Xiyan Luo

The CRISPR-based detection methods have been widely applied, yet they remain limited by the non-universal nature of one-pot diagnostic approaches. Here, we report a universal one-pot fluorescent method for the detection of epidemic pathogens, delivering results within 15-20 min. This method uses heparin sodium to precisely tunes the cis-cleavage capability of Cas12 via interference with the Cas12a-crRNA binding process, thereby generating significant fluorescence due to the accumulation of isothermal amplification products. Additionally, this universal assay accommodates both classic and suboptimal PAMs, as well as various Cas12a subtypes such as LbCas12a, AsCas12a, and AapCas12b. Such a robust method demonstrates sensitivity and specificity exceeding 95% in the detection of monkeypox pseudovirus, influenza A virus, and SARS-CoV-2 from saliva or wastewater samples, when compared with qPCR or RT-qPCR. Moreover, the cost of heparin sodium per thousand uses is $0.01 to $0.04 only. Collectively, this universal and fast one-pot approach based on heparin sodium offers potential possibilities for point-of-care testing. CRISPR-based detection has long been challenged by the separation of amplification and detection steps. In this study, Cheng et al., show that heparin sodium modulates Cas12 cleavage activity and apply a one pot system to detect clinical influenza A virus and SARS-CoV-2 in wastewater.

Dissemination of antibiotic resistance genes (ARGs) in urban water bodies has become a significant environmental and health concern. Many approaches based on real-time quantitative PCR (qPCR) have been developed to offer rapid and highly specific detection of ARGs in water environments, but the complicated and time-consuming procedures have hindered their widespread use. Herein, we developed a facile one-step approach for rapid detection of ARGs by leveraging the trans-cleavage activity of Cas12a and recombinase polymerase amplification (RPA). This efficient method matches the sensitivity and specificity of qPCR and requires no complex equipment. The results show a strong correlation between the prevalence of four ARG markers (ARGs: sul1, qnrA-1, mcr-1, and class 1 integrons: intl1) in tap water, human urine, farm wastewater, hospital wastewater, municipal wastewater treatment plants (WWTPs), and proximate natural aquatic ecosystems, indicating the circulation of ARGs within the urban water cycle. Through monitoring the ARG markers in 18 WWTPs in 9 cities across China during both peak and declining stages of the COVID epidemic, we found an increased detection frequency of mcr-1 and qnrA-1 in wastewater during peak periods. The ARG detection method developed in this work may offer a useful tool for promoting a sustainable urban water cycle.

Bacterial wilt, caused by Ralstonia solanacearum, is a destructive disease that causes significant loss in ginger productivity. This disease is notoriously difficult to control by chemical measures, prompting the exploration of biological control as a more sustainable and effective alternative strategy. In our research, we aimed to develop effective microbial agents to combat R. solanacearum-induced bacterial wilt in ginger. Initially, 307 Bacillus-like strains were isolated from ginger rhizosphere soil. Among these, ten strains exhibited significant antibacterial activity after extensive in vitro screening, with Bacillus-like isolate F18 being the most effective. Further tests in a co-culture assay revealed six isolates, notably L70, that significantly reduced the pathogen's fitness. To further assess their effectiveness, six isolates of L3, L72, L73, F18, HC-5, and L70 were then subjected to a hydroponics assay, where they substantially lowered disease incidence in ginger seedlings, especially strains F18, HC-5, and L73. These three strains were identified as Bacillus velezensis, exhibiting beneficial traits such as IAA production, siderophore secretion, and phosphate and potassium solubilization. Additionally, they also harbored genes related to the production of the dipeptide bacilysin and the polyketides macrolactin, bacillaene and difficidin. In greenhouse experiments, these three B. velezensis strains, particularly F18 and HC-5, demonstrated remarkable efficacy in controlling bacterial wilt. Tubers soaked in these biocontrol agents showed reduced disease severity and pathogen population. Moreover, the treatments promoted ginger growth, notably in tuber fresh weight, with HC-5 showing the greatest improvement. Conclusively, this study suggests a highly efficient strategy for biologically controlling ginger bacterial wilt, with B. velezensis strains HC-5, L73, and F18 emerging as promising biocontrol agents.

The tomato pinworm, Tuta absoluta, or Phthorimaea absouta, is native to South America, but quickly spread to other regions of world, including Europe, Africa, and Asia, devastating to global tomato production. However, a lack of high-quality genome resources makes it difficult to understand its high invasiveness and ecological adaptation. Here, we sequenced the genome of the tomato pinworm using Nanopore platforms, yielding a genome assembly of 564.5 Mb with contig N50 of 3.33 Mb. BUSCO analysis demonstrated that this genome assembly has a high-level completeness of 98.0% gene coverage. In total, 310 Mb are repeating sequences accounting for 54.8% of genome assembly, and 21,979 protein-coding genes are annotated. Next, we used the Hi-C technique to anchor 295 contigs to 29 chromosomes, yielding a chromosome-level genome assembly with a scaffold N50 of 20.7 Mb. In sum, the high-quality genome assembly of the tomato pinworm is a useful gene resource that contributes to a better understanding of the biological characteristics of its invasiveness and will help in developing an efficient control policy.