CN102286612A - Fast detection reagent kit for pathogenic microorganism - Google Patents
Fast detection reagent kit for pathogenic microorganism Download PDFInfo
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- CN102286612A CN102286612A CN2010102028380A CN201010202838A CN102286612A CN 102286612 A CN102286612 A CN 102286612A CN 2010102028380 A CN2010102028380 A CN 2010102028380A CN 201010202838 A CN201010202838 A CN 201010202838A CN 102286612 A CN102286612 A CN 102286612A
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Abstract
The invention discloses a fast detection method for pathogenic microorganism and a fast detection reagent kit for microorganism. The method and reagent disclosed by the invention can be used for detecting various kinds of pathogenic microorganism in a short time and have the characteristics that the operation is simple, convenient and fast, the sensitivity is high, the specificity is strong, the cost is low, the required quantity of samples is small, high flux can be realized, the method and the reagent kit are applicable to in-site screening, and the like. The method and the reagent kit can be used for the fast detection of food-borne poisoning and food safety, and the defects of the current fast microorganism detection technology and equipment are overcome.
Description
Technical field
The invention belongs to biological technical field; More specifically, the present invention relates to a kind of pathogenic microorganism quick detection kit.
Background technology
In recent years, great food safety incident takes place frequently, and causes people's great attention.To fundamentally solve food-safety problem, just must be to each link enforcement whole process control and monitoring such as production, processing, circulation and sale of food, this just needs can satisfy in a large number quick, convenient, accurate, the sensitive Food Safety Analysis detection technique of this requirement.
Along with science and technology development, the effect of food safety quick analytic detection method aspect inspection for food hygiene is more and more important.From long term growth, subject development such as immunology, molecular biology, computer technology and automatization have greatly promoted the Food Safety Analysis detection method and have developed to sensitiveer direction more easily, set up food safety quick analytic detection method efficiently, the quality control in foodstuff production, transportation, the sales process is had crucial meaning; In a single day the poisoning of food-borne pathogenic microorganism taking place, also need finish detection to formulate scientific and reasonable treatment plan in the shortest time, wins treatment time simultaneously.Applying of these real-time analysis detection techniques, be not only one of traditional Food Safety Analysis detection technique is improved and improves, also make food quality safety that further assurance has been arranged, thereby promote foodstuffs industry healthy more, advance apace, constantly satisfy the needs that the people improve health conditions.
More existing food-borne pathogens Fast Detection Technique are to utilize immunological method mostly at present, this method instrumentation is simple, detection speed is fast, sensitivity is higher, specificity is better and the sample aequum is few, is difficult to preparation, uses and manyly anti-ly easily to produce cross reactions and false positive occurs and once can only detect shortcoming and defect such as one or more pathogenic bacterium but also exist as the pathogenic bacterium monoclonal antibody.Therefore, also need to develop at present the new technology that can detect pathogenic bacterium apace, efficiently.
Summary of the invention
The object of the present invention is to provide a kind of pathogenic microorganism method for quick and test kit.
In a first aspect of the present invention, a kind of method for rapid inspecting animalcule is provided, described method comprises:
(1), determines the specific target gene respectively at 1-50 kind to be detected (preferably 2-30 kind, as 10 kinds, 15 kinds, 20 kinds) microorganism;
(2) each target gene of determining according to step (1), determine its specific pair of inside primer (primer (Fi) and reverse inboard primer (Ri) just to the inside) and pair of outside primer (primer (Fo) and reverse outside primer (Ro) just laterally) respectively, the sequence of described inboard primer can be complementary with corresponding target gene part, again can complementary with corresponding super primer part (promptly the super primer part of primer and forward be complementary just to the inside, reverse inboard primer and the partly complementation of reverse super primer); Described super primer is that a primer is right, and the super primer sequence of forward is shown in SEQ ID NO:1, and reverse super primer sequence is shown in SEQ ID NO:2; Mix above-mentioned primer, obtain primer mixture;
(3) with the testing sample being pcr template, is that primer carries out the PCR reaction with the primer mixture of step (2), obtains the pcr amplification system;
(4) add the probe have detectable signal in the pcr amplification system of step (3), described probe can be specifically and corresponding target gene complementation, and at different target gene detectable signal differences;
(5) identify detectable signal, thereby determine the kind of microorganism.
In a preference, described microorganism is pathogenic bacterium (not comprising virus).
In another preference, 5 ' end of described reverse super primer has a discernible signal; It preferably is biotin labeling.
In another preference, in the step (3), the amount of the super primer of forward (bar number) be each target gene correspondence primer 3-10 doubly; It preferably is 4-6 times; More preferably be 5 times, the amount of reverse super primer (bar number) be each target gene correspondence primer 10-50 doubly; It preferably is 10-30 times; More preferably be 20 times.
In another preference, the amount of reverse super primer is 4 times of the super primer of forward.
In another preference, the primer and the probe of described microorganism target gene and correspondence thereof are as follows:
Listeria monocytogenes (Listeria monocytogenes) is induced Actin muscle assembling precursor protein gene, and primer is shown in SEQ ID NO:8-11, and probe is shown in SEQ ID NO:12;
Shigellae (Shigella) aggressive plasmid H antigen gene, primer is shown in SEQ ID NO:13-16, and probe is shown in SEQ ID NO:17;
Salmonellas (Salmonella) invasin protein A gene, primer is shown in SEQ ID NO:18-21, and probe is shown in SEQ ID NO:22;
Bacillus proteus (Bacillus proleus) F type ATP enzyme beta subunit gene, primer is shown in SEQ ID NO:23-26, and probe is shown in SEQ ID NO:27;
Bacillus cereus (Bacillus cereus) enterotoxin T gene, primer is shown in SEQ ID NO:33-36, and probe is shown in SEQ ID NO:37;
Intestinal bacteria ETEC (Escherichia coli ETEC) heat-labile toxin AB subunit gene, primer is shown in SEQ ID NO:43-46, and probe is shown in SEQ ID NO:47;
Streptococcus aureus (Staphylococcus aureus) thermostability nuclease gene, primer is shown in SEQ ID NO:53-56, and probe is shown in SEQ ID NO:57;
Campylobacter jejuni (Campylobacter jejuni) gyrase A subunit gene, primer is shown in SEQ IDNO:63-66, and probe is shown in SEQ ID NO:67;
Escherichia coli O 157 (Escherichia coli O157) O-antigen-specific gene, primer is shown in SEQ IDNO:73-76, and probe is shown in SEQ ID NO:77;
Campylobacter coli (Campylobacter.Coli) siderophore transfer related protein gene, primer is shown in SEQ ID NO:83-86, and probe is shown in SEQ ID NO:87;
Yersinia enterocolitica (Yersinia enterocolitica) is sticked the invasin protein gene, and primer is shown in SEQ ID NO:88-91, and probe is shown in SEQ ID NO:92;
Vibrio cholerae (Vibrio cholerae) hemolysin protein A gene, primer is shown in SEQ ID NO:93-96, and probe is shown in SEQ ID NO:97; Or
The heat-resisting hemolytic toxin gene of Vibrio parahemolyticus (Vibrio parahaemolyticu), primer is shown in SEQID NO:98-101, and probe is shown in SEQ ID NO:102.
In another preference, when detecting multiple microorganism simultaneously, be used for the PCR reaction after mixing at the primer of the target gene of multiple microorganism.
In another preference, in the step (3), the PCR reaction process is as follows:
(a) 90-99 ℃ is carried out 15 ± 2min;
(b) 90-99 ℃ is carried out 30 ± 3sec → 50-65 ℃ and carries out 2 ± 0.5min → 72 and ℃ carry out 60 ± 5sec; 10-30 circulation (preferably 12-20 circulation) altogether;
(c) 90-99 ℃ is carried out 30 ± 3sec → 65-72 ℃ and carries out 90 ± 5sec; 3-20 circulation (preferably 5-8 circulation) altogether;
(d) 90-99 ℃ is carried out 20 ± 2sec → 50-65 ℃ and carries out 20 ± 2sec → 72 and ℃ carry out 30sec; 25-45 circulation (preferable 30-40 circulation) altogether;
(e)72℃，3±0.5min。
In another preference, in the step (4), described detectable signal is a fluorescent microsphere; It preferably is nanometer fluorescent microspheres.
In another aspect of this invention, provide a kind of microbial rapid detection reagent combination, described agent combination comprises:
Super primer, described super primer is that a primer is right, and the super primer sequence of forward is shown in SEQ ID NO:1, and reverse super primer sequence is shown in SEQ ID NO:2;
Primer at 1-50 kind microorganism target gene to be detected, primer corresponding to arbitrary target gene comprises pair of inside primer and pair of outside primer, the sequence of described inboard primer can be complementary with corresponding target gene part, again can complementary with corresponding super primer part (promptly the super primer part of primer and forward be complementary just to the inside, promptly reverse inboard primer and the partly complementation of reverse super primer); And
At the probe of 1-50 kind microorganism target gene to be detected, described probe is can be specifically complementary with corresponding target gene and have detectable signal, and at different target gene detectable signal differences.
In a preference, be selected from the primer and the probe of microorganism target gene to be detected:
Induce Actin muscle to assemble the primer and the probe of precursor protein gene corresponding to Listeria monocytogenes, wherein primer is shown in SEQ ID NO:8-11, and probe is shown in SEQ ID NO:12;
Corresponding to the primer and the probe of Shigellae aggressive plasmid H antigen gene, wherein primer is shown in SEQ IDNO:13-16, and probe is shown in SEQ ID NO:17;
Corresponding to the primer and the probe of Salmonellas invasin protein A gene, wherein primer is shown in SEQ ID NO:18-21, and probe is shown in SEQ ID NO:22;
Corresponding to the primer and the probe of Bacillus proteus F type ATP enzyme beta subunit gene, wherein primer is shown in SEQ IDNO:23-26, and probe is shown in SEQ ID NO:27;
Corresponding to the primer and the probe of bacillus cereus enterotoxin T gene, wherein primer is shown in SEQ ID NO:33-36, and probe is shown in SEQ ID NO:37;
Corresponding to the primer and the probe of intestinal bacteria ETEC heat-labile toxin AB subunit gene, wherein primer is shown in SEQ ID NO:43-46, and probe is shown in SEQ ID NO:47;
Corresponding to the primer and the probe of streptococcus aureus thermostability nuclease gene, wherein primer is shown in SEQ ID NO:53-56, and probe is shown in SEQ ID NO:57;
Corresponding to the primer and the probe of campylobacter jejuni gyrase A subunit gene, wherein primer is shown in SEQ IDNO:63-66, and probe is shown in SEQ ID NO:67;
Corresponding to the primer and the probe of Escherichia coli O 157 O-antigen-specific gene, wherein primer is shown in SEQID NO:73-76, and probe is shown in SEQ ID NO:77;
Corresponding to the primer and the probe of campylobacter coli siderophore transfer related protein gene, wherein primer is shown in SEQ ID NO:83-86, and probe is shown in SEQ ID NO:87;
Stick the primer and the probe of invasin protein gene corresponding to Yersinia enterocolitica, wherein primer is shown in SEQ ID NO:88-91, and probe is shown in SEQ ID NO:92;
Corresponding to the primer and the probe of vibrio cholerae hemolysin protein A gene, wherein primer is shown in SEQ ID NO:93-96, and probe is shown in SEQ ID NO:97; Or
Corresponding to the primer and the probe of the heat-resisting hemolytic toxin gene of Vibrio parahemolyticus, wherein primer is shown in SEQ IDNO:98-101, and probe is shown in SEQ ID NO:102.
In another aspect of this invention, provide the purposes of described agent combination, be used to prepare the test kit that detects microorganism.
In another aspect of this invention, provide a kind of microbial rapid detection reagent box, contain
Container; And
Be sub-packed in the described agent combination in the container.
In a preference, also contain fluorescent microsphere, colouring reagents, archaeal dna polymerase, thinner, working instructions etc. in the described test kit.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1, multiplex PCR nested primers amplification procedure synoptic diagram.
Fig. 2, food-borne pathogens multiplex PCR testing process synoptic diagram.
Embodiment
In order to overcome the defective that present food security analysis and detection technology and equipment exist, the inventor is through deep research, develop a kind of novel pathogenic bacterium method for quick and supporting reagent or test kit first, described method and reagent can detect various pathogens in a short period of time, but have quick, highly sensitive, high specificity easy and simple to handle, with low cost, the few high-throughput of sample aequum, be fit to characteristics such as field screening.
Microorganism and target gene
" microorganism " of the present invention can be the various microorganisms that are present in the food, preferably is meant for human body or the deleterious bacterium of animal body, i.e. pathogenic bacterium.Preferably, described microorganism does not comprise virus.
" target gene (or being called goal gene) " of the present invention is meant the gene that is present in corresponding microorganism to be measured specifically, also be that described target gene is that its corresponding Institute of Micro-biology is distinctive, the existence of this microorganism is specifically pointed in the existence of a certain target gene.
Thereby the target gene that in detecting mixed system, exists determine microorganism have situation the time, the selection of target gene is an outbalance, consider not cause non-specific binding (can reduce the accuracy rate of detection).The inventor is through repetition test, at the microorganism that needs detect, found representative and is unlikely the target gene that causes non-specific binding.As optimal way of the present invention, the target gene of described pathogenic bacterium and correspondence thereof is as follows:
The pathogenic bacterium target gene
Listeria monocytogenes (Listeria monocytogenes) is induced Actin muscle assembling precursor protein gene (actA);
Shigellae (Shigella) aggressive plasmid H antigen gene (ipaH);
Salmonellas (Salmonella) invasin protein A gene (invA);
Bacillus proteus (Bacillus proleus) F type ATP enzyme beta subunit gene (atpD);
Bacillus cereus (Bacillus cereus) enterotoxin T gene (bceT);
Intestinal bacteria ETEC (Escherichia coli ETEC) heat-labile toxin AB subunit gene (hle AB);
Streptococcus aureus (Staphylococcus aureus) thermostability nuclease gene (nuc);
Campylobacter jejuni (Campylobacter jejuni) gyrase A subunit (gyrA);
Escherichia coli O 157 (Escherichia coli O157) O-antigen-specific (rfbE);
Campylobacter coli (Campylobacter.Coli) siderophore transfer related protein (ceuE);
(Yersinia sticks invasin protein (ail) to Yersinia enterocolitica;
enterocolitica)
Vibrio cholerae (Vibrio cholerae) hemolysin albumin A (hlyA);
Vibrio parahemolyticus (Vibrio parahaemolyticu) heat-resisting hemolytic toxin (tlh).
Preferably, described target gene can be selected from above one or more.Specifically select according to the kind of required detected microorganism.
Detection reagent and test kit
At the target gene of required detected microorganism, design suitable amplimer and probe.The method of design of primer and probe can be with reference to existing technology.Yet owing to need detect the target gene of multiple microorganism simultaneously, PCR reaction system complexity when therefore designing primer, also needs to consider to reduce the generation probability of non-specific binding and good expanding effect.
In the present invention, in same PCR reaction system, add primer more than two pairs, can amplify a plurality of nucleic acid fragments simultaneously, thereby measure a plurality of target genes efficiently.At the target gene of the food-borne pathogens of intend measuring, be each target spot design two pairs of nido multiplex PCR Auele Specific Primer (being inboard primers F i/Ri and outside primers F o/Ro), the target gene (goal gene) of the specific bacteria that is used to increase is with reference to Fig. 1.In the PCR detection architecture, the addition of Auele Specific Primer is extremely low, only is used for the target spot that increases in incipient several cycles, and this primer has sequence label (can be complementary with super primer part), can be discerned by super primer.Described part complementation is meant 20-100% just to the inside between the primer and the super primer of forward (30-80% preferably, as 40%, 50%, 60%) sequence be complementary, it is complementary that the sequence of 20-100% (preferably 30-80%, as 40%, 50%, 60%) is arranged between reverse inboard primer and the reverse super primer.
The sequence label that carries at Auele Specific Primer designs super primer (super primer of forward and reverse super primer).In reaction system, add the super primer enough satisfy exponential amplification, this primer can be after Auele Specific Primer begins to increase several cycles the quick identification sequence label, purpose fragment in batches further increases.The super primer that uses for every kind of pathogenic bacterium is all identical on sequence, thereby significantly reduces the background interference of detected result, improves detection sensitivity.Because super primer is with regard to pair of sequences, therefore as can be known: when detecting multiple target gene, each of a plurality of target gene correspondences just to the inside primer one section sequence is arranged is identical.
By repetition test, the inventor has found the super primer of ideal, at the primer and the probe of each target gene, as listed in the table 2.Described super primer, target gene primer and probe have been formed the agent combination that detects microorganism.
Preferably, described reverse super primer also carries a discernible signal, is used for relative quantification ground and determines quantitative relation between the contained multiple microorganism of sample.More preferably, described discernible signal is a biotin labeling.More preferably, described discernible signal is positioned at 5 ' end of reverse super primer.
The present invention also provides the test kit that detects microorganism, contains container; And be sub-packed in described agent combination in the container.Preferably, use, also contain the reagent of operations such as being useful on microorganism culturing, DNA extracting, pcr amplification or signal detection in the described test kit for the ease of those skilled in the art; Fluorescent microsphere for example, colouring reagents, archaeal dna polymerase, thinner, substratum etc.In addition, also contain working instructions etc. in the described test kit.
Detection method
The invention provides at same sample system and detect or identify multiple method of microorganism quickly and efficiently.
Among the present invention, unless otherwise indicated, described " sample " is generalized, it can be anyly need therefrom identify material or the system that whether has microorganism, as being but be not limited to various liquid or solid-state food, medicine, body fluid, tissue, this sample can also be secretory product or the movement of Mammals (comprising the people), as vomitus, ight soil, saliva, urine etc.
Whether method of the present invention is based on existing a certain target gene to identify in the microorganism to be detected.Therefore, testing sample need carry out suitable pre-treatment so that cell discharges target gene, thereby makes target gene to be identified.The method that makes cell discharge target gene is well-known to those skilled in the art, can adopt the method for lysing cell routinely.As a kind of optimal way of the present invention, can handle sample by boiling water bath, make microorganism discharge the nucleic acid of target gene.Usually, at Gram-negative bacteria, can crack cell with the method for boiling; And to gram-positive microorganism, then also need to add granulated glass sphere, and thermal agitation just can crack cell.
Detection method of the present invention can detect multiple microorganism simultaneously, can carry out the fractionation and the combination of different microorganisms according to demand.Generally be the 1-50 kind, 2-30 kind preferably is as 10 kinds, 15 kinds, 20 kinds.Preferably, detect one or more microorganisms that are selected from Escherichia coli O 157, campylobacter coli, intestinal bacteria ETEC, Salmonellas, Listeria monocytogenes, streptococcus aureus, Shigellae, campylobacter jejuni, Bacillus proteus, bacillus cereus, Vibrio parahemolyticus, vibrio cholerae, enterocolitis, Yersinia; Preferably, detect mentioned microorganism simultaneously.
Determine at least a specific target gene at each microorganism, design primer respectively, when being used to increase, all target gene primers are merged, and merge, be used to carry out pcr amplification with super primer according to each target gene.
The amount of super primer is abundant, with the purpose fragment that increases in large quantity.As optimal way of the present invention, with respect to each target gene, the amount of super primer (bar number) be each target gene correspondence primer 3-10 doubly, preferably be 4-6 times, more preferably be 5 times.As optimal way of the present invention, the amount of reverse super primer is 4 times of the super primer of forward, can effectively reduce the competition between the primer like this, thereby improves the sensitivity that detects, and reduces the influence that causes because of background interference.
Method of the present invention also provides preferable PCR reaction conditions, is divided into 5 step by step, comprising: (a) 90-99 ℃ is carried out 15 ± 2min; (b) 90-99 ℃ is carried out 30 ± 3sec → 50-65 ℃ and carries out 2 ± 0.5min → 72 and ℃ carry out 60 ± 5sec; 10-30 circulation altogether; (c) 90-99 ℃ is carried out 30 ± 3sec → 65-72 ℃ and carries out 90 ± 5sec; 3-20 circulation altogether; (d) 90-99 ℃ is carried out 20 ± 2sec → 50-65 ℃ and carries out 20 ± 2sec → 72 and ℃ carry out 30sec; 25-45 circulation altogether; (e) 72 ℃, 3 ± 0.5min.Utilize this reaction conditions to carry out pcr amplification, amplification efficiency height and non-specific binding are few.
Behind the pcr amplification that is through with, the pcr amplification product that obtains is detected, with qualitative or determine the situation that exists of target gene quantitatively.As optimal way of the present invention, in the pcr amplification system, add the probe have detectable signal, described probe can be specifically and corresponding target gene complementation, and at different target gene detectable signal differences; Be tested and appraised detectable signal, thereby determine the kind of microorganism.
As optimal way of the present invention, described detectable signal is a fluorescent microsphere, preferably is nanometer fluorescent microspheres.Fluorescent microsphere can be the multicolour coding, is directed to each probe and sets a kind of fluorescent microsphere with unique color-code, thereby distinguish the pairing target gene of this probe by colour developing.
Method of the present invention can be applicable to many-sided detection applications, includes but not limited to: detect the microorganism in the food and medicine; Detect the secretory product of Mammals (comprising the people), as whether having microorganism in vomitus, ight soil, saliva, the urine etc.Preferably, method of the present invention is used for detecting the microorganism of food and medicine and the microorganism in the poisoning of food source property, can be applicable to the microorganism detection of each intermediate links of food safety management.
Major advantage of the present invention is:
(1) high efficiency can detect multiple pathogenic micro-organism simultaneously or a plurality of goal gene are carried out somatotype in same PCR reaction tubes.And multiple microorganism detects in same reaction tubes simultaneously, and operation steps is simple, elapsed time well below before detection technique commonly used, can save the detection cost greatly.
(2) systematicness, method of the present invention are suitable for the detection of pathogenic agent in groups, as enteron aisle pathogenic bacteria and no gemma anerobe.
(3) accuracy, the present invention has optimized various reaction conditionss, has avoided many uncertain factors of existing in the complex operations process, has improved the accuracy and the sensitivity that detect greatly.
(4) detection method of the present invention is low for equipment requirements, meets the clinical practice needs especially.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
Materials and methods
Bacterial classification
Details such as the table 1 of 13 kinds of food-borne pathogens strain name that detected among the embodiment and bacterium number.
Table 1
Reagent
HotStarTaq Plus Master Mix Kit; The multiple PCR primer mixture; The microballon mixed solution of probe mark; Liquid-phase chip detection damping fluid (5M TMAC, 20%Sarkosyl, 1M Tris-HCl, 0.5MEDTA); Streptavidin-PE; The detection reaction stop buffer.
Enumeration
With the to be measured bacterium liquid overnight culture dilution 10 of aseptic technique with abundant mixing
-1-10
-8Times, and select 10
-6, 10
-7, 10
-83 each 1ml of extent of dilution are in aseptic plate, and 2 of each extent of dilution parallel (repeating repeatedly) in time with the plate count agar substratum pour plate that is cooled to 46 ℃ about 15ml, and rotate plate it is mixed.After agar solidifies, flat board upset is cultivated 12h ± 2h for 37 ℃, select the flat board of colony number between 30-300CFU to carry out enumeration, and the mean number of getting 2 flat boards is as a dilution colony number.And calculate by following formula:
Colony-forming unit in every milliliter (cfu)=3 average colony number * extension rates of multiple of same extent of dilution.
II. embodiment
Embodiment 1, primer design
Selected target bacteria target gene to be detected is determined its specific and conserved sequence by the Blast analysis software, utilizes Oligo 6 primer-design softwares design primer, and each detects the many covers of bacterium design primer, to select best combination of primers.Selected super primer sees Table 2, and selected and the specific detection primer sequence of food to be measured source property microorganism detection target gene see Table 2.
Table 2
Embodiment 2, multi-PCR detection method
(1) nucleic acid extraction
Get the centrifugal 5min of bacterium liquid 10000rpm, bacterial sediment is collected in centrifugal back, and bacterial sediment is resuspended in the aseptic ddH of 100 μ l
2Among the O, boiling water bath 15min smudge cells; After fully shaking mixing, the centrifugal 5min of 10000rpm; Get supernatant liquor as nucleic acid-templated, carry out PCR.
(2)PCR
According to table 3 ratio preparation PCR reaction solution.
Table 3
| Reagent | μL |
| ddH 2O | 14 |
| Mix primer | 6 |
| Taq?Master?Mix | 25 |
| Nucleic acid-templated | 5 |
| Cumulative volume | 50 |
In the table 3, contain food to be measured source property microorganism detection target gene specific in the mix primer and detect primer (2 pairs of primers of each target gene) and super primer, wherein with respect to arbitrary target gene, the amount of the super primer of forward is about 5 times that arbitrary target gene specific detects the primer amount, and the amount of reverse super primer is about 20 times of the super primer of forward.In the PCR reaction system of each 50 μ L, the amount of every target gene specific primer is 1 ρ mol, and the amount of the super primer of forward is 5 ρ mol, and the amount of reverse super primer is 20 ρ mol.
The PCR reaction process sees Table 4.
Table 4
| a | 95℃，15min | |
| b | 94℃，30sec→55℃，2min→72℃，60sec | 15 circulations |
| c | 94℃，30sec→70℃，90sec | 6 circulations |
| d | 94℃，20sec→55℃，20sec→72℃，30sec | 35 circulations |
| e | 72℃，3min |
Embodiment 3, liquid-phase chip detect
The process that probe is coupled on the microballoon is as follows:
(1) get the naked pearl liquid of 0.5ml, 12, centrifugal 3 minutes of 000rpm;
(2) remove supernatant, add the 0.1M MES solution (pH4.5 is available from Sigma) of 50 μ L, vortex is after 10 seconds, and 12, centrifugal 3 minutes of 000rpm;
(3) remove supernatant, use 0.1M MES solution (pH4.5) to clean once again, add the probe solution of the 100pmol/L of 2.5 μ L behind the vibration mixing, vortex mixing in 10 second;
(4) with 0.1M MES solution (pH4.5) preparation EDC solution, making its concentration is 10mg/mL, gets 2.5 μ L after preparing and joins in the mixed solution in the step 3, and 10 seconds mixings of vortex left standstill 30 minutes under room temperature lucifuge condition;
(5) add the freshly prepared EDC solution of 2.5 μ L (available from Sigma) again, 10 seconds mixings of vortex left standstill 30 minutes under room temperature lucifuge condition again;
(6) after EDC finishes dealing with, add 1mLTween-20 solution (0.02%), behind the vortex mixing, 12, centrifugal 3 minutes of 000rpm;
(7) remove supernatant, add 1mL SDS solution (0.1%), behind the vortex mixing, 12, centrifugal 3 minutes of 000rpm;
(8) remove supernatant, add 125 μ L TE damping fluids (pH8), 4 ℃ of preservations behind the mixing.
Carry out microballoon according to table 5 ratio and detect the liquid preparation.
Table 5
| Reagent | μL |
| The microballoon mixed solution | 10 |
| Detect damping fluid | 35 |
| Cumulative volume | 45 |
Every hole adds 45 μ L microballoons detection liquid in 96 orifice plates, fully behind the mixing, adds 5 μ L PCR liquid to be measured, blows and beats mixing repeatedly; Hatch 10min for 52 ℃; Streptavidin-PE detects liquid according to the preparation of table 6 ratio.
Table 6
| Reagent | μL |
| Streptavidin-PE | 0.6 |
| Detect damping fluid | 9.4 |
| Cumulative volume | 10μL |
Every hole adds 10 μ L Streptavidin-PE again and detects liquid, hatches 5min for 52 ℃; Every hole adds 120 μ L stop buffers, and liquid-phase chip detects.
Embodiment 4, sensitivity detect
A. detect target to determine its minimum detectability to detect bacterium DNA
Extract the DNA of 13 kinds of pathogenic bacterium respectively, be diluted to following gradient with sterilized water: 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L and 0.001ng/ μ L, each DNA dilution gradient of each detection bacterium is got 1 μ L volume and is used for PCR reaction (with reference to embodiment 2), carry out liquid-phase chip subsequently and detect (as embodiment 3), to determine the minimum detectability of its dna level.
DNA of bacteria detection sensitivity qualification result sees Table 7.
Table 7
| Bacteria types | Can detected low DNA amount (ng) | |
| 1 | Bacillus?cereus | 0.1 |
| 2 | Bacillus?proleus | 0.1 |
| 3 | Campylobacter?coli | 0.1 |
| 4 | Campylobacter?jejuni | 0.1 |
| 5 | Escherichia?coli(ETEC?strain) | 0.1 |
| 6 | Escherichia?coli(O157?strain) | 0.1 |
| 7 | Listeria?monocytogenes | 0.1 |
| 8 | Salmonella | 0.1 |
| 9 | Shigella | 0.1 |
| 10 | Staphylococcus?aureus | 0.1 |
| 11 | Vibrio?cholerae | 0.1 |
| 12 | Vibrio?parahaemolyticus | 0.1 |
| 13 | Yersinia?enterocolitica | - |
For the single target gene of different DNA amounts, carry out multiplex PCR detection and liquid-phase chip detection with corresponding mix primer (comprising super primer and target gene primer), the results are shown in Table 8.Each numerical value is the relative microballoon amount that liquid-phase chip instrument (luminex company) is measured in the table.According to every result's negative mean value and standard deviation, consider factors such as error, pcr amplification efficient simultaneously, determine that numerical value 240 is threshold value, surpass the positive findings that is decided to be of this value.Disturb bacterium E.coli 25922 (FDA provides by Shanghai City) and H with the SDA specified standards
2O is as negative control.In the table, the digitized representation relevant detection result of bold Italic mark is positive.As seen, designed primer specificity is good, can detect the target gene DNA of low levels.
Table 8
B. serve as to detect target to determine its minimum detectability to detect the bacterium colony number
It is 10 that pathogenic bacterium bacterium liquid is diluted respectively
1Cfu/ml, 10
2Cfu/ml, 10
3Cfu/ml, 10
4The bacteria suspension of cfu/ml extracts DNA respectively, carries out multiplex PCR and liquid-phase chip and detects, to determine its lowest detectable limit.
Carry out CFU sensitivity at Bacillus cereus, Bacillus proleus, Escherichia coli (ETEC strain), Listeria monocytogenes, Salmonella, Shigella, StaphylococcuS aureus, Vibrioparahaemolyticus and these 9 kinds of pathogenic bacterium of Yersinia enterocolitica and detect, the result is as shown in table 9.
Table 9
| Bacterial species | Can detected minimum bacterial count (CFU) |
| ?Bacillus?cereus | 10 2 |
| ?Bacillus?proleus | 10 2 |
| ?Escherichia?coli(ETEC?strain) | 10 3 |
| ?Listeria?monocytogenes | l0 2-10 3 |
| ?Salmonella | 10 2-10 3 |
| ?Shigella | 10 2-10 3 |
| ?Staphylococcus?aureus | 10 2 |
| ?Vibrio?parahaemolyticus | 10 2 |
| ?Yersinia?enterocolitica | 10 2-10 3 |
After extracting DNA respectively for the bacteria suspension of different bacterium colony concentration, carry out multiplex PCR and liquid-phase chip detects with corresponding mix primer (comprising super primer and target gene primer), every kind of bacterium colony repeats repeatedly result such as table 10.Each numerical value is the relative microballoon amount that liquid-phase chip instrument (luminex company) is measured in the table.According to every result's negative mean value and standard deviation, consider factors such as error, pcr amplification efficient simultaneously, determine that numerical value 240 is threshold value, surpass the positive findings that is decided to be of this value.With H
2O is as negative control.Wherein, the digitized representation relevant detection result of bold Italic mark is positive.As seen, designed primer specificity is good, the detection accuracy height, and detected result is reliable and stable.
Table 10
Embodiment 5, multiplicity test experience
From 13 kinds of bacteriums, randomly draw 8 kinds of bacteriums, extract DNA and the balanced mix of these 8 kinds of bacteriums respectively.The DNA mixed solution is diluted to 8ng/ μ L, 0.8ng/ μ L and 0.08ng/ μ L with sterilized water, and guarantees that the dna content of every kind of bacterium reaches 1ng/ μ L, 0.1ng/ μ L and 0.01ng/ μ L respectively in total DNA mixed solution gradient.The DNA mixed solution of getting the different gradients of 1 μ L volume respectively is used for the PCR reaction, carries out liquid-phase chip subsequently and detects, and liquid-phase chip result and actual value are compared, and detects performance to verify its multiplicity.
The results are shown in Table 11.Each numerical value is the relative microballoon amount that liquid-phase chip instrument (luminex company) is measured in the table.According to every result's negative mean value and standard deviation, consider factors such as error, pcr amplification efficient simultaneously, determine that numerical value 240 is threshold value, surpass the positive findings that is decided to be of this value.With H
2O is as negative control.As can be seen from Table 11, when DNA mixed solution total amount is 8ng, 8 kinds of bacteriums can be detected simultaneously,, its accuracy can be confirmed by contrasting with randomly drawing raw data.When DNA mixed solution total amount was reduced to 0.8ng, Vibrio p can't detect, and when DNA mixed solution total amount was reduced to 0.08ng, E.coli ETEC, E.coli O157, Vibrio p and Y.enterocolitica can't detect.This shows, in the present invention, when lowest detection is limited to 1ng, in same reaction system, can detect at least 8 kinds of bacteriums simultaneously.Wherein, the digitized representation relevant detection result of bold Italic mark is positive.
Table 11
Embodiment 6, full pattern mark-on detect
With Salmonellas, proteus vulgaris and streptococcus aureus serves as to detect bacterium, utilize combination of primers of the present invention to carry out the experiment of full pattern mark-on, detailed process is as follows: get 3 kinds of solid food samples (25g) and 3 kinds of liquid food samples (25ml) respectively, add the 225ml substratum, add the thalline liquid of gradient detection object bacteria, the concentration gradient of object bacteria bacterium liquid is 10
2, 10
3, 10
4, 10
5, 10
6, 10
7, 10
8Cfu; 37 ℃, 2h is cultivated in 260 commentaries on classics/min concussion; The preliminary filtration got nutrient solution 200ml, the centrifugal 5min of 10000rpm after the removal food debris; Bacterial sediment is resuspended in the aseptic ddH of 100 μ l
2Among the O, boiling water bath 15min broken cell; After fully shaking mixing, the centrifugal 5min of 10000rpm; Get supernatant liquor as nucleic acid-templated, carry out PCR, and then carry out liquid-phase chip and detect (referring to Fig. 2 flow process).
In full pattern mark-on test experience, owing to be subjected to the interference of food impurity, the lowest detectable limit of selected three kinds of bacteriums in this detection is compared with its CFU detectability and all to be raised an order of magnitude to some extent, and promptly the Bacillusproleus lowest detection is limited to 10
4CFU, the Salmonella lowest detection is limited to 10
3CFU, the Staphylococcusaureus lowest detection is limited to 10
4CFU, concrete data and detected result are as shown in table 12.Yet this detection lower bound has satisfied the existing requirement that food microorganisms are detected fully, and (existing detection requires generally 10
5CFU is following).
Table 12
Multiplex PCR detected result such as table 13.Each numerical value is the relative microballoon amount that liquid-phase chip instrument (luminex company) is measured in the table.According to every result's negative mean value and standard deviation, consider factors such as error, pcr amplification efficient simultaneously, determine that numerical value 240 is threshold value, surpass the positive findings that is decided to be of this value.With H
2O is as negative control.Wherein, the digitized representation relevant detection result of bold Italic mark is positive.As seen, no matter at liquid or solid, method detection sensitivity height of the present invention, the result is reliable and stable.
Table 13
In general, the liquid-phase chip detection method is as a kind of novel molecular biology Fast Detection Technique, but have characteristics such as quick, highly sensitive, high specificity easy and simple to handle, inexpensive, the few high-throughput of sample aequum, detection by quantitative and suitable field screening, can be applied to great public food safety and food poisoning rapid detection project.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Inst. of Life Science, CAS
＜120〉a kind of pathogenic microorganism quick detection kit
<130>100284Z1
<160>107
<170>PatentIn?version?3.3
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ttctttgcgt?tatgtctctg 20
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<212>DNA
＜213〉primer
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gtgaacatgg?agaatgggcc 20
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caggccacgt?tttgtcatgc?gatgttgcta?cccatgttcc 40
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ttctttgcgt?tatgtctctg?aaagcttctc?gcctgcctcc 40
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<212>DNA
＜213〉primer
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tgtgaggcgg?aatcaaaagg 20
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tctatgttcc?tggaaggacg 20
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cagctgagct?atgtgcgatg 20
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caggccacgt?tttgtcatgc?cacttgaata?tcctaactcc 40
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ttctttgcgt?tatgtctctg?tatgtgtatg?gtaaagaaac 40
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<212>DNA
＜213〉primer
<400>11
ggtcaacttt?taatcctgac 20
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ttgttaaatt?atccagtgta 20
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actctcagag?ggtggctgac 20
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<400>14
caggccacgt?tttgtcatgc?ggctgaaagc?atcaggtctg 40
<210>15
<211>40
<212>DNA
＜213〉primer
<400>15
ttctttgcgt?tatgtctctg?gctgttcagt?ctcacgcatc 40
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<212>DNA
＜213〉primer
<400>16
cagtcagctg?acggtaaatc 20
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<211>20
<212>DNA
＜213〉probe
<400>17
cagagggaag?ccggtgcaca 20
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<211>20
<212>DNA
＜213〉primer
<400>18
gatccccgca?ttgctgattg 20
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<212>DNA
＜213〉primer
<400>19
caggccacgt?tttgtcatgc?gattagtgcc?ggttttatcg 40
<210>20
<211>40
<212>DNA
＜213〉primer
<400>20
ttctttgcgt?tatgtctctg?acagctgcgt?catgatattc 40
<210>21
<211>20
<212>DNA
＜213〉primer
<400>21
ccaatacaaa?tgggttgttc 20
<210>22
<211>20
<212>DNA
＜213〉probe
<400>22
taaatggcga?tacggataat 20
<210>23
<211>21
<212>DNA
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<400>23
ccaacctacg?aagagttatc?a 21
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<211>41
<212>DNA
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<400>24
caggccacgt?tttgtcatgc?ctcacaagaa?ctgcttgaaa?c 41
<210>25
<211>40
<212>DNA
＜213〉primer
<400>25
ttctttgcgt?tatgtctctg?agtaacctga?gtgctcgatc 40
<210>26
<211>19
<212>DNA
＜213〉primer
<400>26
ccaacaccag?caaatacag 19
<210>27
<211>19
<212>DNA
＜213〉probe
<400>27
tgtccgtttg?ctaaaggtg 19
<210>28
<211>19
<212>DNA
＜213〉primer
<400>28
gaatgagcca?ccaggaaac 19
<210>29
<211>40
<212>DNA
＜213〉primer
<400>29
caggccacgt?tttgtcatgc?cactgacggg?tctgactatg 40
<210>30
<211>39
<212>DNA
＜213〉primer
<400>30
ttctttgcgt?tatgtctctg?tggtaaccta?ccgctgatg 39
<210>31
<211>19
<212>DNA
＜213〉primer
<400>31
cttgcagaac?acccatctc 19
<210>32
<211>19
<212>DNA
＜213〉probe
<400>32
tgtccgtttg?ctaaaggtg 19
<210>33
<211>20
<212>DNA
＜213〉primer
<400>33
aacatttaga?cgcaacaatc 20
<210>34
<211>40
<212>DNA
＜213〉primer
<400>34
caggccacgt?tttgtcatgc?tgcaataaat?gatctaggac 40
<210>35
<211>40
<212>DNA
＜213〉primer
<400>35
ttctttgcgt?tatgtctctg?gatggtacgc?ttattacttc 40
<210>36
<211>20
<212>DNA
＜213〉primer
<400>36
tcaataacag?catccagttc 20
<210>37
<211>20
<212>DNA
＜213〉probe
<400>37
aagtgaagat?ttaagtggcc 20
<210>38
<211>20
<212>DNA
＜213〉primer
<400>38
caaactagca?ttaaaagttc 20
<210>39
<211>40
<212>DNA
＜213〉primer
<400>39
caggccacgt?tttgtcatgc?aaatacgtca?gatagtaatg 40
<210>40
<211>40
<212>DNA
＜213〉primer
<400>40
ttctttgcgt?tatgtctctg?tgtaagtaag?cacgtcctgg 40
<210>41
<211>20
<212>DNA
＜213〉primer
<400>41
taaatttcat?tattcccaac 20
<210>42
<211>20
<212>DNA
＜213〉probe
<400>42
ttaaaaacgc?cagatgctgc 20
<210>43
<211>20
<212>DNA
＜213〉primer
<400>43
gatgtattag?gcgtatacag 20
<210>44
<211>40
<212>DNA
＜213〉primer
<400>44
caggccacgt?tttgtcatgc?ctcacccata?tgaacaggag 40
<210>45
<211>40
<212>DNA
＜213〉primer
<400>45
ttctttgcgt?tatgtctctg?aaattaacac?gataccatcc 40
<210>46
<211>20
<212>DNA
＜213〉primer
<400>46
aatcgttcat?caatcacacc 20
<210>47
<211>20
<212>DNA
＜213〉probe
<400>47
taggtggaat?accatattct 20
<210>48
<211>20
<212>DNA
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<400>48
tcatatacgg?aatcgatggc 20
<210>49
<211>40
<212>DNA
＜213〉primer
<400>49
caggccacgt?tttgtcatgc?ctcacccata?tgaacaggag 40
<210>50
<211>40
<212>DNA
＜213〉primer
<400>50
ttctttgcgt?tatgtctctg?tttttttggg?agtctatatg 40
<210>51
<211>20
<212>DNA
＜213〉primer
<400>51
tccttcatcc?tttcaatggc 20
<210>52
<211>20
<212>DNA
＜213〉probe
<400>52
ttctttgcgt?tatgtctctg 20
<210>53
<211>20
<212>DNA
＜213〉primer
<400>53
cgtaaataga?agtggttctg 20
<210>54
<211>40
<212>DNA
＜213〉primer
<400>54
caggccacgt?tttgtcatgc?gatccaacag?tatatagtgc 40
<210>55
<211>40
<212>DNA
＜213〉primer
<400>55
ttctttgcgt?tatgtctctg?cattaattta?accgtatcac 40
<210>56
<211>20
<212>DNA
＜213〉primer
<400>56
atgtcattgg?ttgacctttg 20
<210>57
<211>20
<212>DNA
＜213〉probe
<400>57
aaaaaattac?ataaagaacc 20
<210>58
<211>20
<212>DNA
＜213〉primer
<400>58
gaaaaatgta?actgttcagg 20
<210>59
<211>40
<212>DNA
＜213〉primer
<400>59
caggccacgt?tttgtcatgc?gttggatctt?caagcaagac 40
<210>60
<211>40
<212>DNA
＜213〉primer
<400>60
ttctttgcgt?tatgtctctg?accttcccat?caaaaacatc 40
<210>61
<211>20
<212>DNA
＜213〉primer
<400>61
aacacgatta?atcccctctg 20
<210>62
<211>20
<212>DNA
＜213〉probe
<400>62
ggaaaaatat?aatttatata 20
<210>63
<211>20
<212>DNA
＜213〉primer
<400>63
tgctgttata?ggtcgttatc 20
<210>64
<211>40
<212>DNA
＜213〉primer
<400>64
caggccacgt?tttgtcatgc?tccacatgga?gatacagcag 40
<210>65
<211>40
<212>DNA
＜213〉primer
<400>65
ttctttgcgt?tatgtctctg?gccttgtcct?gtaatacttg 40
<210>66
<211>20
<212>DNA
＜213〉primer
<400>66
caccatctat?agatccaaag 20
<210>67
<211>20
<212>DNA
＜213〉probe
<400>67
ttggttagaa?tggctcaaga 20
<210>68
<211>20
<212>DNA
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<400>68
caacaagttg?attttgaagc 20
<210>69
<211>42
<212>DNA
＜213〉primer
<400>69
caggccacgt?tttgtcatgc?attaatgctt?taaaacctga?tc 42
<210>70
<211>42
<212>DNA
＜213〉primer
<400>70
ttctttgcgt?tatgtctctg?ttaaaaaatt?tgcattatca?ag 42
<210>71
<211>20
<212>DNA
＜213〉primer
<400>71
accataaagt?tttgcaacgc 20
<210>72
<211>18
<212>DNA
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<400>72
ctccaacttt?atttgtag 18
<210>73
<211>20
<212>DNA
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<400>73
ggagatgaag?ttattgttcc 20
<210>74
<211>40
<212>DNA
＜213〉primer
<400>74
caggccacgt?tttgtcatgc?acactgacat?atatagcatc 40
<210>75
<211>40
<212>DNA
＜213〉primer
<400>75
ttctttgcgt?tatgtctctg?gtttcattat?ctgaatcaac 40
<210>76
<211>20
<212>DNA
＜213〉primer
<400>76
gtcactaaca?gacatttgcc 20
<210>77
<211>20
<212>DNA
＜213〉probe
<400>77
tataaaatac?acaggagcca 20
<210>78
<211>20
<212>DNA
＜213〉primer
<400>78
aggattagct?gtacataggc 20
<210>79
<211>40
<212>DNA
＜213〉primer
<400>79
caggccacgt?tttgtcatgc?ggcatgacgt?tataggctac 40
<210>80
<211>40
<212>DNA
＜213〉primer
<400>80
ttctttgcgt?tatgtctctg?tgttctaact?gggctaatcc 40
<210>81
<211>20
<212>DNA
＜213〉primer
<400>81
cgtgatataa?aatcatcagc 20
<210>82
<211>21
<212>DNA
＜213〉probe
<400>82
gacaaatatc?tgcgctgcta?t 21
<210>83
<211>20
<212>DNA
＜213〉primer
<400>83
caacaagttg?attttgaagc 20
<210>84
<211>42
<212>DNA
＜213〉primer
<400>84
caggccacgt?tttgtcatgc?attaatgctt?taaaacctga?tc 42
<210>85
<211>42
<212>DNA
＜213〉primer
<400>85
ttctttgcgt?tatgtctctg?ttaaaaaatt?tgcattatca?ag 42
<210>86
<211>20
<212>DNA
＜213〉primer
<400>86
accataaagt?tttgcaacgc 20
<210>87
<211>18
<212>DNA
＜213〉probe
<400>87
ctccaactat?gtttgtag 18
<210>88
<211>20
<212>DNA
＜213〉primer
<400>88
tatacattgg?ataatgaccc 20
<210>89
<211>40
<212>DNA
＜213〉primer
<400>89
caggccacgt?tttgtcatgc?aaggttttaa?cctgaagtac 40
<210>90
<211>40
<212>DNA
＜213〉primer
<400>90
ttctttgcgt?tatgtctctg?tagaagaaat?cgtatccctg 40
<210>91
<211>20
<212>DNA
＜213〉primer
<400>91
gtgaccaaac?ttattactgc 20
<210>92
<211>20
<212>DNA
＜213〉probe
<400>92
tatggcagta?ataagtttgg 20
<210>93
<211>20
<212>DNA
＜213〉primer
<400>93
attacaatgc?tgctgattgg 20
<210>94
<211>40
<212>DNA
＜213〉primer
<400>94
caggccacgt?tttgtcatgc?aagccgaaga?caacgcatta 40
<210>95
<211>40
<212>DNA
＜213〉primer
<400>95
ttctttgcgt?tatgtctctg?accaaaacgc?gtttttgctg 40
<210>96
<211>20
<212>DNA
＜213〉primer
<400>96
tcactgatct?gactgaaatc 20
<210>97
<211>20
<212>DNA
＜213〉probe
<400>97
agctgcgcga?tttggtgatt 20
<210>98
<211>20
<212>DNA
＜213〉primer
<400>98
gttctacacc?aacacgtcgc 20
<210>99
<211>40
<212>DNA
＜213〉primer
<400>99
caggccacgt?tttgtcatgc?aacgttatcc?gtcagcgttg 40
<210>100
<211>40
<212>DNA
＜213〉primer
<400>100
ttctttgcgt?tatgtctctg?agcgattgtc?agcggcgaag 40
<210>101
<211>20
<212>DNA
＜213〉primer
<400>101
agatcgtgtg?gttgtatgag 20
<210>102
<211>20
<212>DNA
＜213〉probe
<400>102
acattagatt?tggcgaacga 20
<210>103
<211>20
<212>DNA
＜213〉primer
<400>103
gttctacacc?aacacgtcgc 20
<210>104
<211>40
<212>DNA
＜213〉primer
<400>104
caggccacgt?tttgtcatgc?aacgttatcc?gtcagcgttg 40
<210>105
<211>40
<212>DNA
＜213〉primer
<400>105
ttctttgcgt?tatgtctctg?agcgattgtc?agcggcgaag 40
<210>106
<211>20
<212>DNA
＜213〉primer
<400>106
agatcgtgtg?gttgtatgag 20
<210>107
<211>40
<212>DNA
＜213〉probe
<400>107
acattagatt?tggcgaacga?acattagatt?tggcgaacga 40
Claims (12)
1. a method for rapid inspecting animalcule is characterized in that, described method comprises:
(1), determines the specific target gene respectively at 1-50 kind microorganism to be detected;
(2) each target gene of determining according to step (1) is determined its specific pair of inside primer and pair of outside primer respectively, and the sequence of described inboard primer can be complementary with corresponding target gene part, again can be complementary with corresponding super primer part; Described super primer is that a primer is right, and the super primer sequence of forward is shown in SEQ ID NO:1, and reverse super primer sequence is shown in SEQ ID NO:2; Mix above-mentioned primer, obtain primer mixture;
(3) with the testing sample being pcr template, is that primer carries out the PCR reaction with the primer mixture of step (2), obtains the pcr amplification system;
(4) add the probe have detectable signal in the pcr amplification system of step (3), described probe can be specifically and corresponding target gene complementation, and at different target gene detectable signal differences; With
(5) identify detectable signal, thereby determine the kind of microorganism.
2. the method for claim 1 is characterized in that, 5 ' end of described reverse super primer has a discernible signal.
3. the method for claim 1 is characterized in that, in the step (3), the amount of the super primer of forward be each target gene correspondence primer 3-10 doubly, and the amount of reverse super primer be each target gene correspondence primer 10-50 doubly.
4. the method for claim 1 is characterized in that, the primer and the probe of described microorganism target gene and correspondence thereof are as follows:
Listeria monocytogenes (Listeria monocytogenes) is induced Actin muscle assembling precursor protein gene, and primer is shown in SEQ ID NO:8-11, and probe is shown in SEQ ID NO:12;
Shigellae (Shigella) aggressive plasmid H antigen gene, primer is shown in SEQ ID NO:13-16, and probe is shown in SEQ ID NO:17;
Salmonellas (Salmonella) invasin protein A gene, primer is shown in SEQ ID NO:18-21, and probe is shown in SEQ ID NO:22;
Bacillus proteus (Bacillus proleus) F type ATP enzyme beta subunit gene, primer is shown in SEQ ID NO:23-26, and probe is shown in SEQ ID NO:27;
Bacillus cereus (Bacillus cereus) enterotoxin T gene, primer is shown in SEQ ID NO:33-36, and probe is shown in SEQ ID NO:37;
Intestinal bacteria ETEC (Escherichia coli ETEC) heat-labile toxin AB subunit gene, primer is shown in SEQ ID NO:43-46, and probe is shown in SEQ ID NO:47;
Streptococcus aureus (Staphylococcus aureus) thermostability nuclease gene, primer is shown in SEQ ID NO:53-56, and probe is shown in SEQ ID NO:57;
Campylobacter jejuni (Campylobacter jejuni) gyrase A subunit gene, primer is shown in SEQ IDNO:63-66, and probe is shown in SEQ ID NO:67;
Escherichia coli O 157 (Escherichia coli O157) O-antigen-specific gene, primer is shown in SEQ IDNO:73-76, and probe is shown in SEQ ID NO:77;
Campylobacter coli (Campylobacter.Coli) siderophore transfer related protein gene, primer is shown in SEQ ID NO:83-86, and probe is shown in SEQ ID NO:87;
Yersinia enterocolitica (Yersinia enterocolitica) is sticked the invasin protein gene, and primer is shown in SEQ ID NO:88-91, and probe is shown in SEQ ID NO:92;
Vibrio cholerae (Vibrio cholerae) hemolysin protein A gene, primer is shown in SEQ ID NO:93-96, and probe is shown in SEQ ID NO:97; Or
The heat-resisting hemolytic toxin gene of Vibrio parahemolyticus (Vibrio parahaemolyticu), primer is shown in SEQID NO:98-101, and probe is shown in SEQ ID NO:102.
5. the method for claim 1 is characterized in that, in the step (3), the PCR reaction process is as follows:
(a) 90-99 ℃ is carried out 15 ± 2min;
(b) 90-99 ℃ is carried out 30 ± 3sec → 50-65 ℃ and carries out 2 ± 0.5min → 72 and ℃ carry out 60 ± 5sec; 10-30 circulation altogether;
(c) 90-99 ℃ is carried out 30 ± 3sec → 65-72 ℃ and carries out 90 ± 5sec; 3-20 circulation altogether;
(d) 90-99 ℃ is carried out 20 ± 2sec → 50-65 ℃ and carries out 20 ± 2sec → 72 and ℃ carry out 30sec; 25-45 circulation altogether;
(e)72℃，3±0.5min。
6. the method for claim 1 is characterized in that, in the step (4), described detectable signal is a fluorescent microsphere.
7. the method for claim 1 is characterized in that, described microorganism is pathogenic bacterium.
8. method as claimed in claim 7 is characterized in that described pathogenic bacterium are food-borne pathogens.
9. microbial rapid detection reagent combination is characterized in that described agent combination comprises:
Super primer, described super primer is that a primer is right, and the super primer sequence of forward is shown in SEQ ID NO:1, and reverse super primer sequence is shown in SEQ ID NO:2;
Primer at 1-50 kind microorganism target gene to be detected, primer corresponding to arbitrary target gene comprises pair of inside primer and pair of outside primer, the sequence of described inboard primer can be complementary with corresponding target gene part, again can be complementary with corresponding super primer part; And
At the probe of 1-50 kind microorganism target gene to be detected, described probe is can be specifically complementary with corresponding target gene and have detectable signal, and at different target gene detectable signal differences.
10. agent combination as claimed in claim 9 is characterized in that, is selected from the primer and the probe of microorganism target gene to be detected:
Induce Actin muscle to assemble the primer and the probe of precursor protein gene corresponding to Listeria monocytogenes, wherein primer is shown in SEQ ID NO:8-11, and probe is shown in SEQ ID NO:12;
Corresponding to the primer and the probe of Shigellae aggressive plasmid H antigen gene, wherein primer is shown in SEQ IDNO:13-16, and probe is shown in SEQ ID NO:17;
Corresponding to the primer and the probe of Salmonellas invasin protein A gene, wherein primer is shown in SEQ ID NO:18-21, and probe is shown in SEQ ID NO:22;
Corresponding to the primer and the probe of Bacillus proteus F type ATP enzyme beta subunit gene, wherein primer is shown in SEQ IDNO:23-26, and probe is shown in SEQ ID NO:27;
Corresponding to the primer and the probe of bacillus cereus enterotoxin T gene, wherein primer is shown in SEQ ID NO:33-36, and probe is shown in SEQ ID NO:37;
Corresponding to the primer and the probe of intestinal bacteria ETEC heat-labile toxin AB subunit gene, wherein primer is shown in SEQ ID NO:43-46, and probe is shown in SEQ ID NO:47;
Corresponding to the primer and the probe of streptococcus aureus thermostability nuclease gene, wherein primer is shown in SEQ ID NO:53-56, and probe is shown in SEQ ID NO:57;
Corresponding to the primer and the probe of campylobacter jejuni gyrase A subunit gene, wherein primer is shown in SEQ IDNO:63-66, and probe is shown in SEQ ID NO:67;
Corresponding to the primer and the probe of Escherichia coli O 157 O-antigen-specific gene, wherein primer is shown in SEQID NO:73-76, and probe is shown in SEQ ID NO:77;
Corresponding to the primer and the probe of campylobacter coli siderophore transfer related protein gene, wherein primer is shown in SEQ ID NO:83-86, and probe is shown in SEQ ID NO:87;
Stick the primer and the probe of invasin protein gene corresponding to Yersinia enterocolitica, wherein primer is shown in SEQ ID NO:88-91, and probe is shown in SEQ ID NO:92;
Corresponding to the primer and the probe of vibrio cholerae hemolysin protein A gene, wherein primer is shown in SEQ ID NO:93-96, and probe is shown in SEQ ID NO:97; Or
Corresponding to the primer and the probe of the heat-resisting hemolytic toxin gene of Vibrio parahemolyticus, wherein primer is shown in SEQ IDNO:98-101, and probe is shown in SEQ ID NO:102.
11. the purposes of claim 9 or 10 described agent combination is used to prepare the test kit that detects microorganism.
12. a microbial rapid detection reagent box is characterized in that, contains
Container; And
Be sub-packed in claim 9 or 10 described agent combination in the container.
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| Publication number | Priority date | Publication date | Assignee | Title |
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-
2010
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| CN1898395A (en) * | 2003-10-13 | 2007-01-17 | 格纳科生物医疗产品公司 | Method and kit for primer based amplification of nucleic acids |
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