PCR-based Method Research Articles

Two-Dimensional (2D) materials in the detection of SARS-CoV-2

The SARS-CoV-2 pandemic has resulted in a devastating effect on human health in the last three years. While tremendous effort has been devoted to the development of effective treatment and vaccines against SARS-CoV-2 and controlling the spread of it, collective health challenges have been encountered along with the concurrent serious economic impacts. Since the beginning of the pandemic, various detection methods like PCR-based methods, isothermal nucleic acid amplification-based (INAA) methods, serological methods or antibody tests, and evaluation of X-ray chest results have been exploited to diagnose SARS-CoV-2. PCR-based detection methods in these are considered gold standards in the current stage despite their drawbacks, including being high-cost and time-consuming procedures. Furthermore, the results obtained from the PCR tests are susceptible to sample collection methods and time. When the sample is not collected properly, obtaining a false result may be likely. The use of specialized lab equipment and the need for trained people for the experiments pose additional challenges in PCR-based testing methods. Also, similar problems are observed in other molecular and serological methods. Therefore, biosensor technologies are becoming advantageous with their quick response, high specificity and precision, and low-cost characteristics for SARS-CoV-2 detection. In this paper, we critically review the advances in the development of sensors for the detection of SARS-CoV-2 using two-dimensional (2D) materials. Since 2D materials including graphene and graphene-related materials, transition metal carbides, carbonitrides, and nitrides (MXenes), and transition metal dichalcogenides (TMDs) play key roles in the development of novel and high-performance electrochemical (bio)sensors, this review pushes the sensor technologies against SARS-CoV-2 detection forward and highlights the current trends. First, the basics of SARS-CoV-2 detection are described. Then the structure and the physicochemical properties of the 2D materials are explained, which is followed by the development of SARS-CoV-2 sensors by exploiting the exceptional properties of the 2D materials. This critical review covers most of the published papers in detail from the beginning of the outbreak.

A simple and rapid CRISPR-Cas12a based detection test for diastatic Saccharomyces cerevisiae

Diastatic Saccharomyces cerevisiae is a common contaminant in the brewing industry. Currently available detection methods are either time consuming or require specialised equipment. The aim of this study was to develop a new rapid and simple assay for the detection of diastatic yeast from samples of beer and yeast. More specifically, the aim was to develop a simple and rapid assay that requires minimal laboratory equipment or training, and yields results as accurate as PCR-based methods. The assay consists of three main steps: DNA extraction, pre-amplification of DNA, and CRISPR-Cas12a based detection and visualisation. Different pre-amplification and visualisation techniques were compared, and the final assay involved a one-pot reaction where LAMP and Cas12a were consecutively used to pre-amplify and detect a fragment from the STA1 gene in a single tube. These reactions required a heat block, a pipette, and a centrifuge with the assay result visualised on a lateral flow strip. The assay was used to monitor an intentionally contaminated brewing fermentation and was shown to yield results as accurate as PCR with previously published primers. Furthermore, the assay yielded results in approximately 75 minutes. The developed assay offers reliable and rapid quality control for breweries of all sizes and can be performed without expensive laboratory equipment. It is suggested that the assay will be particularly useful for smaller breweries without well-equipped laboratories who are looking to implement better quality control.

Clinical application of next generation sequencing-based haplotype linkage analysis in the preimplantation genetic testing for germline mosaicisms

BackgroundPreimplantation genetic testing (PGT) for monogenic disorders (PGT-M) for germline mosaicism was previously highly dependent on polymerase chain reaction (PCR)-based directed mutation detection combined with linkage analysis of short tandem repeats (STRs). However, the number of STRs is usually limited. In addition, designing suitable probes and optimizing the reaction conditions for multiplex PCR are time-consuming and laborious. Here, we evaluated the effectiveness of next generation sequencing (NGS)-based haplotype linkage analysis in PGT of germline mosaicism.MethodsPGT-M with NGS-based haplotype linkage analysis was performed for two families with maternal germline mosaicism for an X-linked Duchenne muscular dystrophy (DMD) mutation (del exon 45–50) or an autosomal TSC1 mutation (c.2074C > T). Trophectoderm biopsy and multiple displacement amplification (MDA) were performed for a total of nine blastocysts. NGS and Sanger sequencing were performed in genomic DNA of family members and embryonic MDA products to detect DMD deletion and TSC1 mutation, respectively. Single nucleotide polymorphism (SNP) sites closely linked to pathogenic mutations were detected with NGS and served in haplotype linkage analysis. NGS-based aneuploidy screening was performed for all embryos to reduce the risk of pregnancy loss.ResultsAll nine blastocytes showed conclusive PGT results. Each family underwent one or two frozen-thawed embryo transfer cycles to obtain a clinical pregnancy, and the prenatal diagnosis showed that the fetus was genotypically normal and euploid for both families.ConclusionsNGS-SNP could effectively realize PGT for germline mosaicism. Compared with PCR-based methods, the NGS-SNP method with increased polymorphic informative markers can achieve a greater diagnostic accuracy. Further studies are warranted to verify the effectiveness of NGS-based PGT of germline mosaicism cases in the absence of surviving offsprings.

Clinical outcomes of core binding factor acute myeloid leukemia in the modern era.

e19054 Background: Acute myeloid leukemia (AML) with alterations in core binding factor (CBF) has a favorable prognosis. The impact of complex cytogenetics or ELN 2022 adverse genetic risk markers — such as ASXL1, RUNX1, EZH2, SRSF2, (and the rest of the secondary-type mutations [STMs]) and TP53 — in CBF-AML is unclear. Additionally, treatment outcomes in CBF-AML using front-line novel approaches, such as a hypomethylating agent (HMA; decitabine or azacitidine) with venetoclax, are unknown. This study analyzed outcomes of CBF-AML with respect to higher-risk molecular and cytogenetic cohorts and treatment approaches. Methods: We identified 58 consecutive patients with CBF-AML treated at VCU Massey Cancer Center from January 1, 2013 to January 1, 2022. We used Fisher’s exact test for between-group comparisons. We analyzed survival by the Kaplan-Meier method and compared groups with the log-rank test. The date of death was used to calculate overall survival; patients were censored at the date of the last contact. Measurable residual disease (MRD) was assessed using PCR-based methods or multiparameter flow cytometry with a minimum sensitivity of 10-3. Results: We analyzed 58 patients with inv(16) or t(8;21) AML treated in the front-line setting. Seven of these patients had concomitant genetic or molecular abnormalities typically considered high risk, including complex cytogenetics or TP53mut. Forty-nine (84.5%) patients received intensive chemotherapy (IC) with FLAG, CPX-351, or conventional 7+3 with or without gemtuzumab ozogamicin; nine patients (15.5%) received lower-intensity therapies with HMAs with or without venetoclax. No differences in overall survival were seen between the patients with CBF-AML with or without complex cytogenetics or TP53mut (not reached for either cohort, median follow-up time of 6.1 y. and 5.0 y., respectively, p = 0.969). There was no difference in the rates of MRD negativity in CBF-AML with or without complex cytogenetics or TP53mut ( p = 0.544). The presence of secondary-type mutations also did not appear to significantly impact survival in CBF-AML ( p = 0.654). There was no difference in the rates of MRD negativity in CBF-AML with STMs and those without STMs ( p = 0.500). Patients that were ineligible for IC and were treated with venetoclax + HMA or HMA monotherapy had significantly reduced composite complete remission rates (CRR; CR + CRi; 25% vs 91.3%, p = 0.007) and median overall survival (3.7 m. versus not reached, p = 0.038). Conclusions: Our data confirm the superior outcomes in induction candidates of CBF-AML treated with IC. The presence of complex cytogenetics, TP53mut, or secondary-type mutations did not appear to influence the rates of MRD negativity or overall survival in CBF-AML.

Triplex qPCR assay for Campylobacter jejuni and Campylobacter coli monitoring in wastewater

Campylobacter spp. is one of the most frequent pathogens of bacterial gastroenteritis recorded worldwide. Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) are the two major disease-associated species, accounting for >95 % of infections, and thus have been selected for disease surveillance. Monitoring temporal variations in pathogen concentration and diversity excreted from community wastewater allows the early detection of outbreaks. Multiplex real-time/quantitative PCR (qPCR) enables multi-target quantification of pathogens in various types of samples including wastewater. Also, an internal amplification control (IAC) is required for each sample when adopting PCR-based methods for pathogen detection and quantification in wastewater to exclude the inhibition of the wastewater matrix. To achieve reliable quantification of C. jejuni and C. coli towards wastewater samples, this study developed and optimized a triplex qPCR assay by combining three qPCR primer-probe sets targeting Campylobacter jejuni subsp. jejuni, Campylobacter coli, and Campylobacter sputorum biovar sputorum (C. sputorum), respectively. This triplex qPCR assay not only can directly and simultaneously detect the concentration of C. jejuni and C. coli in wastewater but also can achieve the PCR inhibition control using C. sputorum primer-probe set. This is the first developed triplex qPCR assay with IAC for C. jejuni and C. coli, to be used in the wastewater-based epidemiology (WBE) applications. The optimized triplex qPCR assay enables the detection limit of the assay (ALOD100%) and wastewater (PLOD80%) as 10 gene copy/μL and 2 log10 cells/mL (2 gene copies/μL of extracted DNA), respectively. The application of this triplex qPCR to 52 real raw wastewater samples from 13 wastewater treatment plants demonstrated its potential as a high-throughput and economically viable tool for the long-term monitoring of C. jejuni and C. coli prevalence in communities and the surrounding environments. This study provided an accessible methodology and a solid foundation for WBE-based monitoring of Campylobacter spp. relevant diseases and paved the road for future WBE back-estimation of C. jejuni and C. coli prevalence.

Towards understanding host–pathogen dynamics of cetacean poxvirus: attainable approach through the application of a repetitive non-invasive skin sampling in bottlenose dolphins (Tursiops truncatus) under human care

Nowadays, zoos and aquariums, along with the constant advancement of sociocultural moral values, are proactively committed to ensuring and safeguarding cetacean health standards. This entails developing new approaches to health assessments by embracing minimally invasive sampling methods and enhanced animal handling and management, among other aspects. Hence, in the present survey, to appraise skin diseases, the implementation of cytology cell samplers as a non-invasive skin sampling device on 18 bottlenose dolphins housed in two facilities in the Canary Islands during the months of April, October, and December 2019 was performed to isolate cetacean poxvirus in tattoo-like lesions through a real-time PCR-based method using the DNA polymerase gene. Samples were repeatedly collected over time from eleven tattoo-like lesions and from apparently healthy skin to serve as a control for all study animals. From a total of 55 skin samples, detection of the poxvirus was attained in 31 (56.36%); specifically, on 20 of 21 samples collected from tattoo-like lesions (95.23%) and on 11 of 34 samples acquired from apparently healthy skin (32.35%). Correspondingly, the current study constitutes the first report of the isolation of cetacean poxvirus in skin samples without macroscopical signs of tattoo lesions in cetaceans. Likewise, ten of the eleven dolphins that showed tattoo lesions housed in Facility 1 were positive for tattoo skin disease, while four dolphins held in Facility 2 were positive for cetacean poxvirus without ever showing clinical evidence of the disease. This raises the question of whether this pathogen can produce latent infections and whether progression of the disease may depend on environmental stimuli, viral load, or the good health/immunological status of individual animals. Accordingly, further scientific research on cetaceans under human care could provide the knowledge, skills, and resources to understand the host–pathogen dynamics of cetacean poxviruses and their effect on cetaceans’ health.

Miniaturized Rapid Electrochemical Immunosensor Based on Screen Printed Carbon Electrodes for Mycobacterium tuberculosis Detection.

In 2019, over 21% of an estimated 10 million new tuberculosis (TB) patients were either not diagnosed at all or diagnosed without being reported to public health authorities. It is therefore critical to develop newer and more rapid and effective point-of-care diagnostic tools to combat the global TB epidemic. PCR-based diagnostic methods such as Xpert MTB/RIF are quicker than conventional techniques, but their applicability is restricted by the need for specialized laboratory equipment and the substantial cost of scaling-up in low- and middle-income countries where the burden of TB is high. Meanwhile, loop-mediated isothermal amplification (LAMP) amplifies nucleic acids under isothermal conditions with a high efficiency, helps in the early detection and identification of infectious diseases, and can be performed without the need for sophisticated thermocycling equipment. In the present study, the LAMP assay was integrated with screen-printed carbon electrodes and a commercial potentiostat for real time cyclic voltammetry analysis (named as the LAMP-Electrochemical (EC) assay). The LAMP-EC assay was found to be highly specific to TB-causing bacteria and capable of detecting even a single copy of the Mycobacterium tuberculosis (Mtb) IS6110 DNA sequence. Overall, the LAMP-EC test developed and evaluated in the present study shows promise to become a cost-effective tool for rapid and effective diagnosis of TB.

DNA Polymerases for Whole Genome Amplification: Considerations and Future Directions.

In the same way that specialized DNA polymerases (DNAPs) replicate cellular and viral genomes, only a handful of dedicated proteins from various natural origins as well as engineered versions are appropriate for competent exponential amplification of whole genomes and metagenomes (WGA). Different applications have led to the development of diverse protocols, based on various DNAPs. Isothermal WGA is currently widely used due to the high performance of Φ29 DNA polymerase, but PCR-based methods are also available and can provide competent amplification of certain samples. Replication fidelity and processivity must be considered when selecting a suitable enzyme for WGA. However, other properties, such as thermostability, capacity to couple replication, and double helix unwinding, or the ability to maintain DNA replication opposite to damaged bases, are also very relevant for some applications. In this review, we provide an overview of the different properties of DNAPs widely used in WGA and discuss their limitations and future research directions.

Microsatellite instability in non-endometrioid ovarian epithelial tumors: a study of 400 cases comparing immunohistochemistry, PCR, and NGS based testing with mutation status of MMR genes

Testing of microsatellite instability is not only used as a triage for possible Lynch syndrome, but also to predict immunotherapy treatment response. The aim of this study was to assess the frequency of mismatch repair deficiency (MMR-D) / microsatellite instability (MSI) in 400 cases of non-endometrioid ovarian tumors (high-grade serous, low-grade serous, mucinous and clear cell), to compare different methodological approaches of testing, and to assess the optimal approach for next generation sequencing (NGS) MSI testing. For all tumors, we evaluated immunohistochemical (IHC) expression of MMR proteins and assessed microsatellite markers by PCR-based method. Except for high-grade serous carcinoma, we correlated the findings of IHC and PCR with NGS-based MSI testing. We compared the results with somatic and germline mutation in MMR genes. Among the whole cohort, seven MMR-D cases, all clear cell carcinomas (CCC), were found. On PCR analysis, six cases were MSI-high and one was MSS. In all cases, mutation of an MMR gene was found; in two cases, the mutation was germline (Lynch syndrome). An additional five cases with a mutation in MMR gene(s) with MSS status and without MMR-D were identified. We further utilized sequence capture NGS for MSI testing. Employing 53 microsatellite loci provided high sensitivity and specificity. Our study shows that MSI occurs in 7% of CCC while it is rare or absent in other non-endometrioid ovarian neoplasms. Lynch syndrome was present in 2% of patients with CCC. However, some cases with MSH6 mutation can evade all testing methods, including IHC, PCR, and NGS-MSI.

Rapid Bacteriophage Quantification by Digital Biosensing on a SlipChip Microfluidic Device.

The rise of antimicrobial resistance (AMR) is a major global public health concern, and it is urgent to develop new antimicrobial drugs and alternative therapies. There has been growing interest in the use of phage therapy as an alternative to treat AMR, and it has shown promising results in early studies and clinical trials. Phage quantification is a crucial step in the development and application of phage therapy. The traditional double-layer plaque assay requires cumbersome manual operations and typically takes up to 18 h to yield a rough phage estimation. Spectrophotometry, flow cytometry, and PCR-based methods cannot distinguish between infectious and noninfectious phages. Here, we developed a digital biosensing method for rapid bacteriophage quantification on a digital phage SlipChip (dp-SlipChip) microfluidic device containing 2304 microdroplets in 3 nL. By compartmentalizing the phages and bacteria in nanoliter droplets and analyzing the growth profile of bacteria at 3 h, the number of infectious phages can be precisely quantified. The results from the dp-SlipChip were consistent with the traditional double-layer plaque assay method and exhibited higher consistency and repeatability. The dp-SlipChip does not require a complex fluidic handling instrument to generate and manipulate droplets. This SlipChip-based digital biosensing method not only provides a promising tool for rapid phage quantification, which is important for the use of phages in clinical practice to treat antimicrobial-resistant bacteria, but can also be used as an ultrasensitive, high-specificity method to detect bacteria. Furthermore, this approach can be applied to other digital biology studies that require analysis at the single-object level.

Trypanosomatids in Bloodsucking Diptera Insects (Ceratopogonidae and Simuliidae) Wild-Caught at Raptor Bird Nests in Temperate Forests

Trypanosomatids are a huge group of vertebrate, invertebrate and plant parasites that can cause severe illnesses in hosts. Although avian trypanosomes are widely spread throughout the world, information about their transmission and vector–host relationships is still scarce. This study aimed to investigate Trypanosoma prevalence in bloodsucking Diptera collected at tree-nesting raptor birds’ nests. Insects were collected in temperate forests of Eastern-Central Europe, in Lithuania, using UV light traps hung near common buzzard (Buteo buteo) and lesser spotted eagle (Clanga pomarina) nests at about 15 m height above the ground. A total of 1248 Culicoides (Ceratopogonidae) females and 3 blackflies (Simuliidae) were collected and tested for the presence of trypanosomatids using PCR-based methods. The blood of 85 nestlings, belonging to three different species (Buteo buteo, Clanga pomarina and Haliaeetus albicilla) was collected and tested using the same methods. We found that 11.1% of the investigated insects (one Simulium female and Culicoides biting midges belonging to five species) were infected with Trypanosoma parasites (Trypanosoma sp., T. bennetti group, T. avium) and monoxenous trypanosomatids (Crithidia sp., Obscuromonas sp.). Only one common buzzard nestling was found to be infected with Trypanosoma avium. The phylogenetic relationships of detected parasites were determined. Our findings supplement information on the ornithophilic behavior of Culicoides females, potential vector species of avian Trypanosoma, and produce some new information on the detection of monoxenous trypanosomatids (Crithidia sp. and Obscuromonas sp.) in Culicoides.

Candidatus Rickettsia Vini DNA in Ticks Collected from Nest Burrows of the European Sand Martin (Riparia riparia) in Sweden.

Background: Birds can cross geographical and environmental barriers and thereby facilitate dispersal of tick-borne pathogens both as carriers of infected ticks and as reservoirs of pathogenic microorganisms. Ixodes lividus (Ixodida: Ixodidae) is an endophilic tick in the Palearctic region that is highly specialized on its host, the European sand martin Riparia riparia. The purpose of this study was to determine whether I. lividus ticks sampled from sand martin nests in Sweden carry vector-borne pathogens. Materials and Methods: Fed ticks were collected in the autumns of 2017 and 2019 from the nests of a European sand martin colony in southern Sweden. Ticks were identified morphologically to developmental stage and species and were tested for tick-borne pathogens using PCR-based methods. Results: None of the 41 ticks tested positive for five tick-borne pathogens including Borrelia spp., tick-borne encephalitis virus (TBEV), Neoehrlichia mikurensis, Anaplasma phagocytophilum, and Babesia spp. Thirty-seven (13 females, 23 nymphs and 1 larva) of the 41 ticks tested positive for the gltA gene of Rickettsia spp. The sequences of the 17 kDa and gltA genes were most closely related to Candidatus Rickettsia vini. Conclusion: Our study confirms other reports that I. lividus ticks associated with the European sand martin have high infection prevalence of Ca. R. vini.

Diagnosis of Soybean Diseases Caused by Fungal and Oomycete Pathogens: Existing Methods and New Developments

Soybean (Glycine max) acreage is increasing dramatically, together with the use of soybean as a source of vegetable protein and oil. However, soybean production is affected by several diseases, especially diseases caused by fungal seed-borne pathogens. As infected seeds often appear symptomless, diagnosis by applying accurate detection techniques is essential to prevent propagation of pathogens. Seed incubation on culture media is the traditional method to detect such pathogens. This method is simple, but fungi have to develop axenically and expert mycologists are required for species identification. Even experts may not be able to provide reliable type level identification because of close similarities between species. Other pathogens are soil-borne. Here, traditional methods for detection and identification pose even greater problems. Recently, molecular methods, based on analyzing DNA, have been developed for sensitive and specific identification. Here, we provide an overview of available molecular assays to identify species of the genera Diaporthe, Sclerotinia, Colletotrichum, Fusarium, Cercospora, Septoria, Macrophomina, Phialophora, Rhizoctonia, Phakopsora, Phytophthora, and Pythium, causing soybean diseases. We also describe the basic steps in establishing PCR-based detection methods, and we discuss potentials and challenges in using such assays.

Methodological comparison of bronchoalveolar lavage fluid-based detection of respiratory pathogens in diagnosis of bacterium/fungus-associated pneumonia in critically ill patients.

Bacterium/fungus-associated pneumonia (BAP/FAP) is the prominent cause of high mortality and morbidity with important clinical impacts globally. Effective diagnostic methods and proper specimen types hopefully facilitate early diagnosis of pneumonia and prevent spread of drug-resistant bacteria/fungi among critically ill patients. In the present study, 342 bronchoalveolar lavage fluid (BALF) samples were collected from critically ill patients with pulmonary infections between November 2020 and March 2021. The BALF materials were comparatively employed to screen BAP/FAP through microscopy, culture, antigenic marker and PCR-based methods. The limit of detection (LOD) of cultures and PCR for bacteria/fungi was determined by serial dilution assays. Specimen slides were prepared with Gram staining for microscopic examinations. Microbial cultures and identifications underwent routine clinical protocols with the aid of mass spectrometry. (1,3)-β-D-glucan and galactomannan tests with BALF were carried out accordingly. Direct detection of pathogens in BALF was achieved through PCR, followed by sequencing and BLAST in GenBank database for pathogenic identification. The subjects' demographic and clinical characteristics were well evaluated. BAP/FAP was identified in approximately 47% of the subjects by the BALF-based PCR. The PCR-based diagnostic methods showed improved detection performance for fungi with good LOD, but performed similarly for bacteria, when compared to the cultures. There was poor agreement among traditional microscopy, culture and PCR assays for bacterial detections (kappa value, 0.184 to 0.277). For overall bacterial/fungal detections, the microscopy showed the lowest detecting rate, followed by the cultures, which displayed a slightly higher sensitivity than the microscopy did. The sensitivity of PCR was much higher than that of the other means of interest. However, the traditional cultures rather than antigenic marker-based approaches were moderately consistent with the PCR-based methods in fungal species identification, particularly for Candida and Aspergillus spp. Our findings further revealed that the age, length of hospital stay, invasive procedures and cerebral diseases were likely considered as main risk factors for BAP/FAP. Screening for BALF in critically ill patients with suspected pneumonia pertaining high risk factors using combined PCR-based molecular detection strategies would hopefully contribute to early diagnosis of BAP/FAP and improved prognosis of the patients.

Host Cells of Leucocytozoon (Haemosporida, Leucocytozoidae) Gametocytes, with Remarks on the Phylogenetic Importance of This Character.

Leucocytozoon parasites remain poorly investigated in comparison to other haemosporidians. The host cell inhabited by their blood stages (gametocytes) remains insufficiently known. This study aimed to determine the blood cells inhabited by Leucocytozoon gametocytes in different species of Passeriformes and to test if this feature has a phylogenetic importance. We microscopically analyzed blood films stained with Giemsa from six different bird species and individuals and used PCR-based methods for parasite lineage identification. The DNA sequences obtained were applied for phylogenetic analysis. Leucocytozoon parasite from the song thrush Turdus philomelos (cytochrome b lineage STUR1), the blackbird Turdus merula (undetermined lineage), the garden warbler Sylvia borin (unknown lineage) inhabited erythrocytes, a parasite from the blue tit Cyanistes caeruleus (PARUS4) infects lymphocytes, while in the wood warbler Phylloscopus sibilatrix (WW6) and the common chiffchaff Phylloscopus collybita (AFR205) they were found inhabiting thrombocytes. Parasites infecting thrombocytes were closely related, while the parasites infecting erythrocytes were placed in three different clades, and the one found in lymphocytes was placed in a separate clade. This shows that the determination of host cells inhabited by Leucocytozoon parasites can be phylogenetically important and should be considered in future species descriptions. Noteworthy, phylogenetic analysis might be used for the prediction of which host cells parasite lineages might inhabit.

A systematic survey on prevalence of grapevine trunk disease pathogens in Oregon vineyards.

Grapevine trunk diseases (GTDs) are found in vineyards worldwide and can be caused by different fungal pathogens. To characterize types of GTDs in Oregon vineyards, and how the GTD pathogens' prevalence is affected by two geographical regions, a survey was conducted in which grapevine trunk samples were collected from 15 and 14 wine grape (Vitis vinifera) vineyards in southern and northern Oregon respectively. Fungal species were identified through culture and PCR based methods. GTD pathogens that were identified included Botryosphaeriaceae spp. and Phaeoacremonium spp. from 72 and 21% of the surveyed vineyards respectively; Phaeomoniella chlamydospora, Cryptovalsa ampelina, Truncatella angustata, Seimatosporium lichenicola, Hormonema viticola from 7% of the surveyed vineyards; and Dactylonectria macrodidyma, and Pestaloptiopsis sp. from 3% of the surveyed vineyards. Pathogens were identified in both regions and in young and mature vineyards. The presence of GTD from the Botryosphaeria dieback complex was significantly affected by regions (P=0.021) with pathogens being significantly more abundant in Willamette Valley (northern region) compared to Rogue Valley (southern region) vineyards. Some differences among other tested variables such as vineyard age, cultivars, rootstocks, and pruning methods were observed for all disease complexes; however the differences were not statistically significant. Our study summarizes that Botryosphaeria dieback and Esca disease complexes are the most prevalent diseases infecting grapevines in Oregon vineyards and management practices need to be geared towards these economically important diseases. In addition, pathogens from other disease complexes are also present suggesting a need for regular disease monitoring and following practices to limit the spread of these pathogens.

Site-specific analysis of ribosomal 2'O-methylation by quantitative reverse transcription PCR under low deoxynucleotide triphosphate concentrations.

Ribose 2'O-methylation (Nm, ribomethylation) is the most abundant RNA modification present in rRNA. It has been shown that alterations in ribosomal 2'O-methylation at individual Nm sites likely reflect regulated cellular processes. Although several analytical approaches for Nm detection and profiling have been developed, a simple and affordable method for the screening and measurement of individual Nm sites in large numbers of tissue samples is required to examine their potential for clinical translation. Here, we describe a new quantitative reverse transcription PCR-based method that can sensitively assess ribomethylation levels at specific rRNA sites at single-nucleotide resolution in low input amounts of total RNA.

Optimized Workflow for Whole Genome and Transcriptome Next‐Generation Sequencing of Single Cells or Limited Nucleic Acid Samples

Whole genome and whole transcriptome sequencing require orders of magnitude more of starting nucleic acid than what is found in single cells or other extremely limited samples. High fidelity amplification of this minute amount of nucleic acids is essential to overcome the limitations caused by the low input, degradation and contamination, and to ensure a sufficient amount of DNA for preparation of high complex and high quality next-generation sequencing (NGS) libraries. Recent technical advances in multiple displacement amplification (MDA) enable studies of rare cell types, heterogeneity of body fluids, tissues, environmental samples, and organisms that cannot be cultured. Several strategies for amplification of limiting amounts of nucleic acid have been described, with PCR being popular. However, PCR-based methods result in high error rates, lower library complexity, and lower coverage uniformity. In this article, a HiFi MDA is used to accurately amplify the limited material and to allow library preparation starting from high input, while reducing PCR cycling to achieve sufficient library yields. This article describes a complete workflow from cells and small quantities of DNA or RNA to NGS libraries for Illumina sequencing instruments. © 2023 QIAGEN GmbH. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Whole genome amplification from single cells Support Protocol 1: PicoGreen™ quantification of MDA amplified DNA Support Protocol 2: Purification of amplified DNA after MDA Basic Protocol 2: Whole transcriptome amplification from single cells Alternate Protocol: Whole transcriptome amplification from purified RNA Basic Protocol 3: Enrichment of complete small genomes using target-specific primers in MDA Basic Protocol 4: Complete viral RNA amplification using target-specific primers in MDA Basic Protocol 5: Enzymatic fragmentation and adapter ligation of MDA amplified material Basic Protocol 6: Normalization of library concentration using magnetic beads.

A Simple and Affordable Method to Create Nonsense Mutation Clones of p53 for Studying the Premature Termination Codon Readthrough Activity of PTC124.

(1) Background: A premature termination codon (PTC) can be induced by a type of point mutation known as a nonsense mutation, which occurs within the coding region. Approximately 3.8% of human cancer patients have nonsense mutations of p53. However, the non-aminoglycoside drug PTC124 has shown potential to promote PTC readthrough and rescue full-length proteins. The COSMIC database contains 201 types of p53 nonsense mutations in cancers. We built a simple and affordable method to create different nonsense mutation clones of p53 for the study of the PTC readthrough activity of PTC124. (2) Methods: A modified inverse PCR-based site-directed mutagenesis method was used to clone the four nonsense mutations of p53, including W91X, S94X, R306X, and R342X. Each clone was transfected into p53 null H1299 cells and then treated with 50 μM of PTC124. (3) Results: PTC124 induced p53 re-expression in H1299-R306X and H1299-R342X clones but not in H1299-W91X and H1299-S94X clones. (4) Conclusions: Our data showed that PTC124 more effectively rescued the C-terminal of p53 nonsense mutations than the N-terminal of p53 nonsense mutations. We introduced a fast and low-cost site-directed mutagenesis method to clone the different nonsense mutations of p53 for drug screening.

Analysis of Italian isolates of Pantoea stewartii subsp. stewartii and development of a real-time PCR-based diagnostic method.

Pantoea stewartii subsp. stewartii (Pss) causes Stewart's vascular wilt of maize, and it is responsible for serious crop losses. Pss is indigenous to North America and spreads with maize seeds. The presence of Pss has been notified in Italy since 2015. The risk assessment of the entry of Pss in the EU from the United States through seed trade is in the order of magnitude of hundred introductions per year. Several molecular or serological tests were developed for the detection of Pss and used as official analysis for the certification of commercial seeds. However, some of these tests lack adequate specificity, not allowing to correctly discriminate Pss from P. stewartii subsp. indologenes (Psi). Psi is occasionally present in maize seeds and is avirulent for maize. In this study, several Italian isolates of Pss recovered in 2015 and 2018 have been characterized by molecular, biochemical, and pathogenicity tests; moreover, their genomes have been assembled through MinION and Illumina-sequencing procedures. Genomic analysis reveals multiple introgression events. Exploiting these results, a new primer combination has been defined and verified by real-time PCR, allowing the development of a specific molecular test able to detect the presence of Pss down to the concentration of 103 CFU/ml in spiked samples of maize seed extracts. Due to the high analytical sensitivity and specificity achieved with this test, the detection of Pss has been improved disentangling the inconclusive results in Pss maize seed diagnosis, overcoming its misidentification in place of Psi. Altogether, this test addresses the critical issue associated with maize seeds imported from regions where Stewart's disease is endemic.