg?gag?gca?gca?gta?ggg?????49
aat?attg
com31?????????cac?act?gga?act?gag?aca?cgg?tcg?aga?ctc?cta?cgg?ga??????????38
Bru-2?????????cgt?acc?att?tgc?tac?gga?ata?act?cag?gga?aac?ttg?tg??????????38
B-6???????????tgc?tag?ttg?aat?aag?ctg?gca?cct?tga?cg??????????????????????29
Negative probe contrast ccg ctg tat cac aag ggc tgg tac c 25
Array probe contrast fluorescent primer reverse complementary sequence 25
3. pathogen enterobacteria oligonucleotide chip specific detection
Optimizing under the good two double pcr amplifications of cover and hybridization condition, detecting the standard pathogen enterobacteria of 146 strain different serotypes.Wherein included common pathogen enterobacteria: Escherichia coli O 157: H7W933,882364; Totally 146 strains such as enteroinvasive E 48017, intestinal bacteria, cholera vibrio O 139, O1 type, streptococcus aureus 6538, Salmonellas, Yersinia, Bacillus cereus, Shigellae, listeria spp, pseudomonas, sex change bacillus, brucella, Campylobacter, clostridium perfringens, Vibrio parahemolyticus, citrobacter, tarda, Vibrio vulnificus, klebsiella, Serratia.With the DNA of bacteria is that template is carried out two cover double pcr amplification, product and chip hybridizations.Wherein occur the signal in the results of hybridization of whole bacteriums except that positive control probe common21 and common31, other the probe of signal occurs, and all the bacterium with its hybridization is corresponding, and the hybridization of some negative bacterias does not then have any signal generation (seeing Table 2-2).Selected being used to of this explanation differentiates that the probe of above-mentioned 17 kinds of pathogen enterobacterias is special, illustrates that also this experiment is based upon on the double PCR of the two covers basis, and the gene chip of 17 kind pathogen enterobacterias of qualitative detection is reliable simultaneously.
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 (ABI8909).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, the equal 0.5 μ M of forward and reverse primer concentration, dNTP is 100 μ M, 1U Taq archaeal dna polymerase, 1 * PCR reaction buffer, 50-100ng template DNA; Amplification condition is: and pre-sex change (94 ℃, 5min); 35 circulations: sex change (94 ℃, 30sec), annealing (56 ℃, 30sec) extend (72 ℃, 30sec); Extend (72 ℃, 5min); 4 ℃, ∞.
In the reaction of multiple asymmetric PCR, the change of concentration has tangible influence to the amplification efficiency of goal gene between the four 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 four products all can reach close and amplification efficiency and hybridization signal preferably.The result who optimizes is: the forward primer concentration of 16S rDNA, ipaH and 23S rDNA, vipR gene is respectively 0.25 μ M, 0.25 μ M in the double PCR reaction of two covers, and oppositely fluorescent dye primer concentration is respectively 0.75 μ M, 0.75 μ M.In the 20ul reaction system, dNTP is 200 μ M, 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. common pathogen enterobacteria oligonucleotide chip prepares oligonucleotide probe, and (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 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.
4. common pathogen enterobacteria oligonucleotide chip hybridization and detection
4.1 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.
4.2 hybridization and the sex change of the fluorescently-labeled PCR product of hybridization aftertreatment (98 ℃, 5min, ice bath, 5min) back and hybridization solution (5 * SSC, 5% methane amide, 0.1%SDS) mixing is got 12 μ 1 and is transferred to the chip reaction zone, and chip places in the hybridizing box, together with reaction in 50 ℃ of water-baths of hybridizing box immersion 60 minutes, chip after the hybridization successively 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.
4.3 scanning and result judge that chip scans with chip scanner GenePix 4000B, with GenePix Pro 4.0 software analysis results.
Table 2-2: applying gene chip detection 146 strain entero-bacte results
| ??I-2 | ??VP1 | ??VP23 | ??H7-1 | ??H7-2 | ??SS5 | ??CP1 | ??Cam5 | ??pr2 | ??B6 | ??CB1 | ??Bru1 | ??Bru2 | ??Neg | ??COM21 |
| ??- | ??- | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??- | ??- | ??- | ??+ |
| ??- | ??- | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??- | ??- | ??- | ??+ |
| ??- | ??- | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??- | ??- | ??- | ??+ |
| ??- | ??- | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??- | ??- | ??- | ??+ |
| ??- | ??- | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??- | ??- | ??- | ??+ |
| ??- | ??- | ??- | ??- | ??- | ??+ | ??- | ????- | ??- | ??- | ??- | ??- | ??- | ??- | ??+ |
| ??- | ??- | ??- | ??- | ??- | ??+ | ??- | ????- | ??- | ??- | ??- | ??- | ??- | ??- | ??+ |
| ??- | ??- | ??- | ??- | ??- | ??+ | ??- | ????- | ??- | ??- | ??- | ??- | ??- | ??- | ??+ |
| ??+ | ??- | ??- | ??- | ??- | ??+ | ??- | ????- | ??- | ??- | ??- | ??- | ??- | ??- | ??+ |
| ??- | ??+ | ??+ | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??- | ??- | ??- | ??+ |
| ??- | ??- | ??- | ??+ | ??+ | ??+ | ??- | ????- | ??- | ??- | ??- | ??- | ??- | ??- | ??+ |
| ??- | ??- | ??- | ??- | ??- | ??- | ??+ | ????- | ??- | ??- | ??- | ??- | ??- | ??- | ??+ |
| ??- | ??- | ??- | ??- | ??- | ??- | ??- | ????+ | ??- | ??- | ??- | ??- | ??- | ??- | ??+ |
| ??- | ??- | ??- | ??- | ??- | ??- | ??- | ????- | ??+ | ??- | ??- | ??- | ??- | ??- | ??+ |
| ??- | ??- | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??+ | ??- | ??- | ??- | ??+ |
| ??- | ??- | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??+ | ??+ | ??- | ??+ |
| ??- | ??- | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??+ | ??- | ??- | ??- | ??- | ??+ |
| ??- | ??- | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??- | ??- | ??- | ??+ |
| ??- | ??- | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??- | ??- | ??- | ??- |
| Bacterial strain | Quantity | ??SA5 | ??LI2 | ??LI3 | ??VC1 | ??YE1 | ??ye-2e | ??SHI1 | ??V1 | ??V2 | ??SAL1 | ??I-1 |
| Streptococcus aureus | ??+ | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??- | ??- | |
| Listeria spp | ??- | ??+ | ??+ | ??- | ??- | ????- | ??- | ??- | ??- | ??- | ??- | |
| Vibrio cholerae | ??- | ??- | ??- | ??+ | ??- | ????- | ??- | ??- | ??- | ??- | ??- | |
| Yersinia | ??- | ??- | ??- | ??- | ??+ | ????- | ??- | ??- | ??- | ??- | ??- | |
| Yersinia entero-colitica | ??- | ??- | ??- | ??- | ??+ | ????+ | ??- | ??- | ??- | ??- | ??- | |
| Salmonellas | ??- | ??- | ??- | ??- | ??- | ????- | ??+ | ??- | ??- | ??- | ??- | |
| Salmonella typhi | ??- | ??- | ??- | ??- | ??- | ????- | ??+ | ??+ | ??+ | ??- | ??- | |
| Shigellae | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??+ | ??- | |
| Dysentery bacterium | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??+ | ??+ | |
| Vibrio parahemolyticus | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??- | ??- | |
| ??O157:H7 | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??- | ??- | |
| Clostridium perfringens | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??- | ??- | |
| Campylobacter | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??- | ??- | |
| Bacillus proteus | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??- | ??- | |
| Clostridium botulinum | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??- | ??- | |
| Brucella | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??- | ??- | |
| Cured sample gemma Pseudomonas | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??- | ??- | |
| Other entero-bacte | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??+ | ??- | |
| Other non-entero-bacte | ??- | ??- | ??- | ??- | ??- | ????- | ??- | ??- | ??- | ??- | ??- |
Sequence table
＜110〉Institute of Radiation Medicine, Academy of Military Medical Sciences, PLA
＜120〉cover is used to oligonucleotide probe that detects common pathogen enterobacteria and uses thereof
<160>91
<210>1
<211>33
<212>DNA
＜213〉artificial sequence
<400>1
aac?gga?cga?gga?gct?tgc?ttc?tct?gat?gtt?agc?33
<210>2
<211>35
<212>DNA
＜213〉artificial sequence
<400>2
caa?aag?tga?aag?acg?gtc?ttg?ctg?tca?ctt?ata?ga?35
<210>3
<211>36
<212>DNA
＜213〉artificial sequence
<400>3
aaa?ctc?tgt?tat?tag?gga?aga?aca?tat?gtg?taa?gta?36
<210>4
<211>36
<212>DNA
＜213〉artificial sequence
<400>4
gtg?cac?atc?ttg?acg?gta?cct?aat?cag?aaa?gcc?acg?36
<210>5
<211>36
<212>DNA
＜213〉artificial sequence
<400>5
aca?tat?gtg?taa?gta?act?gtg?cac?atc?ttg?acg?gta?36
<210>6
<211>36
<212>DNA
＜213〉artificial sequence
<400>6
tga?cta?ctt?gta?agc?aca?cgg?ttt?cag?gtt?cta?ttt?36
<210>7
<211>33
<212>DNA
＜213〉artificial sequence
<400>7
aat?att?ttg?aac?cgc?atg?gtt?caa?aag?tga?aag?33
<210>8
<211>18
<212>DNA
＜213〉artificial sequence
<400>8
tac?gac?caa?ata?cta?aac?18
<210>9
<211>20
<212>DNA
＜213〉artificial sequence
<400>9
caa?taa?tca?tca?ctt?gag?gc?20
<210>10
<211>33
<212>DNA
＜213〉artificial sequence
<400>10
aac?gga?gga?aga?gct?tgc?tct?tcc?aaa?gtt?agt?33
<210>11
<211>36
<212>DNA
＜213〉artificial sequence
<400>11
tgt?tgt?tag?aga?aga?aca?agg?ata?aga?gta?act?gct?36
<210>12
<211>36
<212>DNA
＜213〉artificial sequence
<400>12
tag?aga?aga?aca?agg?ata?aga?gta?act?gct?tgt?ccc?36
<210>13
<211>22
<212>DNA
＜213〉artificial sequence
<400>13
gtg?gca?tgc?gcc?aca?ctt?tat?c?22
<210>14
<211>36
<212>DNA
＜213〉artificial sequence
<400>14
aaa?cga?gtt?atc?tga?acc?ttc?ggg?gaa?cga?taa?cgg?36
<210>15
<211>24
<212>DNA
＜213〉artificial sequence
<400>15
ggt?acg?cag?tca?cag?gac?aaa?gcc?24
<210>16
<211>33
<212>DNA
＜213〉artificial sequence
<400>16
cag?cac?aga?gga?act?tgt?tcc?ttg?ggt?ggc?gag?33
<210>17
<211>33
<212>DNA
＜213〉artificial sequence
<400>17
gcg?gca?gcg?gga?agt?agt?tta?cta?ctt?tgc?cgg?33
<210>18
<211>30
<212>DNA
＜213〉artificial sequence
<400>18
agg?ggt?tga?gtt?taa?tac?gct?caa?tca?ttg?30
<210>19
<211>31
<212>DNA
＜213〉artificial sequence
<400>19
cat?aaa?ggt?taa?taa?cct?ttg?tga?ttg?acg?t?31
<210>20
<211>30
<212>DNA
＜213〉artificial sequence
<400>20
ggg?agt?aaa?gtt?aat?acc?ttt?gct?cat?tga?30
<210>21
<211>24
<212>DNA
＜213〉artificial sequence
<400>21
ctg?gcc?tcc?gaa?tga?tat?cta?ttt?24
<210>22
<211>38
<212>DNA
＜213〉artificial sequence
<400>22
tat?cta?ttt?cgg?ggt?tgg?agc?tgc?tgg?cat?tat?tga?ag?38
<210>23
<211>38
<212>DNA
＜213〉artificial sequence
<400>23
aca?ggc?tgt?agc?gat?tta?ggt?tca?caa?aca?aat?aat?tt?38
<210>24
<211>25
<212>DNA
＜213〉artificial sequence
<400>24
cgt?tat?cat?ttc?aag?ttc?gcg?act?a?25
<210>25
<211>33
<212>DNA
＜213〉artificial sequence
<400>25
cgg?caa?cat?cag?tcc?agg?ata?tta?tca?gaa?atg?33
<210>26
<211>30
<212>DNA
＜213〉artificial sequence
<400>26
ggt?gtt?gtg?gtt?aat?aac?cgc?agc?aat?tga?30
<210>27
<211>40
<212>DNA
＜213〉artificial sequence
<400>27
gaa?ggc?ctt?ttc?gat?aat?gat?acc?ggc?gct?ctg?ctc?tcc?c?40
<210>28
<211>42
<212>DNA
＜213〉artificial sequence
<400>28
gga?ccg?tgt?cgc?gct?cac?atg?gaa?caa?tct?ccg?gaa?aac?cct?42
<210>29
<211>28
<212>DNA
＜213〉artificial sequence
<400>29
agt?ctt?tcg?ctg?ttg?ctg?ctg?atg?cca?c?28
<210>30
<211>35
<212>DNA
＜213〉artificial sequence
<400>30
cct?ctg?cgg?agc?ttc?gac?agc?agt?ctt?tcg?ctg?tt?35
<210>31
<211>35
<212>DNA
＜213〉artificial sequence
<400>31
ata?ccg?tct?ctg?cac?gca?ata?cct?ccg?gat?tcc?gt?35
<210>32
<211>32
<212>DNA
＜213〉artificial sequence
<400>32
tcc?gga?ttc?cgt?gaa?cag?gtc?gct?gca?tgg?ct?32
<210>33
<211>23
<212>DNA
＜213〉artificial sequence
<400>33
gca?gtc?acc?cca?taa?aag?agg?ct?23
<210>34
<211>27
<212>DNA
＜213〉artificial sequence
<400>34
tca?ccc?cat?aaa?aga?ggc?tcc?cac?tgc?27
<210>35
<211>26
<212>DNA
＜213〉artificial sequence
<400>35
act?cct?gct?taa?cac?aag?ttg?agt?ag?26
<210>36
<211>24
<212>DNA
＜213〉artificial sequence
<400>36
tcg?gtg?tag?gat?gag?act?ata?tag?24
<210>37
<211>40
<212>DNA
＜213〉artificial sequence
<400>37
act?cct?gct?taa?cac?aag?ttg?agt?agg?gaa?agt?ttt?tcg?g?40
<210>38
<211>27
<212>DNA
＜213〉artificial sequence
<400>38
ggt?ata?gtt?aat?ctg?ccc?tac?aca?aga?27
<210>39
<211>41
<212>DNA
＜213〉artificial sequence
<400>39
ctc?ctt?ttc?tta?ggg?aag?aat?tct?gac?ggt?acc?taa?gga?at?41
<210>40
<211>32
<212>DNA
＜213〉artificial sequence
<400>40
act?cct?gct?taa?cac?aag?ttg?agt?agg?gaa?ag?32
<210>41
<211>36
<212>DNA
＜213〉artificial sequence
<400>41
gat?ttg?cct?gta?taa?cct?cct?acg?acc?tta?gac?tag?36
<210>42
<211>29
<212>DNA
＜213〉artificial sequence
<400>42
cgg?agt?aaa?tcc?taa?tac?aaa?gct?aac?ca?29
<210>43
<211>31
<212>DNA
＜213〉artificial sequence
<400>43
ttc?tat?cca?tcc?gaa?gac?ttc?aaa?aag?cct?t?31
<210>44
<211>40
<212>DNA
＜213〉artificial sequence
<400>44
act?cct?gct?taa?cac?aag?ttg?agt?agg?gaa?agt?ttt?tcg?g?40
<210>45
<211>33
<212>DNA
＜213〉artificial sequence
<400>45
cat?ttt?gaa?ccg?cat?ggt?tcg?aaa?ttg?aaa?ggc?33
<210>46
<211>30
<212>DNA
＜213〉artificial sequence
<400>46
gga?taa?cat?ttt?gaa?ccg?cat?ggt?tcg?aaa?30
<210>47
<211>28
<212>DNA
＜213〉artificial sequence
<400>47
ggc?tgt?cac?tta?tgg?atg?gac?ccg?cgt?c?28
<210>48
<211>24
<212>DNA
＜213〉artificial sequence
<400>48
tgc?tag?ttg?aat?aag?ctg?gca?cct?24
<210>49
<211>34
<212>DNA
＜213〉artificial sequence
<400>49
ggc?ttc?ggc?tgt?cac?tta?tgg?atg?gac?ccg?cgt?c?34
<210>50
<211>29
<212>DNA
＜213〉artificial sequence
<400>50
tgc?tag?ttg?aat?aag?ctg?gca?cct?tga?cg?29
<210>51
<211>24
<212>DNA
＜213〉artificial sequence
<400>51
tgc?gta?gcc?gac?ctg?aga?ggg?tgt?24
<210>52
<211>16
<212>DNA
＜213〉artificial sequence
<400>52
cga?gct?caa?agt?atg?t?16
<210>53
<211>18
<212>DNA
＜213〉artificial sequence
<400>53
gcc?gag?ctc?aaa?gta?tgt?18
<210>54
<211>24
<212>DNA
＜213〉artificial sequence
<400>54
gcc?gag?ctc?aaa?gta?tgt?ggc?att?24
<210>55
<211>22
<212>DNA
＜213〉artificial sequence
<400>55
act?act?cat?act?cga?cgc?tag?c?22
<210>56
<211>27
<212>DNA
＜213〉artificial sequence
<400>56
gtg?ata?agg?ttc?ggt?aag?cgc?tat?gcc?27
<210>57
<211>27
<212>DNA
＜213〉artificial sequence
<400>57
ggc?ata?gcg?ctt?acc?gaa?cct?tat?cgc?27
<210>58
<211>34
<212>DNA
＜213〉artificial sequence
<400>58
aag?atg?gca?tca?tca?ttc?aac?caa?agg?agc?aat?c?34
<210>59
<211>40
<212>DNA
＜213〉artificial sequence
<400>59
aag?atg?gca?tca?tca?ttc?aac?caa?agg?agc?aat?cgc?tat?g?40
<210>60
<211>36
<212>DNA
＜213〉artificial sequence
<400>60
gaa?aga?tgg?cat?cat?cat?tca?acc?aaa?gga?gca?atc?36
<210>61
<211>48
<212>DNA
＜213〉artificial sequence
<400>61
atg?gca?tca?tca?ttc?aac?caa?agg?agc?aat?ccg?ctatga?gat?gga?ccc?48
<210>62
<211>45
<212>DNA
＜213〉artificial sequence
<400>62
atg?gca?tca?tca?ttc?aac?caa?agg?agc?aat?ccg?cta?tga?gat?gga?45
<210>63
<211>25
<212>DNA
＜213〉artificial sequence
<400>63
cca?gac?tcc?tac?ggg?agg?cag?cag?t?25
<210>64
<211>27
<212>DNA
＜213〉artificial sequence
<400>64
ctc?cta?cgg?gag?gca?gca?gta?ggg?aat?27
<210>65
<211>23
<212>DNA
＜213〉artificial sequence
<400>65
cac?act?gga?act?gag?aca?cgg?tc?23
<210>66
<211>49
<212>DNA
＜213〉artificial sequence
<400>66
act?gag?aca?cgg?tcc?aga?ctc?cta?cgg?gag?gca?gca?gta?ggg?aat?attg?49
<210>67
<211>38
<212>DNA
＜213〉artificial sequence
<400>67
cac?act?gga?act?gag?aca?cgg?tcg?aga?ctc?cta?cgg?ga?38
<210>68
<211>25
<212>DNA
＜213〉artificial sequence
<400>68
tgt?ctg?gga?aac?tgc?ctg?atg?gag?g?25
<210>69
<211>31
<212>DNA
＜213〉artificial sequence
<400>69
cgt?cgc?aag?acc?aaa?gag?ggg?gac?ctt?cgg?g?31
<210>70
<211>31
<212>DNA
＜213〉artificial sequence
<400>70
ggt?ttc?agg?ttc?ttt?ttc?act?ccc?ctc?gcc?g?31
<210>71
<211>43
<212>DNA
＜213〉artificial sequence
<400>71
aac?ctg?ccc?atg?gct?aga?tca?ccg?ggt?ttc?ggg?tct?ata?ccc?t?43
<210>72
<211>39
<212>DNA
＜213〉artificial sequence
<400>72
aat?ttt?tca?aca?tta?gtc?ggt?tcg?gtc?ctc?cag?tta?gtg?39
<210>73
<211>27
<212>DNA
＜213〉artificial sequence
<400>73
cct?ctt?gcc?atc?gga?tgt?gcc?cag?atg?27
<210>74
<211>39
<212>DNA
＜213〉artificial sequence
<400>74
tat?aag?aga?atc?gca?tga?ttt?tct?tat?cca?aag?att?tat?39
<210>75
<211>32
<212>DNA
＜213〉artificial sequence
<400>75
atg?aag?ctt?cct?tcg?gga?agt?gga?tta?gcg?gc?32
<210>76
<211>30
<212>DNA
＜213〉artificial sequence
<400>76
ggg?ata?gcc?ttc?cga?aag?gaa?gat?taa?tac?30
<210>77
<211>33
<212>DNA
＜213〉artificial sequence
<400>77
gaa?cgg?taa?cag?gaa?gca?gct?tgc?tgt?ttg?ctg?33
<210>78
<211>21
<212>DNA
＜213〉artificial sequence
<400>78
aac?gtc?gca?aga?cca?aag?agg?21
<210>79
<211>46
<212>DNA
＜213〉artificial sequence
<400>79
gtg?ccc?ttc?ggg?gga?aag?att?tat?cgg?caa?atg?atc?ggc?ccg?cgt?t?46
<210>80
<211>38
<212>DNA
＜213〉artificial sequence
<400>80
cgt?acc?att?tgc?tac?gga?ata?act?cag?gga?aac?ttg?tg?38
<210>81
<211>30
<212>DNA
＜213〉artificial sequence
<400>81
taa?cag?gag?aaa?gct?tgc?ttt?ctt?gct?gac?30
<210>82
<211>30
<212>DNA
＜213〉artificial sequence
<400>82
cgt?cga?gtt?cac?aat?aac?agc?atc?ttc?aga?30
<210>83
<211>36
<212>DNA
＜213〉artificial sequence
<400>83
cga?gcg?gta?aca?gga?gaa?agc?ttg?ctt?tct?tgc?tga?36
<210>84
<211>28
<212>DNA
＜213〉artificial sequence
<400>84
cga?gcg?gta?aca?gga?gaa?agc?ttg?ctt?t?28
<210>85
<211>35
<212>DNA
＜213〉artificial sequence
<400>85
gta?aca?gga?gaa?agc?ttg?ctt?tct?tgc?tga?cga?gc?35
<210>86
<211>36
<212>DNA
＜213〉artificial sequence
<400>86
gtc?tac?gga?cca?aag?cag?ggg?ctc?ttc?gga?cct?tgc?36
<210>87
<211>28
<212>DNA
＜213〉artificial sequence
<400>87
gtg?ata?aag?tta?ata?cct?tta?tca?att?g?28
<210>88
<211>36
<212>DNA
＜213〉artificial sequence
<400>88
att?gcg?taa?cag?aga?gaa?agc?ttg?ctt?tct?tgc?tga?36
<210>89
<211>28
<212>DNA
＜213〉artificial sequence
<400>89
att?gcg?taa?cag?aga?gaa?agc?ttg?ctt?t?28
<210>90
<211>36
<212>DNA
＜213〉artificial sequence
<400>77
taa?cag?aga?gaa?agc?ttg?ctt?tct?tgc?tga?ctg?agc?36
<210>91
<211>28
<212>DNA
＜213〉artificial sequence
<400>91
gtg?ata?aag?tta?ata?cct?ttg?tca?att?g?28
Claims (7)
1. a cover is used to detect the oligonucleotide probe of common pathogen enterobacteria, 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 25-50 base.
A. the 16S rDNA gene test probe of streptococcus aureus
B. the 16S rDNA gene test probe that belongs to of listeria spp
C. the 23S rDNA gene test probe of enterorrhagia Bacillus coil 0157: H7
D. the 16S rDNA gene test probe of vibrio cholerae
E. the 16S rDNA gene test probe of Yersinia
F. the 16S rDNA gene test probe of yersinia entero-colitica
G. the 16S rDNA gene test probe of salmonella
H. salmonella typhi plays the gene VipR detection probes of positive regulating and controlling effect to Vi antigen presentation gene ViaB
I. the 16S rDNA gene test probe of Shigella
J. coding is attacked plasmid antigen H gene ipaH detection probes on the big plasmid of dysentery bacterium
K. the 16S rDNA gene test probe of Vibrio parahemolyticus
L. the 23S rDNA gene test probe of Vibrio parahemolyticus
M. the total 16S rDNA gene test probe of Shigella, salmonella and intestinal bacteria
N. the 16S rDNA gene test probe of clostridium perfringens
O. the 16S rDNA gene test probe of Campylobacter
P. the 16S rDNA gene test probe of Bacillus proteus
Q. the 16S rDNA gene test probe that belongs to of wax-like bacillus
R. the 16S rDNA gene test probe of Clostridium botulinum
S. the general 16S rDNA gene test probe of eubacterium
T. brucellar 16S rDNA gene test probe.
2. a cover oligonucleotide probe as claimed in claim 1, wherein
The a probe is selected from the following oligonucleotide sequence 1 or several
SA1????aac?gga?cga?gga?gct?tgc?ttc?tct?gat?gtt?agc
SA2????caa?aag?tga?aag?acg?gtc?ttg?ctg?tca?ctt?ata?ga
SA3????aaa?ctc?tgt?tat?tag?gga?aga?aca?tat?gtg?taa?gta
SA4????gtg?cac?atc?ttg?acg?gta?cct?aat?cag?aaa?gcc?acg
SA5????aca?tat?gtg?taa?gta?act?gtg?cac?atc?ttg?acg?gta
SA-6???tga?cta?ctt?gta?agc?aca?cgg?ttt?cag?gtt?cta?ttt
SA-7???aat?att?ttg?aac?cgc?atg?gtt?caa?aag?tga?aag
SA-8????tac?gac?caa?ata?cta?aac
SA-9????caa?taa?tca?tca?ctt?gag?gc
The b probe is selected from the following oligonucleotide sequence 1 or several
LI1?????aac?gga?gga?aga?gct?tgc?tct?tcc?aaa?gtt?agt
LI2?????tgt?tgt?tag?aga?aga?aca?agg?ata?aga?gta?act?gct
LI3?????tag?aga?aga?aca?agg?ata?aga?gta?act?gct?tgt?ccc
Lis-23??gtg?gca?tgc?gcc?aca?ctt?tat?c
The c probe is selected from the following oligonucleotide sequence 1 or several
H7-1????gca?gtc?acc?cca?taa?aag?agg?ct
H7-2????tca?ccc?cat?aaa?aga?ggc?tcc?cac?tgc
The d probe is selected from the following oligonucleotide sequence 1 or several
VC1?????cag?cac?aga?gga?act?tgt?tcc?ttg?ggt?ggc?gag
The e probe is selected from the following oligonucleotide sequence 1 or several
YE1?????gcg?gca?gcg?gga?agt?agt?tta?cta?ctt?tgc?cgg
ye-3????agg?ggt?tga?gtt?taa?tac?gct?caa?tca?ttg
The f probe is selected from the following oligonucleotide sequence 1 or several
ye-2e???cat?aaa?ggt?taa?taa?cct?ttg?tga?ttg?acg?t
The g probe is selected from the following oligonucleotide sequence 1 or several
SHI1????ggg?agt?aaa?gtt?aat?acc?ttt?gct?cat?tga
The h probe is selected from the following oligonucleotide sequence 1 or several
V-1?????aaa?tag?ata?tca?ttc?gga?ggc?cag
V-2?????ctt?caa?taa?tgc?cag?cag?ctc?caa?ccc?cga?aat?aga?ta
V-3?????aca?ggc?tgt?agc?gat?tta?ggt?tca?caa?aca?aat?aat?tt
V-4?????tag?tcg?cga?act?tga?aat?gat?aac?g
V-5?????cat?ttc?tga?taa?tat?cct?gga?ctg?atg?ttg?ccg
The i probe is selected from the following oligonucleotide sequence 1 or several
SAL1????ggt?gtt?gtg?gtt?aat?aac?cgc?agc?aat?tga
The j probe is selected from the following oligonucleotide sequence 1 or several
I-1?????gaa?ggc?ctt?ttc?gat?aat?gat?acc?ggc?gct?ctg?ctc?tcc?c
I-2?????gga?ccg?tgt?cgc?gct?cac?atg?gaa?caa?tct?ccg?gaa?aac?cct
I-3??????agt?ctt?tcg?ctg?ttg?ctg?ctg?atg?cca?c
I-4??????cct?ctg?cgg?agc?ttc?gac?agc?agt?ctt?tcg?ctg?tt
I-5??????ata?ccg?tct?ctg?cac?gca?ata?cct?ccg?gat?tcc?gt
I-6??????tcc?gga?ttc?cgt?gaa?cag?gtc?gct?gca?tgg?ct
The k probe is selected from the following oligonucleotide sequence 1 or several
VP1??????aaa?cga?gtt?atc?tga?acc?ttc?ggg?gaa?cga?taa?cgg
1 probe is selected from the following oligonucleotide sequence 1 or several
VP23?????ggt?acg?cag?tca?cag?gac?aaa?gcc
The m probe is selected from the following oligonucleotide sequence 1 or several
SS3??????tgt?ctg?gga?aac?tgc?ctg?atg?gag?g
SS4??????cgt?cgc?aag?acc?aaa?gag?ggg?gac?ctt?cgg?g
SS5??????ggt?ttc?agg?ttc?ttt?ttc?act?ccc?ctc?gcc?g
SS6??????aac?ctg?ccc?atg?gct?aga?tca?ccg?ggt?ttc?ggg?tct?ata?ccc?t
SS7??????aat?ttt?tca?aca?tta?gtc?ggt?tcg?gtc?ctc?cag?tta?gtg
SS8??????cct?ctt?gcc?atc?gga?tgt?gcc?cag?atg
The n probe is selected from the following oligonucleotide sequence 1 or several
CP-1?????cga?gct?caa?agt?atg?t
CP-2?????gcc?gag?ctc?aaa?gta?tgt
CP-3?????gcc?gag?ctc?aaa?gta?tgt?ggc?att
CP-4?????act?act?cat?act?cga?cgc?tag?c
CP-5?????gtg?ata?agg?ttc?ggt?aag?cgc?tat?gcc
CP-6?????ggc?ata?gcg?ctt?acc?gaa?cct?tat?cac
cp-16????aag?atg?gca?tca?tca?ttc?aac?caa?agg?agc?aat?c
CP-16-1??aag?atg?gca?tca?tca?ttc?aac?caa?agg?agc?aat?cgc?tat?g
CP1??????gaa?aga?tgg?cat?cat?cat?tca?acc?aaa?gga?gca?atc
CP-new-1?atg?gca?tca?tca?ttc?aac?caa?agg?agc?aat?ccg?cta?tga?gat?gga?ccc
CP-new-2?atg?gca?tca?tca?ttc?aac?caa?agg?agc?aat?ccg?cta?tga?gat?gga
The o probe is selected from the following oligonucleotide sequence 1 or several
Cam-1????act?cct?gct?taa?cac?aag?ttg?agt?ag
Cam-2????tcg?gtg?tag?gat?gag?act?ata?tag
Cam-3????act?cct?gct?taa?cac?aag?ttg?agt?agg?gaa?agt?ttt?tcg?g
Cam-4????ggt?ata?gtt?aat?ctg?ccc?tac?aca?aga
Cam-5????ctc?ctt?ttc?tta?ggg?aag?aat?tct?gac?ggt?acc?taa?gga?at
Cam-6????act?cct?gct?taa?cac?aag?ttg?agt?agg?gaa?ag
Cam-7????gat?ttg?cct?gta?taa?cct?cct?acg?acc?tta?gac?tag
Cam-8????cgg?agt?aaa?tcc?taa?tac?aaa?gct?aac?ca
Cam-9????ttc?tat?cca?tcc?gaa?gac?ttc?aaa?aag?cct?t
Cam-3-1??act?cct?gct?taa?cac?aag?ttg?agt?agg?gaa?agt?ttt?tcg?g
Cam-1????act?cct?gct?taa?cac?aag?ttg?agt?ag
The p probe is selected from the following oligonucleotide sequence 1 or several
PR1??????taa?cag?gag?aaa?gct?tgc?ttt?ctt?gct?gac
PR-2?????cgt?cga?gtt?cac?aat?aac?agc?atc?ttc?aga
pr-2?????cga?gcg?gta?aca?gga?gaa?agc?ttg?ctt?tct?tgc?tga
pr-3?????cga?gcg?gta?aca?gga?gaa?agc?ttg?ctt?t
pr-4?????gta?aca?gga?gaa?agc?ttg?ctt?tct?tgc?tga?cga?gc
pr-5?????gtc?tac?gga?cca?aag?cag?ggg?ctc?ttc?gga?cct?tgc
pr-6?????gtg?ata?aag?tta?ata?cct?tta?tca?att?g
pr-7?????att?gcg?taa?cag?aga?gaa?agc?ttg?ctt?tct?tgc?tga
pr-8?????att?gcg?taa?cag?aga?gaa?agc?ttg?ctt?t
pr-9?????taa?cag?aga?gaa?agc?ttg?ctt?tct?tgc?tga?ctg?agc
pr-10????gtg?ata?aag?tta?ata?cct?ttg?tca?att?g
The q probe is selected from the following oligonucleotide sequence 1 or several
B-1??????cat?ttt?gaa?ccg?cat?ggt?tcg?aaa?ttg?aaa?ggc
B-2??????gga?taa?cat?ttt?gaa?ccg?cat?ggt?tcg?aaa
B-3??????ggc?tgt?cac?tta?tgg?atg?gac?ccg?cgt?c
B-4??????tgc?tag?ttg?aat?aag?ctg?gca?cct
B-5??????ggc?ttc?ggc?tgt?cac?tta?tgg?atg?gac?ccg?cgt?c
B-6??????tgc?tag?ttg?aat?aag?ctg?gca?cct?tga?cg
B-41?????tgc?gta?gcc?gac?ctg?aga?ggg?tgt
The r probe is selected from the following oligonucleotide sequence 1 or several
CB-1?????tat?aag?aga?atc?gca?tga?ttt?tct?tat?cca?aag?att?tat
CB-2?????atg?aag?ctt?cct?tcg?gga?agt?gga?tta?gcg?gc
CB-3???????ggg?ata?gcc?ttc?cga?aag?gaa?gat?taa?tac
The s probe is selected from the following oligonucleotide sequence 1 or several
common-1???cca?gac?tcc?tac?ggg?agg?cag?cag?t
common-2???ctc?cta?cgg?gag?gca?gca?gta?ggg?aat
common-3???cac?act?gga?act?gag?aca?cgg?tc
common-2-1?act?gag?aca?cgg?tcc?aga?ctc?cta?cgg?gag?gca?gca?gta?ggg?aat?attg
common-3-1?cac?act?gga?act?gag?aca?cgg?tcg?aga?ctc?cta?cgg?ga
The t probe is selected from the following oligonucleotide sequence 1 or several
Bru-1??gtg?ccc?ttc?ggg?gga?aag?att?tat?cgg?caa?atg?atc?ggc?ccg?cgt?t
Bru-2??cgt?acc?att?tgc?tac?gga?ata?act?cag?gga?aac?ttg?tg?。
3. a cover oligonucleotide probe as claimed in claim 1 is characterized in that comprising simultaneously 20 groups of probes of a～t.
4. a cover oligonucleotide probe as claimed in claim 3, wherein a probe is SA5, the b probe is LI2 and LI3, the c probe is O157:H7-1 and O157:H7-2, the d probe is VC1, and the e probe is YE1, and the f probe is ye-2e, the g probe is SHI1, the h probe is V2 and V3, and the i probe is SAL1, and the j probe is I-1 and I-2, the k probe is VP1, the l probe is VP23, and the m probe is SS5, and the n probe is CP-new-1, the o probe is Cam-5, the p probe is PR-2, and the q probe is B-6, and the r probe is CB-1, the s probe is common-2-1 and common-3-1, and the t probe is Bru-2.
5. the purposes of the described arbitrary oligonucleotide probe of claim 1～4 is characterized in that utilizing probe can the corresponding gene of the common pathogen enterobacteria in the sample be detected, and is particularly useful for the detection based on biochip technology.
6. the purposes of the described oligonucleotide probe of claim 5 is characterized in that utilizing probe can detect 17 kinds of common pathogen enterobacterias and dysentery bacterium and salmonella typhi is distinguished.
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 import and export food quarantine, bacteriology classification and epidemiology survey, biological warfare agent detection etc.
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