微生物学

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

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.

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:

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.

The COVID-19 pandemic requires mass screening to identify those infected for isolation and quarantine. Individually screening large populations for the novel pathogen, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is costly and requires a lot of resources. Sample pooling methods improve the efficiency of mass screening and consume less reagents by increasing the capacity of testing and reducing the number of experiments performed, and are therefore especially suitable for under-developed countries with limited resources. Here, we propose a simple, reliable pooling strategy for COVID-19 testing using clinical nasopharyngeal (NP) and/or oropharyngeal (OP) swabs. The strategy includes the pooling of 10 NP/OP swabs for extraction and subsequent testing via quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR), and may also be applied to the screening of other pathogens.

Hepatitis B virus (HBV) is the major cause of liver diseases and liver cancer worldwide. After infecting hepatocytes, the virus establishes a stable episome (covalently closed circular DNA, or cccDNA) that serves as the template for all viral transcripts. Specific and accurate quantification of cccDNA is difficult because infected cells contain abundant replicative intermediates of HBV DNA that share overlapping sequences but arranged in slightly different forms. HBV cccDNA can be detected by Southern blot or qPCR methods which involve enzymatic digestion. These assays are laborious, have limited sensitivity, or require degradation of cellular DNA (which precludes simple normalization). The method described in this protocol, cccDNA inversion quantitative (cinq)PCR, instead uses a series of restriction enzyme-mediated hydrolysis and ligation reactions that convert cccDNA into an inverted linear amplicon, which is not amplified or detected from other forms of HBV DNA. Importantly, cellular DNA remains quantifiable during sample preparation, allowing normalization and markedly improving precision. Further, a second linear fragment (derived from enzymatic digestion of a separate region of the HBV DNA genome and is present in all forms of HBV DNA) can be used to simultaneously quantify total HBV levels.

Graphic abstract:

Selective detection of HBV cccDNA and total HBV DNA using cinqPCR (Reproduced from Tu et al., 2020a).

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; initially named 2019-nCoV) is responsible for the recent coronavirus disease (COVID-19) pandemic, and polymerase chain reaction (PCR) is the current standard method for diagnosis from patient samples. As PCR assays are prone to sequence mismatches due to mutations in the viral genome, it is important to verify the genomic variability at primer/probe binding regions periodically. This step-by-step protocol describes a bioinformatics approach for an extensive evaluation of the sequence variability within the primer/probe target regions of the SARS-CoV-2 genome. The protocol can be applied to any molecular diagnostic assay of choice using freely available software programs and the ready-to-use multiple sequence alignment (MSA) file provided.

Graphic abstract:


Overview of the sequence tracing protocol. The figure was created using the Library of Science and Medical Illustrations from somersault18:24 licensed under a CC BY-NC-SA 4.0 license (https://creativecommons.org/licenses/by-nc-sa/4.0/).

Video abstract: https://youtu.be/M1lV1liWE9k

The efficiency of cleavage of individual CRISPR/Cas9-sgRNAs remains difficult to predict based on the CRISPR target sequence alone. Different intracellular environments (dependent on cell type or cell cycle state for example) may affect sgRNA efficiency by altering accessibility of genomic DNA through DNA modifications such as epigenetic marks and DNA-binding proteins (e.g., histones) as well as alteration of the chromatin state of genomic DNA within the nucleus.

We recently reported a multi-step screening method for the identification of efficient sgRNAs targeting the Herpes simplex virus (HSV-1) genome and reported a differential mechanism for viral inhibition by CRISPR-Cas9 in the latent versus lytic phase. The screening platform detailed in this protocol allows step-by-step testing of the efficiency of cleavage in a cell-free system and in the context of viral target cells such as human foreskin fibroblasts followed by functional testing of the effects of CRISPR/sgRNA on viral protein expression, replication, and reactivation. This strategy could be readily applied to other target cells such as pluripotent stem cell-derived human sensory neurons or other human DNA viruses.

We present a safe and low-cost method suitable for DNA extraction from mycelium and tree tissue samples. After sample preparation, the extraction takes about 60 min. Method performance was tested by extracting DNA from various tree tissue samples and from mycelium grown on solid and liquid media. DNA was extracted from juvenile and mature host material (Picea abies, Populus trichocarpa, Pseudotsuga menziesii) infected with different pathogens (Heterobasidion annosum, Heterobasidion parviporum, Leptographium wagenerii, Sphaerulina musiva). Additionally, DNA was extracted from pure cultures of the pathogens and several endophytic fungi. PCR success rate was 100% for young poplar material and fungal samples, and 48-72% for conifer and mature broadleaved plant samples. We recommend using 10-50 mg of fresh sample for the best results. The method offers a safe and low-cost DNA extraction alternative to study tree-fungus interactions, and is a potential resource for teaching purposes.

Many RNA viruses are found in protozoan parasites. They can be responsible for more serious pathology or treatment failure. For the detection of viral double-stranded RNA (dsRNA), sequence-dependent and -independent methods are available, such as quantitative real-time PCR and immunofluorescence, dot blot, ELISA or sequencing. The technique presented here is sequence-independent and is well detailed in the following protocol, taking the example of Leishmania RNA virus (LRV) in Leishmania guyanensis (Lgy) species. To summarise, the protocol is divided into four major steps: RNA extraction from the parasites, RNA purification, enzymatic digestions with DNase I and Nuclease S1, and visualization by gel electrophoresis. This method can be used to detect other viral dsRNA in other parasites. It provides an additional tool, complementary to other techniques previously cited and it is easy and quite fast to achieve.