Protocols in Current Issue
Protocols in Past Issues
0 Q&A 511 Views Jun 20, 2024

Foot-and-mouth disease (FMD) is a severe and extremely contagious viral disease of cloven-hoofed domestic and wild animals, which leads to serious economic losses to the livestock industry globally. FMD is caused by the FMD virus (FMDV), a positive-strand RNA virus that belongs to the genus Aphthovirus, within the family Picornaviridae. Early detection and characterization of FMDV strains are key factors to control new outbreaks and prevent the spread of the disease. Here, we describe a direct RNA sequencing method using Oxford Nanopore Technology (ONT) Flongle flow cells on MinION Mk1C (or GridION) to characterize FMDV. This is a rapid, low cost, and easily deployed point of care (POC) method for a near real-time characterization of FMDV in endemic areas or outbreak investigation sites.

0 Q&A 578 Views Jan 5, 2024

Fusarium oxysporum can cause many important plant diseases worldwide, such as crown rot, wilt, and root rot. During the development of strawberry crown rot, this pathogenic fungus spreads from the mother plant to the strawberry seedling through the stolon, with obvious characteristics of latent infection. Therefore, the rapid and timely detection of F. oxysporum can significantly help achieve effective disease management. Here, we present a protocol for the recombinase polymerase amplification– lateral flow dipstick (RPA–LFD) detection technique for the rapid detection of F. oxysporum on strawberry, which only takes half an hour. A significant advantage of our RPA–LFD technique is the elimination of the involvement of professional teams and laboratories, which qualifies it for field detection. We test this protocol directly on plant samples with suspected infection by F. oxysporum in the field and greenhouse. It is worth noting that this protocol can quickly, sensitively, and specifically detect F. oxysporum in soils and plants including strawberry.

Key features

• This protocol is used to detect whether plants such as strawberry are infected with F. oxysporum.

• This protocol has potential for application in portable nucleic acid detection.

• It can complete the detection of samples in the field within 30 min.

Graphical overview

0 Q&A 850 Views Nov 5, 2023

The precise and rapid detection of fungi is important in various fields, including clinics, industry, and agriculture. While sequencing universal DNA barcodes remains the standard method for species identification and phylogenetic analysis, a significant bottleneck has been the labor-intensive and time-consuming sample preparation for genomic DNA extraction. To address this, we developed a direct PCR method that bypasses the DNA extraction steps, facilitating efficient target DNA amplification. Instead of extracting genomic DNA from fungal mycelium, our method involves adding a small quantity of mycelium directly to the PCR mixture, followed by a heat shock and vortexing. We found these simple adjustments to be sufficient to lyse many filamentous fungal cells, enabling target DNA amplification. This paper presents a comprehensive protocol for executing direct PCR in filamentous fungi. Beyond species identification, this direct PCR approach holds promise for diverse applications, such as diagnostic PCR for genotype screening without fungal DNA extraction. We anticipate that direct PCR will expedite research on filamentous fungi and diagnosis of fungal diseases.

Key features

• Eliminates the time-consuming genomic DNA extraction step for PCR, enhancing the speed of molecular identification.

• Adds a small quantity of mycelium directly into the PCR mix.

• Emphasizes the crucial role of heat shock and vortexing in achieving efficient target DNA amplification.

• Accelerates the molecular identification of filamentous fungi and rapid diagnosis of fungal diseases.

Graphical overview

Direct PCR using filamentous fungal biomass

0 Q&A 440 Views Sep 5, 2023

In the field of molecular genetics, DNA extraction protocols and kits are sample-specific and proprietary, preventing lateral distribution among similar facilities from different sectors to alleviate supply shortages during a crisis. Expanding upon previous fast extraction protocols such as alkaline- and detergent-based ones, the use of boiling-hot water to rupture cells, virions, and nuclei, as proposed during the COVID-19 pandemic, might alleviate shortages and costs. Different soft, relatively abundant (highly enriched), and uncomplicated (genomically homogenous and with few inhibitors) biosamples are collected in 1.5 mL tubes, mixed with boiling-hot water, and stirred vigorously, so as to have membranes lysed and proteins deactivated; mechanical disruption may be used as well if necessary. Incubation in boiling water bath for 20–30 min follows. Depending on sample type and quantity, which affects the total extraction volume, 2–5 μL are pipetted off for direct PCR and the same volume for two decimal serial dilutions. The latter are intended to optimize the crude extract to a workable DNA/inhibitor concentration balance for direct PCR. Uncomplicated, highly enriched samples such as mycelial growth in fruits and human swab samples can be processed, contrary to complicated samples such as blood and physically unyielding samples such as plant tissue. The extract can be used for immediate PCR in both benchtop and portable thermocyclers, thus allowing nucleic acid amplification tests (NAAT) being performed in resource-limited settings with low cost and waste footprint or during prolonged crises, where supply chain failures may occur.

Key features

• DNA extraction from different sample types using only boiling water and occasional mechanical assistance.

• Crude extract serially diluted twice, 10- and 100-fold, to bypass purification and quantification steps.

• Direct PCR for 2–10 μL of crude lysate and dilutions (conditional to sample type and quantity) to enhance probability of workable DNA-inhibitors’ concentrations.

• Lowers the cost and curtails the overall footprint of testing to increase sustainability in field operations and in standard lab environments under supply chain derailment.

0 Q&A 414 Views May 20, 2023

Fast and accurate detection of pathogenic bacterial infection in patients with severe pneumonia is significant to its treatment. The traditional culture method currently used by most medical institutions relies on a time-consuming culture process (over two days) that is unable to meet clinical needs. Rapid, accurate, and convenient species-specific bacterial detector (SSBD) has been developed to provide timely information on pathogenic bacteria. The SSBD was designed based on the fact that Cas12a indiscriminately cleaves any DNA following the binding of the crRNA-Cas12a complex to the target DNA molecule. SSBD involves two processes, starting with PCR of the target DNA using primers specific for the pathogen, followed by detection of the existence of pathogen target DNA in the PCR product using the corresponding crRNA and Cas12a protein. Compared to the culture test, the SSBD can obtain accurate pathogenic information in only a few hours, dramatically shortening the detection time and allowing more patients to benefit from timely clinical treatment.

0 Q&A 1304 Views Nov 20, 2022

Membrane transporters and soluble binding proteins recognize particular nutrients, metabolites, vitamins, or ligands. By modifying genetically engineered single cysteine residues near the active sites of such proteins with extrinsic maleimide fluorophores, the approaches that we report create sensitive fluorescent sensors that detect, quantify, and monitor molecules that are relevant to the biochemistry, physiology, microbiology, and clinical properties of pro- and eukaryotic organisms.

Graphical abstract:

0 Q&A 2036 Views Apr 20, 2022

The absence of long term, primary untransformed in vitro models that support hepatitis B virus (HBV) infection and replication have hampered HBV pre-clinical research, which was reflected in the absence of a curative therapy until recently. One of the limitations for in vitro HBV research has been the absence of high titer and pure recombinant HBV stocks, which, as we describe here, can be generated using simple, and reproducible protocols. In addition to infection of more conventional in vitro and in vivo liver model systems, recombinant high titer purified HBV stocks can also be used to efficiently infect differentiated human liver organoids, whose generation, maintenance, and infection is discussed in detail in a companion organoid protocol. Here, we also describe the protocols for the detection of specific viral read-outs, including HBV DNA in the supernatant of the cultures, covalently closed circular DNA (cccDNA) from intracellular DNA preparations, and HBV viral proteins and viral RNA, which can be detected within the cells, demonstrating the presence of a complete viral replication cycle in infected liver organoids. Although an evolving platform, the human liver organoid model system presents great potential as an exciting new tool to study HBV infection and progression to hepatocellular carcinoma (HCC) in primary cells, when combined with the use of high-titer and pure recombinant HBV stock for infection.

Graphical abstract:

0 Q&A 1507 Views Mar 5, 2022

The impact of viral diseases on human health is becoming increasingly prevalent globally with the burden of disease being shared between resource-rich and poor areas. As seen in the global pandemic caused by SARS-CoV-2, there is a need to establish viral detection techniques applicable to resource-limited areas that provide sensitive and specific testing with a logistically conscious mindset. Herein, we describe a direct-to-PCR technology utilizing mechanical homogenization prior to viral PCR detection, which allows the user to bypass traditional RNA extraction techniques for accurate detection of human coronavirus. This methodology was validated in vitro, utilizing human coronavirus 229E (HCoV-229E), and then clinically, utilizing patient samples to test for SARS-CoV-2 infection. In this manuscript, we describe in detail the protocol utilized to determine the limit of detection for this methodology with in vitro testing of HCoV-229E.

0 Q&A 1832 Views Feb 5, 2022

Biofilms serve as a bacterial survival strategy, allowing bacteria to persist under adverse environmental conditions. The non-pathogenic Listeria innocua is used as a surrogate organism for the foodborne pathogen Listeria monocytogenes, because they share genetic and physiological similarities and can be used in a Biosafety Level 1 laboratory. Several methods are used to evaluate biofilms, including different approaches to determine biofilm biomass or culturability, viability, metabolic activity, or other microbial community properties. Routinely used methods for biofilm assay include the classical culture-based plate counting method, biomass staining methods (e.g., crystal violet and safranin red), DNA staining methods (e.g., Syto 9), methods that use metabolic substrates to detect live bacteria (e.g., tetrazolium salts or resazurin), and PCR-based methods to quantify bacterial DNA. The NanoLuc (Nluc) luciferase biofilm assay is a viable alternative or complement to existing methods. Functional Nluc was expressed in L. innocua using the nisin-inducible expression system and bacterial detection was performed using furimazine as substrate. Concentration dependent bioluminescence signals were obtained over a concentration range greater than three log units. The Nluc bioluminescence method allows absolute quantification of bacterial cells, has high sensitivity, broad range, good day-to-day repeatability, and good precision with acceptable accuracy. The advantages of Nluc bioluminescence also include direct detection, absolute cell quantification, and rapid execution.

Graphic abstract:

Engineering Listeria innocua to express NanoLuc and its application in bioluminescence assay.

1 Q&A 2949 Views Dec 20, 2021

Arabidopsis thaliana-Pseudomonas syringae pathosystem has been used as an important model system for studying plant-microbe interactions, leading to many milestones and breakthroughs in the understanding of plant immune system and pathogenesis mechanisms. Bacterial infection and plant disease assessment are key experiments in the studies of plant-pathogen interactions. The hypersensitive response (HR), which is characterized by rapid cell death and tissue collapse after inoculation with a high dose of bacteria, is a hallmark response of plant effector-triggered immunity (ETI), one layer of plant immunity triggered by recognition of pathogen-derived effector proteins. Here, we present a detailed protocol for bacterial disease and hypersensitive response assays applicable to studies of Pseudomonas syringae interaction with various plant species such as Arabidopsis, Nicotiana benthamiana, and tomato.

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