PCR Protocol Research Articles

Mapping and quantification of potato virus A RNA genomes within viral particles and polysomes in infected plant cells.

Potato virus A belongs to the genus Potyvirus, a group of single-stranded positive sense RNA viruses infecting crops worldwide. To initiate infection in a host, its genome takes part in different activities, viz., translation, replication, encapsidation during the infection cycle. Extensive research has been carried out to scrutinize the stages of potyviral infection cycle and decipher the strategies it employs to cause disease. Nonetheless, the amount of viral RNA taking part in translation and virion formation, at a given time point, is missing. In this study, we quantified the percentage of viral RNA that exists as virions and those that associates with host polysome, relative to total viral RNA in infected plant tissue. We employed a revised version of immune-capture reverse transcription PCR and polysome profiling to address our queries. We tested three different coating antibody concentrations and further optimized the immuno-capture reverse transcription PCR protocol to address its limitation of binding and retaining viral particles. Our results indicate that most of the viral RNA (69%) exists as encapsidated genomes, while 3% of total viral RNA associates with host polysomes. These findings are crucial for correct interpretation of quantitative translational studies in which correlation must be made between the number of polysome-associated transcripts and the amount of protein synthesized.

Profiling of 24-nt phasiRNAs in Rice Anther by Massively Parallel Sequencing and Stem-Loop Reverse Transcription PCR.

Small RNAs are highly abundant and play important roles in plant reproduction. Profiling of small RNAs in reproductive tissues is a critical step in understanding their biology. Here, we describe a protocol for small RNA profiling in rice anthers, with a focus on an abundantly expressed but little-understood reproductive small RNA class named 24-nucleotide phased secondary small interfering RNAs (24-nt phasiRNAs). The protocol details small RNA library preparation steps for high-throughput sequencing, with a subsequent bioinformatic analysis framework. A low-throughput stem-loop reverse transcription PCR protocol for quick semi-quantification of small RNAs is also included as a complementary approach. The collective protocol, although focused on 24-nt phasiRNAs, should be amenable to other small RNA classes in various plant reproductive tissues.

Identification of a novel GP60 subtype family of Cryptosporidium wrairi from capybaras (Hydrochoerus hydrochaeris) inhabiting urban areas in the state of Mato Grosso do Sul, Brazil

Capybaras (Hydrochoerus hydrochaeris) are hosts for several parasites of public health importance, including Cryptosporidium spp. Therefore, this study aimed to perform the molecular characterization of Cryptosporidium spp. in fecal samples from capybaras inhabiting urban areas. We analyzed 401 fecal samples from capybaras in two municipalities of the state of Mato Grosso do Sul, Brazil. Fecal samples were purified using centrifugal sedimentation with ethyl acetate. They were then screened for Cryptosporidium spp. by malachite green negative staining and a nested PCR protocol targeting the 18S rRNA gene. Samples positive by microscopy or PCR were examined by PCR protocols targeting the actin, HSP-70, and GP60 genes. Amplicons from all PCR protocols were subjected to genetic sequencing. Microscopic screening and 18S rRNA gene-targeted PCR identified 0.25 % (1/401) and 0.5 % (2/401) of samples, respectively, as positive for Cryptosporidium spp. The genetic sequences of the 18S rRNA, HSP-70, actin, and GP60 genes showed genetic similarity to Cryptosporidium wrairi sequences of 99.07 %, 99.69 %, 99.57 %, and 91.51 %, respectively. Genetic sequencing and phylogenetic analyses identified the novel GP60 subtype family VIIbA13 of C. wrairi. In conclusion, we report in this study a low prevalence of the novel GP60 subtype family VIIbA13 of C. wrairi in free-living capybaras from urban areas of the state of Mato Grosso do Sul, Brazil. We propose that capybaras act as a novel host for C. wrairi.

Existencia/ausencia de los genes <i>stx2e</i> y <i>f18</i> en aislados de<i> Escherichia coli </i>de cerdos libres de diarrea

The aim of the study was to evaluate Escherichia coli isolates for the presence or absence of the f18 and stx2e genes. In total, 186 isolates obtained from piglets under 50 days of age that did not have diarrhoea at the time of sampling were studied. The animals were from four high-tech breeding farms located in the Lima region. Sampling was done in 2019 as part of a preliminary study using rectal swabs. A commercial purification kit was used for DNA extraction and a conventional PCR protocol was used for gene detection. No PCR product for stx2e or f18 could be detected in any of the E. coli isolated from these piglets.

Dynamics of Amphibian Pathogen Detection Using Extended Museum Specimens.

Natural history collections have long served as the foundation for understanding our planet's biodiversity, yet they remain a largely untapped resource for wildlife disease studies. Extended specimens include multiple data types and specimen preparations that capture the phenotype and genotype of an organism and its symbionts-but preserved tissues may not always be optimized for downstream detection of various pathogens. Frogs are infected by an array of pathogens including Batrachochytrium dendrobatidis (Bd), Ranavirus (Rv), and Amphibian Perkinsea (Pr), which provides the opportunity to study differences in detection dynamics across tissue types. We used quantitative PCR protocols to screen two tissue types commonly deposited in museum collections, toe clips and liver, from two closely related host species, Rana catesbeiana and Rana clamitans. We compared Bd, Rv, and Pr infection prevalence and intensity between species and tissue types and found no significant difference in prevalence between species, but Bd intensity was higher in R. clamitans than R. catesbeiana. Toe tissue exhibited significantly higher Bd infection loads and was more useful than liver for detecting Bd infections. In contrast, Rv was detected from more liver than toe tissues, but the difference was not statistically significant. Our results support the use of extended specimen collections in amphibian disease studies and demonstrate that broader tissue sampling at the time of specimen preparation can maximize their utility for downstream multipathogen detection.

Development of molecular diagnostic protocols for simultaneous identification of common bed bugs (Cimex lectularius) and tropical bed bugs (Cimex hemipterus)

BackgroundThe resurgence of two bed bug species, the common bed bug (Cimex lectularius Linnaeus, 1758) and tropical bed bug (Cimex hemipterus Fabricius, 1803), in the same geographical regions has been frequently reported recently. Consequently, the rapid identification of these species is crucial for implementing targeted capture traps and tailored pyrethroid resistance diagnosis, due to differences in genetic and physiological traits.MethodsTo develop molecular diagnostic methods, distinct protocols were established for multiplex PCR and loop-mediated isothermal amplification (LAMP) using species-specific primers based on species-specific segments of internal transcribed spacer 2 sequences. These methods were optimized for rapid and accurate identification of the two bed bug species.ResultsBoth multiplex PCR and LAMP protocols were effective in simultaneously identifying the two bed bug species, even when utilizing DNA released from dead specimens. Notably, the straightforward procedure and minimal time commitment of LAMP suggest its potential for rapid and accurate diagnosis of bed bugs in the field. The diagnostic accuracy of these methods was validated through a blind test.ConclusionsThe multiplex PCR and LAMP protocols lay the foundation for rapid and accurate field identification of bed bug species, enabling the use of appropriate traps and the detection of species-specific pyrethroid resistance mutations. This approach ensures effective management tailored to the unique characteristics of each bed bug species.Graphical

Molecular Survey of Parasitic Contamination of Frozen Berries.

Berries represent healthy dietary options and contain bioactive compounds associated with a decreased risk of diseases. Despite representing healthy food choices, these products can be contaminated by pathogenic microorganisms, including parasites. Among foodborne parasites, Giardia duodenalis, Cryptosporidium parvum, Cyclospora cayetanensis, Toxoplasma gondii, and Echinococcus multilocularis are of significant public health importance and have been recently detected in fresh berries in Europe, including Italy. Berries can be purchased fresh or frozen, and it is worrying that even frozen berries could represent a risk for the consumer. In fact, several parasites can resist freezing temperatures and have been responsible for outbreaks of infection. The aim of this study was to investigate the presence of G. duodenalis, C. parvum, C. cayetanensis, T. gondii, and E. multilocularis in frozen berries with simplex and multiplex real-time PCR protocols. A total of 108 packages of mixed frozen berries were bought from supermarkets located in a south-eastern region of Italy. The samples were tested using two simplex real-time PCR protocols targeting C. parvum and G. duodenalis, respectively, and a multiplex real-time PCR targeting C. cayetanensis, T. gondii, and E. multilocularis. None of the investigated parasites were detected in the frozen berry samples tested. This research topic is still unexplored and of great current interest. These results represent a first attempt to investigate parasitic contamination of frozen berries sold on the Italian market, but further large-scale surveys are required.

A diagnostic gap to fill: Development of molecular tools to distinguish the cyst-forming nematode species Heterodera carotae and Heterodera cruciferae

The carrot cyst nematode, Heterodera carotae, is an important pest causing qualitative and quantitative production losses across several countries worldwide. In culture crops, Heterodera carotae and Heterodera cruciferae, can be found in mixture and are virtually unrecognizable from a morphological point of view. However, only H. carotae is able to develop on carrot. To monitor carrot production and set up suitable H. carotae control plans, simple and reliable diagnostic methods to detect and identify these two species are needed. In this study, we developed and successfully tested two real-time PCR protocols (SYBR Green singleplex and TaqMan multiplex real-time PCRs) against both species: H. carotae and H. cruciferae. Using two specific sets of primers, we managed, whatever the methods, to distinguish the two species. Moreover, we showed a higher specificity using the TaqMan assays but a better sensitivity with the SYBR Green assays. In this study, we are proposing two useful and reliable diagnostic tools to enable farmers and scientists to rapidly detect two morphologically and genetically closely related species and supervise agricultural management.

Molecular screening of selected vector-borne pathogens circulating in owned dogs in the Caribbean archipelago of Guadeloupe (France)

Vector-borne diseases represent a major health challenge, both because of the complexity of their control, their common zoonotic nature, or the pathology they can cause in the individual. In tropical areas, surveillance of these diseases is even more important, since the activity of vectors is usually continuous throughout the year. To develop effective prophylaxis and surveillance programs, it is important to know the identity and prevalence of these pathogens as well as their distribution in a given territory. In Guadeloupe, a French archipelago located in the Lesser Antilles of the Caribbean, no information exists about vector-borne diseases in companion animals. With this aim, blood samples were obtained from 46 owned dogs with outdoor access from five different veterinary clinics located in the two mainland islands, and the presence of DNA of the main canine vector-borne pathogens (CVBP) was investigated through diverse PCR protocols. At least one pathogen was detected in 30.4 % of the dogs. The most frequently detected CVBP was Coxiella burnetii (17.4 %), followed by Dirofilaria immitis (8.7 %), and Candidatus Mycoplasma haematoparvum, Hepatozoon canis and Rickettsia spp. (2.2 % in all cases). One dog was coinfected with Candidatus M. haematoparvum and D. immitis. All samples were negative for Anaplasma spp., Ehrlichia spp., Bartonella spp., Borrelia burgdorferi sensu lato, piroplasmids, and Leishmania spp. No significant differences in pathogen occurrence were observed between the two main islands or according to the dog's sex and age groups. This study contributes to filling a relevant gap in the knowledge of vector-borne diseases in the Caribbean.

Evidence of G1-G3 Complex Life Cycle of Echinococcus granulosus sensu stricto Through Molecular Characterization (Genotyping) in Dogs, Sheep, Goats and Humans of North-western Himalayas, India

Background: The helminthic zoonotic disease echinococcosis is brought on by tapeworms that are members of the genus (Family Taeniidae). Currently, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Echinococcus oligarthrus and Echinococcus shiquicus are identified species. Of them, E. granulosus and E. multilocularisare important zoonotic organisms that cause cystic echinococcosis and alveolar echinococcosis in humans, respectively (Bowles and McManus, 1993). Echinococcus granulosus is mainly spread by dogs and a few other intermediate hosts, although it is hardly ever a part of a naturally occurring predator-prey cycle that is not caused by humans. Methods: In a present study, 1254 small ruminants (723 sheep and 531 goats) and 305 dogs, including 122 pet dogs visiting small animal clinics, 183 stray dogs in Jammu division, for a period of 12 months (July, 2021- June, 2022) were taken for the detection of the prevalence of cystic echinococcosis and molecular detection. Result: Among sheep and goats, 209 animals (sheep = 137 and goats = 72) were found infected with hydatidosis with an overall infection rate of 16.67% (sheep = 18.95% and goats = 13.56%). Molecular prevalence based on copro PCR was conducted in dogs, in which the overall prevalence of Echinococcus was found to be 14.09% [stray (22.95%); pet (0.82%)]. For the identification of Echinococcus species from cyst of sheep, goats and humans, a PCR protocol employing the Cox1 primers was used. The amplified products of PCR were electrophoretically separated on agar gels (1%) and analysed in a gel documentation system. Clear amplification was noticed at 440 bp for sheep, goat andhuman. The generated Cox1 partial sequences were assigned theaccession numbers as: dog (ON427821), goat (ON427822), sheep (ON427823), human 1(OP215294) and human 2 (OP215295). All the Cox1 sequences from dog, goat, sheep and humans were clustering in same clade, irrespective of their geographical location, representingthe cycle of transmission in Jammu region as Echinococcus granulosus G1-G3 complex.

First Report of Cacao Mild Mosaic Virus Associated with Cacao (Theobroma cacao) in Hawai'i, USA.

Cacao, Theobroma cacao, is an important tropical agricultural crop and the key ingredient of chocolate, which has an international trade value of $103 billion/year (Marelli et al. 2019). Cacao mild mosaic virus (CaMMV) is a badnavirus that causes mild symptoms compared with some of the closely related species of Cacao swollen shoot virus (CSSV), the latter of which are currently only found in West Africa (Marelli et al. 2019; Ullah et al. 2021). CaMMV was recently found in symptomatic commercial cacao trees in Mayagüez, Puerto Rico (Puig et al. 2020), and subsequently detected in a USDA ARS (Agricultural Research Service) quarantine greenhouse in Miami, FL (Puig 2021). The USDA ARS germplasm repository in Hilo, Hawai'i, USA serves as a backup collection for Puerto Rico's cacao germplasm, and field trials have been established from cacao germplasm from Miami to evaluate select varieties. To determine if CaMMV is present in the collection in Hilo, greenhouse and field accessions were tested. Using an optimized sampling and PCR protocol established by Puig (2021), three young cacao petioles per tree or seedling were collected and pooled, and DNA was extracted using the NucleoSpin Plant II commercial kit (Macherey-Nagel, Düren, Germany) following the manufacturer's instructions. Samples were molecularly identified via end point PCR, gel electrophoresis, and Sanger sequencing. PCR amplification of CaMMV using the virus-specific primer set Mia-1396F (5'-ACCGTGTCTAYCAGCACTGGA-3') and Mia-1667R (5'-GACCACCGTCAGCCAGAC-3') produced 289 bp amplicons. Of 230 plants sampled, 26 CaMMV positive detections were discovered in greenhouse and field plantings in Hilo. Most CaMMV+ plants contained some form of leaf chlorosis (96.2%). The sequenced PCR products from Hawai'i were deposited in GenBank (accession nos. OQ692890-OQ692891) and showed 99.2% nucleotide identity to CaMMV accessions from Puerto Rico (MW052520; n = 23) and 98.1% nucleotide identity to CaMMV accessions from Florida, USA (MZ409692; n = 3) in BLASTn analysis. For species-level confirmation, the RT-RNase H domain was amplified from 9 isolates using CaMMV-specific primers (Mia5385F, 5'-AGGACAACGGCTTTCTTGGT-3'/Mia6616R, 5'-GAGACTAACTTGGTTAGGGCT-3'), sequenced, and deposited in GenBank (accession nos. PP997461-PP997462). Sequences matched most closely to GenBank isolates from Puerto Rico (MT253659; 98.0%; n = 7) and Trinidad and Tobago (NC_033738; 97.1%; n = 2). CaMMV, previously known as cacao Trinidad virus A, was first reported in 1943 and was associated with 7 to 33% yield reduction, loss of vigor, and tree decline (Posnette 1944; Swarbrick 1961). CaMMV is known to exist in the Americas and was most recently detected in Brazil and Indonesia (Kandito et al. 2022; Ramos‑Sobrinho et al. 2021). To the best of our knowledge, this is the first report of CaMMV infecting cacao in Hawai'i. The cacao industry continues to expand in Hawai'i, and cultivation occurs on at least four of the main islands including Hawai'i Island, O'ahu, Maui, and Kaua'i. To develop disease management strategies, further investigation is needed to define CaMMV symptomology, and determine the distribution and effect this virus has on production in Hawai'i. In the meantime, cacao will be screened regardless of visible symptomology to maintain pathogen-free accessions and avoid the transfer of virus-containing germplasm.

Towards a Standardized Operating Procedure for eDNA‐Based Biomonitoring in Coastal Marine Salmon Aquaculture

ABSTRACTThe marine aquaculture industry and regulators are in the process of implementing environmental DNA (eDNA) metabarcoding of microbial communities for compliance monitoring. This requires standardization of sampling, laboratory, and data analysis protocols. Towards this goal, we in this study completed two further milestones using samples collected from two Scottish salmon farms: (i) We tested the effect of using two different PCR protocols (i.e., different DNA polymerases, master mixes, and annealing temperatures), which are frequently being used in eDNA biomonitoring of aquaculture installations, for the amplification of the taxonomic marker gene (V3‐V4 hypervariable region of the bacterial 16S rRNA gene). (ii) We quantified sampling background noise obtained from eDNA samples and statistically compared results with the sampling bias observed in macrofaunal samples from the same source sediments. We detected differences in bacterial community structures resulting from the performance of different PCR protocols, profoundly influencing the interpretation of biomonitoring results. Furthermore, we found that sampling‐induced errors for eDNA samples were similar to errors for macrofaunal samples collected according to compliance monitoring protocol (~25% variability in both cases). Finally, we showed that within‐grab variances of microbial community structures were in the same order of magnitude (less than 10× difference in all cases) as the one obtained from replicate grabs collected from the same locale (impact category). Based on our findings, we suggest using a consistent PCR protocol for biomonitoring efforts to improve the comparability of results, especially when different service providers are conducting the biomonitoring. We propose a sampling scheme to be considered in eDNA biomonitoring that includes taking three replicate grabs at each locale, with one replicate sample from each grab. This minimizes sampling‐induced errors and makes upcoming eDNA‐based monitoring results comparable with previous compliance monitoring results obtained from macrofaunal data.

Detection of HBV DNA by direct PCR protocol and Nested PCR protocol and comparing them in chronic patients and healthy carriers of Hepatitis B virus.

Hepatitis B virus is a major public health problem, and the outcome of infection depends on the effectiveness of the virus's interaction with the host, and in particular on the strength of the natural and conditioned response of humoral and cellular immunity. This study was conducted to assess the clinical status, genetic immunity and viral (viral) traits of Iraqi patients with chronic hepatitis B virus and healthy carriers of hepatitis B surface antigen, as well as to show the relationship between the immune status and the chronic condition of the disease. The study included (60) patients with chronic hepatitis B virus type (B) and (60) healthy hepatitis B surface antigen carriers who attended the Teaching Hospital for Gastrointestinal and Liver Diseases in Nasiriya City Al-Hussein Teaching Hospital, Thalassemia Center and the Central Blood Bank in Nasiriya Citysouthern of Iraq. for the period from the first period. From February 2022 until the end of February 2022, the average age of patients infected with chronic hepatitis B virus was 64.5 years, and the average age of healthy carriers of hepatitis B surface antigen was 35. 1 year, and the ratio of males to females was (12.2:) for chronic patients and (13.6:) for carriers of the disease. As for the deoxyribonucleic acid, it was (100%) in chronic patients and healthy carriers using the Nested type polymerase chain reaction, and at a rate of (30.0%) and (46.7%) in chronic patients and healthy carriers, respectively using the polymerase chain reaction technique. direct with a significant difference between the two interactions. The current results confirm that a nested PCR protocol to detect HBV DNA is a more sensitive way of detecting HBV in Iraq CHB patients and carriers. Recent advanced techniques such as nucleic acid assays, quantitative assays for viral load and RT-PCR for Iraqi viral hepatitis laboratories are recommended.

Detecting life by behavior, the overlooked sensitivity of behavioral assays

Detecting life has driven research and exploration for centuries, but recent attempts to compile and generate a framework that summarizes life features, aimed to develop strategies for life detection missions beyond planet Earth, have disregarded a key life feature: behavior. Yet, some behaviors such as biomineralization or motility have occasionally been proposed as biosignatures to detect life. Here, we capitalize on a specific taxis’ motility behavior, magnetotaxis, to experimentally provide insights in support of behavior as an unambiguous, sensitive biosignature, and magnetic forces as a prescreening option. Using a magnetotactic bacterial species, Magnetospirillum magneticum, we conducted a lab sensitivity experiment comparing PCR with the hanging drop behavioral assay, using a dilution series. The hanging drop behavioral assay visually shows the motility of MTB toward magnetic poles. Our findings reveal that the behavioral assay exhibits higher sensitivity in the detection of M. magneticum when compared to the established PCR protocol. While both methods present similar detection sensitivities at high concentrations, at ≥ 10–7 fold dilutions, the behavioral method proved more sensitive. The behavioral method can detect bacteria even when samples are diluted at 10–9. Comparable results were obtained with environmental samples from the Hula Valley. We propose behavioral cues as valuable biosignatures in the ongoing efforts of life detection in unexplored aquatic habitats on Earth and to stimulate and support discussions about how to detect extant life beyond Earth. Generic and robust behavioral assays can represent a methodological revolution.

Testosterone with Silymarin Improves Diabetes-obesity Comorbidity Complications by Modulating Inflammatory Responses and CYP7A1/ACC Gene Expressions in Rats.

The co-morbidity of DMOB has become increasingly problematic among the world's population because of a high-calorie diet and sedentary lifestyle. DMOB is associated with lower testosterone (TN) levels, the male sex hormone. The phytochemical compound silymarin (SN) exerts antidiabetic activity by modifying β-cells and anti-obesity activity by inhibiting adipogenesis by methylxanthine. The goal of this study was to find out how well testosterone (TN) with silymarin (SN) protects against oxidative stress and inflammation in the liver of the experimental rats with type 2 diabetes (T2D) and obesity (DMOB). The present study evaluates the efficacy of TN and SN combination (TNSN) on the levels of the potential parameters, such as body mass, serum marker enzymes, fasting glucose levels, HbA1c levels, lipid profile, enzymatic and non-enzymatic antioxidants, proinflammatory cytokines, gene expression pathways, and histopathology in a DMOB comorbidity rat model. Male Sprague-Dawley (SD) rats were fed a high-fat diet (HFD) for 20 weeks with an administration of a single dose of streptozotocin (STZ) i.p. injection (30 mg/kg) on the 9th week of the study. The procedure was to develop the DMOB co-morbidity model in the experimental animals. Co-treatment of TN and SN administration were followed throughout the experiment. Rats were sacrificed after overnight fasting to collect serum and liver tissue samples. Samples were analyzed using a clinical chemistry automated analyzer, spectrophotometry, and quantitative real-time PCR (qPCR) methods and protocols. Analyses of body mass changes, serum marker enzymes, fasting glucose levels, HbA1c levels, lipid profiles, enzymatic and non-enzymatic antioxidants, TNF-α, IL-6, adiponectin, CYP7A1, ACC expression pathways, and histopathology showed significant abnormal levels (P ≤ 0.05) in the pathological group. These were efficiently treated to normal by the administration of TNSN. These results concluded that TNSN exerted protective efficacy against the liver abnormalities in the co-morbidity of the DMOB rat model.

Comparison of diagnostic methods for laboratory diagnosis of the zoonotic tapeworm Dipylidium caninum in cats

The tapeworm Dipylidium caninum is the most widely distributed cestode infecting dogs, cats, and sometimes humans, worldwide. The diagnosis of the infection caused by D. caninum is achieved via the visualization of proglottids in feces or with traditional microscopic tests, but both lack sensitivity. The present study has evaluated and compared the diagnostic performance of a PCR protocol on different feline biological samples to detect D. caninum. A sample of feces, a Scotch tape test from the perianal area, and a rectal swab were collected from a total of 100 privately owned cats from Italy and Greece. All fecal samples were subjected to macroscopic examination and to floatation. Based on the results of the above tests the cats were divided in three groups, i.e. (i) cats positive for D. caninum (regardless of positivity for other endoparasites (Group A; n = 50 cats), (ii) cats negative for D. caninum but infected by other helminths (Group B; n = 25 cats), and (iii) cats negative for intestinal endoparasites (Group C; n = 25 cats). For each sample, the DNA was extracted from feces, floatation supernatant, Scotch tape test and rectal swabs and subjected to PCR. For 33 cats from Group A, at least one sample type scored positive at PCR. Of these, all were PCR-positive in the floatation aliquot, while nine and one cats were positive by PCR on feces and Scotch tape test, respectively. Swabs were negative by PCR for all the cats. None of the samples from cats of Groups B and C was positive by any PCR. Sequences obtained from amplicons generated from samples of cats enrolled in Italy had 99–100 % identity with the recently described D. caninum feline genotype. The data presented here suggest that PCR could be a useful tool for diagnosing D. caninum infections, under certain circumstances, e.g. when proglottids are unidentified, unseen or overlooked, even though it has limitations, e.g. false negative results due to fecal PCR inhibitors, uneven distribution of parasitic elements, or to intermittent proglottid and/or egg shedding. Thus, it may not be, currently, the best diagnostic choice for dipylidiosis.

Evaluation of mtDNA common deletion in esophageal squamous cell carcinoma.

Mitochondrial defects are thought to play a role in cancer initiation and progression for a long time. Because of the absence of protective histones and an inefficiency in the DNA repair process, mitochondrial DNA is known to be prone to mutations. The deletion of 4977bp is one of the most common mutations in human cancers. This study aimed to investigate the relationship between 4977bp common deletion and Esophageal Squamous Cell Carcinoma Disease (SCC) to provide prognostic information. By using a PCR protocol, this study identified the 4977bp deletion of mtDNA. A PCR method was used on tumor samples from 41 squamous cell carcinoma patients and blood samples from 50 healthy individuals to detect DNA. Among the 41 tumor samples (80.5%), 33 were found to have the 4977bp deletion, while none of the blood samples from healthy individuals contained it. It is shown that the deletion of 4977bp of mtDNA correlates significantly with SCC in this study. A 4977bp deletion could be used as an effective cancer screening indicator and biomarker for early diagnosis and prevention of cancer.

Development of a technique for molecular typing of <i>Bacillus anthracis</i> strains using new VNTR and INDEL markers

Introduction. Bacillus anthracis, the pathogen of a particularly dangerous zoonotic disease known as anthrax, requires strict epidemiological control and is characterized by high genetic homogeneity, which necessitates the development of genotyping methods. The aim of the study were to to find and characterize the VNTR and INDEL loci of B. anthracis and to develop on their basis a genotyping technique by PCR with electrophoretic detection of the results. Materials and methods. Marker search and phylogenetic analysis were performed on a sample of 388 genomes of B. anthracis strains, 322 from the GenBank collection (RefSeq) and 66 from the collection of the Stavropol Anti-Plague Institute of Rospotrebnadzor. Phylogenetic analysis was performed on the basis of SNP crustal alignment using the Parsnp program. The search for markers was carried out using the Mauve program and author's scripts in Python. PCR was performed using a ScreenMix-HS kit (CJSC "Eurogen", Russia). Results. Genomic variations of B. anthracis strains (SNP — 25,664, SNR — 14,387, VNTR — 693, INDEL — 14,667) were found, bioinformatic analysis of which revealed nine new VNTR and six INDEL molecular markers most suitable for genotyping. The genetic (allelic) variants of the markers are described. Primers were selected for the found markers and a PCR protocol with detection by electrophoresis in agarose gel was developed. When typing using VNTR markers was applied, the strains were divided into nine clusters: A.Br.Ames, A.Br.001/002, A.Br.Aust94, A.Br.005/006, A.Br.008/009 (Tsiankovskii), A.Br.008/009 (STI), A.Br.008/009 (A.Br.125), A.Br.008/009 (strain 228/269), B.Br.001/002. When typing using INDEL markers, the strains were divided into six clusters: A.Br.Ames, A.Br.001/002, A.Br.Aust94, A.Br.008/009(Tsiankovskii), B.Br.001/002(B.Br.014), as well as a cluster comprising several genetic lineages: A.Br.008/009 (STI), A.Br.008/009 (A.Br.125), A.Br.005/006 и B.Br.001/002. Conclusion. The use of the developed methodology for the identification of variable VNTR and INDEL loci makes it possible to reliably determine the phylogenetic position of B. anthracis strains and is promising for use in the epidemiological investigation of anthrax outbreaks.

F2c.*C20209T mutation in patients with a history of thrombosis: A case report, retrospective 2 site-results and review of the literature.

G20210A (c.*97G>A) prothrombin gene variant, found in white population has been associated with an increased risk of venous thromboembolism (VTE). Other rare polymorphisms in F2 gene (C20209T) have been reported, more rare and touching black people, but its potential association with VTE remain uncertain. About a 69 years-old Caucasian woman presenting an unprovoked deep venous thrombosis of the leg, we analyzed retrospectively 25.000 thrombophilia tests on a 11-year period of time (2007-2018), at Nice and Marseille University Hospitals, and performed extensive review of the literature. Genetic determination included a similar PCR protocol and sequencing. Twenty-one heterozygous cases out of 25.585 determinations (0.08%) was found. The C20209T mutation detected in our Caucasian patient is rare, with a frequency that differed from what was reported in the previous literature, mainly in non-Caucasian patients (Africans, Africans-Americans, and Caribbeans). One hundred and thirteen patients with this mutation have been described in the literature, of which only one homozygous. This study is the most important on C20209T mutation performed at present, allowing to precise its frequency and its potential role in venous thromboembolism.

Comparison of different molecular protocols for the detection of Uncinaria stenocephala infection in dogs

The present study aims to assess the performance of different molecular targets using various matrices of samples for the detection of Uncinaria stenocephala (US) in hookworm infected dogs. To this end, the DNA extraction was performed on the following matrices of samples: (i) larvae of US obtained from experimentally infected dogs with US with different larvae counts per microliter (µl); (ii) pure US eggs suspension in distilled water with different egg counts per µl; (iii) spiked dog fecal samples with different US eggs per gram (EPG) of feces; (iv) feces from dogs naturally infected with hookworm eggs; (v) fecal suspension with hookworm eggs recovered from the FLOTAC apparatus. All the samples were tested with four different PCR protocols targeting specific regions for the detection of both hookworms US and AC as follows: Protocol A (ITS1, 5.8 S, ITS2) and Protocol B (18 S) for the detection of both species, Protocol C (ITS1) for the detection of AC and Protocol D (ITS1) for the detection of US. The best results were obtained with DNA extracted from US larvae matrix obtained from experimentally infected dogs, showing a detection limit of 3.5 larvae/ml for the protocols A, B and D. A moderate correlation was found between the FLOTAC technique and PCR protocols B and D with respect to fecal samples from dogs naturally infected with hookworms. Indeed, PCR protocols B (18 S) and D (ITS1) gave the best results for feces and fecal suspension from naturally infected dogs. However, all the PCR protocols used showed lower sensitivity than FLOTAC technique. Perhaps, isolating US eggs in advance could help to obtain better quality and quantity of DNA, avoiding some notable factors such as inhibitors present in faecal samples. However, a further study is needed to evaluate and standardise a protocol for the recovery of parasitic elements, that could be applied prior to DNA extraction. Therefore, this could lead to a better amplification of US eggs DNA. In conclusion, our results showed that the type of sample (sample-matrix) used for the DNA extraction samples is crucial, as this affects the diagnostic sensitivity of the technique.