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The use of environmental DNA (eDNA) as a tool for monitoring represents a major innovative advance in environmental science, one that enables the detection of species without the need to observe or capture them. This article assesses the state of play of eDNA research targeting crayfish. We found a total of 41 peer‐reviewed articles published between 2014 and 2023 on both native and invasive species. Most studies focused on invasive species (or a native/invasive species co‐detection assessment) (65.8%). There was also a clear geographical bias across studies, with more than half conducted in Europe (51.2%) and a quarter in the United States (26.8%). In contrast, there were none conducted in Africa. The relatively large number of published studies has led to an interesting diversity of protocols designed or utilized, with most favouring the development of their own assays (69.33%). That said, filtration (as an eDNA capture method) was common (80.5%), along with the use of commercially available eDNA extraction kits (69.8%). The COI gene also appeared to be the preferred target region (89.33%). Such range of protocols is interesting, but is it optimal? Are the best protocols always being utilized? Or is the chance for novel application hampering our ability to explore larger trends across studies? [ABSTRACT FROM AUTHOR]

The potential for a donor‐derived transmission of porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) to the recipient has been recognized since pigs were considered candidate donors for xenotransplantation. This review gives a short description of the viral properties and summarizes the current evidence of the effects of PCMV/PRV transmission in preclinical xenotransplantation. Despite evidence that PCMV/PRV does not infect human and non‐human primate cells, activation in the transplanted organ and detrimental systemic complications have been described. As PCMV/PRV is a herpesvirus able to establish latency, the importance of adequate screening of donor pigs is emphasized, as no efficient treatment is available. Furthermore, easy and successful ways of elimination of PCMV/PRV from pig herds are indicated. [ABSTRACT FROM AUTHOR]

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Cereals are found in many foods. Testing their authenticity is necessary to comply with the rules of labeling and to avoid unfair competition. To protect consumers, European Union (EU) law requires labeling of ingredients that cause allergies or intolerances, especially for cereals containing gluten, such as wheat (including common wheat, durum wheat, spelled wheat), rye, barley, oats or their hybridized strains and products thereof (OJEU, 2011; OJEU, 2014). Thus, the identification of cereals in a product is of paramount importance, not only to prevent the risks related to food safety in sensitive or allergic persons but also to avoid economic fraud. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) is a worldwide health problem affecting humans, animals, and the environment within the framework of the “One Health” concept. The aim of our study was to evaluate the prevalence of pathogenic strains of the species Escherichia coli (E. coli), their AMR profile, and biofilm-forming potential. The isolated strains from three swine faeces and free lagoons (ISO 16654:2001/Amd 1:2017) were confirmed using Phoenix M50 and 16S rDNA PCR. The antibiotic sensitivity to 34 clinically applied antibiotics was determined by Phoenix M50 and the disc diffusion method, according to the protocols of the CLSI and EUCAST. We confirmed the presence of 16 E. coli isolates, of which 87.5% were multi-drug-resistant and 31.25% performed strong biofilms. The possibility for the carrying and transmission of antibiotic-resistance genes to quinolones (qnr), aminoglycosides (aac(3)), β-lactamase-producing plasmid genes ampC, and blaSHV/blaTEM was investigated. We confirmed the carrying of blaSHV/blaTEM in one and ampC in seven isolates. The strains were negative for the virulence genes (ETEC (LT, STa, and F4), EPEC (eae), and STEC/VTEC (stx and stx2all)). The results should contribute to the development of effective measures for limitation and control on the use of antibiotics, which is a key point in the WHO action plan.

In order to characterize native strains of Bacillus thuringiensis of the Colombian Caribbean with toxic effect against insect vectors, 28 samples of bacteria identified as B. thuringiensis were isolated from different soils and muds around the city of Valledupar. Using a biological test, five isolates of B. thuringiensis showed toxic effect against larvae of Aedes aegypti. PCR methods were used to detect cry1, cry2, cry4B, cry10 and cyt1 genes. Cry1 and cry2 genes were detected in 35.7% and 32.1% of the 28 isolates analyzed, respectively. Surprisingly, reduced lengths of cry4B gene segments were detected in 28.6% of B. thuringiensis samples. The presence of cry10 or cyt1 was not detected in any of the 28 samples of B. thuringiensis, despite the high sensitivity of the assays used. The results show that B. thuringiensis samples from the Colombian Caribbean have atypical characteristics compared to those of Latin America and elsewhere in the world, which is consistent with the idea that the geographic origin of B. thuringiensis samples is associated with their biological and genetic characteristics.

Phytophthora fragariae var. rubi (Wilcox & Duncan), a causal agent of raspberry root rot, is a serious soil-borne pathogen listed by EPPO as an A2 quarantine pest. Root samples were collected from badly diseased raspberry plants showing a variety of characteristic and often dramatic symptoms during surveys carried out in western Serbia in 2002. Identification of the causal agent was performed in collaboration work with the Scottish Crop Research Institute (S.C.R.I.), Dundee, UK. Necrotic roots were plated on selective French bean agar (incorporating ampicilin, ryfamicin, bavistin and hymexasol). Detection of isolates was based on cultural and morphological features compared with referent cultures. DNA was extracted directly from the sampled roots using extraction buffer (200 mM Tris- HCl pH 8.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS), purified by multi spin separation columns [Thistle Scientific (Axygen)] or in 24:1 mixture of chlorophorm- iso-amyl alcohol and amplified by nested PCR (ITS4 and DC6 for first round, DC1and DC5 for second round). Diluted DNA extracts were also amplified by conventional PCR with modified ”universal” Phytophthora primers (ITS 6, ITS 7 and ITS 8, Cooke et al., 2000) and digested with Msp1. Digestion patterns of the universal primers PCR product from infected roots matched those of Scottish strains. P. fragariae var. rubi occured on 8 out of 14 sites. Our results indicate that nested PCR (ITS4 and DC6 for first round, DC1 and DC5 for second round) or digestion of the ”universal” Phytophthora primers PCR product for detection of P. fragariae var. rubi are more sensitive and less time-consuming and therefore recommended for use.

Neutropenic patients are at risk of the development of hyalohyphomycosis and mucormycosis. Correct identification is essential for the initiation of the specific treatment, but concomitant mold infections are rarely reported. We report one unprecedented case of concomitant mucormycosis and fusariosis in a neutropenic patient with acute myeloid leukemia. The patient developed rhino-orbital infection by Rhizopus arrhizus and disseminated infection by Fusarium solani. The first culture from a sinus biopsy grew Rhizopus, which was consistent with the histopathology report of mucormycosis. A second sinus biopsy collected later during the patient's clinical deterioration was reported as hyalohyphomycosis, and the culture yielded F. solani. Due to the discordant reports, the second biopsy was reviewed and two hyphae types suggestive of both hyalohyphomycetes and mucormycetes were found. The dual mold infection was confirmed by PCR assays from paraffinized tissue sections. Increased awareness of the existence of dual mold infections in at-risk patients is necessary. PCR methods in tissue sections may increase the diagnosis of dual mold infections. In case of sequential biopsies showing discrepant results, mixed infections have to be suspected. [ABSTRACT FROM AUTHOR]

Extracted DNA from basidiomata of frozen field-collected and herbarium specimens collected in different periods and maintained in a variety of ways was evaluated according to general condition, color, spectral absorbance ratio, final concentration and PCR amplification, using two distinct and most common extraction methods: SDS (Sodium Doecyl Sulphate) and CTAB (Cetyl Trimetyl Ammonium Bromide). Most of the frozen fieldcollected basidiomata presented a very good to good DNA condition and no degradation. All the herbarium specimens exhibited poor DNA condition with low molecular weight bands. Practically all the DNA solutions of the samples were colored, and spectral absorbance ratios were considerably variable, with lower values observed in darker ones. All the samples were successfully PCR-amplified. DNA extraction of Herbarium specimens and also specimens with high polysaccharide content was only achievable using the CTAB method. For an all-purpose DNA extraction from field-collected and dehydrated coriaceous and woody basidiomata bearing a high content of secondary metabolites and/or polysaccharides, the CTAB-based method is definitely best the choice of method.

The prevalence and diversity of malaria in natural populations of birds are important in ecological and evolutionary studies. Applying PCR-based methods we studied diversity and prevalence of avian haemosporidian parasites in 3 species of Phylloscopidae in Caucasus. Only 20 individuals of 95 captured birds were infected. We had one sample with multiple infections: it carried one Plasmodium and two Haemoproteus lineages. 20 infected samples resulted 15 unique haplotypes and include parasites from all three genera (Plasmodium, Haemoproteus, Leucocytozoon). 5 haplotypes were not recorded before. We also compared our results with published data. Despite our data visually differ from the published data, to obtain statistically reliable data for comparative analyses more samples are needed. [ABSTRACT FROM AUTHOR]

Wolbachia is a large monophyletic genus of intracellular bacteria, traditionally detected using PCR assays. Its considerable phylogenetic diversity and impact on arthropods and nematodes make it urgent to assess the efficiency of these screening protocols. The sensitivity and range of commonly used PCR primers and of a new set of 16S primers were evaluated on a wide range of hosts and Wolbachia strains. We show that certain primer sets are significantly more efficient than others but that no single protocol can ensure the specific detection of all known Wolbachia infections. [ABSTRACT FROM AUTHOR]

The amplified fragment length polymorphism (AFLP) technique is a widely used multi-purpose DNA fingerprinting tool. The ability to size-separate fluorescently labelled AFLP fragments on a capillary electrophoresis instrument has provided a means for high-throughput genome screening, an approach particularly useful in studying the molecular ecology of nonmodel organisms. While the 'per-marker-generated' costs for AFLP are low, fluorescently labelled oligonucleotides remain costly. We present a cost-effective method for fluorescently end-labelling AFLPs that should make this tool more readily accessible for laboratories with limited budgets. Both standard fluorescent AFLPs and the end-labelled alternatives presented here are repeatable and produce similar numbers of fragments when scored using both manual and automated scoring methods. While it is not recommended to combine data using the two approaches, the results of the methods are qualitatively comparable, indicating that AFLP end-labelling is a robust alternative to standard methods of AFLP genotyping. For researchers commencing a new AFLP project, the AFLP end-labelling method outlined here is easily implemented, as it does not require major changes to PCR protocols and can significantly reduce the costs of AFLP studies. [ABSTRACT FROM AUTHOR]

Biogenic amines are low molecular weight organic bases frequently found in wine. Several toxicological problems resulting from the ingestion of wine containing biogenic amines have been described. In wine, histamine, tyramine, and putrescine are mainly produced by the decarboxylation of the amino acid histidine, tyrosine, and ornithine, respectively, by lactic acid bacteria action. The bacterial ability to decarboxylate amino acids is highly variable, and therefore the detection of bacteria possessing amino acid decarboxylase activity is important to prevent biogenic amine accumulation in wine. Molecular methods for the early and rapid detection of these producer bacteria are becoming an alternative to traditional culture methods. Moreover, quantitative PCR methods are useful to enumerate biogenic amine-producer bacteria on wine. Molecular methods detect potential biogenic amine risk formation in wine before the amine is produced. This review will cover the molecular methods proposed in the literature for the detection of biogenic amine-producing bacteria in wine. These methods could improve winemaking control in order to avoid biogenic amine production. [ABSTRACT FROM AUTHOR]

A prospective study estimating antiepileptic and antiviral regimens administered to infants with symptomatic epilepsy and human cytomegalovirus (HCMV) neuroinfection followed for at least 4 years. Thirty-two infants (19 female, 13 male) with epileptic seizures and HCMV neuroinfection diagnosed during the first year of life. Detection of HCMV DNA by qualitative polymerase chain reaction (PCR) method in cerebrospinal fluid (CSF), blood leukocytes, and urine confirmed the diagnosis. Infants were treated with intravenous ganciclovir (GCV) and different antiepileptic drugs. All had multiple electroencephalographic and neuroimaging examinations. Outcome of seizures was assessed using Engel classification system in the child’s fourth year of life. Cessation of seizures was achieved in 19 infants (59.4%). In 11 children (34.4%) it was possible to withdraw administration of AEDs after 30–36 months. No infantile spasms, generalized tonic–clonic seizures, or polymorphic seizures were observed. They remained seizure-free for 1–6 years without relapse and their psychomotor development was normal. Four patients with intractable epilepsy (class V) had the longest GCV treatment (median 8 weeks). GCV treatment was implemented at the time or within 1 month after the onset of epileptic seizures in 10 of 11 infants withdrawn from AEDs. Early introduction of antiepileptic and antiviral GCV regimens in epilepsy and CMV neuroinfection may result in discontinuation of antiepileptic treatment and normal psychomotor development in infants. [ABSTRACT FROM AUTHOR]

A non-destructive procedure based on nested RT-PCR and dot-blot hybridization has been developed for the detection of asymptomatic IPNV-carrier fish. The pair of primers designed for RT-PCR amplified a 599-bp fragment of the pVP2 region within the polyprotein gene, resulting in the detection of IPNV genotype III.1. The use of a nested RT-PCR allowed the amplification of IPNV genotypes III.1 and I.2. In addition, a 191-bp probe was designed for hybridization studies used in combination with the nested RT-PCR. The application of the nested RT-PCR to analyse blood samples from asymptomatic redbanded seabream, Pagrus auriga, and common seabream, P. pagrus, specimens showed a 53.1% and 77.8% prevalence of IPNV-carriers, respectively. The combination of nested RT-PCR and dot-blot hybridization increased the detection rates up to 100% for redbanded seabream and 94.4% for common seabream. Therefore, the protocol described in this study is highly sensitive and specific for the detection of IPNV in asymptomatic carrier fish, and, in addition, the results demonstrate the carrier state in two newly cultured sparid species in southern Spain. [ABSTRACT FROM AUTHOR]

In the times of industrial food production, regional and traditional food articles provide an attractive alternative for people looking for unforgettable sensory impressions. Regional or traditional food, commonly recognized as palatable and healthy, is also, for many consumers, a unique, sentimental journey back to tastes from childhood times. A gradual increase of demand for this type of food articles as well as relatively high prices of these products may generate among unscrupulous food manufacturers a number of imp-proper production practices, e.g. replacement of a more expensive meat by a less expen-sive alternative. Species composition of meat products can be verified using chroma-tographic, immunological, electrophoretic, or genetic methods. One of the genetic methods applied in examining the authenticity of food composition, in-cluding meat and its products, is the polymerase chain reaction (PCR). This paper pre-sents the most important techniques utilizing this technology to identify the origin of spe-cific meat components constituting part of regional or traditional food articles. It was demonstrated that PCR techniques, in combination with species-specific primers, PCR- -RFLP, PCR-SSCP and real-time PCR, allow identification of meat species occurring in-ependently or in mixtures with other meat species as well as meat subjected to thermal treatment or other technological processes in the course of industrial production. The only exception is the PCR-RAPD method that fails to identify meat species in the case of strong DNA degradation or in complex meat mixtures. [ABSTRACT FROM AUTHOR]

Simple Summary: Human cytomegalovirus (HCMV) infects 40–70% of adult populations in developed countries and this is thought to be involved in breast cancer progression; however, reports of detection of the viral genome in breast tumors ranges from 0–100%. We optimized a method that is both sensitive and specific to detect HCMV DNA in tissues from Canadian breast cancer patients. Only ~42% of HCMV-seropositive patients expressed viral DNA in their breast tumors. Viral transcription was not detected in any HCMV-infected breast tumors, indicating a latent infection; however, HCMV seropositivity and the presence of latent infections in breast tumors were independently, and in combination, associated with increased metastasis. HCMV DNA-positive tumors were also associated with lower relapse-free survival. Therefore, HCMV infection status should be accounted for during the monitoring and treatment of breast cancer patients. Prevention or reducing the effects of HCMV infection could decrease morbidity and mortality from metastatic disease. Human cytomegalovirus (HCMV) infects 40–70% of adults in developed countries. Detection of HCMV DNA and/or proteins in breast tumors varies considerably, ranging from 0–100%. In this study, nested PCR to detect HCMV glycoprotein B (gB) DNA in breast tumors was shown to be sensitive and specific in contrast to the detection of DNA for immediate early genes. HCMV gB DNA was detected in 18.4% of 136 breast tumors while 62.8% of 94 breast cancer patients were seropositive for HCMV. mRNA for the HCMV immediate early gene was not detected in any sample, suggesting viral latency in breast tumors. HCMV seropositivity was positively correlated with age, body mass index and menopause. Patients who were HCMV seropositive or had HCMV DNA in their tumors were 5.61 (CI 1.77–15.67, p = 0.003) or 5.27 (CI 1.09–28.75, p = 0.039) times more likely to develop Stage IV metastatic tumors, respectively. Patients with HCMV DNA in tumors experienced reduced relapse-free survival (p = 0.042). Being both seropositive with HCMV DNA-positive tumors was associated with vascular involvement and metastasis. We conclude that determining the seropositivity for HCMV and detection of HCMV gB DNA in the breast tumors could identify breast cancer patients more likely to develop metastatic cancer and warrant special treatment. [ABSTRACT FROM AUTHOR]

This paper reviews aspects relevant to detection and quantification of genetically modified (GM) material within the feed/food chain. The GM crop regulatory framework at the international level is evaluated with reference to traceability and labelling. Current analytical methods for the detection, identification, and quantification of transgenic DNA in food and feed are reviewed. These methods include quantitative real-time PCR, multiplex PCR, and multiplex real-time PCR. Particular attention is paid to methods able to identify multiple GM events in a single reaction and to the development of microdevices and microsensors, though they have not been fully validated for application. [ABSTRACT FROM AUTHOR]

The taxonomy and ecology of wild-caught Anopheles marajoara mosquitoes derived from rice fields in Frederick Settlement, Trinidad, were studied in the laboratory using specimens identified with species-specific random amplified polymorphic DNA (RAPD) profiles and recently developed rDNA ITS2 polymerase chain reaction methods. Adults were collected using Shannon traps and human bait in 2 houses over a 1-year period. All mosquitoes collected were taken to the laboratory, where they were identified, wing-length measured, and parity rates determined using standard methods. In addition, 25 females were blood fed and subsequently offered a blood meal every 2 h for a 60-h period. Based on the morphological keys and molecular tools used, the presence of An. marajoara is confirmed in Trinidad for the first time. Analysis of the seasonal distribution of An. marajoara revealed that over 58% were collected during the rainy season. The wing length of 660 females measured averaged 2.90 ± 0.130 mm, with no significant differences being observed among the parous and nulliparous females' wing sizes (2.90 and 2.92 mm, respectively). In addition, the monthly parous rate was not significantly correlated with mean wing-length over time (r = 0.157, df = 16, P > 0.07). Results from the blood feeding studies showed 85% of females blood fed immediately (hour 0) after capture in the field. However, blood feeding declined thereafter until 24 h later, when over 40% refed. This study clearly identified the presence of An. marajoara in Trinidad and provides information of the seasonal abundance and blood-feeding behaviors. These results suggest that this species can play a significant role in the transmission of malaria within its geographical range in the Neotropics. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) is a worldwide health problem affecting humans, animals, and the environment within the framework of the "One Health" concept. The aim of our study was to evaluate the prevalence of pathogenic strains of the species Escherichia coli (E. coli), their AMR profile, and biofilm-forming potential. The isolated strains from three swine faeces and free lagoons (ISO 16654:2001/Amd 1:2017) were confirmed using Phoenix M50 and 16S rDNA PCR. The antibiotic sensitivity to 34 clinically applied antibiotics was determined by Phoenix M50 and the disc diffusion method, according to the protocols of the CLSI and EUCAST. We confirmed the presence of 16 E. coli isolates, of which 87.5% were multi-drug-resistant and 31.25% performed strong biofilms. The possibility for the carrying and transmission of antibiotic-resistance genes to quinolones (qnr), aminoglycosides (aac(3)), β-lactamase-producing plasmid genes ampC, and blaSHV/blaTEM was investigated. We confirmed the carrying of blaSHV/blaTEM in one and ampC in seven isolates. The strains were negative for the virulence genes (ETEC (LT, STa, and F4), EPEC (eae), and STEC/VTEC (stx and stx2all)). The results should contribute to the development of effective measures for limitation and control on the use of antibiotics, which is a key point in the WHO action plan. [ABSTRACT FROM AUTHOR]

DNA diagnosis of hemophilias has received much attention in recent years, and different methods are presently available to analyze the coagulation factor IX and VIII genes. This review is intended to serve as a brief guide to current and emerging diagnostic strategies. [ABSTRACT FROM AUTHOR]

New methodologies based on the principle of "sewage epidemiology" have been successfully applied before in the detection of illegal drugs. The study describes the idea of early detection of a virus, e.g., SARS-CoV-2, in wastewater in order to focus on the area of virus occurrence and supplement the results obtained from clinical examination. By monitoring temporal variation in viral loads in wastewater in combination with other analysis, a virus outbreak can be detected and its spread can be suppressed early. The use of biosensors for virus detection also seems to be an interesting application. Biosensors are highly sensitive, selective, and portable and offer a way for fast analysis. This manuscript provides an overview of the current situation in the area of wastewater analysis, including genetic sequencing regarding viral detection and the technological solution of an early warning system for wastewater monitoring based on biosensors.

In order to characterize native strains of Bacillus thuringiensis of the Colombian Caribbean with toxic effect against insect vectors, 28 samples of bacteria identified as B. thuringiensis were isolated from different soils and muds around the city of Valledupar. Using a biological test, five isolates of B. thuringiensis showed toxic effect against larvae of Aedes aegypti. PCR methods were used to detect cry1, cry2, cry4B, cry10 and cyt1 genes. Cry1 and cry2 genes were detected in 35.7% and 32.1% of the 28 isolates analyzed, respectively. Surprisingly, reduced lengths of cry4B gene segments were detected in 28.6% of B. thuringiensis samples. The presence of cry10 or cyt1 was not detected in any of the 28 samples of B. thuringiensis, despite the high sensitivity of the assays used. The results show that B. thuringiensis samples from the Colombian Caribbean have atypical characteristics compared to those of Latin America and elsewhere in the world, which is consistent with the idea that the geographic origin of B. thuringiensis samples is associated with their biological and genetic characteristics.