A polyethylene glycol enhanced ligation-triggered self-priming isothermal amplification for the detection of SARS-CoV-2 D614G mutation.

We presented a polyethylene glycol (PEG) enhanced ligation-triggered self-priming isothermal amplification (PEG-LSPA) for the detection D614G mutation in S-glycoprotein of SARS-CoV-2. PEG was employed to improve the ligation efficiency of this assay by constructing a molecular crowding environment. Two hairpin probes (H1 and H2) were designed to contain 18 nt and 20 nt target binding site at their 3' end and 5' end, respectively. In presence of target sequence, it complemented with H1 and H2 to trigger ligation by ligase under molecular crowding condition to form ligated H1-H2 duplex. Then 3' terminus of the H2 would be extended by DNA polymerase under isothermal conditions to form a longer extended hairpin (EHP1). 5' terminus of EHP1 with phosphorothioate (PS) modification could form hairpin structure due to the lower Tm value. The resulting 3' end overhang would also fold back as a new primer to initiate the next round of polymerization, resulting in the formation of a longer extended hairpin (EHP2) containing two target sequence domains. In the circle of LSPA, long extended hairpin (EHPx) containing numerous target sequence domains was produced. The resulting DNA products can be monitored in real-time fluorescence signaling. Our proposed assay owns an excellent linear range from 10 fM to 10 nM with a detection limit down to 4 fM. Thus, this work provides a potential isothermal amplification method for monitoring mutations in SARS-CoV-2 variants.

Spectrophotometric Detection of the BRCA1 Gene via Exponential Isothermal Amplification and Hybridization Chain Reaction of Surface-Bound Probes.

In this work, we demonstrated an ultrasensitive approach with a dual-amplification strategy for DNA assay based on isothermal exponential amplification (EXPAR) and the hybridization chain reaction (HCR). In the presence of target DNA, the hairpin probe DNA (HP1) recognized and partially hybridized with the target DNA to form double-stranded structures containing the full recognition sequences for nicking endonuclease and then initiated EXPAR. Under the reaction of EXPAR, a large number of single-stranded DNA (ssDNA) was produced in the circle of nicking, polymerization, and strand displacement. The resulting ssDNA can bind to the surface-bound probe on the well of the microplate and trigger the hybridization chain reaction, resulting in the production of numerous double-stranded DNA concatamers with biotin labeling. In the presence of streptavidin-conjugated horseradish peroxidase (HRP), the amplified signal can be detected by a spectrophotometer via HRP-catalyzed substrate 3,3'5,5'-tetramethylbenzidine (TMB). This proposed dual-amplification method provides a detection limit of 74.48 aM, which also exhibits good linearity ranging from 0.1 fM to 100 pM.

Universal point-of-care detection of proteins based on proximity hybridization-mediated isothermal exponential amplification.

We have developed a point-of-care (POC) lateral flow biosensor (LFB) for universal protein detection based on a proximity hybridization-mediated protein-to-DNA signal transducer, isothermal exponential amplification (EXPAR) and catalytic hairpin assembly (CHA) with high sensitivity and specificity. In this assay, a protein signal transducer was employed to convert the input protein to the output DNA signal. Antibody conjugated DNA1 was firstly hybridized with the output DNA (DNA3). The binding of antibody conjugated DNA1 and DNA2 to the same protein was able to increase the local concentrations, resulting in strand displacement between DNA3 and DNA2. DNA3 with nicking endonuclease recognition sequences at the 5' end then hybridized with hairpin probe 1 to mediate EXPAR in the presence of nicking endonuclease and polymerase. A large number of single strand DNA were produced in the circle of nicking, polymerization and strand displacement. The resulting ssDNA products were further amplified by CHA to generate double-stranded DNA products. The double-stranded DNA products were detected with a lateral flow biosensor within 5 min. This proposed assay has very high sensitivity and selectivity with a dynamic response ranging from 1 fM to 100 nM, and the detection limit was 0.74 fM. This work provides a universal and simple method for protein detection.