Combination Of Hybridization Chain Reaction Research Articles

Expanded expression of pro-neurogenic factor SoxB1 during larval development of gastropod Lymnaea stagnalis suggests preadaptation to prolonged neurogenesis in Mollusca.

The remarkable diversity observed in the structure and development of the molluscan nervous system raises intriguing questions regarding the molecular mechanisms underlying neurogenesis in Mollusca. The expression of SoxB family transcription factors plays a pivotal role in neuronal development, thereby offering valuable insights into the strategies of neurogenesis. In this study, we conducted gene expression analysis focusing on SoxB-family transcription factors during early neurogenesis in the gastropod Lymnaea stagnalis. We employed a combination of hybridization chain reaction in situ hybridization (HCR-ISH), immunocytochemistry, confocal microscopy, and cell proliferation assays to investigate the spatial and temporal expression patterns of LsSoxB1 and LsSoxB2 from the gastrula stage to hatching, with particular attention to the formation of central ring ganglia. Our investigation reveals that LsSoxB1 demonstrates expanded ectodermal expression from the gastrula to the hatching stage, whereas expression of LsSoxB2 in the ectoderm ceases by the veliger stage. LsSoxB1 is expressed in the ectoderm of the head, foot, and visceral complex, as well as in forming ganglia and sensory cells. Conversely, LsSoxB2 is mostly restricted to the subepithelial layer and forming ganglia cells during metamorphosis. Proliferation assays indicate a uniform distribution of dividing cells in the ectoderm across all developmental stages, suggesting the absence of distinct neurogenic zones with increased proliferation in gastropods. Our findings reveal a spatially and temporally extended pattern of SoxB1 expression in a gastropod representative compared to other lophotrochozoan species. This prolonged and widespread expression of SoxB genes may be interpreted as a form of transcriptional neoteny, representing a preadaptation to prolonged neurogenesis. Consequently, it could contribute to the diversification of nervous systems in gastropods and lead to an increase in the complexity of the central nervous system in Mollusca.

An electrochemical apta-assay based on hybridization chain reaction and aflatoxin B1-driven Ag-DNAzyme as amplification strategy

Herein, a novel electrochemical apta-assay based on hybridization chain reaction (HCR) and aflatoxin B1-driven Ag-DNAzyme was prepared. The combination of HCR and Ag-DNAzyme was designed for the first time as a dual signal amplification strategy for the detection of mycotoxins. The substrate DNA (sDNA) was fixed to the electrode surface, which contained the RNA A (rA) site and HCR initiation sequence. The sDNA opened the hairpin DNA structures and triggered a cascade of hybridization events. The DNA double strands formed by the HCR bound large amounts of methylene blue (MB). Aptamer and complementary DNA bound to Ag+ by C-Ag+-C complexes. AFB1 can drive Ag+ shedding, and Ag+ induced Ag-DNAzyme to shear the rA site of sDNA. The amount of binding MB decreased and the current signal decreased. Replaced biological enzymes with metal ion-mediated DNAzyme enhanced the stability of the prepared sensors while reducing the preparation cost. An adequate determination of AFB1 in corn flour, walnut powder, and other actual samples are validated, which indicated the good accuracy and potential application in real samples. The strategy is characterized by simple operation, good stability, and low preparation cost, and has good application prospects in food safety and quality control.

The helper oligonucleotides enable detection of folded single-stranded DNA by lateral flow immunoassay after HCR signal amplification

A combination of Hybridization Chain Reaction (HCR) and Lateral Flow Immunoassay (LFIA) is an attractive strategy for a simple signal amplification DNA/RNA detection. The present study aimed to report a strategy used to solve a problem encountered when the target DNA contained folded secondary structure during HCR, enabling HCR hairpin probes to easily access the target site. The 24-nt conserved sequence within 3′-UTR, present only in dengue virus genome but not in other species, is an ideal target to use as a probe binding site for pan-dengue virus detection. Thus, the 105-nt target containing the 24-nt target sequence was chosen as a target with secondary structures.The 24-nucleotide (nt) synthetic target DNA successfully induced HCR reaction within 5 min at room temperature. However, the HCR detection of the 105-nt synthetic target DNA with secondary structures was problematic. The probe hybridization was prevented by the secondary structures of the target, resulting in a failure to generate HCR product. To solve this problem, two helper oligonucleotides (helper1 and helper2) were designed to linearize the folded structure of the 105-nt target through strand-displacement mechanism, allowing the HCR hairpin probes to easily access the target site. The HCR product with the labeled helper oligonucleotides and the labeled probes were successfully detected by LFIA. With this strategy, the combination of the helper-enhanced HCR and LFIA exhibited a limit of detection (LOD) in a nanomolar range of the 105-nt DENV synthetic target DNA. Our study demonstrated that signal amplification by the combination of HCR and LFIA could successfully detect the target DNA with secondary structure, but not target RNA with secondary structure.In summary, this work provided a proof of concept of two main issues including probe hybridization enhancement by helper oligonucleotide for the target with complicated secondary structure and the advantage of a combination of labeled helper and HCR probes design for LFIA to overcome the false positive result from HCR probe leakage. Our findings on the use of helper oligonucleotides may be beneficial for the development of other isothermal amplification, since the secondary structure of the target is one of the major obstacles among hybridization-based methods.

An aptamer biosensor based dual signal amplification system for the detection of salmonella typhimurium

Salmonella, a typical foodborne pathogen, always seriously threatens the health and even life of both humans and animals. However, highly sensitive and fast quantitative methods for its detection are remaining to be challenged. Herein, we presented an efficient method with dual signal amplification strategy by combining immune hybridization chain reaction (HCR) with surface enhanced Raman scattering (SERS) to high sensitively detect Salmonella typhimurium in food. After sample preparation, S. typhimurium were specifically captured by immunomagnetic beads (IMBs), then aptamers and hairpin-probes were added to trigger HCR to form nicked dsDNA, finally 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was incubated with HCR products and then the whole system was mixed with AgNP colloid to detect the SERS intensity at 1610 cm−1. As a result, a good linear relationship was achieved between SERS intensities and corresponding concentrations of S. typhimurium ranging from 10 to 105 CFU/mL, with a limit of detection (LOD) of 6 CFU/mL in 3.5 h. The proposed method has been successfully applied to capture and detect the S. typhimurium in spiked milk samples, and the results were consistent with those of the traditional plate counting method. The method, with combination of HCR and SERS, achieves double amplification of the detection signal and significantly improves the detection sensitivity of S. typhimurium. And it also shows good application potential for the highly sensitive detection of other contaminants in food.

Fluorescence detection for DNA using hybridization chain reaction with enzyme-amplification

A signal amplification based on a combination of hybridization chain reaction (HCR) and enzyme-enhancement was applied in the study of a novel biosensor. The target sequence was added as catalyst which was simply transmitted but did not disappear as the DNA double strand grew on the surface of magnetic beads (MBs).