-
Analytical Chemistry Jun 2024DNA biosynthesis, a focus of fundamental and applied research, typically involves DNA polymerases by using templates, primers, and dNTPs. Some polymerases can polymerize...
DNA biosynthesis, a focus of fundamental and applied research, typically involves DNA polymerases by using templates, primers, and dNTPs. Some polymerases can polymerize dNTPs for DNA de novo synthesis, although this is generally to occur randomly. This novel synthesis method has garnered our attention and practical use. Herein, we observed that the addition of endonuclease significantly enhances the efficiency of the de novo synthesis reaction catalyzed by the DNA polymerase. We further investigated the reaction conditions that influence this efficiency. Building on the optimal reaction conditions, we developed a rapid and efficient strategy for preparing DNA hydrogel. Further, coupled with the CRISPR-Cas system, we developed a nucleic acid signal amplification system characterized by versatility, sensitivity, specificity, and no risk of aerosol contamination. We successfully detected viral nucleic acids in clinical samples. In summary, our study demonstrates the significant potential of DNA polymerase- and endonuclease-catalyzed DNA de novo synthesis in diverse applications.
Topics: DNA-Directed DNA Polymerase; DNA; Nucleic Acid Amplification Techniques; CRISPR-Cas Systems; Endonucleases; Humans; Hydrogels
PubMed: 38768388
DOI: 10.1021/acs.analchem.4c01964 -
Molecular Plant Pathology Nov 2023Begomoviruses and criniviruses, vectored by whiteflies (Bemisia tabaci), are important threats to crops worldwide. In recent years, the spread of cucurbit leaf crumple...
Begomoviruses and criniviruses, vectored by whiteflies (Bemisia tabaci), are important threats to crops worldwide. In recent years, the spread of cucurbit leaf crumple virus (CuLCrV), cucurbit yellow stunting disorder virus (CYSDV) and cucurbit chlorotic yellows virus (CCYV) on cucurbit crops has been reported to cause devastating crop losses in many regions of the world. In this study, a multiplex recombinase polymerase amplification (RPA) assay, an isothermal technique for rapid and simultaneous detection of DNA and RNA viruses CuLCrV, CYSDV and CCYV was developed. Highly specific and sensitive multiplex RPA primers for the coat protein region of these viruses were created and evaluated. The sensitivity of the multiplex RPA assay was examined using serially diluted plasmid containing the target regions. The results demonstrated that multiplex RPA primers have high sensitivity with a detection limit of a single copy of the viruses. The multiplex RPA primers were specific to the target as indicated by testing against other begomoviruses, potyviruses and an ilarvirus, and no nonspecific amplifications were noted. The primers simultaneously detected mixed infection of CCYV, CYSDV and CuLCrV in watermelon and squash crude extracts. This study is the first report of a multiplex RPA assay for simultaneous detection of mixed infection of DNA and RNA plant viruses.
Topics: Animals; Recombinases; Coinfection; Hemiptera; Plant Viruses; Crops, Agricultural; DNA
PubMed: 37462133
DOI: 10.1111/mpp.13380 -
Annals of Clinical Microbiology and... Mar 2024Neisseria meningitidis can cause life-threatening meningococcal meningitis and meningococcemia. Old standard microbiological results from CSF/blood cultures are time...
BACKGROUND
Neisseria meningitidis can cause life-threatening meningococcal meningitis and meningococcemia. Old standard microbiological results from CSF/blood cultures are time consuming. This study aimed to combine the sensitivity of loop-mediated isothermal nucleic acid amplification (LAMP) with the specificity of CRISPR/Cas12a cleavage to demonstrate a reliable diagnostic assay for rapid detection of N. meningitidis.
METHODS
A total of n = 139 samples were collected from patients with suspected meningococcal disease and were used for evaluation. The extracted DNA was subjected to qualitative real-time PCR, targeting capsular transporter gene (ctrA) of N. meningitidis. LAMP-specific primer pairs, also targeting the ctrA, were designed and the LAMP products were subjected to CRISPR/Cas12 cleavage reaction. the readout was on a lateral flow strip. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of LAMP-CRISPR/Cas was compared with real-time PCR assays. The limit of detection (LOD) was established with serial dilutions of the target N. meningitidis DNA and calculated by Probit regression analysis.
RESULTS
Six LAMP assay-specific primers were developed targeting the ctrA gene of N. meningitidis, which is conserved in all meningococcal serogroups. The LAMP primers did not amplify DNA from other bacterial DNA tested, showing 100% specificity. The use of 0.4 M betaine increased the sensitivity and stability of the reaction. LAMP-CRISPR/Cas detected meningococcal serogroups (B, C, W). The assay showed no cross-reactivity and was specific for N. meningitidis. The LOD was 74 (95% CI: 47-311) N. meningitidis copies. The LAMP-CRISPR/Cas performed well compared to the gold standard. In the 139 samples from suspected patients, the sensitivity and specificity of the test were 91% and 99% respectively.
CONCLUSION
This developed and optimized method can complement for the available gold standard for the timely diagnosis of meningococcal meningitis and meningococcemia.
Topics: Humans; Neisseria meningitidis; Meningitis, Meningococcal; Meningococcal Infections; Sensitivity and Specificity; DNA, Bacterial; Sepsis
PubMed: 38555443
DOI: 10.1186/s12941-024-00688-1 -
Molecular Ecology Resources Aug 2023Detection of invasive species is critical for management but is often limited by challenges associated with capture, processing and identification of early life stages....
Detection of invasive species is critical for management but is often limited by challenges associated with capture, processing and identification of early life stages. DNA metabarcoding facilitates large-scale monitoring projects to detect establishment early. Here, we test the use of DNA metabarcoding to monitor invasive species by sequencing over 5000 fishes in bulk ichthyoplankton samples (larvae and eggs) from four rivers of ecological and cultural importance in southern Canada. We were successful in detecting species known from each river and three invasive species in two of the four rivers. This includes the first detection of early life-stage rudd in the Credit River. We evaluated whether sampling gear affected the detection of invasive species and estimates of species richness, and found that light traps outperform bongo nets in both cases. We also found that the primers used for the amplification of target sequences and the number of sequencing reads generated per sample affect the consistency of species detections. However, these factors have less impact on detections and species richness estimates than the number of samples collected and analysed. Our analyses also show that incomplete reference databases can result in incorrectly attributing DNA sequences to invasive species. Overall, we conclude that DNA metabarcoding is an efficient tool for monitoring the early establishment of invasive species by detecting evidence of reproduction but requires careful consideration of sampling design and the primers used to amplify, sequence and classify the diversity of native and potentially invasive species.
Topics: Animals; Introduced Species; Biodiversity; DNA Barcoding, Taxonomic; Fishes; Larva; DNA; DNA Primers
PubMed: 37101312
DOI: 10.1111/1755-0998.13803 -
Parasitology Research Dec 2023Ticks are important vectors involved in the transmission of pathogens of zoonotic and veterinary importance. In this study, ticks were collected from cattle in Navrongo,...
Ticks are important vectors involved in the transmission of pathogens of zoonotic and veterinary importance. In this study, ticks were collected from cattle in Navrongo, Kintampo, and Kumasi and screened for pathogen DNA using PCR and Sanger sequencing. A total of 454 ticks were collected, morphologically identified and confirmed using primers that target the 660-bp segment of the mitochondrial COI gene. The predominant tick species was Amblyomma variegatum (70.26%). DNA was extracted from 85 tick pools and screened for the presence of Rickettsia DNA based on the 639 bp of the outer membrane protein A (ompA) gene, Ehrlichia/Anaplasma DNA based on the 345 bp fragment of the 16SrRNA gene and Babesia/ Theileria DNA based on the 560 bp fragment of the ssrRNA gene. From the 85 tick pools, the DNA of pathogens detected were Rickettsia africae (36.47%), Rickettsia aeschlimannii (16.47%), Ehrlichia canis (2.35%), Babesia occultans (1.18%), Theileria velifera (1.18%) and a symbiont Candidatus Midichloria mitochondrii (8.24%). This study reports the first molecular detection of Candidatus Cryptoplasma californiense (1.18%) in Ghana. Coinfections were recorded in 8.24% of the tick pools. The findings of this study highlight the importance of tick species in Ghana and the need to adopt effective control measures to prevent pathogen spread.
Topics: Animals; Cattle; Ghana; Tick-Borne Diseases; Cattle Diseases; Rickettsia; Rhipicephalus; Theileria; DNA
PubMed: 38095712
DOI: 10.1007/s00436-023-08071-3 -
Molecular Biology Reports Dec 2023The COI mitochondrial gene has been chosen as the "DNA barcode in animals" and the large quantity of genetic information in public databanks gives solid support for the...
FishDNAIDs: DNA barcoding as a tool in the development and validation in silico and in vitro of detection systems to four species of Characiformes of commercial importance in the Brazilian Amazon.
BACKGROUND
The COI mitochondrial gene has been chosen as the "DNA barcode in animals" and the large quantity of genetic information in public databanks gives solid support for the use of DNA barcoding as a promising tool for the development of a specific molecular detection system.
METHODS AND RESULTS
The present study aimed to develop a Specific Molecular Detection System (SMDS: FishDNAIDs) (primers and probe sets) for the following four target species: Prochilodus nigricans, Potamorhina altamazonica, Psectrogaster rutiloides and Triportheus angulatus, in qPCR assays. In silico and in vitro tests (using gDNA) were performed to test these sets. The database generated contained the cytochrome c oxidase subunit I (COI) nucleotide sequence for 183 specimens of Characiformes, distributed in 34 species representing eight families. In silico, primers designed for the target species amplified different species from the same genus, except for P. rutiloides, which amplified only the target species. In the in vitro test, using the SYBRGreen and TaqMan® fluorescence systems, both sets detected the respective target species (P. nigricans, P. altamazonica, P. rutiloides and T. angulatus). In the qPCR assays using SYBRGreen, species considered to be related were also detected, in addition to the target species, with the exception of P. amazonica and P. essequibensis (correlated to P. rutiloides). All target species were detected in the qPCR assays using the TaqMan® system; however, with the SMDS PALT, the target species P. altamazonica was detected with low C values (22.21 ± 0.17) as well as the correlates of P. latior and P. pristigaster, though with high C values (23.51 ± 0.19 and 30.21 ± 0.95). This assay uniquely identifies known adult tissue samples from all four species.
CONCLUSIONS
The primers and probe sets developed can act as powerful tools for detecting the target Characiformes species.
Topics: Humans; Animals; Characiformes; DNA Barcoding, Taxonomic; Brazil; DNA; DNA Primers; Phylogeny
PubMed: 37962704
DOI: 10.1007/s11033-023-08872-w -
Journal of Medical Virology Feb 2024Metagenomic next-generation sequencing (mNGS) is a valuable technique for identifying pathogens. However, conventional mNGS requires the separate processing of DNA and...
Metagenomic next-generation sequencing (mNGS) is a valuable technique for identifying pathogens. However, conventional mNGS requires the separate processing of DNA and RNA genomes, which can be resource- and time-intensive. To mitigate these impediments, we propose a novel method called DNA/RNA cosequencing that aims to enhance the efficiency of pathogen detection. DNA/RNA cosequencing uses reverse transcription of total nucleic acids extracted from samples by using random primers, without removing DNA, and then employs mNGS. We applied this method to 85 cases of severe acute respiratory infections (SARI). Influenza virus was identified in 13 cases (H1N1: seven cases, H3N2: three cases, unclassified influenza type: three cases) and was not detected in the remaining 72 samples. Bacteria were present in all samples. Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii were detected in four influenza-positive samples, suggesting coinfections. The sensitivity and specificity for detecting influenza A virus were 73.33% and 95.92%, respectively. A κ value of 0.726 indicated a high level of concordance between the results of DNA/RNA cosequencing and SARI influenza virus monitoring. DNA/RNA cosequencing enhanced the efficiency of pathogen detection, providing a novel capability to strengthen surveillance and thereby prevent and control infectious disease outbreaks.
Topics: Humans; Influenza, Human; RNA; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H3N2 Subtype; Pneumonia; High-Throughput Nucleotide Sequencing; Sensitivity and Specificity; DNA; Metagenomics
PubMed: 38373115
DOI: 10.1002/jmv.29406 -
International Journal of Molecular... Dec 2023Red algae (Rhodophyta) are a heterogeneous group of marine algal species that have served as a source of high-value molecules, including antioxidants and scaffolds, for...
Red algae (Rhodophyta) are a heterogeneous group of marine algal species that have served as a source of high-value molecules, including antioxidants and scaffolds, for novel drug development. However, it is challenging to identify Rhodophytes through morphological features alone, and in most instances, that has been the prevailing approach to identification. Consequently, this study undertook the identification of red algae species in Kenton-on-Sea, South Africa, as a baseline for future research on red algae biodiversity and conservation. The identification was achieved by designing, analysing, and using a set of universal primers through DNA barcoding of the L gene. The PCR products of the L gene were sequenced, and 96% of the amplicons were successfully sequenced from this set and matched with sequences on BOLD, which led to these species being molecularly described. Amongst these species are medicinally essential species, such as and , and potential cryptic species. This calls for further investigation into the biodiversity of the studied region. Meanwhile, the availability of these primers will ease the identification process of red algae species from other coastal regions.
Topics: DNA Barcoding, Taxonomic; DNA; DNA Primers; Carboxy-Lyases; Rhodophyta; Seaweed; Pentoses
PubMed: 38203228
DOI: 10.3390/ijms25010058 -
The Science of the Total Environment Dec 2023Animal detection through DNA present in environmental samples (eDNA) is a valuable tool for detecting rare species, that are difficult to observe and monitor. eDNA-based... (Review)
Review
Animal detection through DNA present in environmental samples (eDNA) is a valuable tool for detecting rare species, that are difficult to observe and monitor. eDNA-based tools are underpinned by molecular evolutionary principles, key to devising tools to efficiently single out a targeted species from an environmental sample. Here, we present a comprehensive review of the use of eDNA-based methods for the detection of targeted animal species, such as rare, endangered, or invasive species, through the analysis of 549 publications (2008-2022). Aquatic ecosystems have been the most surveyed, in particular, freshwaters (74 %), and to a less extent marine (14 %) and terrestrial systems (10 %). Vertebrates, in particular, fish (38 %), and endangered species, have been the focus of most of these studies, and Cytb and COI are the most employed markers. Among invertebrates, assays have been mainly designed for Mollusca and Crustacea species (21 %), in particular, to target invasive species, and COI the most employed marker. Targeted molecular approaches, in particular qPCR, have been the most adopted (75 %), while eDNA metabarcoding has been rarely used to target single or few species (approx. 6 %). However, less attention has been given in these studies to the effects of environmental factors on the amount of shed DNA, the differential amount of shed DNA among species, or the sensitivity of the markers developed, which may impact the design of the assays, particularly to warrant the required detection level and avoid false negatives and positives. The accuracy of the assays will also depend on the availability of genetic data and vouchered tissue or DNA samples from closely related species to assess both marker and primers' specificity. In addition, eDNA-based assays developed for a particular species may have to be refined for use in a new geographic area taking into account site-specific populations, as well as any intraspecific variation.
Topics: Animals; Ecosystem; Vertebrates; DNA, Environmental; Fishes; DNA; Introduced Species; Biodiversity; Environmental Monitoring
PubMed: 37647964
DOI: 10.1016/j.scitotenv.2023.166675 -
Veterinary Pathology Jun 2024Fixation and demineralization protocols for bone marrow (BM) across diagnostic laboratories are not standardized. How different protocols affect histomorphology and DNA...
Fixation and demineralization protocols for bone marrow (BM) across diagnostic laboratories are not standardized. How different protocols affect histomorphology and DNA amplification is incompletely understood. In this study, 2 fixatives and 3 demineralization methods were tested on canine BM samples. Twenty replicate sternal samples obtained within 24 hours of death were fixed overnight in either acetic acid-zinc-formalin (AZF) or 10% neutral-buffered formalin (NBF) and demineralized with formic acid for 12 hours. Another 53 samples were fixed in AZF and demineralized with hydrochloric acid for 1-hour, formic acid for 12 hours, or ethylenediamine tetraacetic acid (EDTA) for 24 hours. Histologic sections were scored by 4 raters as of insufficient, marginal, good, or excellent quality. In addition, DNA samples extracted from sections treated with the different fixation and demineralization methods were amplified with 3 sets of primers to conserved regions of and genes. Amplification efficiency was graded based on review of capillary electrophoretograms. There was no significant difference in the histomorphology scores of sections fixed in AZF or NBF. However, EDTA-based demineralization yielded higher histomorphology scores than demineralization with hydrochloric or formic acid, whereas formic acid resulted in higher scores than hydrochloric acid. Demineralization with EDTA yielded DNA amplification in 29 of 36 (81%) samples, whereas demineralization with either acid yielded amplification in only 2 of 72 (3%) samples. Although slightly more time-consuming and labor-intensive, tissue demineralization with EDTA results in superior morphology and is critical for polymerase chain reaction (PCR) amplification with the DNA extraction method described in this article.
PubMed: 38842072
DOI: 10.1177/03009858241257920