Methods Of Disease Testing

With a growing number of pets and the increasing demand for purebred pets, more and more purebred dogs and cats began to enter the pet families. At the same time, the market's love for purebred dogs and cats has also changed the overall health level of pets to a certain extent. Among them, dog and cat genetic diseases are a common potential risk threatening the life and health of pets. According to statistics, the average incidence rate of genetic diseases in dogs and cats is as high as 21%. Once there is a risk, genetic diseases will inevitably appear in an animal's life sooner or later, and will torture them until the end of life.

Screening for genetic diseases in advance is to grasp the golden period of treatment. At present, there are more than 500 known genetic diseases of dogs and cats, and the number of diseases that can be determined by detection is nearly 200. In every lazy and charming cat and every happy running dog, there may be genetic diseases that are ready to show up. At present, there are a variety of disease detection methods in use, which is conducive to expanding the choice of pet owners.

Fluorescence Quantitative PCR Technique

Fluorescence quantitative PCR (realtime-PCR) technology is a new technology based on ordinary PCR technology in recent years. It detects the PCR product with the help of fluorescent signals, and marks and tracks the PCR product through fluorescent dye or fluorescent-labeled specific probe. In the amplification process, after each cycle, the fluorescence quantitative PCR method only needs to detect whether the sample has an amplification signal, and the PCR reaction has a high efficiency of nucleic acid amplification, which can detect small mutations. According to different probe markers, it can be divided into the probe-based method and arms method. The sensitivity of arms method is higher than probe-based method, and it is easier to select and enrich low concentration DNA mutations from a large number of wild-type DNA.

ARMS-PCR Technology

Amplification refractory mutation system (ARMS) is the development of PCR technology, also known as allele characteristic PCR (AS-PCR), which is used to detect known mutant genes. In this method, two 5' end primers are designed, one is complementary to normal DNA and the other is complementary to mutant DNA. For homozygous mutation, these two primers and 3' end primers are added respectively for two parallel PCR. Only primers completely complementary to mutant DNA can be extended and PCR expanded products can be obtained. If the mismatch is located at the 3' end of the primer, the PCR cannot be extended. ARMS-PCR is a commonly used gene mutation detection method in the laboratory. ARMS-PCR has high sensitivity and can detect mutant genes with a mutation ratio of 1% or even lower in cells.

Sanger Sequencing Technology

The basic principle of the Sanger sequencing method is to add the mixture of dNTP / ddNTP to the end of specific primers under the action of DNA polymerase to produce oligonucleotide chains with different lengths and fluorescent markers corresponding to four bases. When the fluorescent-labeled oligonucleotide chain mixture is separated by capillary electrophoresis of gene analyzer, the software can tell the corresponding base according to the fluorescent color, and determine the position of the corresponding base in the sequence according to the sequence of fluorescent signals, so as to obtain the DNA sequence of specific gene location points and fragments and achieve the goal of differential diagnosis of molecular pathogenesis and guiding clinical disease treatment.

Next-Generation Sequencing (NGS)

NGS, also known as mass parallel sequencing (MPS), contains a variety of sequencing technologies that can produce a large number of digital gene sequences at one time. It is a revolutionary progress of Sanger sequencing. Using the concept of parallel sequencing, NGS can sequence millions or even billions of DNA molecules at the same time, realizing the goal of large-scale and next-generation sequencing. The product sequencing principles of different manufacturers are different, mainly including sequencing while synthesis (SBS), mass parallel sequencing based on "DNA cluster" and reversible terminal termination, continuous ligation reaction sequencing of 4-color fluorescent-labeled oligonucleotides and semiconductor chip sequencing. The next-generation sequencing technology can not only be used for large-scale genome sequencing, but also for gene expression analysis, identification of noncoding small RNA, screening of transcription factor target genes and DNA methylation.

Next-generation sequencing technology has three advantages that the traditional Sanger sequencing method does not have. To begin with, it uses chips for sequencing, which can read sequencing at millions of points at the same time. Secondly, next-generation sequencing technology has a quantitative function. The number of times DNA in the sample is sequenced reflects the abundance of this DNA in the sample. And thirdly, compared with the traditional Sanger sequencing method, the sequencing cost of NGS technology is very low.

Fluorescence in situ hybridization

The basic principle of fluorescence in situ hybridization (FISH) is the same as that of chromogenic in situ hybridization. The difference is that FISH uses a fluorescein-labeled probe to react with the nucleic acid to be tested in tissue cells to form a hybrid, and the signal expression is observed by a fluorescence microscope or laser confocal microscope. In order to detect multiple targets at the same time, a new technology, multicolor fluorescence in situ hybridization, has been developed based on FISH. It uses several fluorescein-labeled probes with different colors for in situ hybridization, which can detect multiple genes at the same time, and expands the clinical application of FISH technology. FISH technology is mainly used in pathological diagnosis at chromosome and DNA levels. Compared with in situ hybridization, FISH is safer, more rapid, specific, and accurate.

DNA hybridization

Although the raising knowledge and scientific data involved in pet genetic diseases are complex, we suggest pet owners use a simple genetic disease test to evaluate the risk of genetic diseases in their pet's life in advance. BioVenic provides a variety of genetic testing services for common genetic diseases of dogs and cats.

References

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