CN109825618B - Dual PCR detection method for detecting bee-brood cocci and bee larva bacillus - Google Patents
Dual PCR detection method for detecting bee-brood cocci and bee larva bacillus Download PDFInfo
- Publication number
- CN109825618B CN109825618B CN201910258230.0A CN201910258230A CN109825618B CN 109825618 B CN109825618 B CN 109825618B CN 201910258230 A CN201910258230 A CN 201910258230A CN 109825618 B CN109825618 B CN 109825618B
- Authority
- CN
- China
- Prior art keywords
- bee
- bacillus
- melissococcus
- detecting
- bees
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a dual PCR detection method for detecting melissococcus apis and bacillus larvae of bees, which is used for detecting the melissococcus apis and the bacillus larvae of the bees in bees and bee products. The method has high specificity for detecting the melissococcus nidus and the bacillus larvae of the bees, has no cross reaction with each other and other bee pathogenic bacteria, and has good repeatability; the sensitivity for detecting the two pathogenic bacteria can reach about 1 pg/mu L; expensive instruments and reagents are not needed in the whole operation, two pathogens can be detected simultaneously by one operation, and the whole reaction is completed within 2 hours. The invention provides a new technical means for rapidly detecting the bee brood cocci and the bee larval bacillus in the bees and bee products, and has important practical significance and practical application value for protecting the health and safety of domestic bee colonies and promoting the health development of the international trade of bee products.
Description
Technical Field
The invention relates to a dual PCR detection method for detecting meliococcus alvei and bacillus larvae of bees, belongs to the technical field of biology, and is suitable for detecting the meliococcus alvei and the bacillus larvae of the bees in bees and bee products.
Background
American Foulbrood (AFB) and European Foulbrood (EFB) are two bacterial, acute and destructive infectious diseases which can cause death of bee larvae, can cause massive death of the bee larvae and finally cause death of the whole bee colony, and have huge harm. The latest famous book of epidemic disease of imported animals of the people's republic of China, which is jointly made by the Ministry of agriculture and the quality inspection Bureau, classifies the Ministry of agriculture and the quality inspection Bureau as two types of infectious diseases. The world animal health Organization (OIE) also lists the bee products in a quarantine directory as import and export bees and bee product quarantine objects.
American foulbrood is one of the most major bacterial diseases which endanger the growth of bee larvae at present, and mild people reduce the reproductive and collecting capacity of bee colonies, and severe people cause the overbearing of bee colonies. The disease is spread rapidly and seriously, is difficult to cure after the disease occurs, is difficult to eradicate, and is recurrent frequently, especially 1 day old bee larvae are very sensitive to the larval bacillus and die quickly once infected.
European foul brood is a malignant infectious disease of bee larvae, has rapid spread and great harm, and can cause the death of the larvae in large quantities. The sick bee colony can not collect honey and breed normally, and the bee colony is weakened or even covered. Through epidemiological investigation, which kind of bees are resistant to European foul brood is not found at present, eastern bees are more susceptible than western bees, and particularly, the disease attack of Chinese bees is more serious.
The causative agent of American foul brood is Bacillus Larvae (Paenibacillus Larvae, PL), a gram-positive bacterium in the form of a spore. The generated spores are surrounded by a 7-layer structure, and the special structure ensures that the spores of the larval bacillus have extremely strong vitality and extremely strong resistance to heat, chemical substances and the like, and can survive for at least 35 years in severe environments such as high-temperature drying and the like. The spores suspended in honey survive for 4-6 weeks under sunlight. The extra-strong resistance of the larval bacillus to the external environment enables the larval bacillus to survive for a long time in honey, royal jelly, propolis and bee pollen. Bee European foul brood is caused by the bee hives, i.e., melissococcus Pluton (MP), which is a gram-positive bacterium but has unstable staining characteristics, and sometimes may be a gram-negative bacterium and is facultative anaerobic. The melissococcus meliae has extremely strong resistance to external adverse environment, can survive for about 1 year in honeycomb or honey, can survive for 17 months at room temperature and under dry conditions, can also survive in honey, royal jelly, propolis and bee pollen, and can be transmitted by bee and bee products.
In recent years, as the international trade of bees and bee products is increased, the safety problem of bee blight caused by the increase is receiving attention from all parties.
The research on bee diseases in China is rare, and besides some researches on the biology and epidemiology of the important epidemic diseases of bees and the diagnosis of the bee colony epidemic diseases, the research on the detection of bee pathogens in bee products is almost blank. According to the requirements of the original national quality control bureau, the detection of bee pathogens in bee products is enhanced in the future, and particularly, the quarantine of American foulbrood and European foulbrood of bees is enhanced to prevent the bee pathogens from being transmitted into China through the products.
Aiming at the detection of imported bee products, the quality safety problems such as drug residues, production and processing sanitation, adulteration and the like are mainly focused at present, especially, the control on the antibiotic residues and the artificial pollution in the production and processing process is strict, and sufficient attention is not paid to bee pathogens possibly carried in the bee products. Because of the control of the use of antibiotics for bees, bee epidemic diseases are caused by poor management of some bee farms with poor sanitary management conditions, and some pathogens can be transmitted through products such as honey, so that the risk of pathogen transmission is increased. Meanwhile, as the import of bee products is increased in recent years, a considerable part of the bee products are raw honey, and the possibility of spreading bee diseases is higher. Therefore, it is very necessary to detect the bee epidemic disease of imported bee products and to develop an effective detection method.
Therefore, the invention establishes a double PCR detection method for detecting the bee brood cocci and the bee larva bacillus, can simultaneously detect the two most important pathogenic bacteria of the bees, is applied to the rapid detection of the bees and bee products, and has important practical significance for protecting the healthy development of the domestic bee-keeping industry, preventing the spread of epidemic diseases and promoting the development of the international trade of the bee products.
Disclosure of Invention
The invention aims to provide a dual PCR detection method for detecting bee hive coccus and bee larva bacillus, which is used for detecting bee hive coccus and bee larva bacillus in bees and bee products, has the characteristics of high sensitivity, strong specificity, economy and convenience, and can meet the current requirements of bee epidemic disease diagnosis and quarantine of import and export bee products.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows:
a dual PCR primer for detecting melissococcus nidus and bacillus larvae comprises an upstream primer MP-F and a downstream primer MP-R for amplifying specific fragments of the melissococcus nidus, an upstream primer PL-F and a downstream primer PL-R for amplifying specific fragments of the bacillus larvae, and the nucleotide sequences of the primers are as follows:
an upstream primer MP-F:5 'AGAAGAGTGGCGGCGGACGGGTGA-3';
the downstream primer MP-R:5 'ACCGTCACGAGGAAAACAGTTATCCAC-3';
an upstream primer PL-F:5 'GCCAAGGAAGAACGGCCAGGG-3';
the downstream primer PL-R:5' CCACCTCTGCGTGGCAA-.
The invention also aims to provide a detection method based on the dual PCR primers for detecting the cryptococcus apis cerana and the bacillus larvae, which comprises the following steps:
(1) Double PCR reaction system configuration: 2 XPCR Master Mix 12.5 uL, 10 umol/L upstream and downstream primers MP-F and MP-R are 0.5 uL respectively, 10 umol/L upstream and downstream primers PL-F and PL-R are 0.75 uL respectively, 2 uL of sample DNA to be detected is added, and then 8.0 uL of deionized water is added to make the total reaction volume be 25.0 uL.
(2) Double PCR reaction procedure: pre-denaturation at 95 ℃ for 3 min; 95. denaturation at 45 s, annealing at 64 ℃ for 30 s, and denaturation at 72 ℃ for 45 s for 35 cycles; 72. extending at 5 deg.C for 5 min, and keeping at 4 deg.C.
(3) Detecting, analyzing and judging a PCR amplification product: taking 8 mu L of PCR amplification product, carrying out electrophoresis on 1.5% agarose gel added with 0.01% GelRed nucleic acid dye, detecting the PCR amplification product on a gel imaging system, and then judging the result:
when the double PCR amplification product contains a strip with the size of 401 bp, the sample to be detected contains the melissococcus nidus;
when the double PCR amplification product contains a strip with the size of 706 bp, the sample to be detected contains the bacillus larvae of bees;
when the double PCR amplification product contains a strip with the size of 401 bp and a strip with the size of 706 bp at the same time, the sample to be detected contains the melissococcus apis and the bacillus larvae;
when the double PCR amplification product does not contain a strip with the size of 401 bp and does not contain a strip with the size of 706 bp, the sample to be detected does not contain the honey bee cocci and does not contain the bacillus larvae.
The invention has the following advantages:
(1) Good stability and specificity: according to the invention, through a large amount of Blast comparison analysis, highly conserved and specific gene sequences of the melissococcus nidus and the bacillus larvae are screened; two pairs of optimal primers are selected by comparing a plurality of groups of primers; the reaction specificity is further improved by adjusting the annealing temperature, the method has high specificity for detecting the melissococcus apis and the bacillus larvae of the bees, has no cross reaction with each other and other bee pathogenic bacteria, and has better repeatability;
(2) The sensitivity is high: the invention further improves the sensitivity by optimizing the proportion of the primers and the annealing temperature for multiple times, the sensitivity for detecting two target genes can reach 1 pg/mu L, and the synchronism is good;
(3) Dual detection, labour saving and time saving detects with low costsly: the bee hive cryptococcus and the bee larva bacillus are used as the most important and the most common pathogenic bacteria of the bees, are difficult to distinguish according to clinical symptoms and need to be identified through pathogeny, and can detect two kinds of pathogenic bacteria at one time by utilizing the invention, and the whole reaction is completed within 2 hours, thereby saving time and labor. Meanwhile, the invention does not need expensive fluorescent PCR instrument, and does not need to synthesize expensive probe and corresponding reagent, the detection cost is low, the operation is simple, and the invention is beneficial to the application and popularization of laboratories in the basic department.
Drawings
FIG. 1 shows the result of electrophoresis after the step annealing temperature PCR amplification of the specific primers of Apis mellifera L.in example 1. Wherein M:100bp Ladder DNA Marker; the annealing temperatures of 1-8 are 50 deg.C, 51 deg.C, 53 deg.C, 55.9 deg.C, 59.3 deg.C, 62.1 deg.C, 64.1 deg.C, 65 deg.C, respectively.
FIG. 2 shows the electrophoresis results of the Bacillus larvae-specific primers of example 1 after gradient annealing temperature PCR amplification. Wherein M:100bp Ladder DNA Marker; the annealing temperatures of 1-8 are 50 deg.C, 51 deg.C, 53 deg.C, 55.9 deg.C, 59.3 deg.C, 62.1 deg.C, 64.1 deg.C, and 65 deg.C, respectively.
FIG. 3 shows the optimized results of the primer ratio and annealing temperature for the double PCR detection method of Apiscooccus mellifera and Bacillus larvae. Primer concentration: 10. mu mol/L of upstream and downstream primers MP-F and MP-R1-20 are both 0.5 mu L; 10. mu mol/L of upstream and downstream primers PL-F and PL-R: 0.6 muL for 1-5, 0.75 muL for 6-10, 0.9 muL for 11-15, and 1.0 muL for 16-20. Annealing temperature: 1. 6, 11 and 16 are 58 ℃; 2. 7, 12 and 17 are 60 ℃; 3. 8, 13 and 18 are 62 ℃; 4. 9, 14 and 19 are 64 ℃; 5. 10, 15 and 20 are 66 ℃.
FIG. 4 shows the results of the specificity determination of the double PCR detection method for M.alvei and B.larvae in example 4. Wherein 1: melissococcus alvei; 2: melissococcus nidus + ascosphaera apis; 3: melissococcus nidus + paratyphus melitensis; 4: melissococcus nidus and aspergillus flavus; 5: nidus Apis cocci + Apis cerana microsporidiana; 6: the bee hive is the mixture of the meliococcus apis and the bacillus larvae; 7: beehmeria alvei and Bacillus larvae model bacteria; 8: bacillus larvae of bees; 9: bacillus larvae plus ascosphaera apis; 10: bacillus larvae of bees and Bacillus paratyphi of bees; 11: bacillus larvae plus Aspergillus flavus; 12: bacillus larvae of bees and microsporidian of oriental bees.
FIG. 5 shows the results of the sensitivity measurements of the double PCR detection method for M.alvei and B.larvae of bee of example 5. Wherein M:100bp Ladder DNA Marker I; 1-8 concentrations of the template DNAs of the melissococcus apis and the bacillus larvae are respectively 1 pg/muL, 100 ng/muL, 10 ng/muL, 1 ng/muL, 100 pg/muL, 10 pg/muL, 1 pg/muL and 0.1 pg/muL.
Detailed Description
In order to further illustrate the invention, but not to limit it, reference is made to the following examples. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagent and the biological material can be obtained from commercial sources without special instructions except that the strains such as the melissococcus apis and the bacillus larvae of the bees are provided by special institutions.
Example 1:
a dual PCR primer for detecting melissococcus nidus and bacillus larvae comprises an upstream primer MP-F and a downstream primer MP-R for amplifying specific fragments of the melissococcus nidus, an upstream primer PL-F and a downstream primer PL-R for amplifying specific fragments of the bacillus larvae, and the nucleotide sequences of the primers are as follows:
an upstream primer MP-F:5 'AGAAGAGTGGCGGCGGACGGGTGA-3';
the downstream primer MP-R:5 'ACCGTCACGAGGAAAACAGTTATCCAC-3';
an upstream primer PL-F:5 'GCCAAGGAAGAACGGCCAGGG-3';
the downstream primer PL-R:5' CCACCTCTGCGTGGCAA-.
As shown in FIGS. 1 and 2, both of them have good amplification effects at an annealing temperature of 50 ℃ to 65 ℃.
Example 2:
the primer proportion and annealing temperature optimization result of the double PCR detection method for the melissococcus nidus and the bacillus larvae:
(1) Double PCR reaction system configuration: 2 XPCR Master Mix 12.5 mul, 10 mul mol/L upstream and downstream primers MP-F and MP-R each 0.5 mul, 10 mul mol/L upstream and downstream primers PL-F and PL-R each 0.60 mul-0.75 mul, 2 mul of sample DNA to be tested, then adding deionized water to make the total reaction volume 25.0 mul.
(2) Double PCR reaction procedure: pre-denaturation at 95 ℃ for 3 min; 95. denaturation at 58-66 deg.C for 45 s, annealing at 58-66 deg.C for 30 s, and denaturation at 72 deg.C for 45 s for 35 cycles; 72. extending at 5 deg.C for 5 min, and keeping at 4 deg.C.
(3) Optimizing the result: the results of the optimization of the primer ratio and the annealing temperature are shown in FIG. 3, when the primer ratio is 1: 1.5 and the annealing temperature is 64 ℃, the amplification effects of the primer ratio and the annealing temperature are consistent, and the electrophoresis bands are bright, so that the primer amounts of the melissococcus apis and the bacillus Apis in the reaction system are respectively 0.5. Mu.L and 0.75. Mu.L, and the optimal annealing temperature is 64 ℃.
Example 3:
the detection method for optimizing the dual PCR primers for detecting the melissococcus apis and the bacillus larvae comprises the following steps:
(1) Double PCR reaction system configuration: 2 XPCR Master Mix 12.5 uL, 10 umol/L upstream and downstream primers MP-F and MP-R are 0.5 uL respectively, 10 umol/L upstream and downstream primers PL-F and PL-R are 0.75 uL respectively, 2 uL of sample DNA to be detected is added, and then 8.0 uL of deionized water is added to make the total reaction volume be 25.0 uL.
(2) Double PCR reaction procedure: pre-denaturation at 95 ℃ for 3 min; 95. 35 cycles of denaturation at 45 s, annealing at 64 ℃ for 30 s and denaturation at 72 ℃ for 45 s; 72. extending for 5 min at 4 deg.C and keeping the temperature.
(3) Detecting, analyzing and judging a PCR amplification product: taking 8 mu L of PCR amplification product, carrying out electrophoresis on 1.5% agarose gel added with 0.01% GelRed nucleic acid dye, detecting the PCR amplification product on a gel imaging system, and then judging the result:
if the double PCR amplification product contains a strip with the size of 401 bp, the sample to be detected contains the melissococcus nidus;
if the double PCR amplification product contains a strip with the size of 706 bp, the sample to be detected contains the bacillus larvae of the bees;
if the double PCR amplification product does not contain a strip with the size of 401 bp and does not contain a strip with the size of 706 bp, the sample to be detected does not contain the melissococcus nidulans and does not contain the bacillus larvae of the bees.
Example 4:
specificity determination of the double PCR detection method of the melissococcus apiae and the bacillus larvae:
respectively taking 4 common bee pathogenic bacteria (parasites) such as ascosphaera apis, paratyphoid bacillus apis, aspergillus flavus, microsporidia apis cerana and the like as samples, extracting DNA by a conventional method, respectively matching with the melissococcus apis or the bacillus apis cerana to sample, carrying out PCR reaction and result judgment by the method of the embodiment 3, and as can be seen from the figure 4, only the melissococcus apis and the bacillus apis cerana (model bacteria) generate specific target bands, which indicates that the kit has strong specificity.
Example 5:
and (3) measuring the sensitivity of the double PCR detection method of the melissococcus hiveis and the bacillus larvae of the bees:
the plasmids containing the target fragments of the melissococcus apiae and the bacillus larvae are respectively used as templates, 10 times of the plasmids are gradually increased and diluted to corresponding concentrations, and then the plasmids are added into a reaction system, the method of the embodiment 3 is used for PCR reaction and result judgment, and as can be seen from figure 5, the 1 pg/muL template of the melissococcus apiae and the bacillus larvae can still amplify obvious DNA bands after the PCR products are subjected to 1.5% agarose gel electrophoresis, thereby proving that the sensitivity of 2 target targets in the double PCR detection method can reach 1 pg/muL and the double PCR detection method has better sensitivity.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> inspection and quarantine technology center of Fujian entry-exit inspection and quarantine bureau
<120> double PCR detection method for detecting bee-brood cocci and bee larva bacillus
<130> 4
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> Artificial sequence
<400> 1
agaagagtgg cggacgggtg a 21
<210> 2
<211> 25
<212> DNA
<213> Artificial sequence
<400> 2
accgtcacga ggaaaacagt tactc 25
<210> 3
<211> 21
<212> DNA
<213> Artificial sequence
<400> 3
gccaaggaag aacggccagg g 21
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence
<400> 4
Claims (1)
1. A double PCR detection method for detecting the bee hive coccus and the bee larva bacillus for the purpose of non-disease diagnosis and treatment is characterized by comprising the following steps:
(1) Double PCR reaction system configuration: 2 XPCR Master Mix 12.5 muL, 10 mumol/L upstream and downstream primers MP-F and MP-R used for amplifying specific segments of the melissococcus meliae are 0.5 muL respectively, 10 mumol/L upstream and downstream primers PL-F and PL-R used for amplifying specific segments of the bacillus meliae are 0.75 muL respectively, 2 muL of sample DNA to be detected is added, and 8.0 muL of deionized water is added to ensure that the total reaction volume is 25.0 muL;
(2) Double PCR reaction procedure: 95. pre-denaturation at deg.C for 3 min; 95. 35 cycles of denaturation at 45 s, annealing at 64 ℃ for 30 s and denaturation at 72 ℃ for 45 s; 72. extending at 5 deg.C for 5 min, and keeping the temperature at 4 deg.C;
(3) Detection, analysis and judgment of PCR amplification products: taking 8 mu L of PCR amplification product, carrying out electrophoresis on 1.5% agarose gel added with 0.01% GelRed nucleic acid dye, detecting the PCR amplification product on a gel imaging system, and then judging the result:
when the double PCR amplification product contains a strip with the size of 401 bp, the sample to be detected contains the melissococcus nidus;
when the double PCR amplification product contains a strip with the size of 706 bp, the sample to be detected contains the bacillus larvae;
when the double PCR amplification product contains a strip with the size of 401 bp and a strip with the size of 706 bp at the same time, the sample to be detected contains the melissococcus apiae and the bacillus larvae;
the upstream primer MP-F and the downstream primer MP-R for amplifying the specific segment of the melissococcus nidus, and the upstream primer PL-F and the downstream primer PL-R for amplifying the specific segment of the bacillus larvae of the bees are as follows in nucleotide sequence:
an upstream primer MP-F:5 'AGAAGAGTGGCGGCGGACGGGTGA-3';
the downstream primer MP-R:5 'ACCGTCACGAGGAAAACAGTTATCCK-3';
an upstream primer PL-F:5 'GCCAAGGAAGAACGGCCAGGG-3';
the downstream primer PL-R:5 'CCACCTCTGCGTGGCTGGCAA-3'.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910258230.0A CN109825618B (en) | 2019-04-01 | 2019-04-01 | Dual PCR detection method for detecting bee-brood cocci and bee larva bacillus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910258230.0A CN109825618B (en) | 2019-04-01 | 2019-04-01 | Dual PCR detection method for detecting bee-brood cocci and bee larva bacillus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109825618A CN109825618A (en) | 2019-05-31 |
| CN109825618B true CN109825618B (en) | 2022-11-22 |
Family
ID=66873880
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910258230.0A Active CN109825618B (en) | 2019-04-01 | 2019-04-01 | Dual PCR detection method for detecting bee-brood cocci and bee larva bacillus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109825618B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112795670A (en) * | 2021-01-27 | 2021-05-14 | 重庆市畜牧科学院 | Specific PCR amplification primer pair for detecting tricholoma alveorum, detection method and application |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107190081A (en) * | 2017-07-11 | 2017-09-22 | 伊犁职业技术学院 | Bee venoms TaqMan fluorescent quantitative PCR detection methods |
| CN107177690B (en) * | 2017-07-11 | 2020-11-24 | 伊犁出入境检验检疫局综合技术服务中心 | Dual TaqMan fluorescence quantitative PCR (polymerase chain reaction) detection method for pathogen of bee larva putrid disease |
| KR102146359B1 (en) * | 2017-09-08 | 2020-08-21 | 대한민국 | A primer for the diagnosis of parasitic and bacterial diseases of bees, a method and kit for diagnosing parasitic and bacterial diseases of bees using the same |
| JP2019062745A (en) * | 2017-09-28 | 2019-04-25 | 株式会社Ｄｎａチップ研究所 | Method of detecting causal bacterium of bee disease and detection kit of the same |
-
2019
- 2019-04-01 CN CN201910258230.0A patent/CN109825618B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN109825618A (en) | 2019-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Boyle et al. | Rapid quantitative detection of chytridiomycosis (Batrachochytrium dendrobatidis) in amphibian samples using real-time Taqman PCR assay | |
| CN109913565B (en) | Kit, primer pair, probe and method for detecting vibrio parahaemolyticus | |
| Nakanishi et al. | A simple identification method of saliva by detecting Streptococcus salivarius using loop‐mediated isothermal amplification | |
| CN105018628B (en) | Differentiate the kit of brucella A19 vaccine strains and street strain | |
| Grudlewska-Buda et al. | Comparison of the intensity of biofilm formation by Listeria monocytogenes using classical culture-based method and digital droplet PCR | |
| CN108676910A (en) | A kind of LAMP detection primer of fusarium prolifertum and its application | |
| CN108265120B (en) | 16-linked bovine mastitis pathogenic bacterium nucleic acid typing kit and detection method thereof | |
| CN108531627A (en) | One kind is for detecting the streptococcic RPA fluorescent quantitations primer pair of B races, probe, kit and detection method | |
| Amoupour et al. | Differentiation of Brucella abortus and B. melitensis biovars using PCR-RFLP and REP-PCR | |
| CN109825618B (en) | Dual PCR detection method for detecting bee-brood cocci and bee larva bacillus | |
| CN106434935A (en) | Composition and method for identifying pasteurella multocida and/or haemophilus parasuis | |
| CN106801103B (en) | Detection primer group, detection kit and multiplex PCR detection method for streptococcus agalactiae | |
| CN104911269A (en) | Primers, probe and kit for identifying Brucella A19 vaccine strain in aerosol | |
| CN110093446B (en) | Primer and probe combination for detecting phytophthora cinnamomi by recombinase-mediated isothermal amplification-lateral flow chromatography technology and application of primer and probe combination | |
| KR101846182B1 (en) | Primer sets for simultaneous detection of Staphylococcus aureus, Bacillus cereus and Salmonella spp., polymerase chain reaction kit thereof | |
| AU2020103778A4 (en) | Primer Set for Detection of Streptococcus agalactiae, Detection Kit and Multiplex PCR Detection Method | |
| JP3525259B2 (en) | Detection of Pectinatus spp. | |
| EP2999798A1 (en) | Method for simultaneous detection of bacteria and fungi in a biological preparation by pcr, primers as well as bacteria and fungi detection kit | |
| KR101752274B1 (en) | Primer set for high sensitive real-time multiplex loop-mediated isothermal amplification reaction for determining type of shiga toxin genes stx1 and stx2 of Enterohemorrhagic Escherichia coli, and method for determining type of shiga toxin genes of Enterohemorrhagic Escherichia coli using the same | |
| CN104561343B (en) | Distinguish the primer and kit of detection low pathogenicity and highly pathogenic babesia motasi | |
| KR101395938B1 (en) | Pcr diagnosis using specific primer for bacteria that cause diseases of allomyrina dichotoma | |
| CN111518935A (en) | Kit, primer pair, probe and method for detecting enterobacter sakazakii | |
| CN107177690B (en) | Dual TaqMan fluorescence quantitative PCR (polymerase chain reaction) detection method for pathogen of bee larva putrid disease | |
| CN110184370B (en) | Specific primer for detecting Acinetobacter johnsonii, method and application | |
| Shivachandra et al. | Detection of Pasteurella multocida in experimentally infected embryonated chicken eggs by PCR assay |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |