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2021 ◽  
Vol 8 ◽  
Author(s):  
Jasmin Nicole Nessler ◽  
Wendy Karen Jo ◽  
Albert D. M. E. Osterhaus ◽  
Martin Ludlow ◽  
Andrea Tipold

Meningoencephalitis of unknown origin (MUO) describes a group of meningoencephalitides in dogs with a hitherto unknown trigger. An infectious agent has been suggested as one possible trigger of MUO but has not been proven so far. A relatively new method to screen for viral RNA or DNA is next-generation sequencing (NGS) or deep sequencing. In this study, a metagenomics analysis of the virome in a sample is analyzed and scanned for known or unknown viruses. We examined fresh-frozen CSF of 6 dogs with MUO via NGS using a modified sequence-independent, single-primer amplification protocol to detect a possible infectious trigger. Analysis of sequencing reads obtained from the six CSF samples showed no evidence of a virus infection. The inability to detect a viral trigger which could be implicated in the development of MUO in the examined population of European dogs, suggests that the current techniques are not sufficiently sensitive to identify a possible virus infection, that the virus is already eliminated at the time-point of disease outbreak, the trigger might be non-infectious or that there is no external trigger responsible for initiating MUO in dogs.


2021 ◽  
Author(s):  
Klaudia Chrzastek ◽  
Holly S Sellers ◽  
Darrell Kapczynski

Background. The Reoviridae family represents the largest family of double-stranded RNA (dsRNA) viruses, and the members have been isolated from a wide range of mammals, birds, reptiles, fishes, insects, plants. Orthoreoviruses, one of the 15 recognized genera in the Reoviridae family, can infect humans and nearly all mammals, and birds. Genomic characterization of reoviruses has not been adopted on a large-scale due to the complexity of obtaining sequences for all 10 segments. Results. In this study, we developed a time-efficient, and practical method to enrich reovirus sequencing reads from isolates that allowed for full genome recovery using single-primer amplification method coupled with next generation sequencing. We refer to this protocol as reovirus-Single Primer Amplification (R-SPA). Our results demonstrated that most of the genes were covered with at least 500 reads per base space. Furthermore, R-SPA covered both 5' and 3' end of each reovirus genes. Conclusion. A universal and fast amplification protocol that yields double-stranded cDNA in sufficient abundance and facilitates and expedites the whole genome sequencing of reoviruses was presented in this study.


Author(s):  
Ahmed Babiker ◽  
Katherine Immergluck ◽  
Samuel D. Stampfer ◽  
Anuradha Rao ◽  
Leda Bassit ◽  
...  

To provide an accessible and inexpensive method to surveil for SARS-CoV-2 mutations, we developed a multiplex real-time RT-PCR (the Spike SNP assay) to detect specific mutations in the spike receptor binding domain. A single primer pair was designed to amplify a 348 bp region of spike , and probes were initially designed to detect K417, E484K, and N501Y. The assay was evaluated using characterized variant sample pools and residual nasopharyngeal samples. Variant calls were confirmed by SARS-CoV-2 genome sequencing in a subset of samples. Subsequently, a fourth probe was designed to detect L452R. The lower limit of 95% detection was 2.46 to 2.48 log 10 GE/mL for the three initial targets (∼1-2 GE/reaction). Among 253 residual nasopharyngeal swabs with detectable SARS-CoV-2 RNA, the Spike SNP assay was positive in 238 (94.1%) samples. All 220 samples with Ct values < 30 for the SARS-CoV-2 N2 target were detected, whereas 18/33 samples with N2 Ct values ≥ 30 were detected. Spike SNP results were confirmed by sequencing in 50/50 samples (100%). Addition of the 452R probe did not affect performance for the original targets. The Spike SNP assay accurately identifies SARS-CoV-2 mutations in receptor binding domain, and it can be quickly modified to detect new mutations that emerge.


mSphere ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Jacquelynn Benjamino ◽  
Benjamin Leopold ◽  
Daniel Phillips ◽  
Mark D. Adams

ABSTRACT Current sequencing-based methods for profiling microbial communities rely on marker gene (e.g., 16S rRNA) or metagenome shotgun sequencing (mWGS) analysis. We present an approach based on a single-primer extension reaction using a highly multiplexed oligonucleotide probe pool. This approach, termed MA-GenTA (microbial abundances from genome tagged analysis), enables quantitative, straightforward, cost-effective microbiome profiling that combines desirable features of both 16S rRNA and mWGS strategies. The use of multiple probes per target genome and rigorous probe design criteria enabled robust determination of relative abundance. To test the utility of the MA-GenTA assay, probes were designed for 830 genome sequences representing bacteria present in mouse stool specimens. Comparison of the MA-GenTA data with mWGS data demonstrated excellent correlation down to 0.01% relative abundance and a similar number of organisms detected per sample. Despite the incompleteness of the reference database, nonmetric multidimensional scaling (NMDS) clustering based on the Bray-Curtis dissimilarity metric of sample groups was consistent between MA-GenTA, mWGS, and 16S rRNA data sets. MA-GenTA represents a potentially useful new method for microbiome community profiling based on reference genomes. IMPORTANCE New methods for profiling the microbial communities can create new approaches to understanding the composition and function of those communities. In this study, we combined bacterial genome-specific probe design with a highly multiplexed single primer extension reaction as a new method to profile microbial communities, using stool from various mouse strains as a test case. This method, termed MA-GenTA, was benchmarked against 16S rRNA gene sequencing and metagenome sequencing methods and delivered similar relative abundance and clustering data. Since the probes were generated from reference genomes, MA-GenTA was also able to provide functional pathway data for the stool microbiome in the assayed samples. The method is more informative than 16S rRNA analysis while being less costly than metagenome shotgun sequencing.


2021 ◽  
Vol 5 (1) ◽  
pp. 008-012
Author(s):  
Bahador Davood ◽  
Mohammadi Ashraf ◽  
Foroughi Abolhasan ◽  
Alirezaie Behnam

The sequence-independent, single-primer amplification (SISPA) enables the random amplification of nucleic acids, allowing the detection and genome sequencing of different viral agents. This feature of SISPA method provides evidence for application of it in monitoring the presence of adventitious RNA viruses in cell cultures. We evaluated SISPA method for the detection of a challenge RNA virus representing adventitious agent in cell cultures. Besides, by optimizing the SISPA method in our laboratory, we found false-positive results on negative control lanes in electrophoresis gels. To investigate the sources of contamination, false-positive results of SISPA were cloned into Escherichia coli cells, sequenced, and phylogenetically analyzed. This data revealed that the SISPA method can be used as an adjunct method to confirm the absence of unexpected adventitious RNA viruses in cell cultures. The phylogenetic analysis of SISPA contaminant sequences showed that the false-positive results were caused by nucleic acid amplification of commercial cDNA synthesis kit reagents, probably tracing back to expression plasmids and host ribosomal sequences, used for the production of enzymes. Therefore, laboratories using random amplification methods must be constantly aware of the potentials of such contaminations, yielding false-positive results and background noise in the final NGS reads.


2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Jiao Chen ◽  
Pansong Zhang ◽  
Haixia Wang ◽  
Yanjing Shi

Abstract Adulteration of beef with cheap chicken has become a growing problem worldwide. In this study, a quick, single primer-triggered isothermal amplification (SAMP) combined with a fast nucleic acid extraction method was employed to detect the chicken meat in adulterated beef. Chicken from adulterated beef was identified using the chicken species-specific primer designed according to the Gallus gallus mitochondrial conserved sequences. Our SAMP method displayed good specificity and sensitivity in detecting chicken and beef meat DNA–the limit of detection (LOD) of SAMP is 0.33 pg/μL of chicken and beef total DNA and 2% w/w chicken meat in beef. The whole work flow from DNA extraction to signal detection can be finished within 1 h, fulfilling the requirement of on-site meat species identification.


North East India is extremely rich in natural resources and is a biodiversity hotspot. Many plant species, including Citrus are known to have originated from this region. Citus macroptera Mont. is a wild, endangered species which have been found to exist naturally in various parts of this region, including Meghalaya. To access the genetic variability among genotypes and their phylogeny, 30 genotypes of wild C. macroptera Mont. were collected from Garo Hills of Meghalaya. Single primer based DNA markers viz. RAPD, ISSR, DAMD were utilized to ascertain genetic diversity. The percentage polymorphic bands for RAPD, ISSR, DAMD were found to be 97.71%, 94.67% and 100% respectively. ISSR showed the highest values for both RP (7.67) and MI (5.03) highlighting its efficacy in determining genetic variations. A concatenated approach, Single Primer Amplification Reaction (SPAR) was also followed to assess their genetic diversity. Dendogram generated from SPAR data showed that the South-West Garo Hills population is the most recently evolved amongst all others while West Garo Hills collections retain an ancestral position in the evolutionary time-frame. Population genetics parametres such as Gene flow (Nm) and the diversity among populations (GST) were found to be 1.9894 and 0.2009 respectively. Gene flow estimates (Nm>1) suggests appreciable gene flow in the populations. AMOVA data further supported this with high percentage of variations (92%) within populations whereas variations among populations were about 8% only. Shannon’s information index (I) values and Nei’s gene diversity (h) varied between 0.303-0.423and 0.201-0.285 respectively. The use of SPAR method yields a clear and concise picture of the underlying genetic variabilities, and a detailed and comprehensive data analysis will help conceive efficient and sustainable conservation strategies for this important plant.


2021 ◽  
Vol 8 (3) ◽  
Author(s):  
Vanessa Smilansky ◽  
Aurelie Chambouvet ◽  
Mari Reeves ◽  
Thomas A. Richards ◽  
David S. Milner

Alveolate protists within the phylum Perkinsea have been found to infect amphibians across a broad taxonomic and geographic range. Phylogenetic analysis has suggested the existence of two clades of amphibian Perkinsea: a putatively non-pathogenic clade linked to asymptomatic infections of tadpoles in Africa, Europe and South America, and a putatively pathogenic clade linked to disease and mass mortality events of tadpoles in North America. Here, we describe the development of a duplex TaqMan qPCR assay to detect and discriminate between rDNA sequences from both clades of Perkinsea in amphibian tissues. The assay uses a single primer pair to target an 18S small subunit (SSU) ribosomal RNA (rRNA) gene region shared between the two clades, and two dual-labelled probes to target a region within this fragment that is diagnostic for each clade. This assay enables rapid screening for each of the two Perkinsea groups, allowing for detection, primarily of the phylogenetic group associated with disease outbreaks, and secondarily for the phylogenetic group with no current disease relationship identified. Incorporation of our novel qPCR assay into the routine surveillance of amphibian populations will allow for the assessment of the incidence of each protist clade, thereby providing an improved understanding of Perkinsea infection pervasiveness and a method to underpin future conservation planning.


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