Nucleic Acid Extraction Research Articles

Active soil microbial composition and proliferation are directly affected by the presence of biocides from building materials

Combinations of biocides are commonly added to building materials to prevent microbial growth and thereby cause degradation of the façades. These biocides reach the environment by leaching from façades posing an environmental risk. Although ecotoxicity to the aquatic habitat is well established, there is hardly any data on the ecotoxicological effects of biocides on the soil habitat. This study aimed to characterize the effect of the biocides terbutryn, isoproturon, octhilinone, and combinations thereof on the total and metabolically active soil microbial community composition and functions. Total soil microbial community was retrieved directly from the nucleic acid extracts, while the DNA of the active soil microbial community was separated after bromodeoxyuridine labeling. Bacterial 16S rRNA gene and fungal internal transcribed spacer region gene-based amplicon sequencing was carried out for both active and total, while gene copy numbers were quantified only for the total soil microbial community. Additionally, soil respiration and physico-chemical parameters were analyzed to investigate overall soil microbial activity. The bacterial and fungal gene copy numbers were significantly affected by single biocides and combined biocide soil treatment but not soil respiration and physico-chemical parameters. While the total soil microbiome experienced only minor effects from single and combined biocide treatment, the active soil microbiome was significantly impacted in its diversity, richness, composition, and functional patterns. The active bacterial richness was more sensitive than fungal richness. However, the adverse effects of the biocide combination treatments on soil bacterial richness were highly dependent on the identities of the biocide combination. Our results demonstrate that the presence of biocides frequently used in building materials affects the active soil microbiome. Thereby, the approach described herein can be used as an ecotoxicological measure for the effect on complex soil environments in future studies.

Preparation of norovirus GII loop mediated isothermal amplification freeze-drying microsphere reagents and its application in an on-site integrated rapid detection platform

Norovirus is an infectious disease that can cause non-bacterial gastroenteritis, which has a low infectious dose, rapid onset, and strong transmission ability; therefore, rapid and sensitive detection is essential to reduce the transmission of gastroenteritis. In the study, a norovirus GII loop-mediated isothermal amplification assay was developed and prepared into freeze-drying microspheres, and a closed-cassette-based, integrated, reagent-ambient storage, on-site instant detection platform for norovirus GII was constructed using a commercial, fully automated nucleic acid analyzer with integrated magnetic bearing based nuclear acid extraction and nucleic acid detection, with a sensitivity of 10 copies/μL, with no cross-reactivity with other 5 viruses. For 28 simulated samples, the integrated assay platform was consistent with the experimental results of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays after conventional laboratory nucleic acid extraction. The entire process can be finished in about 1 h, which is ideal for immediate rapid detection.

Detection of Reverse Transcriptase LAMP-Amplified Nucleic Acid from Oropharyngeal Viral Swab Samples Using Biotinylated DNA Probes through a Lateral Flow Assay.

This study focuses on three key aspects: (a) crude throat swab samples in a viral transport medium (VTM) as templates for RT-LAMP reactions; (b) a biotinylated DNA probe with enhanced specificity for LFA readouts; and (c) a digital semi-quantification of LFA readouts. Throat swab samples from SARS-CoV-2 positive and negative patients were used in their crude (no cleaning or pre-treatment) forms for the RT-LAMP reaction. The samples were heat-inactivated but not treated for any kind of nucleic acid extraction or purification. The RT-LAMP (20 min processing time) product was read out by an LFA approach using two labels: FITC and biotin. FITC was enzymatically incorporated into the RT-LAMP amplicon with the LF-LAMP primer, and biotin was introduced using biotinylated DNA probes, specifically for the amplicon region after RT-LAMP amplification. This assay setup with biotinylated DNA probe-based LFA readouts of the RT-LAMP amplicon was 98.11% sensitive and 96.15% specific. The LFA result was further analysed by a smartphone-based IVD device, wherein the T-line intensity was recorded. The LFA T-line intensity was then correlated with the qRT-PCR Ct value of the positive swab samples. A digital semi-quantification of RT-LAMP-LFA was reported with a correlation coefficient of R2 = 0.702. The overall RT-LAMP-LFA assay time was recorded to be 35 min with a LoD of three RNA copies/µL (Ct-33). With these three advancements, the nucleic acid testing-point of care technique (NAT-POCT) is exemplified as a versatile biosensor platform with great potential and applicability for the detection of pathogens without the need for sample storage, transportation, or pre-processing.

Rapid, sensitive, and convenient detection of Plasmodium falciparum infection based on CRISPR and its application in detection of asymptomatic infection

Rapid and convenient detection of the Plasmodium in clinically diagnosed individuals and asymptomatically infected populations is essential for global malaria eradication, especially in malaria-endemic African countries where medical equipment and professionals are relatively deficient. Here, we described a CRISPR-based diagnostic for the detection of Plasmodium falciparum, the deadliest and most prevalent species of malaria parasite in Africa, via lateral flow strip readout without the need of nucleic acid extraction. The assay exhibited 100% sensitivity on clinical samples (5 P falciparum) and significant consistency with qPCR test on asymptomatic infection samples (49 P falciparum and 51 non-P. falciparum, Kappa=0.839). An artemisinin-resistant P. falciparum strain and 4 other laboratory-cultured strains can also be detected through this assay, whereas no cross-reactivity with Plasmodium vivax was observed. A 0.001% parasitaemia (corresponding to ∼60 parasites/μL) below the “low parasite density” test threshold (200 parasites/µL) is detectable. Our study demonstrated that direct malaria detection using whole blood on the spot and the detection of both clinical and asymptomatic infections of P. falciparum are feasible. This method is expected to be employed for clinical testing and large-scale community screening in Africa and possibly other places, contributing to the accurate diagnosis and control of malaria.

Study on monitoring nucleic acid contamination in laboratory and its application

Monitoring nucleic acid contamination in laboratories is essential for ensuring the accuracy and reliability of polymerase chain reaction (PCR) assay results. Compared to traditional chemical reagent-based extraction, magnetic bead extraction is a targeted adsorption method for extracting DNA, offering greater efficiency and minor hazard. However, the effectiveness of this method in detecting nucleic acid contaminations in laboratory remains unclear. This study aimed to develop a PCR-based laboratory environmental monitoring method to investigate the effectiveness of magnetic bead extraction for detecting nucleic acid contamination. To mimic nucleic acid contamination, Staphylococcus aureus was aerosolized in a PCR laboratory. Following sampling, nucleic acid extraction was performed using the magnetic bead extraction method. Samples were analyzed using quantitative real-time fluorescence PCR (qPCR), and the concordance between the magnetic bead extraction results and theoretical results was assessed. This study presents a PCR laboratory nucleic acid contamination monitoring protocol. The results demonstrated that the nucleic acid detection outcomes obtained using the magnetic bead extraction method were consistent with the theoretical results across all regions tested using this protocol. The magnetic bead extraction method demonstrated comparable detection outcomes to the theoretical results in monitoring laboratory environments. This approach provides a more efficient and environmental friendly method of monitoring the laboratory environment.

Retracted: Functionalized Ferroferric Oxide Nanomagnetic Beads for Extraction of Nucleic Acid and Its Application in Early Screening of Colorectal Cancer.

[This retracts the article DOI: 10.1155/2021/5230666.].

The imperative for quality control programs in Monkeypox virus DNA testing by PCR: CIBERINFEC quality control.

To evaluate molecular assays for Mpox diagnosis available in various clinical microbiology services in Spain through a quality control (QC) approach. A total of 14 centers from across Spain participated in the study. The Reference Laboratory dispatched eight serum samples and eight nucleic acid extracts to each participating center. Some samples were spiked with Mpox or Vaccinia virus to mimic positive samples for Mpox or other orthopox viruses. Participating centers provided information on the results obtained, as well as the laboratory methods used. Among the 14 participating centers seven different commercial assays were employed, with the most commonly used kit being LightMix Modular Orthopox/Monkeypox (Mpox) Virus (Roche®). Of the 12 centers conducting Mpox determinations, concordance ranged from 62.5% (n = 1) to 100% (n = 11) for eluates and from 75.0% (n = 1) to 100% (n = 10) for serum. Among the 10 centers performing Orthopoxvirus determinations, a 100% concordance was observed for eluates, while for serum, concordance ranged from 87.5% (n = 6) to 100% (n = 4). Repeatedly, 6 different centers reported a false negative in serum samples for Orthopoxvirus diagnosis, particularly in a sample with borderline Ct = 39. Conversely, one center, using the TaqMan™ Mpox Virus Microbe Detection Assay (Thermo Fisher), reported false positives in Mpox diagnosis for samples spiked with vaccinia virus due to cross-reactions. We observed a positive correlation of various diagnostic assays for Mpox used by the participating centers with the reference values. Our results highlight the significance of standardization, validation, and ongoing QC in the microbiological diagnosis of infectious diseases, which might be particularly relevant for emerging viruses.

Diagnosis of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) by RT-PCR using different extraction protocols

RT-PCR method was performed using four different nucleic acid extraction methods to identify viroids; peach latent mosaic viroid (PLMVd) and hop stunt viroid (HSVd) which causes serious damage to stone fruits. Leaf samples were collected from fruit orchards showing viroid-like symptoms in İzmir province. Silica capture, citric buffer, lithium chloride, and ames buffer methods were used to extract total nucleic acids. The four extraction methods were compared using samples collected during the vegetation period from naturally infected trees (plum, apricot, and peach). They were evaluated with RT-PCR tests. In 64 stone fruit tree samples, only the silica capture method gave reliable and accurate results in RT-PCR molecular tests for the detection of PLMVd and HSVd. The other wielded nucleic acid extraction methods were found to be ineffective for the isolation of the viroid RNAs.

Fungicides and insecticides can alter the microbial community on the cuticle of honey bees.

Honey bees are crucial for our ecosystems as pollinators, but the intensive use of plant protection products (PPPs) in agriculture poses a risk for them. PPPs do not only affect target organisms but also affect non-targets, such as the honey bee Apis mellifera and their microbiome. This study is the first of its kind, aiming to characterize the effect of PPPs on the microbiome of the cuticle of honey bees. We chose PPPs, which have frequently been detected in bee bread, and studied their effects on the cuticular microbial community and function of the bees. The effects of the fungicide Difcor® (difenoconazole), the insecticide Steward® (indoxacarb), the combination of both (mix A) and the fungicide Cantus® Gold (boscalid and dimoxystrobin), the insecticide Mospilan® (acetamiprid), and the combination of both (mix B) were tested. Bacterial 16S rRNA gene and fungal transcribed spacer region gene-based amplicon sequencing and quantification of gene copy numbers were carried out after nucleic acid extraction from the cuticle of honey bees. The treatment with Steward® significantly affected fungal community composition and function. The fungal gene copy numbers were lower on the cuticle of bees treated with Difcor®, Steward®, and PPP mix A in comparison with the controls. However, bacterial and fungal gene copy numbers were increased in bees treated with Cantus® Gold, Mospilan®, or PPP mix B compared to the controls. The bacterial cuticular community composition of bees treated with Cantus® Gold, Mospilan®, and PPP mix B differed significantly from the control. In addition, Mospilan® on its own significantly changed the bacterial functional community composition. Cantus® Gold significantly affected fungal gene copy numbers, community, and functional composition. Our results demonstrate that PPPs show adverse effects on the cuticular microbiome of honey bees and suggest that PPP mixtures can cause stronger effects on the cuticular community than a PPP alone. The cuticular community composition was more diverse after the PPP mix treatments. This may have far-reaching consequences for the health of honey bees.

Photofabricated channel-digital microfluidics (pCDMF): A promising lab-on-a-chip platform for fully integrated digital PCR

Microfluidics-based digital PCR (dPCR) is a potent technique capable of absolute quantification of nucleic acids with ultrahigh sensitivity. However, an indispensable process, nucleic acid extraction (NAE) remains hard to be integrated with dPCR into a monolithic device, because both of them involve complicated processes and exquisite fluid manipulation. An ideal solution to this issue is to integrate the two main microfluidic platforms, channel-based microfluidics (CMF) and digital microfluidics (DMF), which, however, remains very challenging. To address these challenges, we herein develop a photofabricated channel-digital microfluidic chip (pCDMF) with significant advantages including: the fabrication of pCDMF is very convenient due to the flexible material choices of photocuring, without tedious process of hydrophobic coating; a parallel-plate configuration can be easily formed, enabling all basic functions of DMF; various channels and microstructures can be freely designed, allowing for fluidic functions of CMF. It is therefore the first example of full integration of DMF and CMF as a monolithic device, with capabilities to precisely handle both discrete droplets and continuous fluids. Finally, an all-in-one device for sample-to-answer digital PCR has been successfully demonstrated. We also envisioned that the pCDMF may open an alternative route toward the fully integrated lab-on-a-chip for various biological applications.

Multiplexed on-site sample-in-result-out test through microfluidic real-time PCR (MONITOR) for the detection of multiple pathogens causing influenza-like illness.

The global COVID-19 pandemic and frequent outbreaks of other infectious diseases, such as influenza and monkeypox, often initially manifest with non-specific influenza-like symptoms. Portability to the outbreak site and accurate identification of the various pathogens causing influenza-like illness are crucial for the rapid implementation of effective control measures. Utilizing real-time PCR and microfluidic technology, a multiplexed on-site sample-in-result-out test through microfluidic real-time PCR (MONITOR) has been developed for detecting the pathogens responsible for influenza-like illness. The operator simply needs to introduce the sample to a highly integrated microfluidic chip (requiring ≤1 minute), and the system can autonomously execute sample pre-processing, nucleic acid extraction, and PCR amplification and deliver results for eight pathogens within 85 minutes. The detection limit of MONITOR for the eight pathogens ranges from 0.78 to 6.25 copies/µL. Standard curves demonstrate notable linearity and amplification efficiency. A simulated clinical sample test reveals MONITOR's sensitivity, specificity, and accuracy at 97.5%, 100%, and 98%, respectively. Bland-Altman analysis demonstrates strong agreement between the cycle threshold of positive MONITOR samples and quantitative polymerase chain reaction (qPCR) (R2 = 0.952), suggesting MONITOR's ability to indirectly quantify pathogen load. The fully enclosed structure of the system chip minimizes sample and environmental cross-contamination, rendering the assay independent of a specialized laboratory. The portable, fully automated MONITOR facilitates on-site, comprehensive detection of multiple pathogens, even non-specialized operators with minimal training. This presents a promising approach for the rapid and precise diagnosis of influenza-like illness in grassroots communities and underserved rural areas lacking centralized laboratories.IMPORTANCEThis study combines quantitative polymerase chain reaction (qPCR) and microfluidics to introduce MONITOR, a portable field detection system for multiple pathogens causing influenza-like illness. MONITOR can be rapidly deployed to enable simultaneous sample-in-result-out detection of eight common influenza-like illness (ILI) pathogens with heightened sensitivity and specificity. It is particularly well suited for communities and regions without centralized laboratories, offering robust technical support for the prompt and accurate monitoring and detection of ILI. It holds the potential to be a potent tool in the early detection and prevention of infectious diseases.

RPA-CRISPR/Cas12a-Based Detection of Haemophilus parasuis.

Haemophilus parasuis (H. parasuis, HPS) is a prominent pathogenic bacterium in pig production. Its infection leads to widespread fibrinous inflammation in various pig tissues and organs, often in conjunction with various respiratory virus infections, and leads to substantial economic losses in the pig industry. Therefore, the rapid diagnosis of this pathogen is of utmost importance. In this study, we used recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR) technology to establish a convenient detection and analysis system for H. parasuis that is fast to detect, easy to implement, and accurate to analyze, known as RPA-CRISPR/Cas12a analysis. The process from sample to results can be completed within 1 h with high sensitivity (0.163 pg/μL of DNA template, p < 0.05), which is 104 -fold higher than the common PCR method. The specificity test results show that the RPA-CRISPR/Cas12a analysis of H. parasuis did not react with other common pig pathogens, including Streptococcus suis type II and IX, Actinobacillus pleuropneumoniae, Escherichia coli, Salmonella, Streptococcus suis, and Staphylococcus aureus (p < 0.0001). The RPA-CRISPR/Cas12a assay was applied to 15 serotypes of H. parasuis clinical samples through crude extraction of nucleic acid by boiling method, and all of the samples were successfully identified. It greatly reduces the time and cost of nucleic acid extraction. Moreover, the method allows results to be visualized with blue light. The accurate and convenient detection method could be incorporated into a portable format as point-of-care (POC) diagnostics detection for H. parasuis at the field level.

Analytical Validation of an Automated Semiconductor-Based Next-Generation Sequencing Assay for Detection of DNA and RNA Alterations in Myeloid Neoplasms

Myeloid neoplasms are heterogeneous tumors derived from early hematopoietic progenitors. Most international guidelines, including the European LeukemiaNet 2022 update, recommend testing a comprehensive set of genes, most within a 3- to 5-day period for optimal treatment decisions. Next-generation sequencing gene panels are essential for identifying genetic alterations, risk stratification, and determining targeted therapies for myeloid malignancies. This study describes the analytical validation of the Oncomine Myeloid Assay GX v2 (Myeloid GX v2) in combination with the Ion Torrent Genexus System using commercial controls, 16 variant-negative samples, and 130 clinical samples of myeloid neoplasms. The Myeloid GX v2 panel detected single nucleotide variants (SNVs), insertions/deletions (indels) (allele frequency >5%), and gene fusions (minimum 11 fusion copies/μL) in synthetic controls with a sensitivity of 100%. Specificity for detection of SNVs, indels, or fusions in 16 variant-negative samples was 100%. Sensitivity for detection of SNVs, indels, and gene fusions in 130 clinical samples was 99%, 97%, and 100%, respectively. Overall precision was 100% for SNVs, 96% for indels, and 100% for fusions. The average turnaround time from nucleic acid extraction to results was 2 days. The Myeloid GX v2 panel is highly accurate and reproducible for the detection of SNVs, indels, and gene fusions in myeloid neoplasms. The ability to deliver clinically relevant results in a short time is key to providing personalized treatments.

Simultaneous extraction and detection of DNA and RNA from viruses, prokaryotes, and eukaryotes in wastewater using a modified COPMAN

Wastewater surveillance can offer a comprehensive grasp of infectious disease prevalence and human health because wastewater contains various human-derived microbial pathogens, including viruses, bacteria, and fungi. However, methods capable of simultaneous detection of multiple groups of targets in the automated systems and large-scale surveillance are still under development. Here, we demonstrated the modification, involving the addition of bead-beating, to the existing COPMAN (COagulation and Proteolysis method using MAgnetic beads for detection of Nucleic acids in wastewater) enabled enhanced detection of various microorganisms, including SARS-CoV-2. The modified method, termed bead-beating COPMAN (BB-COPMAN), was evaluated through spike-and-recovery experiments and comparative analysis against three previously reported methods for simultaneous DNA/RNA detection. Our study targeted a range of microorganisms, including enveloped and non-enveloped RNA viruses (SARS-CoV-2, PMMoV), a DNA virus (crAssphage), archaea, gram-negative and gram-positive bacteria (E. coli, Lachnospiraceae), antibiotic resistance gene (ampC), and fungi (Candida albicans). The recovery rates of BB-COPMAN for gram-negative and gram-positive bacteria were 17 and 2.1-fold higher, respectively, compared to the method for DNA/RNA detection. Additionally, BB-COPMAN exhibited the highest extraction efficiency among the tested methods, achieving 1.2–5.7 times more DNA and 1.1–69 times more RNA yield on average. BB-COPMAN allowed the detection of SARS-CoV-2 from all nine samples and PMMoV at concentrations 39–97 times higher than other methods. Moreover, BB-COPMAN detected larger amounts of DNA for four out of six DNA targets than the previously reported DNA/RNA detection method. These results demonstrated that BB-COPMAN enables enhanced detection of multiple targets in a single flow of nucleic acid extraction, making the method well-suited for automated systems. In conclusion, BB-COPMAN is a promising method in wastewater surveillance for assessing the prevalence of wide range of pathogenic microorganisms.

Clinical performance of the novel full-genotyping OncoPredict HPV Quantitative Typing assay using the VALGENT framework.

Clinical validation of human papillomavirus (HPV) assays according to international criteria is prerequisite for their implementation in cervical cancer screening. OncoPredict HPV Quantitative Typing (QT) assay (Hiantis Srl, Milan, Italy) is a novel full-genotyping multiplex real-time PCR quantitative assay targeting E6/E7 genes, allowing individual viral load determination of 12 high-risk (HR) HPV types. Quality controls for sample adequacy, efficiency of nucleic acid extraction and PCR inhibition are included in the assay. Clinical performance of OncoPredict HPV QT test was assessed as part of the "Validation of HPV Genotyping Tests" (VALGENT-2) framework, consisting of 1300 cervical liquid-based cytology (LBC) samples of women aged between 20 and 60 years who had originally attended for routine cervical screening in Scotland. The clinical accuracy of the OncoPredict HPV QT (index test) for the detection of CIN2+ was assessed relative to the GP5+/6+ Enzyme ImmunoAssay (GP5+/6+ EIA) (comparator test), using noninferiority criteria. Intra- and interlaboratory reproducibility of the assay was assessed on a subpopulation, comprising 526 samples. The relative sensitivity and specificity for OncoPredict HPV QT vs GP5+/6+-PCR-EIA were 1.01 (95% CI: 0.99-1.03) and 1.03 (95% CI: 1.0-1.06) respectively. The P-values for noninferiority were ≤0.001. The intra- and inter-laboratory reproducibility demonstrated a high concordance (>98.7%) with kappas for individual types ranging from 0.66 to 1.00. OncoPredict HPV QT fulfills the international validation criteria for the use of HPV tests in cervical cancer screening.

Rapid SERS detection of diverse filamentous fungi by superparamagnetic particles and flexible silver nanowires

In clinical practice, infections caused by Aspergillus species are common and associated with high mortality rates. However, isolating Aspergillus from clinical samples and cultures is challenging, and serological tests cannot differentiate between strains. Furthermore, the complex cell wall structure in Aspergillus species hinders DNA extraction and subsequent nucleic acid detection. Aspergillus fumigatus, Aspergillus flavus, and Aspergillus niger are common and frequently encountered pathogenic species. This study aimed to establish an efficient and rapid method for detecting Aspergillus species in clinical samples. Fe3O4@PEI.NH2 nanoparticles with large particle sizes were employed to capture Aspergillus from the samples. Flexible silver nanowires (AgNWs) were prepared and used as the surface-enhanced Raman scattering (SERS) substrate for Aspergillus detection. Through SERS spectroscopy, the identification of captured Aspergillus species was confirmed. Furthermore, the SERS spectrum underwent principal component analysis employing orthogonal partial least-squares discrimination analysis (OPLS-DA) as a multivariate analysis technique to distinguish between the various Aspergillus species effectively. After 10-fold cross-validation, the trained model achieved a testing accuracy of 99.15%, indicating excellent classification performance. By utilizing pre-prepared Fe3O4@PEI.NH2 and AgNWs, the detection process was completed within 60 min without disrupting the cell wall of Aspergillus.

Border disease virus and chlamydophila abortus co-infection in aborted sheep foetuses

Abortion in sheep and goats is one of the most important reproductive problems that affect small ruminants breeding. Non-infectious factors and infectious agents such as bacterial, viral, mycotic and parasitic infectious agents can cause abortion in sheep and goats. A high rate of abortion (43.75%) was observed in two sheep flocks in Niğde Province in Turkey during the lambing season in 2016. To determine the reason of abortion, aborted sheep foetuses (n = 3) and EDTA whole blood samples (n = 3) from mother of the foetuses were collected from two sheep flocks. Aborted foetuses and buffy coat cells of the EDTA whole blood samples were used for total nucleic acid extraction. Extracted nucleic acids were analysed for akabane virus (AKAV), bluetongue virus (BTV), peste des petits ruminants virus (PPRV), pestiviruses, Schmallenberg virus (SBV), Brucella spp., Chlamydophila abortus (C. abortus), Coxiella burnetii and Listeria monocytogenes. Border disease virus (BDV) RNA and C. abortus DNA were detected in three aborted sheep foetuses whereas other investigated infectious agents were not detected. Additionally, EDTA whole blood samples from mother of the foetuses were also found BDV positive. To the best of my knowledge, this is the first report on BDV and C. abortus co-infection in aborted sheep foetuses.

The detection of Bursaphelenchus xylophilus via accelerated strand exchange amplification: An ultra‐rapid and accurate method

AbstractOne of the most damaging pathogens of pinewood is the pinewood nematode, Bursaphelenchus xylophilus, which could cause an adverse effect on the ecosystems of forests and the commerce of timber. Therefore, it is crucial to realize rapid and accurate B. xylophilus detection. In this work, an accelerated strand exchange amplification method (ASEA) was established to detect B. xylophilus for the first time. By integrating with fast nucleic acid extraction, the whole detection procedure could be finished within 30 min, dramatically shortened the detection time. The ASEA method exhibited high specificity towards B. xylophilus and the detection limit for B. xylophilus plasmid DNA was as low as 1.0 × 100 copies/μL. Furthermore, the ASEA approach also exhibited accurate detection for B. xylophilus when applied to actual pinewood samples, meeting the demand of B. xylophilus detection in realistic scenario. We believe the ASEA method has significant potential for B. xylophilus detection, and it will be helpful for controlling forest pest and quarantine regulations.

Fast preparation of high-quality viral dsRNA from fungal tissue by commercial nucleic acid extraction kits

The genomes of most known mycoviruses consist of double stranded RNA (dsRNA) or single stranded RNA (ssRNA). Therefore, for all aspects of mycovirology, the research is highly dependent on the quality and quantity of RNA either by the extraction of genomic dsRNA or dsRNA as a replicating intermediate. A common procedure to extract dsRNA is its binding on a cellulose matrix after a phenol/chloroform purification step. A commercial kit for dsRNA extraction facilitated the researchers´ daily work, but is not available anymore. To extract nucleic acids in a standardized good quality and quantity from small amounts of starting material, we compared commercial kits for gDNA extraction to the kits for RNA extraction using fungal material with a high and a low virus titer. Here we show that viral dsRNA can be extracted using commercial gDNA kits from fungal tissue with a high and a low virus titer in the same quality and quantity as it was done with the discontinued dsRNA extraction kit.

A NitroPure Nitrocellulose Membrane-Based Grapevine Virus Sampling Kit: Development and Deployment to Survey Japanese Vineyards and Nurseries.

We developed a NitroPure Nitrocellulose (NPN) membrane-based method for sampling and storing grapevine sap for grapevine virus detection. We devised an efficient nucleic acid extraction method for the NPN membrane, resulting in 100% amplification success for grapevine leafroll-associated virus 2 (GLRaV2) and 3 (GLRaV3), grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine virus A, grapevine virus B, and grapevine red blotch virus (GRBV). This method also allowed the storage of recoverable nucleic acid for 18 months at room temperature. We created a sampling kit to survey GLRaV2, GLRaV3, and GRBV in Japanese vineyards. We tested the kits in the field in 2018 and then conducted mail-in surveys in 2020-2021. The results showed a substantial prevalence of GLRaV3, with 48.5% of 132 sampled vines being positive. On the other hand, only 3% of samples tested positive for GLRaV2 and none for GRBV.