[go: up one dir, main page]

CN117305477A - Fluorescence detection kit for genotyping of cat blood group - Google Patents

Fluorescence detection kit for genotyping of cat blood group Download PDF

Info

Publication number
CN117305477A
CN117305477A CN202311584665.7A CN202311584665A CN117305477A CN 117305477 A CN117305477 A CN 117305477A CN 202311584665 A CN202311584665 A CN 202311584665A CN 117305477 A CN117305477 A CN 117305477A
Authority
CN
China
Prior art keywords
sequence
blood
primer
sequence table
probe
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.)
Granted
Application number
CN202311584665.7A
Other languages
Chinese (zh)
Other versions
CN117305477B (en
Inventor
赵方圆
巩玉洁
赵荣茂
毕铭钰
曹英芳
张琼林
陈娟
杨晓霞
袁婷婷
魏单平
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.)
Beijing Nabai Bio Tech Co ltd
Original Assignee
Beijing Nabai Bio Tech Co ltd
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 Beijing Nabai Bio Tech Co ltd filed Critical Beijing Nabai Bio Tech Co ltd
Priority to CN202311584665.7A priority Critical patent/CN117305477B/en
Publication of CN117305477A publication Critical patent/CN117305477A/en
Application granted granted Critical
Publication of CN117305477B publication Critical patent/CN117305477B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • 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

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The embodiment of the invention discloses a fluorescence detection kit for genotyping a cat blood group, which comprises a primer probe combination, wherein the primer probe combination comprises 4 primer probe groups: the primer probe group 1 consists of a primer M Blood RF-179-2A shown in a sequence 1 of a sequence table, a primer M Blood RF-179-2C shown in a sequence 2 of the sequence table, a primer M Blood RR-179-2 shown in a sequence 3 of the sequence table and a probe M Blood RP-179-2 shown in a sequence 4 of the sequence table. The kit for detecting the cat blood group genotyping by using the Taqman probe fluorescence PCR technology for the first time has the advantages of simpler operation, higher sensitivity and specificity, rapid response and high accuracy, solves the problem of noninvasive detection of cats, and is easy to interpret and visual in result.

Description

Fluorescence detection kit for genotyping of cat blood group
Technical Field
The embodiment of the invention relates to the technical field of biology, in particular to a fluorescence detection kit for genotyping a cat blood group.
Background
Similar to the ABO blood group system common to humans, the blood group of cats also mainly includes A, B, AB and unshaped (or called blood group M). Among these, type a blood is most common, accounting for about 90% or more of the total number of cats worldwide (siamesed, burmese, russian blue, oxes, eastern shorthair cats are all essentially type a blood). Type B blood is rare (the most abundant varieties of type B blood are deven cat, british cat, ke Nisi cat, puppet cat, turkish cat, foreign cat, etc.). AB type blood is less common and has a lower proportion than panda blood in humans. Unlike dogs, cats have antibodies to "non-self" or foreign erythrocytes, which can elicit a fatal immune response. For example, a strong accessory anti-type a antibody in a type B cat causes a strong immune response when blood from a type a cat is delivered to a type B cat, and the half-life of the delivered red blood cells is only a few minutes to a few hours, which in turn causes acute or subacute hemolysis of the transfusion, resulting in death of the transfused cat. In addition, it is also particularly important to determine the blood group of cats during breeding, and clinical hemolysis of newborn cats occurs because the milk of the female cats contains antibodies of 'non-self' blood group, and when the female cats of the B blood group breast-feed a or AB blood type kittens, the kittens can generate hemolysis, jaundice and brown urine symptoms within two weeks, and even die. Thus, the identification of the blood group of cats is of great importance.
The existing cat blood type detection mode in the market at present is mostly a blood sampling agglutination method, and although the detection speed is high, the sensitivity is poor, and a professional is required to take blood, so that the blood sample has a short preservation time, and meanwhile, stress response is easily caused to cats. However, other methods are fewer, and the detection method for noninvasively detecting the cat blood type is only the SNaPshot method at present. Although the sensitivity is higher, the operation process is complex, and the detection time is longer. Therefore, a detection method with simple and rapid operation, moderate price and high accuracy is needed in the field.
Disclosure of Invention
Therefore, the embodiment of the invention provides a fluorescence detection kit for genotyping the blood group of cats.
In order to achieve the above object, the embodiment of the present invention provides the following technical solutions:
the invention provides a primer probe combination, which comprises the following 4 primer probe groups:
the primer probe set 1 consists of a primer M Blood RF-179-2A shown in a sequence 1 of a sequence table, a primer M Blood RF-179-2C shown in a sequence 2 of the sequence table, a primer M Blood RR-179-2 shown in a sequence 3 of the sequence table and a probe M Blood RP-179-2 shown in a sequence 4 of the sequence table;
the primer probe group 2 consists of a primer M Blood RF-268-2T shown in a sequence 5 of a sequence table, a primer M Blood RF-268-2A shown in a sequence 6 of the sequence table, a primer M Blood RR-268-2 shown in a sequence 7 of the sequence table and a probe M Blood RP-268-2 shown in a sequence 8 of the sequence table;
the primer probe set 3 consists of a primer M Blood FF-364-1T shown in a sequence 9 of a sequence table, a primer M Blood FF-364-1C shown in a sequence 10 of the sequence table, a primer M Blood FR-364-1 shown in a sequence 11 of the sequence table and a probe M Blood FP-364-1 shown in a sequence 12 of the sequence table;
the primer probe set 4 consists of a primer M Blood FF-1322-1del shown in a sequence 13 of a sequence table, a primer M Blood FF-1322-1wt shown in a sequence 14 of the sequence table, a primer M Blood FR-1322-1-01 shown in a sequence 15 of the sequence table and a probe M Blood FP-1322-1-02 shown in a sequence 16 of the sequence table.
The invention also provides application of the primer probe combination in cat blood group gene detection.
The invention also provides a kit containing the primer probe combination, and the kit is used for identifying the cat blood group genes.
In one embodiment of the invention, the kit further comprises an internal set of primer probes;
the inner reference primer probe set comprises a primer M Blood IC124-F (364F) shown in a sequence table 17, a primer M Blood IC3-F (179F) shown in a sequence table 18, a probe M Blood IC124-P (364) shown in a sequence table 19 and a probe M Blood IC3-P (179) shown in a sequence table 20.
In another aspect, the present invention provides a method for identifying the blood group of a cat comprising the steps of: detecting genotypes of the cats to be detected at the 4 SNP loci, and determining blood types of the cats to be detected according to the genotypes;
the 4 SNP sites are shown below:
a first SNP locus, the 179 th nucleotide of the sequence 21 from the 5' end in the sequence table, wherein the SNP is a G/T polymorphism;
a second SNP locus, the 268 th nucleotide of the sequence 21 from the 5' end in the sequence table, wherein the SNP is a T/A polymorphism;
a third SNP locus, 364 th nucleotide from the 5' end of the sequence 21 in the sequence table, wherein the SNP is C/T polymorphism;
and a fourth SNP locus, wherein the 1322 th nucleotide of the sequence 21 in the sequence table is from the 5' end, and the SNP is a T/delT polymorphism.
The invention finally provides a method for identifying cat blood types, which comprises the following steps: extracting genome DNA of a cat to be detected;
detecting 4 SNP loci of the genome DNA by using the primer probe combination, so as to obtain genotype data of the cat to be detected on the 4 SNP loci;
and determining the blood type of the cat to be tested according to the genotype data.
In the invention, a Taqman probe fluorescence PCR technology is utilized, namely, a fluorescence labeling probe matched with a target sequence is added into a PCR reaction system, the probe is an oligonucleotide with a 5 '-end report group and a 3' -end quenching group, and when the probe is complete in the PCR amplification process, the quenching group is close to the report group, so that fluorescence emitted by the report group is absorbed by the quenching group and does not emit a fluorescence signal. When the primer extends, the probe combined with the template is cut off by Taq enzyme (5 '. Fwdarw.3' exonuclease activity), the reporter group is separated from the quenching group, a fluorescent signal is generated, the whole PCR process is monitored in real time by utilizing the accumulation of the fluorescent signal, and finally the initial template is subjected to qualitative analysis or quantitative analysis through a standard curve. The method has the advantages of strong specificity, high sensitivity, high automation degree, no pollution and the like.
The embodiment of the invention has the following advantages:
the kit for detecting the cat blood group genotyping by using the Taqman probe fluorescence PCR technology for the first time has the advantages of simpler operation, higher sensitivity and specificity, rapid response and high accuracy, solves the problem of noninvasive detection of cats, and is easy to interpret and visual in result.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.
The structures, proportions, sizes, etc. shown in the present specification are shown only for the purposes of illustration and description, and are not intended to limit the scope of the invention, which is defined by the claims, so that any structural modifications, changes in proportions, or adjustments of sizes, which do not affect the efficacy or the achievement of the present invention, should fall within the scope of the invention.
Fig. 1 is a graph of the detection result of the detection hole 1 provided in embodiment 3 of the present invention;
fig. 2 is a graph of the detection result of the detection hole 2 provided in embodiment 3 of the present invention;
fig. 3 is a graph of the detection result of the detection hole 3 according to embodiment 3 of the present invention;
fig. 4 is a graph of the detection result of the detection hole 4 provided in embodiment 3 of the present invention;
fig. 5 is a graph of the detection result of the detection hole 5 according to embodiment 3 of the present invention;
FIG. 6 is a graph of the detection result of the detection hole 6 according to embodiment 3 of the present invention;
fig. 7 is a graph of the detection result of the detection hole 7 according to embodiment 3 of the present invention;
fig. 8 is a graph of the detection result of the detection hole 8 according to embodiment 3 of the present invention.
Detailed Description
Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 Cat genomic DNA extraction
Step one, collecting and processing samples, and collecting a cat oral swab: unsealing the sampling swab packaged in a sealing way, and taking out the oral swab.
And secondly, stretching one end with the sampling crown into the oral cavity, tightly abutting against the inner cheek at one side of the oral cavity, slightly forcefully scraping back and forth for more than 10 times, preferably more than 30 seconds, and rotating the swab while scraping.
And thirdly, after scraping, the sampling swab can be held by one hand, the sampling tube is opened, the thumb is used for propping against the sampling push rod, and the sampling crown is propped into the sampling tube. The sampling tube cover is screwed down, so that the sampling tube can be stored at normal temperature. The loop bar and the push rod are thrown into the medical garbage can.
And step four, extracting and processing the nucleic acid sample, extracting total DNA from the acquired nucleic acid sample by using a commercial Kit (TIANamp Virus DNA/RNA Kit), and storing the extracted total DNA at-80 ℃ for later use.
Example 2 primer-probe set design for detection of feline blood group determining genotype
1. Primer probe combination design
According to the cat blood group determining Gene CMAH (Gene ID: 100750220) which can be inquired on NCBI website, the nucleotide sequence of the first 1400 of the Gene is shown as sequence 21 in a sequence table, and four single nucleotide mutation sites (CMAH 179G > T, CMAH268T > A, CMAH364C > T and CMAH1322 delT) of the cat blood group determining Gene CMAH are shown as sequence 21 in the sequence table, and primer 5.0 software is adopted to design specific primers and Taqman probes as shown in the table 1, and internal index primers and probes.
Table 1 primer and probe sequence table
Wherein the nucleotide sequence of sequence 21 is:
TCACGTATAACACCTGAAACGGAAACCCTAGGGAATCTTCCACAGGCGGGGAAGCGGGAAAGACCAACACGTCTACAGCGACAACCAATACAAAGCCATCCGGAGCCTGAACTATGACTTCCCTTACTCCTTTCTGGCTTCTCTGAGTTTTCTCCATGGCTGCTGTGCATCTCTGTTAGCTGAGGAGGATTTTAAGGCAACACCCAAAAACCTCTTCCCAGGTAGGTGATCCTGAGCCTATAAACTGCCAGTTTGTCCTGCTTTATGAATAAGATCTTGTGGAAGAGGTGCCAGGCTATCCAACTGGGGCAACTCTGTACGAGGCGAGTGTCTCTGTCGCTTGGAAAGGTTCAATCTGATTCCTCCAGCTGGGTGTGGTTTCCAGTGTTTCCCGTGTGAATTTCTGTGGCGGGGGTGGGGGGGGCAGGTGATGGCGGGGCCTGGGCTGCCCCACCCTGCCCCAGCCCTGCCCCCAGGGCTCTCCCCATGGAGCTTTGATGCCGATAGCATCCCGTCTGTGGGCAGGTCCCTGACCCTGGAGGGGATGGGAAGGGTGGTCCCGGGCAGGCTCTGCAGGACCTTCCCAAGCCACCCCACATCTTCCATCAGCTGCGAGTACTGGGCTTGGAGGAGTGAAGAAGCGCCCAGAGAATCCCCTGGGTTTCTGACATGGATTTCACCGGCCCCCTGGCCACCACCCACATGGTATCAGGATCCGTCACCCCGGCCACAGAGCACTCCCCTTCCATGACCTTTTTTCAGGGGTGGGAGGAGCCCAAAAGGTCAAGTGCTTGATTCAGATTCCAATTAAAACGAGTCATTGTTCTTGGCATTGAGACCCCCAAACCGACATGGCTCAGAGCTGTGAGGATGGGAAGCAAATGTCTCTCAGCTGAGTCGTTAGAGGTAAGAATGTGGGAGAAACAGCCCCTTGTATCTGTTACTATATATCTGATAGACTCTTTCCTGTTCTCAACTGGCACCATTCATAGCTGCATTTTTAGTAAGCCTGTTACTTTGACCTTGCAAGGCTTGTCACCGTGATCTATTTTCCAGACGGCTTTGGGTTACCTGGTAAAACCAGTGAATTCTGTGGGCAAGAGTCCACTGCTGTGCTTCTTTTTTTTTTTTTTTTCGCTGTGGAATGAACGGATGAGGAGCAGTGCTGTATGGAATGGGATGCTGGTGGGTAAGGGTTTCCGCATTTTCACAGCTGATGGTGCTGGCTATTACAAGTTCTGGAAGGAAAATCTAACCCATATTGAGAATAGTGTCTGTTGTGATGAGGACAGATCACTTGCTTTTGTAAGATGGAAGGGGTCCAGTATAATCCATCTGGTGGGTGGCTTCTCCCTGGGAAATGGGGCAGTATTGGAGAGTCGGTGTTGGTCTCAGCTATT。
2. cat blood group determining gene CMAH genotyping
And (3) preparing a fluorescent PCR reaction system, wherein each sample is required to simultaneously carry out 8-hole independent fluorescent PCR reactions, wherein the upstream primer and the downstream primer in the reaction solution are respectively 10 mu mol/L, the probe concentration is 10 mu mol/L, and 5 mu L of the genome DNA to be detected prepared in the example 1 is taken. The specific configuration is shown in table 2.
TABLE 2 fluorescent PCR reaction System configuration Table
3. Fluorescent PCR reaction procedure
The specific reaction procedure is shown in Table 3 for fluorescent PCR.
TABLE 3 fluorescent PCR reaction procedure
4. Analysis and interpretation of fluorescent PCR test results
The nucleic acids extracted in example 1 were amplified simultaneously using 8 systems according to the procedure shown in Table 3. Based on the Ct values and curve conditions of FAM and VIC for each well, the validity of the sample results was determined according to Table 4. And judging genotypes of the 4 SNP loci according to whether each hole has an amplification curve, ct values and delta Ct, and judging cat blood types and genotype judgment standards according to a genotype comparison table 5 of the 4 SNP loci.
TABLE 4 interpretation of specific detection results of fluorescence PCR of cat blood group
Table 5 cat blood group determination criteria
Example 3 use of the primer probe set of example 2 for detection of blood group in cats
Genomic DNA extracted from cats to be tested is added into 8 detection holes according to specified amounts, and corresponding detection primer probe sets, internal index primers and probes are added according to the requirements of Table 2.
Fluorescent PCR was performed according to the PCR reaction procedure of example 2 table 3. The fluorescence PCR detection results are shown in FIGS. 1 to 8, the detection result patterns of 8 detection holes, the determination results are shown in Table 6 according to the requirements of tables 4 and 5 of example 2, the genotypes of 4 SNP loci of the cat to be tested of this example, and the determined blood type of the cat to be tested.
TABLE 6 blood group and genotype of cats to be tested
Example 4 compliance verification of the Cat blood group fluorescent Probe detection kit of the invention
The detection kit using the fluorescent probe combination of the embodiment 2 of the invention and the test strip of the commercial blood drawing agglutination method are used for simultaneous detection, wherein the detection kit using the fluorescent probe combination detects 110 oral swabs, the commercial blood drawing agglutination method test strip detects 110 EDTA anticoagulated whole blood samples from animal sources, and the results are compared, so that the coincidence rate of the detection kit prepared by the primer probe set of the embodiment 2 of the invention and the whole sample of the outsourcing test strip is 100%, which shows good correspondence with the comparison, and the specific results are shown in the coincidence verification results of the fluorescent probe detection kit and the competitive product detection of the invention in Table 7.
TABLE 7 compliance verification results
Sample numbering Agglutination method (competing brand) Results of bidding products Agglutination method (Nabai) PCR
M293 Blessing wound A A A
M294 Blessing wound A A A
M295 Blessing wound A A A
M296 Blessing wound A A A
M297 Blessing wound A A A
M298 Blessing wound A A A
M299 Blessing wound A A A
M300 Blessing wound A A A
M301 Blessing wound A A A
M302 Blessing wound A A A
M303 Blessing wound A A A
M304 Blessing wound A A A
M305 Blessing wound A A A
M306 Blessing wound A A A
M307 Blessing wound A A A
M308 Blessing wound A A A
M309 Blessing wound A A A
M310 Blessing wound A A A
M311 Blessing wound A A A
M312 Blessing wound A A A
M313 Taiwan Baiwei A A A
M314 Taiwan Baiwei A A A
M315 Taiwan Baiwei A A A
M317 Blessing wound A A A
M318 Blessing wound A A A
M319 Blessing wound A A A
M320 Blessing wound A A A
M321 Blessing wound A A A
M322 Blessing wound A A A
M323 Blessing wound A A A
M324 Blessing wound A A A
M325 Blessing wound A A A
M326 Blessing wound A A A
M327 / / Hemolysis is not measured A
M328 Taiwan Baiwei A A A
M329 Taiwan Baiwei A A A
M330 Taiwan Baiwei A A A
M331 Taiwan Baiwei A A A
M332 / / A A
M333 / / A A
M335 / / A A
M336 / / A A
M337 / / A A
M338 / / A A
M341 / / A A
M344 / / A A
M345 / / A A
M348 / / A A
M351 / / A A
M352 / / A A
M355 / / A A
M356 / / A A
M358 / / A A
M360 / / A A
M361 / / B B
M362 / / A A
M363 / / A A
M364 / / A A
M282 / / B B
M365 / / A A
M366 / / A A
M367 Taiwan Baiwei B B B
M368 Taiwan Baiwei A A A
M369 Taiwan Baiwei A A A
M370 Taiwan Baiwei A A A
M371 Taiwan Baiwei A A A
M372 / / A A
M373 / / A A
M374 / / A A
M375 / / A A
M376 / / A A
M377 / / A A
M378 / / A A
M379 / / A A
M380 / / A A
M381 / / A A
M382 / / A A
M383 / / A A
M384 / / A A
M385 / / A A
M386 / / A A
M387 / / A A
M388 / / A A
M389 / / A A
M390 / / A A
M391 / / A A
M392 / / A A
M393 / / A A
M394 / / A A
M395 / / A A
M396 / / A A
M397 / / A A
M398 / / A A
M399 / / A A
M400 / / A A
M401 / / A A
M402 / / A A
M403 / / A A
M404 / / A A
M405 / / A A
M406 / / A A
M407 / / A A
M408 / / A A
M409 / / A A
M410 / / A A
M411 / / A A
M412 / / A A
M413 / / A A
M414 / / A A
M415 / / A A
While the invention has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (6)

1. The primer probe combination comprises the following 4 primer probe groups:
the primer probe set 1 consists of a primer M Blood RF-179-2A shown in a sequence 1 of a sequence table, a primer M Blood RF-179-2C shown in a sequence 2 of the sequence table, a primer M Blood RR-179-2 shown in a sequence 3 of the sequence table and a probe M Blood RP-179-2 shown in a sequence 4 of the sequence table;
the primer probe group 2 consists of a primer M Blood RF-268-2T shown in a sequence 5 of a sequence table, a primer M Blood RF-268-2A shown in a sequence 6 of the sequence table, a primer M Blood RR-268-2 shown in a sequence 7 of the sequence table and a probe M Blood RP-268-2 shown in a sequence 8 of the sequence table;
the primer probe set 3 consists of a primer M Blood FF-364-1T shown in a sequence 9 of a sequence table, a primer M Blood FF-364-1C shown in a sequence 10 of the sequence table, a primer M Blood FR-364-1 shown in a sequence 11 of the sequence table and a probe M Blood FP-364-1 shown in a sequence 12 of the sequence table;
the primer probe set 4 consists of a primer M Blood FF-1322-1del shown in a sequence 13 of a sequence table, a primer M Blood FF-1322-1wt shown in a sequence 14 of the sequence table, a primer M Blood FR-1322-1-01 shown in a sequence 15 of the sequence table and a probe M Blood FP-1322-1-02 shown in a sequence 16 of the sequence table.
2. Use of the primer probe combination of claim 1 in the detection of feline blood group genes.
3. A kit comprising the primer probe combination of claim 1 for use in identifying a cat blood group gene.
4. The kit according to claim 3, wherein,
the kit further comprises an internal index primer probe set;
the inner reference primer probe set comprises a primer M Blood IC124-F (364F) shown in a sequence table 17, a primer M Blood IC3-F (179F) shown in a sequence table 18, a probe M Blood IC124-P (364) shown in a sequence table 19 and a probe M Blood IC3-P (179) shown in a sequence table 20.
5. A method of identifying a feline blood group comprising the steps of: detecting genotypes of the cats to be detected at the 4 SNP loci, and determining blood types of the cats to be detected according to the genotypes;
the 4 SNP sites are shown below:
a first SNP locus, the 179 th nucleotide of the sequence 21 from the 5' end in the sequence table, wherein the SNP is a G/T polymorphism;
a second SNP locus, the 268 th nucleotide of the sequence 21 from the 5' end in the sequence table, wherein the SNP is a T/A polymorphism;
a third SNP locus, 364 th nucleotide from the 5' end of the sequence 21 in the sequence table, wherein the SNP is C/T polymorphism;
and a fourth SNP locus, wherein the 1322 th nucleotide of the sequence 21 in the sequence table is from the 5' end, and the SNP is a T/delT polymorphism.
6. A method of identifying a feline blood group comprising the steps of:
extracting genome DNA of a cat to be detected;
detecting 4 SNP loci of the genome DNA by using the primer probe combination of claim 1, thereby obtaining genotype data of the cat to be detected on the 4 SNP loci;
and determining the blood type of the cat to be tested according to the genotype data.
CN202311584665.7A 2023-11-27 2023-11-27 Fluorescence detection kit for genotyping of cat blood group Active CN117305477B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202311584665.7A CN117305477B (en) 2023-11-27 2023-11-27 Fluorescence detection kit for genotyping of cat blood group

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202311584665.7A CN117305477B (en) 2023-11-27 2023-11-27 Fluorescence detection kit for genotyping of cat blood group

Publications (2)

Publication Number Publication Date
CN117305477A true CN117305477A (en) 2023-12-29
CN117305477B CN117305477B (en) 2024-03-08

Family

ID=89255485

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202311584665.7A Active CN117305477B (en) 2023-11-27 2023-11-27 Fluorescence detection kit for genotyping of cat blood group

Country Status (1)

Country Link
CN (1) CN117305477B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117805402A (en) * 2024-03-01 2024-04-02 北京纳百生物科技有限公司 Cat blood typing detection method and kit
CN117820483A (en) * 2024-03-01 2024-04-05 北京纳百生物科技有限公司 Cat N-terminal brain natriuretic peptide precursor protein monoclonal antibody, detection test strip and application

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080220427A1 (en) * 2006-12-21 2008-09-11 Regents Of The University Of California Test for determining blood type in the cat
CN103898213A (en) * 2014-03-14 2014-07-02 南方医科大学 Nested asymmetric PCR reagent kit for detecting alpha 2 globin gene point mutation
KR101684832B1 (en) * 2016-06-22 2016-12-08 (주)한국유전자정보센터 A composition for determining the feline blood types by realtime PCR and a detection method using it
CN108048547A (en) * 2017-12-22 2018-05-18 美因健康科技（北京）有限公司 The method that blood Direct PCR carries out Taqman partings
CN111118138A (en) * 2019-12-13 2020-05-08 重庆浦洛通基因医学研究院有限公司 Kit and method for detecting polymorphism of folate metabolism ability genes MTHFR and MTRR
CN113736923A (en) * 2021-10-15 2021-12-03 安徽农业大学 Primer and kit for real-time fluorescent quantitative PCR (polymerase chain reaction) detection of Chapparvovirus
CN115717170A (en) * 2022-12-26 2023-02-28 北京格致博雅生物科技有限公司 Method for detecting blood type of cat by using SNaPshot technology
CN115927670A (en) * 2022-12-19 2023-04-07 北京格致博雅生物科技有限公司 Kit for detecting six hair colors of cat by using fluorescence ARMS-PCR capillary electrophoresis technology

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080220427A1 (en) * 2006-12-21 2008-09-11 Regents Of The University Of California Test for determining blood type in the cat
CN103898213A (en) * 2014-03-14 2014-07-02 南方医科大学 Nested asymmetric PCR reagent kit for detecting alpha 2 globin gene point mutation
KR101684832B1 (en) * 2016-06-22 2016-12-08 (주)한국유전자정보센터 A composition for determining the feline blood types by realtime PCR and a detection method using it
CN108048547A (en) * 2017-12-22 2018-05-18 美因健康科技（北京）有限公司 The method that blood Direct PCR carries out Taqman partings
CN111118138A (en) * 2019-12-13 2020-05-08 重庆浦洛通基因医学研究院有限公司 Kit and method for detecting polymorphism of folate metabolism ability genes MTHFR and MTRR
CN113736923A (en) * 2021-10-15 2021-12-03 安徽农业大学 Primer and kit for real-time fluorescent quantitative PCR (polymerase chain reaction) detection of Chapparvovirus
CN115927670A (en) * 2022-12-19 2023-04-07 北京格致博雅生物科技有限公司 Kit for detecting six hair colors of cat by using fluorescence ARMS-PCR capillary electrophoresis technology
CN115717170A (en) * 2022-12-26 2023-02-28 北京格致博雅生物科技有限公司 Method for detecting blood type of cat by using SNaPshot technology

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ALEXANDRA KEHL等: "Molecular characterization of blood type A, B, and C (AB) in domestic cats and a CMAH genotyping scheme", PLOS ONE, vol. 13, no. 9, 20 September 2018 (2018-09-20), pages 0204287 *
EVA SPADA等: "Evaluating the association between blood genotype or phenotype and haemoplasma infection in UK and Italian cats", VET REC., vol. 192, no. 12, 12 October 2022 (2022-10-12), pages 2282 *
S TASKER等: "Feline blood genotyping versus phenotyping, and detection of non-AB blood type incompatibilities in UK cats", J SMALL ANIM PRACT., vol. 55, no. 4, 30 April 2014 (2014-04-30), pages 185 - 189, XP055550392, DOI: 10.1111/jsap.12180 *
佚名: "点突变精准检测技术-ARMS PCR", pages 1, Retrieved from the Internet <URL:https://mp.weixin.qq.com/s?__biz=MzA5OTUwOTE0Ng==&mid=2650648178&idx=2&sn=39b8e81c9486cf3235d865af2e077f0d&chksm=88883887bfffb191bc7a3982160fbec485c8d693614a43a75d56accffebdda6136d76556a4d9&scene=2720220616> *
朱小亚等: "基于Taqman荧光ARMS-PCR技术检测CYP3A5基因多态性方法的建立", 分子诊断与治疗杂志, vol. 978, no. 1, pages 1 - 15 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117805402A (en) * 2024-03-01 2024-04-02 北京纳百生物科技有限公司 Cat blood typing detection method and kit
CN117820483A (en) * 2024-03-01 2024-04-05 北京纳百生物科技有限公司 Cat N-terminal brain natriuretic peptide precursor protein monoclonal antibody, detection test strip and application
CN117820483B (en) * 2024-03-01 2024-05-28 北京纳百生物科技有限公司 Cat N-terminal brain natriuretic peptide precursor protein monoclonal antibody, detection test strip and application
CN117805402B (en) * 2024-03-01 2024-05-28 北京纳百生物科技有限公司 Cat blood typing detection method and kit

Also Published As

Publication number Publication date
CN117305477B (en) 2024-03-08

Similar Documents

Publication Publication Date Title
CN117305477B (en) Fluorescence detection kit for genotyping of cat blood group
CN108624659B (en) Real-time quantitative PCR method for detecting components of meat products
CN107794304B (en) Genotyping detection kit for yak individual identification and paternity test
CN107760789B (en) A genotyping detection kit for yak paternity testing and individual identification
CN113278711B (en) Gene chip, molecular probe combination, kit and application for sheep germplasm resource identification and pedigree reconstruction
CN114959059B (en) A combination of SNP loci related to the coefficient of variation of wool fiber diameter of fine-wool sheep and its application
CN106367491A (en) Kit for detecting deafness susceptibility genes
CN113265476A (en) Gene chip, molecular probe combination, kit and application for analyzing milk production performance of sheep
CN113265475A (en) Gene chip for analyzing sheep fat tail, molecular probe combination, kit and application
CN116904607A (en) SNP molecular marker combination for screening meat quality traits of Guan Ling cattle and application thereof
CN109355376A (en) Fragile X mental retardation FMR1 genetic test primer, kit and detection method
CN115717170A (en) Method for detecting blood type of cat by using SNaPshot technology
CN118406775B (en) Application of a SNP molecular marker associated with the chest girth trait of beef cattle at 3 months of age
CN111088327A (en) A kind of method and application of SIKE1 gene CNV marker-assisted detection of cattle body size traits
CN111073957A (en) Method for calibrating real-time fluorescent quantitative PCR instrument
CN115478112B (en) A detection method and application of yak MOGAT2 gene CNV marker
CN111154892A (en) Primer pair, kit, method and application for detecting sheep BMPR1B gene insertion/deletion polymorphism
CN111088369A (en) Detection method, primer pair and application of sheep RORA gene insertion/deletion polymorphism
CN112501320B (en) Snake origin component rapid detection kit and application thereof
WO2023087791A1 (en) Dna barcode for identifying origin of floccularia luteovirens, primer group, and application
CN110894531A (en) STR locus set for pig and application
CN110343766A (en) A method of based on calf-derived Cyclospora in QPCR quantitative detection livestock meat
CN116555402B (en) Telomere detection kit and telomere length detection method for non-disease diagnosis purpose
CN117265139B (en) Weight-related SNP (single nucleotide polymorphism) marker of safucke sheep as well as screening method and application thereof
CN111518918B (en) A kind of method for screening dairy cows with different lactoferrin content and test kit used therefor

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