CN102242218B - 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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- CN102242218B CN102242218B CN201110198185.8A CN201110198185A CN102242218B CN 102242218 B CN102242218 B CN 102242218B CN 201110198185 A CN201110198185 A CN 201110198185A CN 102242218 B CN102242218 B CN 102242218B
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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 histamine entero-bacte.
Background technology
Biogenic amine is the low-molecular weight nitrogen-containing compounds 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 and have the nitrosamine of obvious mutagenicity and potential carinogenicity and indirect carcinogenesis with the food preservatives such as nitrite.
Histamine in food is mainly by the microorganisms of carrying amino acid decarboxylase.Be applied at present histamine determination method in food and mainly contain microbiology method, chromatography, molecular biology method.The ultimate principle of microbiology method is according to histamine, to produce the specificity substratum of condition and strain growth characteristics design.The fast and reliable substratum that do not rely on of molecular biology method, has made up the deficiency of additive method.Polymerase chain reaction (PCR) is as common method in molecular biology, have easy, fast, sensitivity, the feature such as single-minded has become the detection method of important target gene.Food usually can be subject to the pollution of entero-bacte, and this wherein can exist and produce amine bacterium, and the generation bacterium that detects at present histamine mainly lays particular emphasis on milk-acid bacteria, for entero-bacte, rarely has report.
Summary of the invention
The object of this invention is to provide a kind of auxiliary detection and identify the method for producing 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), DNA shown in the sequence 2 of DNA shown in the sequence 1 of sequence table and sequence table, consists of.This special primer is to only can specific amplification to producing the genomic dna of histamine entero-bacte.
Assistant identification provided by the invention is produced the test kit of histamine entero-bacte, comprise described special primer to universal primer pair; Described universal primer (expanding the 16S rDNA fragment of DCPTA bacterium) forms DNA shown in the sequence 4 of DNA shown in the sequence 3 by sequence table and sequence table.Described test kit also comprises can differential medium; The pH of described differential medium is 5.3, water and solute, consists of; Described in every 100mL, in differential medium, contain following solute: 0.5g Tryptones, 0.5g yeast extract, 1.0g L-Histidine, 0.5g sodium-chlor, 0.1g calcium carbonate, 3.0g agar and 0.006g purpurum bromocresolis.At the solid bacteria differential medium that contains Bromocresol purple and Histidine, cultivate, produce 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 histamine entero-bacte.
Described special primer is to producing the test kit of histamine entero-bacte to can be used for preparing assistant identification with described universal primer.
The present invention also protects a kind of assistant identification histamine to produce the method for bacterium; comprise the steps: with described special primer the genomic dna of bacterium to be measured is carried out to pcr amplification; if obtain the product histamine entero-bacte that pcr amplification product bacterium to be measured is candidate, if do not obtain described pcr amplification product bacterium to be measured for candidate's non-product histamine entero-bacte.Described method also can comprise the steps: to take that the genomic dna of described candidate's product histamine entero-bacte is template, with described universal primer, to carrying out, after pcr amplification, checks order, and 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 to cultivate with differential medium, and single bacterium colony of picking purple extracts described genomic dna.With described special primer, when the genomic dna of bacterium to be measured is carried out to 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.With described special primer, when the genomic dna of bacterium to be measured is carried out to pcr amplification, the program of described pcr amplification is: 94 ℃ of 5min; Then carry out 35 circulations, the program of each circulation is 94 ℃ of 30s, 52 ℃ of 45s, 72 ℃ of 40s; After loop ends, 72 ℃ of 7min.With described universal primer, when carrying 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.With described universal primer, when carrying out pcr amplification, the program of described pcr amplification is: 94 ℃ of 5min; Then carry out 35 circulations, the program of each circulation is 94 ℃ of 30s, 56 ℃ of 45s, 72 ℃ of 40s; After 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: with described special primer the genomic dna of sample to be tested is carried out to pcr amplification; if obtain pcr amplification product sample to be tested, be candidate's the sample that produces histamine entero-bacte that contains, if do not obtain the sample that does not contain product histamine entero-bacte that described pcr amplification product sample to be tested is candidate.Described method also can comprise the steps: to take that described candidate's the genomic dna that contains the sample that produces histamine entero-bacte is template, with described universal primer, to carrying out, after pcr amplification, check order, according to sequencing result, judge described candidate's the kind of producing histamine entero-bacte in the sample that produces histamine entero-bacte that contains.The size of described pcr amplification product specifically can be 420bp.Described method also comprises the steps:, by VRBGA substratum, each entero-bacte bacterial strain in sample to be tested is carried out to 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, water and solute, consists of; In every liter of described VRBGA substratum, contain following solute: 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.With described special primer, when the genomic dna of sample to be tested is carried out to 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.With described special primer, when the genomic dna of sample to be tested is carried out to pcr amplification, the program of described pcr amplification is: 94 ℃ of 5min; Then carry out 35 circulations, the program of each circulation is 94 ℃ of 30s, 52 ℃ of 45s, 72 ℃ of 40s; After loop ends, 72 ℃ of 7min.With described universal primer, when carrying out pcr amplification, the system of described pcr amplification is: 10 * Ex Taq Buffer (Mg
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.With described universal primer, when carrying out pcr amplification, the program of described pcr amplification is: 94 ℃ of 5min; Then carry out 35 circulations, the program of each circulation is 94 ℃ of 30s, 56 ℃ of 45s, 72 ℃ of 40s; After 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 produces the product histamine entero-bacte in histamine entero-bacte or assistant identification sample to be tested to can be used for assistant identification.
Described special primer is to producing the product histamine entero-bacte in histamine entero-bacte or assistant identification sample to be tested with described universal primer to can be used for assistant identification.
Utilize primer pair provided by the invention or method not only can the product histamine entero-bacte in leavened food (as soy sauce, fermented bean curd etc.) to be detected, can also apply to any to produce 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 histamine entero-bacte and do not produce histamine bacterium, and by using molecular biological method further to identify, more accurate, reliably.The present invention is specially adapted to assess the risk that in leavened food, histamine forms, and to daily production, can carry out quality control effectively, timely, is a kind of very promising food hygiene detection technology.
Accompanying drawing explanation
Fig. 1 is positive bacterium colony and the negative bacterium colony on differential medium flat board; 1 represents negative bacterium; 2 represent positive bacteria.
Fig. 2 is the agarose electrophoresis result figure of PCR method test set propylhomoserin decarboxylase gene; 1,2 be accredited as positive bacterial strain for the plate assay positive and PCR; 3 for plate assay positive but PCR is accredited as negative bacterial strain.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.
The design of embodiment 1, primer and preparation
According to the conservative region of the L-Histidine decarboxylase. encoding gene (hdc gene) of the various product histamine entero-bacte on NCBI, design special primer to (being formed by primer LB1 and primer LB2).Selection for the universal primer of the 16S rDNA of various product histamine bacterium to (being formed by primer B-for and primer B-for).Synthetic special primer to universal primer pair.
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 '.
In embodiment 2, leavened food, produce the detection and identification of histamine entero-bacte
One, sample process
Asepticly take commercially available soy sauce and become bent 20g, shred and be placed in the triangular flask that 180ml sterile saline is housed under aseptic condition, fully mix, (extent of dilution is 10 on homogenizer, to jolt 10min
-1); Then with sterile saline, carry out 10 times of gradient dilutions, as each sample diluent, (extent of dilution is followed successively by 10
-2, 10
-3...).
Two, separated entero-bacte from sample
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, adjusts pH to 7.4 ± 0.2.
Sample diluent is coated to VRBGA substratum, cultivate 24h for 37 ℃; Single bacterium colony streak inoculation that each form of picking is different from substratum, in VRBGA substratum, is cultivated 24h for 37 ℃; Carry out continuously transferring on new VRBGA substratum for 2 times bacterium colony (streak inoculation) adopt the same terms to cultivate, obtains bacterial strain 16 strains of purifying.
Three, utilize differential medium primary dcreening operation
The preparation of differential medium: 5g Tryptones, 5g yeast extract, 10.0g L-Histidine, 5g sodium-chlor, 1g calcium carbonate, 30.0g agar and 0.06g purpurum bromocresolis water are settled to 1000mL, adjust pH to 5.3 ± 0.1; 121 ℃ of sterilizing 10min.
The bacterial strain that step 2 is obtained adopts respectively an inoculation method to be seeded to differential medium, cultivates 36h for 30 ℃.Fig. 1 is shown in by photo, the product histamine entero-bacte bacterium colony that the bacterium colony of purple is candidate.Totally 3 strain bacterial strains are shown as purple bacterium colony.
Four, PCR identifies
The 3 strain bacterial strains that picking step 3 obtains respectively, extract genomic dna, take genomic dna as template, with the primer pair that LB1 and LB2 form, carry 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; Then carry out 35 circulations, the program of each circulation is 94 ℃ of 30s, 52 ℃ of 45s, 72 ℃ of 40s; After loop ends, 72 ℃ of 7min; Be cooled to 4 ℃, finish.
Pcr amplification product is carried out to 1% agarose gel electrophoresis.The results are shown in Figure 2, the bacterial strain that shows the specific band of is that PCR identifies positive 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 to sequence verification, and result shows that this specific band is 420bp.
Four, Sequence Identification
The genomic dna that respectively PCR is accredited as to 2 positive strain bacterial strains is template, with the primer pair that B-for and B-rev form, carries 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; Then carry out 35 circulations, the program of each circulation is 94 ℃ of 30s, 56 ℃ of 45s, 72 ℃ of 40s; After loop ends, 72 ℃ of 7min, are cooled to 4 ℃, finish.
PCR is accredited as the sequencing result of 2 positive bacterial strains and sees respectively the sequence 5 of sequence table and the sequence 6 of sequence table, all the highest with the homology of separating ornithine Raoul bacterium (Raoultella ornithinolytica) (GENBANK ACCESSION NO.HM231274.1), be 99%.Sequencing result shows, 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 (9)
1. assistant identification is produced the special primer pair of histamine entero-bacte, DNA shown in the sequence 2 of DNA shown in the sequence 1 of sequence table and sequence table, consists of.
2. assistant identification is produced the test kit of histamine entero-bacte, comprise special primer claimed in claim 1 to universal primer pair; Described universal primer forms DNA shown in the sequence 4 of DNA shown in the sequence 3 by sequence table and sequence table.
3. following (a) or (b) application in the test kit of preparation assistant identification product histamine entero-bacte:
(a) special primer pair described in claim 1;
(b) described in claim 1 special primer to universal primer pair; Described universal primer forms DNA shown in the sequence 4 of DNA shown in the sequence 3 by sequence table and sequence table.
4. assistant identification is produced the method for histamine entero-bacte, comprise the steps: with special primer described in claim 1 genomic dna of bacterium to be measured is carried out to pcr amplification, if obtain the product histamine entero-bacte that pcr amplification product bacterium to be measured is candidate, if do not obtain described pcr amplification product bacterium to be measured for candidate's non-product histamine entero-bacte; Described method is non-disease treatment or diagnostic method.
5. method as claimed in claim 4, it is characterized in that: described method also comprises the steps: to take that the genomic dna of described candidate's product histamine entero-bacte is template, with universal primer, to carrying out, after pcr amplification, check order, according to sequencing result, judge the kind of described candidate's product histamine entero-bacte; Described universal primer forms DNA shown in the sequence 4 of DNA shown in the sequence 3 by sequence table and sequence table.
6. the method as described in claim 4 or 5, is characterized in that: described method also comprises the steps: described bacterium to be measured to cultivate with differential medium, and single bacterium colony of picking purple extracts described genomic dna; The pH of described differential medium is 5.3, water and solute, consists of; Described in every 100mL, in differential medium, contain following solute: 0.5g Tryptones, 0.5g yeast extract, 1.0g L-Histidine, 0.5g sodium-chlor, 0.1g calcium carbonate, 3.0g agar and 0.006g purpurum bromocresolis.
7. the method for the product histamine entero-bacte in assistant identification sample to be tested, comprise the steps: with special primer described in claim 1 genomic dna of sample to be tested is carried out to pcr amplification, if obtain pcr amplification product sample to be tested, be candidate's the sample that produces histamine entero-bacte that contains, if do not obtain the sample that does not contain product histamine entero-bacte that described pcr amplification product sample to be tested is candidate; Described method is non-disease treatment and diagnostic method.
8. method as claimed in claim 7, it is characterized in that: described method also comprises the steps: to take that described candidate's the genomic dna that contains the sample that produces histamine entero-bacte is template, with universal primer, to carrying out, after pcr amplification, check order, according to sequencing result, judge described candidate's the kind of producing histamine entero-bacte in the sample that produces histamine entero-bacte that contains; Described universal primer forms DNA shown in the sequence 4 of DNA shown in the sequence 3 by sequence table and sequence table.
9. method as claimed in claim 7 or 8, is characterized in that: described method also comprises the steps:, by VRBGA substratum, each entero-bacte bacterial strain in sample to be tested is carried out to 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, water and solute, consists of; In every liter of described VRBGA substratum, contain following solute: 3.0g yeast extract, 7.0g peptone, 5.0g sodium-chlor, No. 3 cholate of 1.5g, 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, water and solute, consists of; Described in every 100mL, in differential medium, contain following solute: 0.5g Tryptones, 0.5g yeast extract, 1.0g L-Histidine, 0.5g sodium-chlor, 0.1g calcium carbonate, 3.0g agar and 0.006g purpurum bromocresolis.
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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 (4)
| Title |
|---|
| 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;Hajime Takahashi等;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;20030531;第69卷(第5期);2568–2579 * |
| Emmanuel Coton等.Multiplex PCR for colony direct detection of Gram-positive histamine- and tyramine-producing bacteria.《Journal of Microbiological Methods》.2005,第63卷296– 304. |
| HajimeTakahashi等.CloningandSequencingoftheHistidineDecarboxylaseGenesofGram-Negative Histamine-Producing Bacteria and Their Application in Detection and Identification of These Organisms in Fish.《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》.2003 |
| Multiplex PCR for colony direct detection of Gram-positive histamine- and tyramine-producing bacteria;Emmanuel Coton等;《Journal of Microbiological Methods》;20050601;第63卷;296– 304 * |
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