Isothermal Strand-displacement Polymerization Reaction Research Articles

Dual-labeling ratiometric electrochemical strategy initiated with ISDPR for accurate screening MecA gene

An outstanding dual-labeling ratiometric electrochemical biosensor based on isothermal strand displacement polymerization reaction (ISDPR) for highly sensitive and selective detection of mecA gene has been proposed. Concretely, in the presence of mecA gene, the addition of methylene blue (MB)-labeled primer and polymerase induced recycling amplification to change the structure of the ferrocene (Fc)-labeled hairpin probe, thereby releasing abundant target gene to realize the signal amplification and dual-signal output. Through this process, the electrochemical responses of Fc (IFc) and MB (IMB) were both substantially reduced and increased proportionally, ensuring that the value of IMB/IFc can accurately reflect the true detection level of mecA gene. Benefiting from the “signal-on/off” strategy, the fabricated biosensor exhibited outstanding sequence specificity to discriminate mismatched mecA gene, which verified to be 2.72 times that of single-label detection for perfect match/single base mismatch (PM/MM) discrimination ratio. This strategy effectively integrated the advantages of signal amplification and ratiometric modes, making the biosensor exhibit a broad working range with 10 fM - 3000 pM and a limit of detection (LOD) with 3.33 fM (S/N = 3). Moreover, the proposed biosensor has good feasibility for mecA gene determination in water samples due to acceptable recoveries (95–115%) and repeatability relative standard deviations (RSD) value of 4%. This will provide a powerful sensing platform for improving accuracy and decreasing background signal of sensor for ARGs screening in environmental monitoring.

Rapid and Sensitive Isothermal Detection of Nucleic-acid Sequence by Multiple Cross Displacement Amplification.

We have devised a novel amplification strategy based on isothermal strand-displacement polymerization reaction, which was termed multiple cross displacement amplification (MCDA). The approach employed a set of ten specially designed primers spanning ten distinct regions of target sequence and was preceded at a constant temperature (61–65 °C). At the assay temperature, the double-stranded DNAs were at dynamic reaction environment of primer-template hybrid, thus the high concentration of primers annealed to the template strands without a denaturing step to initiate the synthesis. For the subsequent isothermal amplification step, a series of primer binding and extension events yielded several single-stranded DNAs and single-stranded single stem-loop DNA structures. Then, these DNA products enabled the strand-displacement reaction to enter into the exponential amplification. Three mainstream methods, including colorimetric indicators, agarose gel electrophoresis and real-time turbidity, were selected for monitoring the MCDA reaction. Moreover, the practical application of the MCDA assay was successfully evaluated by detecting the target pathogen nucleic acid in pork samples, which offered advantages on quick results, modest equipment requirements, easiness in operation, and high specificity and sensitivity. Here we expounded the basic MCDA mechanism and also provided details on an alternative (Single-MCDA assay, S-MCDA) to MCDA technique.

Amplified electrochemical detection of mecA gene in methicillin-resistant Staphylococcus aureus based on target recycling amplification and isothermal strand-displacement polymerization reaction

Rapid detection of drug-resistant bacteria can facilitate the early diagnosis of various infections and provide essential guidance for appropriate usage of antibiotics. An ultrasensitive electrochemical DNA (E-DNA) sensor based on isothermal strand-displacement polymerization reaction (ISDPR) for detection of mecA gene in methicillin-resistant Staphylococcus aureus (MRSA) was proposed in this study. Methylene blue (MB)-labeled hairpin probes were self-assembled on a gold electrode to confine MB molecules close to the electrode surface for efficient electron transfer. The hairpin probes were then hybridized with complementary target DNA and undergone conformational changes, which led to a decreased electrochemical response. The primers annealed with the opened stems of hairpin probes were extended by DNA polymerase, which in turn released the target DNA to trigger the next polymerization cycle. Finally, each target DNA underwent through many rounds, resulting in a mass of MB molecules moved away from the electrode surface, which generated significant amplified current suppression for mecA gene detection. With this target recycling amplification strategy, the proposed DNA biosensor could offer excellent analytical performance for the detection of mecA gene and provide a new electrochemical method for the early diagnosis of drug-resistant bacteria.

Visual detection of glial cell line-derived neurotrophic factor based on a molecular translator and isothermal strand-displacement polymerization reaction.

BackgroundGlial cell line-derived neurotrophic factor (GDNF) is a small protein that potently promotes the survival of many types of neurons. Detection of GDNF is vital to monitoring the survival of sympathetic and sensory neurons. However, the specific method for GDNF detection is also un-discovered. The purpose of this study is to explore the method for protein detection of GDNF.MethodsA novel visual detection method based on a molecular translator and isothermal strand-displacement polymerization reaction (ISDPR) has been proposed for the detection of GDNF. In this study, a molecular translator was employed to convert the input protein to output deoxyribonucleic acid signal, which was further amplified by ISDPR. The product of ISDPR was detected by a lateral flow biosensor within 30 minutes.ResultsThis novel visual detection method based on a molecular translator and ISDPR has very high sensitivity and selectivity, with a dynamic response ranging from 1 pg/mL to 10 ng/mL, and the detection limit was 1 pg/mL of GDNF.ConclusionThis novel visual detection method exhibits high sensitivity and selectivity, which is very simple and universal for GDNF detection to help disease therapy in clinical practice.

A new photoelectrochemical biosensors based on DNA conformational changes and isothermal circular strand-displacement polymerization reaction

We report a strategy for the transduction of DNA hybridization into a readily detectable photoelectrochemical signal by means of a conformational change analogous to electrochemical DNA (E-DNA) approach. To demonstrate the effect of distance change for photosensitizer to the surface of electrode on the change of photocurrent, photosensitizer Ru(bpy)2(dcbpy)2+ tagged DNA stem–loop structures were self-assembled onto a nanogold modified ITO electrode. Hybridization induced a large conformational change in DNA structure, which in turn significantly altered the electron-transfer tunneling distance between the electrode and photosensitizer. The resulting change in photocurrent was proportional to the concentration of DNA in the range of 1.0×10−10–8.0×10−9M. In order to improve the sensitivity of the photoelectrochemical biosensor, an amplified detection method based on isothermal strand displacement polymerization reaction was employed. With multiple rounds of isothermal strand replication, which led to strand displacement and constituted consecutive signal amplification, a detection limit of 9.4×10−14M target DNA was achieved.

Amplified electrochemiluminescence detection of nucleic acids by hairpin probe-based isothermal amplification

Here, we present a straightforward method for isothermal amplified detection of nucleic acids. In this proof-of-concept study, a specific DNA sequence is amplified through hairpin probe-based isothermal strand-displacement polymerization reaction and then detected via a sensitive and commercially available ECL detection platform. Results show that the DNA sequence derived from the Listeria monocytogenes hly gene can be detected down to 10 pM in solution, together with correlation of the detected signal with the initial concentration of target DNA. Moreover, the designed stem-loop structured hairpin probe shows single-base variation differentiating ability. Considering the superior sensitivity and specificity, as well as the simple-to-implement features, the developed assay demonstrates a great potential of becoming a first-line tool for quantitative analysis of nucleic acids for biomedical research.

Sensitive fluorescence detection of nucleic acids based on isothermal circular strand-displacement polymerization reaction

Here we have developed a sensitive DNA amplified detection method based on isothermal strand-displacement polymerization reaction. This method takes advantage of both the hybridization property of DNA and the strand-displacement property of polymerase. Importantly, we demonstrate that our method produces a circular polymerization reaction activated by the target, which essentially allows it to self-detect. Functionally, this DNA system consists of a hairpin fluorescence probe, a short primer and polymerase. Upon recognition and hybridization with the target ssDNA, the stem of the hairpin probe is opened, after which the opened probe anneals with the primer and triggers the polymerization reaction. During this process of the polymerization reaction, a complementary DNA is synthesized and the hybridized target is displaced. Finally, the displaced target recognizes and hybridizes with another probe, triggering the next round of polymerization reaction, reaching a target detection limit of 6.4 × 10−15 M.