CN117344042A - Specific primer and kit for detecting sea tail worms and detection method thereof - Google Patents

Specific primer and kit for detecting sea tail worms and detection method thereof Download PDF

Info

Publication number
CN117344042A
CN117344042A CN202311489093.4A CN202311489093A CN117344042A CN 117344042 A CN117344042 A CN 117344042A CN 202311489093 A CN202311489093 A CN 202311489093A CN 117344042 A CN117344042 A CN 117344042A
Authority
CN
China
Prior art keywords
sea
kit
probe
specific primer
tail
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
Application number
CN202311489093.4A
Other languages
Chinese (zh)
Inventor
谢尚微
陈万东
詹子锋
徐奎栋
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanji Islands National Marine Nature Reserve Management Bureau
Institute of Oceanology of CAS
Original Assignee
Nanji Islands National Marine Nature Reserve Management Bureau
Institute of Oceanology of CAS
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Nanji Islands National Marine Nature Reserve Management Bureau, Institute of Oceanology of CAS filed Critical Nanji Islands National Marine Nature Reserve Management Bureau
Priority to CN202311489093.4A priority Critical patent/CN117344042A/en
Publication of CN117344042A publication Critical patent/CN117344042A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6841—In situ hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/90—Protozoa ; Processes using protozoa

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Analytical Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention belongs to the field of aquatic animal disease science, and particularly relates to a specific primer and a kit for detecting sea fish ciliates and a detection method thereof. The specific primer sequence for detecting the sea tail worm is 5'-TGCCGATTAAAGATCAACATAC-3'. The specific primer is used as a probe in detection of sea tail filarial parasitic to sea fish. The invention designs a specific DNA probe aiming at the sea tail worm, establishes a corresponding in-situ fluorescence hybridization reaction system and realizes quick identification of the sea tail worm. The technology and the kit have the characteristics of simplicity in operation, sensitivity, rapidness and the like.

Description

Specific primer and kit for detecting sea tail worms and detection method thereof
Technical Field
The invention belongs to the field of aquatic animal disease science, and particularly relates to a specific primer and a kit for detecting sea fish ciliates and a detection method thereof.
Background
With the rapid development of the fish farming industry, various diseases which are difficult to cure have been continuously brought about with great challenges to the control and management work of diseases, such as diseases caused by ciliates of the peltate-type. The shield fiber ciliates are widely distributed in natural water bodies and culture water bodies and are important components of micro-food loops of the water bodies, wherein the marine tail worms Uronema marinum are one of the most common facultative parasites causing serious diseases in marine fishes. Despite the ecological and economic dual importance, diagnosis of such ciliates is still very difficult.
Morphological methods are the traditional means of identifying ciliates of the class of scutellarioides, however, in the aquaculture industry, their application is limited by 4 aspects: 1) Most of the shield fiber living body forms are quite similar, and for non-taxonomic specialists, the silver-dyed forms are still difficult to distinguish, and the marine tail worms and other non-parasitic shield fiber ciliates are difficult to distinguish; 2) The shield fibers of spore morphology are difficult to identify with an optical microscope; 3) Silver staining techniques for morphological identification are very complex and time consuming; 4) Worldwide, there are very few protist taxonomies that can serve the aquaculture industry. Thus, new methods and kits for rapid, simple and accurate identification of the shield fibers remain a pressing need. In recent years, the emerging fluorescent in situ hybridization technology is considered as an effective detection technology, and has been widely used in environmental microorganism detection. The fluorescent in situ hybridization technology is to use fluorescent labeled specific oligonucleotide fragments as probes to hybridize with biological genome DNA molecules in a target environment according to DNA sequences specific to the populations on different classification levels of known organisms, and detect the existence and abundance of the specific biological populations. However, this technique has not been sufficiently used for detection of fish-disease ciliates.
Disclosure of Invention
The invention aims to provide a specific primer, a kit and a detection method for detecting sea tail worms.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a specific primer for detecting the sea tail worm has a sequence 5'-TGCCGATTAAAGATCAACATAC-3'.
The application of the specific primer is that the specific primer is used as a probe in the detection of sea tail worms parasitizing sea fish.
The probe is a cyanine dye Cy-3 marked at the 5' end of the primer, namely, a probe Um1280.
A kit for detecting ciliates of marine fishes, which comprises the specific primer.
And the 5' -end of the specific primer sequence is marked with cyanine dye Cy-3 as a target probe.
The kit comprises formamide hybridization solution containing target probes, eluent and a staining agent.
The formamide hybridization solution comprises 20% of formamide (formamide) by mass, 30 ng/. Mu.l of probe Um1280 concentration, 0.01% of SDS (sodium dodecyl sulfate) by mass, and 0.9M, TRIS-HCl molar concentration of 20mM of NaCl;
the eluent is SDS (sodium dodecyl sulfate) mass fraction 0.01%, naCl molar concentration 0.9M, TRIS-HCl molar concentration 20mM and EDTA molar concentration 5mM;
the stain was 1.5ng/ml DAPI solution.
The application of the kit in fluorescence in-situ hybridization detection of sea tail worms of sea fish.
In a darkroom, dropwise adding formamide hybridization containing a target probe into a sample to be detected fixed by a fixing solution, incubating for 2 hours at 46 ℃, eluting for 10 minutes by an eluent water bath at 48 ℃ after incubation, performing contrast dyeing on the eluted air-dried sample at room temperature and a 4',6-Diamidino-2-Phenylindole (DAPI) solution, washing the air-dried sample after dyeing, and observing the fluorescence intensity under a fluorescence microscope.
The detection is carried out by a fluorescence microscope, and the detection of orange red fluorescence signals indicates that the sample to be detected contains the sea tail worm
The invention has the advantages that:
the invention takes common disease ciliates parasitic in large yellow croaker, fugu rubripes, cynoglossus semilaevis, large yellow croaker and turbot fish, namely sea tail filarial as test objects, and realizes the rapid detection of the group by developing specific nucleic acid probes and kits for the test objects and combining with fluorescent in situ hybridization technology; the detection method has the characteristics of simplicity in operation, high specificity, sensitivity, rapidness and the like.
Drawings
FIG. 1 is a photograph showing fluorescence of a living body (A), universal probe (B, I, J), hybridization target probe Um1280 (C, H, K, L), um626 probe (D, E), negative probe (F) and chimeric 4',6-Diamidino-2-Phenylindole (DAPI) (E, G, M) of a sample ciliate provided in the examples of the present invention. A. A live photograph of a sea tail worm; B. color development photo of the universal probe for hybridization of the sea tail filarial worm, and the worm body presents red fluorescence; C. photograph of a hybridization Um1280 probe of the sea tail filarial worm, wherein the concentration of formamide hybridization solution is 30%, and the worm body presents red fluorescence; d, E, hybridizing a Um626 probe and a DAPI photo of the sea tail worm with the worm body, (D) is a photo under a Cy3 excitation module, the concentration of formamide hybridization solution is 30%, the worm body is non-fluorescent, and (E) the cell nucleus of the worm body is blue fluorescent; f, G, the photographs of the sea tail filarial worm with a worm hybridization negative probe and DAPI, wherein (F) is a photograph under a Cy3 excitation module, the concentration of formamide hybridization solution is 30%, the worm is non-fluorescent, and (G) the photograph under the DAPI excitation module shows blue fluorescence; H. photograph of a hybridization Um1280 probe of the sea tail filarial worm, wherein the concentration of formamide hybridization solution is 30%, and the worm body presents red fluorescence; i, J is a fluorescence photograph of a positive control probe hybridized with sea tail worm (I) and another shield fiber ciliate-water drop pseudokan ciliate Pseudocohnilembus persalinus (J), and red fluorescence is emitted to indicate that the FISH experiment is successful; k, L is a fluorescent photograph of a target probe Um1280 hybridized with sea tail filarial and pseudohealthy filarial drop, K is a photograph of a probe Um1280 hybridized with sea tail filarial, the body is red fluorescent, which indicates that the probe Um1280 successfully detects the target body, and L is free of red fluorescent, which indicates that Um1280 cannot detect pseudohealthy filarial drop; m is a photograph of L insect bodies under the DAPI excitation module, and the cell nuclei of the insect bodies are blue fluorescent, so that the positions of the insect bodies are shown.
Detailed Description
The invention is further described below with reference to the drawings.
Example 1
Preparation of specific probes
(1) The worm sample was derived from the sea tail worm of the institute of science, china, national academy of sciences (FIG. 1A). The insect body is subjected to expansion culture by taking cooked shrimp meat as food, and then genome is extracted by utilizing DNeasy Tissue kit (Qiagen) according to the requirements of the instruction; PCR amplification of the SSU rRNA gene sequence was performed using ciliate universal primers (5'-AAC CTG GTT GAT CCT GCC AGT-3' and 5'-TGA TCC TTCTGC AGG TTC ACC TAC-3'), and a 25. Mu.l amplification reaction system comprised: about 50ng of template DNA, 0.7U Taq DNA polymerase, 1. Mu.M (final concentration) of 5 'and 3' primers, 0.2mM (final concentration) of dNTPs, mgCl 2 2mM (final concentration), 2.5. Mu.l of Takara PCR buffer, and an appropriate amount of sterilized double distilled water (completed to 25. Mu.l of the whole reaction system), the reaction procedure was as follows: denaturation at 94℃for 5 min, then denaturation at 94℃for 1 min, annealing at 60℃for 1 min, extension at 70℃for 2 min, total of 35 cycles, and extension for 10 min. The PCR products were purified and sequenced by sequencing companies, which matched the existing records.
(2) After obtaining SSU rRNA gene sequences of the sea tail filarial, downloading homologous sequences of all populations of GenBank and SSU rRNA gene sequences of all scurf ciliates, comparing sequences by using Mafft software, and screening out regions of the sea tail filarial different from other scurf ciliates from the comparison sequences. For these regions, alternative primers were designed using Primer5 software. The design criteria of the primer are as follows: a) At least 2 base mismatches compared to other species sequences; b) Identical to the sequence of the species (including other species sequences); c) The length is 20-30bp. Because not every primer can be combined with the SSU rRNA gene of the target sample with high efficiency, the implementation refers to the reported probe hybridization fluorescence brightness distribution diagram of the eukaryotic Saccharomyces cerevisiae Saccharomyces cerevisiae, and selects a sequence in 2 areas with stronger fluorescence brightness as the target primer, and marks cyanine dye Cy-3 (C 31 H 37 KN 2 O 8 S 2 ) Synthesizing the candidate probe. The names and sequences of the alternative probes are: a) Um1280, sequence 5'-TGCCGATTAAAGATCAACATAC-3'; b) Um626, sequence 5'-AACTAGCTTTGCAAGCGGATGTA-3'.
(3) The detection effect of the candidate probes Um1280 and Um626 is evaluated by using the Fluorescence In Situ Hybridization (FISH) effect of the known eukaryote universal probe Euk1209R (5'-GGGCATCACAGACCTG-3') as a positive control probe and the complementary sequence of the positive control probe as a negative control probe (both labeled with Cy-3 fluorescent labels). The experimental steps are as follows:
the insect suspension and the Boen solution obtained in the step (1) are prepared according to the following steps of 1:1 volume was mixed and fixed for 10 minutes, after which the insect suspension was low pressure filtered using a 1.2 μm pore size microporous filter (Millipore, diameter 25 mm). After washing the filter membrane 5 times with 1ml of sterile water, the filter membrane was placed in a clean petri dish, naturally air-dried, and the air-dried sample 1 was divided into 4 pieces. The 4 probes (containing 2 control probes and 2 alternative probes) were each equipped with 30% Formamide (FA) hybridization solution and eluent (formulation see table 1). Each hybridization solution was covered with only 1 filter membrane, and the filters were incubated in a 46℃hybridization box for 3 hours, and after each filter membrane incubation, the probe that did not hybridize successfully was eluted by an eluent water bath at 48℃for 15 minutes. Each filter was then air-dried in the dark after rinsing with distilled water, and then counterstained with 50. Mu.l of 4',6-Diamidino-2-Phenylindole (DAPI) solution at a concentration of 1. Mu.g/ml for 3 minutes. After dyeing, each filter membrane is sequentially rinsed with distilled water and 80% alcohol, and air dried. Finally, the filters were placed on slides and blocked with anti-fluorescence quenching gel Vecta Shield mountant (Burlingame, CA).
The blocked slides were visualized using a fluorescence microscope (Axiostar Plus, carl Zeiss GmbH). In order to directly compare dyeing effects, microscope parameters of various observations and shooting are kept consistent, wherein the exposure time of a Cy-3 fluorescent signal is 10ms, and a high fluorescence transmittance FISH filter disc group is a Cy3 excitation module (orange color, an excitation value of 550nm and emission of 570 nm); the exposure time of the DAPI fluorescent signal is 0.4ms, and the high fluorescence transmittance FISH filter disc group is a DAPI excitation module (blue, excitation value 340nm and emission 570 nm).
FIG. 1B is a photograph of a universal probe for hybridization of a sea tail worm (positive control), wherein the worm body shows red fluorescence, and the success of the worm body FISH hybridization test is shown; FIG. 1C is a photograph of a probe of hybridization of sea tail worm with Um1280, and the worm body shows red fluorescence, which indicates that the probe Um1280 successfully detects the target worm body; FIG. 1D, E is a photograph of a sea tail worm hybridized with a Um626 probe and DAPI, (D) is a photograph under a Cy3 excitation module, the worm has almost no fluorescence, which indicates that the efficacy of the Um626 probe is low, (E) is a photograph under a DAPI excitation module, the nucleus of the worm is blue fluorescence, which indicates the position of the worm; FIG. 1F, G is a photograph of a sea tail worm hybridized with a negative control probe, (F) is a photograph under a Cy3 excitation module, the worm has almost no fluorescence, and (G) the dapI excitation module shows blue fluorescence of the nucleus of the worm, showing the position of the worm.
The detection result of the fluorescence in situ hybridization shows that only Um1280 in the alternative probes can be used as an effective specific probe for detecting the sea tail worm.
Example 2
Fluorescent in situ hybridization detection system optimization
(1) The hybridization solution concentration of optimal Formamide (FA) content is established. The lower the concentration of formamide in the hybridization solution, the higher the fluorescence intensity of the probe, but the higher the fluorescence intensity, the less clear the insect body contour, so the lowest FA concentration with obvious fluorescence effect of the target probe is selected as the optimal concentration by comparison. Hybridization buffer and eluent were formulated at concentrations of hybridization solution with a gradient of formamide concentration ranging from 0% to 50% (formulation see Table 1). After hybridization, microscopic examination (the specific steps are carried out according to the steps (2) and (3) in the preparation of the specific probe), the optimal formamide hybridization solution concentration containing the target probe is 20% (the effect is shown in figure 1H), and the dosages of all the components are as follows: formamide (formamide) 20% by mass, probe Um1280 concentration 30 ng/. Mu.l, SDS (sodium dodecyl sulfate) 0.01% by mass, naCl 0.9M, TRIS-HCl 20mM.
The corresponding eluent comprises the following components in percentage by weight: SDS (sodium dodecyl sulfate) mass fraction 0.01%, naCl molar concentration 0.9M, TRIS-HCl molar concentration 20mM, EDTA molar concentration 5mM.
Table 1. Hybridization buffer and elution buffer formulations at different Formamide (FA) concentrations.
Example 3
(1) The body of large yellow croaker, which is a parasitic sea tail on the body surface, is purchased from the yellow croaker society of the south suede island, and the body sample 1 required for the experiment is washed out from the ulcerated tissue on the body surface, and simultaneously, in order to further verify the specificity of the target probe, another species of ciliate of the species of the marine institute of the national academy of sciences, namely, pseudokon drop Pseudocohnilembus persalinus, is used as the sample 2 to be tested (see Zhan, z., stoeck, t., dunthornb, M. & Xu, k.2014.Identification of the pathogenic ciliate Pseudocohnilembus persalinus (oligomerophra: scilicocilia) by fluorescence in situ hybridization. European Journal of Protistology,50,16-24.).
The probes used in the experiment are a positive control probe Euk1209R (5'-GGGCATCACAGACCTG-3') and a target probe Um1280 (5'-TGCCGATTAAAGATCAACATAC-3'), and the specific steps are as in the steps (2) and (3) in the preparation of the specific probe.
FIGS. 1I-L show the results of the FISH detection. FIGS. 1I and J are fluorescent photographs of a positive control probe hybridized with sea tail worm (I) and another shield fiber ciliate- -water drop pseudokang ciliate Pseudocohnilembus persalinus (J), red fluorescence is emitted to indicate that a FISH experiment is successful, FIGS. 1K and L are fluorescent photographs of a target probe Um1280 hybridized with sea tail worm and water drop pseudokang ciliate, (K) are photographs of a probe Um1280 hybridized with sea tail worm, and a worm body presents red fluorescence to indicate that the probe Um1280 successfully detects the target worm body; (L) no red fluorescence indicates that Um1280 is unable to detect water drop pseudokanbrix; FIG. 1M is a photograph of the insect body of FIG. 1L under a DAPI excitation module, and the nucleus of the insect body shows blue fluorescence, showing the position of the insect body.
In this embodiment, the effect of detecting the sea tail worm by using the target probe is obvious (as shown in fig. 1K), and the target probe does not react positively with the shield fiber ciliate having a similar shape (as shown in fig. 1L), which verifies that the target probe has the characteristic of high specificity.
5 fields are randomly selected on the filter membrane containing the target probe, the number of the insects observed by the white light source is the actual number of the insects in the fields, and the number of the insects with positive reaction is counted. As a result, all insects can be detected to have obvious red fluorescence, which indicates that the technical scheme has the characteristic of high sensitivity.
In addition, the method does not need professional personnel (classification expert) to operate, and compared with the traditional morphological identification which is complex and time-consuming, the method has the advantages of simplicity in operation and short time consumption, and is suitable for common production personnel to use.

Claims (10)

1. A specific primer for detecting sea tail worm, which is characterized in that: the specific primer sequence for detecting the sea tail worm is 5'-TGCCGATTAAAGATCAACATAC-3'.
2. The use of a specific primer according to claim 1, wherein: the specific primer is used as a probe in detection of sea tail filarial parasitic to sea fish.
3. Use of a specific primer according to claim 2, wherein: the probe is Cy-3, namely, a probe Um1280, which is obtained by labeling cyanine dye at the 5' -end of the primer of claim 1.
4. A kit for detecting ciliates of marine fish, characterized in that: a kit comprising the specific primer according to claim 1.
5. A kit for detecting ciliates in marine fish as recited in claim 4 wherein: and the 5' -end of the specific primer sequence is marked with cyanine dye Cy-3 as a target probe.
6. A kit for detecting ciliates in marine fish as in claim 4 or 3 wherein: the kit comprises formamide hybridization solution containing target probes, eluent and a staining agent.
7. A kit for detecting ciliates in marine fish as defined in claim 6 wherein:
the formamide hybridization solution comprises 20% of formamide (formamide) by mass, 30 ng/. Mu.l of probe Um1280 concentration, 0.01% of SDS (sodium dodecyl sulfate) by mass, and 0.9M, TRIS-HCl molar concentration of 20mM of NaCl;
the eluent is SDS (sodium dodecyl sulfate) mass fraction 0.01%, naCl molar concentration 0.9M, TRIS-HCl molar concentration 20mM and EDTA molar concentration 5mM;
the stain was 1.5ng/ml DAPI solution.
8. Use of the kit of claim 4, wherein: the kit is applied to fluorescence in situ hybridization detection of sea tail filarial of sea fish.
9. Use of a kit according to claim 8, characterized in that: in a darkroom, dropwise adding formamide hybridization containing a target probe into a sample to be detected fixed by a fixing solution, incubating for 2 hours at 46 ℃, eluting for 10 minutes by an eluent water bath at 48 ℃ after incubation, performing contrast dyeing on the eluted air-dried sample at room temperature and a 4',6-Diamidino-2-Phenylindole (DAPI) solution, washing the air-dried sample after dyeing, and observing the fluorescence intensity under a fluorescence microscope.
10. Use of a kit according to claim 9, characterized in that: and detecting by a fluorescence microscope, and finding out an orange-red fluorescence signal to indicate that the sample to be detected contains the sea tail worm.
CN202311489093.4A 2023-11-09 2023-11-09 Specific primer and kit for detecting sea tail worms and detection method thereof Pending CN117344042A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202311489093.4A CN117344042A (en) 2023-11-09 2023-11-09 Specific primer and kit for detecting sea tail worms and detection method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202311489093.4A CN117344042A (en) 2023-11-09 2023-11-09 Specific primer and kit for detecting sea tail worms and detection method thereof

Publications (1)

Publication Number Publication Date
CN117344042A true CN117344042A (en) 2024-01-05

Family

ID=89369304

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202311489093.4A Pending CN117344042A (en) 2023-11-09 2023-11-09 Specific primer and kit for detecting sea tail worms and detection method thereof

Country Status (1)

Country Link
CN (1) CN117344042A (en)

Similar Documents

Publication Publication Date Title
US11473129B2 (en) Multiplex labeling of molecules by sequential hybridization barcoding
Ji et al. Single molecule fluorescent in situ hybridization (smFISH) of C. elegans worms and embryos
US20060257884A1 (en) Methods for preparing and analyzing cells having chromosomal abnormalities
DeLong Single-cell identification using fluorescently labeled, ribosomal RNA-specific probes
CN106636318B (en) Nucleic acid signal amplification detection kit
CN105385757B (en) Method and kit for room temperature in situ detection of target nucleic acid in biological sample
Pradillon et al. Species identification of marine invertebrate early stages by whole-larvae in situ hybridisation of 18S ribosomal RNA
JPH07177900A (en) Method for rapid diagnosis of microorganism by in-situ hybridization in watery suspension
CN106434921B (en) A kind of microbial sources tracking molecular marked compound and its high-flux detection method detected for a variety of fecal pollution sources
Dyková et al. Testate amoeba Rhogostoma minus Belar, 1921, associated with nodular gill disease of rainbow trout, Oncorhynchus mykiss (Walbaum)
Whiley et al. Detection of Legionella species in potting mixes using fluorescent in situ hybridisation (FISH)
CN114891902A (en) Primer-probe combination for rapidly detecting five virulent pathogenic bacteria based on liquid drop digital PCR and application method thereof
Bolková et al. Quantitative gene expression analysis in Caenorhabditis elegans using single molecule RNA FISH
CA3179280A1 (en) Systems, devices and methods for analysis
CN108624653A (en) A kind of kit of quantum dot detection of nucleic acids for bloodstream infection pathogen
CN117344042A (en) Specific primer and kit for detecting sea tail worms and detection method thereof
CN112322793A (en) RNA virus rapid detection method based on CCP-FRET
CN104372069B (en) The specific probe of a kind of quick detection water droplet puppet health fibre worm and detection method thereof and application
Chen et al. Development of rRNA and rDNA-targeted probes for fluorescence in situ hybridization to detect Heterosigma akashiwo (Raphidophyceae)
KR20140019760A (en) Molecular methods for identification of oyster species using species-specific probes, dna chip, and molecular kits
US20110269118A1 (en) Methods for identifying cells by combinatorial fluorescence imaging
Pita et al. FISH methods in cytogenetic studies
CN103451310B (en) Gene chip capable of simultaneously detecting various vibrios and method for detecting vibrios
Podushkina et al. Utilizing multiplex fluor LAMPs to illuminate multiple gene expressions in situ
KR102121571B1 (en) DNA repeat based pre-labelled oligoprobes and primers for rapid and efficient FISH in genus Senna

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