CN115710604A - Detection primer composition and detection method of milelia - Google Patents
Detection primer composition and detection method of milelia Download PDFInfo
- Publication number
- CN115710604A CN115710604A CN202211014380.5A CN202211014380A CN115710604A CN 115710604 A CN115710604 A CN 115710604A CN 202211014380 A CN202211014380 A CN 202211014380A CN 115710604 A CN115710604 A CN 115710604A
- Authority
- CN
- China
- Prior art keywords
- primer
- detecting
- amplification
- pairs
- primer composition
- 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.)
- Pending
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 17
- 238000001514 detection method Methods 0.000 title abstract description 39
- 230000003321 amplification Effects 0.000 claims abstract description 31
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 22
- 201000010099 disease Diseases 0.000 claims abstract description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 6
- 238000012408 PCR amplification Methods 0.000 claims description 21
- 239000012634 fragment Substances 0.000 claims description 14
- 238000012360 testing method Methods 0.000 claims description 10
- 238000001962 electrophoresis Methods 0.000 claims description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 4
- 238000000137 annealing Methods 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 238000012257 pre-denaturation Methods 0.000 claims description 3
- 239000011543 agarose gel Substances 0.000 claims description 2
- 238000003745 diagnosis Methods 0.000 claims 1
- 244000052769 pathogen Species 0.000 abstract description 9
- 230000001717 pathogenic effect Effects 0.000 abstract description 5
- 241000219470 Mirabilis Species 0.000 abstract description 4
- 230000002265 prevention Effects 0.000 abstract description 4
- 244000000010 microbial pathogen Species 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 35
- 241000270934 Rana catesbeiana Species 0.000 description 12
- 241000894006 Bacteria Species 0.000 description 6
- 208000002177 Cataract Diseases 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 238000010586 diagram Methods 0.000 description 5
- 241000269350 Anura Species 0.000 description 4
- 241000589565 Flavobacterium Species 0.000 description 4
- 244000052616 bacterial pathogen Species 0.000 description 4
- 230000004888 barrier function Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000607528 Aeromonas hydrophila Species 0.000 description 3
- 241000270959 Pelophylax nigromaculatus Species 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 241000588626 Acinetobacter baumannii Species 0.000 description 2
- 241000607574 Aeromonas veronii Species 0.000 description 2
- 108020000946 Bacterial DNA Proteins 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 241001560470 Quasipaa spinosa Species 0.000 description 2
- 241000269435 Rana <genus> Species 0.000 description 2
- 241000607715 Serratia marcescens Species 0.000 description 2
- 210000005228 liver tissue Anatomy 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 241000233866 Fungi Species 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 241000653900 Micrantha Species 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 206010040047 Sepsis Diseases 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 241000191940 Staphylococcus Species 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 239000013256 coordination polymer Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 231100000206 health hazard Toxicity 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000007403 mPCR Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 244000039328 opportunistic pathogen Species 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 238000001853 pulsed-field electrophoresis Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 208000019206 urinary tract infection Diseases 0.000 description 1
- 238000012070 whole genome sequencing analysis Methods 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of pathogenic microorganism detection, and discloses a primer composition and a method for detecting milelia, wherein the primer composition comprises at least one of three pairs of primers EM02F/R, EM03cF/R and EM04cF/R, and a method for detecting milelia is constructed on the basis of the primer composition. For the three pairs of specific primers, both single-pair primer amplification and random two-pair primer combination and three-pair primer combination can specifically distinguish the milelia mirabilis, and the detection method has high accuracy and low cost, and lays a foundation for the rapid detection, identification and prevention control of the frog askew white disease pathogen milelia mirabilis.
Description
Technical Field
The invention belongs to the technical field of pathogenic microorganism detection, and particularly relates to a primer composition and a method for detecting Erizabeth micrantha.
Background
Elizabeth miesha (Elizabethkkingia miricola) is an important species of the genus Elizabeth, and was first isolated from condensate water at Mir space stations (Kim et al 2005). The opportunistic pathogens pose a significant health hazard to humans and can cause arthritis, urinary tract infection, pneumonia and sepsis in humans. It has also been reported in amphibians that frogs infected with elizala milrina develop neurological diseases that cause damage to the central nervous system and symptoms similar to meningitis (Huang et al 2019; lei et al 2019; trimpert et al, 2021). The frogs infected by the bacterium milrinia have high mortality, high transmission speed and serious repeated morbidity (Trimpert et al 2020).
In recent years, symptoms of milrinia elizasa infection have been reported in a variety of non-urocanic amphibians including rana nigromaculata, rana spinosa, rana frogs, rana spinosa and the like, and diseased frogs often have symptoms such as head distortion and cataracts (Hu et al, 2017 lei et al, 2019 trimpert et al.2020. The research on the pathogens of the bullfrog with crooked heads and the white inner barrier has a long history. The classification status of pathogenic bacteria of the genus elizabetum and the classification system are merely problematic, and the classification status of pathogenic bacteria of bullfrog wry head and white barrier disease has not been clarified for a long time. For example, flavobacterium pyomeningitidis has been reported to cause frogs and cataract in Chen Guangming (1994). Chenfubo et al (2000) found that the causative agent of the Bullfrog wry head cataract was Flavobacterium. Aeromonas hydrophila was found in 2009 in Ponkanhao, and Staphylococcus luteus and Pseudomonas shallownsis were found to be present in bullfrog tissue with wry head cataract in 2010, respectively. Therefore, the establishment of an accurate and efficient rapid detection method for the bullfrog pathogens suffering from the crooked heads and the cataract is of great importance for the treatment, prevention and control of the bullfrog pathogens suffering from the crooked heads and the cataract.
Zhan et al (2020) established a fluorescent quantitative PCR detection method for milelizabeth, and can be used for rapid identification of milelizabeth isolates from different sources. In Hurui snow (2020), the molecular epidemiology of different Elizabeth isolates was studied by pulsed field electrophoresis and Rep-PCR. In addition, methods of whole genome sequencing have also been applied to species identification of elizasa millerii (shayang, 2018). These methods for identifying Elizabeth are efficient and accurate, but require a certain amount of equipment and high costs. At present, no report of analyzing and identifying the bullfrog Elizabeth by adopting a multiple PCR method exists at home and abroad, so that the development of a simple multiple PCR primer for quickly identifying the bullfrog wry head white barrier pathogen Mierelisha white fungus and the application of the multiple PCR primer in clinical detection have important significance for healthy breeding and disease control of bullfrogs.
Disclosure of Invention
In view of this, the present invention aims to provide a multiplex PCR primer and a method for rapidly identifying a pathogenic bacterium, namely, elizabethan milneri, so as to achieve simple, low-cost, accurate and efficient determination of the pathogenic bacterium.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the invention firstly develops three pairs of specific PCR primers in a gene coding region with unknown functions based on the whole genome sequence analysis of a bullfrog milbemyces elizakii strain Mir-N11 obtained by separation, wherein the sequences of the three pairs of specific PCR primers are shown as follows:
EM02F:5’-GCTTCCGCTTCTGATAATTGAC-3’，
EM02R:5’-GGAGACGAGCTATTAGTGTATCG-3’；
EM03cF:5’-AACTACTGTCACTAATC-3’，
EM03cR:5’-GAATTGTTAGCTGTTGAA-3’；
EM04cF:5’-CCAGTTTTTAGAGTTGAAAT-3’，
EM04cR:5’-GACATACACATACTTTGT-3’。
the three pairs of specific PCR primers can be used independently, can be used by combining two primer pairs, or can be used by combining three pairs of primers, and can be used for specifically distinguishing the milelia. Optimally, three primer sets were selected for use and their molar ratio was 1.
The invention further provides a detection kit for detecting the milbemyces elizakii, the detection kit comprises at least one pair of the three pairs of primers, and the detection kit can also comprise other reagents for PCR amplification.
The invention also provides a method for detecting the milelizabeth, which specifically comprises the following steps: extracting DNA of a sample to be detected, carrying out PCR amplification by using the DNA as a template and adopting any one pair, any two pairs or three pairs of the three pairs of primers, and carrying out agarose gel electrophoresis detection on an amplification product.
In the above detection scheme, the reaction system for PCR amplification is preferably: 30 mu.L total, 2 XTaq PCR Master Mix 15 mu.L, template DNA 2 mu.L, final concentration of each primer 1 mu mol/L, ddH 2 And the balance of O.
In the above detection scheme, the reaction procedure of PCR amplification is preferably: pre-denaturation at 95 ℃ for 3min; denaturation at 95 ℃ for 30s, annealing at 47 ℃ for 30s, extension at 72 ℃ for 1min for 30s, and amplification for 34 cycles; extension at 72 ℃ for 10min.
In the above detection scheme, the amplification product is preferably detected by electrophoresis on a 0.8% agarose gel.
In the above detection scheme, the sizes of the specific fragments detected by agarose gel electrophoresis are specifically: the length of the amplified fragment of the primer pair EM02F/R is 473bp, the length of the amplified fragment of the primer pair EM03cF/R is 870bp, and the length of the amplified fragment of the primer pair EM04cF/R is 617bp.
The invention has the beneficial effects that: when the detection primer composition and the constructed method combining the multiple PCR identification and the single primer amplification product sequencing are applied to the detection of clinical pathogens, the detection primer composition has the advantages of high accuracy, simplicity in operation, low cost and the like; moreover, the three pairs of specific primers can specifically distinguish the milelia mirabilis no matter the single pair of primers is amplified, or the random two pairs of primer combinations and the three pairs of primer combinations, which lays a foundation for the rapid detection, identification and prevention control of the frog wry white disease pathogen milelia mirabilis.
Drawings
FIG. 1 is an electrophoretogram of genomic DNA of Erizabeth miers amplified by three pairs of primers provided by the present invention;
FIG. 2 is a diagram showing the results of the sensitivity detection of the single PCR amplification of the three pairs of primers provided by the present invention;
FIG. 3 is a diagram showing the results of the sensitivity detection of the double-primer combination PCR amplification in the three pairs of primers provided by the present invention;
FIG. 4 is a diagram showing the results of the sensitivity detection of PCR amplification with three primer combinations provided by the present invention;
FIG. 5 is an electrophoretogram of PCR amplification of genomic DNA of different bacteria with three pairs of primers provided by the present invention;
FIG. 6 is an electrophoresis diagram of the double primers in the three pairs of primers provided by the present invention for amplifying genomic DNA of different bacteria;
FIG. 7 is an electrophoresis diagram of three pairs of primers provided by the present invention for amplifying genomic DNA of different bacteria.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In the following examples, unless otherwise specified, all the methods are conventional; the reagents and materials are commercially available unless otherwise specified.
Example 1
In this example, three pairs of specific primers were designed based on the whole gene sequence (GenBank registration sequence number CP 090369.1) of the wry head and white inner barrier pathogen Mir-N11 strain isolated from Bullfrog-diseased tissue in the gene coding region with unknown function (the GenBank sequence numbers are UIO96951.1, UIO97127.1 and UIO96598.1, respectively) as follows:
EM02F：5’-GCTTCCGCTTCTGATAATTGAC-3’(SEQ ID NO.1)，
EM02R：5’-GGAGACGAGCTATTAGTGTATCG-3’(SEQ ID NO.2)；
EM03cF：5’-AACTACTGTCACTAATC-3’(SEQ ID NO.3)，
EM03cR：5’-GAATTGTTAGCTGTTGAA-3’(SEQ ID NO.4)；
EM04cF：5’-CCAGTTTTTAGAGTTGAAAT-3’(SEQ ID NO.5)，
EM04cR：5’-GACATACACATACTTTGT-3’(SEQ ID NO.6)；
wherein the length of the amplified fragment of the primer pair EM02F/R is 473bp, the length of the amplified fragment of the primer pair EM03cF/R is 870bp, and the length of the amplified fragment of the primer pair EM04cF/R is 617bp.
Example 2
Based on the primer pair obtained in example 1, a detection system for elizasa milbeli was constructed:
the extracted genomic DNA of Elizabeth milrinia was diluted to 1 ng/. Mu.L. Respectively amplifying gene segments by using a single primer pair amplification method, a pairwise primer pair combined amplification method and a three-pair primer combined amplification method.
The PCR amplification reaction system is as follows: 2 XTaq PCR Master Mix 15. Mu.L (CWBIO), template DNA (1 ng/ul) 2. Mu.L, final concentration of each primer 1. Mu. Mol/L, supplemented with appropriate amount of ddH 2 O to 30. Mu.L.
The PCR amplification procedure was: pre-denaturation at 95 ℃ for 3min; denaturation at 95 ℃ for 30s, annealing at 47 ℃ for 30s, extension at 72 ℃ for 1min for 30s, and amplification for 34 cycles; extension at 72 ℃ for 10min.
As shown in FIG. 1, lane 1 shows triple PCR amplification products, lane 2 shows double PCR amplification products of a combination of primer pair EM02F/R and primer pair EM04cF/R, lane 3 shows double PCR amplification products of a combination of primer pair EM02F/R and primer pair EM03cF/R, lane 4 shows double PCR amplification products of a combination of primer pair EM03cF/R and primer pair EM04cF/R, lane 5 shows amplification products of primer pair EM02F/R, lane 6 shows amplification products of primer pair EM04cF/R, lane 7 shows amplification products of primer pair EM03cF/R, lane 8 shows a sterilized water control, and M shows DNA Marker.
As can be seen from FIG. 1, in the same reaction system, 3 bands with molecular weights of 473bp, 617bp and 870bp can be specifically amplified by using three pairs of primer combinations; in the same reaction system, two specific bands with molecular weight of 473bp and 617bp can be specifically amplified by using the duplex PCR amplification of the combination of the primer pair EM02F/R and the primer pair EM04 cF/R; the duplex PCR using the combination of the primer pair EM02F/R and the primer pair EM03cF/R can specifically amplify two specific bands with molecular weights of 473bp and 870 bp; the double PCR amplification using the combination of the primer pair EM03cF/R and the primer pair EM04cF/R can specifically amplify two specific bands with the molecular weights of 617bp and 870 bp; and a specific band with the molecular weight of 470bp, 617bp and 870bp is obtained by single primer pair EM02F/R, EM04cF/R and EM03cF/R amplification respectively.
Example 3
Based on the detection system constructed in example 2, this example further determines the amplification sensitivity of the three primer pair combination:
and (3) carrying out gradient dilution on template DNA of the milelia leucotricha (Em), and then respectively carrying out single primer, double primer combination and three primer combination amplification sensitivity tests.
The single primer test result is shown in FIG. 2, and the detection results of the primer pairs EM2F/R, EM03cF/R and EM04cF/R for amplifying Em genome DNA are sequentially shown from left to right; the test result of the double-primer combination is shown in FIG. 3, the test result of the EM03cF/R + EM2F/R, EM2F/R + EM04cF/R and EM03cF/R + EM04cF/R two-pair primer combination amplification Em genome DNA is sequentially shown from left to right, and FIG. 4 is the test result of the EM2F/R, EM03cF/R and EM04cF/R three-pair primer combination amplification. In FIGS. 2-4, M is DNA Marker, lanes 1-8 are sequentially DNA sample concentration to be measured of 1ng/μ L and 10 -1 ng/μL，10 -2 ng/μL，10 -3 ng/μL，10 - 4 ng/μL，10 -5 ng/μL，10 -6 ng/μL，10 -7 ng/μL。
As can be seen from FIGS. 2 to 4, the reaction system and reaction conditions in example 2 were diluted to 10 -6 ng/ul DNA template still can amplify clear specific bands, which indicates that the primer combination has higher sensitivity under the amplification condition.
Example 4
Based on the detection system constructed in example 2, this example further detected the specificity of each primer combination:
genomic DNAs of Elizabethigia miricola (Em), serratia marcescens (Sm), aeromonas veronii (Av), aeromonas hydrophila (Ah), flavobacterium Cucumidis (Chrnehighly, ch) and Acinetobacter baumannii (Ab) were extracted using a bacterial genome extraction kit (Tiangen Biotech Co., ltd.), and the DNA concentrations of the respective samples were adjusted to 1 ng/. Mu.L as a template for PCR amplification. And amplifying the genome DNA of each bacterium by using a single primer pair amplification method, a pairwise primer pair combined amplification method and a three-pair primer combined amplification method respectively.
The single primer test result is shown in FIG. 5, and the detection results of the primer pairs EM2F/R, EM04cF/R and EM03cF/R for amplifying Em genome DNA are sequentially shown from left to right; the test result of the double-primer combination is shown in FIG. 6, and the test result of the EM04cF/R + EM2F/R, EM03cF/R + EM04cF/R and EM03cF/R + EM2F/R two pairs of primer combination amplification Em genome DNA is sequentially obtained from left to right; FIG. 7 shows the results of the combined amplification of three pairs of primers EM2F/R, EM03cF/R and EM04 cF/R. In FIGS. 5 to 7, M is DNA Marker, lane 1 is EM genomic DNA, lane 2 is Sm genomic DNA, lane 3 is Av genomic DNA, lane 4 is Ah genomic DNA, lane 5 is Ch genomic DNA, lane 6 is Ab genomic DNA, lane 7 is ddH genomic DNA 2 And (4) O negative control.
As can be seen from FIG. 5, the single primer pairs EM02F/R, EM04cF/R and EM03cF/R can only amplify Elizabeth milrinia, and a specific band with molecular weight of 470bp, 617bp and 870bp is obtained. There was no amplification product of the genomic DNA derived from Serratia marcescens (Sm), aeromonas veronii (Av), aeromonas hydrophila (Ah), flavobacterium Cucumidis (Chysereuterium cuumeris, ch), acinetobacter baumannii (Ab).
As can be seen from FIG. 6, the duplex PCR amplification using the combination of primer pair EM02F/R and primer pair EM04cF/R can specifically amplify two specific bands having molecular weights of 473bp and 617bp from the genome of Elizabeth miehei, while the other detected bacterial DNAs do not amplify the bands; the double PCR using the combination of the primer pair EM02F/R and the primer pair EM03cF/R can specifically amplify two specific bands with molecular weights of 473bp and 870bp, and other detection bacterial DNAs do not have amplified bands; the double PCR amplification of the combination of the primer pair EM03cF/R and the primer pair EM04cF/R can specifically amplify two specific bands with the molecular weights of 617bp and 870bp, and other detection strains do not amplify the bands;
as can be seen from FIG. 7, using all three primer pairs, only 3 specific bands were amplified in genomic DNA of Elizabeth miehei, while no band was amplified in genomic DNA of other test bacteria.
Example 5
Based on the detection system constructed in example 2, this example further verifies the accuracy of the PCR amplification result:
water samples (30 parts), soil samples (30 parts) and bullfrog liver tissues (30 parts) from a natural farm were collected. In addition, 22 different isolates of Elizabalata miehei derived from Rana nigromaculata and Rana nikoensis were incubated overnight at 30 ℃ in a room. And extracting total genome DNA of each sample and strain, amplifying DNA of 90 samples by using all three pairs of primer pair combinations and a single primer pair EM02F/R, and judging the conformity of triple PCR amplification and single pair primer amplification according to an amplification map. And then recovering and purifying the band amplified by the EM02F/R single primer pair, then sending the band to a biotechnology company for sequence determination, and comparing the sequence obtained by sequencing with the sequence of the target amplified fragment to determine the positive amplification accuracy. The results are shown in the following table:
22 rana nigromaculata and bullfrog Irisella melissa isolates with different sources can be specifically amplified to obtain specific banding patterns by using triple PCR and single primer pair PCR; in 30 parts of water sample DNA, 9 positive samples can be detected by using triple PCR or EM02F/R single primer pair, and no positive sample is detected in a soil sample or frog liver tissue; compared with the known corresponding gene fragments after the products amplified by the EM02F/R single primer are recovered and sequenced, the positive samples tested by the result are all the specific gene fragments of the Elizabeth miers, and the accuracy rate reaches 100 percent.
In conclusion, the detection system constructed by matching the three pairs of detection primers provided by the invention can realize the accurate and specific detection of the milbemyces elizakii, has low detection limit and low detection cost, and lays a foundation for the rapid detection, identification and prevention control of the frog wry white disease pathogen milbemyces elizakii.
The above-described embodiments of the present invention should not be construed as limiting the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. The primer composition for detecting the Erizaeus miehei comprises at least one of the following three pairs of primers:
EM02F：5’-GCTTCCGCTTCTGATAATTGAC-3’，
EM02R：5’-GGAGACGAGCTATTAGTGTATCG-3’；
EM03cF：5’-AACTACTGTCACTAATC-3’，
EM03cR：5’-GAATTGTTAGCTGTTGAA-3’；
EM04cF：5’-CCAGTTTTTAGAGTTGAAAT-3’，
EM04cR：5’-GACATACACATACTTTGT-3’。
2. the primer composition for detecting elizabeth milbemyces according to claim 1, wherein the three pairs of primers are included in the primer composition.
3. The primer composition for detecting Aeschynia oryzae according to claim 1, wherein the molar ratio of the three pairs of primers is 1.
4. A kit for detecting Elizabeth milrinia, which comprises the primer composition according to claim 1.
5. The test kit of claim 4, comprising reagents for PCR amplification.
6. A method for detecting Elizabeth milrinia for non-disease diagnosis and treatment, which comprises extracting DNA from a sample to be detected, amplifying the DNA using the DNA as a template by using the primer composition according to claim 1, and detecting the amplified product by agarose gel electrophoresis.
7. The method for detecting Aerma oryzae according to claim 6, wherein the reaction system for amplification is: 30 mu.L of 2 XTaq PCR Master Mix 15 mu.L of template DNA, the final concentration of each primer is 1 mu mol/L, ddH 2 And the balance of O.
8. The method for detecting elizamide according to claim 6, wherein the amplification reaction is performed by: pre-denaturation at 95 ℃ for 3min; denaturation at 95 ℃ for 30s, annealing at 47 ℃ for 30s, extension at 72 ℃ for 1min for 30s, and amplification for 34 cycles; extension for 10min at 72 ℃.
9. The method for detecting elizamide according to claim 6, wherein the specific fragment size obtained by agarose gel electrophoresis is specifically: the length of the amplified fragment of the primer pair EM02F/R is 473bp, the length of the amplified fragment of the primer pair EM03cF/R is 870bp, and the length of the amplified fragment of the primer pair EM04cF/R is 617bp.
10. The method of claim 6, wherein the amplification product is detected by electrophoresis on a 0.8% agarose gel.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211014380.5A CN115710604A (en) | 2022-08-23 | 2022-08-23 | Detection primer composition and detection method of milelia |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211014380.5A CN115710604A (en) | 2022-08-23 | 2022-08-23 | Detection primer composition and detection method of milelia |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115710604A true CN115710604A (en) | 2023-02-24 |
Family
ID=85230694
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211014380.5A Pending CN115710604A (en) | 2022-08-23 | 2022-08-23 | Detection primer composition and detection method of milelia |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115710604A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20100111120A (en) * | 2009-04-06 | 2010-10-14 | 목원대학교 산학협력단 | Novel elizabethkingia miricola fbf-7 having keratinolytic activity and the protease therefrom |
| WO2019226992A1 (en) * | 2018-05-24 | 2019-11-28 | The Trustees Of Columbia University In The City Of New York | Bacterial capture sequencing platform and methods of designing, constructing and using |
| CN110684855A (en) * | 2019-10-21 | 2020-01-14 | 华中农业大学 | Real-time fluorescent quantitative RT-PCR detection method for Iressta micrantha |
| CN113278559A (en) * | 2021-06-11 | 2021-08-20 | 江西省农业科学院畜牧兽医研究所 | Erizabeth milrina from rana nigromaculata and application thereof |
-
2022
- 2022-08-23 CN CN202211014380.5A patent/CN115710604A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20100111120A (en) * | 2009-04-06 | 2010-10-14 | 목원대학교 산학협력단 | Novel elizabethkingia miricola fbf-7 having keratinolytic activity and the protease therefrom |
| WO2019226992A1 (en) * | 2018-05-24 | 2019-11-28 | The Trustees Of Columbia University In The City Of New York | Bacterial capture sequencing platform and methods of designing, constructing and using |
| CN110684855A (en) * | 2019-10-21 | 2020-01-14 | 华中农业大学 | Real-time fluorescent quantitative RT-PCR detection method for Iressta micrantha |
| CN113278559A (en) * | 2021-06-11 | 2021-08-20 | 江西省农业科学院畜牧兽医研究所 | Erizabeth milrina from rana nigromaculata and application thereof |
Non-Patent Citations (7)
| Title |
|---|
| PARAKRITI GUPTA等: "Elizabethkingia miricola :A rare non-fermenter causing urinary tract infection", WORLD JOURNAL OF CLINICAL CASES, no. 05, 16 May 2017 (2017-05-16), pages 187 - 190 * |
| 佚名: "CP090369.1", NCBI, 11 January 2022 (2022-01-11), pages 1 - 4 * |
| 刘春等: "牛蛙米尔伊丽莎白菌的分离、鉴定与特性分析", 仲恺农业工程学院学报, vol. 35, 30 June 2022 (2022-06-30), pages 8 - 14 * |
| 向圣汉: "牛蛙源米尔伊丽莎白菌的分离鉴定及其对宿主免疫应答的影响", 中国优秀硕士学位论文, 15 February 2024 (2024-02-15), pages 1 - 74 * |
| 向圣汉等: "牛蛙源米尔伊丽莎白菌的分离鉴定与药敏试验", 水产学杂志, vol. 36, 31 August 2023 (2023-08-31), pages 83 - 88 * |
| 张琦: "米尔伊丽莎白菌快速分子检测方法的建立与应用", 中国优秀硕士学位论文全文数据库, 15 February 2021 (2021-02-15), pages 1 - 68 * |
| 李川北等: "黑斑蛙歪头病病原米尔伊丽莎白菌的分离、 鉴定及特性分析", 淡水渔业, vol. 52, 31 May 2022 (2022-05-31), pages 66 - 73 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Dec et al. | 16S-ARDRA and MALDI-TOF mass spectrometry as tools for identification of Lactobacillus bacteria isolated from poultry | |
| Lee et al. | A multiplex real-time PCR for differential detection and quantification of Salmonella spp., Salmonella enterica serovar Typhimurium and Enteritidis in meats | |
| Tanigawa et al. | Identification and typing of Lactococcus lactis by matrix-assisted laser desorption ionization-time of flight mass spectrometry | |
| Adhikari et al. | Pathogenic and genetic diversity of Xanthomonas translucens pv. undulosa in North Dakota | |
| Frosth et al. | Development and comparison of a real-time PCR assay for detection of Dichelobacter nodosus with culturing and conventional PCR: harmonisation between three laboratories | |
| Luedtke et al. | Evaluation of real time PCR assays for the detection and enumeration of enterohemorrhagic Escherichia coli directly from cattle feces | |
| CN113621717A (en) | Streptococcus suis rapid visualization RPA detection kit based on CRISPR-Cas12a and application thereof | |
| US20110287965A1 (en) | Methods and compositions to detect clostridium difficile | |
| CN106801103B (en) | Detection primer group, detection kit and multiplex PCR detection method for streptococcus agalactiae | |
| JP5610395B2 (en) | Genetic methods for species identification of Campylobacter | |
| Badouei et al. | Prevalence and clonal distribution of avian Escherichia coli isolates harboring increased serum survival (iss) gene | |
| CN115710604A (en) | Detection primer composition and detection method of milelia | |
| CN103911446A (en) | Gastric juice multiple real-time polymerase chain reaction (PCR) detection-based helicobacter pylori (HP) individualized treatment auxiliary diagnosis method | |
| JP5395674B2 (en) | Genetic methods for species identification of Campylobacter | |
| CN109517911B (en) | Dual PCR detection primer and detection method for melon bacterial fruit blotch and leaf blotch | |
| AU2020103778A4 (en) | Primer Set for Detection of Streptococcus agalactiae, Detection Kit and Multiplex PCR Detection Method | |
| Gehring et al. | Identification of Erwinia species isolated from apples and pears by differential PCR | |
| RU2737775C1 (en) | Method for identifying yersinia pestis and yersinia pseudotuberculosis and simultaneous differentiation of yersinia pestis of main and central asian subspecies by multiplex pcr | |
| JP2023106944A (en) | Primer set which specifically detects lactic acid bacteria lq80 strain, kit including primer set, and detection method using primer set | |
| CN116334081A (en) | Method for detecting pathogenic microorganisms based on CRISPR technology | |
| CN103122383B (en) | Horizontal fluorescent quantitation polymerase chain reaction (PCR) detection method of streptococcus suis strain | |
| Takajo et al. | Development of a simple and practical method of discrimination between Vibrio furnissii and V. fluvialis based on single-nucleotide polymorphisms of 16S rRNA genes observed in V. furnissii but not in V. fluvialis | |
| JP2022533456A (en) | Method for detecting levels of H. pylori in fecal samples | |
| Fardsanei et al. | The Study of Antimicrobial Resistance among Salmonella enterica Strains Isolated from Children with Gastroenteritisin in Tehran, Iran | |
| López et al. | Evaluation of the efficiency of a conventional PCR protocol for the diagnosis of bacterial spot disease caused by Xanthomonas arboricola pv. pruni in stone fruits and almond |
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 |