CRISPR/Cas13a-Responsive and RNA-Bridged DNA Hydrogel Capillary Sensor for Point-of-Care Detection of RNA.

Disease diagnostics and surveillance increasingly highlight the importance of portable, cost-effective, and sensitive point-of-care (POC) detection of nucleic acids. Here, we report a CRISPR/Cas13a-responsive and RNA-bridged DNA hydrogel capillary sensor for the direct and visual detection of specific RNA with high sensitivity. The capillary sensor was simply prepared by loading RNA-cross-linking DNA hydrogel film (∼0.2 mm ± 0.02 mm) at the end of a capillary. When CRISPR/Cas13a specifically recognizes the target RNA, the RNA bridge in the hydrogel film is cleaved by the trans-cleavage activity of CRISPR/Cas13a, increasing the permeability of the hydrogel film. Different concentrations of target RNA activate different amounts of Cas13a, cleaving different amounts of the RNA bridge in the hydrogel and causing corresponding changes in the permeability of the hydrogel. Therefore, samples containing different amounts of the target RNA travel to different distances in the capillary. Visual reading of the distance provides quantitative detection of the RNA target without the need for any nucleic acid amplification or auxiliary equipment. The technique was successfully used for the determination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in clinical nasopharyngeal (NP) swab and saliva samples. Easily quantifiable distance using a ruler eliminates the need for any optical or electrochemical detection equipment, making this assay potentially useful for POC and on-site applications.

Dual CRISPR/Cas13a Cascade Strand Displacement-Triggered Transcription for Point-of-Care Detection of Plasmodium in Asymptomatic Malaria.

Asymptomatic infections of Plasmodium parasites are major obstacles to malaria control and elimination. A sensitive, specific, and user-friendly method is urgently needed for point-of-care (POC) Plasmodium diagnostics in asymptomatic malaria, especially in resource-limited settings. In this work, we present a POC method (termed Cas13a-SDT) based on the cascade sequence recognition and signal amplification of dual Cas13a trans-cleavage and strand displacement-triggered transcription (SDT). Cas13a-SDT not only achieves exceptional specificity in discriminating the target RNA from nontarget RNAs with any cross-interaction but also meets the sensitivity criterion set by the World Health Organization (WHO) for effective malaria detection. Remarkably, this novel method was successfully applied to screen malaria in asymptomatic infections from clinical samples. The proposed method provides a user-friendly and visually interpretable output mode while maintaining high accuracy and reliability comparable to RT-PCR. These excellent features demonstrate the significant potential of Cas13a-SDT for POC diagnosis of Plasmodium infections, laying a vital foundation for advancing malaria control and elimination efforts.

Single-microbead space-confined digital quantification strategy (SMSDQ) for counting microRNAs at the single-molecule level.

Quantification of microRNAs (miRNAs) at the single-molecule level is of great significance for clinical diagnostics and biomedical research. The challenges lie in the limits to transforming single-molecule measurements into quantitative signals. To address these limits, here, we report a new approach called a Single Microbead-based Space-confined Digital Quantification (SMSDQ) to measure individual miRNA molecules by counting gold nanoparticles (AuNPs) with localized surface plasmon resonance (LSPR) light-scattering imaging. One miRNA target hybridizes with the alkynyl-modified capture DNA probe immobilized on a microbead (60 µm) and the azide-modified report DNA probe anchored on AuNP (50 nm), respectively. Through the click reaction between the alkynyl and azide group, a single microbead can covalently link the AuNPs in the confined space within the view of the microscope. By digitally counting the light-scattering spots of AuNPs, we demonstrated the proposed approach with single-molecule detection sensitivity and high specificity of single-base discrimination. Taking the advantages of ultrahigh sensitivity, specificity, and the digital detection manner, the approach is suitable for evaluating cell heterogeneity and small variations of miRNA expression and has been successfully applied to direct quantification of miRNAs in one-tenth single-cell lysates and serum samples without RNA-isolated and nucleic acid amplification steps.

Multiple-probe-assisted DNA capture and amplification for high-throughput African swine fever virus detection.

African swine fever (ASF) is one of the most devastating infectious diseases affecting domestic pigs and wild boar. The grave socio-economic impact of African swine fever infection at a global level makes large-scale rapid and robust diagnosis a critical step towards effective control. Here, we describe multiple-probe-assisted DNA capture and amplification technology (MADCAT) - a novel, sensitive, simple, and high-throughput method for detecting ASFV directly from whole blood or other complex matrices. Through a unique DNA capture approach which specifically captures the target DNA onto 96-well plate for subsequent amplification, MADCAT abandons the complicated extraction protocol and achieves ultrafast and high-throughput detection. The sample-to-result time for 96 samples is about 90 min, as compared with the 3-4 h time of the conventional real-time qPCR method. The limit of detection (LOD) of MADCAT is 0.5 copies/µL blood and is 5 times more sensitive than an extraction-based qPCR assay when testing serially diluted whole blood samples. The assay is 100% specific against other common swine pathogens. In the clinical diagnosis of 96 field samples, all 22 positive samples were correctly identified with lower Ct values than extraction-based qPCR, confirming its high diagnostic sensitivity (100%). Owing to its high-throughput, specific high sensitivity, and direct detection features, MADCAT shows great potential for use in large-scale ASFV surveillance and monitoring for effective disease control. KEY POINTS: • No nucleic acid extraction, 100% capture efficiency, and high-throughput • Ultra-high sensitivity of 0.5 DNA copies/µL or 6 DNA copies/reaction • The sample-to-answer time for 96 samples is about 90 min.

One-by-one single-molecule counting method for digital quantification of SARS-CoV-2 RNA.

Digital counting individual nucleic acid molecule is of great significance for fundamental biological research and accurate diagnosis of genetic diseases, which is hard to achieve with existing single-molecule detection technologies. Herein, we report a novel one-by-one single-molecule counting method for digital quantification of SARS-Cov-2 RNA. This method uses one fluorescent micromotor functionalized with peptide nucleic acids (PNAs) to specially capture one target RNA molecule. The RNA-micromotors can be propelled by the electric field to target district and accurately counted. Moreover, the method can also clearly discriminate one-base mutation in the target RNAs, indicating the great potential for clinical diagnostics and virus traceability survey.

Effectiveness of joint 3 + 1 malaria strategy along China-Myanmar cross border areas.

BACKGROUND: Cross-border malaria in Laiza City of Myanmar seriously affected Yingjiang County of China and compromised reaching the goal of malaria elimination by 2020. Since 2017, a pilot project on 3 + 1 strategy of joint cross-border malaria prevention and control was carried out for building a malaria buffer in these border areas. Here, 3 were the three preventive lines in China where different focalized approaches of malaria elimination were applied and + 1 was a defined border area in Myanmar where the integrated measures of malaria control were adopted. METHODS: A 5-year retrospective analysis (2015 to 2019) was conducted that included case detection, parasite prevalence and vector surveillance. Descriptive statistics was used and the incidence or rates were compared. The annual parasite incidence and the parasite prevalence rate in + 1 area of Myanmar, the annual importation rate in Yingjiang County of China and the density of An. minimus were statistically significant indictors to assess the effectiveness of the 3 + 1 strategy. RESULTS: In + 1 area of Myanmar from 2015 to 2019, the averaged annual parasite incidence was (59.11 ± 40.73)/1000 and Plasmodium vivax accounted for 96.27% of the total confirmed cases. After the pilot project, the annual parasite incidence dropped 89% from 104.77/1000 in 2016 to 12.18/1000 in 2019, the microscopic parasite prevalence rate dropped 100% from 0.34% in 2017 to zero in 2019 and the averaged density of An. Minimus per trap-night dropped 93% from 1.92 in June to 0.13 in September. The submicroscopic parasite prevalence rate increased from 1.15% in 2017 to 1.66% in 2019 without significant difference between the two surveys (P = 0.084). In Yingjiang County of China, neither indigenous nor introduced case was reported and 100% cases were imported from Myanmar since 2017. The averaged annual importation rate from 2015 to 2019 was (0.47 ± 0.15)/1000. After the pilot project, the annual importation rate dropped from 0.59/1000 in 2016 to 0.28/1000 in 2019 with an overall reduction of 53% in the whole county. The reduction was 67% (57.63/1000 to 18.01/1000) in the first preventive line, 52% (0.20/1000 to 0.10/1000) in the second preventive line and 36% (0.32/1000 to 0.22/1000) in the third preventive line. The averaged density of An. Minimus per trap-night in the first preventive line dropped 94% from 2.55 in June to 0.14 in September, without significant difference from that of + 1 area of Myanmar (Z value = - 1.18, P value = 0.24). CONCLUSION: The pilot project on 3 + 1 strategy has been significantly effective in the study areas and a buffer zone of border malaria was successfully established between Laiza City of Myanmar and Yingjiang County of China.

Photothermal mediated rolling circle amplification toward specific and direct in situ mRNA detection.

Rolling circle amplification (RCA) had the prospect of assisting clinic diagnosis with advantage in in situ mRNA detection at single cell level. However, for direct mRNA detection, RCA had relatively low detection specificity and efficiency. Here, we introduced 4-(10, 15, 20-Triphenylporphyrin-5-yl)phenylamine (TPP) modified Au nanoparticle (Au-TPP) to improve the specificity of in-situ RCA. Through photothermal effect, Au-TPP acted as the specific heat source upon irradiation of 635 nm laser. The photothermal mediated RCA would be initiated only when the Au-TPP as well as the padlock anchored adjacently on the same target mRNA. Furthermore, we introduced 'C' form target-specific oligonucleotide linker probes to make generic padlock and Au-TPP for different mRNA targets, so that for a new mRNA target one does not have to redesign the padlock and the Au-TPP probe. By these strategies, we successfully developed a specific and photothermal mediated hyperbranched rolling circle amplification for direct in situ mRNA detection, suitable for both formalin-fixed paraffin-embedded (FFPE) tissue section and frozen tissue section.

Identification of a selective DNA ligase for accurate recognition and ultrasensitive quantification of N6-methyladenosine in RNA at one-nucleotide resolution.

N6-Methyladenosine (m6A) is the most frequent post-transcriptional modification in RNA, and it plays a critical role in biological processes. The functions of m6A remain largely unexplored due to a lack of highly sensitive methods to quantitatively determine the m6A modification fraction at a precise location. Here, we first reveal that T3 DNA ligase has significant selectivity towards the m6A modification. On the basis of the new finding, we establish an ultrasensitive quantitation assay for accurately determining m6A at one-nucleotide resolution in RNA. With the proposed assay, as low as 4 fM RNA containing m6A can be determined and the selectivity is up to 54.1-fold to discriminate m6A against unmodified adenosine (A). The sensitivity has been improved about 106-fold so the proposed method can be successfully employed to accurately determine m6A in real biological samples, even in low abundance RNA.