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Parasites & Vectors Feb 2024The house fly, Musca domestica, is a significant carrier of diseases that can impact public health. Repeated use of pyrethroid insecticides may act as a selection...
BACKGROUND
The house fly, Musca domestica, is a significant carrier of diseases that can impact public health. Repeated use of pyrethroid insecticides may act as a selection pressure for mutations and amino acid substitutions in the house fly voltage-sensitive sodium channel (VSSC), which ultimately confers resistance. The objectives of this study were to determine the presence of knockdown resistance (kdr) mutations using molecular tools and to set up a CDC bottle bioassay specific for house flies in the United Arab Emirates (UAE) to screen for deltamethrin resistance.
METHODS
Adult flies were collected from 19 locations in Abu Dhabi, UAE, and DNA was extracted, followed by PCR amplification of specific alleles (PASA) and conventional PCR using several primers to amplify regions of the VSSC gene. Sanger sequencing was performed on PCR products. We also designed primers that detect four kdr mutations using complementary DNA (cDNA) in reverse transcriptase (RT)-PCR followed by Sanger sequencing. Additionally, a CDC bottle bioassay was set up for detecting deltamethrin resistance in adult house flies.
RESULTS
In PASA, the primers successfully amplified the target bands (480, 280 and 200 bp). The kdr allele was found in flies collected from 18 of the 19 locations, at the highest and lowest prevalence of 46.9% and 9.4%, respectively. Resistant homozygous (RR) insects constituted 5.0% of the tested populations, and heterozygous (RS) insects accounted for 36.5%. The RR genotype was prevalent in house flies collected at 10 of 19 sampling locations. House fly populations were mostly in Hardy-Weinberg equilibrium, except in three locations. In addition to verifying the presence of the previously identified kdr mutation L1014F, in this study we detected two kdr mutations, L1014H and T929I, that have not previously been reported in the UAE. Also, for the first time in the UAE, a CDC bottle bioassay for deltamethrin resistance was used, which found that 60 min and 4.5 µg/ml were the diagnostic time and dose, respectively. Using this assay, we detected deltamethrin resistance in house flies from two of 16 locations, with a resistance level of 12.5%.
CONCLUSIONS
Using DNA sequencing, we confirmed the presence of a known kdr mutation and uncovered two new kdr mutations in house flies from Abu Dhabi. Additionally, we detected deltamethrin resistance in these flies using a CDC bottle bioassay. Further research is recommended to comprehensively identify more kdr mutations in UAE house fly populations and assess their impacts on control strategies.
Topics: Animals; Insecticides; Diptera; United Arab Emirates; Pyrethrins; Houseflies; Mutation; Insecticide Resistance; Nitriles
PubMed: 38302967
DOI: 10.1186/s13071-024-06128-5 -
Plant Disease Aug 2023Vernonia cinerea (L.)Less. is an annual herbaceous plant of the Asteraceae family, which is widely distributed throughout Southeast Asia, India, and the tropical and...
Vernonia cinerea (L.)Less. is an annual herbaceous plant of the Asteraceae family, which is widely distributed throughout Southeast Asia, India, and the tropical and subtropical regions of China. This herb is known to contain various bioactive compounds and is commonly used in traditional system of medicine in China and India (Singh et al. 2021). During the spring of 2022 and 2023, powdery mildew symptoms were observed on 70% of V. cinerea plants on the Hainan Medical University campus (19° 58' 53″ N; 110° 19' 47″ E) in Haikou, Hainan Province, China. Powdery mildew colonies covered the leaf surfaces and stems of affected plants, causing discoloration and defoliation. Mycelia were superficial and hyphal appressoria were nipple-shaped. Conidiophores (n =30) were unbranched, cylindrical, 104 to 188 × 11 to 15 µm, and produced three to five immature conidia in chains with a crenate outline. Foot cells (n =30) were cylindrical, straight or sometimes curved at the base, and 32 to 57 µm long. Conidia (n =100) were ellipsoid-ovoid to doliiform, 19 to 45 ×16 to 26 m (length/width ratio = 1.2 to 2.4), with well-developed fibrosin bodies, and produced germ tubes from the lateral position. Based on these morphological characteristics, the pathogen was provisionally identified as Podosphaera xanthii (Braun and Cook 2012). The teleomorph was not observed. A specimen was deposited in the Hainan Medical University Plant Pathology Herbarium as HMVC-22. To confirm the genus identification and ascertain a putative species, genomic DNA was extracted from mycelium, conidiophores, and conidia using a fungal DNA kit (Omega Bio-Tek, USA). The rDNA internal transcribed spacer (ITS) region was amplified with primers ITS1/ITS4 (White et al. 1990) and sequenced directly. The resulting 577-bp sequence was deposited in GenBank (accession no. OP765400). A BLASTn search in GenBank of this sequence showed 100% similarity with the ITS sequences of P. xanthii isolates from China (MT260063, MT739423 and MT131253), Thailand (LC270780), and Vietnam (KM260731). Additionally, the 28S rDNA region was amplified using the primer pairs NL1 and NL4 (O´Donnell 1993; accession no. OP765401). This region shared 100% similarity with P. xanthii isolates (LC371333, LC270780, and AB936277) as well. Powdery mildew from Hainan sample belonged to the P. xanthii group with strong bootstrap values support 99% in maximum likelihood phylogenetic tree based on ITS and 28S gene sequences. To confirm pathogenicity, five healthy potted plants of V. cinerea were inoculated by gently pressing a powdery mildew-infected leaf onto 15 young leaves. Five non-inoculated plants served as controls. All plants were maintained in a greenhouse at 24 to 30°C, 70% relative humidity, with a 16-h photoperiod. After 7 days, inoculated leaves showed powdery mildew symptoms whereas no symptoms were observed on control plants. The fungal colonies observed on inoculated plants were morphologically identical to those found on the originally infected leaves collected from Hainan Province. Based on the morphological characteristics and molecular identification, the fungus was identified as P. xanthii. In different countries and regions, Erysiphe cichoracearum has been previously reported on some Vernonia species, including V. Galamensis from southern parts of Ethiopia (Hundesa and Mekonnen 2017). To our knowledge, this is the first record of P. xanthii infecting V. cinerea in China. We are concerned that the pathogen could become a threat to the widespread planting of V. cinerea in the future.
PubMed: 37610364
DOI: 10.1094/PDIS-05-23-0944-PDN -
PeerJ 2023In recent years, environmental DNA (eDNA) technology has become an accepted approach for investigating rare and endangered species because of its economic efficiency,...
In recent years, environmental DNA (eDNA) technology has become an accepted approach for investigating rare and endangered species because of its economic efficiency, high sensitivity, and non-invasiveness. The Asian giant softshell turtle () is a first-class protected aquatic animal in China, and traditional resource survey methods have not identified its natural populations for many years. In this study, primers and a TaqMan probe targeting were designed, reaction conditions were optimized, a standard curve was constructed using synthetic DNA, and an eDNA quantitative PCR (qPCR) detection method was established. The eDNA detection technology for revealed that the number of species in the experimental pools showed a significant linear relationship with the eDNA concentration ( < 0.05). The eDNA concentration was negatively correlated with the length of time after the removal of and retention in the water body for 9 days. The qPCR detection method for eDNA established in this study can be applied to the qualitative detection of in water bodies, as well as to preliminary evaluation of its relative biomass. This can serve as a baseline for the investigation of natural population and the evaluation of its wild release effects.
Topics: Animals; DNA, Environmental; Biomass; Turtles; Reptiles; Water
PubMed: 37810767
DOI: 10.7717/peerj.16218 -
Infectious Agents and Cancer Dec 2023The role of high-risk human papillomaviruses (hr-HPVs) in cervical cancer (CC) pathogenesis has long been established. Knowledge about the involvement of hr-HPVs in the...
BACKGROUND
The role of high-risk human papillomaviruses (hr-HPVs) in cervical cancer (CC) pathogenesis has long been established. Knowledge about the involvement of hr-HPVs in the etiology of nasopharyngeal cancers (NPC) was not well appreciated until the early 2000s when a clear link began to emerge. However, it is not clear whether HPV oncogenesis in the different epithelial cancers is associated with L1 gene and long-control region (LCR) sequences variation. This study aimed to investigate the HPV18 L1 gene and LCR sequences variation in cervical and nasopharyngeal biopsies, and assessed E6 and E7 genes expression level in both cancers.
METHOD
Four-hundred and three (403) formalin-fixed paraffin-embedded tissues originating from nasopharyngeal (NPC) (279) and cervical (CC) (124) sites were collected from a pathology laboratory, Pathologist Without Borders, Accra, Ghana. Haematoxylin and eosin staining was carried out to confirm the presence of cancer on prepared biopsy sections. DNA was extracted from the confirmed cancer biopsies, followed by PCR using MY09/GP5+ /6+ primers to detect the presence of HPV and specific primers for the amplification of L1 gene and LCR. Sanger sequencing was carried out to determine HPV genotypes, and L1 and LCR sequences variant of HPV18s in CC and NPC biopsies. The HPV18 E6/E7 mRNA expression pattern in both cancers was determined using RT-qPCR.
RESULTS
Most of the NPC (45%) and CC (55%) biopsies were HPV18 positive. Comparison of HPV18 L1 sequences obtained from cervical and nasopharyngeal cancer tissues, the L1 sequences from the NPC were highly dissimilar with a 59-100% variation among themselves, and in relation to the reference strains. However, the L1 sequences from the CC were more similar with a 91.0-100% variation among the amplified sequences. Also, the LCR sequences from CC were quite different relative to that of NPC. Results for the differential expression of E6/E7 in the two cancers showed a higher fold change in E6 expression in the CC tissues than the NPC tissues while a reverse expression pattern was found for E7 gene.
CONCLUSION
The current study reports for the first-time variations in HPV18 L1 and LCR sequences, and differential expression of E6/E7 genes in NPC compared to CC, suggesting a possible adaptation mechanism of the virus at different cancer sites.
PubMed: 38037052
DOI: 10.1186/s13027-023-00560-5 -
Genes Sep 2023Dahlia () is a widely cultivated ornamental and medicinal plant in China. Recently, dahlia plants with symptoms of leaf mottling and distortion were collected in Hohhot,...
Dahlia () is a widely cultivated ornamental and medicinal plant in China. Recently, dahlia plants with symptoms of leaf mottling and distortion were collected in Hohhot, Inner Mongolia, China. The presence of dahlia common mosaic virus (DCMV), an unassigned species in the genus , was confirmed by high-throughput sequencing. Three fragments of DCMV Inner Mongolia isolate (DCMV-IN) were PCR-amplified with specific primers, sequenced and assembled into the complete genome sequence with a GenBank accession number of OR494328. The double-stranded circular DNA genome of DCMV-IN consists of 7949 bp and contains six open reading frames (ORFs). Sequence analysis showed that DCMV-IN shared high sequence identities with other DCMV isolates available in the GenBank database. Phylogenetic analysis of DCMV isolates and other representative caulimoviruses based on genome sequence clustered four DCMV isolates to a single branch which was closest to dahlia mosaic virus (DMV). No recombination event was detected among the four DCMV isolates.
Topics: Caulimovirus; Dahlia; Phylogeny; Genome, Viral; Polymerase Chain Reaction
PubMed: 37895182
DOI: 10.3390/genes14101833 -
Infection and Drug Resistance 2023The currently used conventional susceptibility testing for drug-resistant is limited due to being time-consuming and having low efficiency. Herein, we propose the use...
BACKGROUND
The currently used conventional susceptibility testing for drug-resistant is limited due to being time-consuming and having low efficiency. Herein, we propose the use of a microfluidic-based method to rapidly detect drug-resistant gene mutations using Kompetitive Allele-Specific PCR (KASP).
METHODS
A total of 300 clinical samples were collected, and DNA extraction was performed using the "isoChip" Mycobacterium detection kit. Phenotypic susceptibility testing and Sanger sequencing were performed to sequence the PCR products. Allele-specific primers targeting 37 gene mutation sites were designed, and a microfluidic chip (KASP) was constructed using 112 reaction chambers to simultaneously detect multiple mutations. Chip validation was performed using clinical samples.
RESULTS
Phenotypic susceptibility of clinical isolates revealed 38 rifampicin (RIF)-resistant, 64 isoniazid (INH)-resistant, 48 streptomycin (SM)-resistant and 23 ethambutol (EMB)-resistant strains, as well as 33 multi-drug-resistant TB (MDR-TB) strains and 20 strains fully resistant to all four drugs. Optimization of the chip-based detection system for drug resistance detection showed satisfactory specificity and maximum fluorescence at a DNA concentration of 1×10 copies/µL. Further analysis revealed that 76.32% of the RIF-resistant strains harbored gene mutations (sensitivity, 76.32%; specificity 100%), 60.93% of the INH-resistant strains had gene mutations (sensitivity, 60.93%; specificity, 100%), 66.66% of the SM-resistant strains carried drug resistance gene mutations (sensitivity, 66.66%; specificity, 99.2%), and 69.56% of the EMB-resistant strains had gene mutations (sensitivity, 69.56%; specificity, 100%). Further, the overall agreement between the microfluidic chip and Sanger sequencing was satisfactory, with a turnaround time of the microfluidic chip was approximately 2 hours, much shorter than the conventional DST method.
CONCLUSION
The proposed microfluidic-based KASP assay provides a cost-effective and convenient method for detecting mutations associated with drug resistance in . It represents a promising alternative to the traditional DST method, with satisfactory sensitivity and specificity and a much shorter turnaround time.
PubMed: 37424666
DOI: 10.2147/IDR.S410779 -
Scientific Reports Jul 2023The practise of restocking and stock improvement as a means of managing fisheries and aquaculture has been widely used. However, it is difficult to claim that fish...
The practise of restocking and stock improvement as a means of managing fisheries and aquaculture has been widely used. However, it is difficult to claim that fish stocking is effective due to a number of challenges. One of those is the lack of suitable monitoring and assessment methods, although all assessment approaches have their strengths and weaknesses. If the full benefits of fisheries and their long-term sustainability are to be realised, it is necessary to examine the effectiveness of restocking and stock enhancement. Therefore, effective, rapid, and dependable monitoring techniques are necessary. In this study, we used an eDNA-based method to identify G. cambodgiensis at 14 sites throughout Thailand's restocking and stock enhancement programme. eDNA from this species was identified in water samples using quantitative polymerase chain reaction (qPCR) tests with primers and a probe specific to G. cambodgiensis. A successful stocking would show positive eDNA results in water samples collected from the studied sites. Only five of the studied sites returned positive eDNA readings, which could be considered a successful stocking. The locations that contained G. cambodgiensis eDNA were either confirmed to be natural habitats or were regularly stocked with a large number of hatchery fish. In this study, we demonstrated that eDNA is a reliable, fast and accurate alternative method for measuring stock improvement.
Topics: Animals; Fisheries; DNA, Environmental; Aquaculture; DNA Primers; Water
PubMed: 37438384
DOI: 10.1038/s41598-023-38218-2 -
Iranian Journal of Parasitology 2023We aimed to determine species of liver fluke that predominately cause fascioliasis in sheep, goats, and cattle in the Sulaymaniyah Province, Iraq using the molecular...
BACKGROUND
We aimed to determine species of liver fluke that predominately cause fascioliasis in sheep, goats, and cattle in the Sulaymaniyah Province, Iraq using the molecular technique of DNA sequencing and restriction fragment length polymorphism (RFLP).
METHODS
The samples were collected from November 2021 to May 2022. The flukes were collected from infected livers of livestock at the slaughterhouse of Sulaymaniyah Governorate, Iraq. A total of 205 flukes were collected from 56 hosts, cattle (n=22), sheep (n=28), and goats (n=6). The specific primers for FCOX1 and 28S rDNA gene amplification were used. The PCR products were subjected to restriction fragment polymorphism (RFLP) assay using and restriction enzymes, besides DNA sequencing.
RESULTS
The results showed the genetic polymorphisms among the flukes. Three patterns of RFLP were observed and , where 28 of them displayed (sheep, n=14, goat, n=3 and cattle, n= 11), whereas 24 samples displayed the (sheep, n=12, goat, n=3 and cattle, n= 9), and only four samples belonged to (sheep n=3 and cattle, n=1). In addition, the result of the ribosomal DNA (28S rDNA) sequencing confirmed that the isolated flukes belonged to , and .
CONCLUSION
All three main species are present in the study area and predominated among the animal species in this area also, our results concluded that PCR-RFLP is a rapid and reliable method for liver fluke species identification.
PubMed: 38169619
DOI: 10.18502/ijpa.v18i4.14264 -
Pediatric Research Oct 2023Interleukin-17F (IL-17F), one of the cytokines, is crucial in the pathophysiology of juvenile idiopathic arthritis (JIA). Therefore, we aimed to determine the relation...
BACKGROUND
Interleukin-17F (IL-17F), one of the cytokines, is crucial in the pathophysiology of juvenile idiopathic arthritis (JIA). Therefore, we aimed to determine the relation between IL17F 7488A/G and IL17F 7383A/G single-nucleotide polymorphisms and JIA susceptibility and to explain their impact on the disease activity.
METHODS
Genomic DNA of 70 patients with JIA and 70 age and sex-matched controls were extracted and typed for IL17F 7488A/G and IL17F 7383A/G single-nucleotide polymorphisms, using polymerase chain reaction with sequence-specific primers method, and compared between patients and controls.
RESULTS
When compared to AA participants, children with the AG genotype of the IL17F 7488A/G and IL17F 7383A/G polymorphisms showed a substantially greater risk of JIA. Furthermore, children with the G allele were 2.8 folds more likely to have JIA than the A allele for IL17F 7488A/G polymorphism and 3.72 folds for IL17F 7383A/G polymorphism. Children with AG genotype of IL17F 7383A/G polymorphism were far more likely to have high activity JIA.
CONCLUSIONS
The G allele of both IL17F 7488A/G and IL17F7383 A/G polymorphisms is associated with increased JIA susceptibility, and JIA at High Disease Activity was more likely to develop in AG subjects of the IL17F 7383 A/G polymorphism.
IMPACT
The relationship between Interleukin-17F 7488A/G and 7383A/G polymorphisms and risk for JIA has not been recognized before. Impact of Interleukin-17F 7488A/G and 7383A/G genotypes on JIA disease activity. The G allele of both IL17F 7488A/G and IL17F7383 A/G polymorphisms are associated with increased JIA susceptibility. AG genotype of Interleukin-17F 7383 A/G polymorphism compared to AA patients, had a higher probability of developing JIA at a High Disease Activity (HDA) level.
Topics: Child; Humans; Arthritis, Juvenile; Case-Control Studies; Genetic Predisposition to Disease; Genotype; Interleukin-17; Polymorphism, Single Nucleotide
PubMed: 36068342
DOI: 10.1038/s41390-022-02288-1 -
Pathogens (Basel, Switzerland) May 2024has a complex lifecycle with multiple intermediate and definitive hosts and influenced by environmental factors. The disease causes significant morbidity in children...
A PCR Test Using the Mini-PCR Platform and Simplified Product Detection Methods Is Highly Sensitive and Specific to Detect DNA Mixed in Human Stool, Snail Tissue, and Water DNA Specimens.
has a complex lifecycle with multiple intermediate and definitive hosts and influenced by environmental factors. The disease causes significant morbidity in children and its prevalent worldwide. There is lack of data about distribution and burden of the disease in endemic regions, owing to poor efficacy of the different diagnostic methods used. A novel PCR-based test was developed by using a portable mini-PCR platform to detect sp. DNA and interpret the results via a fluorescence viewer and smartphone image analyzer application. Human stool, snail tissue, and water samples were used to extract DNA. Primers targeting the ITS-1 of the 18S rDNA gene of sp. were used. The limit of detection of the mini-PCR test was 1 fg/μL for DNA samples diluted in water, 10 fg/μL for /snail DNA scramble, and 100 fg/μL for /stool DNA scramble. The product detection by agarose gel, direct visualization, and image analyses showed the same sensitivity. The Fh mini-PCR had a sensitivity and specificity equivalent to real-time PCR using the same specimens. Testing was also done on infected human stool and snail tissue successfully. These experiments demonstrated that Fh mini-PCR is as sensitive and specific as real time PCR but without the use of expensive equipment and laboratory facilities. Further testing of multiple specimens with natural infection will provide evidence for feasibility of deployment to resource constrained laboratories.
PubMed: 38921738
DOI: 10.3390/pathogens13060440