CN102286612B - Fast detection reagent kit for pathogenic microorganism - Google Patents
Fast detection reagent kit for pathogenic microorganism Download PDFInfo
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- CN102286612B CN102286612B CN201010202838.0A CN201010202838A CN102286612B CN 102286612 B CN102286612 B CN 102286612B CN 201010202838 A CN201010202838 A CN 201010202838A CN 102286612 B CN102286612 B CN 102286612B
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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 fast detection reagent kit for pathogenic microorganism.
Background technology
In recent years, great food safety affair takes place frequently, and causes people's great attention.To fundamentally solve food-safety problem, just must implement whole process control and monitoring to each link such as the production of food, processing, circulation and sale, this just needs can meet in a large number quick, convenient, accurate, the sensitive Food Safety Analysis detection technique of this requirement.
Along with scientific and technical development, the effect of food safety quick analytic detection method aspect inspection for food hygiene is more and more important.From long term growth, the development pole the earth of the subjects such as immunology, molecular biology, computer technology and automatization has promoted Food Safety Analysis detection method to sensitiveer future development more easily, set up efficient food safety quick analytic detection method, the quality control tool in foodstuff production, transportation, sales process is of great significance; Once there is food-borne pathogenic microorganism poisoning simultaneously, also need within the shortest time, complete and detect to formulate scientific and reasonable treatment plan, win treatment time.Applying of these real-time analysis detection techniques, be not only one of traditional Food Safety Analysis detection technique is improved, also make Safety of Food Quality have further assurance, thereby promote foodstuffs industry more healthy, advance rapidly, constantly meet the needs that the people improve health conditions.
More existing food-borne pathogens Fast Detection Technique are to utilize immunological method mostly at present, the method instrumentation is simple, detection speed is fast, sensitivity compared with high, specificity better and sample aequum few, but also exist, be difficult to preparation, use and manyly anti-ly easily produce cross reactions and occur false positive and once can only detect the shortcoming and defect such as one or more pathogenic bacterium as pathogenic bacterium monoclonal antibody.Therefore, also need exploitation can detect rapidly, efficiently the new technology of pathogenic bacterium at present.
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), for 1-50 kind to be detected (preferably 2-30 kind, as 10 kinds, 15 kinds, 20 kinds) microorganism, determine respectively specific target gene;
(2) each target gene of determining according to step (1), determine respectively its specific pair of inside primer (primer (Fi) and reverse inner side primer (Ri) just to the inside) and pair of outside primer (primer (Fo) and oppositely outside primer (Ro) just laterally), the sequence of described inner side primer can be complementary with corresponding target gene part, again can complementary with corresponding super primer part (the super primer part of primer and forward be complementary just to the inside, oppositely inner side primer and the partly complementation of reverse super primer); Described super primer is a primer pair, and the super primer sequence of forward is as shown in SEQ ID NO:1, and reverse super primer sequence is as shown in SEQ ID NO:2; Mix above-mentioned primer, obtain primer mixture;
(3) take testing sample as pcr template, take the primer mixture of step (2) as primer carries out PCR reaction, obtain pcr amplification system;
(4) in the pcr amplification system of step (3), add the probe with detectable signal, described probe can be complementary with corresponding target gene specifically, and different for different target gene detectable signals;
(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 is with a discernible signal; It is preferably biotin labeling.
In another preference, in step (3), the amount of the super primer of forward (number) is 3-10 times of the primer that each target gene is corresponding; It is preferably 4-6 times; Be more preferably 5 times, the amount of reverse super primer (number) is 10-50 times of the primer that each target gene is corresponding; It is preferably 10-30 times; Be more preferably 20 times.
In another preference, the amount of reverse super primer is 4 times of the super primer of forward.
In another preference, primer and the probe of described microorganism target gene and correspondence thereof are as follows:
Listeria monocytogenes (Listeria monocytogenes) induction Actin muscle assembling precursor protein gene, primer is as shown in SEQ ID NO:8-11, and probe is as shown in SEQ ID NO:12;
Shigellae (Shigella) aggressive plasmid H antigen gene, primer is as shown in SEQ ID NO:13-16, and probe is as shown in SEQ ID NO:17;
Salmonellas (Salmonella) invasin protein A gene, primer is as shown in SEQ ID NO:18-21, and probe is as shown in SEQ ID NO:22;
Bacillus proteus (Bacillus proleus) F type ATP enzyme beta subunit gene, primer is as shown in SEQ ID NO:23-26, and probe is as shown in SEQ ID NO:27;
Bacillus cereus (Bacillus cereus) enterotoxin T gene, primer is as shown in SEQ ID NO:33-36, and probe is as shown in SEQ ID NO:37;
Intestinal bacteria ETEC (Escherichia coli ETEC) heat-labile toxin AB subunit gene, primer is as shown in SEQ ID NO:43-46, and probe is as shown in SEQ ID NO:47;
Streptococcus aureus (Staphylococcus aureus) thermostability nuclease gene, primer is as shown in SEQ ID NO:53-56, and probe is as shown in SEQ ID NO:57;
Campylobacter jejuni (Campylobacter jejuni) gyrase A subunit gene, primer is as shown in SEQ IDNO:63-66, and probe is as shown in SEQ ID NO:67;
Escherichia coli O 157 (Escherichia coli O157) O-antigen-specific gene, primer is as shown in SEQ IDNO:73-76, and probe is as shown in SEQ ID NO:77;
Campylobacter coli (Campylobacter.Coli) siderophore transfer related protein gene, primer is as shown in SEQ ID NO:83-86, and probe is as shown in SEQ ID NO:87;
Yersinia enterocolitica (Yersinia enterocolitica) is sticked invasin protein gene, and primer is as shown in SEQ ID NO:88-91, and probe is as shown in SEQ ID NO:92;
Vibrio cholerae (Vibrio cholerae) hemolysin protein A gene, primer is as shown in SEQ ID NO:93-96, and probe is as shown in SEQ ID NO:97; Or
The heat-resisting hemolytic toxin gene of Vibrio parahemolyticus (Vibrio parahaemolyticu), primer is as shown in SEQID NO:98-101, and probe is as shown in SEQ ID NO:102.
In another preference, while detecting multiple-microorganism, after mixing for the primer of the target gene of multiple-microorganism, for PCR, react simultaneously.
In another preference, in step (3), 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 (better 30-40 circulation) altogether;
(e)72℃，3±0.5min。
In another preference, in step (4), described detectable signal is fluorescent microsphere; It is preferably 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 a primer pair, and the super primer sequence of forward is as shown in SEQ ID NO:1, and reverse super primer sequence is as shown in SEQ ID NO:2;
Primer for 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 inner side primer can be complementary with corresponding target gene part, again can be complementary with corresponding super primer part (the super primer part of primer and forward is complementary just to the inside, oppositely inner side primer and the partly complementation of reverse super primer); And
For the probe of 1-50 kind microorganism target gene to be detected, described probe is can be specifically complementary with corresponding target gene and with detectable signal, and different for different target gene detectable signals.
In a preference, for primer and the probe of microorganism target gene to be detected, be selected from:
Corresponding to primer and the probe of Listeria monocytogenes induction Actin muscle assembling precursor protein gene, wherein primer is as shown in SEQ ID NO:8-11, and probe is as shown in SEQ ID NO:12;
Corresponding to primer and the probe of Shigellae aggressive plasmid H antigen gene, wherein primer is as shown in SEQ IDNO:13-16, and probe is as shown in SEQ ID NO:17;
Corresponding to primer and the probe of Salmonellas invasin protein A gene, wherein primer is as shown in SEQ ID NO:18-21, and probe is as shown in SEQ ID NO:22;
Corresponding to primer and the probe of Bacillus proteus F type ATP enzyme beta subunit gene, wherein primer is as shown in SEQ IDNO:23-26, and probe is as shown in SEQ ID NO:27;
Corresponding to primer and the probe of bacillus cereus enterotoxin T gene, wherein primer is as shown in SEQ ID NO:33-36, and probe is as shown in SEQ ID NO:37;
Corresponding to primer and the probe of intestinal bacteria ETEC heat-labile toxin AB subunit gene, wherein primer is as shown in SEQ ID NO:43-46, and probe is as shown in SEQ ID NO:47;
Corresponding to primer and the probe of streptococcus aureus thermostability nuclease gene, wherein primer is as shown in SEQ ID NO:53-56, and probe is as shown in SEQ ID NO:57;
Corresponding to primer and the probe of campylobacter jejuni gyrase A subunit gene, wherein primer is as shown in SEQ IDNO:63-66, and probe is as shown in SEQ ID NO:67;
Corresponding to primer and the probe of Escherichia coli O 157 O-antigen-specific gene, wherein primer is as shown in SEQID NO:73-76, and probe is as shown in SEQ ID NO:77;
Corresponding to primer and the probe of campylobacter coli siderophore transfer related protein gene, wherein primer is as shown in SEQ ID NO:83-86, and probe is as shown in SEQ ID NO:87;
The primer and the probe that corresponding to Yersinia enterocolitica, stick invasin protein gene, wherein primer is as shown in SEQ ID NO:88-91, and probe is as shown in SEQ ID NO:92;
Corresponding to primer and the probe of vibrio cholerae hemolysin protein A gene, wherein primer is as shown in SEQ ID NO:93-96, and probe is as shown in SEQ ID NO:97; Or
Corresponding to primer and the probe of the heat-resisting hemolytic toxin gene of Vibrio parahemolyticus, wherein primer is as shown in SEQ IDNO:98-101, and probe is as shown in SEQ ID NO:102.
In another aspect of this invention, the purposes of the agent combination described in providing, for the preparation of 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 container.
In a preference, in described test kit, also contain fluorescent microsphere, colouring reagents, archaeal dna polymerase, thinner, working instructions etc.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
Fig. 1, multiplex PCR nested primers amplification procedure schematic diagram.
Fig. 2, food-borne pathogens multiplex PCR testing process schematic diagram.
Embodiment
The defect existing in order to overcome present food security analysis and detection technology and equipment, the inventor is through deep research, develop first a kind of novel pathogenic bacterium method for quick and supporting reagent or test kit, described method and reagent can detect various pathogens in a short period of time, there is quick, highly sensitive, high specificity easy and simple to handle, with low cost, sample aequum is few, can high-throughput, be applicable to the features such as field screening.
Microorganism and target gene
" microorganism " of the present invention can be the various microorganisms that are present in food, preferably refers to for human body or the harmful 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 refers to the gene that is present in specifically corresponding microorganism to be measured, 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 existing in detecting mixed system determine microorganism there is situation time, the selection of target gene is outbalance, consider and not cause non-specific binding (can reduce the accuracy rate of detection).The inventor is through repetition test, and the microorganism detecting for needs has been found representative and has been 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:
Pathogenic bacterium target gene
Listeria monocytogenes (Listeria monocytogenes) induction 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 according to the kind of required detected microorganism, select.
Detection reagent and test kit
For 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 to detect the target gene of multiple-microorganism simultaneously, PCR reaction system is complicated, when therefore designing primer, also need to consider to reduce the Probability of non-specific binding and good expanding effect.
In the present invention, in same PCR reaction system, add more than two pairs primer, can amplify a plurality of nucleic acid fragments simultaneously, thereby measure efficiently a plurality of target genes.For the target gene of the food-borne pathogens of intending measuring, for two pairs of Nest multiplex PCR Auele Specific Primers of each shot design (i.e. inner side primers F i/Ri and outside primers F o/Ro), for the target gene (goal gene) of the specific bacteria that increases, with reference to Fig. 1.In PCR detection system, the addition of Auele Specific Primer is extremely low, only in incipient several circulations, is used for the target spot that increases, and this primer, with sequence label (can be complementary with super primer part), can be identified by super primer.Described part complementation refers to 20-100% just to the inside between primer and the super primer of forward (30-80% preferably, as 40%, 50%, 60%) sequence be complementary, it is complementary oppositely between inner side primer and reverse super primer, having the sequence of 20-100% (preferably 30-80%, as 40%, 50%, 60%).
The sequence label carrying for Auele Specific Primer designs super primer (the super primer of forward and reverse super primer).In reaction system, add the super primer that enough meets exponential amplification, this primer can be after Auele Specific Primer starts to increase several circulations identification tag sequence fast, object fragment in batches further increases.The super primer using for every kind of pathogenic bacterium is all identical in sequence, thereby greatly reduces the background interference of detected result, improves detection sensitivity.Because super primer is with regard to pair of sequences, therefore known: when detecting multiple target gene, corresponding each of a plurality of target genes just to the inside primer to have one section of sequence be identical.
By repetition test, the inventor has found desirable super primer, for primer and the probe of each target gene, as listed in table 2.Described super primer, target gene primer and probe have formed the agent combination that detects microorganism.
Preferably, described reverse super primer also carries a discernible signal, for relative quantification determine the quantitative relation between the contained multiple-microorganism of sample.More preferably, described discernible signal is 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 the described agent combination in container.Preferably, for the ease of those skilled in the art, use, in described test kit, also contain the reagent of operations such as being useful on microorganism culturing, DNA extracting, pcr amplification or signal detection; Fluorescent microsphere for example, colouring reagents, archaeal dna polymerase, thinner, substratum etc.In addition, in described test kit, also contain working instructions etc.
Detection method
The invention provides the method that detects quickly and efficiently or identify multiple-microorganism for same sample system.
In the present invention, unless otherwise indicated, described " sample " is broad sense, it can be anyly need to 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 people), as vomitus, ight soil, saliva, urine etc.
In the microorganism of method of the present invention based on to be detected, whether exist a certain target gene to identify.Therefore, testing sample need to carry out suitable pre-treatment so that cell discharges target gene, thereby target gene can be identified.The method that makes cell discharge target gene is well-known to those skilled in the art, can adopt routinely the method for lysing cell.As a kind of optimal way of the present invention, can pass through boiling water bath processing sample, make Microbiological release go out the nucleic acid of target gene.Typically, for Gram-negative bacteria, by the method for boiling, can crack cell; And to gram-positive microorganism, 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 according to demand fractionation and the combination of different microorganisms.Be generally 1-50 kind, 2-30 kind preferably, 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.
For each microorganism, determine at least one specific target gene, according to each target gene, design respectively primer, when all target gene primers being merged when increasing, and merge with super primer, for carrying out pcr amplification.
The amount of super primer is abundant, with the object fragment that increases in large quantity.As optimal way of the present invention, with respect to each target gene, the amount of super primer (number) is 3-10 times of the primer that each target gene is corresponding, is preferably 4-6 times, is more preferably 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 primer like this, thereby improves the sensitivity detecting, and reduces the impact causing because of background interference.
Method of the present invention also provides preferably 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 is high and non-specific binding is few.
After the pcr amplification that is through with, the pcr amplification product obtaining is detected, with qualitative or determine quantitatively the situation that exists of target gene.As optimal way of the present invention, in pcr amplification system, add the probe with detectable signal, described probe can be complementary with corresponding target gene specifically, and different for different target gene detectable signals; Be tested and appraised detectable signal, thereby determine the kind of microorganism.
As optimal way of the present invention, described detectable signal is fluorescent microsphere, is preferably nanometer fluorescent microspheres.Fluorescent microsphere can be multicolour coding, is directed to each probe and sets a kind of fluorescent microsphere with unique color-code, thereby distinguish the corresponding 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 food and medicine; Detect the secretory product of Mammals (comprising people), as whether there is microorganism in vomitus, ight soil, saliva, urine etc.Preferably, method of the present invention is the microorganism in poisoning for detection of the microorganism in food and medicine and 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 microorganisms simultaneously or a plurality of goal gene be carried out to somatotype in same PCR reaction tubes.And multiple-microorganism detects in same reaction tubes simultaneously, operation steps is simple, elapsed time well below before conventional detection technique, can greatly save testing cost.
(2) systematicness, method of the present invention is suitable for the detection of pathogenic agent in groups, as enteron aisle pathogenic bacteria with without gemma anerobe.
(3) accuracy, the present invention has optimized various reaction conditionss, has avoided many uncertain factors of existing in complex operations process, has greatly improved the accuracy and the sensitivity that detect.
(4) detection method of the present invention is low for equipment requirements, meets especially clinical practice needs.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition as people 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 all specialties of using in literary composition and scientific words and one skilled in the art are familiar.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
Materials and methods
Bacterial classification
The details of 13 kinds of food-borne pathogens strain name that detect in embodiment and bacterium number is as table 1.
Table 1
Reagent
HotStarTaq Plus Master Mix Kit; Multiple PCR primer mixture; The microballon mixed solution of probe mark; Liquid-phase chip detects damping fluid (5M TMAC, 20%Sarkosyl, 1M Tris-HCl, 0.5MEDTA); Streptavidin-PE; Detection reaction stop buffer.
Enumeration
With aseptic technique, the bacterium liquid overnight culture to be measured fully mixing is diluted to 10
-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 (repeatedly), are in time cooled to the plate count agar substratum pour plate of 46 ℃ by 15ml left and right, and rotate plate it is mixed.After agar solidifies, 37 ℃ of Flat plate turnovers are cultivated to 12h ± 2h, select the flat board of colony number between 30-300CFU to carry out enumeration, and get 2 dull and stereotyped mean numbers as a dilution colony number.And calculate by following formula:
Average colony number * extension rate that colony-forming unit in every milliliter (cfu)=same extent of dilution 3 times repeats.
II. embodiment
The design of embodiment 1, primer
Selected target bacteria target gene to be detected, determines its specific and conserved sequence by Blast analysis software, utilizes Oligo 6 primer-design software design primers, and each detects the many covers of bacterium design primer, to select best combination of primers.Selected super primer is in Table 2, and the selected and specific detection primer sequence of food to be measured source property microorganism detection target gene is in Table 2.
Table 2
Embodiment 2, multi-PCR detection method
(1) nucleic acid extraction
Get the centrifugal 5min of bacterium liquid 10000rpm, centrifugal rear collection bacterial sediment, bacterial sediment is resuspended in the aseptic ddH of 100 μ l
2in O, boiling water bath 15min smudge cells; After fully concussion mixes, 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 table 3, in mix primer, contain food to be measured source property microorganism detection target gene specific 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 approximately 5 times that arbitrary target gene specific detects primer amount, and the amount of reverse super primer is approximately 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.
PCR reaction process is in 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 microballoon is as follows:
(1) get the naked pearl liquid of 0.5ml, centrifugal 3 minutes of 12,000rpm;
(2) remove supernatant, add the 0.1M MES solution (pH4.5, purchased from Sigma) of 50 μ L, vortex is after 10 seconds, centrifugal 3 minutes of 12,000rpm;
(3) remove supernatant, then use 0.1M MES solution (pH4.5) to clean once, add the probe solution of the 100pmol/L of 2.5 μ L after vibration mixes, vortex mixes for 10 seconds;
(4) with 0.1M MES solution (pH4.5) preparation EDC solution, making its concentration is 10mg/mL, gets 2.5 μ L and join in the mixed solution in step 3 after preparing, and vortex mixes for 10 seconds, under room temperature lucifuge condition standing 30 minutes;
(5) add the 2.5 freshly prepared EDC solution of μ L (purchased from Sigma), vortex mixes for 10 seconds again, under room temperature lucifuge condition standing 30 minutes again;
(6), after EDC finishes dealing with, add 1mLTween-20 solution (0.02%), after vortex mixes, centrifugal 3 minutes of 12,000rpm;
(7) remove supernatant, add 1mL SDS solution (0.1%), after vortex mixes, centrifugal 3 minutes of 12,000rpm;
(8) remove supernatant, add 125 μ L TE damping fluids (pH8), mix rear 4 ℃ of preservations.
According to table 5 ratio, carry out microballoon and detect liquid preparation.
Table 5
| Reagent | μL |
| Microballoon mixed solution | 10 |
| Detect damping fluid | 35 |
| Cumulative volume | 45 |
In 96 orifice plates, every hole adds 45 μ L microballoons to detect liquid, after fully mixing, adds 5 μ L PCR liquid to be measured, and piping and druming mixes repeatedly; Hatch 10min for 52 ℃; According to the preparation of table 6 ratio, Streptavidin-PE detects liquid.
Table 6
| Reagent | μL |
| Streptavidin-PE | 0.6 |
| Detect damping fluid | 9.4 |
| Cumulative volume | 10μL |
Every hole adds 10 μ L Streptavidin-PE to detect liquid again, hatches 5min for 52 ℃; Every hole adds 120 μ L stop buffers, and liquid-phase chip detects.
Embodiment 4, sensitivity detect
A. the detection bacterium DNA of take determines its minimum detectability as detecting target
Extract respectively the DNA of 13 kinds of pathogenic bacterium, with sterilized water, be diluted to Gradient: 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L and 0.001ng/ μ L, each detects each DNA dilution gradient of bacterium and gets 1 μ L volume for PCR reaction (with reference to embodiment 2), carry out subsequently liquid-phase chip detection (as embodiment 3), to determine the minimum detectability of its DNA level.
DNA of bacteria detection sensitivity qualification result is in Table 7.
Table 7
| Bacteria types | The low DNA amount (ng) that can detect | |
| 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, with corresponding mix primer (comprising super primer and target gene primer), carry out multiplex PCR detection and liquid-phase chip detection, the results are shown in Table 8.In table, each numerical value is the relative microballoon amount that liquid-phase chip instrument (luminex company) is measured.According to negative mean value and the standard deviation of every result, consider the factors such as error, pcr amplification efficiency simultaneously, determine that numerical value 240 is for threshold value, surpass the positive findings that is decided to be of this value.With SDA specified standards, disturb bacterium E.coli 25922 (FDA provides by Shanghai City) and H
2o is as negative control.In table, the corresponding detected result of the digitized representation of bold Italic mark is positive.Visible, designed primer specificity is good, the target gene DNA of low levels can be detected.
Table 8
B. the detection bacterium colony number of take is determined its minimum detectability as detecting target
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 respectively DNA, carries out multiplex PCR and liquid-phase chip and detects, to determine its lowest detectable limit.
For 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, carry out CFU sensitivity detection, result is as shown in table 9.
Table 9
| Bacterial species | The minimum bacterial count (CFU) that can detect |
| 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 |
Bacteria suspension for different bacterium colony concentration extracts respectively after DNA, with corresponding mix primer (comprising super primer and target gene primer), carries out multiplex PCR and liquid-phase chip detection, and repeatedly, result is as table 10 for every kind of bacterium colony.In table, each numerical value is the relative microballoon amount that liquid-phase chip instrument (luminex company) is measured.According to negative mean value and the standard deviation of every result, consider the factors such as error, pcr amplification efficiency simultaneously, determine that numerical value 240 is for threshold value, surpass the positive findings that is decided to be of this value.With H
2o is as negative control.Wherein, the corresponding detected result of the digitized representation of bold Italic mark is positive.Visible, designed primer specificity is good, and detection accuracy is high, 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 respectively DNA the balanced mix of these 8 kinds of bacteriums.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 respectively 1ng/ μ L, 0.1ng/ μ L and 0.01ng/ μ L in total DNA mixed solution gradient.The DNA mixed solution of getting respectively the different gradients of 1 μ L volume reacts for PCR, carries out subsequently liquid-phase chip detection, and liquid-phase chip result and actual value are contrasted, to verify that its multiplicity detects performance.
The results are shown in Table 11.In table, each numerical value is the relative microballoon amount that liquid-phase chip instrument (luminex company) is measured.According to negative mean value and the standard deviation of every result, consider the factors such as error, pcr amplification efficiency simultaneously, determine that numerical value 240 is for 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, by contrasting with randomly drawing raw data, its accuracy can be confirmed.When DNA mixed solution total amount is reduced to 0.8ng, Vibrio p cannot detect, and when DNA mixed solution total amount is reduced to 0.08ng, E.coli ETEC, E.coli O157, Vibrio p and Y.enterocolitica cannot detect.As can be seen here, in the present invention, when lowest detection is limited to 1ng, in same reaction system, at least 8 kinds of bacteriums can be detected simultaneously.Wherein, the corresponding detected result of the digitized representation of bold Italic mark is positive.
Table 11
Embodiment 6, full pattern mark-on detect
Take Salmonellas, proteus vulgaris and streptococcus aureus as detecting 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 respectively 3 kinds of solid food samples (25g) and 3 kinds of liquid food samples (25ml), add 225ml substratum, add gradient to detect the thalline liquid of 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 turn/min concussions; Preliminary filtration, gets nutrient solution 200ml, the centrifugal 5min of 10000rpm after removal food debris; Bacterial sediment is resuspended in the aseptic ddH of 100 μ l
2in O, boiling water bath 15min broken cell; After fully concussion mixes, the centrifugal 5min of 10000rpm; Get supernatant liquor as nucleic acid-templated, carry out PCR, and then carry out liquid-phase chip detection (referring to Fig. 2 flow process).
In full pattern mark-on test experience, owing to being subject to the interference of food impurity, the lowest detectable limit of selected three kinds of bacteriums in this detection, compares and all raises to some extent an order of magnitude with its CFU detectability, and Bacillusproleus lowest detection is limited to 10
4cFU, Salmonella lowest detection is limited to 10
3cFU, 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 met that existing (existing testing requirement is generally 10 to the requirement of Microbiological detection of foods completely
5cFU is following).
Table 12
Multiplex PCR detected result is as table 13.In table, each numerical value is the relative microballoon amount that liquid-phase chip instrument (luminex company) is measured.According to negative mean value and the standard deviation of every result, consider the factors such as error, pcr amplification efficiency simultaneously, determine that numerical value 240 is for threshold value, surpass the positive findings that is decided to be of this value.With H
2o is as negative control.Wherein, the corresponding detected result of the digitized representation of bold Italic mark is positive.Visible, no matter for liquid or solid, method detection sensitivity of the present invention is high, and result is reliable and stable.
Table 13
In general, liquid-phase chip detection method is as a kind of novel molecular biology Fast Detection Technique, there is quick, highly sensitive, high specificity easy and simple to handle, inexpensive, sample aequum is few, can high-throughput, the feature such as detection by quantitative and applicable field screening, can be applied to great public food safety and food poisoning rapid detection project.
All documents of mentioning in the present invention are all quoted as a reference in this application, just as each piece of document, are quoted as a reference separately.In addition should be understood that 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 limited range equally.
Sequence table
<110> Shanghai Inst. of Life Science, CAS
<120> fast detection reagent kit for pathogenic microorganism
<130>100284Z1
<160>107
<170>PatentIn version 3.3
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ttctttgcgt tatgtctctg aaagcttctc gcctgcctcc 40
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caggccacgt tttgtcatgc cacttgaata tcctaactcc 40
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ttctttgcgt tatgtctctg tatgtgtatg gtaaagaaac 40
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ttctttgcgt tatgtctctg tgtaagtaag cacgtcctgg 40
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Claims (9)
1. a non-diagnostic method for rapid inspecting animalcule, is characterized in that, described method comprises:
(1), for following microorganism to be detected, determine respectively specific target gene;
(2) each target gene of determining according to step (1), determines respectively its specific pair of inside primer and pair of outside primer, and the sequence of described inner side primer can be complementary with corresponding target gene part, again can be complementary with corresponding super primer part; Described super primer is a primer pair, and the super primer sequence of forward is as shown in SEQ ID NO:1, and reverse super primer sequence is as shown in SEQ ID NO:2; Mix above-mentioned inner side primer, outside primer, the super primer of forward, reverse super primer, obtain primer mixture;
(3) take testing sample as pcr template, take the primer mixture of step (2) as primer carries out PCR reaction, obtain pcr amplification system; Wherein, the amount of the super primer of forward is 3-10 times of the primer that each target gene is corresponding, and the amount of reverse super primer is 10-50 times of the primer that each target gene is corresponding;
(4) in the pcr amplification system of step (3), add the probe with detectable signal, described probe can be complementary with corresponding target gene specifically, and different for different target gene detectable signals; With
(5) identify detectable signal, thereby determine the kind of microorganism;
Primer and the probe of described microorganism, its target gene and correspondence thereof are as follows:
Listeria monocytogenes (Listeria monocytogenes): induction Actin muscle assembling precursor protein gene, primer is as shown in SEQ ID NO:8-11, and probe is as shown in SEQ ID NO:12;
Shigellae (Shigella): aggressive plasmid H antigen gene, primer is as shown in SEQ ID NO:13-16, and probe is as shown in SEQ ID NO:17;
Salmonellas (Salmonella): invasin protein A gene, primer is as shown in SEQ ID NO:18-21, and probe is as shown in SEQ ID NO:22;
Bacillus proteus (Bacillus proleus): F type ATP enzyme beta subunit gene, primer is as shown in SEQ ID NO:23-26, and probe is as shown in SEQ ID NO:27;
Bacillus cereus (Bacillus cereus): enterotoxin T gene, primer is as shown in SEQ ID NO:33-36, and probe is as shown in SEQ ID NO:37;
Intestinal bacteria ETEC (Escherichia coli ETEC): heat-labile toxin AB subunit gene, primer is as shown in SEQ ID NO:43-46, and probe is as shown in SEQ ID NO:47;
Streptococcus aureus (Staphylococcus aureus): thermostability nuclease gene, primer is as shown in SEQ ID NO:53-56, and probe is as shown in SEQ ID NO:57;
Campylobacter jejuni (Campylobacter jejuni): gyrase A subunit gene, primer is as shown in SEQ ID NO:63-66, and probe is as shown in SEQ ID NO:67;
Escherichia coli O 157 (Escherichia coli O157): O-antigen-specific gene, primer is as shown in SEQ ID NO:73-76, and probe is as shown in SEQ ID NO:77;
Campylobacter coli (Campylobacter coli): siderophore transfer related protein gene, primer is as shown in SEQ ID NO:83-86, and probe is as shown in SEQ ID NO:87;
Yersinia enterocolitica (Yersinia enterocolitica): stick invasin protein gene, primer is as shown in SEQ ID NO:88-91, and probe is as shown in SEQ ID NO:92;
Vibrio cholerae (Vibrio cholerae): hemolysin protein A gene, primer is as shown in SEQ ID NO:93-96, and probe is as shown in SEQ ID NO:97; With
Vibrio parahemolyticus (Vibrio parahaemolyticu): heat-resisting hemolytic toxin gene, primer is as shown in SEQ ID NO:98-101, and probe is as shown in SEQ ID NO:102.
2. the method for claim 1, is characterized in that, 5 ' end of described reverse super primer is with a discernible signal.
3. the method for claim 1, is characterized in that, in step (3), 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。
4. the method for claim 1, is characterized in that, in step (4), described detectable signal is fluorescent microsphere.
5. the method for claim 1, is characterized in that, described microorganism is pathogenic bacterium.
6. method as claimed in claim 5, is characterized in that, described pathogenic bacterium are food-borne pathogens.
7. a microbial rapid detection reagent composition, is characterized in that, described reagent composition comprises:
Super primer, described super primer is a primer pair, and the super primer sequence of forward is as shown in SEQ ID NO:1, and reverse super primer sequence is as shown in SEQ ID NO:2;
For the primer of microorganism target gene to be detected, corresponding to the primer of each target gene, comprise pair of inside primer and pair of outside primer, the sequence of described inner side primer can be complementary with corresponding target gene part, again can be complementary with corresponding super primer part; And
For the probe of microorganism target gene to be detected, described probe is can be specifically complementary with corresponding target gene and with detectable signal, and different for different target gene detectable signals;
Wherein, primer and the probe for microorganism target gene to be detected is:
Corresponding to primer and the probe of Listeria monocytogenes induction Actin muscle assembling precursor protein gene, wherein primer is as shown in SEQ ID NO:8-11, and probe is as shown in SEQ ID NO:12;
Corresponding to primer and the probe of Shigellae aggressive plasmid H antigen gene, wherein primer is as shown in SEQ ID NO:13-16, and probe is as shown in SEQ ID NO:17;
Corresponding to primer and the probe of Salmonellas invasin protein A gene, wherein primer is as shown in SEQ ID NO:18-21, and probe is as shown in SEQ ID NO:22;
Corresponding to primer and the probe of Bacillus proteus F type ATP enzyme beta subunit gene, wherein primer is as shown in SEQ ID NO:23-26, and probe is as shown in SEQ ID NO:27;
Corresponding to primer and the probe of bacillus cereus enterotoxin T gene, wherein primer is as shown in SEQ ID NO:33-36, and probe is as shown in SEQ ID NO:37;
Corresponding to primer and the probe of intestinal bacteria ETEC heat-labile toxin AB subunit gene, wherein primer is as shown in SEQ ID NO:43-46, and probe is as shown in SEQ ID NO:47;
Corresponding to primer and the probe of streptococcus aureus thermostability nuclease gene, wherein primer is as shown in SEQ ID NO:53-56, and probe is as shown in SEQ ID NO:57;
Corresponding to primer and the probe of campylobacter jejuni gyrase A subunit gene, wherein primer is as shown in SEQ ID NO:63-66, and probe is as shown in SEQ ID NO:67;
Corresponding to primer and the probe of Escherichia coli O 157 O-antigen-specific gene, wherein primer is as shown in SEQ ID NO:73-76, and probe is as shown in SEQ ID NO:77;
Corresponding to primer and the probe of campylobacter coli siderophore transfer related protein gene, wherein primer is as shown in SEQ ID NO:83-86, and probe is as shown in SEQ ID NO:87;
The primer and the probe that corresponding to Yersinia enterocolitica, stick invasin protein gene, wherein primer is as shown in SEQ ID NO:88-91, and probe is as shown in SEQ ID NO:92;
Corresponding to primer and the probe of vibrio cholerae hemolysin protein A gene, wherein primer is as shown in SEQ ID NO:93-96, and probe is as shown in SEQ ID NO:97; With
Corresponding to primer and the probe of the heat-resisting hemolytic toxin gene of Vibrio parahemolyticus, wherein primer is as shown in SEQ ID NO:98-101, and probe is as shown in SEQ ID NO:102;
Wherein, the amount of the super primer of forward is 3-10 times of the primer that each target gene is corresponding, and the amount of reverse super primer is 10-50 times of the primer that each target gene is corresponding.
8. the purposes of microbial rapid detection reagent composition claimed in claim 7, for the preparation of the test kit that detects microorganism.
9. a microbial rapid detection reagent box, is characterized in that, contains
Container; And
Be sub-packed in the following reagent in container:
Super primer, described super primer is a primer pair, and the super primer sequence of forward is as shown in SEQ ID NO:1, and reverse super primer sequence is as shown in SEQ ID NO:2;
For the primer of microorganism target gene to be detected, corresponding to the primer of each target gene, comprise pair of inside primer and pair of outside primer, the sequence of described inner side primer can be complementary with corresponding target gene part, again can be complementary with corresponding super primer part; And
For the probe of microorganism target gene to be detected, described probe is can be specifically complementary with corresponding target gene and with detectable signal, and different for different target gene detectable signals;
Wherein, primer and the probe for microorganism target gene to be detected is:
Corresponding to primer and the probe of Listeria monocytogenes induction Actin muscle assembling precursor protein gene, wherein primer is as shown in SEQ ID NO:8-11, and probe is as shown in SEQ ID NO:12;
Corresponding to primer and the probe of Shigellae aggressive plasmid H antigen gene, wherein primer is as shown in SEQ ID NO:13-16, and probe is as shown in SEQ ID NO:17;
Corresponding to primer and the probe of Salmonellas invasin protein A gene, wherein primer is as shown in SEQ ID NO:18-21, and probe is as shown in SEQ ID NO:22;
Corresponding to primer and the probe of Bacillus proteus F type ATP enzyme beta subunit gene, wherein primer is as shown in SEQ ID NO:23-26, and probe is as shown in SEQ ID NO:27;
Corresponding to primer and the probe of bacillus cereus enterotoxin T gene, wherein primer is as shown in SEQ ID NO:33-36, and probe is as shown in SEQ ID NO:37;
Corresponding to primer and the probe of intestinal bacteria ETEC heat-labile toxin AB subunit gene, wherein primer is as shown in SEQ ID NO:43-46, and probe is as shown in SEQ ID NO:47;
Corresponding to primer and the probe of streptococcus aureus thermostability nuclease gene, wherein primer is as shown in SEQ ID NO:53-56, and probe is as shown in SEQ ID NO:57;
Corresponding to primer and the probe of campylobacter jejuni gyrase A subunit gene, wherein primer is as shown in SEQ ID NO:63-66, and probe is as shown in SEQ ID NO:67;
Corresponding to primer and the probe of Escherichia coli O 157 O-antigen-specific gene, wherein primer is as shown in SEQ ID NO:73-76, and probe is as shown in SEQ ID NO:77;
Corresponding to primer and the probe of campylobacter coli siderophore transfer related protein gene, wherein primer is as shown in SEQ ID NO:83-86, and probe is as shown in SEQ ID NO:87;
The primer and the probe that corresponding to Yersinia enterocolitica, stick invasin protein gene, wherein primer is as shown in SEQ ID NO:88-91, and probe is as shown in SEQ ID NO:92;
Corresponding to primer and the probe of vibrio cholerae hemolysin protein A gene, wherein primer is as shown in SEQ ID NO:93-96, and probe is as shown in SEQ ID NO:97; With
Corresponding to primer and the probe of the heat-resisting hemolytic toxin gene of Vibrio parahemolyticus, wherein primer is as shown in SEQ ID NO:98-101, and probe is as shown in SEQ ID NO:102;
Wherein, the amount of the super primer of forward is 3-10 times of the primer that each target gene is corresponding, and the amount of reverse super primer is 10-50 times of the primer that each target gene is corresponding.
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| CN102936621B (en) * | 2012-08-27 | 2014-06-11 | 上海交通大学 | Bacillus cereus detection method and kit |
| CN103484537A (en) * | 2013-07-17 | 2014-01-01 | 新疆农业大学 | PCR detection kit used for salmonella abortus equi, and detection method thereof |
| CN103484546B (en) * | 2013-09-17 | 2015-07-01 | 北京卓诚惠生生物科技有限公司 | Fourteen-food-borne pathogenic bacterium multiplex PCR detection primer set and kit |
| CN103602739A (en) * | 2013-11-13 | 2014-02-26 | 浙江省水产技术推广总站 | Quick typing kit for major serotypes of listeria monocytogenes and application of quick typing kit |
| CN105986029B (en) * | 2015-05-11 | 2019-12-24 | 中国动物卫生与流行病学中心 | Liquid chip method for identifying pig source food source bacteria |
| CN105331610B (en) * | 2015-10-22 | 2018-09-18 | 大连民族大学 | The five weight PCR primers of pathogenic bacteria and probe and kit in a kind of detection fresh and live agricultural product |
| CN106011297A (en) * | 2016-07-30 | 2016-10-12 | 天津市农业质量标准与检测技术研究所 | Real-time fluorescence PCR based method for rapid screening of pathogenic microorganisms in cucumber |
| CN107058463B (en) * | 2016-08-30 | 2020-11-27 | 郑之北 | Primer for identifying salmonella H antigen |
| CN106811535B (en) * | 2017-03-08 | 2020-01-14 | 山东众合天成检验有限公司 | Primer probe composition for simultaneously detecting five pathogenic bacteria and multiple real-time fluorescence PCR method |
| CN106868160A (en) * | 2017-03-21 | 2017-06-20 | 杭州迪安生物技术有限公司 | Primer and its application of various diarrhoea pathogenic bacterias are detected simultaneously |
| CN106995805A (en) * | 2017-03-26 | 2017-08-01 | 海南大学 | A kind of engineering bacteriophage quick detection microorganism of lysozyme mark |
| CN114934045B (en) * | 2022-06-06 | 2024-06-21 | 福建省长汀盼盼食品有限公司 | Probe, chip, kit and method for detecting food microorganisms |
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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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