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Primer Thermostatic Amplification Technology

Publish:2017-11-13 11:50:32  Views:1106

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Nucleic acid amplification is the key technology for nucleic acid molecule diagnosis. According to the nucleic acid amplification reaction temperature change requirements, nucleic acid amplification techniques can be divided into two categories: one is PCR nucleic acid amplification technology, and the other is constant temperature nucleic acid amplification technology.

PCR technology refers to the three steps of DNA amplification by controlling temperature changes: template denaturation (eg 95 ° C) - primer hybridization (eg 58 ° C) -DNA synthesis (eg 72 ° C). This repeated cycle of temperature changes (eg, 35 repetitions) is usually controlled by a sophisticated and complex instrument (PCR instrument). Nucleic Acid Isothermal Amplification (NAIA) is the whole process of amplification reaction carried out at the same temperature, without the need to undergo dozens of cyclic changes in temperature as PCR reaction. This feature makes them greatly simplify the requirements of the instrument for amplification, the reaction time is greatly reduced, which has great application value, has become a hot spot in the development of molecular diagnostics.

The current NAIA technologies include Rolling Circle Amplification (RCA), Transcription Mediated Amplification (TMA), Nucleic Acid Sequence Based Amplification (NASBA), strand displacement Strand displacement amplification (SDA), Loop-Mediated Isothermal Amplification (LAMP) and Helicase Dependent Amplification (HDA). The various nucleic acid amplification techniques are owned by foreign companies.

Crossing Priming Amplification (CPA) is a new thermostatic nucleic acid amplification technology completely developed by Ustar alone. It is also the first nucleic acid amplification technology with independent intellectual property rights in China. CPA combines with other technologies of YST Company (such as rapid nucleic acid extraction technology and nucleic acid test strip detection technology) to form a complete on-site rapid molecular detection platform, which can develop many thermostatic amplification detection kits and is widely used in Molecular diagnosis, epidemic prevention and quarantine, biomedical research, individualized treatment and other fields. The technology has been applied for invention patents in China and the United States, in the United States, "Molecular Diagnostics" and other magazines published two articles, and adopted the Bill Gates Foundation to apply for funding.

In addition to the Bst DNA polymerase containing the strand displacement function, the CPA amplification system mainly includes an amplification primer and two cross-primers. These oligonucleotide strands can rely on the highly active strand displacement properties of Bst DNA polymerase, enabling continuous cycling of DNA. CPA amplification consists of the following steps:
    PFs in the cross forward primer CPF are complementary to PFa in the template DNA, initiating DNA synthesis such that PRa is introduced into the amplified product;
    The peripheral primer DP1s, which is complementary to the DP1a front of PFa, is extended forward by strand displacement DNA polymerase while replacing the single-stranded product (structure 3) synthesized by CPF that binds to CPR and DP2a, forming a double-stranded product with the template DNA Structure 2);
    In structure 3, DP2a is extended forward by the strand displacement type DNA polymerase, displacing the single-stranded product (structure 5) extended by CPR while synthesizing the single-stranded DNA produced by the CPF extension in step 2 to form a double-stranded product (Structure 4), while the structure 5 relative to the initial DNA template, more PRs and PFs two fragment sequences;
    The 3-end PFa and PRs in single-stranded structure 5 can complementarily bind PFs in CPF and PRa in CPR, respectively, to extend and replace the corresponding single-stranded product under the action of strand displacement DNA polymerase. An additional PFs area (Structure 8) is added to the extension relative to structure 5;
    Therefore, the continuous hybridization and extension of the amplification primers CPF and CPR not only allowed the length of structure 5 to be continuously lengthened, thus introducing more complementary regions of CPR and 3 'end of CPR, but also replaced various structures that can be combined with CPF and CPR3 End complementary single-stranded product;
    Through continuous hybridization of CPF and CPR and DNA polymerase strand displacement, making the DNA copy number continued to increase, so as to achieve the effect of gene amplification.

The nucleic acid extraction of clinical biological samples using the device is cost-effective and can be applied to primary hospitals and economically underdeveloped areas with less on-site testing or experimental resources in only a few minutes. The nucleic acid can also meet the needs of large hospitals in specific situations, such as emergency Or bedside nucleic acid rapid diagnosis. The technology has applied for patents for inventions, and get Gates Foundation research and development funding.

The basic principle of CPA is as follows:
 
  Figure 2 CPA amplification reaction diagram

Compared with the widely used fluorescent PCR technology, CPA technology has the following advantages:
    Fast reaction time: about 1 hour reaction time, so that the kit can be used for inspection and quarantine, detection and monitoring of unexpected infectious diseases on-site testing or hospital bedside diagnosis(Point-of-Testing, POCT);
     Low detection cost: At present, the fluorescence quantitative PCR instrument is expensive and can not be popularized in many small and medium-sized hospitals. CPA amplification requires only a centrifuge and a simple thermostat, such as ordinary water bath, metal bath, can be amplified.
    Simple operation: the operator's skill requirements are not high, the vast majority of people through simple training or self-control can grasp for the wide range of nucleic acid detection reagents to create the conditions.