Development of a rapid, sensitive detection method for SARS-CoV-2 and influenza virus based on recombinase polymerase amplification combined with CRISPR-Cas12a assay.

Respiratory tract infections are associated with the most common diseases transmitted among people and remain a huge threat to global public health. Rapid and sensitive diagnosis of causative agents is critical for timely treatment and disease control. Here, we developed a novel method based on recombinase polymerase amplification (RPA) combined with CRISPR-Cas12a to detect three viral pathogens, including SARS-CoV-2, influenza A, and influenza B, which cause similar symptom complexes of flu cold in the respiratory tract. The detection method can be completed within 1 h, which is faster than other standard detection methods, and the limit of detection is approximately 102 copies/µL. Additionally, this detection system is highly specific and there is no cross-reactivity with other common respiratory tract pathogens. Based on this assay, we further developed a more simplified RPA/CRISPR-Cas12a system combined with lateral flow assay on a manual microfluidic chip, which can simultaneously detect these three viruses. This low-cost detection system is rapid and sensitive, which could be applied in the field and resource-limited areas without bulky and expensive instruments, providing powerful tools for the point-of-care diagnostic.

Rapid quantification of live/dead lactic acid bacteria in probiotic products using high-sensitivity flow cytometry.

A laboratory-built high-sensitivity flow cytometer (HSFCM) was employed for the rapid and accurate detection of lactic acid bacteria (LAB) and their viability in probiotic products. LAB were stained with both the cell membrane-permeable SYTO 9 green-fluorescent nucleic acid stain and the red-fluorescent nucleic acid stain, propidium iodide, which penetrates only bacteria with compromised membranes. The side scatter and dual-color fluorescence signals of single bacteria were detected simultaneously by the HSFCM. Ultra-high temperature processing milk and skim milk spiked with Lactobacillus casei were used as the model systems for the optimization of sample pretreatment and staining. The viable LAB counts measured by the HSFCM were in good agreement with those of the plate count method, and the measured ratios between the live and dead LAB matched well with the theoretical ratios. The established method was successfully applied to the rapid quantification of live/dead LAB in yogurts and fermented milk beverages of different brands. Moreover, the concentration and viability status of LAB in ambient yogurt, a relatively new yet popular milk product in China, are also reported.

Probing minority population of antibiotic-resistant bacteria.

The evolution and spread of antibiotic-resistant pathogens has become a major threat to public health. Advanced tools are urgently needed to quickly diagnose antibiotic-resistant infections to initiate appropriate treatment. Here we report the development of a highly sensitive flow cytometric method to probe minority population of antibiotic-resistant bacteria via single cell detection. Monoclonal antibody against TEM-1 ß-lactamase and Alexa Fluor 488-conjugated secondary antibody were used to selectively label resistant bacteria green, and nucleic acid dye SYTO 62 was used to stain all the bacteria red. A laboratory-built high sensitivity flow cytometer (HSFCM) was applied to simultaneously detect the side scatter and dual-color fluorescence signals of single bacteria. By using E. coli JM109/pUC19 and E. coli JM109 as the model systems for antibiotic-resistant and antibiotic-susceptible bacteria, respectively, as low as 0.1% of antibiotic-resistant bacteria were accurately quantified. By monitoring the dynamic population change of a bacterial culture with the administration of antibiotics, we confirmed that under the antimicrobial pressure, the original low population of antibiotic-resistant bacteria outcompeted susceptible strains and became the dominant population after 5hours of growth. Detection of antibiotic-resistant infection in clinical urine samples was achieved without cultivation, and the bacterial load of susceptible and resistant strains can be faithfully quantified. Overall, the HSFCM-based quantitative method provides a powerful tool for the fundamental studies of antibiotic resistance and holds the potential to provide rapid and precise guidance in clinical therapies.

Trace detection of specific viable bacteria using tetracysteine-tagged bacteriophages.

Advanced methods are urgently needed to determine the identity and viability of trace amounts of pathogenic bacteria in a short time. Existing approaches either fall short in the accurate assessment of microbial viability or lack specificity in bacterial identification. Bacteriophages (or phages for short) are viruses that exclusively infect bacterial host cells with high specificity. As phages infect and replicate only in living bacterial hosts, here we exploit the strategy of using tetracysteine (TC)-tagged phage in combination with biarsenical dye to the discriminative detection of viable target bacteria from dead target cells and other viable but nontarget bacterial cells. Using recombinant M13KE-TC phage and Escherichia coli ER2738 as a model system, distinct differentiation between individual viable target cells from dead target cells was demonstrated by flow cytometry and fluorescence microscopy. As few as 1% viable E. coli ER2738 can be accurately quantified in a mix with dead E. coli ER2738 by flow cytometry. With fluorescence microscopic measurement, specific detection of as rare as 1 cfu/mL original viable target bacteria was achieved in the presence of a large excess of dead target cells and other viable but nontarget bacterial cells in 40 mL artificially contaminated drinking water sample in less than 3 h. This TC-phage-FlAsH approach is sensitive, specific, rapid, and simple, and thus shows great potential in water safety monitoring, health surveillance, and clinical diagnosis of which trace detection and identification of viable bacterial pathogens is highly demanded.

High-throughput single-cell analysis of low copy number ß-galactosidase by a laboratory-built high-sensitivity flow cytometer.

Single-cell analysis is vital in providing insights into the heterogeneity in molecular content and phenotypic characteristics of complex or clonal cell populations. As many essential proteins and most transcription factors are produced at a low copy number, analytical tools with superior sensitivity to enable the analysis of low abundance proteins in single cells are in high demand. ß-galactosidase (ß-gal) has been the standard cellular reporter for gene expression in both prokaryotic and eukaryotic cells. Here we report the development of a high-throughput method for the single-cell analysis of low copy number ß-gal proteins using a laboratory-built high-sensitivity flow cytometer (HSFCM). Upon fluorescence staining with a fluorogenic substrate, quantitative measurements of the basal and near-basal expression of ß-gal in single Escherichia coli BL21(DE3) cells were demonstrated. Statistical distribution can be determined quickly by analyzing thousands of individual cells in 1-2min, which reveals the heterogeneous expression pattern that is otherwise masked by the ensemble analysis. Combined with the quantitative fluorometric assay and the rapid bacterial enumeration by HSFCM, the ß-gal expression distribution profile could be converted from arbitrary fluorescence units to protein copy numbers per cell. The sensitivity and speed of the HSFCM offers great capability in quantitative analysis of low abundance proteins in single cells, which would help gaining a deeper insight into the heterogeneity and fundamental biological processes in microbial populations.

A targetable nanogenerator of nitric oxide for light-triggered cytotoxicity.

Nanoscale-vesicles that can target pathogens are valuable for biomedical applications. In this study, a photo-responsive nanogenerator of nitric oxide (NO) comprised of a hydrophobic core of 3-trifluoromethyl-4-nitroaniline (TFNA) and a hydrophilic shell of mannosylated poly[styrene-alter-(maleic acid)] was constructed to target and kill lectin-expressing cells. The release of NO from the nanogenerator (T@P-M) was effectively induced by luminol-derived chemiluminescence (CL), leading to high-efficiency killing of Escherichia coli (E. coli) treated with T@P-M. In addition, the uptake of T@P-M by Raw 264.7 macrophages was achieved by cell surface lectin-mediated endocytosis, enabling the intracellular release of NO from the internalized T@P-M upon the induction of extracellular chemiluminescence. Because in vivo-generated CL can overcome the limited penetration of exogenous light into biological tissues, T@P-M has potential uses as a targetable photo-activatable microbicide to combat pathogens bearing lectins or residing in macrophages.

Detection and quantification of bacterial autofluorescence at the single-cell level by a laboratory-built high-sensitivity flow cytometer.

Cellular autofluorescence can affect the sensitivity of fluorescence microscopic or flow cytometric assays by interfering with or even precluding the detection of low-level specific fluorescence. Here we developed a method to detect and quantify bacterial autofluorescence in the green region of the spectrum at the single-cell level using a laboratory-built high-sensitivity flow cytometer (HSFCM). The detection of the very weak bacterial autofluorescence was confirmed by analyzing polystyrene beads of comparable and larger size than bacteria in parallel. Dithionite reduction and air re-exposure experiments verified that the green autofluorescence mainly originates from endogenous flavins. Bacterial autofluorescence was quantified by calibrating the fluorescence intensity of nanospheres with known FITC equivalents, and autofluorescence distribution was generated by analyzing thousands of bacterial cells in 1 min. Among the eight bacterial strains tested, it was found that bacterial autofluorescence can vary from 80 to 1400 FITC equivalents per cell, depending on the bacterial species, and a relatively large cell-to-cell variation in autofluorescence intensity was observed. Quantitative measurements of bacterial autofluorescence provide a reference for the background signals that can be expected with bacteria, which is important in guiding studies of low-level gene expression and for the detection of low-abundance biological molecules in individual bacterial cells. This paper presents the first quantification of bacterial autofluorescence in FITC equivalents.

Size differentiation and absolute quantification of gold nanoparticles via single particle detection with a laboratory-built high-sensitivity flow cytometer.

Employing single nanoparticle detection with a laboratory-built high-sensitivity flow cytometer, we developed a simple and versatile platform that is capable of detecting the surface plasmon resonance scattering of gold nanoparticles (GNPs) as small as 24 nm, differentiating GNPs of different sizes, and providing accurate quantification of GNPs. Low-concentration samples (fM to pM) in small volumes (microL) can be measured in minutes with an analysis rate of up to 100-200 GNPs per second. Among these features, absolute quantification provides a distinct advantage because it does not require standard samples.