Label-Free and DNAzyme-Mediated Biosensor with a High Signal-to-Noise Ratio for a Lumpy Skin Disease Virus Assay.

(LSDV) is a severe and highly contagious form of cowpox. As LSDV continues to mutate and there is no vaccine and treatment in nonendemic countries, early detection of LSDV becomes an important basis for epidemic prevention and control, especially for detection of conserved sequences. A new label-free and sensitive fluorescence method was developed based on a light-up RNA aptamer for detecting LSDV.

Completely Free from PAM Limitations: Asymmetric RPA with CRISPR/Cas12a for Nucleic Acid Assays.

Experimentally, Cas12a can recognize multiple protospacer adjacent motif (PAM) sequences and is not restricted to the "TTTN". However, the application of the CRISPR/Cas12a system is still limited by the PAM for double-stranded DNA (dsDNA). Here, we developed asymmetric RPA (Asy-RPA) to completely break the limitations of PAM.

Field-friendly and ultra-fast detection platform without nucleic acid extraction for virus detection.

The polymeric chain reaction (PCR) has come under fire for being time-consuming, requiring expensive equipments, and requiring the extraction and purification of nucleic acids. Here, an ultra-fast and sensitive detection platform without nucleic acid extraction solved the above problems. Firstly, the RoomTemp Sample Lysis Kit released the nucleic acid in 3 min and removed the inhibition to facilitate the amplification reaction.

Hybridization chain reaction circuit controller: CRISPR/Cas12a conversion amplifier for miRNA-21 sensitive detection.

MicroRNA (miRNA) is crucial to the diagnose of various diseases. However, the accurate detection of miRNA has been challenging due to its short length and low abundance. Here, we designed a hybridization chain reaction (HCR) circuit controller to initiate the CRISPR/Cas12a conversion amplifier (HCR-Cas12a controller) for sensitive detection of miRNA-21 (miR-21).

The End of the Gray Zone: One-Tube Nested Recombinase Polymerase Amplification with Ultrahigh Signal-to-Noise Ratio for Precisely Detecting and Surveilling Viruses.

The samples were difficult to accurately determine positive or negative between 35 and 40 cycles by real-time quantitative PCR (qPCR) as the standard method. Here, we developed one-tube nested recombinase polymerase amplification (ONRPA) technology with CRISPR/Cas12a to overcome this difficulty. ONRPA broke the amplification plateau to substantially enhance the signals, which considerably improved the sensitivity and eliminated the problem of gray area.

Multiple accurate and sensitive arrays for Capripoxvirus (CaPV) differentiation.

Capripoxvirus (CaPV) contains three viruses that have caused massive losses in the livestock and dairy industries. Accurate CaPV differentiation has far-reaching implications for effectively controlling outbreaks. However, it has a great challenge to distinguishing three viruses due to high homology of 97%.

A light-up fluorescence platform based DNA: RNA hybrid G-quadruplet for detecting single nucleotide variant of ctDNA and miRNA-21.

The nucleic acid assay is an area of great concern in the diagnosis and treatment of breast cancer. Here, we developed a DNA: RNA hybrid G-quadruplet (HQ) detection platform based on strand displacement amplification (SDA) and Baby Spinach RNA aptamer for single nucleotide variant (SNV) of circulating tumor DNA (ctDNA) and miRNA-21. This was the first in vitro construction of HQ for the biosensor.

Rapid and Ultrasensitive Approach for the Simultaneous Detection of Multilocus Mutations to Distinguish Rifampicin-Resistant .

The untested empirical medications exacerbated the development of multidrug-resistant (MDR-TB). Here, we develop a rapid and specific method based on loop-mediated isothermal amplification and duplex-specific nuclease for distinguishing rifampicin-resistant . Three probes were designed for the codons 516, 526, and 531 on the RNA polymerase β-subunit (rpoB) gene.

Carbon nanodots combined with loop-mediated isothermal amplification (LAMP) for detection of African swine fever virus (ASFV).

The spread of African swine fever virus (ASFV) caused huge economic costs, so early detection is particularly important. Here, we established a fluorescence biosensor based on carbon nanodots (CNDs) and loop-mediated isothermal amplification (LAMP) to ultra-sensitively detect ASFV. LAMP with high efficiency produced a large amount of pyro phosphoric acid and caused pH change in a short time.

Non-nucleic acid extraction and ultra-sensitive detection of African swine fever virus via CRISPR/Cas12a.

Early diagnosis of the African swine fever virus (ASFV) is the main preventive measure for ASFV. Here, we developed a fluorescent biosensor and lateral flow assay (LFA) strip based on direct PCR combined with CRISPR/Cas12a system for ASF. Direct PCR can simultaneously split samples and efficiently amplify without sacrificing sensitivity, which eliminated the steps of nucleic acid extraction.

One-pot platform for rapid detecting virus utilizing recombinase polymerase amplification and CRISPR/Cas12a.

The livestock industry has been deeply affected by African swine fever virus (ASFV) and Capripoxvirus (CaPV), which caused an enormous economic damage. It is emergent to develop a reliable detection method. Here, we developed a rapid, ultra-sensitive, and one-pot DNA detection method combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a for ASFV and CaPV, named one-pot-RPA-Cas12a (OpRCas) platform.

Automated, portable, and high-throughput fluorescence analyzer (APHF-analyzer) and lateral flow strip based on CRISPR/Cas13a for sensitive and visual detection of SARS-CoV-2.

COVID-19 has erupted and quickly swept across the globe, causing huge losses to human health and wealth. It is of great value to develop a quick, accurate, visual, and high-throughput detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we developed a biosensor based on CRISPR/Cas13a combined with recombinase polymerase amplification (RPA) to detect S and Orf1ab genes of SARS-CoV-2 within 30 min.

Simultaneous detection of CaMV35S and T-nos utilizing CRISPR/Cas12a and Cas13a with multiplex-PCR (MPT-Cas12a/13a).

Here, we established a strategy (MPT-Cas12a/13a) that combined CRISPR/Cas12a and Cas13a for simultaneously detecting CaMV35S and T-nos based on multiplex PCR (M-PCR) and transcription. It realized a simultaneous detection mode with different signals in the same space. The MPT-Cas12a/13a had excellent sensitivity with the limit of detection as low as 11 copies of T-nos and 13 copies of CaMV35S and it had outstanding specificity and anti-interference ability in actual sample analysis.

The fluorescent biosensor for detecting N methyladenine FzD5 mRNA and MazF activity.

N methyladenine (mA) modification of the FzD5 mRNA, an important post-transcriptional regulation in eukaryotes, is closely related to the occurrence and development of breast cancer. Here, we developed an ultra-sensitive biosensor based on MazF combining with cascaded strand displacement amplification (C-SDA) and CRISPR/Cas12a to detect mA FzD5 mRNA. MazF toxin protein is a vital component of the bacterial mazEF toxin-antitoxin system that is sensitive to mA RNA.

Single-nucleotide variant of PIK3CA gene assay by CRISPR/Cas12a combined with rolling circle amplification.

PIK3CA gene plays an important role in the PI3K/Akt/mTOR signaling pathway, and its mutation is closely related to the occurrence and development of breast cancer and Lipoblastoma. Therefore, it is of great value to detect the PIK3CA mutant gene. Here, an analytical method coupled CRISPR/Cas12a with rolling circle amplification (RCA) technology was constructed for ultra-sensitive and specific detection of the single-nucleotide variant (SNV) of the PIK3CA gene.

Ultrasensitive detection of gene-PIK3CA mutation based on cascaded strand displacement amplification and trans-cleavage ability of CRISPR/Cas12a.

Low abundance gene-PIK3CA mutation detection is crucial for the clinical diagnosis and treatment of breast cancer. Here, a fluorescent biosensor which combines cascaded strand displacement amplification (C-SDA) and trans-cleavage ability of CRISPR/Cas12a was established to ultra-sensitively detect gene-PIK3CA mutation. The mutated gene-PIK3CA can combine with complementary sequence to form an intact recognition site for endonuclease FspI.

Ultra-sensitive MicroRNA-21 detection based on multiple cascaded strand displacement amplification and CRISPR/Cpf1 (MC-SDA/CRISPR/Cpf1).

MicroRNA-21 (miR-21) has been considered as a potential biomarker for cancer diagnosis and prognosis due to its high expression in tumors. Here, an analytical method which integrates the multiple cascaded strand displacement amplification and CRISPR/Cpf1 (MC-SDA/CRISPR/Cpf1) was proposed to ultra-sensitively detect it.

An ultrasensitive and point-of-care sensor for the telomerase activity detection.

Telomerase owns great application potential in diagnosis, therapy, prognosis, and drug screening of cancers. Thus, the ultrasensitive and point-of-care detection of telomerase activity meets the clinical demands extremely. Here, a sensor based on telomerase extends activators to unlock the ssDNase activity of CRISPR/Cas12a was created for the first time to detect the telomerase activity.

Colorimetric and fluorescent dual-identification of glutathione based on its inhibition on the 3D ball-flower shaped Cu-hemin-MOF's peroxidase-like activity.

Hemin as the organic linker ligand and Cu (II) as the metal center were applied to prepare a copper-metal-organic framework (Cu-hemin-MOF) via one-step hydrothermal method. Characterization using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and X-ray powder diffraction (XRD) demonstrate that the acquired Cu-hemin-MOF possesses the appearance of 3D ball-flower shape with the existence of C, N, O, Fe, and Cu on the surface. Further study found that this 3D ball-flower shaped Cu-hemin-MOF exhibits peroxidase-like activity, which can catalyze the peroxidase substrate of o-phenylenediamine (OPD) to generate 2,3-diaminophenazine (DAP) in the presence of HO.

Terminal deoxynucleotidyl transferase induced activators to unlock the trans-cleavage of CRISPR/Cpf 1 (TdT-IU- CRISPR/Cpf 1): An ultrasensitive biosensor for Dam MTase activity detection.

DNA adenine methylation methyltransferase (Dam MTase) plays an important role in gene expression and cell development, and involves in some development of tumors. There are many methods to detect Dam MTase activity, yet they have some shortcomings such as high cost, limited detection limit and complicated operation. Here, a new fast and simple multiple amplification strategy based on terminal deoxynucleotidyl transferase induced activators to unlock the collateral cleavage activities (trans-cleavage) of CRISPR/Cpf 1 (TdT-IU-CRISPR/Cpf 1) was firstly established.

A Methodology for Ultrasensitive Detection of Sequence-Specific DNA or Uracil-DNA Glycosylase Activity.

Ultrasensitive detection of sequence-specific DNA and uracil-DNA glycosylase (UDG) activity shows great practical significance in clinical diagnostic and biomedical studies. Here, a methodology based on a CRISPR/Cas12a system coupled with enhanced strand displacement amplification (E-SDA) was innovatively established for sequence-specific DNA or UDG activity detection. Sequence-specific DNA or DNA primers processed by UDG and Endonuclease IV can initiate E-SDA, generating auxiliary DNA chains, which act as activators to unlock the indiscriminate collateral cleavage activities (trans-cleavage) of the CRISPR/Cas12a.