Primer and probe composition for cat blood type detection, detection method, and use

Patent metadata

Field	Value
Publication number	US-12480153-B2
Application number	US-202418938479-A
Country	US
Kind code	B2
Filing date	Nov 6, 2024
Priority date	Nov 21, 2023
Publication date	Nov 25, 2025
Grant date	Nov 25, 2025

Abstract

Provided are a primer and probe composition for cat blood type detection, a detection method, and use. The primer and probe composition for detecting the wild-types and the mutants of the above four sites is designed according to the single nucleotide polymorphism (SNP) mutation of genotyping of 179G>T, 268T>A, 364C>T and 1322delT four sites of a cat cytidine monophospho-N-acetylneuraminic acid (CMAH) gene; an amplification curve is plotted by utilizing a real-time fluorescence polymerase chain reaction (PCR) technology, and the recognition of bases of four SNP sites is achieved by analysis of the amplification curve, so as to determine cat blood types. The detection method of the present disclosure is carried out entirely in a closed space, thereby avoiding the problem of environmental aerosol pollution.

First claim

What is claimed is:

1 . A cat blood type detection method, comprising:
performing PCR reaction using a nucleic acid extracted from a cat oral swab sample as template DNA, employing a primer and probe composition, and directly performing fluorescence collection without taking out a PCR reaction product after the PCR reaction is completed; and detecting and analyzing whether a cat cytidine monophospho-N-acetylneuraminic acid (CMAH) gene has the single nucleotide polymorphism (SNP) mutation of 179G>T, 268T>A, 364C>T and 1322delT four sites by using a real time fluorescence PCR technology, thereby achieving the cat blood type detection;
the primer and probe composition comprising four groups of primers and four groups of probes:
a first group of primers comprising a forward primer comprising a sequence of SEQ ID NO. 1, and a reverse primer comprising a sequence of SEQ ID NO.2;
a second group of primers comprising a forward primer comprising a sequence of SEQ ID NO.5, and a reverse primer comprising a sequence of SEQ ID NO.6;
a third group of primers comprising a forward primer comprising a sequence of SEQ ID NO.9 and a reverse primer comprising a sequence of SEQ ID NO.10;
a fourth group of primers comprising a forward primer comprising a sequence of SEQ ID NO. 13, and a reverse primer comprising a sequence of SEQ ID NO.14;
a first group of probes comprising a wild-type probe comprising a sequence of SEQ ID NO.3, and a mutant probe comprising a sequence of SEQ ID NO.4;
a second group of probes comprising a wild-type probe comprising a sequence of SEQ ID NO.7, and a mutant probe comprising a sequence of SEQ ID NO.8;
a third group of probes comprising a wild-type probe comprising a sequence of SEQ ID NO.11, and a mutant probe comprising a sequence of SEQ ID NO.12; and
a fourth group of probes comprising a wild-type probe comprising a sequence of SEQ ID NO.15, and a mutant probe comprising a sequence of SEQ ID NO.16.

2 . The cat blood type detection method according to claim 1 , further comprising: determining a detection result of the sample to be negative if an amplification signal is not obtained during the PCR reaction by utilizing real-time fluorescence PCR, conversely, determining the detection result of the sample to be positive if the amplification signal is obtained by utilizing the real-time fluorescence PCR.

3 . The cat blood type detection method according to claim 1 , further comprising the following steps of:
preparing a primer and probe in the primer and probe composition for detecting a wild-type site into a wild-type detection solution, and preparing a primer and probe for detecting a mutant site into a mutant detection solution; and respectively mixing a to-be-detected sample containing the template DNA with the wild-type detection solution and the mutant detection solution, then respectively performing the detection of a wild-type site and a mutant site by utilizing the real-time fluorescence PCR technology, and determining the cat blood type according to the two obtained detection results of the wild-type site and the mutant site.

4 . The cat blood type detection method according to claim 3 , further comprising:
respectively preparing a wild-type positive reference and a mutant positive reference into a wild-type positive quality control product and a mutant positive quality control product at a final concentration of 10 5 -10 7 copies/ml; wherein the wild-type positive reference comprises a wild-type specific plasmid fragment comprising a sequence of SEQ ID NO.17, and the mutant positive reference comprises a mutant specific plasmid fragment comprising a sequence of SEQ ID NO.18; the wild-type detection solution detects the wild-type positive reference, and the detection results are as follows: four fluorescence channels FAM, VIC, ROX and CY5 are all positive; the wild-type detection solution detects the mutant positive reference, and the detection results are as follows: four fluorescence channels FAM, VIC, ROX and CY5 four fluorescence channels are all negative; the mutant detection solution detects the wild-type positive reference, and the detection results are as follows: four fluorescence channels FAM, VIC, ROX and CY5 are all negative; the mutant detection solution detects the mutant positive reference, and the detection results are as follows: four fluorescence channels FAM, VIC, ROX and CY5 are all positive; the wild-type positive reference and the mutant positive reference are respectively applied as the wild-type positive quality control product and the mutant positive quality control product for determining whether experimental results are under control.

5 . The cat blood type detection method according to claim 4 , further comprising:
respectively mixing the to-be-detected sample, the wild-type positive reference, the mutant positive reference and a blank control sample with the wild-type detection solution and the mutant detection solution to prepare multiple groups of samples; respectively placing the multiple groups of samples in a real-time fluorescence PCR instrument which runs according to the following set procedures: step {circle around (1)}: 95° C.×5 min, running for 1 cycle; step {circle around (2)}: 95° C.×5 s; 60° C.×20 s; running for 45 cycles; in step {circle around (2)}, respectively setting the FAM channel, the VIC channel, the ROX channel and the CY5 channel when 60° C.×20 s for fluorescence collection, wherein the sites of a cat CMAH gene detected by the VIC channel, the ROX channel and the CY5 channel respectively correspond to 179G >T, 268T>A, 364C>T and 1322delT; and analyzing fluorescence collection results, including: the detection result is interpreted as positive when Ct≤40 in the detection result; the detection result is interpreted as negative when Ct is not detected in the detection result.

6 . A cat blood type detection reagent adopting the cat blood type detection method according to claim 1 ,
wherein the cat blood type detection reagent comprises primer and probe compositions as shown in SEQ ID NO.1-SEQ ID NO.16, and the primer and probe compositions comprise:
a first group of primers comprising a forward primer comprising a sequence of SEQ ID NO.1 and a reverse primer comprising a sequence of SEQ ID NO.2;
a second group of primers comprising a forward primer comprising a sequence of SEQ ID NO.5 and a reverse primer comprising a sequence of SEQ ID NO.6;
a third group of primers comprising a forward primer comprising a sequence of SEQ ID NO.9 and a reverse primer comprising a sequence of SEQ ID NO.10;
a fourth group of primers comprising a forward primer comprising a sequence of SEQ ID NO.13 and a reverse primer comprising a sequence of SEQ ID NO.14;
a first group of probes comprising a wild-type probe comprising a sequence of SEQ ID NO.3 and a mutant probe comprising a sequence of SEQ ID NO.4;
a second group of probes comprising a wild-type probe comprising a sequence of SEQ ID NO.7 and a mutant probe comprising a sequence of SEQ ID NO.8;
a third group of probes comprising a wild-type probe comprising a sequence of SEQ ID NO.11 and a mutant probe comprising a sequence of SEQ ID NO.12; and
a fourth group of probes comprising a wild-type probe having comprising a sequence of SEQ ID NO.15 and a mutant probe comprising a sequence of SEQ ID NO.16.

7 . The cat blood type detection reagent according to claim 6 , further comprising a positive reference; the positive reference comprising a wild-type positive reference and a mutant positive reference; the wild-type positive reference comprising a wild-type specific plasmid fragment comprising a sequence of SEQ ID NO.17; and the mutant positive reference comprising a mutant specific plasmid fragment comprising a sequence of

Assignees

• Univ Soochow
• Suzhou Sundia Innovation Biotechnology Co Ltd

Inventors

• Fei Min
• Wu Yingchao
• Li Weiwei
• Cao Yifeng
• Lu Kaiye
• Yan Waner
• Fei Zhaoqin
• Zhu Dongning
• Lu Lijun

Classifications

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• C12Q2600/124

  Animal traits, i.e. production traits, including athletic performance or the like · CPC title

• C12Q1/6876 Primary

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title