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55 results for “multiplex PCR”
Development of thrips barcode database and multiplex real-time PCR assay for quarantine and agriculture pest species
<p>Thrips (Order Thysanoptera) species are agriculturally important as plant sap sucking pests and vectors of several plant diseases. They are very small insects and commonly associated with imported commodities at New Zealand border in all life stages. Morphological identification of thrips is mainly performed on adults, but the available identification keys for immature stages do not include many species and are inadequate, thus DNA barcode was regularly used for thrips identification, here, we have generated DNA barcode data for over 29 thrips species from over 100 individuals. At New Zealand border,<em> Frankliniella occidentalis </em>is the dominant species intercepted, followed by <em>F. panamensis</em>, <em>Thrips palmi</em> and <em>T. tabaci </em>and several other thrips species. Hence, we have also developed a multiplex real time PCR assay, targeting the four thrips species to facilitate the identification of quarantine interceptions with more accurate and faster diagnostic method for any developmental stages. The DNA barcode database further assists in thrip identification. The assay showed high specificity for all the four target species and could detect 10 copies/ µL of the target DNA. Linear responses and high correlation coefficients between the amount of DNA and <em>C</em><sub>q</sub> values for each species were also achieved. The method was tested on single egg, larva and adult and proved to be applicable for all life stages of the four species. This study has demonstrated the assay is a useful biosecurity tool for rapid and reliable identification of the target thrips species. </p>
Figure 5 in Multiplex-PCR differentiation of two Hyalomma and two Haemaphysalis species (Acari: Ixodidae)
Figure 5. One percent agarose gel electrophoresis stained with Cyber Safe® showing ITS2 fragments amplified using primer pairs Fanas/Ran for Hyalomma anatolicum (amplicon size 749 bp) and Fanas/Ras for Hy. asiaticum (amplicon size 408 bp) (A), COI fragments amplified using primer pairs Fsul/Rpun and Fsul/Rsul for Haemaphysalis punctata (amplicon size 524 bp) and Ha. sulcata (amplicon size 614 bp) (B). (100 bp DNA ladder).
Figure 2 in Multiplex-PCR differentiation of two Hyalomma and two Haemaphysalis species (Acari: Ixodidae)
Figure 2. The ventral morphological aspect of representative male specimens of Haemaphysalis punctata (A) collected from Mazandaran province and Ha. sulcata (B) collected from Lorestan province; white arrows showing position of character spur of coxa IV (size of specimens was not considered).
Figure 1 in Multiplex-PCR differentiation of two Hyalomma and two Haemaphysalis species (Acari: Ixodidae)
Figure 1. The dorsal morphological aspect of representative male specimens of Hyalomma anatolicum (A) and Hy. asiaticum (B) both collected from Lorestan province; white and red arrows showing position of characters cervical grooves and dorsal posterior margin of the basis capituli, respectively (size of specimens was not considered).
Figure 4 in Multiplex-PCR differentiation of two Hyalomma and two Haemaphysalis species (Acari: Ixodidae)
Figure 4. Fourth female coxal spur of Haemaphysalis sulcata (A) and Ha. punctata (B), both collected from Mazandaran province, Iran.
Figure 3 in Multiplex-PCR differentiation of two Hyalomma and two Haemaphysalis species (Acari: Ixodidae)
Figure 3. General schema of representative female dorsal scutum of Hyalomma anatolicum (A) and Hy. asiaticum (B), both collected from Lorestan province (size of specimens was not considered).
Figure 2 in Molecular identification of Trichinella species by multiplex PCR: new insight for Trichinella murrelli
Figure 2. Electrophoretic profiles of Trichinella murrelli uniplex PCR amplifications.DNA from T. murrelli (isolate code ISS35) reference larvae was used. Lane L = 50 bp ladder. The genes targeted were the Expansion Segment V (ESV, lane 1), Internal Transcribed Spacer 1 II (ITS1 II, Lane 2), ITS1 III (lane 3), ITS2 IV (lane 4), and ITS2 V (lane 5).
Figure 3 in Molecular identification of Trichinella species by multiplex PCR: new insight for Trichinella murrelli
Figure 3. Alignment of the 256 bp fragment of ITS1 II of Trichinella murrelli obtained by uniplex PCR.BLAST analysis revealed 99.6% identity with different clones of T. murrelli, including clone 5 (Accession number KC006421).
Figure 1 in Molecular identification of Trichinella species by multiplex PCR: new insight for Trichinella murrelli
Figure 1. Electrophoretic profiles of Trichinella murrelli and T. britovi larva amplicons after multiplex PCR amplification.DNA extracts from 1 and 10 larvae of T. murrelli (isolate code ISS35) in lane 1 and lanes 2–4, respectively; and of T. britovi (isolate code ISS235) larva in lane 5. Lane L1 = 100 bp ladder.
Fig. 1 in Molecular diagnostic technique for the differentiation of the Formosan subterranean termite, Coptotermes formosanus (Isoptera: Rhinotermitidae) from other subterranean termites by multiplex-PCR
Fig. 1. Ethidium bromide-stained agarose gel (2%) illustrating a common amplicon of 262 bp from the mtDNA 16S gene for various termite species and unique amplicon of 221 bp specific for the Formosan subterranean termite.
Fig. 2. Multiplex PCR gel showing the 716 in Molecular diagnostics of the honey bee parasites Lotmaria passim and Crithidia spp. (Trypanosomatidae) using multiplex PCR
Fig. 2. Multiplex PCR gel showing the 716 to 724 bp amplicon for Lotmaria passim and Crithidia species, the L. passim specific 499 bp amplicon, and the Crithidia specific 245 bp amplicon.
Fig. 1 in Molecular diagnostics of the honey bee parasites Lotmaria passim and Crithidia spp. (Trypanosomatidae) using multiplex PCR
Fig. 1. Bayesian molecular phylogenetic tree showing relationship of 2 Hawaiian Lotmaria passim positive samples relative to other trypanosomes from Gen- Bank for a 608 bp region of the rDNA SSU gene.
Fig. 1 in Molecular identification of Atlantic goliath grouper Epinephelus itajara (Lichtenstein, 1822) (Perciformes: Epinephelidae) and related commercial species applying multiplex PCR
Fig. 1. Phylogrambasedontheamplificationofthesequence of the Cytochrome Oxidase I gene of the commerciallyexploited species of fishes of the families Epinephelidae and Polyprion americanus (Polyprionidae). The samples collected in the present study are underlined and those obtained from the GenBank database appear together with their accession numbers.
Dataset for Multiplex-PCR detection and Nanopore-based genotyping of fish pathogens
<p>This is a revised zip file contains scripts, initial fastq files, assembled amplicon (public and from this study) as well as bioinformatics intermediate files used for this study.</p> <p>Changelog:</p> <p>1. Fixed a bug in the 02_consensus.sh to enable proper removal of amplicons with zero depth</p> <p>2. Added a script (06_unclassified_read.sh) to extract and annotate reads that previously could not align to the 4 reference gene segment. Now the previously unclassified reads will be re-align (raw fastq) back to the gene segments as well as an additional tilapia genome assembly to gauge amount of reads mapping to the host genome. Furthermore, any read that still fail to align with minimap2 was subsequently aligned using blastn (-word_size 15 -evalue 0.01) against the same sequences.</p> <p>File Structure and Descriptions</p> <p>├── 01_process.sh : primer trimming, length-based filtering, read alignment, alignment filtering (unique hit) and extraction of uniquely hit reads for consensus generation<br> ├── 02_consensus.sh : [need artic conda env] Generation of consensus based on uniquely-mapped reads and minimal read depth of 20x required to call a variant (or it will be masked)<br> ├── 03_cleanup.sh: General folder and intermediate file re-organization<br> ├── 04_filter.sh: [need quast conda env] statistic of consensus generated and filtering of consensus with one or more ambiguous base (N), not suitable for haplotype<br> ├── 05_cluster.sh: clustering of consensus based on 100% identity threshold to generate putative haplotype<br> ├── 06_unclassified_read.sh: Extraction and annotation of unclassified reads using lenient criteria and with host reference genome as added reference<br> ├── Amplicon_FastQ folder: uniquely mapped fastq files for consensus generation<br> ├── BAM: alignment files generated from minimap2 used as input for the artic pipeline to identify variants<br> ├── Cluster_Rep.txt: Consensus sequences that were chosen to represent each haplotype<br> ├── Consensus folder: consensus fasta files generated for each sample containing sequences for each specific pathogen<br> ├── Coverage folder: coverage and base-level read depth for each sample and each pathogen reference genes<br> ├── Filter: individual fasta sequences (only 1 sequence per file) for each pathogen and each sample without any ambiguous base for subsequent clustering analysis<br> ├── Full_Haplotype.fasta: all possible haplotype sequences generated for each pathogen<br> ├── Gap_Analysis.tsv: Table with percentage of gap (0-100%) for each consensus sequence generated (used for filtering)<br> ├── Haplotype folder: Intermediate file and sample-level haplotype used to infer final haplotype and generate haplotype summary<br> ├── Haplotype_summary.tsv: Table with sample ID and their respectively pathogen haplotype<br> ├── Minimap2_PAF: Intermediate alignment generated from minimap2 used to generate the count table<br> ├── FailMinimap2 folder: FastQ files that didn't align using minimap2. Will be subsequently aligned using blastN (more sensitive) against the same reference sequences as minimap2<br> ├── Host_4Pathogen.fasta: Fasta file containing the tilapia genome and 4 pathogen (primer binding site included)<br> ├── Original: fastq with original naming prior to renaming based on sampleID. a script (rename.sh) was included to show renaming scheme<br> ├── primer.fasta: Primer sequences used for identifying and trimming reads with flanking primer sequence<br> ├── primer.fasta.fai: the index file for primer.fasta<br> ├── PrimerTrim folder: Primer-trimmed reads<br> ├── quast_results: consensus statistics generated by quast<br> ├── RawCount.tsv: Count table generated that can used as a input to generate figure<br> ├── RawFastq folder: Raw reads that have been renamed to reflect sample information<br> ├── readme.md: The current readme file<br> ├── ref_full_latest.fasta: Reference sequence of (gene segments) 4 pathogens e.g. TilV, ISKNV, SAG (Streptococcus agalactiae), FNO (Francisella noatunensis subsp. orientalis)<br> ├── ref_full_latest.primer.fasta: Same as above but with their primer binding sequence trimmed similar to the processed reads<br> ├── ref_full_latest.primer.fasta.fai<br> ├── RenameHaplotype: Script to perform reorganization of cdhit output<br> ├── Seq.stat.tsv: Sequencing statistics<br> ├── Uniq_PAF: Minimap2 alignment file for raw reads that initially failed quality check (no primer present and/or less than 80% query coverage / not unique alignment)<br> ├── Unmap: Raw reads that initial failed quality check (no primer on both ends / less than 80% query coverage / not unique alignment) <br> └── VCF: VCF files from medaka variant calling used to generate the final consensus</p>
Multiplexing PCR allows the identification of within-species genetic diversity in ancient eDNA
<p><span>Sedimentary ancient DNA (<em>seda</em>DNA) has rarely been used to obtain population-level data due to either a lack of taxonomic resolution for the molecular method used, limitations in the reference material or inefficient methods. Here, we present the potential of multiplexing different PCR primers to retrieve population-level genetic data from <em>seda</em>DNA samples. <em>Vaccinium</em> <em>uliginosum</em> (Ericaceae) is a widespread species with a circumpolar distribution and three lineages for present-day populations. We searched 18 plastid genomes for intraspecific variable regions and developed 61 primers to target these. Initial multiplex PCR testing resulted in a final set of 38 primers. These primers were used to analyse 20 lake <em>seda</em>DNA samples (11,200 cal. yr BP to present) from five different localities in northern Norway, the Alps and the Polar Urals. All known V<em>. uliginosum</em> lineages in these regions and all primers could be recovered from the <em>seda</em>DNA data, where for each sample 28.1 primers containing 34.15 variant sequences were obtained on average. All sediment samples were dominated by a single lineage, except three alpine samples which had co-occurrence of two different lineages. Furthermore, lineage turnover was observed in the Alps and northern Norway, suggesting that present-day phylogeographical studies may overlook past genetic patterns. Multiplexing primers is a promising tool for generating population-level genetic information from <em>seda</em>DNA. The relatively simple method, combined with high sensitivity, provides a scalable method that will allow researchers to track populations through time and space using environmental DNA.</span></p>
Efficacy of a Multiplex BangasureTM RT-PCR Kit for the Detection of COVID-19
ClinicalTrials.gov study NCT05190016. IPD Sharing: NO. Countries: 1. Publications: 14.
Improvements Through the Use of a Rapid Multiplex PCR Enteric Pathogen Detection Kit in Children With Hematochezia
ClinicalTrials.gov study NCT03362970. IPD Sharing: NO. Countries: 1. Publications: 5.
Multiplexing PCR allows the identification of within-species genetic diversity in ancient eDNA
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Data from: Multiplex preamplification PCR and microsatellite validation allows accurate single nucleotide polymorphism (SNP) genotyping of historical fish scales
Incorporating historical tissues into the study of ecological, conservation, and management questions can broaden the scope of population genetic research by enhancing our understanding of evolutionary processes and anthropogenic influences on natural populations. Genotyping historical and low-quality samples has been plagued by challenges associated with low amounts of template DNA and the potential for preexisting DNA contamination among samples. We describe a two-step process designed to (i) accurately genotype large numbers of historical low-quality scale samples in a high-throughput format and (ii) screen samples for preexisting DNA contamination. First, we describe how an efficient multiplex preamplification PCR of 45 single nucleotide polymorphisms (SNPs) can generate highly accurate genotypes with low failure and error rates in subsequent SNP genotyping reactions of individual historical scales from sockeye salmon (Oncorhynchus nerka). Second, we demonstrate how the method can be modified for the amplification of microsatellite loci to detect preexisting DNA contamination. A total of 760 individual historical scale and 182 contemporary fin clip samples were genotyped and screened for contamination. Genotyping failure and error rates were exceedingly low and similar for both historical and contemporary samples. Preexisting contamination in 21% of the historical samples was successfully identified by screening the amplified microsatellite loci. The potential for automation, low failure and error rates, and ability to multiplex both the preamplification and subsequent genotyping reactions combine to make the protocol ideally suited for efficiently genotyping large numbers of potentially contaminated low-quality sources of DNA.
FIGURE 2 in A multiplex PCR method for identification of two common true cutworm species (Lepidoptera: Noctuidae) tested in the central plain of Guilan province, Iran
FIGURE 2. Multiplex PCR by combination of three equimolar primers for COI (IpsiloF1/ XestiaF1/LepR1). 1: 100 bp DNA ladder; 2,3: Agrotis ipsilon; 4,5: Xestia c-nigrum, 6–9, Agrotis ipsilon. 6,7: larvae specimens collected from Chaf (Langroud), 8,9: larvae specimens collected from Rasht.
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