Point-of-care COVID-19 testing: colorimetric diagnosis using rapid and ultra-sensitive ramified rolling circle amplification.

In this paper, we report a molecular diagnostic system-combining a colorimetric probe (RHthio-CuSO4) for pyrophosphate sensing and isothermal gene amplification (ramified rolling circle amplification)-that operates with high selectivity and sensitivity for clinical point-of-care diagnosis of SARS-CoV-2. During the polymerase phase of the DNA amplification process, pyrophosphate was released from the nucleotide triphosphate as a side product, which was then sensed by our RHthio-CuSO4 probe with a visible color change. This simple colorimetric diagnostic system allowed highly sensitive (1.13 copies/reaction) detection of clinical SARS-CoV-2 within 1 h, while also displaying high selectivity, as evidenced by its discrimination of two respiratory viral genomes (human rhino virus and respiratory syncytial virus) from that of SARS-CoV-2. All of the reactions in this system were performed at a single temperature, with positive identification being made by the naked eye, without requiring any instrumentation. The high sensitivity and selectivity, short detection time (1 h), simple treatment (one-pot reaction), isothermal amplification, and colorimetric detection together satisfy the requirements for clinical point-of-care detection of SARS-CoV-2. Therefore, we believe that this combination of a colorimetric probe and isothermal amplification will be useful for point-of-care testing to prevent the propagation of COVID-19.

Dual-site ligation-assisted loop-mediated isothermal amplification (dLig-LAMP) for colorimetric and point-of-care determination of real SARS-CoV-2.

A probing system has been developed based on dual-site ligation-assisted loop-mediated isothermal amplification (dLig-LAMP) for the selective colorimetric detection of SARS-CoV-2. This approach can induce false-positive and -negative detection in real clinical samples; dLig-LAMP operates with improved selectivity. Unlike RT-LAMP, the selectivity of dLig-LAMP is determined in both the ligation and primer binding steps, not in the reverse transcription step. With this selective system in hand, we developed a colorimetric signaling system for point-of-care detection. We also developed a colorimetric probe for sensing pyrophosphate, which arises as a side product during the LAMP DNA amplification. Thus, dLig-LAMP appears to be an alternative method for improving the selectivity problems associated with reverse transcription. In addition, combining dLig-LAMP with colorimetric pyrophosphate probing allows point-of-care detection of SARS-CoV-2 within 1 h with high selectivity.

RNA Polymerase-Mediated Stepwise RNA-Primed RNA Polymerization for Site-Specific Multiple Labeling into RNA: A Fluorescence Resonance Energy Transfer Probe Detects the Structural Change of an RNA G-Quadruplex.

In this paper, we report a stepwise RNA-primed RNA polymerization method for the site-specific incorporation of multiple fluorescent moieties into RNA, mediated by an RNA polymerase. A screen of several RNA polymerases revealed that T7 RNA polymerase was the only one that functioned in the RNA-primed RNA polymerization. In the first fluorescence labeling step, a fluorescent rUthioTP residue was incorporated directly into the RNA using T7 RNA polymerase; the second fluorescence labeling step was performed using a post-labeling strategy: directly introducing an rUamiTP residue into RNA, using T7 RNA polymerase, and then reacting with ylidenemalononitrile enamine (P3). The whole process for the site-specific introduction of the multiple labeled moieties was performed through stepwise RNA-primed RNA polymerization. Interestingly, the resulting multiple-labeled RNA exhibited fluorescence resonance energy transfer (FRET) between the two fluorescent labels in the RNA. We optimized the FRET-breaking point in the RNA by changing of distance between the two fluorescent labels and then used this property for the detection of the structural change of the RNA. The FRET signal increased in intensity upon the transformation of the RNA from a single-strand structure to the G-quadruplex. This approach for site-specific FRET labeling into RNA using RNA polymerase suggests the possibility of performing other diverse site-specific modifications at predefined positions in RNA.

Rapid and highly sensitive hairpin structure-mediated colorimetric detection of miRNA.

Herein, we report a novel hairpin structure-mediated diagnostic method for the simple and rapid colorimetric detection of miRNA through the sensing of pyrophosphate. When the hairpin structure of the template DNA (h-Probe) was hybridized with the primer, the DNA primer extension mediated by nPfu special enzyme was blocked. However, this h-Probe was extended using nPfu special enzyme, upon the structural change of the template DNA, from a hairpin to a linear structure, in the presence of the target miRNA. The miRNA-hybridized template DNA sequence was cleaved by a duplex-specific nuclease (DSN), which cleaved the DNA from the RNA-DNA hybrid, thereby allowing the target miRNA to be recycled. Primer extension using nPfu special enzyme produced pyrophosphate when nucleotide triphosphate was incorporated into the DNA; this pyrophosphate was sensed in terms of a color change, from pink to colorless, when using pp Probe, a probe developed previously by our group. This novel system for the colorimetric detection of target miRNA operated with high sensitivity (LOD = 132 aM) and selectivity, with the whole detection process requiring only 30 min. Furthermore, this system could also detect miRNA fluorimetrically with similar sensitivity (LOD = 105 aM), highlighting the dual-sensing properties of pp Probe. This unique, extremely simple, and rapid system for the detection of miRNA through a highly sensitive color change would presumably be useful in applications requiring point-of-care detection.

Combined recombinase polymerase amplification/rkDNA-graphene oxide probing system for detection of SARS-CoV-2.

The development of rapid, highly sensitive, and selective methods for the diagnosis of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) should help to prevent the spread of this pandemic virus. In this study, we combined recombinase polymerase amplification (RPA), as a means of isothermal DNA amplification, with an rkDNA-graphene oxide (GO) probe system to allow the rapid detection of SARS-CoV-2 with high sensitivity and selectivity. We used in situ enzymatic synthesis to prepare an rkDNA probe that was complementary to an RPA-amplified sequence of the target N-gene of SARS-CoV-2. The fluorescence of this rkDNA was perfectly quenched in the presence of GO. When the quenched rkDNA-GO system was added to the RPA-amplified sequence of the target SARS-CoV-2, the fluorescence recovered dramatically. The combined RPA/rkDNA-GO system exhibited extremely high selectivity (discrimination factor: 17.2) and sensitivity (LOD = 6.0 aM) for the detection of SARS-CoV-2. The total processing time was only 1.6 h. This combined RPA/rkDNA-GO system appears to be a very efficient and simple method for the point-of-care detection of SARS-CoV-2.

rkDNA-graphene oxide as a simple probe for the rapid detection of miRNA21.

In this study we developed a novel diagnostic tool for the detection of miRNA21, based on the fluorescent nucleotide morpholine naphthalimide deoxyuridine (dUrkTP). We incorporated dUrkTP into DNA through primer extension to obtain rkDNA displaying high fluorescence. We then used lambda exonuclease, a specific nuclease for 3́-monophosphate-containing DNA, to separate rkDNA from its complementary sequence. The fluorescence of the free rkDNA was quenched dramatically upon interacting with graphene oxide (GO). Our rkDNA-GO fluorescence probing system exhibited high sensitivity and selectivity for the detection of miRNA21. This inexpensive probing system, employing simple primer extension and exonuclease degradation, required only 30 min to detect its target miRNA. This strategy appears suitable for the detection of diverse types of miRNA.

Structural Effects of Magnetostrictive Materials on the Magnetoelectric Response of Particulate CZFO/NKNLS Composites.

In this study, magnetostrictive powders of CoFe2O4 (CFO) and Zn-substituted CoFe2O4 (CZFO, Zn = 0.1, 0.2) were synthesized in order to decrease the optimal dc magnetic field (Hopt.), which is required to obtain a reliable magnetoelectric (ME) voltage in a 3-0 type particulate composite system. The CFO powders were prepared as a reference via a typical solid solution process. In particular, two types of heterogeneous CZFO powders were prepared via a stepwise solid solution process. Porous-CFO and dense-CFO powders were synthesized by calcination in a box furnace without and with pelletizing, respectively. Then, heterogeneous structures of pCZFO and dCZFO powders were prepared by Zn-substitution on calcined powders of porous-CFO and dense-CFO, respectively. Compared to the CFO powders, the heterogeneous pCZFO and dCZFO powders exhibited maximal magnetic susceptibilities (χmax) at lower Hdc values below ±50 Oe and ±10 Oe, respectively. The Zn substitution effect on the Hdc shift was more dominant in dCZFO than in pCZFO. This might be because the Zn ion could not diffuse into the dense-CFO powder, resulting in a more heterogeneous structure inducing an effective exchange-spring effect. As a result, ME composites consisting of 0.948Na0.5K0.5NbO3⁻0.052LiSbO3 (NKNLS) with CFO, pCZFO, and dCZFO were found to exhibit Hopt. = 966 Oe (NKNLS-CFO), Hopt. = 689⁻828 Oe (NKNLS-pCZFO), and Hopt. = 458⁻481 Oe (NKNLS-dCZFO), respectively. The low values of Hopt. below 500 Oe indicate that the structure of magnetostrictive materials should be considered in order to obtain a minimal Hopt. for high feasibility of ME composites.