CN102242218A - Method for assisting in detecting and identifying histamine-producing enterobacteria - Google Patents
Method for assisting in detecting and identifying histamine-producing enterobacteria Download PDFInfo
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- CN102242218A CN102242218A CN2011101981858A CN201110198185A CN102242218A CN 102242218 A CN102242218 A CN 102242218A CN 2011101981858 A CN2011101981858 A CN 2011101981858A CN 201110198185 A CN201110198185 A CN 201110198185A CN 102242218 A CN102242218 A CN 102242218A
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Abstract
The invention discloses a method for assisting in detecting and identifying histamine-producing enterobacteria. In the method, a specific primer pair for assisting in identifying the histamine-producing enterobacteria is provided, consists of deoxyribonucleic acids (DNAs) shown as a sequence 1 and a sequence 2 in a sequence table and can specifically amplify the genomic DNA of the histamine-producing enterobacteria. The method comprises the following steps of: culturing a sample in a solid bacterial differential medium containing bromcresol purple and histidine, and screening potential histamine-producing enterobacteria; amplifying a histidine decarboxylase (hdc) gene, and further identifying; and amplifying the 16S ribosomal DNA (rDNA) segment of the histamine-producing enterobacteria to identify the type of the histamine-producing enterobacteria. The method is simple and quick, and the histamine-producing enterobacteria are easy to identify, can be distinguished from non-histamine-producing enterobacteria on the same flat plate, and is further identified by a molecular biological method to achieve higher accuracy and reliability; and the method is particularly suitable for evaluating the risk of histamine formed in fermented food, can be used for performing quality control on daily production effectively and timely, and is a food hygiene detection technology with a wide application prospect.
Description
Technical field
The present invention relates to a kind of auxiliary detection and identify the method for producing the histamine entero-bacte.
Background technology
Biogenic amine is the lower molecular weight nitrogenous compound that a class is mainly formed by amino acid decarboxylase or aldehyde and ketone amination.Mainly contain putrescine, cadaverine, phenylethylamine, spermine, spermidine, histamine, tryptamines and tyrasamine.When the biogenic amine of human body excess intake, can cause the damage of nerve system of human body and cardiovascular systems.In addition, biogenic amine also can generate nitrosamine with obvious mutagenicity and potential carinogenicity and indirect carcinogenesis with food preservatives such as nitrite.
Histamine in the food mainly is by the microorganisms of carrying amino acid decarboxylase.Be applied at present that histamine check and analysis method mainly contains microbiology method, chromatography, molecular biology method in the food.The ultimate principle of microbiology method is the specificity substratum that produces condition and strain growth characteristics design according to histamine.The fast and reliable substratum that do not rely on of molecular biology method has remedied the deficiency of additive method.Polymerase chain reaction (PCR) is as common method in the molecular biology, have easy, fast, sensitivity, characteristics such as single-minded have become the detection method of important target gene.Food usually can be subjected to the pollution of entero-bacte, and wherein can there be product amine bacterium in this, and the generation bacterium that detects histamine at present mainly lays particular emphasis on milk-acid bacteria, then rarely has report for entero-bacte.
Summary of the invention
The purpose of this invention is to provide a kind of auxiliary detection and identify the method for producing the histamine entero-bacte.
Assistant identification provided by the invention is produced the special primer of histamine entero-bacte to (amplification L-Histidine decarboxylase. hdc gene), is made up of DNA shown in the sequence 2 of DNA shown in the sequence 1 of sequence table and sequence table.This special primer is to only can specific amplification to the genomic dna that produces the histamine entero-bacte.
Assistant identification provided by the invention is produced the test kit of histamine entero-bacte, comprises that described special primer is to right with universal primer; Described universal primer (expanding the 16S rDNA fragment of DCPTA bacterium) is to being made up of DNA shown in the sequence 4 of DNA shown in the sequence 3 of sequence table and sequence table.But described test kit also comprises differential medium; The pH of described differential medium is 5.3, is made up of water and solute; Contain following solute in the described differential medium of every 100mL: 0.5g Tryptones, 0.5g yeast extract, 1.0g L-Histidine, 0.5g sodium-chlor, 0.1g lime carbonate, 3.0g agar and 0.006g purpurum bromocresolis.Cultivate at the solid bacteria differential medium that contains purpurum bromocresolis indicator and Histidine, produce the histamine bacterium and be shown as purple.
Described special primer is to can be used for preparing the test kit that assistant identification is produced the histamine entero-bacte.
Described special primer is to producing the test kit of histamine entero-bacte with described universal primer to can be used for preparing assistant identification.
The present invention also protects a kind of assistant identification histamine to produce the method for bacterium; comprise the steps: the genomic dna to bacterium to be measured to be carried out pcr amplification with described special primer; if obtain the product histamine entero-bacte of pcr amplification product bacterium to be measured, if do not obtain the non-product histamine entero-bacte of described pcr amplification product bacterium to be measured for the candidate for the candidate.Described method can comprise the steps: that also the genomic dna with described candidate's product histamine entero-bacte is a template, checks order behind the pcr amplification to carrying out with described universal primer, judges the kind of described candidate's product histamine entero-bacte according to sequencing result.The size of described pcr amplification product specifically can be 420bp.Described method also can comprise the steps: described bacterium to be measured is cultivated with differential medium, and single bacterium colony of picking purple extracts described genomic dna.When with described special primer the genomic dna to bacterium to be measured being carried out pcr amplification, the system of described pcr amplification is: 10 * Ex TaqBuffer (Mg
2+Plus) 5 μ L, dNTP Mixture 4 μ L, every primer 2 μ L, Ex Taq (5U/ml) 0.4 μ L, template 1 μ L, ddH
2O 35.6 μ L.When with described special primer the genomic dna to bacterium to be measured being carried out pcr amplification, the program of described pcr amplification is: 94 ℃ of 5min; Carry out 35 circulations then, each round-robin program is 94 ℃ of 30s, 52 ℃ of 45s, 72 ℃ of 40s; After the loop ends, 72 ℃ of 7min.When carrying out pcr amplification, the system of described pcr amplification is: 10 * Ex Taq Buffer (Mg with described universal primer
2+Plus) 5 μ L, dNTP Mixture 4 μ L, every primer 2 μ L, Ex Taq (5U/ml) 0.4 μ L, template 1 μ L, ddH
2O 35.6 μ L.When carrying out pcr amplification, the program of described pcr amplification is: 94 ℃ of 5min with described universal primer; Carry out 35 circulations then, each round-robin program is 94 ℃ of 30s, 56 ℃ of 45s, 72 ℃ of 40s; After the loop ends, 72 ℃ of 7min.Described bacterium to be measured specifically can be separates ornithine Raoul bacterium (Raoultella ornithinolytica).
The present invention also protects the method for the product histamine entero-bacte in a kind of assistant identification sample to be tested; comprise the steps: the genomic dna to sample to be tested to be carried out pcr amplification with described special primer; if obtain the contain sample that produce histamine entero-bacte of pcr amplification product sample to be tested, if do not obtain the sample that do not contain product histamine entero-bacte of described pcr amplification product sample to be tested for the candidate for the candidate.Described method can comprise the steps: that also the genomic dna that contains the sample that produces the histamine entero-bacte with described candidate is a template, check order behind the pcr amplification to carrying out with described universal primer, judge described candidate's the kind of producing the histamine entero-bacte in the sample that produces the histamine entero-bacte that contains according to sequencing result.The size of described pcr amplification product specifically can be 420bp.Described method also comprises the steps: by the VRBGA substratum each the entero-bacte bacterial strain in the sample to be tested to be carried out purifying, obtains the bacterial strain of pure culture; The bacterial strain of each pure culture is cultivated with described differential medium respectively, and single bacterium colony of picking purple extracts described genomic dna; The pH of described VRBGA substratum is in 7.4 ± 0.2 scopes, is made up of water and solute; Contain following solute in every liter of described VRBGA substratum: 3.0g yeast extract, 7.0g peptone, 5.0g sodium-chlor, 1.5g3 cholate, 10.0g lactose, 10.0g glucose, 0.03g toluylene red, 0.002g Viola crystallina, 15.0g agar.When with described special primer the genomic dna to sample to be tested being carried out pcr amplification, the system of described pcr amplification is: 10 * Ex Taq Buffer (Mg
2+Plus) 5 μ L, dNTP Mixture 4 μ L, every primer 2 μ L, Ex Taq (5U/ml) 0.4 μ L, template 1 μ L, ddH
2O 35.6 μ L.When with described special primer the genomic dna to sample to be tested being carried out pcr amplification, the program of described pcr amplification is: 94 ℃ of 5min; Carry out 35 circulations then, each round-robin program is 94 ℃ of 30s, 52 ℃ of 45s, 72 ℃ of 40s; After the loop ends, 72 ℃ of 7min.When carrying out pcr amplification, the system of described pcr amplification is: 10 * Ex Taq Buffer (Mg with described universal primer
2+Plus) 5 μ L, dNTP Mixture4 μ L, every primer 2 μ L, Ex Taq (5U/ml) 0.4 μ L, template 1 μ L, ddH
2O 35.6 μ L.When carrying out pcr amplification, the program of described pcr amplification is: 94 ℃ of 5min with described universal primer; Carry out 35 circulations then, each round-robin program is 94 ℃ of 30s, 56 ℃ of 45s, 72 ℃ of 40s; After the loop ends, 72 ℃ of 7min.Described sample to be tested specifically can be and contains the sample of separating ornithine Raoul bacterium (Raoultella ornithinolytica).
Described special primer is to can be used for the product histamine entero-bacte in assistant identification product histamine entero-bacte or the assistant identification sample to be tested.
Described special primer is to producing product histamine entero-bacte in histamine entero-bacte or the assistant identification sample to be tested with described universal primer to can be used for assistant identification.
Utilize primer provided by the invention to or method not only can the product histamine entero-bacte in the leavened food (as soy sauce, fermented bean curd etc.) be detected, can also apply to any with produce the histamine entero-bacte and differentiate relevant detection.The inventive method is simple, fast, be easy to identification, can on same flat board, distinguish and produce the histamine entero-bacte and do not produce the histamine bacterium, and by using molecular biological method further to identify, accurate more, reliably.The present invention is specially adapted to assess the risk that histamine forms in the leavened food, can carry out quality control effectively, timely to daily production, is a kind of very promising food hygiene detection technology.
Description of drawings
Fig. 1 is positive bacterium colony and the negative bacterium colony on the differential medium flat board; 1 represents negative bacterium; 2 represent positive bacteria.
Fig. 2 is the agarose electrophoresis figure as a result of PCR method test set propylhomoserin decarboxylase gene; 1,2 is that the dull and stereotyped positive and the PCR of identifying is accredited as the male bacterial strain; 3 are accredited as the positive but PCR is accredited as negative bacterial strain for dull and stereotyped.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.
According to the conservative region design special primer of the L-Histidine decarboxylase. encoding gene (hdc gene) of the various product histamine entero-bacte on the NCBI to (forming) by primer LB1 and primer LB2.Selection at the universal primer of the 16S rDNA of various product histamine bacterium to (forming) by primer B-for and primer B-for.Synthetic special primer is to right with universal primer.
LB1 (sequence 1 of sequence table): 5 '-AAGATACCCATTACTCCGTG-3 ';
LB2 (sequence 2 of sequence table): 5 '-TCTACAGAGATCCGATCGAC-3 '.
B-for (sequence 3 of sequence table): 5 '-AGAGTTTGATCCTGGCTCAG-3 ';
B-rev (sequence 4 of sequence table): 5 '-AAGGAGGTGATCCAGCCGCA-3 '.
Produce the detection and the evaluation of histamine entero-bacte in embodiment 2, the leavened food
One, sample process
Asepticly take by weighing commercially available soy sauce and become bent 20g, shred under the aseptic condition and be placed in the triangular flask that the 180ml sterile saline is housed, abundant mixing, (extent of dilution is 10 to jolt 10min on homogenizer
-1); Carry out 10 times of gradient dilutions with sterile saline then, (extent of dilution is followed successively by 10 as each sample diluent
-2, 10
-3...).
Two, from sample, separate entero-bacte
The preparation of Viola crystallina toluylene red cholate glucose agar medium (VRBGA substratum): 3.0g yeast extract, 7.0g peptone, 5.0g sodium-chlor, 1.5g3 cholate, 10.0g lactose, 10.0g glucose, 0.03g toluylene red, 0.002g Viola crystallina, 15.0g agar are settled to 1000mL with distilled water, heating for dissolving is transferred pH to 7.4 ± 0.2.
The sample diluent is coated the VRBGA substratum, cultivate 24h for 37 ℃; Single bacterium colony streak inoculation that each form of picking is different from the substratum is cultivated 24h for 37 ℃ in the VRBGA substratum; Carry out continuously transferring on new VRBGA substratum for 2 times bacterium colony (streak inoculation) and adopt the same terms to cultivate obtains bacterial strain 16 strains of purifying.
Three, utilize the differential medium primary dcreening operation
The preparation of differential medium: 5g Tryptones, 5g yeast extract, 10.0g L-Histidine, 5g sodium-chlor, 1g lime carbonate, 30.0g agar and 0.06g purpurum bromocresolis water are settled to 1000mL, transfer pH to 5.3 ± 0.1; 121 ℃ of sterilization 10min.
The bacterial strain that step 2 is obtained adopts an inoculation method to be seeded to differential medium respectively, cultivates 36h for 30 ℃.Photo is seen Fig. 1, and the bacterium colony of purple is candidate's a product histamine entero-bacte bacterium colony.Totally 3 strain bacterial strains are shown as the purple bacterium colony.
Four, PCR identifies
The 3 strain bacterial strains that obtain of picking step 3 extract genomic dna respectively, are template with the genomic dna, with the primer of LB1 and LB2 composition to carrying out pcr amplification.
Pcr amplification system (50 μ L): 10 * Ex Taq Buffer (Mg2+plus), 5 μ L, dNTP Mixture (each 2.5mM) 4 μ L, each 2 μ L of LB1 and LB2, Ex Taq (5U/ml) 0.4 μ L, genomic dna 1 μ L, ddH
2O35.6 μ L.
Pcr amplification program: 94 ℃ of 5min; Carry out 35 circulations then, each round-robin program is 94 ℃ of 30s, 52 ℃ of 45s, 72 ℃ of 40s; After the loop ends, 72 ℃ of 7min; Be cooled to 4 ℃, finish.
Pcr amplification product is carried out 1% agarose gel electrophoresis.The results are shown in Figure 2, the bacterial strain that shows one specific band is that PCR identifies the male bacterial strain.In the bacterial strain of the 3 strain pure cultures that step 2 obtains, there are 2 strain PCR to be accredited as the positive.Pcr amplification product is carried out sequence verification, and the result shows that this specific band is 420bp.
Four, Sequence Identification
The genomic dna that respectively PCR is accredited as male 2 strain bacterial strains is a template, and the primer of forming with B-for and B-rev is to carrying out pcr amplification, and the product of pcr amplification is checked order.
Pcr amplification system (50 μ L): 10 * Ex Taq Buffer (Mg
2+Plus) 5 μ L, dNTP Mixture (each 2.5mM) 4 μ L, each 2 μ L of B-for and B-for, Ex Taq (5U/ml) 0.4 μ L, template 1 μ L, ddH
2O35.6 μ L.
Pcr amplification program: 94 ℃ of 5min; Carry out 35 circulations then, each round-robin program is 94 ℃ of 30s, 56 ℃ of 45s, 72 ℃ of 40s; After the loop ends, 72 ℃ of 7min are cooled to 4 ℃, finish.
PCR is accredited as the sequencing result of 2 bacterial strains of male and sees the sequence 5 of sequence table and the sequence 6 of sequence table respectively, all the highest with the homology of separating ornithine Raoul bacterium (Raoultella ornithinolytica) (GENBANK ACCESSION NO.HM231274.1), be 99%.Sequencing result shows that two strain bacterium are Klebsiella ornithinolytica (Raoultella ornithinolytica).Through form, Physiology and biochemistry and Molecular Identification, this two strains bacterium is really for separating ornithine Raoul bacterium (Raoultella ornithinolytica).
Claims (10)
1. the special primer of assistant identification product histamine entero-bacte is right, is made up of DNA shown in the sequence 2 of DNA shown in the sequence 1 of sequence table and sequence table.
2. assistant identification is produced the test kit of histamine entero-bacte, comprises that the described special primer of claim 1 is to right with universal primer; Described universal primer is to being made up of DNA shown in the sequence 4 of DNA shown in the sequence 3 of sequence table and sequence table.
3. following (a) or (b) application in the test kit of preparation assistant identification product histamine entero-bacte:
(a) the described special primer of claim 1 is right;
(b) the described special primer of claim 1 is to right with universal primer; Described universal primer is to being made up of DNA shown in the sequence 4 of DNA shown in the sequence 3 of sequence table and sequence table.
4. assistant identification is produced the method for histamine entero-bacte, comprise the steps: the genomic dna to bacterium to be measured to be carried out pcr amplification with the described special primer of claim 1, if obtain the product histamine entero-bacte of pcr amplification product bacterium to be measured, if do not obtain the non-product histamine entero-bacte of described pcr amplification product bacterium to be measured for the candidate for the candidate.
5. method as claimed in claim 4, it is characterized in that: described method comprises the steps: that also the genomic dna with described candidate's product histamine entero-bacte is a template, check order behind the pcr amplification to carrying out with universal primer, judge the kind of described candidate's product histamine entero-bacte according to sequencing result; Described universal primer is to being made up of DNA shown in the sequence 4 of DNA shown in the sequence 3 of sequence table and sequence table.
6. as claim 4 or 5 described methods, it is characterized in that: described method also comprises the steps: described bacterium to be measured is cultivated with differential medium, and single bacterium colony of picking purple extracts described genomic dna; The pH of described differential medium is 5.3, is made up of water and solute; Contain following solute in the described differential medium of every 100mL: 0.5g Tryptones, 0.5g yeast extract, 1.0g L-Histidine, 0.5g sodium-chlor, 0.1g lime carbonate, 3.0g agar and 0.006g purpurum bromocresolis.
7. the method for the product histamine entero-bacte in the assistant identification sample to be tested, comprise the steps: the genomic dna to sample to be tested to be carried out pcr amplification with the described special primer of claim 1, if obtain the contain sample that produce histamine entero-bacte of pcr amplification product sample to be tested, if do not obtain the sample that do not contain product histamine entero-bacte of described pcr amplification product sample to be tested for the candidate for the candidate.
8. method as claimed in claim 7, it is characterized in that: described method comprises the steps: that also the genomic dna that contains the sample that produces the histamine entero-bacte with described candidate is a template, check order behind the pcr amplification to carrying out with universal primer, judge described candidate's the kind of producing the histamine entero-bacte in the sample that produces the histamine entero-bacte that contains according to sequencing result; Described universal primer is to being made up of DNA shown in the sequence 4 of DNA shown in the sequence 3 of sequence table and sequence table.
9. as claim 7 or 8 described methods, it is characterized in that: described method also comprises the steps: by the VRBGA substratum each the entero-bacte bacterial strain in the sample to be tested to be carried out purifying, obtains the bacterial strain of pure culture; The bacterial strain of each pure culture is cultivated with differential medium respectively, and single bacterium colony of picking purple extracts described genomic dna; The pH of described VRBGA substratum is in 7.4 ± 0.2 scopes, is made up of water and solute; Contain following solute in every liter of described VRBGA substratum: 3.0g yeast extract, 7.0g peptone, 5.0g sodium-chlor, 1.5g3 cholate, 10.0g lactose, 10.0g glucose, 0.03g toluylene red, 0.002g Viola crystallina and 15.0g agar; The pH of described differential medium is 5.3, is made up of water and solute; Contain following solute in the described differential medium of every 100mL: 0.5g Tryptones, 0.5g yeast extract, 1.0g L-Histidine, 0.5g sodium-chlor, 0.1g lime carbonate, 3.0g agar and 0.006g purpurum bromocresolis.
10. following (a) or (b) produce application in the product histamine entero-bacte in histamine entero-bacte or the assistant identification sample to be tested in assistant identification:
(a) the described special primer of claim 1 is right;
(b) the described special primer of claim 1 is to right with universal primer; Described universal primer is to being made up of DNA shown in the sequence 4 of DNA shown in the sequence 3 of sequence table and sequence table.
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| CN201110198185.8A CN102242218B (en) | 2011-07-15 | 2011-07-15 | Method for assisting in detecting and identifying histamine-producing enterobacteria |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101215597A (en) * | 2007-12-26 | 2008-07-09 | 广东省微生物研究所 | Bacillus cereus specificity color biochemical fast detection kit and detection method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101215597A (en) * | 2007-12-26 | 2008-07-09 | 广东省微生物研究所 | Bacillus cereus specificity color biochemical fast detection kit and detection method |
Non-Patent Citations (2)
| Title |
|---|
| EMMANUEL COTON等: "Multiplex PCR for colony direct detection of Gram-positive histamine- and tyramine-producing bacteria", 《JOURNAL OF MICROBIOLOGICAL METHODS》 * |
| HAJIME TAKAHASHI等: "Cloning and Sequencing of the Histidine Decarboxylase Genes of Gram-Negative, Histamine-Producing Bacteria and Their Application in Detection and Identification of These Organisms in Fish", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
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