CN112301119A - Method, primer and kit for screening multiple genetic diseases of cats - Google Patents
Method, primer and kit for screening multiple genetic diseases of cats Download PDFInfo
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- CN112301119A CN112301119A CN201910688040.2A CN201910688040A CN112301119A CN 112301119 A CN112301119 A CN 112301119A CN 201910688040 A CN201910688040 A CN 201910688040A CN 112301119 A CN112301119 A CN 112301119A
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- dna
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- 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
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- 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
- C12Q1/6858—Allele-specific amplification
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- 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
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- 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
- C12Q2600/16—Primer sets for multiplex assays
Abstract
The invention discloses a method for screening multiple genetic diseases of cats, which comprises the following steps: 1) obtaining a sample containing DNA from a cat; 2) extracting DNA from the sample containing DNA; 3) constructing a second generation sequencing library to obtain DNA fragments containing 20 DNA targets to be detected, wherein the following primers are used: SEQ ID No.1-40, wherein each two of said primers in turn constitute a forward primer and a reverse primer; 4) sequencing the library; 5) analyzing sequencing data to obtain DNA mutation conditions of 40 disease sites; 6) predicting or diagnosing disease based on the mutation. The invention also discloses a primer and a kit for screening the multiple genetic diseases of the cats.
Description
Technical Field
The invention belongs to the field of gene detection, and particularly relates to a method and a kit for screening multiple genetic diseases of cats.
Background
Methods for screening feline genetic diseases currently fall into two main categories, first category: binding of Biochemical entities by clinical characterization
The testing method is used for diagnosing the genetic disease of the cats; the second type: the mutation sites causing genetic diseases were qualitatively identified by PCR and Sanger sequencing.
The technical disadvantages of the two types of products are as follows: the first type: 1) can be detected only when the disease is in attack, and the gold time for prevention, delay and treatment is lost; 2) the symptoms of a plurality of genetic diseases in the disease onset stage are very similar, and even after a plurality of clinical characterization examinations or biochemical tests are carried out, the specific diseases are difficult to diagnose; 3) cats do not describe their true feelings in language as humans can, presenting greater challenges to disease diagnosis. The second type: 1) the cost of PCR and Sanger sequencing is high, if cats need to comprehensively screen dozens or even hundreds of genetic diseases, the cost is often too high to realize 2) when various diseases are comprehensively screened, a large amount of cat saliva or blood samples are needed, and sampling is troublesome.
Accordingly, there is a need in the art for a method and kit for screening multiple genetic diseases in cats.
Disclosure of Invention
The invention achieves the detection of 20 disease sites by saliva DNA extraction and a targeted high-throughput sequencing kit. Accordingly, in a first aspect, the present invention provides a method of screening cats for multiple genetic diseases, said method comprising the steps of:
1) obtaining a sample containing DNA from a cat;
2) extracting DNA from the sample containing DNA;
3) constructing a second generation sequencing library to obtain DNA fragments containing 20 DNA targets to be detected, wherein the primers are as follows:
4) sequencing the library;
5) analyzing sequencing data to obtain DNA mutation conditions of 20 disease sites;
6) predicting or diagnosing disease based on the mutation.
In one embodiment, the DNA-containing sample is saliva from a cat.
In one embodiment, the sequencing is performed in step 4) using an Illumina next generation sequencer.
In a second aspect, the invention provides a kit for screening multiple genetic diseases in cats, the kit comprising
The following primers: 1-40, wherein each two of said primers in turn constitute a forward primer and a reverse primer.
In one embodiment, the kit further comprises reagents for obtaining a DNA-containing sample from a cat, preferably saliva.
In one embodiment, the kit further comprises reagents for extracting DNA from the sample.
In one embodiment, the kit further comprises reagents for sequencing the library.
In a third aspect, the invention also provides a primer group for screening multiple genetic diseases of cats, and the primer group
Comprises the following primers: 1-40, wherein each two of said primers in turn constitute a forward primer and a reverse primer.
The invention has the advantages that: compared with the prior scheme I, the DNA information is used for disease diagnosis, so that the method is accurate and predictive, and the disease risk can be detected before the disease is developed. Compared with the existing scheme II, because a plurality of sites can be simultaneously detected by the second-generation sequencing platform, the cost is greatly reduced, the detection speed is improved, and the requirement on the initial DNA sample amount is reduced.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive
Serve as limitations to the claimed invention.
Detailed Description
In the present invention, the present inventors found that 20 sites on the genome of cats are associated with diseases, and in order to detect these 20 sites, the present inventors first obtained 20 DNA sequences by which the corresponding sites, these 20 DNA sequences and the corresponding mutation sites can be uniquely located.
In the present invention, for each site, dozens of primers can be designed using primer design for corresponding
In order to detect the 20 groups of primers in one kit, the invention verifies through complicated experiments that the primers with the length of 18-25 have minimum interference with each other, so that the primers in the length range are selected from the primers in each site for further determination, and 3 pairs of primers closest to the range are selected for determination of the sites without the primers in the range. Meanwhile, the inventors considered the sequence similarity between primers at different sites, and set the average similarity of each sequence to other selected sequences to 20%, and the similarity to any other selected sequence to not more than 40%, the smaller the more preferable. The following sequences were selected:
in the present invention, for SEQ ID NO. X, X is an odd number and is a forward primer, and X is an even number and is a reverse primer.
Compared with the prior scheme I, the method of the invention uses DNA information in saliva or blood to diagnose diseases. Compared with the existing scheme II, the PCR primer which can be amplified in the same reaction is designed, and a plurality of sites are simultaneously detected through a second-generation sequencing platform.
Objects and functions of the present invention and methods for accomplishing the same by reference to the exemplary embodiments
The method will be elucidated. However, the present invention is not limited to the exemplary embodiments disclosed below; it can be implemented in different forms. The nature of the description is merely to assist those skilled in the relevant art in a comprehensive understanding of the specific details of the invention.
Examples
In this example, the experimental procedure was as follows:
saliva samples were taken from 4 cats (cat nos. 1-4).
The procedure for extracting saliva samples is as follows
1) A disposable sampling swab was used. The swab head was inserted between the cat's lip and the upper gum, the upper lip was pressed and the swab head was gently rotated 10 times.
2) The swab is removed without exposing the swab head to any sources of contamination.
3) The swab was air dried for 15 minutes.
And 2, extracting DNA from the saliva samples respectively.
gDNA Buccal Cell Kit using semer fly (Thermo Fisher)
Cat genomic DNA was extracted from cotton swabs (steps referred to product instructions).
3, using the primer of the invention to construct a second-generation sequencing library to obtain the DNA fragment containing 20 DNA targets to be detected (multiple PCR + second-generation sequencing reduces the experiment cost and improves the experiment speed). The primers of the invention are synthesized, and library construction is carried out by using Illumina TrueSeq library prep kit.
And 4, sequencing the library by using an Illumina next generation sequencer.
Sequencing was performed in IMH-bio using an Illumina Hiseq 2000 sequencer.
And 5, analyzing the sequencing data to obtain DNA mutation conditions of 20 disease sites.
Wherein, cat 2 carries pathogenic genes at the site of polycystic kidney; cat 4 carries a causative gene at the site of "vitamin D rickets". The other 19 sites of cats 2 and 4 were normal; cat 1 and cat 3 were normal at all 20 sites.
6, verifying
Samples of 20 site data were selected for validation by PCR and Sanger sequencing.
As a result, the sequencing results of these 20 sites were completely consistent with those obtained in step 5.
Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein
Are both easily conceived and understood. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
Sequence listing
<110> Beijing Chong Gene science and technology services Ltd
Method, primer and kit for screening multiple genetic diseases of cats
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<212> DNA
<213> Artificial sequence (unknown)
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Claims (8)
1. A method of screening cats for multiple genetic diseases, the method comprising the steps of:
1) obtaining a sample containing DNA from a cat;
2) extracting DNA from the sample containing DNA;
3) constructing a second generation sequencing library to obtain DNA fragments containing 20 DNA targets to be detected, wherein the following primers are used:
1-40, wherein each two of the primers in turn constitute a forward primer and a reverse primer;
4) sequencing the library;
5) analyzing sequencing data to obtain DNA mutation conditions of 20 disease sites;
6) predicting or diagnosing disease based on the mutation.
2. The method of claim 1, wherein the DNA-containing sample is saliva from a cat.
3. The method of claim 1, wherein in step 4) the sequencing is performed using an Illumina next generation sequencer.
4. A kit for screening multiple genetic diseases of cats, which comprises the following primers: 1-40, wherein each two of said primers in turn constitute a forward primer and a reverse primer.
5. The kit of claim 4, further comprising reagents for obtaining a DNA-containing sample from a cat, preferably saliva.
6. The kit of claim 4, further comprising reagents for extracting DNA from a sample.
7. The kit of claim 4, further comprising reagents for sequencing the library.
8. A primer group for screening multiple genetic diseases of cats, which comprises the following primers: 1-40, wherein each two of said primers in turn constitute a forward primer and a reverse primer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910688040.2A CN112301119A (en) | 2019-07-29 | 2019-07-29 | Method, primer and kit for screening multiple genetic diseases of cats |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910688040.2A CN112301119A (en) | 2019-07-29 | 2019-07-29 | Method, primer and kit for screening multiple genetic diseases of cats |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112301119A true CN112301119A (en) | 2021-02-02 |
Family
ID=74329890
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910688040.2A Pending CN112301119A (en) | 2019-07-29 | 2019-07-29 | Method, primer and kit for screening multiple genetic diseases of cats |
Country Status (1)
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| CN (1) | CN112301119A (en) |
Citations (6)
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|---|---|---|---|---|
| WO2003074723A2 (en) * | 2002-03-01 | 2003-09-12 | Ravgen, Inc. | Methods for detection of genetic disorders |
| WO2013095935A1 (en) * | 2011-12-19 | 2013-06-27 | Hill's Pet Nutrition, Inc. | Compositions and methods for diagnosing and treating hyperthyroidism in companion animals |
| CN106350589A (en) * | 2016-08-31 | 2017-01-25 | 汪道文 | DNA library for detecting pathogenic genes of genetic vascular diseases and application thereof |
| CN106544431A (en) * | 2016-10-28 | 2017-03-29 | 林巍 | The sequencing library construction method of hyperphenylalaninemia related gene exons mutation |
| CN107868820A (en) * | 2017-10-26 | 2018-04-03 | 深圳深知生物科技有限公司 | Method, primer and the kit of canine multiple genetic disease examination |
| CN110468194A (en) * | 2019-08-12 | 2019-11-19 | 广州万德基因医学科技有限公司 | The multiple PCR primer group and kit in library are built for Inherited Metabolic Disorders high-flux sequence |
-
2019
- 2019-07-29 CN CN201910688040.2A patent/CN112301119A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003074723A2 (en) * | 2002-03-01 | 2003-09-12 | Ravgen, Inc. | Methods for detection of genetic disorders |
| CN1650031A (en) * | 2002-03-01 | 2005-08-03 | 拉瓦格恩公司 | Rapid analysis of variations in a genome |
| WO2013095935A1 (en) * | 2011-12-19 | 2013-06-27 | Hill's Pet Nutrition, Inc. | Compositions and methods for diagnosing and treating hyperthyroidism in companion animals |
| CN104039823A (en) * | 2011-12-19 | 2014-09-10 | 希尔氏宠物营养品公司 | Compositions and methods for diagnosing and treating hyperthyroidism in companion animals |
| CN106350589A (en) * | 2016-08-31 | 2017-01-25 | 汪道文 | DNA library for detecting pathogenic genes of genetic vascular diseases and application thereof |
| CN106544431A (en) * | 2016-10-28 | 2017-03-29 | 林巍 | The sequencing library construction method of hyperphenylalaninemia related gene exons mutation |
| CN107868820A (en) * | 2017-10-26 | 2018-04-03 | 深圳深知生物科技有限公司 | Method, primer and the kit of canine multiple genetic disease examination |
| CN110468194A (en) * | 2019-08-12 | 2019-11-19 | 广州万德基因医学科技有限公司 | The multiple PCR primer group and kit in library are built for Inherited Metabolic Disorders high-flux sequence |
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