CN106148502A - A kind of high throughput method directly detecting food source pathogenic bacteria - Google Patents

Abstract

A kind of high throughput method directly detecting food source pathogenic bacteria of the present invention, relates to the fast detecting method of bacterium high flux based on 16S rRNA and liquid-phase chip.Including using the 16S rRNA of 2-20 kind bacterium as mensuration target, design capture probe and detection probe；2-20 kind microballoon, detection probe and the testing sample mixing processing through cell cracking of specific capture probe, formation " the 16S rRNA-detection probe of microballoon-capture probe-specific bacteria " compound will be attached with；The microballoon identification signal of different microballoons and detectable in the compound that detection is formed, so that it is determined that the amount of the presence or absence of each tested bacteria and existence in testing sample.The method of the present invention eliminates the conventional molecular biological necessary PCR step of detection, make detection become faster, easier, more reasonable, with low cost.The present invention also optimizes the probe set group that can simultaneously detect multiple bacterial strain, and Detection results is good.

Description

A kind of high throughput method directly detecting food source pathogenic bacteria

Technical field

The present invention relates to microorganism detection field, more particularly it relates to based on 16S rRNA and liquid The fast detecting method of bacterium high flux of phase chip.

Background technology

At present, great food safety affair takes place frequently, and causes the great attention of people.Fundamentally to solve food Product safety problem, is necessary for each links such as the production to food, processing, circulation and sale and implements whole process control And monitoring, this is accomplished by disclosure satisfy that in a large number food quick, convenient, accurate, the sensitive peace of this requirement Complete analysis detection technique.

With scientific and technical development, food safety quick analytic detection method is in terms of inspection for food hygiene Act on more and more important.From the point of view of long term growth, immunology, molecular biology, computer technology with automatically The development pole the earth of the subjects such as change has promoted Food Safety Analysis detection method to sensitiveer direction more easily Development, sets up efficient food safety quick analytic detection method, to food production, transport, sales process In quality control tool be of great significance；Once there is food-borne pathogenic microorganism poisoning thing simultaneously Part, it is also desirable to complete to detect to formulate scientific and reasonable therapeutic scheme within the shortest time, win treatment time. The popularization and application of these quick analysis and detection technologies, are not only to traditional Food Safety Analysis detection technique One is improved, and also makes Safety of Food Quality have further guarantee, thus promotes food industry more Add health, advance rapidly, constantly meet the needs that the people improve health conditions.

Some food-borne pathogens Fast Detection Technique existing at present are to utilize immunological method, the party mostly Method instrumentation is simple, detection speed is fast, sensitivity compared with high, specific preferably and sample aequum is few, but There is also pathogenic bacteria monoclonal antibody such as to be difficult to prepare, use and many anti-be easily generated cross reaction false positive occurs And once can only detect the shortcoming and defect such as one or more pathogenic bacteria.Therefore, also needing to exploitation at present can Quickly, efficiently detect the new technology of pathogenic bacteria.

Content of the invention

It is an object of the invention to provide based on 16S rRNA or its pcr amplification product and liquid-phase chip is thin The fast detecting method of bacterium high flux.

In a first aspect of the present invention, providing a kind of fast-bacteria-detection method, described method includes:

(1) using the 16S rRNA of 2-20 kind bacterium or its pcr amplification product as measuring target, design is caught Obtain probe and detection probe；Described capture probe is that 16S rRNA or its PCR for every kind of bacterium expand Increase production the specific probe of thing, and it is attached respectively on 2-20 kind microballoon；Described detection probe be with respectively The probe that on the 16S rRNA of bacterium or its pcr amplification product, consensus sequence is complementary, and it includes that one can examine Survey label；Described 2-20 kind microballoon carries different microballoon identification signals respectively；

(2) by be attached with specific capture probe 2-20 kind microballoon, detection probe and through cell cracking at The testing sample mixing of reason；So that 16S rRNA or its PCR amplification of the specific bacteria in testing sample Product, capture probe corresponding with this specific bacteria and microballoon, and detection probe is formed, and " microballoon-capture is visited The 16S rRNA of pin-specific bacteria or its pcr amplification product-detection probe " compound；

(3) microballoon identification signal and the detectable of different microballoons in the compound that (2) are formed are detected, So that it is determined that the amount of the presence or absence of each tested bacteria and existence in testing sample.

In a preference, described method is used for differentiating 16S rRNA or its pcr amplification product sequence not Same bacterium.

In another preference, in step (2), described testing sample only carries out cell cracking process, does not wraps Include the step of PCR amplification or cell proliferation.

In another preference, in step (1), also include: expand for each bacterial 16 S rRNA or its PCR Consensus sequence on volume increase thing, Design assistant (Helper) sequence, described auxiliary sequencel is for stably opening (in crossover process, high temperature makes 16S rRNA or its PCR amplification produce for 16S rRNA or its pcr amplification product Thing denaturation, secondary structure opens and becomes strand, now helper and 16S rRNA or its pcr amplification product Complementary combination, plays stable effect), strengthen hybridization check signal.It is preferred that described auxiliary sequencel Position is positioned between capture probe and detection probe.

In another preference, 3 ' ends of detection probe connect detectable；Or capture probe is 5 ' End carries out amido modified.

In another preference, described bacterium is pathogenic bacteria.

In another preference, include as the pathogenic bacteria measuring target: Escherichia coli (Escherichia coli), Yersinia enterocolitica (Yersinia enterocolitica), staphylococcus aureus (Staphylococcus aureus), bacillus cereus (Bacillus cereus), monocyte hyperplasia Liszt Salmonella (Listeria monocytogenes), Shigella (Shigella), salmonella (Salmonella), slope Rugged enterobacteria (Enterobacter sakazakii), proteus (Proteus vulgaris), vibrio parahaemolytious (Vibrio parahaemolyticus), campylobacter jejuni (Campylobacter jejuni), Vibrio vulnificus (Vibrio Vulnificus) 2 kinds or two or more.

In another preference, include as the pathogenic bacteria measuring target: Escherichia coli, Shigella, sand Door Salmonella, Enterobacter sakazakii, yersinia enterocolitica, proteus, vibrio parahaemolytious, monokaryon Monocytogenes, staphylococcus aureus, campylobacter jejuni, Bacillus cereus, wound arc Bacterium, the sequence for the capture probe of each pathogenic bacteria is SEQ ID NO:45～SEQ ID NO:56 successively, Or its complementary series；The sequence of detection probe is SEQ ID NO:57, or its complementary series；It is preferred that Also include Helper sequence SEQ ID NO:58, or its complementary series.

In another preference, include as the pathogenic bacteria measuring target: Escherichia coli, enterocolitis Ademilson Salmonella, staphylococcus aureus, bacillus cereus, Listeria Monocytogenes, for The sequence of the capture probe of each pathogenic bacteria is SEQ ID NO:59～SEQ ID NO:63 successively, or it is complementary Sequence；The sequence of detection probe is SEQ ID NO:64 and/or SEQ ID NO:67, or its complementary series； It is preferred that also include Helper sequence SEQ ID NO:65 and SEQ ID NO:66, or its complementary series.

In another preference, include as the pathogenic bacteria measuring target: Escherichia coli, Shigella, sand Door Salmonella, Enterobacter sakazakii, yersinia enterocolitica, proteus, vibrio parahaemolytious, wound Vibrios, the sequence for the capture probe of each pathogenic bacteria is SEQ ID NO:1～SEQ ID NO:8 successively, Or its complementary series；The sequence of detection probe is SEQ ID NO:9, or its complementary series；It is preferred that also Including Helper sequence SEQ ID NO:10, or its complementary series.

In another preference, as measure target pathogenic bacteria include: Enterobacter sakazakii, Escherichia coli and Shigella, yersinia enterocolitica, salmonella, proteus, vibrio parahaemolytious, wound Hindering vibrios, the sequence for the capture probe of each pathogenic bacteria is SEQ ID NO:68～SEQ ID NO successively: 74, or its complementary series；Wherein Escherichia coli and Shigella share capture probe SEQ ID NO:69, Or its complementary series；The sequence of detection probe is SEQ ID NO:75, or its complementary series.

In another preference, include as the pathogenic bacteria measuring target: Escherichia coli, Shigella, sand Door Salmonella, Enterobacter sakazakii, yersinia enterocolitica, proteus, vibrio parahaemolytious, wound Vibrios, the sequence for the capture probe of each pathogenic bacteria is SEQ ID NO:11～SEQ ID NO:18 successively, Or its complementary series；The sequence of detection probe is SEQ ID NO:19, or its complementary series；It is preferred that Also include Helper sequence SEQ ID NO:20, or its complementary series.

In another preference, include as the pathogenic bacteria measuring target: yersinia enterocolitica, Proteus, vibrio parahaemolytious, Listeria Monocytogenes, staphylococcus aureus, jejunum are curved Aspergillus, Bacillus cereus, Vibrio vulnificus, the sequence for the capture probe of each pathogenic bacteria is SEQ successively ID NO:21～SEQ ID NO:28, or its complementary series；The sequence of detection probe is SEQ ID NO:29, Or its complementary series；It is preferred that also include Helper sequence SEQ ID NO:30, or its complementary series.

In another preference, include as the pathogenic bacteria measuring target: Escherichia coli, Shigella, sand Door Salmonella, Enterobacter sakazakii, yersinia enterocolitica, proteus, vibrio parahaemolytious, monokaryon Monocytogenes, Vibrio vulnificus, staphylococcus aureus, campylobacter jejuni, waxy brood cell's bar Bacterium, the sequence for the capture probe of each pathogenic bacteria is SEQ ID NO:31～SEQ ID NO:42 successively, Or its complementary series；The sequence of detection probe is SEQ ID NO:43, or its complementary series；It is preferred that Also include Helper sequence SEQ ID NO:44, or its complementary series.

In another preference, include as the pathogenic bacteria measuring target: Listeria Monocytogenes, Bacillus cereus, staphylococcus aureus, campylobacter jejuni, for the capture probe of each pathogenic bacteria Sequence is SEQ ID NO:78～SEQ ID NO:81 successively, or its complementary series；The sequence of detection probe Row are SEQ ID NO:82～SEQ ID NO:83, or its complementary series.

In another preference, when the pathogenic bacteria as mensuration target include that Escherichia coli and/or will are congratulated simultaneously When Salmonella and yersinia enterocolitica, also include: use the capture probe of SEQ ID NO:77, Or its complementary series；With the detection probe of SEQ ID NO:76, or its complementary series detection enterocolitis Yersinia ruckeri, thus yersinia enterocolitica is distinguished with Escherichia coli and/or Shigella.

In another aspect of this invention, the kit for detecting pathogenic bacteria, described pathogenic bacteria bag are provided Include: Escherichia coli, Shigella, salmonella, Enterobacter sakazakii, yersinia enterocolitica, Proteus, vibrio parahaemolytious, Listeria Monocytogenes, staphylococcus aureus, jejunum Campylobacter spp, Bacillus cereus, Vibrio vulnificus；Described kit includes: for catching of each pathogenic bacteria Obtain probe SEQ ID NO:45～SEQ ID NO:56, or its complementary series；Detection probe SEQ ID NO: 57, or its complementary series；It is preferred that also include auxiliary sequencel SEQ ID NO:58, or its complementary series.

In another aspect of this invention, the kit for detecting pathogenic bacteria, described pathogenic bacteria bag are provided Include: Escherichia coli, yersinia enterocolitica, staphylococcus aureus, bacillus cereus, Listeria Monocytogenes；Described kit includes: for the capture probe SEQ of each pathogenic bacteria ID NO:59～SEQ ID NO:63, or its complementary series；Detection probe SEQ ID NO:64 and/or SEQ ID NO:67, or its complementary series；It is preferred that also include auxiliary sequencel SEQ ID NO:65 and SEQ ID NO:66, or its complementary series.

In another aspect of this invention, the kit for detecting pathogenic bacteria, described pathogenic bacteria bag are provided Include: Escherichia coli, Shigella, salmonella, Enterobacter sakazakii, yersinia enterocolitica, Proteus, vibrio parahaemolytious, Vibrio vulnificus；Described kit includes: for catching of each pathogenic bacteria Obtain probe SEQ ID NO:1～SEQ ID NO:8, or its complementary series, detect probe SEQ ID NO:9, Or its complementary series；It is preferred that also include auxiliary sequencel SEQ ID NO:10, or its complementary series.

In another aspect of this invention, the kit for detecting pathogenic bacteria, described pathogenic bacteria bag are provided Include: Enterobacter sakazakii, Escherichia coli and Shigella, yersinia enterocolitica, salmonella, Proteus, vibrio parahaemolytious, Vibrio vulnificus；Described kit includes: for catching of each pathogenic bacteria Obtain probe SEQ ID NO:68～SEQ ID NO:74, or its complementary series；Detection probe SEQ ID NO: 75, or its complementary series.

In another aspect of this invention, the kit for detecting pathogenic bacteria, described pathogenic bacteria bag are provided Include: Escherichia coli, Shigella, salmonella, Enterobacter sakazakii, yersinia enterocolitica, Proteus, vibrio parahaemolytious, Vibrio vulnificus；Described kit includes: for catching of each pathogenic bacteria Obtain probe SEQ ID NO:11～SEQ ID NO:18, or its complementary series；Detection probe SEQ ID NO: 19, or its complementary series；It is preferred that also include auxiliary sequencel SEQ ID NO:20, or its complementary series.

In another aspect of this invention, the kit for detecting pathogenic bacteria, described pathogenic bacteria bag are provided Include: yersinia enterocolitica, proteus, vibrio parahaemolytious, monocyte hyperplasia Liszt Salmonella, staphylococcus aureus, campylobacter jejuni, Bacillus cereus, Vibrio vulnificus；Described reagent Box includes: for the capture probe SEQ ID NO:21～SEQ ID NO:28 of each pathogenic bacteria, or its Complementary series；Detection probe SEQ ID NO:29, or its complementary series；It is preferred that also include assisting sequence Row SEQ ID NO:30, or its complementary series.

In another aspect of this invention, the kit for detecting pathogenic bacteria, described pathogenic bacteria bag are provided Include: Escherichia coli, Shigella, salmonella, Enterobacter sakazakii, yersinia enterocolitica, Proteus, vibrio parahaemolytious, Listeria Monocytogenes, Vibrio vulnificus, golden yellow grape Coccus, campylobacter jejuni, Bacillus cereus；Described kit includes: for catching of each pathogenic bacteria Obtain probe SEQ ID NO:31～SEQ ID NO:42, or its complementary series；The sequence of detection probe is SEQ ID NO:43, or its complementary series；It is preferred that also include auxiliary sequencel SEQ ID NO:44, Or its complementary series.

In another aspect of this invention, the kit for detecting pathogenic bacteria, described pathogenic bacteria bag are provided Include: Listeria Monocytogenes, Bacillus cereus, staphylococcus aureus, jejunum campylobacter Bacterium；Described kit includes: for the capture probe SEQ ID NO:78～SEQ ID NO of each pathogenic bacteria: 81, or its complementary series；Detection probe SEQ ID NO:82, SEQ ID NO:83, or its complementary series.

In another aspect of this invention, provide the kit for detecting pathogenic bacteria, in described kit also Including: the capture probe of SEQ ID NO:77, or its complementary series；, the detection of SEQ ID NO:76 Probe, or its complementary series；For by yersinia enterocolitica and Escherichia coli and/or will Hayes Bacterium distinguishes.

The other side of the present invention due to this disclosure, be to those skilled in the art aobvious and It is clear to.

Brief description

Fig. 1, technical schematic diagram.

The operating process schematic diagram of the method for Fig. 2, probe design and the present invention.

Fig. 3, detection scheme design diagram.

Fig. 4, detection scheme design diagram.

Detailed description of the invention

The present inventor, through in-depth study, discloses a kind of pathogenic bacteria high flux first and examines technology soon, neither Needing to extract nucleic acid, without PCR amplification, the method for the present invention eliminates conventional molecular biological detection must Must PCR step, make detection become faster, easier, more reasonable, with low cost.The present inventor goes back base In described method, optimizing the probe set group that can simultaneously detect multiple bacterial strain, Detection results is good.These Probe and can also be able to obtain as the specific probe of the PCR primer of 16S rRNA gene magnification Good Detection results.

As used herein, " capture probe ", " Capture DNA ", " Capture Probe " are interchangeable Use, refer to the 16S rRNA or its PCR for being fixed on a kind of bacterium on microballoon, specific binding Amplified production and with this kind of bacterium beyond the 16S rRNA of other target bacterias or its pcr amplification product do not occur In conjunction with probe.

As used herein, " detection probe ", " Detection DNA ", " Detector oligonucletide ", " Detetor " is used interchangeably, and refers on the 16S rRNA with all target bacterias or its pcr amplification product The complementary probe of consensus sequence.

As used herein, described " microballoon identification signal " refers to be positioned on microballoon, is used for distinguishing difference The identification signal of capture probe.For convenience's sake, for the microballoon being fixed with same capture probe, Microballoon identification signal is preferably identical.Preferably, described microballoon identification signal is fluorescence signal, and, For the microballoon being fixed with capture probe not of the same race, the color of fluorescence is different, for being fixed with of the same race catching Obtaining the microballoon of probe, the color of fluorescence is preferably identical.

As used herein, described " detectable " refers to and detects probe is combined or couples, use Send out with corresponding 16S rRNA or its pcr amplification product in display (or as display mark) detection probe The raw signal combining.

In a preferred embodiment of the present invention, described detectable is biotin, and this biotin is as aobvious Indicating will, when the phycoerythrin with streptomycete Avidin coupling, (streptavidin-phycoerythrin, is also called PE Mark streptavidin, SA-R-PE) combine after, produce fluorescence signal by laser excitation.

As used herein, described " target bacteria " refers to as the bacterium measuring target, such as some food In pathogenic bacteria.

The Cleaning Principle of the method for the present invention as shown in Figure 1-2, based on multiple as the bacteriums measuring target 16S rRNA or the sequence of its pcr amplification product, design capture probe and detection probe；Capture probe is For 16S rRNA or the specific probe of its pcr amplification product of every kind of bacterium, and it is attached respectively to On 2-20 kind microballoon；Detection probe is to have sequence on the 16S rRNA with each bacterium or its pcr amplification product Arrange complementary probe, and it includes a detectable；Described 2-20 kind microballoon carries different respectively Microballoon identification signal；The 2-20 kind microballoon of specific capture probe will be attached with, detect probe and through cell The testing sample mixing that cracking is processed；So that the 16S rRNA or its PCR of the specific bacteria in testing sample Amplified production, capture probe corresponding with this specific bacteria and microballoon, and detection probe formed " microballoon- The 16S rRNA of capture probe-specific bacteria or its pcr amplification product-detection probe " compound；Detection The microballoon identification signal of different microballoons and detectable in the compound being formed, so that it is determined that treat test sample The amount of the presence or absence of each tested bacteria and existence in product.

In the present invention, in liquid-phase chip system, including have the microballoon of different microballoon identification signal, Ye Jiwei The species of ball identification signal is identical with the species of target bacteria or the species of unnecessary target bacteria.On described microballoon admittedly Surely there is different capture probes.

In the present invention, capture probe is fixed on the DOP detailed operating procedure on microballoon and can use conventional method, for example Product description or website according to Luminex company: the method described in www.luminexcorp.com Carry out coupled, thus obtain the coupling thing that different microballoon is formed with respective capture probe.

Owing to different microballoon identification signals, making can be distinguished between different microballoon.Excellent in the present invention Selecting in mode, described microballoon identification signal is fluorescence signal, and, the fluorescence signal on microballoon is because fixing The difference of the corresponding capture probe on microballoon and different.

Microballoon of the present invention can use the materials such as polystyrene, polyvinyl chloride, polyethylene to make. Further, the average diameter of described microballoon is 1-100 μm；It is furthermore preferred that the average diameter of described microballoon For 2-50 μm, for example, the average diameter of described microballoon is 2-10 μm.

The present invention also optimizes capture probe and detection probe for various pathogens, to be more than one in detection Improve efficiency and accuracy when planting pathogenic bacteria.

The technical advantage of the present invention be without nucleic acid extraction, without PCR amplification, high flux, identify rapidly The species of the common pathogen in bacterium, particularly some food and relative populations.The bacterium solution that directly will obtain After boiling process, supernatant and detection sequence, the microballoon of coupling specific probe does after hybridizing, can go up machine testing. Whether the signal value finally according to machine detection can contain pathogenic bacteria and pathogenic bacteria content is many by judgement sample Few.

The method of the present invention can also only detect viable bacteria simultaneously, not detect dead bacterium.Dead endobacillary 16S rRNA Easily by the nuclease degradation of self, can't detect signal in theory or signal value is very weak.Have in fresh viable bacteria Substantial amounts of 16S rRNA, can detect very high signal.

In a preferred embodiment of the invention, for common multiple food-borne pathogens: Staphylococcus aureus Bacterium, Listeria Monocytogenes, Shigella, vibrio parahaemolytious, Bacillus cereus, slope are rugged Enterobacteria, campylobacter jejuni, salmonella, enteropathogenic E. Coli group O157:H7, Proteus, Yersinia enterocolitica and the Fast Detection Technique of Vibrio vulnificus.With these pathogenic bacteria as target Thing, with 16S rRNA or its pcr amplification product as target gene, separately designs general detection sequence and spy Pin, by the probe of design and the polystyrene microsphere coupling of carboxylated, is hybridized by one-step method rRNA/DNA, Specifically capture pathogenic bacteria 16S rRNA fragment, thus reach to detect the purpose of object bacteria.

The method of the present invention can be applied not only to the detection of food-borne pathogens, can be also used for environment measuring, The hygienic conditions monitoring of the public arenas such as such as water source detection, enterprise's production environment, hospital and school, and Research of human body micro-ecological environment etc..

The method of the present invention had both possessed that the sample consumption of liquid-phase chip is few, simple to operate, quick, sensitivity The advantages such as high, personalization assembling flexibly, overcome again the necessary PCR step of conventional molecular biological detection, Make detection become faster, easier, less expensive, more reasonable.

Based on the method for the present invention, visit for the capture probe designed by the bacterial species of required mensuration, detection Pin (or may also include Helper) can be placed in kit, is prepared as the product of commercialization, it is simple to people Carry out precisely, bacterium qualitative or quantitative analysis rapidly.

In kit, different capture probes can be connected or be coupled to have different microballoon identification signal Microballoon on.

In kit, described detection probe can be connected with detectable, such as biotin.

The kit of the present invention also can comprise operation instructions, illustrate described kit for bacterial species, And instruct people to carry out examinations according to correct flow process, condition, dosage.

Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are only used for The bright present invention rather than restriction the scope of the present invention.The experiment side of unreceipted actual conditions in the following example Method, generally writes according to normal condition such as J. Pehanorm Brooker etc., Molecular Cloning: A Laboratory guide, the third edition, Science Press, the condition described in 2002, or according to the condition proposed by manufacturer.

I, material and method

1st, experimental strain

Vibrio vulnificus, Escherichia coli, salmonella, bacillus cereus, yersinia enterocolitica, Proteus, Enterobacter sakazakii, Shigella, vibrio parahaemolytious, staphylococcus aureus, monocyte Monocytogenes and jejunum campylobacter Pseudomonas.

Table the 1st, bacterial strain information

2nd, experiment reagent

The microballoon of conjugated probes: Bio-rad company；

LiquiChip 200 instrumental correction microballoon: Qiagen company；

The sheath fluid of LiquiChip 200 instrument: Qiagen company；

Streptavidine-R-PE:Qiagen company；

0.02%Tween 20 (Sigma P9416), 0.1%SDS (Sigma L4522), 1 × TE, pH8.0 (Sigma T9285)；

Detection buffer solution: 5M TMAC (Sigma T3411), 20%Sarkosyl solution (Sigma L7414), 1M Tris-HCl, pH 8.0 (Sigma T3038), 0.5M EDTA, pH8.0 (Invitrogen 15575-020)；

Its root RNA extracts kit；

DNA marker: full scholar gold Trans 2K DNA Marker；

The culture medium of cultivation bacterium: yeast extract (LP0021) OXOID, peptone (LP0042) OXOID, sodium chloride 10019318 (analyzing pure) Chemical Reagent Co., Ltd., Sinopharm Group；

Common LB culture medium (every 100ml): yeast extract 0.5g, peptone 1.0g, sodium chloride 1.0g； Add distilled water to volume 100ml, 121 DEG C of high pressure steam sterilizations 20 minutes.

3%NaCl LB culture medium (every 100ml): yeast extract 0.5g, peptone 1.0g, sodium chloride 3.0g, Add distilled water to volume 100ml, 121 DEG C of high pressure steam sterilizations 20 minutes.

3rd, laboratory apparatus

PCR amplification instrument S1000:Bio-rad company；

LiquiChip 200:Qiagen company；

Gel imaging system: Bio-rad company；

NanoDrop 1000Spectrophotometer。

4th, sequence D etector and specific probe Capture design (Theoretical Design) for every kind of bacterium are detected

(1) specific Pseudomonas all sequences length is downloaded more than 1000bp's from NCBI nucleotide database 16S rRNA gene.

(2) sequence alignment of multiple bacterium and single bacterium is carried out

Multiple Sequence Alignment network address Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/)

(3) select possible conserved sequence, analyze the conserved sequence in 12 kinds of bacterium and variable sequence.According to Variable sequence determines the suitable probe area of 12 kinds of bacterium.

Analyze conservative rate in every kind of Pseudomonas for the selected probe sequence, finally determine probe sequence.

16S rRNA full length gene about the 1500bp of bacterium, the present inventor analyzes through a large amount of, for The 16S rRNA gene complete sequence design of each bacterium of required detection obtains 5 region detection schemes.Often Individual region includes general detection sequence D etector of several bacterium and the specific probe for every kind of bacterium design Capture.The theoretical sequence such as table 2-1 of design, shown in 2-2,2-3,2-4,2-5.

Table 2-1, region 1Detector, helper and Capture sequence

Table 2-2, region 2Detector, helper and Capture sequence

Table 2-3, region 3Detector, helper and Capture sequence

Table 2-4, region 4Detector, helper and Capture sequence

Table 2-5, region 5Detector and Capture sequence

5th, bacterium Total RNAs extraction

The bacterium total serum IgE extraction agent box using sky root extracts total serum IgE, the feasibility of checking probe in detecting.

6th, total rna concentration detection

Use total serum IgE dense to the part bacterial strain extracting for NanoDrop 1000 Spectrophotometer Degree and purity are identified.

Table the 3rd, the quantitative table of part bacterial strain RNA

Sample	Concentration (ng/ul)	A260	A280	260/280
					Enteropathogenic E. Coli O157:H7	48.98	1.225	0.643	1.9
Yersinia enterocolitica	290.85	7.271	3.494	2.08
					Bacillus cereus	314.04	7.851	3.823	2.05
Staphylococcus aureus	214.75	5.369	2.62	2.05

7th, probe sequence, Detector, Helper sequence are designed

The probe sequence of composite part bacterial classification, according to the 16S rRNA full length sequence of each bacterial classification, synthesis inspection Survey the probe sequence (table 4) of each bacterial classification, as hybridization probe after 3 ' Amino End Group modifications, synthesize Detector sequence, As detection sequence (table 5) after 5 ' end biotin modifications.All sequences transfers to the biological (Shanghai) Co., Ltd. of raw work Synthesis.After taking probe, carry out conjugation by the microballoon coupling specification of Bio-rad.

The 4th, table designs probe sequence

Note: in table 4 each probe in table 2-5 design corresponding theory probe sequence is corresponding or base front and rear row Slightly difference on row, table 4 probe is the optimization of corresponding Theoretical Design in table 2-5.

The 5th, table synthesizes Detector, Helper sequence

Title	Sequence (SEQ ID NO :)
		Detector_H	5’-(Biotin)-TTTTTTCGAGCTGACGACARCCATGCASCACCTGTCWC-3’(64)
Helper+oligonucleotides	5’-TTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTC-3’(65)
		Helper-oligonucleotides	5’-CTCACGACACGAGCTGACGACAACCATGCACCACCTGTCAC-3’(66)
Detector oligonucleotide-UD-1082	5’-(Biotin)-TTTTTT GGTTGCGCTCGTTGCGGGACTTAACCCAACAT-3’(67)

Note: in table 5, sequence and the theoretical sequence of design in table 2-5 are substantially in a region.Helper+ Oligonucleotides designs for large intestine, yersinia enterocolitica probe in helper region Helper sequence；Helper-oligonucleotides is for waxy, Liszt, gold Portugal in helper region The Helper sequence of probe design.Detector oligonucleotide-UD-1082 be for Escherichia coli, Yersinia enterocolitica, waxy, Liszt, the general detection sequence of gold Portugal design.

8th, microballoon-probe conjugate experiment

(concentration of microballoon is 1.25 × 10 to vortex microballoon⁷Individual/mL) 5min, 100 μ L microballoons are gone to 1.5ml In centrifuge tube, lucifuge, wash microballoon with 0.1M MES.Centrifugal microballoon 12000rpm, 3min, abandon Clearly, more resuspended with 50 μ L 0.1M MES.A kind of probe reagent of 2 μ L 100umol/L is joined often In one corresponding microballoon (i.e. the microballoon of different coding) suspension.Add 2.5 μ L 10mg/mL EDC immediately Comprise in the mixing tube of probe and microballoon to each, vortex 10 seconds, incubated at room temperature mixed liquor 30 minutes.

For the second time EDC interpolation and hatching (step is ibid).

Hatch twice and add the EDC solution (10mg/mL) that 2.5 μ L 0.1M MES dissolve respectively to probe-micro- In the mixing tube of ball, vortex 10 seconds, hatch 30 minutes, place in room temperature dark place.

Then, with 0.02% polysorbas20 washing microballoon, add 1mL 0.02% polysorbas20 to each coupling In the centrifuge tube of microballoon, vortex 10 seconds, centrifuge 3 minutes with 12000rpm, abandon supernatant.Add 1mL 0.1%SDS Clean, vortex 10 seconds, centrifuge 3 minutes with 12000rpm, abandon supernatant.Eddy diffusion, with 100 μ L 1 × TE Vortex 5 seconds.Lucifuge 4 DEG C preservation.

Operate as described above, the microballoon of each capture probe and respectively different numbering is carried out coupling.

II. embodiment

The specificity experiments of embodiment the 1st, the mixed probe of 5 kinds of probes

1st, the specific detection of 5 kinds of probes

Enteropathogenic E. Coli O157:H7, yersinia enterocolitica, monokaryon in table 4 are synthesized The mixed probe of Monocytogenes, staphylococcus aureus and Bacillus cereus probe and table Detector, Helper sequence in 5.Schematic diagram such as Fig. 3.

The hybridization system utilizing table 6 carries out hybridization reaction.

The 6th, table hybridizes system

Microballoon mixed system (microballoon concentration: 1.25 × 10⁷/ ml): 500 μ L-30 × N=M.

30 expression extension rates；N represents the volume (μ L) of various probe-microspheres solution；M represents 1 × TE's Volume (mol/L).

The preparation of 1 × TE (10mM Tris-HCl, pH=8.0,0.1mM EDTA pH=8.0): 1M Tris-HCl PH=8.0,1mL；0.5M EDTA pH=8.0,0.2mL；

It is settled to 100mL with ultra-pure water.

The processing mode of bacterium solution

(1) take out glycerine bacterial classification from-80 DEG C of refrigerators, cultivate to OD₆₀₀About 0.6.Collect 800 μ L bacterium Liquid is in the new 1.5ml EP pipe polluting without RNase.

(2) centrifuge 12000rpm, after 3min, abandon supernatant, exhaust supernatant, it is to avoid the culture medium pair of residual as far as possible The interference of subsequent experimental.

(3) often pipe bacterium solution precipitation adds the resuspended precipitation of water without RNase for the 600 μ L, centrifugal 12000rpm, 3min After abandon supernatant, exhaust supernatant as far as possible.

(4) often pipe precipitation adds the resuspended mixing of water without RNase for the 100ul, closes the lid, carries out respective markers. Pipe is placed on cursory upper clamping.

(5) open electromagnetic oven, boil by pot dress proper amount of clear water.After water is opened, put clamping the cursory of pipe Enter in pot, boiling water boiling about 5-10min.

(6) pipe is taken out, supernatant is transferred to new without Rnase pollution by centrifugal 12000rpm after 3min In 1.5ml EP pipe, carry out respective markers.Put into-20 DEG C or-80 DEG C of Refrigerator stores.

Hybridization conditions

·50℃10min；

Often pipe adds 1 × S-PE 10 μ L, mixes 50 DEG C and hatches 5min；

Often pipe adds the sterilizing pure water that 120ul is pre-heated to 50 DEG C, mixes, and terminates hybridization；

Upper machine testing.

The testing result of probe specificity such as table 7.

The testing result (fluorescence intensity median MFI) of table the 7th, probe specificity

The result of above-mentioned table 7 is visible:

(1) staphylococcus aureus RNA can be captured signal by gold Portugal probe.By the bacterium solution of enzymatic lysis also Signal can be captured.

(2) bacillus cereus RNA, bacterium solution all can be captured signal by waxy probe.

(3) RNA, the bacterium solution of yersinia enterocolitica can be caught by yersinia genus probe and big intestinal probe Receive signal.

(4) RNA, the bacterium solution of enteropathogenic E. Coli O157:H7 can be by big intestinal probe, small intestine colon Scorching Yersinia ruckeri probe captures signal.

3rd, the impact on hybridization signal for the Helper is tested

1st, the checking of Helper impact

Hybridization system such as table 8-1.

Table 8-1, have Helper hybridization system

Hybridize system without Helper

Hybridization conditions is as follows:

·50℃10min；

Often pipe adds 1 × S-PE 10 μ L, mixes 50 DEG C and hatches 5min；

Often pipe adds the sterilizing pure water that 120ul is pre-heated to 50 DEG C, mixes, and terminates hybridization；

Upper machine testing.

Result such as table 8-2.

Table 8-2, Helper result (Detector UD1082) on hybridization signal impact experiment

Result shows, after removing Helper, and the probe of bacillus cereus and yersinia enterocolitica liquid Signal about decreases half, illustrates that Helper has significant humidification to the capture of hybridization signal.

4th, improvement during Helper is not applied

On the premise of not needing Helper, change the position of Detector, set in the position being originally Helper Meter Detector, changes Detector_H into.Checking Detector, Capture Probe and RNA or bacterium solution direct Step hybridization, if signal detected.Schematic diagram such as Fig. 4.

The 9th, table hybridizes system

Hybridization conditions:50℃10min；Often pipe adds 1 × S-PE 10ul, mixes 50 DEG C and hatches 5min；Often pipe adds 120ul is pre-heated to the sterilizing pure water of 50 DEG C, mixes, and terminates hybridization；Upper machine testing.

When the 10th, table is not added with Helper hybridization, the testing result (Detector uses Detector_H) of part bacterial strain

Find out from the RNA of staphylococcus aureus and bacillus cereus and the testing result of bacterium solution, be not added with In the case of Helper, Detector, Capture Probe and RNA or the direct step hybridization of bacterium solution, can detect To signal.

The specificity experiments of embodiment the 2nd, the mixed probe of 7 kinds of probes

1st, the specific detection of the mixed probe of 7 kinds of probes

The probe sequence of composite part bacterial classification, according to existing bacterial classification 16S rRNA full length sequence, synthesizes following bacterial classification Probe sequence, 5 ' Amino End Group modify after as hybridization probe, synthesize Detector_I-3T sequence, 3 ' end biologies Conduct after element is modified detects sequence.

Through the 16S rDNA full length sequence of Escherichia coli and Shigella each different strains is analyzed, two kinds The 16S rDNA of bacterium is just the same, so Escherichia coli and Shigella share a kind of probe Ecoli_I.

Table the 11st, capture probe and general Detector_I-3T sequence information

Note: the probe of table 11 synthesis is substantially same area with the probe of table 2-1 design, is that table 2-1 is theoretical The optimization of design.Through the checking of previous experiments, helper can be not added with.

1st, probe specificity confirmatory experiment

The 12nd, table hybridizes system

Hybridization conditions:50℃10min；Often pipe adds 1 × S-PE 10ul, mixes 50 DEG C and hatches 5min；Often pipe adds 120ul is pre-heated to the sterilizing pure water of 50 DEG C, mixes, and terminates hybridization；Upper machine testing.

The testing result of table the 13rd, part bacterial strain

Result above shows:

(1) the shared probe Ecoli-I of Escherichia coli and Shigella detects the RNA of Escherichia coli and Shigella During with bacterium solution, signal is all higher, and effect is preferable.

(2), when the probe E.sa-I of Enterobacter sakazakii detects RNA and the bacterium solution of Enterobacter sakazakii, signal all compares Height, effect is preferable.

(3), when the probe Salm-I of salmonella detects RNA and the bacterium solution of salmonella, bacterium solution signal is permissible, Effect is preferable.

(4), when the probe VP-I of vibrio parahaemolytious detects RNA and the bacterium solution of vibrio parahaemolytious, signal is all higher, Effect is more satisfactory.

3rd, the versatility the result to various coli strains for the Escherichia coli probe

The setting of hybridization system and hybridization conditions is the same " 1 ".

Versatility the result such as table 14.

Table 14 coli strain processes bacterium solution checking

From the point of view of the hybridization signal of 14 strain Escherichia coli bacteria liquids, Escherichia coli probe can detect all greatly The signal of intestines bacterium solution.

4th, the versatility the result to various Shigella strains for the Escherichia coli probe

The setting of hybridization system and hybridization conditions is the same " 1 ".

Versatility the result such as table 15.

Table the 15th, Shigella strain processes bacterium solution checking

From the point of view of the hybridization signal of 9 strain Shigella liquid, big intestinal probe can detect all Shigella liquid Signal.

5th, vibrio parahaemolytious, Vibrio vulnificus and yersinia enterocolitica process bacterium solution checking

The setting of hybridization system and hybridization conditions is the same " 1 ".

The result such as table 16.

Table the 16th, vibrio parahaemolytious, Vibrio vulnificus and yersinia enterocolitica process bacterium solution Checking

From the point of view of the hybridization signal of 11 strain vibrio parahaemolytious, 11 strain vibrio parahaemolytious are all by vibrio parahaemolytious Probe in detecting is to signal.But because the homology of vibrio parahaemolytious and Vibrio vulnificus sequence is very high, thus secondary During the detection of hemolysis vibrion bacterium solution, some bacterial strains may detect that the crossbar signal of weak wound probe.

The bacterial strain of 2 strain Vibrio vulnificus can be by wound probe in detecting to signal.

From the point of view of the hybridization signal of Yersinia enterocoltitica liquid, the signal of big intestinal probe is believed than the probe of yersinia genus self Number strong.Two kinds of probes may detect that the signal of yersinia genus bacterium solution.So when detecting unknown sample, only Big intestinal probe is had to have the sample of signal to be probably Escherichia coli or Shigella；If have big intestinal probe and The signal of Ademilson probe, it may be possible to small intestine Yersinia ruckeri.

6th, salmonella processes the checking of bacterium solution versatility

The setting of hybridization system and hybridization conditions is the same " 1 ".

Versatility the result such as table 17.

Table the 17th, salmonella processes bacterium solution checking

From the point of view of the results of hybridization of 13 strain Salmonella bacterium solution, salmonella probe can be with specific detection to sand The signal of door Salmonella liquid.

7th, in the present embodiment, the detection sensitivity of part bacterial classification is tested

Using the probe of table 11, the hybridization system of table 12 and hybridization conditions, as being total to of following groups experiment Same experimental technique.

(1) detection sensitivity of secondary molten probe is verified with vibrio parahaemolytious VP4F13

The RNA concentration of vibrio parahaemolytious VP4F13 is through being determined as 1514ng/ μ L.

By this sample according to gradient dilution, when RNA concentration is 4.73ng/ μ L, signal can be examined. Less than the sample of this concentration, then may can't detect signal.Concrete outcome is shown in Table 18.

Table the 18th, vibrio parahaemolytious detection sensitivity result

(2) detection sensitivity of deformation probe is verified with proteus G1M1205

The RNA concentration of proteus G1M1205 is through being determined as 1116ng/ μ l.

By this sample according to gradient dilution, when RNA concentration is 13.95ng/ μ l, signal can be examined. Less than the sample of this concentration, then may can't detect signal.Concrete outcome is shown in Table 19.

Table the 19th, proteus detection sensitivity result

(3) detection sensitivity of big intestinal probe is verified with Escherichia coli Ecoli35218

The RNA concentration of Escherichia coli Ecoli35218 is through being determined as 1104.2ng/ μ L.

By this sample according to gradient dilution, when RNA concentration is 13.8ng/ μ L, signal can be examined.Low In the sample of this concentration, then may can't detect signal.Concrete outcome is shown in Table 20.

Table the 20th, E. coli detection sensitivity results

(4) detection sensitivity of the rugged probe of slope is verified with Enterobacter sakazakii E.sa296.

The RNA concentration of Enterobacter sakazakii E.sa296 is through being determined as 1273.04ng/ μ L.

By this sample according to gradient dilution, when RNA concentration is 31.83ng/ μ L, signal can be examined. Less than the sample of this concentration, then may can't detect signal.Concrete outcome is shown in Table 21.

Table the 21st, Enterobacter sakazakii detection sensitivity result

(5) detection sensitivity of sramana's probe is verified with salmonella 21484

The RNA concentration of salmonella 21484 is through being determined as 442ng/ μ L.

By this sample according to gradient dilution, when RNA concentration is 22.1ng/ μ L, signal can be examined. Less than the sample of this concentration, then may can't detect signal.Concrete outcome is shown in Table 22.

Table 22 Salmeterol fluticasone propionate sensitivity results

(6) detection sensitivity of wound probe is verified with Vibrio vulnificus VV

The RNA concentration of Vibrio vulnificus VV is through being determined as 438ng/ μ L.

By this sample according to gradient dilution, when RNA concentration is 21.9ng/ μ L, signal can be examined.Low In the sample of this concentration, then may can't detect signal.Concrete outcome is shown in Table 23.

Table the 23rd, Vibrio vulnificus detection sensitivity result

8th, the detection reagent of further good discrimination Escherichia coli and Shigella

In the probe table 11 of design, Escherichia coli probe (sharing with Shigella) Ecoli_I is little in detection During Yersinia enterocolitica bacterium solution, Escherichia coli probe Ecoli_I often ratio enterocolitis yersinia genus The signal of Salmonella probe Yer_I is high.But yersinia enterocolitica probe Yer_I only can detect The signal of yersinia enterocolitica.In order to sufficiently distinguish Escherichia coli and enterocolitis yersinia genus Salmonella, the present inventor devises again a detection probe for yersinia enterocolitica and capture is visited Pin, such as table 24.

The detection detector of table the 24th, yersinia enterocolitica and probe

(1) yersinia enterocolitica probe Yercapture specificity verification experiment

Hybridization system such as table 25.

The hybridization system of table 25 yersinia enterocolitica

Make after working concentration is diluted to for the microballoon of coupling yersinia enterocolitica probe Yercapture With.Take after the microballoon stoste of the 6 good probes of μ l coupling adds 94 μ l TE dilutions and mix, lucifuge.

Hybridization conditions:；50℃10min；Often pipe adds 1 × S-PE 10 μ l, mixes 50 DEG C and hatches 5min；Often pipe adds 120 μ l are pre-heated to the sterilizing pure water of 50 DEG C, mix, and terminate hybridization；Upper machine testing.

Specificity verification result such as table 26.

Table the 26th, yersinia enterocolitica probe Yercapture specificity verification result

Median
		Sample	Yersinia genus probe Yer capture
Yersinia enterocolitica	1742
		Escherichia coli O 157 CICC21530	67.5
Blank	10

(2) yersinia enterocolitica probe Yercapture exclusiveness the result

The bacterial strain and yersinia enterocolitica bacterial strain of various Escherichia coli and Shigella is examined jointly Survey, it was demonstrated that what the probe Yercapture of yersinia enterocolitica can be special distinguishes Escherichia coli, Shigella dysenteriae and yersinia enterocolitica.

According to hybridization system and the experiment condition of table 25, to yersinia enterocolitica probe Yercapture Verify.Result such as table 27.

Table the 27th, yersinia enterocolitica probe Yercapture exclusiveness the result

The strain bacterium and yersinia enterocolitica bacterial strain of various Escherichia coli and Shigella is examined jointly After survey, it was demonstrated that what the probe Yercapture of yersinia enterocolitica can be special distinguishes large intestine bar Bacterium, shigella dysenteriae and yersinia enterocolitica.

So when using the unknown bacterium of this kit detection, using the probe of table 37 and the hybridization system of table 38 When.If the sample signal obtaining is Escherichia coli probe Ecoli_I or Escherichia coli probe Ecoli_I and little The signal of Yersinia enterocolitica probe Yer_I, can individually make such sample enterocolitis again The detection of Yersinia ruckeri probe Yercapture, according to the hybridization system of table 25 yersinia enterocolitica Do one-time authentication with hybridization conditions.If yersinia enterocolitica probe Yercapture has signal, then may be used It is yersinia enterocolitica with sample certainly, if yersinia enterocolitica probe Yercapture There is no signal, then illustrate that this sample is Escherichia coli or Shigella.

Embodiment is the 3rd, for probe checking probe versatility and the detection sensitivity of 4 kinds of bacterium

The probe checking probe versatility of the following 4 kinds of bacterium of synthesis and detection sensitivity.

Table the 28th, 4 kinds of bacterium probes and general Detector

In general Detector Detector_II be Listeria Monocytogenes, Bacillus cereus and The Detector that this 3 kinds of bacterium of staphylococcus aureus share.Detector_H-3 is campylobacter jejuni and enforcement The Listeria Monocytogenes probe in table 4 in example 1, staphylococcus aureus probe and waxy The general Detector of bacillus probe.

So for this 4 kinds of bacterium: Listeria Monocytogenes, Bacillus cereus, golden yellow Portugal Grape coccus and campylobacter jejuni 4 kinds of probes and 2 kinds of Detector:Detector_ II and Detector_H-3, Hybridize simultaneously, reach to detect the purpose of 4 kinds of bacterium.

1st, the specificity verification experiment of 4 kinds of probes

The hybridization system of 4 kinds of bacterium such as table 29.

The hybridization system of table the 29th, 4 kinds of bacterium

Hybridization conditions is as follows:；50℃10min；Often pipe adds 1 × S-PE 10ul, mixes 50 DEG C and hatches 5min；Often Pipe adds the sterilizing pure water that 120ul is pre-heated to 50 DEG C, mixes, and terminates hybridization；Upper machine testing.

4 kinds of bacterium probe specificity the result such as tables 30.

Table the 30th, 4 kinds of bacterium probe specificity the results

1. for the 2 strain staphylococcus aureuses selecting, staphylococcus aureus probe Sa_ I can detect To higher signal value, and do not intersect with other bacterium, illustrate that staphylococcus aureus probe Sa_ I is special Property and exclusiveness effect are preferable.

2. for the 2 strain Listeria Monocytogenes selected, Listeria Monocytogenes Probe List_ I can detect higher signal value, and does not intersect with other bacterium, and instruction sheet nucleus increases The probe List_ I of raw listeria spp is specific and exclusiveness effect is preferable.

3. for Bacillus cereus, Bacillus cereus probe Bacc_ I can detect higher signal Value, and do not intersect with other bacterium, illustrate that Bacillus cereus probe Bacc_ I is specific and exclusiveness is imitated Fruit is preferably.

4. for 3 strain campylobacter jejunis, campylobacter jejuni probe Cj can detect higher signal value, and Do not intersect with other bacterium, illustrate that campylobacter jejuni probe Cj is specific and exclusiveness effect is good.

2nd, versatility confirmatory experiment in probe kind

(1) Bacillus cereus and campylobacter jejuni process bacterium solution the result

Bacillus cereus and campylobacter jejuni process bacterium solution the result such as table 31.

Table the 31st, Bacillus cereus and campylobacter jejuni process bacterium solution the result

1., from the point of view of the hybridization signal of existing 3 strain campylobacter jejunis, campylobacter jejuni probe Cj can detect To higher signal value, and do not intersect with other bacterium, illustrate that campylobacter jejuni probe Cj is specific and exclusive Property effect is preferable.

2. for Bacillus cereus, Bacillus cereus probe Bacc_ I can detect higher signal Value, and do not intersect with other bacterium, illustrate that Bacillus cereus probe Bacc_ I is specific and exclusiveness is imitated Fruit is preferably.

(2) staphylococcus aureus processes bacterium solution the result

It is as shown in table 32 that staphylococcus aureus processes bacterium solution the result.

Table the 32nd, staphylococcus aureus processes bacterium solution the result

From the point of view of the hybridization signal of existing 11 strain staphylococcus aureuses, staphylococcus aureus probe Sa- I can detect higher signal value, and does not intersect with other bacterium, and staphylococcus aureus probe is described Sa-I is specific and exclusiveness effect is preferable.

(3) Listeria Monocytogenes processes bacterium solution checking

Listeria Monocytogenes processes bacterium solution checking as shown in table 33.

Table the 33rd, Listeria Monocytogenes processes bacterium solution checking

From the point of view of the hybridization signal of existing 10 strain Listeria Monocytogenes, monocyte hyperplasia The probe List-I of listeria spp can detect higher signal value, and does not intersect with other bacterium, says The probe List-I of bright Listeria Monocytogenes is specific and exclusiveness effect is preferable.

3rd, the probe in detecting sensitivity experiment of part bacterial classification

(1) campylobacter jejuni detection sensitivity experiment

Verify the detection sensitivity of campylobacter jejuni probe Cj with campylobacter jejuni Bb.

The RNA concentration of campylobacter jejuni Bb is through being determined as 617ng/ μ L.

By this sample according to gradient dilution, when RNA concentration is 15.43ng/ μ L, signal can be examined. Less than the sample of this concentration, then may can't detect signal.Concrete outcome asks for an interview table 3 below 4.

Table the 34th, campylobacter jejuni detection sensitivity result

(2) bacillus cereus detection sensitivity experiment

Verify the detection sensitivity of bacillus cereus probe Bacc-I with bacillus cereus Bacc.

The RNA concentration of bacillus cereus Bacc is through being determined as 516ng/ μ l.

By this sample according to gradient dilution, when RNA concentration is 25.8ng/ μ l, signal can be examined.Low In the sample of this concentration, then may can't detect signal.Concrete outcome is shown in Table 35.

Table the 35th, bacillus cereus detection sensitivity result

(3) staphylococcus aureus detection sensitivity experiment

Verify the detection sensitivity of staphylococcus aureus probe Sa-I with staphylococcus aureus SA29213.

The RNA concentration of staphylococcus aureus SA29213 is through being determined as 1600ng/ μ L.

By this sample according to gradient dilution, when RNA concentration is 80ng/ μ L, signal can be examined.Low In the sample of this concentration, then may can't detect signal.Concrete outcome is shown in Table 36.

Table the 36th, staphylococcus aureus detection sensitivity result

4th, for embodiment, the 1st, the 2nd, the probe in 3 and several general detector hybridize embodiment simultaneously

Probe and general dector such as table 37.

Table the 37th, probe and general detector

Above-mentioned 11 kinds of probes and 3 kinds of Detector are hybridized, the unknown bacterium of detection simultaneously, detection stream can be simplified Journey, saves the preparation respectively of single hybridization system.

The all probes of mixing and Detector is checked to detect the effect of object bacteria such as according to following experimental program What.11 kinds of probe bulk crossing system such as tables 38.

Table the 38th, 11 kinds of probe bulk crossing systems

Beads mixture represents the microballoon mixed liquor of coupling correspondent probe.

Microballoon mixed system (microballoon concentration: 1.25 × 10⁷/ ml): 500 μ L-30 × N=M.

30 expression extension rates；N represents the volume (μ L) of various probe-microspheres solution；M represents 1 × TE's Volume (mol/L).

The preparation of 1 × TE (10mM Tris-HCl, pH=8.0,0.1mM EDTA pH=8.0): 1M Tris-HCl PH=8.0,1mL；0.5M EDTA pH=8.0,0.2mL；It is settled to 100mL with ultra-pure water.

Hybridization conditions:50℃10min；Often pipe adds 1 × S-PE 10 μ l, mixes 50 DEG C and hatches 5min；Often pipe adds 120 μ l are pre-heated to the sterilizing pure water of 50 DEG C, mix, and terminate hybridization；Upper machine testing.

11 kinds of mixed probes are specific and exclusiveness testing result such as table 39.

Table the 39th, 11 kinds of mixed probes are specific and exclusiveness testing result

From mixing all can probe from the point of view of, 12 kinds of object bacteria to be checked are in the situation of all probes of mixing Under, without interference with the detection of object bacteria.And probe specificity used is fine, will not detect that other are miscellaneous Bacterium.

The all documents mentioned in the present invention are incorporated as reference all in this application, just as each literary composition Offer and be individually recited as with reference to like that.In addition, it is to be understood that reading the above-mentioned instruction content of the present invention Afterwards, the present invention can be made various changes or modifications by those skilled in the art, and these equivalent form of values are same Fall within the application appended claims limited range.

Claims (24)

1. a fast-bacteria-detection method, it is characterised in that described method includes:

(1) using the 16S rRNA of 2-20 kind bacterium or its pcr amplification product as measuring target, design Capture probe and detection probe；Described capture probe is the 16S rRNA or its PCR for every kind of bacterium The specific probe of amplified production, and it is attached respectively on 2-20 kind microballoon；Described detection probe is The probe complementary with consensus sequence on the 16S rRNA of each bacterium or its pcr amplification product, and it includes One detectable；Described 2-20 kind microballoon carries different microballoon identification signals respectively；

(2) 2-20 kind microballoon, the detection probe of specific capture probe will be attached with and crack through cell The testing sample mixing processing；So that the 16S rRNA or its PCR of the specific bacteria in testing sample Amplified production, capture probe corresponding with this specific bacteria and microballoon, and detection probe formed " microballoon- The 16S rRNA of capture probe-specific bacteria or its pcr amplification product-detection probe " compound；

(3) microballoon identification signal and the detectable of different microballoons in the compound that (2) are formed are detected, So that it is determined that the amount of the presence or absence of each tested bacteria and existence in testing sample.

2. the method for claim 1, it is characterised in that in step (2), described testing sample Only carry out cell cracking and process the step that can include or not include PCR amplification or cell proliferation.

3. the method for claim 1, it is characterised in that in step (1), also include: for respectively Consensus sequence on bacterial 16 S rRNA or its pcr amplification product, Design assistant sequence, described auxiliary Sequence, for stablizing 16S rRNA or its pcr amplification product of strand, strengthens hybridization check signal.

4. the method for claim 1, it is characterised in that 3 ' end connections of detection probe can detect Label；It is preferred that 3 ' ends of detection probe add biotin modification after connecting 6 T；Or capture probe exists 5 ' ends carry out amido modified.

5. the method for claim 1, it is characterised in that described bacterium is pathogenic bacteria.

6. method as claimed in claim 5, it is characterised in that include as the pathogenic bacteria measuring target: Escherichia coli (Escherichia coli), yersinia enterocolitica (Yersinia enterocolitica), gold Staphylococcus aureus (Staphylococcus aureus), bacillus cereus (Bacillus cereus), monokaryon is thin Born of the same parents' monocytogenes (Listeria monocytogenes), Shigella (Shigella), salmonella (Salmonella), Enterobacter sakazakii (Enterobacter sakazakii), proteus (Proteus vulgaris), Vibrio parahaemolytious (Vibrio parahaemolyticus), campylobacter jejuni (Campylobacter jejuni), wound 2 kinds of vibrios (Vibrio vulnificus) or two or more.

7. method as claimed in claim 6, it is characterised in that include as the pathogenic bacteria measuring target: Escherichia coli, Shigella, salmonella, Enterobacter sakazakii, yersinia enterocolitica, change Shape bacillus, vibrio parahaemolytious, Listeria Monocytogenes, staphylococcus aureus, jejunum are curved Aspergillus, Bacillus cereus, Vibrio vulnificus, the sequence for the capture probe of each pathogenic bacteria is SEQ successively ID NO:45～SEQ ID NO:56, or its complementary series；The sequence of detection probe is SEQ ID NO:57, Or its complementary series；It is preferred that also include auxiliary sequencel SEQ ID NO:58, or its complementary series.

8. method as claimed in claim 6, it is characterised in that include as the pathogenic bacteria measuring target: Escherichia coli, yersinia enterocolitica, staphylococcus aureus, bacillus cereus, monokaryon Monocytogenes, the sequence for the capture probe of each pathogenic bacteria is SEQ ID NO successively: 59～SEQ ID NO:63, or its complementary series；Detection probe sequence be SEQ ID NO:64 and/or SEQ ID NO:67, or its complementary series；It is preferred that also include auxiliary sequencel SEQ ID NO:65 and SEQ ID NO:66, or its complementary series.

9. method as claimed in claim 6, it is characterised in that include as the pathogenic bacteria measuring target: Escherichia coli, Shigella, salmonella, Enterobacter sakazakii, yersinia enterocolitica, change Shape bacillus, vibrio parahaemolytious, Vibrio vulnificus, the sequence for the capture probe of each pathogenic bacteria is SEQ successively ID NO:1～SEQ ID NO:8, or its complementary series；The sequence of detection probe is SEQ ID NO:9, Or its complementary series；It is preferred that also include auxiliary sequencel SEQ ID NO:10, or its complementary series.

10. method as claimed in claim 6, it is characterised in that include as the pathogenic bacteria measuring target: Enterobacter sakazakii, Escherichia coli and Shigella, yersinia enterocolitica, salmonella, change Shape bacillus, vibrio parahaemolytious, Vibrio vulnificus, the sequence for the capture probe of each pathogenic bacteria is SEQ successively ID NO:68～SEQ ID NO:74, or its complementary series；Wherein Escherichia coli share with Shigella and catch Obtain probe SEQ ID NO:69, or its complementary series；The sequence of detection probe is SEQ ID NO:75, Or its complementary series.

11. methods as claimed in claim 6, it is characterised in that include as the pathogenic bacteria measuring target: Escherichia coli, Shigella, salmonella, Enterobacter sakazakii, yersinia enterocolitica, change Shape bacillus, vibrio parahaemolytious, Vibrio vulnificus, the sequence for the capture probe of each pathogenic bacteria is SEQ successively ID NO:11～SEQ ID NO:18, or its complementary series；The sequence of detection probe is SEQ ID NO: 19, or its complementary series；It is preferred that also include auxiliary sequencel SEQ ID NO:20, or its complementary series.

12. methods as claimed in claim 6, it is characterised in that include as the pathogenic bacteria measuring target: Yersinia enterocolitica, proteus, vibrio parahaemolytious, Listeria Monocytogenes, Staphylococcus aureus, campylobacter jejuni, Bacillus cereus, Vibrio vulnificus, for each pathogenic bacteria The sequence of capture probe is SEQ ID NO:21～SEQ ID NO:28 successively, or its complementary series；Inspection The sequence of probing pin is SEQ ID NO:29, or its complementary series；It is preferred that also include auxiliary sequencel SEQ ID NO:30, or its complementary series.

13. methods as claimed in claim 6, it is characterised in that include as the pathogenic bacteria measuring target: Escherichia coli, Shigella, salmonella, Enterobacter sakazakii, yersinia enterocolitica, change Shape bacillus, vibrio parahaemolytious, Listeria Monocytogenes, Vibrio vulnificus, Staphylococcus aureus Bacterium, campylobacter jejuni, Bacillus cereus, the sequence for the capture probe of each pathogenic bacteria is SEQ successively ID NO:31～SEQ ID NO:42, or its complementary series；The sequence of detection probe is SEQ ID NO: 43, or its complementary series；It is preferred that also include auxiliary sequencel SEQ ID NO:44, or its complementary series.

14. methods as claimed in claim 6, it is characterised in that include as the pathogenic bacteria measuring target: Listeria Monocytogenes, Bacillus cereus, staphylococcus aureus, campylobacter jejuni, Sequence for the capture probe of each pathogenic bacteria is SEQ ID NO:78～SEQ ID NO:81 successively, or Its complementary series；The sequence of detection probe is SEQ ID NO:82～SEQ ID NO:83, or it is complementary Sequence.

15. methods as described in claim 6-14 is arbitrary, it is characterised in that when as mensuration target When pathogenic bacteria include Escherichia coli and/or Shigella and yersinia enterocolitica simultaneously, also include: Use the capture probe of SEQ ID NO:77, or its complementary series；Detection with SEQ ID NO:76 is visited Pin, or its complementary series；Detection yersinia enterocolitica, thus by Yersinia enterocolitica Bacterium distinguishes with Escherichia coli and/or Shigella.

16. for detecting the kit of pathogenic bacteria, it is characterised in that described pathogenic bacteria include: large intestine bar Bacterium, Shigella, salmonella, Enterobacter sakazakii, yersinia enterocolitica, proteus, Vibrio parahaemolytious, Listeria Monocytogenes, staphylococcus aureus, campylobacter jejuni, wax Sample bacillus, Vibrio vulnificus；

Described kit includes: for the capture probe SEQ ID NO:45～SEQ ID of each pathogenic bacteria NO:56, or its complementary series；Detection probe SEQ ID NO:57, or its complementary series；It is preferred that Also include auxiliary sequencel SEQ ID NO:58, or its complementary series.

17. for detecting the kit of pathogenic bacteria, it is characterised in that described pathogenic bacteria include: large intestine bar Bacterium, yersinia enterocolitica, staphylococcus aureus, bacillus cereus, monocyte increase Raw listeria spp；

Described kit includes: for the capture probe SEQ ID NO:59～SEQ ID of each pathogenic bacteria NO:63, or its complementary series；Detection probe SEQ ID NO:64 and/or SEQ ID NO:67, or its Complementary series；It is preferred that also include auxiliary sequencel SEQ ID NO:65 and SEQ ID NO:66, or its Complementary series.

18. for detecting the kit of pathogenic bacteria, it is characterised in that described pathogenic bacteria include: large intestine bar Bacterium, Shigella, salmonella, Enterobacter sakazakii, yersinia enterocolitica, proteus, Vibrio parahaemolytious, Vibrio vulnificus；

Described kit includes: for the capture probe SEQ ID NO:1～SEQ ID of each pathogenic bacteria NO:8, or its complementary series；Detection probe SEQ ID NO:9, or its complementary series；It is preferred that also Including auxiliary sequencel SEQ ID NO:10, or its complementary series.

19. for detecting the kit of pathogenic bacteria, it is characterised in that described pathogenic bacteria include: the rugged intestines of slope Bacillus, Escherichia coli and Shigella, yersinia enterocolitica, salmonella, proteus, Vibrio parahaemolytious, Vibrio vulnificus；

Described kit includes: for the capture probe SEQ ID NO:68～SEQ ID of each pathogenic bacteria NO:74, or its complementary series；Detection probe SEQ ID NO:75, or its complementary series.

20. for detecting the kit of pathogenic bacteria, it is characterised in that described pathogenic bacteria include: large intestine bar Bacterium, Shigella, salmonella, Enterobacter sakazakii, yersinia enterocolitica, proteus, Vibrio parahaemolytious, Vibrio vulnificus；

Described kit includes: for the capture probe SEQ ID NO:11～SEQ ID of each pathogenic bacteria NO:18, or its complementary series；Detection probe SEQ ID NO:19, or its complementary series；It is preferred that Also include auxiliary sequencel SEQ ID NO:20, or its complementary series.

21. for detecting the kit of pathogenic bacteria, it is characterised in that described pathogenic bacteria include: small intestine is tied Enteritis Yersinia ruckeri, proteus, vibrio parahaemolytious, Listeria Monocytogenes, golden yellow Look staphylococcus, campylobacter jejuni, Bacillus cereus, Vibrio vulnificus；

Described kit includes: for the capture probe SEQ ID NO:21～SEQ ID of each pathogenic bacteria NO:28, or its complementary series；Detection probe SEQ ID NO:29, or its complementary series；It is preferred that Also include auxiliary sequencel SEQ ID NO:30, or its complementary series.

22. for detecting the kit of pathogenic bacteria, it is characterised in that described pathogenic bacteria include: large intestine bar Bacterium, Shigella, salmonella, Enterobacter sakazakii, yersinia enterocolitica, proteus, Vibrio parahaemolytious, Listeria Monocytogenes, Vibrio vulnificus, staphylococcus aureus, jejunum Campylobacter spp, Bacillus cereus；

Described kit includes: for the capture probe SEQ ID NO:31～SEQ ID of each pathogenic bacteria NO:42, or its complementary series；The sequence of detection probe is SEQ ID NO:43, or its complementary series； It is preferred that also include auxiliary sequencel SEQ ID NO:44, or its complementary series.

23. for detecting the kit of pathogenic bacteria, it is characterised in that described pathogenic bacteria include: monokaryon is thin Born of the same parents' monocytogenes, Bacillus cereus, staphylococcus aureus, campylobacter jejuni；

Described kit includes: for the capture probe SEQ ID NO:78～SEQ ID of each pathogenic bacteria NO:81, or its complementary series；Detection probe SEQ ID NO:82, SEQ ID NO:83, or it is mutual Complementary series.

24. kits as described in claim 16-23 is arbitrary, it is characterised in that in described kit also Including: the capture probe of SEQ ID NO:77, or its complementary series；The detection of SEQ ID NO:76 is visited Pin, or its complementary series；For by yersinia enterocolitica and Escherichia coli and/or Shigella Distinguish.