Principle and application of isothermal amplification of nucleic acids technology
Isothermal amplification of nucleic acids technique is a new type of nucleic acid amplification technique under constant temperature. The method based on this technique is simple to operate, has high sensitivity and specificity, and shows a good application prospect in clinical and scientific research. In this paper, the principles, characteristics and applications of loop-mediated isothermal amplification of nucleic acids (LAMP), Cross primer isothermal amplification of nucleic acids (CPA), chain substitution isothermal amplification of nucleic acids (SDA), nucleic acid sequence-dependent isothermal amplification of nucleic acids (NASBA) and rolling loop isothermal amplification of nucleic acids (RCA) were reviewed.
1.loop-mediated isothermal amplification of nucleic acids.
LAMP was performed at 60~65 ℃, requiring 4 primers and DNA polymerase of Bacillus lipophilus thermophilus with chain replacement function. The outer primer is similar to the PCR primer, while the inner primer contains two sequences. The process is as follows:
a) The inner primer binds the target gene and extends into double strand under the action of BstDNA polymerase. The outer primer binds to the 5 'end of the double-stranded DNA, forming a ring structure at one end. The other end goes through the same process to form a dumbbell-like structure with rings at both ends.
b) Single stranded DNA with dumbbell structure has dual functions of template and primer, and can be extended immediately under the catalysis of Bst polymerase.
c) Inner primers can also bind to annular structures and extend under the action of enzymes.
loop-mediated isothermal amplification of nucleic acids can be used for pathogen detection. Lin et al. used LAMP technology to detect methicillin-resistant Staphylococcus aureus in respiratory samples. The detection limit of 16s RNA gene by this method was 104 CFU/ml (CFU is colony forming unit), and the detection limit of mecA gene was 105 CFU/ml. Ulep et al. developed a droplet LAMP detection platform for the quantitative detection of E. coli O157: H7 in 5% diluted blood samples, with a detection limit of 10 CFU/μl. The modified LAMP is used to detect pneumocystis without cross-reactivity with other common lung infections and is 1,000 times more sensitive than conventional PCR. In addition, LAMP can also be used for human papillomavirus detection and Salmonella enteritidis detection in food [etc.
LAMP can also be used to detect mutated genes in cancer. Itonaga et al detected gene mutations in pancreatic cancer cell lines by means of nucleic acid-locked acid (PNA-LNA) -mediated LAMP. The mutated gene can be detected when the mutated wild ratio is 0.1%. Raack et al. used a LAMP assay mediated by peptide nucleic acid to detect gene mutations in oral swab samples treated with cell lysate and showed better sensitivity than Sanger sequencing.
2.Cross primer isothermal amplification of nucleic acids.
Cross primer isothermal amplification of nucleic acids is performed at around 63 ℃ and is dependent on Bst DNA polymerase, betaine and cross primers. According to the different number of cross primers, it can be divided into double cross primer amplification and single cross primer amplification.
Double cross primers amplification uses two cross primers and two peel primers. The two cross primers were complementary combined with the template strand and extended, and then the peel primers peeled off the newly synthesized single strand under the action of Bst DNA polymerase, and the last two cross primers synthesized a large number of target fragments using the new single strand as the template under the action of Bst DNA polymerase.
Single cross primers amplification uses one cross primer, two stripping primers and two ordinary primers. First, the cross primers bind to the template strand and extend to double strand, while the peel primers separate the new strand from the template under the action of Bst DNA polymerase. Then two single-stranded DNA of different lengths were synthesized by common primers using the new strand as a template. Finally, the two single chains were used as templates and the pairs of cross primers and common primers were used to form an amplification cycle.
Due to its high sensitivity, Cross primer isothermal amplification of nucleic acids is mainly used for the detection of trace pathogen nucleic acids: Qiao et al. detected the coagulase gene of Staphylococcus by CPA and western blotting with a detection limit of 3 627 fg (1 fg=10-15g). Meng et al. used CPA to detect Aeromonas hydrophila, and the detection limit was about 5×103 CFU/g. The reverse transcription cross primers combined with nucleic acid test strips can also be used for the detection of porcine epidemic diarrhea virus.
3.Chain substitution isothermal amplification of nucleic acids.
The reaction temperature of Chain substitution isothermal amplification of nucleic acids was about 37 ℃, and the reaction required restriction endonucrenase, chain replacement DNA polymerase and two pairs of primers, and one pair of primers (P1 and P2) contained endonucrenase recognition sequences. At the beginning of the reaction, P1 and P2 were complementary bound to the template chain, and extended into double chains under the catalysis of polymerase. Then endonucliase recognized the cleavage sites at both ends of the double chain, and cleaved to form sticky ends. The second pair of primers binds to the end of the template chain, synthesizes a new chain under the action of polymerase and replaces a single chain.
Chain substitution isothermal amplification of nucleic acids can be used for protein detection. Zhang et al. used fluorescent SDA method to amplify the specific sequence formed by the combination of biotin and streptavidin to amplify the signal, thus realizing the detection of streptavidin. The detection limit of this method was 92 pmol/L. SDA combined with fluorescent or transverse flow test strips can be used for rapid detection of pathogens. Toley et al.  developed test strips for detection of Staphylococcus aureus using SDA technology, and the detection limit was as low as every 5 copies /μl. Du et al. developed a sensing platform for detecting uracil-DNA glycosylase activity based on SDA, and the detection limit was 1×10-4 U/ml (U is the active unit of enzyme).
4.Nucleic acid sequence-dependent isothermal amplification of nucleic acids.
Nucleic acid sequence-dependent isothermal amplification of nucleic acids technique is an isothermal amplification method for RNA detection, usually performed at about 42 ℃ , which requires AMV (avian myeloblastosis virus) reverse transcriptase, RNase H, T7 RNA polymerase and a pair of primers. The forward primers contain T7 promoter complementary sequences. In the reaction process, the positive primer binds to the RNA strand, which is catalyzed by AMV enzyme to form DNA-RNA double strand. RNA enzyme H digests the RNA in the hybrid double strand and retains the single strand of DNA. DNA double strand containing T7 promoter sequence was formed under the action of reverse primers and AMV enzyme. The transcription process is completed under the action of T7 RNA polymerase and a large amount of target RNA is produced.
Nucleic acid sequence-dependent isothermal amplification of nucleic acids can be used to detect pathogens, especially RNA viruses. NASBA's digital chip based on HIV RNA can be used to quantify viral load in human plasma samples and can detect as low as 10 copies /μl. Clancy et al.  used fluorescent labeled probe to detect the amplification products of triple non-competitive NASBA of Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae in real time for the diagnosis of bacterial meningitis. The specificity of this method was 100%, and the detection limits of the three bacteria were 55.36, 0.99 and 57.24 cell equivalents, respectively. Real-time quantification of NASBA can also be used to amplify and inactivate MIC1 mRNA in the oocyst to detect and distinguish between cryptosporidium parvus and cryptosporidium hominis.
5.Rolling loop isothermal amplification of nucleic acids.
Rolling loop isothermal amplification of nucleic acids borrows from circular DNA replication in nature. The required enzyme, phi29DNA polymerase, was performed at about 37 ℃. The process of common RCA is as follows: primer combines with circular DNA template and extends to generate single DNA strand containing a large number of target genes.
On this basis, a variety of improved techniques have also been developed, such as multi-primer RCA for simultaneous amplification of multiple sites and index RCA for improving amplification efficiency by using two primers.
Rolling loop isothermal amplification of nucleic acids and its improved techniques have a wide range of applications. The RCA product was amplified by nanomaterial signal and combined with biosensor to detect the content of bisphenol A. The detection limit was 5.4×10-17 mol/L. Notch enhanced RCA was used to detect mirnas (micrornas) in combination with biosensors and fluorescence, with a detection limit of 10 pmol/L. By combining molecular beacon and fluorescence, hyperbranched RCA can quantitatively detect miRNA-21 as low as 1×10-18 mol/L. Ciftci et al. designed a detection method based on RCA, which combined with Streptavidin-biotin system, catalyzed H2O2 by glucose oxidase on electrode network, converted chemical signals into current signals, and detected Ebola virus RNA with a detection limit of 10 pmol/L.
The above is the principle and application of isothermal amplification of nucleic acids technology. If you need more detailed understanding, welcome to contact us!