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Veterinary Sciences Dec 2023PCR is the most effective method for detecting difficult-to-cultivate pathogens and pathogens that are part of mixed infections in animals, such as , which causes bird...
PCR is the most effective method for detecting difficult-to-cultivate pathogens and pathogens that are part of mixed infections in animals, such as , which causes bird ornithobacteriosis, or , which causes infectious coryza. In this work, we developed and validated two efficient and sensitive diagnostic assays for the rapid and accurate detection of and DNA in bacterial isolates and clinical samples using real-time PCR with TaqMan-like probes. When designing the PCR assays, we performed in silico analysis, optimized DNA isolation methods and PCR conditions, and assessed the analytical and diagnostic performance of PCR. We designed primers and probes that have no mismatches with published whole-genome sequences of bacteria. The optimization of conditions showed that the PCR assays are sufficiently robust to changes in temperature and oligonucleotide concentration. The validation showed that the developed assays have high analytical and diagnostic sensitivity and specificity. These assays are expected to improve the differential diagnosis of respiratory diseases in chickens and turkeys.
PubMed: 38250913
DOI: 10.3390/vetsci11010007 -
Frontiers in Cellular and Infection... 2023Female sand flies are hematophagous, feeding on animals and in the process serve as vectors for , the parasites that cause leishmaniasis in humans. Leishmaniasis are a...
INTRODUCTION
Female sand flies are hematophagous, feeding on animals and in the process serve as vectors for , the parasites that cause leishmaniasis in humans. Leishmaniasis are a group of parasitic neglected tropical diseases in 98 countries including Nigeria and kills ~60,000 people/year. In Nigeria, Sokoto State is endemic to leishmaniasis but there is a knowledge gap on the identity of the prevalent sand flies and the species they transmit. Hence, this cross-sectional study was designed to take inventory of the species of sand flies in Sokoto using genetic methods.
METHODS
1,260 (310 females) sand flies were collected from three Local Government Areas (L.G.A) of Sokoto State- Wamakko, Sokoto South and Kware. Genomic DNA was extracted from each fly and DNA amplification by polymerase chain reaction (PCR) was carried out on the DNA samples using primers targeting the arthropods mitochondrial cytochrome oxidase subunit 1 () gene, and nested PCR with primers targeting the gene for internal transcribed spacer-1 () of ribosomal RNA . The PCR products were sequenced.
RESULTS
Gene sequence analysis revealed five species of sand flies belonging to the old-world genera namely and . The identified species were (6.45%), (6.45%), (9.7%), (9.7%), (67.7%). Within the sampling period, sand flies were most abundant in the rainy months of August (104/33.5%) and September (116/37.4%) with all the five identified species occurring. Sequence analysis of gene identified in two sand flies (2/310)- (from Sokoto South) and (from Wamakko). BLAST search in NCBI and phylogenetic analysis revealed that the sand fly species are related to the species reported in different parts of Africa, while the is identical to strain reported in Brazil (KY379083.1).
DISCUSSION
and four species belonging to the genus are the most prevalent sand flies in Sokoto State, Nigeria and they harbor solely. The results shed light on why visceral leishmaniasis is the most predominant form of the disease. Therefore, we recommend that adequate care for dogs must be instituted as dogs are the major animal reservoir for .
Topics: Humans; Female; Animals; Dogs; Psychodidae; Nigeria; Cross-Sectional Studies; Phylogeny; Phlebotomus; Leishmania infantum
PubMed: 37719668
DOI: 10.3389/fcimb.2023.1219629 -
Nature Nanotechnology Aug 2023DNA has emerged as an attractive medium for archival data storage due to its durability and high information density. Scalable parallel random access to information is a...
DNA has emerged as an attractive medium for archival data storage due to its durability and high information density. Scalable parallel random access to information is a desirable property of any storage system. For DNA-based storage systems, however, this still needs to be robustly established. Here we report on a thermoconfined polymerase chain reaction, which enables multiplexed, repeated random access to compartmentalized DNA files. The strategy is based on localizing biotin-functionalized oligonucleotides inside thermoresponsive, semipermeable microcapsules. At low temperatures, microcapsules are permeable to enzymes, primers and amplified products, whereas at high temperatures, membrane collapse prevents molecular crosstalk during amplification. Our data show that the platform outperforms non-compartmentalized DNA storage compared with repeated random access and reduces amplification bias tenfold during multiplex polymerase chain reaction. Using fluorescent sorting, we also demonstrate sample pooling and data retrieval by microcapsule barcoding. Therefore, the thermoresponsive microcapsule technology offers a scalable, sequence-agnostic approach for repeated random access to archival DNA files.
Topics: Capsules; DNA; Information Storage and Retrieval; Oligonucleotides; High-Throughput Nucleotide Sequencing
PubMed: 37142708
DOI: 10.1038/s41565-023-01377-4 -
Journal of Biological Methods 2023Over the last decades, PCR and molecular cloning have profoundly impacted various biological areas, from basic to pharmaceutical sciences. Presented in this study is a...
Over the last decades, PCR and molecular cloning have profoundly impacted various biological areas, from basic to pharmaceutical sciences. Presented in this study is a simple and step-by-step protocol that uses PCR to recover a poor-quality ligase product. In fact, a classic step that can be problematic in typical recombinant DNA manipulations can be the recovery of a product from a T4 DNA ligase reaction between two or more suitably prepared DNA fragments (sticky ends, blunt ends, TA cloning, etc.). This reaction can result in poor yields of the ligation product, due to various causes, mainly the preparation of the DNA fragments, and the poor yield can severely invalidate all subsequent steps. To overcome this problem, we designed a pair of PCR primers to amplify the entire ligase product into satisfactory amount. Of course, high-fidelity DNA polymerase must be used to obtain a faithful copy of the DNA of interest. The fragment thus amplified can then be inserted into a suitable vector and propagated by bacterial transformation. We applied this procedure to modify a synthetic gene by adding a His-Tag to its 5' end, and to insert this new construct into an expression cassette. This last step was achieved by employing a PCR cloning system. In our practical example, comprehensive PCR-based protocol with important tips were introduced. This methodological paper can serve as a roadmap for biologists who want to quickly/fully exploit the potential of the PCR-cloning to get desired constructs.
PubMed: 38023773
DOI: 10.14440/jbm.2023.411 -
The Journal of Molecular Diagnostics :... Jul 2023CXCR4 mutations impact disease presentation and treatment outcomes in Waldenström macroglobulinemia. Current techniques used for CXCR4 mutation detection have a number...
CXCR4 mutations impact disease presentation and treatment outcomes in Waldenström macroglobulinemia. Current techniques used for CXCR4 mutation detection have a number of limitations. The aim of the present study was to develop and analytically validate a novel droplet digital PCR (ddPCR) assay for the simultaneous detection of five of the most common CXCR4 mutations in bone marrow (BM). In silico novel primers and probes designed for simultaneous detection of five hotspot mutations of CXCR4 were first performed. Experimental conditions were optimized, and the assay was analytically validated. The developed assay was further applied in 95 BM samples from patients with IgM gammopathy, 7 BM samples from patients with non-IgM gammopathy and 12 PBMCs from healthy donors, whereas a direct comparison study of Sanger sequencing and allele-specific PCR was performed by using 95 and 39 identical patient tumor DNA samples, respectively. The drop-off ddPCR assay is a robust, cost-effective, highly sensitive, and highly specific screening tool for CXCR4 mutations. Of 95 patients with IgM gammopathy samples, 27 had at least one CXCR4 mutation in their BM samples. With Sanger sequencing, 12 of the 95 samples tested positive, whereas the direct comparison of the developed assay with allele-specific PCR revealed substantial agreement. The clinical performance of the developed assay will be prospectively evaluated in a large number of patients, and the applicability of this assay will be further evaluated.
Topics: Humans; Waldenstrom Macroglobulinemia; Mutation; Polymerase Chain Reaction; Alleles; Lymphoma, B-Cell; Receptors, CXCR4
PubMed: 37088135
DOI: 10.1016/j.jmoldx.2023.03.010 -
Food Chemistry. Molecular Sciences Dec 2023Food authentication is a mandatory effort to assure the fair-trade. This study developed a duplex polymerase chain reaction (PCR) from the NADH dehydrogenase subunit 2...
Food authentication is a mandatory effort to assure the fair-trade. This study developed a duplex polymerase chain reaction (PCR) from the NADH dehydrogenase subunit 2 () gene to amplify specific segments of a cattle and porcine DNA. A universal forward primer composed of nineteen base pairs (bp) (3'-CCAAACACAACTCCGAAAA-5') and species-specific reverse primers composed of twenty (3'-CCAAACACAACTCCGAAAA-5') and twenty-one (3'-TGGCAAGAATTAGGACGGTTA-5') bp were used to limit the amplified DNA segment for porcine and cattle. The PCR reaction would generate a product with a profile of 168 and 227 bp, respectively. To investigate the accuracy and limit of detection, an experiment was conducted using simplex and duplex PCR on commercial meatballs randomly purchased from a commercial market in Surakarta, Indonesia. The findings of this study indicated that could be used as an alternative genetic marker for the identification of porcine and beef species in meat-derived products.
PubMed: 37637373
DOI: 10.1016/j.fochms.2023.100181 -
Plant Disease Nov 2023In October 2023, lesions consistent with descriptions of tar spot (Phyllachora maydis) were observed on corn (Zea mays) in Kent and Sussex County, Delaware (DE). Black,...
In October 2023, lesions consistent with descriptions of tar spot (Phyllachora maydis) were observed on corn (Zea mays) in Kent and Sussex County, Delaware (DE). Black, raised stromata were observed on leaves of commercially grown corn hybrids. Plants were at physiological maturity and disease severity was low with symptoms present on 1 to 10% of plants. In collected tissue, individual leaf severities ranged from 1 to 3% of leaf area with lesions. Hyaline conidia measuring approximately 15.5 µm in length and 0.5 µm in width were observed microscopically (n=5). Stromata were excised and sterilized in a 0.825% sodium hypochlorite solution for 30 s, rinsed in sterile deionized water for 30 s, and dried on a sterile paper towel for 30 s. Tissues were ground in a 1.5 mL microcentrifuge tube with a sterile plastic pestle. DNA was extracted using a DNeasy Plant Mini Kit (Qiagen). DNA was amplified at the internal transcribed spacer (ITS) region with ITS4 and ITS5 primers using polymerase chain reaction (PCR). NCBI BLAST search results yielded 100% sequence homology and 100% query cover (350/515 bp) to P. maydis accession MG881848.1 (Moura et al. 2023). Koch's postulates could not be completed due to the obligate nature of P. maydis. Tarspot was initially discovered in the United States in 2016 in Indiana and Illinois (Ruhl et al. 2016).This is the first report of tar spot on corn in DE. Yield losses from P. maydis can range depending on time of infection, environmental factors, and hybrid susceptibility and have been recorded up to 100% (Rocco da Silva et al. 2021). Because the disease did not enter the area until the end of the season, no yield impact was observed for 2023. Monitoring for the progression of disease will be crucial for future seasons (Telenko et al. 2020). High humidity and moisture levels favor disease development. Approximately half of DE corn acreage is irrigated due to sandy soils, current irrigation timing strategies may need to be reevaluated. Fungicide efficacy trials for management of tar spot have been conducted in other regions, but continued research will be needed to assess management options and optimize application timing for farmers in DE and the Mid-Atlantic region.
PubMed: 38037200
DOI: 10.1094/PDIS-11-23-2332-PDN -
BioRxiv : the Preprint Server For... Jul 2023Canine parvovirus (CPV) is a highly pathogenic virus that affects dogs, especially puppies. CPV is believed to have evolved from feline panleukopenia virus (FPV),...
Canine parvovirus (CPV) is a highly pathogenic virus that affects dogs, especially puppies. CPV is believed to have evolved from feline panleukopenia virus (FPV), eventually giving rise to three antigenic types, CPV-2a, 2b, and 2c. CPV-2 is recognized for its resilience in contaminated environments, ease of transmission among dogs, and pathogenicity for puppies. Despite the relevance of the virus, complete genome sequences of CPV available at GenBank, to date, are scarce. In the current study, we have developed a methodology to allow the recovery of complete CPV-2 genomes directly from clinical samples. For this, seven fecal samples from Gurupi, Tocantins, North Brazil, were collected from puppies with clinical signals of viral enteritis, and submitted to viral DNA isolation and amplification. Two multiplex PCR strategies were designed including primers targeting fragments of 400 base pairs (bp) and 1,000 bp along the complete genome. Sequencing was performed with the Nanopore technology and results obtained with the two approaches were compared. Genome assembly revealed that the 400 bp amplicons generated larger numbers of reads, allowing a more reliable coverage of the whole genome than those attained with primers targeting the larger (1000 bp) amplicons. Nevertheless, both enrichment methodologies were efficient in amplification and sequencing. Viral genome sequences were of high quality and allowed more precise typing and subtyping of viral genomes compared to the commonly employed strategy relying solely on the analysis of the VP2 region, which is limited in scope. The CPV-2 genomes recovered in this study belong to the CPV2a and CPV-2c subtypes, closely related to isolates from the neighboring Amazonian region. In conclusion, the technique reported here may contribute to increase the number of full CPV genomes available, which is essential for understanding the genetic mechanisms underlying the evolution and spread of CPV-2.
PubMed: 37502963
DOI: 10.1101/2023.07.12.548703 -
BMC Research Notes Nov 2023DNA Barcoding has proven to be a reliable method for rapid insect identification. The success of this method is based on the amplification of a specific region, the...
OBJECTIVES
DNA Barcoding has proven to be a reliable method for rapid insect identification. The success of this method is based on the amplification of a specific region, the 'Folmer' barcode region at the 5´ start of the cytochrome c oxidase 1 gene (cox1), with universal primers. Previous studies showed failures of standard "universal" primers to amplify this region in psyllids. The aim of the study was the design of a new alternative more reliable primer combination for taxa of the superfamily Psylloidea and its comparison with the performance of the standard "universal" Folmer-primers.
RESULTS
A newly designed degenerate forward primer LCOP-F was developed following comparison of the sequence alignment of the priming site of "universal" primer LCO1490 and the standard insect forward primer LepF1. When combined with the "universal" reverse primer, HCO2198, this new primer pairing was able to generate barcode sequence for all 36 species in 20 genera across the five families of psyllids tested in this study, and these primers were found to be more universally reliable across psyllid taxa than other primer pairs tested.
Topics: Animals; DNA Barcoding, Taxonomic; Hemiptera; Aphids; DNA Primers; Electron Transport Complex IV
PubMed: 37941051
DOI: 10.1186/s13104-023-06585-8 -
Microorganisms Nov 2023sp. (Piroplasmida: Theileriidae) is one of the most widely known infections transmitted by hard ticks (Acari: Ixodidae) and has been linked to significant economic...
sp. (Piroplasmida: Theileriidae) is one of the most widely known infections transmitted by hard ticks (Acari: Ixodidae) and has been linked to significant economic losses across the globe. The study's main emphasis was theileriosis, a disease that is common in Pakistan and has an incidence ranging from 0.6% to 33%. Through DNA screening of the vector ticks and host blood, this study sought to determine the risk of tick-borne theileriosis in populations of buffalos () and cattle () in Toba Tek Singh district of Punjab, Pakistan. Identified tick species include (35.4%), (30.2%), and (25%). Tick specimens were collected from animals and their respective microenvironments. PCR assays targeting were used to investigate the infection in the DNA extracted from the collected blood samples from large ruminants and salivary glands (SGs) of the ticks. The 18S rRNA of was amplified using specific primers. Positive amplicons were sequenced and verified using BLAST analysis. Overall, 50% of SGs contained DNA. Female ticks, and those collected from cattle and from riverine environments had significantly higher ( < 0.05) rates of infection in their acini. Overall prevalence of infection was 35.9% in blood collected from large ruminants. Cattle had a substantially greater frequency of bovine theileriosis (43.2%) than buffalos (28.7%). Age and sex of large ruminants were significantly positively associated ( < 0.05) with infection. Furthermore, compared to non-riverine cattle (35%) and buffalo (19.5%), riverine cattle (52.2%) and buffalo (36.2%) showed a considerably higher prevalence. The results of this study, which is the first in Pakistan to examine the blood of large ruminants and vectorial function of Ixodid ticks in the transmission of along with associated risk factors, offer an important insight for risk assessment of infection in livestock using vectorial infectivity.
PubMed: 38004696
DOI: 10.3390/microorganisms11112684