CN1982472A - Gene chip for inspecting important intestinal tract peccant germ, its inspecting method and reagent kit - Google Patents
Gene chip for inspecting important intestinal tract peccant germ, its inspecting method and reagent kit Download PDFInfo
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- CN1982472A CN1982472A CN 200510132117 CN200510132117A CN1982472A CN 1982472 A CN1982472 A CN 1982472A CN 200510132117 CN200510132117 CN 200510132117 CN 200510132117 A CN200510132117 A CN 200510132117A CN 1982472 A CN1982472 A CN 1982472A
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Abstract
Gene chip for inspecting enteric important pathomycete, its inspection and reagent kit are disclosed. The gene chip consists of solid-phase carrier and oligonucleotide probe fixed on the carrier, which comprises at least one DNA fragment selected from oligose unit treating enzyme gene or glycosylation transferring enzyme of intestinal bacterium O-antigen gene cluster and DNA fragment selected from colon bacillus 16S rRNA conservative area, Multiplex PCR amplifying for gene set DNA to be inspected by primer, hybridizing with the gene chip, analyzing and appraising by software Bactarray Analyzer. It's efficient and fast, and has excellent sensitivity and specificity. It can be used to discriminate blood serum type and used in science life, medical inspection, food safety, import-export quarantine.
Description
Technical field
The present invention relates to a kind of gene chip and detection method thereof and detection kit, particularly a kind of gene chip and detection method and detection kit that detects important intestinal tract peccant germ.
Background technology
Infectious intestinal disease is one of global important public hygiene problem, and is inferior, non-, draw the area particularly serious.According to the data of WHO, above-mentioned regional children below 5 years old are about more than 5,000,000 because of infectious intestinal disease death person every year, fall ill about 7.5 hundred million～1,000,000,000 person-times.Even also have the problems referred to above in economically developed countries and regions, state such as the U.S., Japan enterohemorrhagic Escherichia coli O 157 for example: the serious popular of H7 enteritis can illustrate this fact.Nineteen eighty-two the U.S.'s reported first hemorrhagic enteritis that causes by EHEC O157:H7 break out.After this, reported this microbial infection all over the world successively, and on the rise.The outbreak of epidemic of the EHEC O157:H7 that took place in Japan in 1996, causing bleeding property diarrhoea, successively involve more than 30 all, mansion, county, infect nearly 10000 people, and cause 12 people's death, caused global concern.China is developing country, the hygienic basis weakness, and economy, culture etc. are still undeveloped, and are in addition vast in territory, populous, and the various places difference is big and development is uneven, thereby brings certain difficulty for the prevention and the control of infectious intestinal disease.According to units such as Shoudu Inst. of Pediatrics 1986～1988 years investigation result estimation of 7 demonstration counties and Beijing at home, China children below 5 years old are annual, and about 1.7 hundred million person-times of enteric infection patient is taken place; Prevent and treat present situation door-to-door survey result according to domestic 21 provinces (city) dysentery in 1987 again and calculate, the annual about 8.36 hundred million person-times of morbidities in the whole nation, wherein children below 5 years old about 2.09 hundred million person-times, and its seriousness has some idea of.1989～1991 years, under WHO supports, health ministry in Shandong, Yunnan, Fujian, Gansu, Beijing and Hunan successively carried out 22 town and country door-to-door surveys, the result shows that the annual morbidity of children below 5 years old is 0.86～3.9 a time/man-year, the mean level (ML) of 6 provinces (city) is 2.5 a times/man-year, and wherein the Rural areas is 2.9 a times/man-year.The morbidity number of year national first, Category B notifiable disease is analyzed surplus nearest 10 after the nineties in 20th century, the morbidity number of respiratory infectious disease, haematogenous and sexually transmitted disease (STD), natural epidemic disease source property and arthropod borne infection, all do not surpass 6 figure places every year, has infectious intestinal disease only and all rank first with 7 figure places every year.Because infectious intestinal disease is one of important factor that causes a large amount of losses of malnourished children, dysplasia and adult labor force, thereby directly endanger people ' s health and continuous development of society economy, must cause enough attention (Wei Chengyu, the basal conditions of China's infectious intestinal disease and Preventing Countermeasures, Chinese public health the 20th the 1st phase of volume of January in 2004).
Pathogen enterobacteria mainly comprises: intestinal bacteria, Salmonellas, Shigellae, vibrio cholerae, Aeromonas, Plesiomonas etc.Pathogen enterobacteria is propagated by water, food and daily living article, and major part is an infecting both domestic animals and human, especially baby and cub (fowl) is often caused serious diarrhoea.China the most commonly intestinal bacteria, Salmonellas and Shigellae, sickness rate accounts for 80% of infantile diarrhea case, accounts for 50% of adult diarrhea case.
Epidemiological survey shows in recent years, and the most common and pathogenic 15 strong type important intestinal tract peccant germs of China comprise at present: 10 serotypes of Shigellae---shigella flexneri 2a type (ShigellaFlexneri 2a) (O-antigen gene bunch sequence from: gb/AY900451), bacillus ceylonensis A 1 type (Shigella sonnei 1) (O-antigen gene bunch sequence from: gb/AF294823), Shigella bogdii O7 (Shigella boydii O7) (O-antigen gene bunch sequence from: the patent No. 03100536.5), Shigella bogdii O9 (Shigella boydii O9) (O-antigen gene bunch sequence from: gb/AF402315), Shigella bogdii O13 (Shigella boydii O13) (O-antigen gene bunch sequence from: number of patent application 03100537.3), Shigella bogdii O16 (Shigella boydiiO16) (O-antigen gene bunch sequence from: number of patent application 200410019049.8), Shigella bogdii O18 (Shigella boydii O18) (O-antigen gene bunch sequence from: number of patent application 200410019051.5), shigella dysenteriae O4 (Shigella dysenteriae O4) (O-antigen gene bunch sequence from: number of patent application 200410019052.x), shigella dysenteriae O8 (Shigelladysenteriae O8) (O-antigen gene bunch sequence from: patent No. 03100539.X), shigella dysenteriae O10 (Shigella dysenteriae O10) (O-antigen gene bunch sequence from: number of patent application 200410019186.1) and colibacillary 5 serotypes---intestinal bacteria O55 (E.coliO55) (O-antigen gene bunch sequence from: gb/AF461121), intestinal bacteria O111 (E.coli O111) (O-antigen gene bunch sequence from: gb/AF078736), intestinal bacteria O114 (E.coli O114) (O-antigen gene bunch sequence from: gb/AF573377), intestinal bacteria O128 (E.coli O128) (O-antigen gene bunch sequence from: gb/AF217096), Escherichia coli O 157 (E.coli O157) (O-antigen gene bunch sequence from: gb/AF061251).
Universal test method to pathogen enterobacteria is to O and the antigenic antiserum(antisera) agglutination reaction of H (mainly being O) now.This method need be separated single bacterium colony again through cultivating earlier, identifies the process of the bacterial species that is infected at last with biochemical identification and serological reaction.The shortcoming of this method is that susceptibility is low, length consuming time, and the loss height, and produce a complete set of antiserum(antisera) that is used to identify without any country in the world.Therefore being badly in need of invention can be quicker, accurate, the method for reliable detection important intestinal tract peccant germ.
Lipopolysaccharides (LPS) is the main surface composition of gram negative bacterium, and keeping its structure and exercise at epicyte has indispensable effect on its biological function, also playing the part of very important role in the interaction of bacterium and outside atmosphere.Lipopolysaccharides is an animal immune system to the primary identification and the target of attack of the Gram-negative bacteria of invasion, also infects in the host cell process from sticking at pathogenic bacteria simultaneously, infects in the immune attack of hiding host cell to play an important role.Typical LPS molecule is by three covalently bound forming of part: O specific polysaccharide chain, core polysaccharide, lipoid A.O specific polysaccharide chain is free on the thalline surface, and its base portion links to each other with core polysaccharide, is positioned at the outermost layer of lipopolysaccharide molecule.Wherein O specific polysaccharide chain is the main surface antigen that causes the host specific antibody response, and O antigen is otherwise known as.O antigen is made up of oligosaccharides repeating unit (≤50), and each repeating unit is made up of three to eight monose usually, the O antigenic structure since constitute repeating unit the monose kind, put in order, the space structure of combination and polysaccharide chain is different and different.This height diversity has determined the specificity between O antigen and the host.According to the antigenic diversity of O, bacterium can be divided into different serotype.For example, common pathogenic bacterium E.coli (comprising Shigella) is divided into 187 O antigen serotypes.
It is O-antigen-specific gene theory (international patent application, WO 98-AU315 and WO99-AU385) that Wang Lei professor and Reeves professor proposed glycosyltransferase gene and oligosaccharide unit treatment enzyme gene in 1998.According to above research theory, we can be at glycosyltransferase gene and the oligosaccharide unit treatment enzyme gene design primer in the O-antigen gene bunch, filter out the gene and the oligonucleotide fragment of high special again by PCR method, reach the purpose that detects pathogenic bacterium with this.The superiority of round pcr itself is given no cause for much criticism, and since 1989 began to be applied to Clinical Laboratory, advantage such as, sensitivity quick, easy with it became a hot technology of clinical experiment diagnostics soon.
If also occur false negative and crossed contamination easily but reaction conditions control is bad, yet the appearance of biochip technology has solved this difficult problem.In July, 1997 Affymetric, (Santa Clara, CA) people such as Thomas R. of company have invented the method (the 6th, 228, No. 575 United States Patent (USP)s) of with biochip technology microorganism being carried out deciding kind and phenotype analytical to Inc..Since nineteen nineties, biochip technology has been set up as a kind of brand-new analysis and detection technology, and along with carrying out progressively of the Human Genome Project grows up.Protocols in Molecular Biology has been used in this technological synthesis, ic manufacturing technology, computer technology, semiconductor technology, confocal laser scanning technique, fluorescent mark technology, make testing process have the susceptibility height, high specificity, large scale integration, automatization, simple and efficient to handle, characteristics such as efficient height are subjected to scientific research personnel's favor, and are widely used at aspects such as genetic expression correlative study and bio-pharmaceuticals researchs.Therefore, identify, not only can simplify technology platform greatly, and be expected to realize high accuracy, highly sensitive, high-throughput, repeatable advantage such as strong if biochip technology is used for bacterium serotype.
In sum, be necessary to invent out a kind of product and the method thereof that can carry out the somatotype detection fast, accurately, reliably to important intestinal tract peccant germ.
Summary of the invention
An object of the present invention is to provide a kind of gene chip that detects important intestinal tract peccant germ, to replenish the deficiency of existing detection technique.
Another object of the present invention provides a kind of said gene chip that utilizes and carries out method easy, quick, the efficient detection important intestinal tract peccant germ.
A further object of the present invention provides a kind of test kit that detects important intestinal tract peccant germ.
For achieving the above object, the present invention has adopted following technical scheme:
The present invention proposes a kind of gene chip that detects important intestinal tract peccant germ, comprise solid phase carrier and be fixed on oligonucleotide probe on this solid phase carrier, wherein the dna fragmentation that contains dna fragmentation that oligosaccharide unit treatment enzyme gene from the O-antigen gene of one or more important intestinal tract peccant germs bunch or glycosyltransferase gene choose at least and choose of the oligonucleotide probe on this gene chip from Shigellae and intestinal bacteria 16S rRNA conserved regions.
In preferred embodiment of the present invention, above-mentioned one or more important intestinal tract peccant germs are selected from shigella flexneri 2a type, bacillus ceylonensis A 1 type, Shigella bogdii O7, Shigella bogdii O9, Shigella bogdii O13, Shigella bogdii O16, Shigella bogdii O18, shigella dysenteriae O4, shigella dysenteriae O8, shigella dysenteriae O10, intestinal bacteria O55, intestinal bacteria O111, intestinal bacteria O114, intestinal bacteria O128 and Escherichia coli O 157.
In preferred embodiment of the present invention, the dna fragmentation that oligosaccharide unit treatment enzyme gene on the O-antigen gene of above-mentioned one or more important intestinal tract peccant germs bunch or glycosyltransferase gene are chosen for example is selected from the dna fragmentation with the base sequence shown in SEQ ID NO:1-NO:48.The dna fragmentation of choosing from Shigellae and intestinal bacteria 16S rRNA conserved regions for example has the base sequence shown in SEQ IDNO:49 and NO:50.
The invention allows for a kind of method that detects important intestinal tract peccant germ, may further comprise the steps:
(1) according to oligosaccharide unit treatment enzyme gene or glycosyltransferase gene and Shigellae and intestinal bacteria 16S rRNA conserved regions sequences Design on the O-antigen gene of one or more important intestinal tract peccant germs bunch and prepare the primer that is used for pcr amplification;
(2) use the described primer of step (1) that the genomic dna of testing sample is carried out pcr amplification;
(3) with amplified production and said gene chip hybridization;
(4) obtain hybridization signal and Analysis and Identification results of hybridization with gene chip scanning instrument.
In preferred embodiment of the present invention, wherein be selected from the oligonucleotide fragment that for example has the base sequence shown in SEQ ID NO:50-NO:80 according to the oligosaccharide unit treatment enzyme gene on the O-antigen gene of one or more important intestinal tract peccant germs bunch or the primer of glycosyltransferase gene design in the above-mentioned steps (1).
In preferred embodiment of the present invention, in the above-mentioned steps (1) according to the primer sequence of Shigellae and intestinal bacteria 16S rRNA conserved regions sequences Design shown in SEQ ID NO:79 and NO:80.
In preferred embodiment of the present invention, for example use interpretation software Bactarray Analyzer Analysis and Identification results of hybridization in the above-mentioned steps (4), step is as follows:
1) validation verification of fluorescent signal
A plurality of strength of signal that repeat a little of every specific probe on the said gene chip are carried out the degree of confidence check, and the strength of signal of rejecting abnormalities is got the strength of signal of the mean value of normal strength of signal as every specific probe;
2) normalization method
Use specific probe overall fluorescent intensity level as the normalization method standard, remove with the normalization method standard, every absolute fluorescence intensity is become relative intensity of fluorescence with every fluorescence intensity;
3) to each specific probe marking
According to relative intensity of fluorescence, each specific probe is provided a score value, the similarity degree of the sequence dna fragment of representative sample and this specific probe sequence;
4) application model is given a mark to the species that may contain in the sample
With the weighted mean of all relevant probe score values of a certain species score value as these species;
5) presumable species in the judgement sample
Handle through above-mentioned steps, program all provides a score value to all possible species in the sample, and the species that the species that are most possible existence that score value is the highest, score value are lower than threshold value can not exist.
The present invention also provides a kind of test kit that detects important intestinal tract peccant germ, this test kit comprises gene chip at least, this gene chip comprises solid phase carrier and the oligonucleotide probe that is fixed on this solid phase carrier, wherein, the oligonucleotide probe dna fragmentation that contains dna fragmentation that oligosaccharide unit treatment enzyme gene from the O-antigen gene of one or more important intestinal tract peccant germs bunch or glycosyltransferase gene choose at least and choose from Shigellae and intestinal bacteria 16S rRNA conserved regions.
In one embodiment of this invention, the test kit of above-mentioned detection important intestinal tract peccant germ also comprises the pcr amplification primer, and these pcr amplification primers are selected from the oligonucleotide fragment with the base sequence shown in SEQ ID NO:51-NO:82.
In one embodiment of this invention, the test kit of above-mentioned detection important intestinal tract peccant germ also comprises the interpretation software Bactarray Analyzer that hybridizing box, hybridization solution and Analysis and Identification result use.
By above-mentioned disclosed technical scheme as seen, utilize gene chip of the present invention can reach the purpose that detects common important intestinal tract peccant germ, because easy and simple to handle, the accuracy height, repeatability is strong, detects so both be applicable to conventional serotype, be applicable to laboratory study again, will have far reaching significance.
This invention has remedied the blank of gene chip according to O-antigen gene bunch last distinguished sequence designing probe detection important intestinal tract peccant germ field, and the method for identifying important pathogen enterobacteria fast and accurately is provided.Shorten detection time greatly, simplified detection method, improved detection accuracy.Domestic gene chip at the pathogenic bacterium context of detection is the discriminating (patent No. CN1536090A) to the bacterium kind at present, and this invention can be differentiated concrete serotype, can instruct pharmaceutical drugs more accurately.In addition because the present invention possesses high sensitivity and specificity, and it detects and the analytical procedure efficient quick, so can be widely used in fields such as life science, medical test, food safety, environment supervision, import and export inspection and quarantine, bio-terrorism strick precaution, epidemiology monitoring.
For above and other objects of the present invention, feature and advantage can be become apparent, below especially exemplified by preferred embodiment, and cooperate Figure of description, be described in detail below.
Description of drawings
Fig. 1 is the profile synoptic diagram of an embodiment of gene chip of the present invention.
Fig. 2 is the synoptic diagram of arranging of 52 probes on the embodiment of gene chip of the present invention.Wherein 0 represents 50%DMSO, and remaining is as shown in table 1.
Fig. 3 represents the scanning result of an embodiment hybridization of Shigella bogdii O13 and gene chip of the present invention.
Fig. 4 represents the scanning result of an embodiment hybridization of shigella dysenteriae O10 and gene chip of the present invention.
Fig. 5 represents the scanning result of an embodiment hybridization of intestinal bacteria O111 and gene chip of the present invention.
Fig. 6 represents the scanning result of an embodiment hybridization of intestinal bacteria O114 and gene chip of the present invention.
Fig. 7 represents the scanning result of an embodiment hybridization of intestinal bacteria O128 and gene chip of the present invention.
Embodiment
Gene chip and detection method thereof in order to further specify detection important intestinal tract peccant germ of the present invention are illustrated especially exemplified by following preferred embodiment, and these embodiment are in order to illustrate rather than limit in any form the present invention.
Embodiment oneThe design of probe and preparation
1. sequence obtains: the wzx and the wzy sequence (shigella boydii O16 is orf5) of putting 15 type important intestinal tract peccant germs that this laboratory records and that obtain from the download of public databases such as GenBank with Artemis software in order.
2. probe design: the gene order that obtains is imported in the OligoArray2.0 software, (length is 40bp ± 5bp to set relevant parameter, the Tm value is 80 ℃ ± 2 ℃), working procedure is online to have designed totally 124 of oligonucleotide probes that are used to detect 15 kinds of serotypes of important intestinal tract peccant germ.
3. probe is selected: carry out the probe screening by 683 hybrid experiments, finally select to obtain length at 40bp ± 5bp from the output result, Tm80 ℃ ± 2 ℃ special, sensitive probe groups becomes the required probe set of gene chip of the present invention.
In preferred embodiment of the present invention, select to have obtained 52 probes as shown in table 1.Wherein 48 of SEQ ID NO:1-NO:48 probe sequences are selected from 15 kinds of common important intestinal tract peccant germs (shigella flexneri 2a type respectively, bacillus ceylonensis A 1 type, Shigella bogdii O7, Shigella bogdii O9, Shigella bogdii O13, Shigella bogdii O16, Shigella bogdii O18, shigella dysenteriae O4, shigella dysenteriae O8, shigella dysenteriae O10, intestinal bacteria O55, intestinal bacteria O111, intestinal bacteria O114, intestinal bacteria O128 and Escherichia coli O 157) O-antigen gene bunch on oligosaccharide unit treatment enzyme gene or glycosyltransferase gene; SEQ ID is the sequence that two probes of NO:49 and NO:50 are selected from Shigellae and intestinal bacteria 16S rRNA conserved regions, as positive control; The probe that probe is numbered n is a poly T fragment, as negative control; The probe that probe is numbered # is a fluorescent probe.
Table 1: all probes and detection effect thereof on the gene chip of detection important intestinal tract peccant germ
| SEQ ID | The probe numbering | Probe sequence (5 '-3 ') | Detectable important intestinal tract peccant germ type |
| NO：1 | 1-1 | 5’-GGGCAGTGTTTCCAAGGTTAAGTAACA TCAAAGACTTTAA-3’ | Shigella flexneri 2a type |
| NO：2 | 1-2 | 5’-CGGTTGGATGACAGTAAGTAATATCAT AAGTCCGGTCATG-3’ | Shigella flexneri 2a type |
| NO：3 | 2-1 | 5’-GGCACTGGATTAGGTGTTGCAAATTAT GTAAAGGCTA-3’ | Bacillus ceylonensis A 1 type |
| NO：4 | 2-2 | 5’-TGATTATCGTCGAGTTGAGTTAGTATTT ATTGGGGTTGAT-3’ | Bacillus ceylonensis A 1 type |
| NO：5 | 2-3 | 5’-TGGTTCTTTTGGTGTCATTTTGCATTGG TAAAGATGAGCT-3’ | Bacillus ceylonensis A 1 type |
| NO：6 | 3-1 | 5’-ATTCCACTTCCATCAATCATATCGTTGA TTATTCCTGCTG-3’ | Shigella bogdii O7 |
| NO：7 | 3-2 | 5’-CCCATTGATTTTGCATCAAATAAGTGGC TTTCTTAAAGGA-3’ | Shigella bogdii O7 |
| NO：8 | 3-3 | 5’-CTTGATACTGATAATGGCGTTGAATAA AGCACTAGTTCCA-3’ | Shigella bogdii O7 |
| NO：9 | 4-1 | 5’-TTATCTTGAGTTTAGTCGAATGCAACGA GTAGTCGCG-3’ | Shigella bogdii O13 |
| NO：10 | 4-2 | 5’-TTATTCTCATATTCGAATGTTACTTTTA CCAGCGCAACTG-3’ | Shigella bogdii O13 |
| NO：11 | 4-3 | 5’-GCTGCGGGGAATAGAAATAAGAATCGT CTGTA TTACTTTA-3’ | Shigella bogdii O13 |
| NO：12 | 5-1 | 5’-TTGTATGTAATCGCAAATACTCGCAGT ACATGTGGA-3’ | Shigella bogdii O16 |
| NO：13 | 5-2 | 5’-CCTGAGCGATTAGAACAACTGTATCTC GAGTCAAGA-3’ | Shigella bogdii O16 |
| NO：14 | 5-3 | 5’-GACATGGGAGTGGAATGAAAACTAAA ATTGCTGAGGCTTT-3’ | Shigella bogdii O16 |
| NO：15 | 5-4 | 5’-GTGAGATTGTTGATTGCTTTAGCCGAG AAGACAAAGATAT-3’ | Shigella bogdii O16 |
| NO：16 | 6-1 | 5’-TTTGTCGTGTCTATTGGTATTGTATTCG GGCAATGG-3’ | Shigella bogdii O18 |
| NO：17 | 6-2 | 5’-TGGAGTTCCATTATCCGAGTGGTATTTT GGTAACAATAAT-3’ | Shigella bogdii O18 |
| NO：18 | 6-3 | 5’-TACTCATTAGGCTTGCTATTTACTGGGC TATTATCAGTTT-3’ | Shigella bogdii O18 |
| NO：19 | 7-1 | 5’-TCGCATTGCTTGGTATTAGAGCTGGTAG TGTTAATT-3’ | Shigella dysenteriae O4 |
| NO：20 | 7-2 | 5’-GCAGCTTCTCTTTACATACGTGCATTAT CAGAAACGGGAA-3’ | Shigella dysenteriae O4 |
| NO：21 | 7-3 | 5’-ACTAGTCTAGATAAAATGGCTAGCTCA GACAATCTTTCTG-3’ | Shigella dysenteriae O4 |
| NO：22 | 7-4 | 5’-TGGATATTTAATTCGGCTATTCTCTATA GCTAGTGAGCCT-3’ | Shigella dysenteriae O4 |
| NO：23 | 8-1 | 5’-GAGGGTGTGTATGGTATGATTGATTAC ATATTGGAGGC-3’ | Shigella dysenteriae O8 |
| NO：24 | 8-2 | 5’-ACTTCAGTTTCTGGGAACGATAAATTC ACACGTTTGC-3’ | Shigella dysenteriae O8 |
| NO：25 | 8-3 | 5’-TTCCTAAATAATCATCCATTTACTGAGG GTGTGTATGGTA-3’ | Shigella dysenteriae O8 |
| NO：26 | 8-4 | 5’-CGCCAATTTATTCTGTGCTATTATCGAA ATTCACTTCAGT-3’ | Shigella dysenteriae O8 |
| NO：27 | 9-1 | 5’-ATGGCACTGATAGTAGCGATAAAACTA TTTTGATCGGG-3’ | Shigella dysenteriae O10 |
| NO：28 | 9-2 | 5’-CGTGCATTAATTGCTATTGTCGTAATTT CTTTCATTGTGG-3’ | Shigella dysenteriae O10 |
| NO：29 | 9-3 | 5’-GGAACACCTTCTTGGGATTATTTTACGC AACCACTTATTA-3’ | Shigella dysenteriae O10 |
| NO：30 | 10-1 | 5′-AGAGTGAGACGAATAATTGGGTGTTAT ATAAGCTCTC-3′ | Intestinal bacteria O55 |
| NO：31 | 10-2 | 5’-GCTTTTGGGGATAACATTATCGATAAT ACCGACCTTTAGA-3’ | Intestinal bacteria O55 |
| NO：32 | 10-3 | 5’-TGAAGCTTTAAAACGTCTAATTATTAGC GGAACTTTCTTG-3’ | Intestinal bacteria O55 |
| NO：33 | 11-1 | 5′-CGGGGATGATATATTATTTGGTTTCACA GCTTGGTG-3′ | Intestinal bacteria O111 |
| NO：34 | 11-2 | 5’-TTACGGTTCTTTATAAGTATTGGTGTGA TAGGAGCATTGG-3’ | Intestinal bacteria O111 |
| NO：35 | 11-3 | 5’-GCTCCTTTCATTGTTGTAAGTTGTTTGT TACTGTTACA-3’ | Intestinal bacteria O111 |
| NO：36 | 11-4 | 5’-TTAAATAACGGCGGACAATATAAGACG TTATATGGACTTC-3’ | Intestinal bacteria O111 |
| NO：37 | 12-1 | 5’-TTATTGTATGCTTGTTAGTGCTTGTGCT GATTTGTTTTCT-3’ | Intestinal bacteria O114 |
| NO：38 | 12-2 | 5’-TGAGATGCTTAAATTAGGTGGATGGAA TGTTAATGGG-3’ | Intestinal bacteria O114 |
| NO：39 | 12-3 | 5’-GTTGTTCATATGCTCAGGGGAAAATCA GAAGAGTATCTAT-3’ | Intestinal bacteria O114 |
| NO：40 | 12-4 | 5’-TGGATGGAATGTTAATGGGTTATTTATT TCAGAAGCATG-3’ | Intestinal bacteria O114 |
| NO：41 | 13-1 | 5′-TCTGATCTTGGATTAAGTAAGATGTACC CAGCAA-3′ | Intestinal bacteria O128 |
| NO：42 | 13-2 | 5’-CGGTGTTTTGCAAGAGATATAAAAGAG TTAGCTTTAGCAT-3’ | Intestinal bacteria O128 |
| NO：43 | 13-3 | 5’-GCTAGGTATTTAGCAAATTCAACAGAT TTGGCTGACTTTG-3’ | Intestinal bacteria O128 |
| NO：44 | 14-1 | 5′-CGATTTCTTTCCGACACCAGAGTTAGAA AAGGAATT-3′ | Escherichia coli O 157 |
| NO：45 | 14-2 | 5’-TAGAGCAAGTTGAAAGTGTTCCATATG TTGTTTCTGAATC-3’ | Escherichia coli O 157 |
| NO：46 | 14-3 | 5’-GTATGCTCGTTGTTTTATCTAAGTTTAG GACAAGACGGAG-3’ | Escherichia coli O 157 |
| NO：47 | 15-1 | 5’-AACTGAGTTCACTTATGGTTCGAGAAC CTTTACTCCATTT-3’ | Shigella bogdii O9 |
| NO：48 | 15-2 | 5’-GGATTTTAATACAACTGAGTTCACTTAT GGTTCGAGAACC-3’ | Shigella bogdii O9 |
| NO：49 | P-1 | 5’-CGGGAACTCAAAGGAGACTGCCAGTGA TAA-3’ | 16S rRNA |
| NO：50 | P-2 | 5’-CGGGAACTCAAAGGAGACTGCCAGTGA TAAACTGGAG-3’ | 16S rRNA |
| n | 5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT- 3’ | Negative contrast | |
| # | 5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTT_ Cy3-3’ | Fluorescent probe |
4. probe is synthetic: entrust probe Synesis Company (Beijing AudioCodes company) synthetic, standby after the 5 ' end of the probe in the table 1 is prolonged 10 T and amination.
Embodiment twoPrimer design and preparation
1. sequence obtains: the wzx and the wzy sequence (shigella boydii O16 is orf5) of putting 15 type important intestinal tract peccant germs that this laboratory records and that obtain from the download of public databases such as GenBank with Artemis software in order.
2. design primer: the gene order that obtains is imported in the primer-design software Primer Premier5.0 software, set relevant parameter (length is 20bp ± 2bp, and Tm value is 50 ℃ ± 5 ℃) working procedure afterwards.
3. primer is selected: choose 50 ℃ ± 5 ℃ of Tm values from the output result, length 20bp ± 2bp comprises probe sequence used in the gene chip at interior primer.Indivedual probes need manual regulation, and primer suitably extends or shortens several bases, makes it comprise probe, meets 50 ℃ ± 5 ℃ of Tm values and other parameter.
In a preferred embodiment of the invention, for adapting to Multiplex PCR,, filter out 32 of suitable primers as shown in table 2 through the information biology primary dcreening operation and by a large amount of Multiplex PCR experiment screenings.
The used whole primers of table 2 amplification
| SEQ ID | The primer numbering | Primer sequence (5 '-3 ') | Amplification effect |
| NO：51 | P-1 | 5’-GTGTTGACTGCTGGATTTC-3’ | Shigella bogdii O7wzx upstream primer |
| NO：52 | P-2 | 5’-CGATATGATTGATGGAAGTG-3’ | Shigella bogdii O7wzx downstream primer |
| NO：53 | P-3 | 5’-GCGTTGGTTGGTGAAAGAG-3’ | Shigella bogdii O9wzy upstream primer |
| NO：54 | P-4 | 5’-TTCCCACAAATCAAACCA-3’ | Shigella bogdii O9wzy downstream primer |
| NO：55 | P-5 | 5’-AAAGATTGGTAGCGTCGG-3’ | Shigella bogdii O13wzy upstream primer |
| NO：56 | P-6 | 5’-TGAAGCCCTGGTAAAGTGC-3’ | Shigella bogdii O13wzy downstream primer |
| NO：57 | P-7 | 5’-CCATACGGATAATGTTGAG-3’ | Shigella bogdii O16orf5 upstream primer |
| NO：58 | P-8 | 5’-TCTTTGTCTTCTCGGCTA-3’ | Shigella bogdii O16orf5 downstream primer |
| NO：59 | P-9 | 5’-CAGGGCACAAACTATCT-3’ | Shigella bogdii O18wzx upstream primer |
| NO：60 | P-10 | 5’-TAATTCGGAATGTGCT-3’ | Shigella bogdii O18wzx downstream primer |
| NO：61 | P-11 | 5’-TTTCTGCTTATTCATTATTG-3’ | Shigella dysenteriae O4wzy upstream primer |
| NO：62 | P-12 | 5’-ACTACCAGCTCTAATACC-3’ | Shigella dysenteriae O4wzy downstream primer |
| NO：63 | P-13 | 5’-TTCCCTCTTGTTGTATTGA-3′ | Shigella dysenteriae O8wzy upstream primer |
| NO：64 | p-14 | 5’-ACCTTTATCAATTGCCTCC-3′ | Shigella dysenteriae O8wzy downstream primer |
| NO：65 | P-15 | 5’-CGCTGTTTCTATATTAATTG-3’ | Shigella dysenteriae O10wzy upstream primer |
| NO：66 | P-16 | 5’-AATTGAAGTGACCAGATAAC-3′ | Shigella dysenteriae O10wzy downstream primer |
| NO：67 | P-17 | 5’-TGAGGTTTCACGTTTCTC-3’ | Bacillus ceylonensis A 1 type wzy upstream primer |
| NO：68 | P-18 | 5’-AATAATCCCTAACTGAGCC-3′ | Bacillus ceylonensis A 1 type wzy downstream primer |
| NO：69 | P-19 | 5’-CACTTGTTGGGTATGCTGG-3’ | Shigella flexneri 2a type wzx upstream primer |
| NO：70 | P-20 | 5’-CCGGCAAACAGATTAGAAA-3’ | Shigella flexneri 2a type wzx downstream primer |
| NO：71 | P-21 | 5’-GGGAGGAGTATTATCATTAC-3′ | Intestinal bacteria O55wzy upstream primer |
| NO：72 | P-22 | 5’-ATCAATCTAAAGGTCGGTA-3′ | Intestinal bacteria O55wzy downstream primer |
| NO：73 | P-23 | 5’-TTAATGCGGA GGATCTATT-3′ | Intestinal bacteria O111wzy upstream primer |
| NO：74 | P-24 | 5’-GTAAGCCCGCAAATCAATC-3′ | Intestinal bacteria O111wzy downstream primer |
| NO：75 | P-25 | 5’-ACTTTCCCAAGCCCATTA-3′ | Intestinal bacteria O114wzy upstream primer |
| NO：76 | P-26 | 5’-CAGCACAAGCACTAACAAG-3′ | Intestinal bacteria O114wzy downstream primer |
| NO：77 | P-27 | 5’-ATGATTTCTTACGGAGTGC-3’ | Intestinal bacteria O128wzy upstream primer |
| NO：78 | P-28 | 5’-CTCTAACCTAATCCCTCCC-3’ | Intestinal bacteria O128wzy downstream primer |
| NO：79 | P-29 | 5’-TTGCTGCTGTAGTTTTATTTCTT-3′ | Escherichia coli O 157 wzy upstream primer |
| NO：80 | P-30 | 5′-TGATGCTTTATTCCCTGTATTCT-3′ | Escherichia coli O 157 wzy downstream primer |
| NO：81 | P-31 | 5′-CACGGGTGAGTAATGTCTGG-3′ | 16S rRNA upstream primer |
| NO：82 | P-32 | 5′-ATCCACGATTACTAGCGATTCC-3′ | 16S rRNA downstream primer |
4. primer is synthetic: it is synthetic, standby to send primer Synesis Company (Beijing AudioCodes) to entrust the primer sequence in the table 2.
Embodiment threeThe chip point sample
Synthetic good probe dry powder pipe (synthetic by AudioCodes) is at first dissolved.Step is as follows: 12000 rev/mins, centrifugal 5 minutes the lid of dry powder pipe (note do not open before centrifugal) adds 50%DMSO (ratio that adds 14 μ l with 1OD adds), with centrifugal fast behind the vibrator mixing, with the liquid on the tube wall from down.After at room temperature placing 1 hour, get and survey OD after 1 μ l dilutes 180 times with 50%DMSO, measure the nucleic acid concentration (ng/ μ l) of this probe (strand), utilizing formula that this probe dilution is become final concentration again is 1 μ g/ μ l.
Formula:
52 probes are joined corresponding position in 384 orifice plates, and every hole adds 10 μ l probes.Utilize automatic point sample instrument with the probe points in 384 orifice plates to slide, form our designed good microarray.This chip utilization be the SpotArray microarry printing system gene chip sample applying instrument (model is SpotArray 72) of PerkiElmer company, use SpotArray control software (tele chemsmp3 stealtypin) to finish whole point samples.(CEL Associates Inc.) is put on the Stage microscope of chip point sample instrument as solid phase carrier, and 384 orifice plates that added probe are put into the sample table place with the clean aldehyde radical slide of 57.5mm * 25.5mm shown in Figure 1 * 1mm (length * wide * height).Start the SpotArray control software, set relevant parameters, the beginning point sample, in the point sample district of 52 oligonucleotide probes with the rule point 3mm * 3mm to the slide that arranges shown in Figure 2, low density dna microarray in the formation, wherein each probe points is the probe (0 expression 50%DMSO) of the probe numbering shown in the correspondence table 1, the rule of arranging of array is identical in four point sample districts on the slide, as shown in Figure 1 wherein between each dot matrix at a distance of 13.5mm, first microarray is apart from slide top 11.5mm, and the 4th microarray is apart from 3 mm of white edging place.In the point sample process, horn control syringe needle, every some sample will be through last sample, pre-point, formal point sample, cleaning, dry plurality of processes.The good chip of point is dried overnight at room temperature, and 45 ℃ of oven dryings are 2 hours then.It is UV-crosslinked to utilize the crosslinked instrument (uvpcl-2000M ultraciolet Crosslinker) of UVP company to carry out again, twice of 600J.Crosslinked good chip is put back in the clean chip cartridges, in order to using.
Embodiment fourDetect the working method of the gene chip of important intestinal tract peccant germ
Utilizing the above-mentioned gene chip for preparing to detect in the method for important intestinal tract peccant germ, the present invention has all done a series of experiment to factors such as detection step, testing conditions and has groped, as each component proportions in the Multiplex PCR reaction mixture, the PCR cycling condition, hybridization temperature, hybridization time etc. and main agents prescription, as hybridization solution, washing lotion etc. (prescription sees following examples for details).Each composition in the PCR reaction mixture wherein, ratio between primer especially, the selection of Buffer, the Taq enzyme concn, the kind of fluorescein and consumption, and template concentrations is repeatedly the optimum combination that obtains behind the contrast experiment.The annealing temperature that PCR adopted, time and extension time, number of cycles is the good condition for selecting after the gradient experiment also.Hybridization temperature is tested through 40 ℃, 50 ℃, 60 ℃ three thermogrades, finally selects 40 ℃.Hybridization time has been done 1.5h, 8h, 14h, 16h, five time gradient experiments of 18h on experiential basis, finally select 16h.The experiment flow of important intestinal tract peccant germ detection chip also for optimizing the back result, is guaranteeing on the basis that the present invention realizes smoothly that present embodiment has finally been selected a step Multiplex PCR method, has shortened the time of detecting greatly, has simplified detection method.Below, specify as follows for utilizing the above-mentioned gene chip for preparing to detect the preferred embodiment of important intestinal tract peccant germ:
1.Multiplex pcr amplification is got genomic dna (Bastin DA, Reeves PR.Sequenceand analysis of the O antigen gene (rfb) cluster of Escherichia coli O111.Gene1995 that 1 μ l (100ng/ μ l) desire detects; 164:17-23.) be added to mixing among the Multiplex PCR Mix (note: this process is wanted lucifuge).Multiplex PCR Mix prescription sees Table 3, and the system cumulative volume is 30 μ l, and wherein dNTP Mixture gives birth to worker D0056 from Shanghai, and Taq enzyme, PCR buffer are from Takara DDR100DM, and Cy3-dutp is from Amersham PA53022.
Table 3.Multiplex PCR Mix prescription
| Composition | Concentration | The application of sample amount |
| The sterilization ultrapure water | ---- | 5.9μl |
| 10×Buffer | 10× | 3μl |
| dNTP | 10mM | 0.3μl |
| P 1～P 30 | About 7 μ M | Each 0.6 μ l |
| P 31～P 32 | About 2.4 μ M | Each 0.6 μ l |
| Taq | 5u/μl | 0.3μl |
| Cy3-dutp | 1mM | 0.3μl |
Above mixed solution is put in the PCR instrument, is carried out following program:
95℃ 5min
95℃ 30sec
50℃ 45sec
72 ℃ of 1min returned for second step and carry out 35 circulations
72℃ 5min
2. hybridization
Above-mentioned pcr amplification product is taken out 15 μ l be put in 65 ℃ of baking ovens oven dry (wanting lucifuge), the oven dry back adds the hybridization solution 13 μ l of 40 ℃ of preheatings, with pipettor it is blown and puts into 98 ℃ of sex change 10min of PCR instrument after even.
Crosslinked good chip is dried up after 1 minute with the flushing of sterilization distilled water, and it is standby to be placed on the lucifuge place.Open hybridizing box and add 50 μ l sterilization ultrapure water in its dark lattice, put into washed chip, that select probe is towards last and build cover plate.(note: cover plate has one of projection to face down, and the well that makes cover plate is sure not to put back towards the operator).The reaction solution that sex change is good takes out from the PCR instrument and adds rapidly in the corresponding well, builds the lid of hybridizing box, and hybridizing box is put into hybridization 16 hours (noting: want lucifuge in the crossover process) in 40 ℃ of water-baths.After chip hybridization finishes, from water-bath, take out hybridizing box, take cover plate away, chip is washed 3min respectively in washing lotion A, wash 3min among the washing lotion B, promptly wash 1.5min in 95% ethanol at washing lotion C at last, be put into lucifuge after drying up and sentence scanning fully.
The prescription of each solution is as follows:
Washing lotion A:1 * SSC, 10%SDS
Washing lotion B:0.05 * SSC
Washing lotion C:95% ethanol
Hybridization solution: 10%dextran Sulfate; 25%formamide;
0.1％SDS；6×SSPE
3. scanning
Open the scanner (Gene Pix 4100A) of AXON company, and made the machine preheating 15 minutes.Chip is put into the lid of building scanner in the scanner after (point has one of probe to face down), starts Gene Pix Pro 6.0 scanning sequences.Select 532 passages for use, the GMT value is set at 650 and scans.Picture with scanning saves as .jpg and two kinds of forms of .GIF respectively.
Hybridization scanning result when utilizing the above-mentioned gene chip difference test sample for preparing for Shigella bogdii O13, shigella dysenteriae O10, intestinal bacteria O111, intestinal bacteria O114 and intestinal bacteria O128 is respectively as Fig. 3, Fig. 4, Fig. 5, Fig. 6 and shown in Figure 7.
4. interpretation of result
The detected result of above-mentioned important intestinal tract peccant germ detection chip is the pictures that scanning obtains, and wherein the results of hybridization of this point is being represented in the brightness of each probe points.For the less chip of probe points, detected result can be judged by naked eyes.Yet, make judgement with the naked eye promptly not objective also unrealistic for the more chip of data point.For objective, the result to detection chip analyzes accurately, the invention provides a computer interpretation program---Bactarray Analyzer.
This computer interpretation program Bactarray Analyzer is a friendly interface, wieldy program based on Microsoft.NET Framwork exploitation.The user only needs the fluorescence data file that chip scanner scans and lasso obtains is imported routine analyzer, clicks processing after simply setting and can obtain analytical results.
After chip hybridization finished, fluorescently-labeled target dna fragment had been fixed on the specific probe of chip.Use chip scanner can see the fluoroscopic image of probe, the power of fluorescence is being represented the quality of this dot blot situation, just the similarity of target dna sequence fragment and this specific probe dna sequence dna.After through the lasso analysis, the fluorescence intensity level of each point is read and the accessible fluorescence intensity file of this program of being stored as.(the scanner naming rule difference of different model, Genpix Personal 4100A type scanner as AXON company is stored as the .GPR file, and the brilliant core LuxScan-10K/A micro-array chip scanner of rich biochip difficult to understand company limited is stored as the .LSR file) processing of this software is all at the fluorescence intensity file.
Because the influence factor that is subjected in the crossover process of chip is a lot, and may there be part difference in patient's target dna fragments sequence and theoretic sequence during actual the use.Therefore, when actual treatment, this software has been done following processing:
1) validation verification of fluorescent signal
For every specific probe, all put some the repetition a little on the chip.When handling, software can carry out the degree of confidence check to these fluorescence intensities that repeat a little earlier, the fluorescence intensity that reasons such as rejecting fluorescein deposition, chip scratch, probe portion comes off cause is unusual, gets the fluorescence signal intensity of the mean value of the fluorescence intensity that remains normal phosphor dot as this specific probe then.
2) normalization method
Before data were further handled, software also needs to eliminate because the difference on the overall fluorescent intensity that DNA amount, fluorescein-labelled situation etc. are brought in the testing sample, promptly to the normalization method of fluorescence intensity data.In actual process, this software uses specific probe overall fluorescent intensity level as the normalization method standard, that is: remove with the normalization method standard with every fluorescence intensity, every absolute fluorescence intensity is become relative intensity of fluorescence, thereby make the test of carrying out under different time, the different condition have comparability.
3) to each specific probe marking
Through above-mentioned processing, the fluorescence intensity of specific probe has become comparable relative intensity of fluorescence, but this software does not simply conclude whether there is this distinguished sequence in the sample, but provide a score value at each specific probe, the similarity degree of the sequence dna fragment of representative sample and this specific probe sequence.
4) application model is given a mark to the species that may contain in the sample
Here " pattern " refers to the schema file of the corresponding relation between the relative specific probe of storage species, and this document is write according to XML (extending mark language) 1.0 standards, and dissimilar chips has different separately patterns.Preserved the corresponding relation between some essential informations of the type chip and the relevant specific probe of all species that this chip can detect in the schema file with it.When species were given a mark, this software can be added up the score value of these all relevant probes of species, and the score value of these species is the weighted mean of all relevant probe score values.
5) presumable species in the judgement sample
Through the processing of above-mentioned steps, program all provides a score value to all possible species in the sample, and the species that the species that are most possible existence that score value is the highest, score value are lower than threshold value can not exist.
Utilize the concrete operations step of the above-mentioned results of hybridization of above-mentioned Bactarray Analyzer software interpretation as follows:
Operation Bactarray Analyzer 1.0 programs, and input user name, password.The newly-built project of " new project " button on the click tools hurdle imports .GPR file to be analyzed by " adding a plurality of files " then.Press Ctrl, can select and open a plurality of files simultaneously.
Choose corresponding file in " listed files ", click the type of " positive control ", " negative control " button appointment this document then, the designator is not defaulted as testing sample etc.Must contain a positive control and two groups of data of a negative control in the project.Can change fields such as " sample source ", " sample number " by actual needs.Click " processing data " button, software will generate the log file of a treating processes automatically.Click filename at " listed files " page, the report page can automatically switch to the analysis result information of this document.Analysis result information is kept under the catalogue of this project place automatically.
Interpretation as a result: report provides information such as censorship file name, sample source, chip type, result, bacterium name, score value, specific probe hybrid rate, reference substance situation, the probable cause analysis that goes wrong, fluorescence intensity, software interpretation date, software operation people.Wherein, " result " item is the important intestinal tract peccant germ type that exists in the test sample.
Embodiment five: gene chip is carried out specificity evaluation and sensitivity detection
1, specificity experiment
Sensing range comprises following three class bacterial strains:
1) belongs to colibacillary Shigellae 19 strains of this 10 type Shigellae and 5 types, intestinal bacteria 11 strains;
2) nearer Shigellae 50 strains, intestinal bacteria 30 strains, Salmonellas 2 strains of 10 type Shigellaes and 5 type intestinal bacteria sibships therewith;
3) other common pathogen enterobacteria: bacillus cereus 2 strains, gold-coloured staphylococci 2 strains, vibrio cholerae DNA2 part.
Do 3 parallel laboratory tests at each bacterial strain.Wherein the hybridization scanning result that should obtain for 10 type Shigellaes in the sensing range and 5 type intestinal bacteria is: as Fig. 3, Fig. 4, Fig. 5, Fig. 6 and the corresponding specific probe of bacterium to be detected shown in Figure 7, over against obtaining positive signal according to probe and fluorescent probe; The computer interpretation program---the sentence read result that Bactarray Analyzer provides is: the bacterium that detects a certain type.
The hybridization scanning result that should obtain for the nearer Shigellae of sibship, intestinal bacteria and Salmonellas is: only over against obtaining positive signal according to probe and fluorescent probe; The computer interpretation program---the sentence read result that Bactarray Analyzer provides is: detect bacterium but not in sensing range.
The hybridization scanning result that other common pathogen enterobacteria should obtain is: have only fluorescent probe to obtain positive signal; The computer interpretation program---the sentence read result that Bactarray Analyzer provides is: do not detect Shigellae and intestinal bacteria.
Detected result: all obtain correct detected result.
2, sensitivity experiment
Sensing range comprises: 19 strain different times of 10 kinds of Shigellaes in the sensing range and the strain isolated that the place is separated to, and 5 kinds of colibacillary 11 strain strain isolateds in the sensing range.Do 3 parallel laboratory tests at each bacterial strain.The template that desire is detected joins with 10ng, 50ng, 100ng and four gradients of 500ng reacts in the PCR system and finally obtains the software sentence read result.The minimum template amount that can access correct and stable detected result is the value of its sensitivity.
Detected result: this cake core is that the detection sensitivity of initial detection sample is 50ng with DNA.
In sum, the present invention designs special nucleotide probe according to oligosaccharide unit treatment enzyme gene or glycosyltransferase gene on the O-antigen gene of important intestinal tract peccant germ bunch, and is fixed on and makes gene chip on the chip carrier.Simultaneously at oligosaccharide unit treatment enzyme gene or glycosyltransferase gene design primer on the O-antigen gene of important intestinal tract peccant germ bunch, genomic dna to detected sample carries out pcr amplification, then amplified production and said gene chip are carried out hybridization, utilize gene chip signal analysis equipment and software at last, just can detect the pathogenic bacterium situation that exists in the sample.The invention has the advantages that: inherit and developed traditional serology detection method, and remedied the deficiency of traditional detection, make that testing process becomes fast, sensitivity and accuracy rate height.
Though the present invention discloses as above with preferred embodiment; right its is not in order to qualification the present invention, any person of ordinary skill in the field, without departing from the spirit and scope of the present invention; can do a little change and improvement, so protection scope of the present invention is as the criterion when looking the claim person of defining.
Sequence table
＜110〉Tianjin Biochip Technology Co., Ltd
＜120〉gene chip and the detection method and the detection kit of detection important intestinal tract peccant germ
<130>5P99011-CN
<160>82
<170>PatentIn version 3.2
<210>1
<211>40
<212>DNA
＜213〉shigella flexneri 2a type (Shigella Flexneri 2a)
<400>1
gggcagtgtt tccaaggtta agtaacatca aagactttaa 40
<210>2
<211>40
<212>DNA
＜213〉shigella flexneri 2a type (Shigella Flexneri 2a)
<400>2
cggttggatg acagtaagta atatcataag tccggtcatg 40
<210>3
<211>37
<212>DNA
＜213〉bacillus ceylonensis A 1 type (Shigella sonnei)
<400>3
ggcactggat taggtgttgc aaattatgta aaggcta 37
<210>4
<211>40
<212>DNA
＜213〉bacillus ceylonensis A 1 type (Shigella sonnei)
<400>4
tgattatcgt cgagttgagt tagtatttat tggggttgat 40
<210>5
<211>40
<212>DNA
＜213〉bacillus ceylonensis A 1 type (Shigella sonnei)
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tggttctttt ggtgtcattt tgcattggta aagatgagct 40
<210>6
<211>40
<212>DNA
＜213〉Shigella bogdii O7 (Shigella boydii O7)
<400>6
attccacttc catcaatcat atcgttgatt attcctgctg 40
<210>7
<211>40
<212>DNA
＜213〉Shigella bogdii O7 (Shigella boydii O7)
<400>7
cccattgatt ttgcatcaaa taagtggctt tcttaaagga 40
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<212>DNA
＜213〉Shigella bogdii O7 (Shigella boydii O7)
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cttgatactg ataatggcgt tgaataaagc actagttcca 40
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<212>DNA
＜213〉Shigella bogdii O13 (Shigella boydii O13)
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ttatcttgag tttagtcgaa tgcaacgagt agtcgcg 37
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<211>40
<212>DNA
＜213〉Shigella bogdii O13 (Shigella boydii O13)
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ttattctcat attcgaatgt tacttttacc agcgcaactg 40
<210>11
<211>40
<212>DNA
＜213〉Shigella bogdii O13 (Shigella boydii O13)
<400>11
gctgcgggga atagaaataa gaatcgtctg tattacttta 40
<210>12
<211>36
<212>DNA
＜213〉Shigella bogdii O16 (Shigella boydii O16)
<400>12
ttgtatgtaa tcgcaaatac tcgcagtaca tgtgga 36
<210>13
<211>36
<212>DNA
＜213〉Shigella bogdii O16 (Shigella boydii O16)
<400>13
cctgagcgat tagaacaact gtatctcgag tcaaga 36
<210>14
<211>40
<212>DNA
＜213〉Shigella bogdii O16 (Shigella boydii O16)
<400>14
gacatgggag tggaatgaaa actaaaattg ctgaggcttt 40
<210>15
<211>40
<212>DNA
＜213〉Shigella bogdii O16 (Shigella boydii O16)
<400>15
gtgagattgt tgattgcttt agccgagaag acaaagatat 40
<210>16
<211>36
<212>DNA
＜213〉Shigella bogdii O18 (Shigella boydii O18)
<400>16
tttgtcgtgt ctattggtat tgtattcggg caatgg 36
<210>17
<211>40
<212>DNA
＜213〉Shigella bogdii O18 (Shigella boydii O18)
<400>17
tggagttcca ttatccgagt ggtattttgg taacaataat 40
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<211>40
<212>DNA
＜213〉Shigella bogdii O18 (Shigella boydii O18)
<400>18
tactcattag gcttgctatt tactgggcta ttatcagttt 40
<210>19
<211>36
<212>DNA
＜213〉shigella dysenteriae O4 (Shigella dysenteriae O4)
<400>19
tcgcattgct tggtattaga gctggtagtg ttaatt 36
<210>20
<211>40
<212>DNA
＜213〉shigella dysenteriae O4 (Shigella dysenteriae O4)
<400>20
gcagcttctc tttacatacg tgcattatca gaaacgggaa 40
<210>21
<211>40
<212>DNA
＜213〉shigella dysenteriae O4 (Shigella dysenteriae O4)
<400>21
actagtctag ataaaatggc tagctcagac aatctttctg 40
<210>22
<211>40
<212>DNA
＜213〉shigella dysenteriae O4 (Shigella dysenteriae O4)
<400>22
tggatattta attcggctat tctctatagc tagtgagcct 40
<210>23
<211>38
<212>DNA
＜213〉shigella dysenteriae O8 (Shigella dysenteriae O8)
<400>23
gagggtgtgt atggtatgat tgattacata ttggaggc 38
<210>24
<211>37
<212>DNA
＜213〉shigella dysenteriae O8 (Shigella dysenteriae O8)
<400>24
acttcagttt ctgggaacga taaattcaca cgtttgc 37
<210>25
<211>40
<212>DNA
＜213〉shigella dysenteriae O8 (Shigella dysenteriae O8)
<400>25
ttcctaaata atcatccatt tactgagggt gtgtatggta 40
<210>26
<211>40
<212>DNA
＜213〉shigella dysenteriae O8 (Shigella dysenteriae O8)
<400>26
cgccaattta ttctgtgcta ttatcgaaat tcacttcagt 40
<210>27
<211>38
<212>DNA
＜213〉shigella dysenteriae O10 (Shigella dysenteriae O10)
<400>27
atggcactga tagtagcgat aaaactattt tgatcggg 38
<210>28
<211>40
<212>DNA
＜213〉shigella dysenteriae O10 (Shigella dysenteriae O10)
<400>28
cgtgcattaa ttgctattgt cgtaatttct ttcattgtgg 40
<210>29
<211>40
<212>DNA
＜213〉shigella dysenteriae O10 (Shigella dysenteriae O10)
<400>29
ggaacacctt cttgggatta ttttacgcaa ccacttatta 40
<210>30
<211>37
<212>DNA
＜213〉intestinal bacteria O55 (E.coli O55)
<400>30
agagtgagac gaataattgg gtgttatata agctctc 37
<210>31
<211>40
<212>DNA
＜213〉intestinal bacteria O55 (E.coli O55)
<400>31
gcttttgggg ataacattat cgataatacc gacctttaga 40
<210>32
<211>40
<212>DNA
＜213〉intestinal bacteria O55 (E.coli O55)
<400>32
tgaagcttta aaacgtctaa ttattagcgg aactttcttg 40
<210>33
<211>36
<212>DNA
＜213〉intestinal bacteria O111 (E.coli O111)
<400>33
cggggatgat atattatttg gtttcacagc ttggtg 36
<210>34
<211>40
<212>DNA
＜213〉intestinal bacteria O111 (E.coli O111)
<400>34
ttacggttct ttataagtat tggtgtgata ggagcattgg 40
<210>35
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<212>DNA
＜213〉intestinal bacteria O111 (E.coli O111)
<400>35
gctcctttca ttgttgtaag ttgtttgtta ctgttaca 38
<210>36
<211>40
<212>DNA
＜213〉intestinal bacteria O111 (E.coli O111)
<400>36
ttaaataacg gcggacaata taagacgtta tatggacttc 40
<210>37
<211>40
<212>DNA
＜213〉intestinal bacteria O114 (E.coli O114)
<400>37
ttattgtatg cttgttagtg cttgtgctga tttgttttct 40
<210>38
<211>37
<212>DNA
＜213〉intestinal bacteria O114 (E.coli O114)
<400>38
tgagatgctt aaattaggtg gatggaatgt taatggg 37
<210>39
<211>40
<212>DNA
＜213〉intestinal bacteria O114 (E.coli O114)
<400>39
gttgttcata tgctcagggg aaaatcagaa gagtatctat 40
<210>40
<211>39
<212>DNA
＜213〉intestinal bacteria O114 (E.coli O114)
<400>40
tggatggaat gttaatgggt tatttatttc agaagcatg 39
<210>41
<211>34
<212>DNA
＜213〉intestinal bacteria O128 (E.coli O128)
<400>41
tctgatcttg gattaagtaa gatgtaccca gcaa 34
<210>42
<211>40
<212>DNA
＜213〉intestinal bacteria O128 (E.coli O128)
<400>42
cggtgttttg caagagatat aaaagagtta gctttagcat 40
<210>43
<211>40
<212>DNA
＜213〉intestinal bacteria O128 (E.coli O128)
<400>43
gctaggtatt tagcaaattc aacagatttg gctgactttg 40
<210>44
<211>36
<212>DNA
＜213〉Escherichia coli O 157 (E.coli O157)
<400>44
cgatttcttt ccgacaccag agttagaaaa ggaatt 36
<210>45
<211>40
<212>DNA
＜213〉Escherichia coli O 157 (E.coli O157)
<400>45
tagagcaagt tgaaagtgtt ccatatgttg tttctgaatc 40
<210>46
<211>40
<212>DNA
＜213〉Escherichia coli O 157 (E.coli O157)
<400>46
gtatgctcgt tgttttatct aagtttagga caagacggag 40
<210>47
<211>40
<212>DNA
＜213〉Shigella bogdii O9 (Shigella boydii O9)
<400>47
aactgagttc acttatggtt cgagaacctt tactccattt 40
<210>48
<211>40
<212>DNA
＜213〉Shigella bogdii O9 (Shigella boydii 09)
<400>48
ggattttaat acaactgagt tcacttatgg ttcgagaacc 40
<210>49
<211>30
<212>DNA
＜213〉Shigellae and intestinal bacteria
<400>49
cgggaactca aaggagactg ccagtgataa 30
<210>50
<211>37
<212>DNA
＜213〉Shigellae and intestinal bacteria
<400>50
cgggaactca aaggagactg ccagtgataa actggag 37
<210>51
<211>19
<212>DNA
＜213〉Shigella bogdii O7 (Shigella boydii O7)
<400>51
gtgttgactg ctggatttc 19
<210>52
<211>20
<212>DNA
＜213〉Shigella bogdii O7 (Shigella boydii O7)
<400>52
cgatatgatt gatggaagtg 20
<210>53
<211>19
<212>DNA
＜213〉Shigella bogdii O9 (Shigella boydii O9)
<400>53
gcgttggttg gtgaaagag 19
<210>54
<211>18
<212>DNA
＜213〉Shigella bogdii O9 (Shigella boydii O9)
<400>54
ttcccacaaa tcaaacca 18
<210>55
<211>18
<212>DNA
＜213〉Shigella bogdii O13 (Shigella boydii O13)
<400>55
aaagattggt agcgtcgg 18
<210>56
<211>19
<212>DNA
＜213〉Shigella bogdii O13 (Shigella boydii O13)
<400>56
tgaagccctg gtaaagtgc 19
<210>57
<211>19
<212>DNA
＜213〉Shigella bogdii O16 (Shigella boydii O16)
<400>57
ccatacggat aatgttgag 19
<210>58
<211>18
<212>DNA
＜213〉Shigella bogdii O16 (Shigella boydii O16)
<400>58
tctttgtctt ctcggcta 18
<210>59
<211>17
<212>DNA
＜213〉Shigella bogdii O18 (Shigella boydii O18)
<400>59
cagggcacaa ac tatct 17
<210>60
<211>16
<212>DNA
＜213〉Shigella bogdii O18 (Shigella boydii O18)
<400>60
taattcggaa tgtgct 16
<210>61
<211>20
<212>DNA
＜213〉shigella dysenteriae O4 (Shigella dysenteriae O4)
<400>61
tttctgctta ttcattattg 20
<210>62
<211>18
<212>DNA
＜213〉shigella dysenteriae O4 (Shigella dysenteriae O4)
<400>62
actaccagct ctaatacc 18
<210>63
<211>19
<212>DNA
＜213〉shigella dysenteriae O8 (Shigella dysenteriae O8)
<400>63
ttccctcttg ttgtattga 19
<210>64
<211>19
<212>DNA
＜213〉shigella dysenteriae O8 (Shigella dysenteriae O8)
<400>64
acctttatca attgcctcc 19
<210>65
<211>20
<212>DNA
＜213〉shigella dysenteriae O10 (Shigella dysenteriae O10)
<400>65
cgctgtttct atattaattg 20
<210>66
<211>20
<212>DNA
＜213〉shigella dysenteriae O10 (Shigella dysenteriae O10)
<400>66
aattgaagtg accagataac 20
<210>67
<211>18
<212>DNA
＜213〉bacillus ceylonensis A 1 type (Shigella sonnei)
<400>67
tgaggtttca cgtttctc 18
<210>68
<211>19
<212>DNA
＜213〉bacillus ceylonensis A 1 type (Shigella sonnei)
<400>68
aataatccct aactgagcc 19
<210>69
<211>19
<212>DNA
＜213〉shigella flexneri 2a type (Shigella Flexneri 2a)
<400>69
cacttgttgg gtatgctgg 19
<210>70
<211>19
<212>DNA
＜213〉shigella flexneri 2a type (Shigella Flexneri 2a)
<400>70
ccggcaaaca gattagaaa 19
<210>71
<211>20
<212>DNA
＜213〉intestinal bacteria O55 (E.coli O55)
<400>71
ggaggagta ttatcattac 20
<210>72
<211>19
<212>DNA
＜213〉intestinal bacteria O55 (E.coli O55)
<400>72
atcaatctaa aggtcggta 19
<210>73
<211>19
<212>DNA
＜213〉intestinal bacteria O111 (E.coli O111)
<400>73
ttaatgcgga ggatctatt 19
<210>74
<211>19
<212>DNA
＜213〉intestinal bacteria O111 (E.coli O111)
<400>74
gtaagcccgc aaatcaatc 19
<210>75
<211>18
<212>DNA
＜213〉intestinal bacteria O114 (E.coli O114)
<400>75
actttcccaa gcccatta 18
<210>76
<211>19
<212>DNA
＜213〉intestinal bacteria O114 (E.coli O114)
<400>76
cagcacaagc actaacaag 19
<210>77
<211>19
<212>DNA
＜213〉intestinal bacteria O128 (E.coli O128)
<400>77
atgatttctt acggagtgc 19
<210>78
<211>19
<212>DNA
＜213〉intestinal bacteria O128 (E.coli O128)
<400>78
ctctaaccta atccctccc 19
<210>79
<211>23
<212>DNA
＜213〉Escherichia coli O 157 (E.coli O157)
<400>79
ttgctgctgt agttttattt ctt 23
<210>80
<211>23
<212>DNA
＜213〉Escherichia coli O 157 (E.coli O157)
<400>80
tgatgcttta ttccctgtat tct 23
<210>81
<211>20
<212>DNA
＜213〉Shigellae and intestinal bacteria
<400>81
cacgggtgag taatgtctgg 20
<210>82
<211>22
<212>DNA
＜213〉Shigellae and intestinal bacteria
<400>82
atccacgatt actagcgatt cc 22
Claims (13)
1. gene chip that detects important intestinal tract peccant germ, comprise solid phase carrier and be fixed on oligonucleotide probe on this solid phase carrier, it is characterized in that the dna fragmentation that oligonucleotide probe on this gene chip contains dna fragmentation that oligosaccharide unit treatment enzyme gene from the O-antigen gene of one or more important intestinal tract peccant germs bunch or glycosyltransferase gene choose at least and chooses from Shigellae and intestinal bacteria 16SrRNA conserved regions.
2. the gene chip of detection important intestinal tract peccant germ according to claim 1 is characterized in that above-mentioned one or more important intestinal tract peccant germs are selected from shigella flexneri 2a type, bacillus ceylonensis A 1 type, Shigella bogdii O7, Shigella bogdii O9, Shigella bogdii O13, Shigella bogdii O16, Shigella bogdii O18, shigella dysenteriae O4, shigella dysenteriae O8, shigella dysenteriae O10, intestinal bacteria O55, intestinal bacteria O111, intestinal bacteria O114, intestinal bacteria O128 and Escherichia coli O 157.
3. gene chip according to claim 1 and 2 is characterized in that the dna fragmentation that oligosaccharide unit treatment enzyme gene from the O-antigen gene of above-mentioned one or more important intestinal tract peccant germs bunch or glycosyltransferase gene are chosen is selected from the dna fragmentation with the base sequence shown in SEQ ID NO:1-NO:48.
4. gene chip according to claim 1 is characterized in that the above-mentioned dna fragmentation of choosing from Shigellae and intestinal bacteria 16SrRNA conserved regions has the base sequence shown in SEQ ID NO:49 and NO:50.
5. gene chip according to claim 1 is characterized in that above-mentioned oligonucleotide probe also comprises negative contrast and fluorescent probe.
6. method that detects important intestinal tract peccant germ is characterized in that may further comprise the steps:
(1) according to oligosaccharide unit treatment enzyme gene or glycosyltransferase gene and Shigellae and intestinal bacteria 16SrRNA conserved regions design on the O-antigen gene of one or more important intestinal tract peccant germs bunch and prepare the primer that is used for pcr amplification;
(2) use the described primer of step (1) that the genomic dna of testing sample is carried out pcr amplification;
(3) with amplified production and the described gene chip hybridization of claim 1;
(4) obtain hybridization signal and Analysis and Identification results of hybridization with gene chip scanning instrument.
7. the method for detection important intestinal tract peccant germ according to claim 6 is characterized in that in the above-mentioned steps (1) being selected from the oligonucleotide fragment with the base sequence shown in SEQ ID NO:51-NO:80 according to the oligosaccharide unit treatment enzyme gene on the O-antigen gene of one or more important intestinal tract peccant germs bunch or the primer of glycosyltransferase gene design.
8. the method for detection important intestinal tract peccant germ according to claim 6 is characterized in that in the above-mentioned steps (1) that primer sequence according to Shigellae and intestinal bacteria 16SrRNA conserved regions design is shown in SEQ ID NO:81 and NO:82.
9. the method for detection important intestinal tract peccant germ according to claim 6 is characterized in that using in the above-mentioned steps (4) interpretation software Bactarray Analyzer Analysis and Identification results of hybridization.
10. the method for detection important intestinal tract peccant germ according to claim 10 is characterized in that using above-mentioned interpretation software Bactarray Analyzer to follow these steps to the Analysis and Identification results of hybridization:
(1) validation verification of fluorescent signal
A plurality of strength of signal that repeat a little of every specific probe on the said gene chip are carried out the degree of confidence check, and the strength of signal of rejecting abnormalities is got the strength of signal of the mean value of normal strength of signal as every specific probe;
(2) normalization method
Use specific probe overall fluorescent intensity level as the normalization method standard, remove with the normalization method standard, every absolute fluorescence intensity is become relative intensity of fluorescence with every fluorescence intensity;
(3) to each specific probe marking
According to relative intensity of fluorescence, each specific probe is provided a score value, the similarity degree of the sequence dna fragment of representative sample and this specific probe sequence;
(4) application model is given a mark to the species that may contain in the sample
With the weighted mean of all relevant probe score values of a certain species score value as these species;
(5) presumable species in the judgement sample
Handle through above-mentioned steps, program all provides a score value to all possible species in the sample, and the species that the species that are most possible existence that score value is the highest, score value are lower than threshold value can not exist.
11. test kit that detects important intestinal tract peccant germ, it is characterized in that comprising at least gene chip, this gene chip comprises solid phase carrier and the oligonucleotide probe that is fixed on this solid phase carrier, wherein, the oligonucleotide probe dna fragmentation that contains dna fragmentation that oligosaccharide unit treatment enzyme gene from the O-antigen gene of one or more important intestinal tract peccant germs bunch or glycosyltransferase gene choose at least and choose from Shigellae and intestinal bacteria 16SrRNA conserved regions.
12. the test kit of detection important intestinal tract peccant germ according to claim 11 is characterized in that also comprising the pcr amplification primer, these pcr amplification primers are selected from the oligonucleotide fragment with the base sequence shown in SEQ ID NO:51-NO:82.
13. the test kit of detection important intestinal tract peccant germ according to claim 11 is characterized in that the interpretation software Bactarray Analyzer that comprises that also hybridizing box, hybridization solution and Analysis and Identification result use.
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| Application Number | Priority Date | Filing Date | Title |
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| CN200510132117A CN1982472B (en) | 2005-12-16 | 2005-12-16 | Gene chip for inspecting important intestinal tract peccant germ, its inspecting method and reagent kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200510132117A CN1982472B (en) | 2005-12-16 | 2005-12-16 | Gene chip for inspecting important intestinal tract peccant germ, its inspecting method and reagent kit |
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| CN1982472A true CN1982472A (en) | 2007-06-20 |
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| CN101407837B (en) * | 2007-10-12 | 2011-09-21 | 天津生物芯片技术有限责任公司 | Gene chip for detecting blood pathogen and reagent kit for detecting |
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| CN1683565A (en) * | 2005-03-15 | 2005-10-19 | 中国人民解放军军事医学科学院放射医学研究所 | Oligonucleotide probe kit for detecting common intestine trac kpathogenic bacteria and its use |
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