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Communications Chemistry Jun 2024Oligonucleotides are advancing as essential materials for the development of new therapeutics, artificial genes, or in storage of information applications. Hitherto, our... (Review)
Review
Oligonucleotides are advancing as essential materials for the development of new therapeutics, artificial genes, or in storage of information applications. Hitherto, our capacity to write (i.e., synthesize) oligonucleotides is not as efficient as that to read (i.e., sequencing) DNA/RNA. Alternative, biocatalytic methods for the de novo synthesis of natural or modified oligonucleotides are in dire need to circumvent the limitations of traditional synthetic approaches. This Perspective article summarizes recent progress made in controlled enzymatic synthesis, where temporary blocked nucleotides are incorporated into immobilized primers by polymerases. While robust protocols have been established for DNA, RNA or XNA synthesis is more challenging. Nevertheless, using a suitable combination of protected nucleotides and polymerase has shown promises to produce RNA oligonucleotides even though the production of long DNA/RNA/XNA sequences (>1000 nt) remains challenging. We surmise that merging ligase- and polymerase-based synthesis would help to circumvent the current shortcomings of controlled enzymatic synthesis.
PubMed: 38890393
DOI: 10.1038/s42004-024-01216-0 -
Parasites & Vectors Jun 2024Malaria transmission in Tanzania is driven by mosquitoes of the Anopheles gambiae complex and Anopheles funestus group. The latter includes An. funestus s.s., an...
BACKGROUND
Malaria transmission in Tanzania is driven by mosquitoes of the Anopheles gambiae complex and Anopheles funestus group. The latter includes An. funestus s.s., an anthropophilic vector, which is now strongly resistant to public health insecticides, and several sibling species, which remain largely understudied despite their potential as secondary vectors. This paper provides the initial results of a cross-country study of the species composition, distribution and malaria transmission potential of members of the Anopheles funestus group in Tanzania.
METHODS
Mosquitoes were collected inside homes in 12 regions across Tanzania between 2018 and 2022 using Centres for Disease Control and Prevention (CDC) light traps and Prokopack aspirators. Polymerase chain reaction (PCR) assays targeting the noncoding internal transcribed spacer 2 (ITS2) and 18S ribosomal DNA (18S rDNA) were used to identify sibling species in the An. funestus group and presence of Plasmodium infections, respectively. Where DNA fragments failed to amplify during PCR, we sequenced the ITS2 region to identify any polymorphisms.
RESULTS
The following sibling species of the An. funestus group were found across Tanzania: An. funestus s.s. (50.3%), An. parensis (11.4%), An. rivulorum (1.1%), An. leesoni (0.3%). Sequencing of the ITS2 region in the nonamplified samples showed that polymorphisms at the priming sites of standard species-specific primers obstructed PCR amplification, although the ITS2 sequences closely matched those of An. funestus s.s., barring these polymorphisms. Of the 914 samples tested for Plasmodium infections, 11 An. funestus s.s. (1.2%), and 2 An. parensis (0.2%) individuals were confirmed positive for P. falciparum. The highest malaria transmission intensities [entomological inoculation rate (EIR)] contributed by the Funestus group were in the north-western region [108.3 infectious bites/person/year (ib/p/y)] and the south-eastern region (72.2 ib/p/y).
CONCLUSIONS
Whereas An. funestus s.s. is the dominant malaria vector in the Funestus group in Tanzania, this survey confirms the occurrence of Plasmodium-infected An. parensis, an observation previously made in at least two other occasions in the country. The findings indicate the need to better understand the ecology and vectorial capacity of this and other secondary malaria vectors in the region to improve malaria control.
Topics: Anopheles; Animals; Tanzania; Mosquito Vectors; Malaria; Humans; RNA, Ribosomal, 18S; Polymerase Chain Reaction; Female; Plasmodium; DNA, Ribosomal Spacer
PubMed: 38886827
DOI: 10.1186/s13071-024-06348-9 -
Plant Disease Jun 2024Avocado (), which is native to Latin America, is mostly planted in southwest China. In November 2021, leaf spot symptoms were observed in a nursery in Chongzuo...
Avocado (), which is native to Latin America, is mostly planted in southwest China. In November 2021, leaf spot symptoms were observed in a nursery in Chongzuo (22.2019°N, 106.4723°E), Guangxi, China. Approximately 90% of avocado seedlings in the nursery were affected. Symptomatic plant fully expanded leaves showed small brown spots that ranged from 1 to 3 mm, with a yellow halo around (Fig.1). Lesions gradually expanded and became nearly round and dark brown. Finally, leaves withered or curled. For pathogen isolation, 15 symptomatic leaves were randomly sampled from different plants of the nursery, five leaves were selected and four samples size 4×4mm were taken from each leaf and were plated on potato glucose agar. Identical fungus colonies were observed in 80% of the samples, and no bacteria were isolated. Single conidial isolation was performed. After 4 days, the colony diameter reached 74.6 mm, colonies appeared gray, and developed aerial hyphae. Conidiophores were mostly solitary with a few clustered erect or slightly curved, knee shaped, and 3.89 to 5.24 µm wide. Conidia were 39.33 -96.88 × 9.96 - 15.59 µm, slightly curved, rarely straight, light brown to yellowish brown, fusoid or navicular, and truncated at the base with 4 to 10 septa. Based on morphological and cultural characteristics, the fungus was identified as sp. (Manamgoda et al. 2014). An isolate named MP211122 was grown on Sachs' ager at 27℃ under 12-h light/dark for 1 week and consistently with Adhikari et al. (2021) no sexual from was observed. To confirm the tentative identification, genomic DNA was extracted, ITS and GAPDH gene were amplified and sequenced using primers ITS1/ITS4 and GPD/GPD2, respectively (Tan et al. 2022). The ITS sequence (GenBank ON248469) shared 100% identity with (MN215632.1), and the GAPDH sequence (ON642344) shared 99.82% identity with (MF490833.1, MK144540.1) and (MK026428.1). A maximum likelihood phylogenetic analysis based on GAPDH and ITS sequences using MEGA 7.0 revealed that the isolate clustered with with 100% bootstrap support(Fig. 2). Healthy 11-month old potted avocado seedlings from disease-free nursery were selected , the conidial suspension (1 × 10 conidia/mL) of MP211122 isolate was prepared by harvesting conidia from a 10-day-old culture on water agar. Conidia were sprayed onto young leaves of six potted plants. Three additional seedlings sprayed with sterile distilled water served as controls. All plants were covered with plastic bags for 3 days to maintain high humidity and then maintained in a greenhouse at 30℃ with a 12-h/12-h light/dark cycle. After 5 days, typical symptoms of small brown spots were observed on all inoculated leaves (Fig.3). All leaves on control plants remained asymptomatic. The reisolated fungus was morphologically identical to the original isolate used for inoculation, fulfilling Koch's postulates. This is the first report of as a pathogen causing leaf spot on avocado in China. This information will facilitate further studies, monitoring and control of the disease as accurate identification of the causal agent is a primary requisite for designing management strategies.
PubMed: 38885029
DOI: 10.1094/PDIS-12-22-2958-PDN -
Plant Disease Jun 2024Datura stramonium L.(jimson weed) is an invasive weed in agricultural fields and a medicinal plant. In April 2022, a leaf disease on D. stramonium was observed in...
Datura stramonium L.(jimson weed) is an invasive weed in agricultural fields and a medicinal plant. In April 2022, a leaf disease on D. stramonium was observed in Zhanjiang (21.17 N, 110.18 E), Guangdong province, China. Early symptoms were small yellow spots on leaves. Later, the spots gradually expanded and turned becoming necrotic with a clear yellow halo and a white center. The disease incidence in the field was 85% (n = 50, about 1 ha). Twenty diseased leaves were collected from the field. The margin of the diseased tissues was cut into 2 mm × 2 mm pieces, surface disinfected with 75% ethanol and 2% sodium hypochlorite for 30 and 60 s, respectively, and rinsed twice with sterile water before isolation. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 ℃. After 2-day incubation, grayish fungal colonies appeared on the PDA, then pure cultures were produced by transferring hyphal tips to new PDA plates. Single-spore isolation method was used to recover pure cultures for three isolates (DSAC-1, DSAC-2, and DSAC-3). The isolates were morphologically identical . They colonies were gray to brownish black. Conidiophores were branched, brown. Conidia were brown, long ellipsoid, had 4-12 transverse and 0-3 longitudinal septa; measured within 67.5-127.8 (average = 105.6) × 12.5-27.8 (average = 20.4) µm (n = 30). Apical beak was longer than conidia body. measured within 40.5-423.5 (average = 365.2) × 2.5-5.8 (average = 3.2) µm (n = 30). Based on morphological characteristics, the three isolates were identified as Alternaria crassa (Sacc.) Rands (Simmons 2007). Molecular identification was performed using the colony polymerase chain reaction method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012) to amplify internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase second largest subunit (RPB2) and translation elongation factor (TEF1) with primers of ITS1/ITS4, GDF1/GDR1, RPB2-5F2/fRPB2-7cR, and EF-1α-F/EF-1α-R, respectively (Walther et al. 2013; Woudenberg et al. 2015; Nishikawa and Nakashima. 2020). Amplicons of the isolates were sequenced and submitted to GenBank (ITS, ON430524-ON430526; GAPDH, ON500656-ON500658; RPB2, ON500659-ON500661; TEF1, ON500662-ON500664). The sequences were 100% identical with those of Alternaria crassa strain CBS 116647 upon BLAST analysis. The sequences were also concatenated for phylogenetic analysis by maximum likelihood. The isolates clustered with A. crassa (CBS 116647, CBS 116648, CBS CBS-110.38, and CBS_103.18 ). Thus, the fungus associated with leaf yellow spot on D. stramonium was identified as A. crassa. Pathogenicity tests were conducted in a greenhouse at 24 ℃-30 ℃ with 80% relative humidity using 3 isolates. Individual plants were grown in pots (n = 5, 1 month old). The unwounded leaflets were inoculated using three isolates (DSAC-1, DSAC-2, and DSAC-3). The fungal mycelium on 5 mm-diameter PDA plugs were placed faced down to the leaves. Sterile PDA was used for mock inoculated comtrols.. The test was performed three times. Disease symptoms were observed on the leaves after 7 days, whereas the controls remained healthy. The pathogen was re-isolated from infected leaves and was morphologically identical to the original isolates, fulfilling Koch's postulates. A. crassa was reported causing leaf spot on D. stramonium in Algeria (Nabahat et al. 2020). To our knowledge, this report is the first report of A. crassa causing leaf yellow spot on D. stramonium in China. This pathogen possesses potential biocontrol properties on the invasive weed, while this study also provides an important reference for the control of the disease of the medicinal plant.
PubMed: 38885028
DOI: 10.1094/PDIS-08-23-1494-PDN -
Plant Disease Jun 2024Psidium guajava L. is widely cultivated in southern China. In May 2021, guava scab on cv. Zhenzhu was observed in Zhanjiang (21.18° N, 110.21° E), Guangdong province,...
Psidium guajava L. is widely cultivated in southern China. In May 2021, guava scab on cv. Zhenzhu was observed in Zhanjiang (21.18° N, 110.21° E), Guangdong province, China. Guava scab was corky with ovoid or round lesions on the surfaces of green fruits. Gradually the lesions sunk. Disease incidence was estimated as 85% in 500 investigated plants in about 50 ha. Twenty diseased fruits were collected from twenty trees in the field. From each fruit the margin of the diseased tissues was cut into 2 mm × 2 mm pieces; surface disinfected with 75% ethanol and 2% sodium hypochlorite for 30 and 60 s, successively; and rinsed thrice with sterile water. The tissues were plated onto potato dextrose agar (PDA) medium and incubated at 28 ℃. Thirty-four isolates were obtained. Single-spore isolation method (Liu et al. 2021) was used to recover pure cultures of three isolates (PGNC-1, PGNC-2, and PGNC-3) . The colonies were initially white with cottony aerial mycelium at 7 days on PDA. Then, these colonies form black acervular conidiomata at 10 days. Conidia were clavate to fusiform, four-septate, straight or slightly curved, and measured 15.8 to 21.2 µm × 4.5 to 6.5 µm (n = 40). The three median cells were versicolored, whereas the basal and apical cells were hyaline. Conidia had a single basal appendage (4.5 to 5.5 µm long; n = 40) and three apical appendages (19.2 to 24.5 µm long; n = 40). The morphological characteristics of the isolates were consistent with the description of Neopestalotiopsis clavispora (Maharachchikumbura et al. 2012). Molecular identification was performed using PCR method with MightyAmp DNA Polymerase (Takara-Bio, Dalian, China) (Lu et al. 2012). Sequences were generated from the isolates using primers for the rDNA ITS (ITS1/ITS4), TEF1-α (EF1-728F/EF1-986R), and β-tubulin (T1/βt2b) loci (Maharachchikumbura et al. 2012). The sequences of the isolates were submitted to GenBank (ITS, OQ996557 to OQ996559; TEF, OR101037 to OR101039; β-tubulin, OR100971 to OR100973). The sequences of the isolates were 100% identical to the type strain MFLUCC12-0281 (accession nos. JX398979, JX399014, and JX399045) through BLAST analysis. The isolates clustered with N. clavispora (MFLUCC12-0280 and MFLUCC12-0281). N. clavispora and Pestalotiopsis clavispora are synonyms. The pathogenicity was tested in vivo. Plants (cv. Zhenzhu) were grown ( 3 years old) in a quarantine orchard at 25 ℃ to 32 ℃ with 60 to 80% relative humidity in May 2022. Disease-free green fruits were inoculated. Sterile cotton balls were immersed in the spore suspension (1 × 105 per mL) and sterile distilled water (control) for about 15 s before they were fixed on the wounded fruits with transparent tape. Five fruits on one plant per isolate were inoculated. Five fruits on one plant severed as control. The test was performed thrice. Disease symptoms were found on the inoculated fruits after 20 days, whereas the controls remained healthy. The pathogen was re-isolated from infected fruits and was phenotypically identical to the original isolates thus fulfilling Koch's postulates. Neopestalotiopsis or Pestalotiopsis spp. were reported to be the causal agents of guava scab in Colombia and in Hawaii (Keith et al. 2006; Solarte et al. 2018). N. clavispora has been reported to cause disease in a broad range of hosts (Ge et al. 2009; Chen et al. 2018), but not in guava. This is the first report of N. clavispora causing guava scab in China. There would be no harvest if this disease is left unmanaged.
PubMed: 38885025
DOI: 10.1094/PDIS-11-23-2357-PDN -
Plant Disease Jun 2024Dollar spot is a major fungal disease affecting turfgrass worldwide and can quickly destroy turfgrass swards. An assimilating probe-based loop-mediated amplification...
Dollar spot is a major fungal disease affecting turfgrass worldwide and can quickly destroy turfgrass swards. An assimilating probe-based loop-mediated amplification (LAMP) assay was developed to detect Clarireedia monteithiana and C. jacksonii, the causal agents of dollar spot within the continental US. Five LAMP primers were designed to target the calmodulin gene with the addition of a 6-carboxyl-fluorescein florescent assimilating probe and the temperature amplification was optimized for C. jacksonii and C. monteithiana identification. The minimum amount purified DNA needed for detection was 0.05 ng µL-1. Specificity assays against host DNA and other turfgrass pathogens were negative. Successful LAMP amplification was also observed for dollar spot infected turfgrass field samples. Further, a DNA extraction technique via rapid heat-chill cycles and visualization of LAMP results via a florescent flashlight was developed and adapted for fast, simple and reliable detection in 1.25 hours. This assimilating probe-based LAMP assay has proved successful as a rapid, sensitive, and specific detection of C. monteithiana and C. jacksonii in pure cultures and from symptomatic turfgrass leaves blades. The assay represents a promising technology to be used in the field for on-site, point-of-care pathogen detection.
PubMed: 38885023
DOI: 10.1094/PDIS-12-23-2608-RE -
Biochemical Society Transactions Jun 2024Mitochondrial DNA replication is initiated by the transcription of mitochondrial RNA polymerase (mtRNAP), as mitochondria lack a dedicated primase. However, the... (Review)
Review
Mitochondrial DNA replication is initiated by the transcription of mitochondrial RNA polymerase (mtRNAP), as mitochondria lack a dedicated primase. However, the mechanism determining the switch between continuous transcription and premature termination to generate RNA primers for mitochondrial DNA (mtDNA) replication remains unclear. The pentatricopeptide repeat domain of mtRNAP exhibits exoribonuclease activity, which is required for the initiation of mtDNA replication in Drosophila. In this review, we explain how this exonuclease activity contributes to primer synthesis in strand-coupled mtDNA replication, and discuss how its regulation might co-ordinate mtDNA replication and transcription in both Drosophila and mammals.
Topics: DNA, Mitochondrial; DNA Replication; Animals; Mitochondria; Humans; DNA-Directed RNA Polymerases; Transcription, Genetic; Drosophila; Exoribonucleases; Drosophila melanogaster
PubMed: 38884788
DOI: 10.1042/BST20230952 -
Applied Microbiology and Biotechnology Jun 2024Mushroom poisoning contributes significantly to global foodborne diseases and related fatalities. Amanita mushrooms frequently cause such poisonings; however,...
Mushroom poisoning contributes significantly to global foodborne diseases and related fatalities. Amanita mushrooms frequently cause such poisonings; however, identifying these toxic species is challenging due to the unavailability of fresh and intact samples. It is often necessary to analyze residues, vomitus, or stomach extracts to obtain DNA sequences for the identification of species responsible for causing food poisoning. This usually proves challenging to obtain usable DNA sequences that can be analyzed using conventional molecular biology techniques. Therefore, this study aimed to develop a DNA mini-barcoding method for the identification of Amanita species. Following the evaluation and optimization of universal primers for DNA mini-barcoding in Amanita mushrooms, we found that the internal transcribed spacer (ITS) gene sequence primer ITS-a was the most suitable DNA barcode primer for identifying Amanita species. Forty-three Amanita samples were subsequently amplified and sequenced. The sequences obtained were analyzed for intra- and inter-species genetic distances, and a phylogenetic tree was constructed. The findings indicated that the designed primers had strong universality among the Amanita samples and could accurately identify the target gene fragment with a length of 290 bp. Notably, the DNA mini-barcode accurately identified the 43 Amanita samples, demonstrating high consistency with the conventional DNA barcode. Furthermore, it effectively identified DNA from digested samples. In summary, this DNA mini-barcode is a promising tool for detecting accidental ingestion of toxic Amanita mushrooms. It may be used as an optimal barcode for species identification and traceability in events of Amanita-induced mushroom poisoning. KEY POINTS: • Development of a DNA mini-barcoding method for Amanita species identification without fresh samples. • The ITS-a primer set was optimized for robust universality in Amanita samples. • The mini-barcode is suitable for screening toxic mushroom species in mushroom poisoning cases.
Topics: Mushroom Poisoning; Amanita; DNA Barcoding, Taxonomic; Phylogeny; DNA, Fungal; DNA Primers; DNA, Ribosomal Spacer; Sequence Analysis, DNA; Humans
PubMed: 38884656
DOI: 10.1007/s00253-024-13219-x -
Journal of Clinical Microbiology Jun 2024The gene has been proposed as a promising phylogenetic marker for bacterial identification, providing theoretically improved species-level resolution compared to the...
The gene has been proposed as a promising phylogenetic marker for bacterial identification, providing theoretically improved species-level resolution compared to the 16S rRNA gene for a range of clinically important taxa. However, its utility in diagnostic microbiology has been limited by the lack of broad-range primers allowing for its amplification from most species with a single PCR assay. Here, we present an assay for broad-range partial amplification and Sanger sequencing of the gene. To reduce cross-reactivity and allow for amplification directly from patient samples, primers were based on the dual priming oligonucleotide principle. The resulting amplicon is ~550 base pairs in length and appropriate for species-level identification. Systematic evaluation of a wide selection of taxa demonstrated improved resolution within multiple important genera, including , , , and species and several genera within the family. Broad-range amplification and Sanger sequencing of 115 bacterial isolates provided unambiguous species-level identification for 97 (84%) isolates, as compared to 57 (50%) using a clinical 16S rRNA gene assay. Several unresolved taxonomic matters disguised by the low resolution of the 16S rRNA gene were revealed using the gene. Using a collection of 33 clinical specimens harboring bacteria and assumed to contain high concentrations of human DNA, the assay identified the pathogen in 29 specimens (88%). Broad-range amplification and sequencing provides a promising tool for bacterial identification, improving discrimination between closely related species and making it amenable for use in culture-based and culture-independent diagnostic approaches.
PubMed: 38884485
DOI: 10.1128/jcm.00266-24 -
Parasites & Vectors Jun 2024The Anopheles dirus complex plays a significant role as a malaria vector in the Greater Mekong Subregion (GMS), with varying degrees of vector competence among species....
BACKGROUND
The Anopheles dirus complex plays a significant role as a malaria vector in the Greater Mekong Subregion (GMS), with varying degrees of vector competence among species. Accurate identification of sibling species in this complex is essential for understanding malaria transmission dynamics and deploying effective vector control measures. However, the original molecular identification assay, Dirus allele-specific polymerase chain reaction (AS-PCR), targeting the ITS2 region, has pronounced nonspecific amplifications leading to ambiguous results and misidentification of the sibling species. This study investigates the underlying causes of these inconsistencies and develops new primers to accurately identify species within the Anopheles dirus complex.
METHODS
The AS-PCR reaction and thermal cycling conditions were modified to improve specificity for An. dirus member species identification. In silico analyses with Benchling and Primer-BLAST were conducted to identify problematic primers and design a new set for Dirus complex species identification PCR (DiCSIP). DiCSIP was then validated with laboratory and field samples of the An. dirus complex.
RESULTS
Despite several optimizations by reducing primer concentration, decreasing thermal cycling time, and increasing annealing temperature, the Dirus AS-PCR continued to produce inaccurate identifications for Anopheles dirus, Anopheles scanloni, and Anopheles nemophilous. Subsequently, in silico analyses pinpointed problematic primers with high Guanine-Cytosine (GC) content and multiple off-target binding sites. Through a series of in silico analyses and laboratory validation, a new set of primers for Dirus complex species identification PCR (DiCSIP) has been developed. DiCSIP primers improve specificity, operational range, and sensitivity to identify five complex member species in the GMS accurately. Validation with laboratory and field An. dirus complex specimens demonstrated that DiCSIP could correctly identify all samples while the original Dirus AS-PCR misidentified An. dirus as other species when used with different thermocyclers.
CONCLUSIONS
The DiCSIP assay offers a significant improvement in An. dirus complex identification, addressing challenges in specificity and efficiency of the previous ITS2-based assay. This new primer set provides a valuable tool for accurate entomological surveys, supporting effective vector control strategies to reduce transmission and prevent malaria re-introducing in the GMS.
Topics: Anopheles; Animals; Polymerase Chain Reaction; DNA Primers; Mosquito Vectors; Malaria; Asia, Southeastern; Sensitivity and Specificity
PubMed: 38880909
DOI: 10.1186/s13071-024-06321-6