Integration of a Cas12a-mediated DNAzyme actuator with efficient RNA extraction for ultrasensitive colorimetric detection of viral RNA.

Developing highly sensitive and specific on-site tests is imperative to strengthen preparedness against future emerging infectious diseases. Here, we describe the construction of a Cas12a-mediated DNAzyme actuator capable of converting the recognition of a specific DNA sequence into an amplified colorimetric signal. To address viral RNA extraction challenges for on-site applications, we developed a rapid and efficient method capable of lysing the viral particles, preserving the released viral RNA, and concentrating the viral RNA. Integration of the DNAzyme actuator with the viral RNA extraction method and loop-mediated isothermal amplification enables a streamlined colorimetric assay for highly sensitive colorimetric detection of respiratory RNA viruses in gargle and saliva. This assay can detect as few as 83 viral particles/100 µL in gargle and 166 viral particles/100 µL in saliva. The entire assay, from sample processing to visual detection, was completed within 1 h at a single controlled temperature. We validated the assay by detecting SARS-CoV-2 in 207 gargle and saliva samples, achieving a clinical sensitivity of 96.3 % and specificity of 100%. The assay is adaptable for detecting specific nucleic acid sequences in other pathogens and is suitable for resource-limited settings.

Investigation of waste alkali-activated cementing material using municipal solid waste incineration fly ash and dravite as precursors: Mechanisms, performance, and on-site application.

The proper treatment of municipal solid waste incineration fly ash (MSWIFA) is a crucial concern due to its hazardous nature and potential environmental harm. To address this issue, this study innovatively utilized dravite and black liquor to solidify MSWIFA. The semi-dry pressing method was employed, resulting in the production of waste alkali-activated cementing material (WACM). This material demonstrated impressive compressive and flexural strength, reaching 45.89 MPa and 6.55 MPa respectively, and effectively solidified heavy metal ions (Pb, Cr, Cu, Cd, and Zn). The leaching concentrations of these ions decreased from 27.15, 10.36, 8.94, 7.00, and 104.4 mg/L to 0.13, 1.05, 0.29, 0.06, and 12.28 mg/L, respectively. The strength of WACM increased by 3 times compared to conventionally produced materials. Furthermore, WACM exhibited excellent long-term performance, with acceptable heavy metal leaching and minimal mechanical degradation. Experimental and theoretical analyses revealed the heavy metal solidification mechanisms, including chemical binding, ion substitution and physical encapsulation. Finally, the on-site application of WACM confirmed its feasibility in meeting both environmental and strength requirements.

Rapid and Visual Detection of Actinidia Chlorotic Ringspot-Associated Virus Using One-Step Reverse-Transcription Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick Assay.

Actinidia chlorotic ringspot-associated virus (AcCRaV) occurs widely in major kiwifruit producing areas of China and is often accompanied by coinfecting viruses, affecting the growth, yield, and quality of kiwifruit. Therefore, a rapid and sensitive detection method is crucial for diagnosing and developing effective AcCRaV management strategies. In this study, a one-step reverse-transcription recombinase polymerase amplification combined with a lateral flow dipstick (RT-RPA-LFD) assay was developed for rapid detection of AcCRaV. Specific primers and a probe were designed based on the conserved region of the coat protein gene sequence of AcCRaV. The one-step RT-RPA reaction can be performed at 35 and 40°C within 10 to 30 min, and the amplification results can be read directly on the LFD within 5 min. The detection limit of the one-step RT-RPA-LFD assay was 10-8 ng (about 20 viral copies), which was equal with one-step RT-qPCR and 100 times more sensitive than one-step RT-PCR. Moreover, the one-step RT-RPA-LFD assay was successfully applied to detect AcCRaV from crude extracts, and the entire detection process can be completed within 40 min. These results indicate that the RT-RPA-LFD assay is a simple, rapid, and sensitive strategy that can be used for accurate diagnosis of AcCRaV-infected kiwifruit plants in the field. To our knowledge, this is the first study applying the one-step RT-RPA-LFD assay to detect a kiwifruit virus.

Development of the yeast and lactic acid bacteria co-culture agent for atmospheric ammonia removing: Genomic features and on-site applications.

Odorous gas (e.g. atmospheric ammonia) in low ventilation public places, such as public toilets and waste transfer stations, causes severe health problems. Many technologies are developed to purify the atmospheric ammonia, among which the microbial agents are supposed to be a green and economical approach. In this study, we developed a yeast, Pichia sp. J1, and a lactic acid bacterium (LAB), Lactobacillus paracasei B1, co-culture agent for atmospheric ammonia removing. The on-site application results indicated the yeast and LAB mixed fermented agent had a maximum ammonia removing efficiency of 98.78%, which is significantly higher than the pure cultures (78.93% for B1 and 75.00% for J1), indicating the co-culture agent is an excellent biological product for ammonia removal. The excellent performance of the agent is closely related to the synergy behaviors between the yeast and LAB. In the co-culture agents, some of the LAB cells adhered closely to the yeast, and the growth and lactic acid producing ability of LAB were significantly promoted by yeast. Genomic analysis indicated the complementary of nutrients, i.e. carbon and nitrogen resources, signal transduction, and adhesion proteins (regulates adhesion behavior) played roles in regulating the synergy effects. Our study offers a novel biological solution of odorous gas purification.

LAMP assay and rapid sample preparation method for on-site detection of flavescence dorée phytoplasma in grapevine.

In Europe the most devastating phytoplasma associated with grapevine yellows (GY) diseases is a quarantine pest, flavescence dorée (FDp), from the 16SrV taxonomic group. The on-site detection of FDp with an affordable device would contribute to faster and more efficient decisions on the control measures for FDp. Therefore, a real-time isothermal LAMP assay for detection of FDp was validated according to the EPPO standards and MIQE guidelines. The LAMP assay was shown to be specific and extremely sensitive, because it detected FDp in all leaf samples that were determined to be FDp infected using quantitative real-time PCR. The whole procedure of sample preparation and testing was designed and optimized for on-site detection and can be completed in one hour. The homogenization procedure of the grapevine samples (leaf vein, flower or berry) was optimized to allow direct testing of crude homogenates with the LAMP assay, without the need for DNA extraction, and was shown to be extremely sensitive.