PCR Protocol Research Articles

First Description of the Occurrence of Slow Bee Paralysis Virus-1 and Deformed Wing Virus B in Apis mellifera ligustica Honeybee in Italy

Among the causes of bee colony death, viruses are among the number of pathogens that can contribute to the ill health of a colony. This study focuses on two of the several honeybee viruses, Slow Bee Paralysis Virus-1 (SBPV-1) and Deformed Wing Virus B (DWV-B), both belonging to the Iflavirus genus. To date, there is limited information available on the presence of these viruses in Italy. For this research project (IZS LT 04/19 RC), funded by the Ministry of Health, the presence and positivity of several honeybee viruses were evaluated in Italy using molecular methods. Convenience sampling was used, and these samples were analyzed for the two viruses and/or other main honeybee viruses, using specific PCR protocols and Sanger sequencing when necessary. A statistical analysis was conducted to study the independence between the viruses. Our data demonstrate for the first time the presence of SBPV-1 in Italian territory with a rather low number in most of the regions investigated, except for Emilia-Romagna and Lazio where it was detected more frequently, while DWV-B was detected at a higher level in all the regions investigated.

Analysis of the relative frequencies of the multipartite BNYVV genomic RNAs in different plants and tissues.

Multipartite virus genomes are composed of two or more segments, each packaged into an independent viral particle. A potential advantage of multipartitism is the regulation of gene expression through changes in the segment copy number. Soil-borne beet necrotic yellow vein virus (BNYVV) is a typical example of multipartism, given its high number of genomic positive-sense RNAs (up to five). Here we analyse the relative frequencies of the four genomic RNAs of BNYVV type B during infection of different host plants (Chenopodium quinoa, Beta macrocarpa and Spinacia oleracea) and organs (leaves and roots). By successfully validating a two-step reverse-transcriptase digital droplet PCR protocol, we show that RNA1 and -2 genomic segments always replicate at low and comparable relative frequencies. In contrast, RNA3 and -4 accumulate with variable relative frequencies, resulting in distinct RNA1 : RNA2 : RNA3 : RNA4 ratios, depending on the infected host species and organ.

Development and validation of a novel detection method for Rickettsia rickettsii using a loop-mediated isothermal amplification assay.

Rickettsia rickettsii is an obligate, intracellular pathogen and the causative agent of Rocky Mountain spotted fever (RMSF). RMSF is an important zoonotic disease due to its high fatal outcome in humans. The difficulty of clinical diagnosis due to the low sensitivity and specificity of current diagnostic methods are a principal setback. We reported the development of a new method for the detection of R. rickettsii in human and tick DNA samples using loop-mediated isothermal amplification (LAMP), as well as the validation of the LAMP test for R. rickettsii in field samples of infected ticks and humans, determining the diagnostic sensitivity and specificity, as well as the reproducibility of the test. This technique uses hydroxy naphthol blue (HNB) as an indicator of the formation of magnesium pyrophosphate, a marker for the presence of DNA. Here, we used a putative R. rickettsii gene as a target for three pairs of primers that specifically amplify R. rickettsii DNA by hairpin-based isothermal amplification technique (LAMP). The sensitivity of the assay was ~1.6-3 pg, which is 10 times more sensitive than PCR. To determine the diagnostics specificity and sensitivity, 103 human DNA samples and 30 tick DNA samples were evaluated. For the human samples, a sensitivity for HNB of 93%, a specificity of 70% and a k of 0.53 were obtained. For electrophoresis the sensitivity was 97% with a specificity of 58% and a k of 0.42. For tick samples, a sensitivity of 80% was obtained, a specificity of 93% for HNB and for electrophoresis the sensitivity and specificity were 87%. The k for both was 0.73. The degree of concordance between HNB and electrophoresis was 0.82 for humans and for ticks, it was 0.87. The result is obtained in shorter time, compared to a PCR protocol, and is visually interpreted by the color change. Therefore, this method could be a reliable tool for the early diagnosis of rickettsiosis.

QPCR assay for detection of Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Elements from CAR-T and TCR-T cells in fresh and formalin-fixed tissue.

As adoptive cellular therapies become more commonplace in cancer care, there is a growing need to monitor site-specific localization of engineered cells-such as chimeric antigen receptor T (CAR-T) cells and T-cell receptor T (TCR-T) cells-in patients' tissues to understand treatment effectiveness as well as associated adverse events. Manufacturing CAR-T and TCR-T cells involves transduction with viral vectors commonly containing the WPRE gene sequence to enhance gene expression, providing a viable assay target unique to these engineered cells. Quantitative PCR (qPCR) is currently used clinically in fresh patient tissue samples and blood with target sequences specific to each immunotherapy product. Herein, we developed a WPRE-targeted qPCR assay that is broadly applicable for detection of engineered cell products in both fresh and archival formalin-fixed paraffin embedded (FFPE) tissues. Using both traditional PCR and SYBR Green PCR protocols, we demonstrate the use of this WPRE-targeted assay to successfully detect two CAR-T cell and two TCR-T cell products in FFPE tissue. Standard curve analysis reported a reproducible limit of detection at 100 WPRE copies per 20μL PCR reaction. This novel and inexpensive technique could provide better understanding of tissue abundance of engineered therapeutic T cells in both tumor and second-site toxicity tissues and provide quantitative assessment of immune effector cell trafficking in archival tissue.

Comparing Genotyping Accuracy Using Buccal Swabs versus Tail Biopsies by PCR in B6;C3-Tg(Prnp-SNCA*A53T)83Vle and B6;C3-Tg(Prnp-SNCA*A53T)83Vle Sncatm1Mjff Mice.

Genotyping is a common and necessary procedure performed on genetically modified animals to distinguish carriers from noncarriers of the variants of interest. Established methods involve collection of tissues such as tips of tails or notches of ears. Noninvasive methods have been described but not widely adopted for reasons including inertia to change, needs to adjust PCR protocols, and the lack of validation; noninvasive genotyping methods are a refinement on animal welfare, but questions remain regarding how they compare with invasive methods in terms of genotyping accuracy rate and reproducibility. To gain answers to these questions, we compared the detection accuracy of the transgene and determination of zygosity in B6;C3-Tg(Prnp-SNCA*A53T)83Vle and B6;C3-Tg(Prnp-SNCA*A53T)83Vle Snca tm1Mjff neonatal mice between tail biopsies and buccal swabs. Moreover, we weighed and observed mice following genotyping to see if any clinical differences can be discerned. Weight data did not support statistically significant differences in mice undergoing different genotyping procedures and control. No statistically significant difference was found between using buccal swabs or tail biopsies for genotyping with PCR or quantitative PCR. None of the pups swabbed was rejected by the dam. Our findings indicate that buccal swabbing is a more humane and feasible alternative to tail biopsies for high-throughput genotyping.

Absence of Leishmania spp. DNA in road-killed wild mammals in Southern Brazil.

Leishmaniasis are neglected diseases transmitted by vectors that affect domestic and wild animals, including humans. Due to its incidence and lethality, this zoonosis is a worrying public health problem, making it essential to identify all links in the transmission chain. Infection of wild mammals by Leishmania spp. remains poorly understood, especially in southern Brazil. Therefore, the objective was to research, using the PCR technique, the presence of Leishmania spp. DNA in road-killed wild mammals in Southern Brazil. Carcasses of 96 animals were collected from highways in the Pelotas microregion, Rio Grande do Sul, southern Brazil and subjected to necropsies. Tissue fragments (spleen, skin, liver, kidney, heart, lung, lymph nodes, bone marrow and blood) were collected and genomic DNA was extracted. PCR protocols targeting the ITS1, kDNA and 18S genes were tested. We found no evidence of Leishmania spp. circulation in the studied population. However, epidemiological studies like this one are of great relevance, as they allow monitoring of the occurrence of pathogens and help identify possible risk areas. As these animals act as epidemiological markers for the presence of the microorganism, studies must be carried out continuously to understand whether there are sources of infection in the region.

Geographical isolation and hyperendemicity of Hepatozoon felis: Epidemiological scenario in Skopelos, Greece, and phylogenetic analysis

Feline hepatozoonosis is a vector-borne disease caused by different species of the genus Hepatozoon, i.e. Hepatozoon felis, Hepatozoon silvestris and Hepatozoon canis. Knowledge on the biology, epidemiology and taxonomy of Hepatozoon spp. is still limited, despite the fact that the number of documented Hepatozoon spp. infections in domestic cats increased in recent years in different countries. This study was carried out to evaluate the prevalence of Hepatozoon spp. in cats living on the island of Skopelos, Greece. Individual blood samples were collected from 54 owned cats and were subjected to Giemsa-stained blood smear examination to investigate the presence of Hepatozoon spp. gamonts and to a specific PCR protocol targeting the 18S rRNA gene of Hepatozoon. A total of 45 cats (83.3%) were found infected by Hepatozoon spp. by at least one of the methods applied. In particular, 43 (79.6%) of the cats were PCR-positive, and in 6 (11.1%) cats gamonts of Hepatozoon spp. were found in the blood smears. A total of 26 H. felis sequences were obtained and the presence of three undescribed single nucleotide polymorphisms were detected. The present results indicate that H. felis species complex may be hyperendemic in isolated/confined areas. In such contexts, geographical isolation may favor the origin of new genotypes or haplotypes or even new species.

Rodents/Shrews and their Ectoparasites are not Associated with the Enzootic Maintenance and Transmission of Coxiella burnetii to Livestock and Humans in Puducherry, India

Objectives: “Q fever,” which is caused by Coxiella burnetii, is endemic in India. In addition to livestock, rodents have also been reported to be associated with enzootic maintenance, favoring pathogen transmission. Currently, however, no data are available on the role of rodents in “Q fever” transmission in India. Methods: A cross-sectional study was undertaken in 39 Puducherry villages to screen Coxiella burnetii in synanthropic rodents (rats and shrews) and their ectoparasites (ticks, mites, and fleas) by real-time and conventional PCR protocols targeting the pathogen specific IS 1111 and com 1 genes. Results: One hundred forty animals were trapped (107 shrews and 33 rats). The ticks, mites, and fleas infesting the rodents were identified as Rhipicephalus sanguineus, Leptotrombidium deliense and Schoengastiella spp., and Xenopsylla cheopis, respectively. PCR screening of the DNA extracted from the rodent/shrew blood samples and their ectoparasites tested negative for C. burnetii. Conclusions: Synanthropic rodents, such as rodents/shrews and their ectoparasites do not have a pivotal part in the enzootic maintenance and spread of Q fever to humans and livestock in Puducherry.

Detection and full genomic sequencing of rare hepatitis E virus genotype 4d in Italian wastewater, undetected by clinical surveillance

Hepatitis E is a liver disease caused by the hepatitis E virus (HEV), primarily transmitted through contaminated water or food. There are four different HEV genotypes in humans, with genotypes 1 and 2 being the most widespread. Genotypes 3 and 4 are found in animals and can also infect humans. Genotype 4 is prevalent in Asia, mainly in China. In Italy, only one outbreak of HEV-4 has been documented, which occurred in 2011, involving five patients. In 2013, HEV G4 was also detected in a pig farm. Since then, no further evidence of HEV genotype 4 has been found in the country. This study describes the first detection of HEV genotype 4, subtype d, in wastewater in central Italy, despite a lack of any clinical case reported in the area. By using a multiplex PCR protocol and two sequencing strategies, Illumina and ONT, the virus's complete genome was sequenced and characterized as subtype 4d. These findings shed light on the potential of environmental surveillance for infectious agents to improve our understanding of epidemiology and support public health efforts.

In Silico Evaluation of the PCR Performance of Different Tests for Detection of WSSV

In this study, the primers of different protocols for the detection of White Spot Syndrome Virus (WSSV) were analyzed in silico to evaluate their potential performance in PCR. As with any biological entity, this virus evolves constantly. Thus, this analysis showed that a few primers, including those recommended by the World Organization for Animal Health (WOAH), might mismatch with some isolates of WSSV, specially with isolates more recently sequenced. Furthermore, a set of primers recommended by WOAH, showed the potential to self-dimer and form hairpin loop structures, which could affect the efficiency of PCR, resulting in an inaccurate diagnostic result. From our perspective, and considering the evolutionary trajectory of this virus, it may be time for the WOAH to update the PCR protocols recommended for WSSV detection, which remains as a highly prevalent and lethal virus.

The Potential of Complementary Sex-Determiner Gene Allelic Diversity for Studying the Number of Patrilines within Honeybee Colonies

Polyandry, a fundamental aspect of honeybee biology, influences genetic diversity within bee colonies. The Csd gene, responsible for sex determination, exhibits a high intraspecific polymorphism, making it a promising candidate for studying patrilines. This study investigates the potential of the complementary sex-determiner gene (Csd) as a marker for genetic studies of honeybee colonies, comparing its efficacy with standard microsatellite markers. A worker bee brood from five colonies was genotyped using both Csd and microsatellite markers. The results showed that Csd alleles offer higher variability than microsatellite markers, providing a superior resolution in genotyping. The number of distinct Csd alleles in each colony surpassed microsatellite markers, demonstrating the potential of Csd for detailed genetic analyses. Despite challenges in Csd amplification efficiency, a two-step nested PCR protocol proved effective. Interestingly, Csd genotyping alone identified more patrilines than the set of five microsatellite markers, emphasizing its efficacy. Combining Csd and microsatellite genotyping enhances the resolution of genetic studies in honeybee colonies, offering valuable insights into genetic diversity, reproductive success, and social dynamics. The Csd gene emerges as a promising tool for advancing genetic studies in honeybee populations.

Prevalence and first molecular identification of Sarcocystis species in feces of domestic dogs (Canis familiaris) in Egypt

BackgroundSarcocystis species are obligatorily heteroxenous protozoan parasites with predator–prey life cycles. Global Knowledge about the epidemiology and the distribution pattern of different Sarcocystis species in dog feces are very scarce. Therefore, the current investigation was conducted to declare the occurrence of Sarcocystis in the fecal specimens of the most common canids in Egypt, the domestic dogs, and to identify the species present using various parasitological and molecular approaches.MethodsA total of 100 dog fecal samples were collected and screened using fecal sugar flotation test for the presence of Sarcocystis oocysts/sporocysts. Additionally, thirty samples were used for genomic DNA extraction. The 18S rRNA gene fragment was the target of primers for a PCR, followed by purification and sequencing of the amplicons.ResultsCurrently, the results obtained reviewed that 4% of fecal samples were positive for Sarcocystis spp. using LM. Additionally, Sarcocystis spp. were verified in sixteen dogs (53.3%, 16/30) using PCR and subsequent sequencing protocols. Statistically, insignificant difference in prevalence of sarcocystosis relative to age and gender was noticed. Morphologically, the detected sporocysts measured 13.2–16.0 × 9.4–11 μm. Based on the 18S rRNA gene, sequencing analysis of amplicons from sporocysts DNA revealed 99.82% nucleotide homology with published S. tenella partial nucleotide sequences from sheep in Iraq and Iran.ConclusionsThis is the first molecular evidence in support of the final host role of domestic dogs in the life cycle of S. tenella in Egypt, which provides a precious diagnostic tool for further epidemiological studies and for the assessment of the effectiveness of control measures for this disease.

Conceptual Design of a Fluid Handling System with Dosing and Mixing for PCR Applications

Typically, samples for PCR instruments are prepared manually by trained laboratory personnel, which in this case refers to the sample preparation, the dispensing of a master mix and a biological sample with subsequent mixing. To reduce the workload, there are approaches towards fully automated PCR systems, but in most cases only proprietary thermal cyclers can be used. Other approaches involve a similarly complex robotic solution. In this work, the spectrum was extended by a method that can be integrated into the workflow of existing PCR protocols. For this purpose, a user-friendly solution using a microfluidic chip was developed. For the sample preparation different mixing concepts were selected as well as a concept for aliquoting. Optimization and evaluation were assisted by utilization of simulations to study system behavior. The systems consisting of a mixer and a flow splitter were manufactured by milling. Subsequently, the mixing and aliquoting capability of the system could be investigated in the laboratory assembly. The testing of this prototype showed moderate results which are attributed to the accuracy of the laboratory assembly.

Occurrence of Hepatozoon in Some Reptiles from Brazilian Biomes with Molecular and Morphological Characterization of Hepatozoon caimani

Amphibians and reptiles represent a considerable proportion of the vertebrate fauna in Brazil. Different blood parasitic infections have been reported in these groups, such as Haemogregarina, Hepatozoon, Trypanosoma and microfilariae. However, insufficient research on interactions between these parasites and their hosts has been carried out in some regions of the country. Samples were collected from populations of wild herpetofauna in different microhabitats throughout Brazil, totaling 111 samples of reptiles from the states of Mato Grosso and Pernambuco. We used an integrative approach, with classical microscopy, morphometry and molecular analysis, in order to identify hemoparasites present in the analyzed fauna. Genomic DNA was extracted for the PCR protocol based on the 18S ribosomal RNA gene for Hepatozoon spp. A total of 53 positives were obtained with molecular screening (47.7%), all confirmed as Hepatozoon spp. using DNA sequencing. Among positive samples, 23 slides were examined, confirming the presence of Hepatozoon spp. in 91.3% of the smears. The phylogenetic analysis performed with sequences from 43 samples resulted in a tree containing several distinct clades. Sequences were generally grouped according to the taxonomic order of the host. Co-infections with microfilariae and Trypanosoma spp. were also found in microscopy analyses. This study describes the presence of Hepatozoon caimani in a new host species (Paleosuchus palpebrosus) that can be a paratenic host in the natural environment. The existence of parasitic co-infections in alligator species underscores the significance of recognizing the impact of infections by various parasitic taxa on the host populations.

Plant Sample Collection and Shipment for Multi-omic Analyses and Phytosanitary Evaluation.

Plant sample preparation for analyses is a fundamental step in high-throughput omics strategies. Especially for plant metabolomics, quenching of hydrolytic enzymes able to affect metabolite concentrations is crucial for the accuracy of results. Given that DNA is usually less labile than metabolites, most sampling and shipment procedures able to preserve the metabolome are also suitable for preventing the degradation of plant DNA or of DNA of pathogens in the plant tissue. In this article, we describe all the steps of sample collection, shipment (including the phytosanitary issues of moving plant samples), and processing for combined genomics and metabolomics from a single sample, as well as the protocols used in our laboratories for downstream approaches for crop plants, allowing collection of multi-omic datasets in large experimental setups. The protocols have been adjusted to apply to both freeze-dried and fresh-frozen material to allow the processing of crop plant samples that will require long-distance transport. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of freeze-dried leaf disks for multiplexed PCR or DArT-Seq genotyping Basic Protocol 2: Medium-throughput preparation of pathogen-free nucleic acids for most genotyping-resequencing applications or pathogen detection Alternate Protocol: Low-throughput extraction of high-quality DNA for resequencing using commercial kits Support Protocol: DNA quality control Basic Protocol 3: Preparation of freeze-dried plant material for metabolomics Basic Protocol 4: Preparation of fresh-frozen plant material for metabolomics Basic Protocol 5: Preparation and shipment of metabolite extracts for metabolomic analyses Basic Protocol 6: Sample shipping and long-term storage.

Review of Rapid Diagnostic Methods for Vibrio Cholerae Detection in the Last Decade (2011-2022).

Cholera epidemic poses a global public health threat, heavily impacting the global economy and societies, with Africa and Asia particularly affected due to factors like; inadequate sanitation, contaminated water, and overcrowding. The associated high rates of morbidity and mortality strain productivity and healthcare costs while complicating control measures. Consequently, the World Health Organization's cholera control initiative and the Diarrhoeal Diseases Laboratory Network advocate for rapid responses to outbreaks and continuous environmental surveillance, since traditional cholera detection methods relying on phenotypic fingerprinting, although considered the gold standard, suffer from labour-intensiveness, time-consumption, and skill requirements. This results in inadequate surveillance and delayed treatment in remote areas lacking well-equipped laboratories. A systematic review was conducted to evaluate the development and performance of cholera rapid diagnostic techniques for detecting cholera in clinical samples and for environmental surveillance purposes over the past decade. Twenty-four commercially produced diagnostics were identified in January 2011. Ten more were mentioned in the literature and yet did not provide enough relevant data due to suspected production withdrawal or fall-back. The vast bulk of tests were discovered to be based on antigen or antibody detection, with DNA accounting for a large proportion of the residual tests. This study revealed a plethora of diagnostic methods, some of which have not yet made it to the commercial market. Promising approaches, such as; Loop-mediated isothermal amplification (LAMP), ELISA, and simplified PCR protocols, are likely to play a significant role in future cholera screening. Findings are herein summarised in tables and figures. Cholera epidemic continues to present a formidable global health challenge with economic and social repercussions. Traditional detection methods fall short in resource-limited areas, necessitating the exploration of advanced molecular techniques, like aptamers, to improve diagnosis, surveillance, and control measures, especially in regions vulnerable to cholera outbreaks.

Prevalence of Leucocytozoon infection in domestic birds in Ghana.

Leucocytozoon is a haemosporidian parasite known to cause leucocytozoonosis in domestic and wild birds in most parts of the world. It is an important pathogen, as some species can be pathogenic, especially in domestic birds. One of the factors affecting poultry health management worldwide is parasitism. However, the study of haemosporidian parasites in Ghana is still lacking. This study sought to assess the prevalence and diversity of Leucocytozoon parasites in domestic birds in Ghana. Blood samples were collected from domestic birds in Ghana's Bono and Eastern regions to screen for Leucocytozoon parasites. Thin blood smears were prepared for microscopy and DNA was extracted from whole blood kept in ethylenediaminetetraacetic acid (EDTA) tubes for PCR. Due to the large number of samples, real-time PCR was performed to amplify the conserved rDNA gene. Two different nested PCR protocols were performed on the positive samples obtained from real-time PCR results, to amplify a partial region of the mitochondrial cytochrome b gene and the amplicons were sequenced. Sequencing revealed six new lineages of Leucocytozoon sp. recovered in 976 individual domestic birds and these sequences were deposited in the National Center for Biotechnology Information (NCBI) GenBank. An overall Leucocytozoon prevalence of 11.6% was reported in all birds sampled. The most prevalent lineage LGHA146 (GenBank accession no. OM643346) (93.8%) was found infecting 3 bird species, Gallus gallus, Meleagris gallopavo, and Anas platyrhynchos. Phylogenetic analysis revealed that the new lineages (GenBank accession nos. OM643342, OM643343, OM643344, OM643345, OM643346, and OM643347), reported in this study were closely related to Leucocytozoon schoutedeni. We suggest that further studies be conducted to evaluate the effect of these parasite species on the general well-being of poultry in Ghana.

Retrospective analysis of Marek's disease virus outbreak in 2013 on chicken farms in North Sumatra, Indonesia

<p class="MDPI17abstract"><strong>Objective:</strong> Marek's disease is lymphoma in chicken caused by Marek's disease virus serotype 1 (MDV-1), characterized by paralysis and tumour formation. The disease has caused enormous economic loss worldwide, including in Indonesia. Unfortunately, molecular data related to MDV-1 from Indonesia is limited. Therefore, this study aimed to identify genetic characteristics of MDV-1 causing outbreaks in commercial farms in North Sumatra, Indonesia, 2013.</p><p class="MDPI17abstract"><strong>Methods:</strong> The amplification of Meq, pp38, gC, and gE genes of the strain MDN2013 was carried out using the PCR protocol. Fragment gene sequencing was accomplished using the Sanger method. Subsequently, the bioinformatic analyses of BLASTN and phylogenetic tree were applied to analyze the molecular characteristics of these gene sequences.</p><p class="MDPI17abstract"><strong>Results:</strong> The Meq, pp38, gC, and gE genes of the strain MDN2013 were successfully sequenced and submitted to the NCBI GenBank database. The Meq genes share a high identity with Asian viruses, mainly those originating from Indonesia, India, and China. The pp38, gC, and gE genes share similarities with other viruses in more diverse regions in Asia, Africa, Europe, and America. The phylogenetic tree analysis of the Meq gene demonstrated specific patterns for pathotype biodiversity where the strain MDN2013 has been categorized as virulent up to very virulent virus.</p><p class="MDPI17abstract"><strong>Conclusion:</strong> In conclusion, the molecular approach is an effective tool for investigating the MDV-1 biodiversity in the field, especially the Meq gene sequence analysis.</p>

Retrospective analysis of Marek's disease virus outbreak in 2013 on chicken farms in North Sumatra, Indonesia

<p class="MDPI17abstract"><strong>Objective:</strong> Marek's disease is lymphoma in chicken caused by Marek's disease virus serotype 1 (MDV-1), characterized by paralysis and tumour formation. The disease has caused enormous economic loss worldwide, including in Indonesia. Unfortunately, molecular data related to MDV-1 from Indonesia is limited. Therefore, this study aimed to identify genetic characteristics of MDV-1 causing outbreaks in commercial farms in North Sumatra, Indonesia, 2013.</p><p class="MDPI17abstract"><strong>Methods:</strong> The amplification of Meq, pp38, gC, and gE genes of the strain MDN2013 was carried out using the PCR protocol. Fragment gene sequencing was accomplished using the Sanger method. Subsequently, the bioinformatic analyses of BLASTN and phylogenetic tree were applied to analyze the molecular characteristics of these gene sequences.</p><p class="MDPI17abstract"><strong>Results:</strong> The Meq, pp38, gC, and gE genes of the strain MDN2013 were successfully sequenced and submitted to the NCBI GenBank database. The Meq genes share a high identity with Asian viruses, mainly those originating from Indonesia, India, and China. The pp38, gC, and gE genes share similarities with other viruses in more diverse regions in Asia, Africa, Europe, and America. The phylogenetic tree analysis of the Meq gene demonstrated specific patterns for pathotype biodiversity where the strain MDN2013 has been categorized as virulent up to very virulent virus.</p><p class="MDPI17abstract"><strong>Conclusion:</strong> In conclusion, the molecular approach is an effective tool for investigating the MDV-1 biodiversity in the field, especially the Meq gene sequence analysis.</p>

Evaluation of human papillomavirus DNA in colorectal cancer and adjacent mucosal tissue samples

BackgroundAlthough the role of viral agents, such as human papillomavirus (e.g. HPV16, HPV18) in colorectal cancer (CRC) has been previously investigated, results remain inconclusive.MethodsTo further evaluate the involvement of oncogenic HPV types in CRC, 40 frozen neoplastic and 40 adjacent colonic tissues collected from Italian patients were analyzed by Luminex-based assays that detect a broad spectrum of HPV types, i.e. Alpha (n = 21), Beta (n = 46) and Gamma HPVs (n = 52). In addition, 125 frozen CRC samples and 70 surrounding mucosal tissues were collected from Czech patients and analyzed by broad spectrum PCR protocols: (i) FAP59/64, (ii) FAPM1 and (iii) CUT combined with Next Generation Sequencing (NGS).ResultsUsing Luminex-basedassays, DNA from HPV16 was detected in 5% (2/40) CRC tissues from Italian patients. One HPV16 DNA-positive CRC case was subsequently confirmed positive for E6*I mRNA. Cutaneous beta HPV types were detected in 10% (4/40) adjacent tissues only, namely HPV111 (n = 3) and HPV120 (n = 1), while gamma HPV168 (n = 1) and HPV199 (n = 1) types were detected in adjacent and in tumor tissues, respectively. The NGS analysis of the CRC Czech samples identified HPV sequences from mucosal alpha-3 (HPV89), alpha-7 (HPV18, 39, 68 and 70) and alpha-10 species (HPV11), as well as cutaneous beta-1 (HPV20, 24, 93, 98, 105,124) beta-2 (HPV23), beta-3 (HPV49) and gamma-1 species (HPV205).ConclusionsOur findings indicate that HPV types belonging to the mucosal alpha, and the ‘cutaneous’ beta and gamma genera can be detected in the colonic mucosal samples with a low prevalence rate and a low number of HPV reads by Luminex and NGS, respectively. However, additional studies are required to corroborate these findings.