Conventional PCR Research Articles

A Novel Approach Based on Real-Time PCR with High-Resolution Melting Analysis for the Simultaneous Identification of Staphylococcus aureus and Staphylococcus argenteus.

Staphylococcus (S.) aureus is a pathogenic bacterium able to cause several diseases in humans and animals as well as foodborne intoxications. S. argenteus, being phenotypically and genotypically related to S. aureus, is part of the so-called S. aureus complex and recently recognized as an emerging pathogen able to cause, like S. aureus, several diseases both in humans and animals, and foodborne poisoning outbreaks. However, it has been reported that the widely used conventional PCR of Brakstad et al. [Journal of Clinical Microbiology, 30(7), 1654-1660, (1992)] targeting the thermostable nuclease gene may provide false-positive S. aureus, as it is able to amplify also S. argenteus. Here, we developed a novel two-step approach that, following the PCR of Brakstad et al. (1992), discriminates S. aureus from S. argenteus by a real-time PCR with high-resolution melting analysis (rt-PCR-HRM). In particular, targeting a polymorphic 137 bp region of the sodA gene, our developed rt-PCR-HRM method clearly discriminated S. aureus from S. argenteus, showing a remarkable difference in their amplification product melting temperatures (approximately 1.3 °C) as well as distinct melting curve shapes. The good sensitivity, reproducibility, user friendliness, and cost effectiveness of the developed method are advantageous attributes that will allow not only its easy employment to correctly identify misidentified isolates present in various collections of S. aureus, but also expand the still lacking knowledge on the prevalence and distribution of S. argenteus.

Development and application of a quantitative real-time PCR method for detection of Decapod iridescent virus 1.

As a newly discovered virus, Decapoda iridovirus 1 (DIV1) can cause a mortality rate of up to 100% in crustaceans, leading to huge economic losses. At present, there is no effective prevention and control measures for this disease. In the present study, the specific primers targeting highly conserved regions of MCP gene were designed, and then a quantitative real-time PCR method was established. The results indicate that DIV1 quantitative real-time PCR established has good specificity and does not cross react with other pathogens including white spot syndrome virus (WSSV), infectious subcutaneous and hematopoietic necrosis virus (IHHNV) and Vibrio parahaemolyticus induced acute hepatopancreatic necrosis disease (VpAHPND). The real-time PCR was capable of detecting DIV1 DNA at a minimum concentration of 10 copies/μL within 34 cycles. The method has good repeatability, with intra group and inter group coefficients of variation both less than 2%. Thirty-two clinical samples were assessed using both the real-time PCR and conventional PCR. The results shown real-time PCR we established are more sensitive than conventional PCR. In conclusion, this method has strong specificity, stable repeatability, and high sensitivity, providing technical support for clinical diagnosis, epidemiology investigation and monitoring of DIV1.

Predominance of blaNDM- and blaIMP-Harboring Escherichia coli Belonging to Clonal Complexes 131 and 23 in a Major University Hospital.

Background and Objectives: Carbapenem resistance is a growing global challenge for healthcare, and, therefore, monitoring its prevalence and patterns is crucial for implementing targeted interventions to mitigate its impact on patient outcomes and public health. This study aimed to determine the prevalence of carbapenem resistance among Escherichia coli (E. coli) strains in the largest tertiary care hospital of the capital territory of Pakistan and to characterize the isolates for the presence of antimicrobial resistance genes. Additionally, the most prevalent sequence types were analyzed. Materials and Methods: A total of 15,467 clinical samples were collected from November 2020 to May 2022, underwent antimicrobial susceptibility testing, and were analyzed for antimicrobial resistance genes through conventional PCR and sequence typing using MLST. Results: In carbapenem-resistant E. coli (CR-EC), 74.19% of isolates harbored the blaNDM gene, with blaNDM-1 (66.96%), blaNDM-5 (12.17%), and blaNDM-7 (20.87%) variants detected. Additionally, blaIMP was found in 25.81% and blaOXA-48 in 35.48% of isolates. The presence of blaCTX-M15 and blaTEM was identified in 83.87% and 73.55% of CR-EC isolates, respectively, while armA and rmtB were detected in 40% and 65.16% of isolates, respectively. Colistin and tigecycline were the most effective drugs against CR-EC isolates, with both showing an MIC50 of 0.5 µg/mL. The MIC90 for colistin was 1 µg/mL, while for tigecycline, it was 2 µg/mL. MLST analysis revealed that the CR-EC isolates belonged to ST131 (24.52%), ST2279 (23.87%), ST3499 (16.13%), ST8051 (15.48%), ST8900 (9.68%), ST3329 (7.10%), ST88 (1.94%), and ST6293 (1.29%). The ST131 complex (70.97%) was the most prevalent, harboring 95.65% of the blaNDM gene, while the ST23 complex (18.06%) harbored 62.50% of the blaIMP gene. Conclusions: Implementing large-scale surveillance studies to monitor the spread of specific pathogens, along with active infection control policies, is crucial for the effective containment and prevention of future epidemics.

Oral Chagas disease outbreak by bacaba juice ingestion: A century after Carlos Chagas' discovery, the disease is still hard to manage.

Orally transmitted acute Chagas disease (ACD) primarily affects low-visibility and low-income individuals in tropical and subtropical zones. Managing ACD remains challenging even after more than 100 years of its discovery. Its spread to non-endemic areas has made it a global health issue. The aim of this work is to demonstrate the difficulties encountered in handling a real-life situation. This report examines an outbreak of 39 cases of ACD due to oral transmission by bacaba juice ingestion that occurred in Pedro do Rosário, Maranhão, Brazil. A clinical and epidemiological investigation, including an entomological search, was conducted. Diagnosis criteria included positive peripheral blood smear (PBS), seroconversion of IgG, and a two-fold increase in IgG titer (laboratory criteria); and clinical findings, epidemiological exposure, and at least one positive IgG test (clinical-epidemiological criteria). In-house conventional polymerase chain reaction (PCR) was performed on 33 samples. All patients were treated with benznidazole. After 4.5 years, IgG levels were reassessed in 26 individuals. The mean age was 33.6 years, with no gender difference. The mean incubation period was 13.8 days, and the mean between symptom onset and treatment was 16.6 days. The most common symptoms were fever and lymphadenopathy (90%). Diagnostic success rates were 66.6% (laboratory criteria), 23% (clinical-epidemiological criteria), and 10.2% (high clinical suspicion despite negative tests). Test positivity rates were 69.7% (PBS), 91.4% (serology), and 100% (PCR). There were no deaths. Serological cure was achieved in 34.6% of cases, and IgG titers decreased in 15.3%. We encountered several barriers in managing ACD, including population vulnerability, reliance on outdated diagnostic techniques, lack of standardized molecular biology methods, and limited therapeutic options. This report underscores the importance of rapid surveillance and early treatment to prevent fatalities. We recommend the standardization of conventional PCR in diagnostic routines.

Pheno- and genotypic epidemiological characterization of Staphylococcus aureus isolated from bulk tank milk in Colombia

Staphylococcus aureus is recognized for causing contagious mastitis in cattle. Elimination of the organism in affected animals is challenging due to its potential antibiotic resistance mechanisms and virulence factors. The aim of the study was the pheno- and genotypic epidemiological characterization of S. aureus isolated from bulk tank milk samples obtained from three municipalities in the northern region of Antioquia. Twenty-one S. aureus isolates from 150 bulk tank milk samples were characterized by evaluating antimicrobial susceptibility to 17 antibiotics using the VITEK 2, and identifying several genes encoding for virulence factors, enterotoxins, and the agr groups by conventional PCR. The clonal relationship between the isolates was assessed using macrorestriction fragment analysis (MRFA) of chromosomal DNA by pulsed-field gel electrophoresis (PFGE). Most of the isolates showed susceptibility to the tested antibiotics; 19% exhibited resistance to tetracycline and all isolates showed beta-lactamases. Molecular typing revealed that 76% carried the agrI and cap5 genes. Seven isolates harbored genes coding for staphylococcal enterotoxins (SE), with seh being the most identified gene. Furthermore, MRFA demonstrated high heterogeneity among the isolates, resulting in the assignment of 18 different MRFA patterns. These results indicate phenotypic resistance to tetracycline and beta-lactams, the presence of genes encoding for staphylococcal enterotoxins, and high heterogeneity among S. aureus isolates from bulk tank milk.

Droplet Digital PCR for Acinetobacter baumannii Diagnosis in Bronchoalveolar Lavage Samples from Patients with Ventilator-Associated Pneumonia.

Advanced diagnostic technologies have made accurate and precise diagnosis of pathogens easy. Herein, we present a new diagnostic method, droplet digital PCR (ddPCR), to detect and quantify Acinetobacter baumannii in mini bronchoalveolar lavage (mini-BAL) samples. A. baumannii causes ventilator-associated pneumonia (VAP), a severe healthcare infection affecting patients' lungs. VAP carries a high risk of morbidity and mortality, making its timely diagnosis crucial for prompt and effective management. Methodology. The assay performance was evaluated by comparing colonization data, quantitative culture results, and different generations of PCR (traditional PCR and Real-Time PCR-qPCR Taqman® and SYBR® Green). The ddPCR and qPCR Taqman® prove to be more sensitive than other molecular techniques. Reasonable analytical specificity was obtained with ddPCR, qPCR TaqMan®, and conventional PCR. However, qPCR SYBR® Green technology presented a low specificity, making the results questionable in clinical samples. DdPCR detected/quantified A. baumanni in more clinical samples than other methods (38.64% of the total samples). This emerging ddPCR technology offers promising advantages such as detection by more patients and direct quantification of pathogens without calibration curves.

Recombinase-aid amplification combined with lateral flow detection assay for sex identification of the great white pelican (Pelecanus onocrotalus)

Sex identification in avian species is essential for biodiversity conservation and ecological studies. However, the sex of nearly half of the birds could not be identified based on their external appearance. It is difficult to visually identify sex to monitor the ecology and conservation of wild populations. In this study, we designed primer pairs for large white pelican using recombinase-based isothermal amplification combined with a lateral flow dipstick (RAA-LFD) assay for chromo-helicase-DNA binding protein (CHD) genes mapped to W chromosomes and an ultra-conserved element (UCE) located on chromosome 6, respectively. Our result showed that the raaW4-RAA-LFD can detect up to 0.1 ng of genomic DNA (gDNA) templates of female pelicans in 30 min at 39 ℃ and accurately distinguish female from male without any cross reactivity. RaaUCE2-RAA-LFD can amplify both male and female pelicans with a detection limit of 25 pg. To further evaluate the assay, 15 white pelicans of unknown sex were tested using the RAA-LFD assay and conventional polymerase chain reaction (PCR). The results of the raaW4-RAA-LFD assay were consistent with those of the conventional PCR. The developed RAA-LFD assay is equipped with field-deployable instruments and offers a field platform for rapid and reliable sex identification in pelicans.

Widespread geographic distribution of Aedes aegypti (Diptera: Culicidae) kdr variants in Panama.

We searched for evidence of knockdown resistance (kdr) mutations in the voltage-gated sodium channel gene of Aedes aegypti (Linnaeus) (Diptera: Culicidae) and Aedes albopictus (Skuse) (Diptera: Culicidae) mosquitoes from Panama. Conventional PCR was performed on 469 Ae. aegypti and 349 Ae. albopictus. We did not discover kdr mutations in Ae. albopictus, but 2 nonsynonymous kdr mutations, V1016I (found in 101 mosquitoes) and F1534C (found in 29 of the mosquitoes with the V1016I), were detected in Ae. aegypti. These kdr mutations were present in all specimens that were successfully sequenced for both IIS5-S6 and IIIS6 regions, which included samples collected from 8 of the 10 provinces of Panama. No other kdr mutations were found in Ae. aegypti, including V1016G, which has already been reported in Panama. Findings suggest that the V1016I-F1534C variant is prevalent in Panama, which might be related to the introduction and passive movement of mosquitoes as part of the used-tire trade. However, we cannot rule out the possibility that selection on de novo replacement of kdr mutations also partially explains the widespread distribution pattern of these mutations. These 2 ecological and evolutionary processes are not mutually exclusive, though, as they can occur in tandem. Research in Panama needs to calculate the genotypic and allelic frequencies of kdr alleles in local Ae. aegypti populations and to test whether some combinations confer phenotypic resistance or not. Finally, future studies will have to track the introduction and spreading of new kdr mutations in both Aedes species.

Molecular, Hematological and Biochemical Investigation of Trypanosoma spp. in Sheep

Background: Trypanosoma spp. is a flagellated unicellular protozoan parasite which caused a disease known as trypanosomiasis in various animals among many areas worldwide resulting in severe economic losses due to morbidities and mortalities. This study was conducted to estimate the prevalence of Trypanosoma spp. in sheep using the molecular assay with detection the impact of infection on various risk factors and blood markers. Methods: Totally, 341 sheep were selected from different areas in Wasit province (Iraq) during June to September (2023) and subjected to draining the venous blood that tested molecularly by the conventional polymerase chain reaction (PCR). Status of different hematological, biochemical and mineral markers were further investigated. Result: Targeting 16S sRNA gene, 7.92% animals were positively infected with trypanosomiasis. Relation to risk factors, positive infection and risk were significantly higher in females than males and in sheep aged greater than 1-3 years than others. Hematological findings reported a significant reduction in RBCs and Hb; while biochemically, significant elevation was reported in concentration of urea. Regarding various minerals, insignificant alteration was seen in infected sheep when compared to healthy ones.

Investigation of Fasciola gigantica in freshwater snail Radix (Lymnaea) spp. In the highly parasite-prevalent area of Nakhon Ratchasima Province, Thailand

ABSTRACT This study investigates the distribution of the Lymnaea (Radix) spp. in Pak Chong district, Nakhon Ratchasima province, northeast Thailand, where a vast cattle farming area is located and has a high prevalence of Fasciola spp. in the cattle. By random selection, 1,414 snails were collected from the natural and man-made ponds. The snails were recorded for morphology and processed for DNA isolation. The snail species were investigated by conventional PCR using a 16S rDNA-specific primer. The result demonstrated that all collected snails were R. (L.) rubiginosa. Moreover, the infection of Fasciola gigantica in the snails was investigated by PCR using a cytochrome c oxidase I (COX1)-specific primer. The results illustrated that the overall prevalence was 22.5% (318/1414), with the highest prevalence in the Nong Sa Rai subdistrict at 73.6% (81/110), which is the highest prevalence of Fasciola gigantica in the snail host that has ever been reported. The lowest prevalence existed in the Pong Ta Long subdistrict at 3.7% (4/109). Our results corresponded to the previous report on the Fasciola spp. infection in the cattle from this area, and the geographical analysis revealed that the most suspected factor would be the earth dam located in these subdistricts, where many animals live freely during the day. Our findings could be helpful for further parasite control and could trigger the study of the biology and associated factors in the future.

Recombinase-aided amplification assay for rapid detection of imipenem-resistant Pseudomonas aeruginosa and rifampin-resistant Pseudomonas aeruginosa.

The indiscriminate use of antibiotics has resulted in a growing resistance to drugs in Pseudomonas aeruginosa. The identification of antibiotic resistance genes holds considerable clinical significance for prompt diagnosis. In this study, we established and optimized a Recombinase-Aided Amplification (RAA) assay to detect two genes associated with drug resistance, oprD and arr, in 101 clinically collected P. aeruginosa isolates. Through screening for the detection or absence of oprD and arr, the results showed that there were 52 Imipenem-resistant P. aeruginosa (IRPA) strains and 23 Rifampin-resistant P. aeruginosa (RRPA) strains. This method demonstrated excellent detection performance even when the sample concentration is 10 copies/μL at isothermal conditions and the results could be obtained within 20 minutes. The detection results were in accordance with the results of conventional PCR and Real-time PCR. The detection outcomes of the arr gene were consistently with the resistance spectrum. However, the antimicrobial susceptibility results revealed that 65 strains were resistant to imipenem, while 49 strains sensitive to imipenem with oprD were identified. This discrepancy could be attributed to genetic mutations. In summary, the RAA has higher sensitivity, shorter time, and lower-cost instrument requirements than traditional detection methods. In addition, to analyze the epidemiological characteristics of the aforementioned drug-resistant strains, we conducted Multilocus Sequence Typing (MLST), virulence gene, and antimicrobial susceptibility testing. MLST analysis showed a strong correlation between the sequence types ST-1639, ST-639, ST-184 and IRPA, while ST-261 was the main subtype of RRPA. It was observed that these drug-resistant strains all possess five or more virulence genes, among which exoS and exoU do not coexist, and they are all multidrug-resistant strains. The non-coexistence of exoU and exoS in P.aeruginosa is related to various factors including bacterial regulatory mechanisms and pathogenic mechanisms. This indicates that the relationship between the presence of virulence genes and the severity of patient infection is worthy of attention. In conclusion, we have developed a rapid and efficient RAA (Recombinase-Aided Amplification) detection method that offers significant advantages in terms of speed, simplicity, and cost-effectiveness (especially in time and equipment aspect). This novel approach is designed to meet the demands of clinical diagnostics.

Development of a RPA-CRISPR/Cas12a based rapid visual detection assay for Porcine Parvovirus 7.

Porcine Parvovirus (PPV) is a significant pathogen in the pig industry, with eight genotypes, including PPV7, identified since its emergence in 2016. Co-infections with viruses such as Porcine Circovirus 2 (PCV2) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) pose serious risks to swine health. Thus, there is an urgent need for rapid, sensitive, and specific detection methods suitable for use in field settings or laboratories with limited resources. We developed a CRISPR/Cas12a-based assay combined with recombinase polymerase amplification (RPA) for the rapid detection of PPV7. Specific RPA primers and five CRISPR RNAs (crRNAs) were designed to target a highly conserved region within the NS1 gene of PPV7. Optimization of crRNA and single-stranded DNA (ssDNA) concentrations was performed to enhance the assay's performance. CrRNA optimization identified crRNA-05 as the optimal candidate for Cas12a-based detection of PPV7, as all synthesized crRNAs demonstrated similar performance. The optimal crRNA concentration was determined to be 200 nM, yielding consistent results across tested concentrations. For ssDNA optimization, the strongest fluorescence signal was achieved with 500 nM of the FAM-BHQ ssDNA receptor. The assay showed a minimal detection limit of 100copies/μl for PPV7, confirmed through fluorescence and lateral flow detection methods. Specificity testing indicated that only PPV7 DNA samples returned positive results, confirming the assay's accuracy. In tests of 50 lung tissue samples from diseased pigs, the RPA-Cas12a assay identified 29 positive samples (58%), surpassing the 22 positive samples (44%) detected by conventional PCR. This highlights the RPA-Cas12a method's enhanced detection capability and its potential utility in clinical surveillance and management of PPV7 in swine populations. The RPA-Cas12a assay effectively detects PPV7 in clinical samples, enhancing disease surveillance and control in pigs. Its adaptability to resource-limited settings significantly improves PPV7 management and prevention strategies, thereby supporting the overall health and development of the pig industry.

Molecular and morphological analysis revealed a new Lipoptena species (Diptera: Hippoboscidae) in southern Spain harbouring Coxiella burnetii and bacterial endosymbionts

Hippoboscid flies (Diptera: Hippoboscidae) are obligate bloodsucking ectoparasites of animals. In Europe, limited research has been conducted on this family until the recent introduction of the deer ked Lipoptena fortisetosa Maa, 1965. A new species of the genus Lipoptena, Lipoptena andaluciensis sp. nov., was found in southern Spain after extensive sampling with carbon-dioxide baited suction traps. A total of 52 females and 32 males were collected at 29 out of 476 sites examined over eight months in 2023. Lipoptena andaluciensis sp. nov. was characterized morphologically and molecularly. The new Lipoptena species can be differentiated from the closely related L. fortisetosa by size, chaetotaxy of the dorsal and ventral thorax, abdominal plates, and genitalia. Based on DNA-barcoding, our specimens showed the highest similarity with Melophagus ovinus (Linnaeus, 1758) (88.4 %) and with L. fortisetosa (86–88 %). Individual screening of Lipoptena specimens (n = 76) for seven important zoonotic pathogens such as bacteria (Anaplasmataceae family: Bartonella spp., Borrelia spp., Coxiella burnetii and Rickettsia spp.) and protozoans (Babesia spp. and Theileria spp.) by conventional PCR and RT-PCR was performed. DNA of C. burnetii was detected in one specimen, while two other specimens harboured Anaplasmataceae (Wolbachia spp., 100 % homology and another endosymbiont probably related to Arsenophonus sp., 95.3 % homology, respectively), all representing the first records of these bacteria in the Lipoptena spp. from Europe. Carbon dioxide traps probed its effectiveness as a reliable passive method for keds surveillance. Our study highlights the existence of a new Lipoptena species, presumably widely distributed in southern Spain. The role of this species in the transmission cycle of pathogens of medical-veterinary relevance needs to be considered in the area.

Comparative analysis of reverse-transcription-polymerase chain reaction for Aichivirus detection.

Aichivirus-A (AiV-A), a member of the Kobuvirus genus of the family Picornaviridae, was first reported in stool samples of patients with non-bacterial gastroenteritis in Aichi Prefecture, Japan, in 1989. AiV has been reported from in various aquatic environments, such as surface water and sewage, can be transmitted via the fecal-oral route through contaminated water. As AiV is known to acute gastroenteritis worldwide, developing methods for AiV detection from contaminated environments and food is required. In the present study, we established an effective polymerase chain reaction (PCR) method to detect AiV. Various real-time reverse transcription (RT)-PCR and conventional PCR methods for AiV detection were compared, and the limit of detection was confirmed by comparing the sensitivity at varied primer concentrations and PCR conditions. The final detection limits were 102 copy/μL in conventional PCR, and 101 copy/μL in the real-time RT-PCR. The optimized method used in this study might aid in detecting AiV contamination.

Innovation and Patenting Activities During COVID-19 and Advancement of Biochemical and Molecular Diagnosis in the Post- COVID-19 Era.

The COVID-19 pandemic is to escalate globally and acquire new mutations quickly, so accurate diagnostic technologies play a vital role in controlling and understanding the epidemiology of the disease. A plethora of technologies acquires diagnosis of individuals and informs clinical management of COVID. Some important biochemical parameters for COVID diagnosis are the elevation of liver enzymes, creatinine, and nonspecific inflammatory markers such as C-reactive protein (CRP) and Interleukin 6 (IL-6). The main progression predictors are lymphopenia, elevated D-dimer, and hyperferritinemia, although it is also necessary to consider LDH, CPK, and troponin in the marker panel of diagnosis. Owing to the greater sensitivity and accuracy, molecular technologies such as conventional polymerase chain reaction (PCR), reverse transcription (RT)-PCR, nested PCR, loop-mediated isothermal amplification (LAMP), and xMAP technology have been extensively used for COVID diagnosis for some time now. To make so many diagnostics accessible to general people, many techniques may be exploited, including point of care (POC), also called bedside testing, which is developing as a portable promising tool in pathogen identification. Some other lateral flow assay (LFA)-centered techniques like SHERLOCK, CRISPR-Cas12a (AIOD-CRISPR), and FNCAS9 editor limited uniform detection assay (FELUDA), etc. have shown auspicious results in the rapid detection of pathogens. More recently, low-cost sequencing and advancements in big data management have resulted in a slow but steady rise of next-generation sequencing (NGS)-based approaches for diagnosis that have potential relevance for clinical purposes and may pave the way toward a better future. Due to the COVID-19 pandemic, various institutions provided free, specialized websites and tools to promote research and access to critically needed advanced solutions by alleviating research and analysis of data within a substantial body of scientific and patent literature regarding biochemical and molecular diagnosis published since January 2020. This circumstance is unquestionably unique and difficult for anyone using patent information to find pertinent disclosures at a specific date in a trustworthy manner.

Molecular genotyping and subgenotyping of duck circovirus at duck farms in Thailand.

Ducks worldwide are infected with duck circovirus (DuCV), which causes feather abnormality, emaciation, and poor growth performance. DuCV is similar to other circoviruses that induce immunosuppression due to the occurrence of the bursae of Fabricius (BF) and spleen atrophies. In Thailand, retarded ducks with feather losses were submitted for disease investigation. The ducks presented low body weight gain, had small BF and spleens, and were consistent with duck-infected DuCV. Our study investigated the possibility of DuCV infection in duck flocks in Thailand. We also analyzed the genetic characteristics of the virus. BF and spleen samples were collected from affected meat and layer ducks from six farms thought to have been infected with DuCV. These tissues were then subjected to histopathological examination and molecular identification using conventional polymerase chain reaction and nucleotide sequencing. To identify DuCV, phylogenetic trees were generated using MEGA version X software. Samples of tissues or swabs were collected to determine whether coinfections with bacteria and viruses existed. Phylogenetic analysis using the entire genome (1995-1996 bp) and cap gene (762 bp) revealed that the DuCV isolates circulating in Thailand belonged to DuCV genotype I, which was further subdivided into two sub-genotypes: sub-genotype I b and an unclassified sub-genotype based on reference sub-genotypes. Thai isolates have variations in 10 amino acid residues in the capsid protein. Ducks infected with Thai DuCV were also coinfected with Riemerella anatipestifer, Escherichia coli, Pasteurella multocida, duck viral enteritis, and duck Tembusu virus, which is consistent with previous DuCV infection studies. Six DuCVs from ducks who were previously found to have feather loss, were underweight, had growth retardation, and had poor body condition were identified in this study as belonging to genotype I and constituting at least two sub-genotypes. Due to the immunosuppressive effects of DuCV, coinfection of bacterial and viral pathogens was typically observed in Thai DuCV-infected ducks.

Development of a highly specific LAMP assay for detection of Sarcocystis tenella and Sarcocystis gigantea in sheep.

Sarcocystis infection in sheep has caused significant economic losses in the livestock industry, and the genetic similarity among Sarcocystis species highlights the need for precise diagnostic methods in sheep. This study developed a loop-mediated isothermal amplification (LAMP) method targeting COX-1 and 28S rRNA genes to detect Sarcocystis tenella and Sarcocystis gigantea, respectively. The LAMP method exhibited high specificity, selectively amplifying target DNA sequences without cross-reactivity with closely related protozoa, such as Toxoplasma gondii and Neospora caninum. Detection limits were determined as 3 × 105 copies/L for S. tenella and 6 × 104 copies/L for S. gigantea, enabling sensitive identification of low-level infections. Comparative analysis with conventional PCR on sheep cardiac tissues demonstrated a higher LAMP detection rate (80.0% vs 66.7%). In conclusion, the LAMP method offers superior sensitivity to conventional PCR, allows visual confirmation of results, and provides a rapid diagnostic tool for identifying S. tenella and S. gigantea infection in sheep. However, due to the limitation of sample availability, we were unable to assess all Sarcocystis species that use sheep as intermediate hosts, which warrants further research.

Using Environmental DNA to Detect and Identify Sweetpotato Whitefly Bemisia argentifolii and Twospotted Spider Mite Tetranychus urticae in Greenhouse‐Grown Tomato Plants

ABSTRACTEnvironmental DNA (eDNA) consists of genetic material shed by living organisms, including those that are deceased, offering a unique opportunity to detect and identify terrestrial insect pests without requiring visual identification. The sweetpotato whitefly, Bemisia argentifolii, and the twospotted spider mite, Tetranychus urticae, are notorious for causing crop losses through virus transmission and direct feeding. Our study aimed to: (1) assess the effectiveness of B. argentifolii literature‐based PCR primers compared to newly developed primers for eDNA amplification, (2) evaluate the sensitivity of conventional PCR (cPCR) and real‐time quantitative PCR (qPCR) for detecting eDNA of B. argentifolii and T. urticae, (3) establish a rapid eDNA processing methodology using the LGC Biosearch Technologies QuickExtract DNA extraction kit and the Qiagen DNeasy Blood and Tissue kit, and (4) test the specificity of the developed primers against non‐target species. B. argentifolii and T. urticae were confined to tomato leaves (Solanum lycopersicum) using clip cages for 24 h, after which eDNA was collected from leaf surfaces using a water spray method, filtered, and processed for DNA amplification. While literature‐based primers showed sufficient sensitivity, their specificity for eDNA applications was inadequate, prompting the design of novel PCR primers for both pest species. Positive eDNA detection was achieved with both amplification methods, with qPCR proving more reliable than cPCR due to the latter's inconsistent performance with positive control samples. We also introduced a rapid eDNA processing approach using the QuickExtract DNA extraction kit, contrasting it with the more conventional Qiagen DNeasy Blood and Tissue kit. We believe that our findings are the first step toward the practical use of eDNA as a highly sensitive, early detection technique.

Diagnostic Value of Conventional Polymerase Chain Reaction for Detecting BRAF V595E Mutation in Liquid and Tissue Specimens of Canine Urothelial and Prostate Carcinomas.

Canine urothelial carcinoma (UC) and prostatic carcinoma (PC) often present diagnostic challenges due to their anatomical locations. The BRAF V595E mutation, analogous to the human BRAF V600E mutation, has been identified in UC and PC. Digital PCR of urine is a non-invasive diagnostic method of mutation detection, but the availability of the necessary equipment is limited. This study aimed to develop a conventional PCR to detect the BRAF V595E mutation in urine and prostatic wash specimens from dogs with UC or PC. Specific primers for detecting wild-type and mutant BRAF V595E genes were validated in 34 formalin-fixed paraffin-embedded (FFPE) tissues, 116 urine samples, and 9 prostatic wash specimens. The results showed that the BRAF V595E mutation detection rate for UC and PC in the tissues was 51.6%. The detection rate in liquid specimens from dogs with lower urinary tract or prostate masses was 53.2%. Of the 41 cases with follow-up, 16 were further diagnosed with UC or PC, with 75% of liquid specimens from these dogs showing the BRAF V595E mutation. This conventional PCR method provides a reliable and non-invasive screening tool for UC and PC in dogs, especially in settings without advanced equipment.

Identificación de <i>Leishmania </i>spp en caninos de tres departamentos del centro de Colombia y su correlación clínica

The species Leishmania infantum and L. chagasi are considered causal agents of visceral leishmaniasis, a relevant disease due to its zoonosis and severity in humans. Canines are the main reservoirs, hence the need to implement precise diagnoses to identify the Leishmania species involved. The aim of the study was to perform a molecular diagnosis of leishmaniasis (conventional PCR and Sanger sequencing) in 29 tissue samples from canines clinically suspected of leishmaniasis, previously diagnosed by non-molecular methods. The presence of the parasite could be identified by PCR in 9/29 samples (L. infantum, n=4; L. chagasi, n=4; L. braziliensis, n=1), evidencing a predominance of L. infantum and L. chagasi in canines with cutaneous and mucocutaneous leishmaniasis. The PCR allowed to clarify previously negative diagnoses; however, limitations were found, such as the absence of Leishmania DNA in some samples and the inability to obtain additional samples due to inaccessibility to patients. Despite these limitations, the study highlights the importance of molecular diagnosis to identify the species causing leishmaniasis.