PCR-based Method Research Articles

The impact of DNA extraction on the quantification of Legionella, with implications for ecological studies.

Monitoring the levels of opportunistic pathogens in drinking water is important to plan interventions and understand the ecological niches that allow them to proliferate. Quantitative PCR is an established alternative to culture methods that can provide a faster, higher-throughput, and more precise enumeration of the bacteria in water samples. However, PCR-based methods are still not routinely applied for Legionella monitoring, and techniques, such as DNA extraction, differ notably between laboratories. Here, we quantify the impact that DNA extraction methods had on downstream PCR quantification and community sequencing. Through a community science campaign, we collected 50 water samples and corresponding shower hoses, and compared two commonly used DNA extraction methodologies to the same biofilm and water phase samples. The two methods showed clearly different extraction efficacies, which were reflected in both the quantity of DNA extracted and the concentrations of Legionella enumerated in both the matrices. Notably, one method resulted in higher enumeration in nearly all samples by about one order of magnitude and detected Legionella in 21 samples that remained undetected by the other method. 16S rRNA amplicon sequencing revealed that the relative abundance of individual taxa, including sequence variants of Legionella, significantly varied depending on the extraction method employed. Given the implications of these findings, we advocate for improvement in documentation of the performance of DNA extraction methods used in drinking water to detect and quantify Legionella, and characterize the associated microbial community.IMPORTANCEMonitoring for the presence of the waterborne opportunistic pathogen Legionella is important to assess the risk of infection and plan remediation actions. While monitoring is traditionally carried on through cultivation, there is an ever-increasing demand for rapid and high-throughput molecular-based approaches for Legionella detection. This paper provides valuable insights on how DNA extraction affects downstream molecular analysis such as the quantification of Legionella through droplet digital PCR and the characterization of natural microbial communities through sequencing analysis. We analyze the results from a risk-assessment, legislative, and ecological perspective, showing how initial DNA processing is an important step to take into account when shifting to molecular-based routine monitoring and discuss the central role of consistent and detailed reporting of the methods used.

The establishment of a molecular diagnostic platform for mitochondrial diseases: from conventional to next-generation sequencing

BackgroundThe aim of this study was to create a molecular diagnostic platform and establish a diagnostic pipeline for patients highly suspected of mitochondrial disorders. The effectiveness of three methods, namely, traditional restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR), Sanger sequencing for hotspot detection and whole mitochondrial DNA (mtDNA), and third-generation (Nanopore) whole mtDNA sequencing, will be compared in diagnosing patients with suspected primary mitochondrial diseases (PMDs). The strengths and limitations of different methods are also discussed. Material and methodsA single-center prospective cohort study was conducted to validate the diagnostic pipeline for suspected mitochondrial diseases. In the first stage, a PCR-based method with five sets of primers was used to screen for eight hotspots (m.3243A>G, m.3460G>A, m.8344A>G, m.8993T>G, m.9185T>C, m.11778G>A, m.13513G>A, and m.4977deletion) using either RFLP or direct Sanger sequencing. Sanger sequencing was also used to confirm the RFLP-positive samples. In the second stage, for samples with negative screening results for the eight hotspots, mitochondrial whole-genome sequencing was performed using Sanger sequencing or third-generation nanopore sequencing. ResultsBetween June 2020 and May 2023, 30 patients from ages 0 to 63 with clinically suspected mitochondrial disease were enrolled. The positive yield for the diagnosis of PMDs was 8/30=26.7%, and the sensitivity of the heteroplasmy level for the RFLP-based method was approximately 5%. The remaining 22 patients who tested negative at the first stage were tested using Sanger sequencing or the third-generation sequencing Nanopore, and all tested negative for pathological mtDNA mutations. Compared to the Sanger sequencing method, the results of RFLP-PCR were compromised by the limitations of incomplete RFLP enzyme digestion. For whole-genome sequencing of mtDNA, Sanger sequencing, instead of nanopore sequencing, is preferred at our institution because of its cost-effectiveness. Conclusions: In our highly selective cohort, most tested positive in the first stage of the 8 hot spots screen. Sanger sequencing is a conventional and accurate method for mitochondrial disease screening, at least for the most common hot spots in the region. The results revealed that Sanger sequencing is an accurate method with the benefit of being more cost-effective. This integral platform of molecular diagnosis bears the advantages of being relatively low cost and having a shorter reporting time, facilitating crucial identification of patients with clinical evidence of such disorders. This diagnostic flowchart has also been translated into routine clinical use in the tertiary hospital.

Improving Consistency and Accuracy: A Novel C. auris Colonization Screening Strategy Using a Nares + Hands Composite Swab

Background: Candida auris is often identified in healthcare settings through bilateral composite of axilla/groin skin swabs screening. Re-screening the same patient has demonstrated inconsistent results over time, complicating the understanding of longitudinal colonization and limiting confidence in negative Results: Previous studies have described identification of colonized patients using other anatomical sites. Here, we compare bilateral composite of nares/hands with bilateral composite of axilla/groin screenings in a cohort of hospitalized patients in Miami, Florida, to assess the use of screening other body sites for C. auris surveillance. Methods: This study took place in a 560-bed academic acute-care facility and included patients previously colonized with C. auris who were cohorted on a 30-bed unit. Bilateral composite samples from both the axilla/groin and nares/hands were obtained simultaneously. Swabs were collected at six different time points at biweekly intervals between March and May 2023 (Figure 1) and sent to the Centers for Disease Control and Prevention for testing with culture and Real-time PCR-based methods. Results: A total of 102 swabs (51 from each swab type) were collected from 19 patients who were each sampled a median of twice (IQR: 1-5). Among the 102 swabs, 35 of 51 (69%) axilla/groin swabs were positive compared with 45 of 51 (88%) nares/hands swabs using culture (Figure 2). Furthermore, 48 of 51 (94%) swabs were positive by culture for both methods, with 15 positive from the nares/hands and one positive from the axilla/groin (Figure 3). Among 11 patients who were tested ≥2 times with nares/hands swabs, 9/11 (81%) tested positive on all sequential swabs via culture and 10/11 (90%) tested positive via PCR (Ct threshold < 3 6.9). Among the same 11 patients but using the axilla/groin swabs, 3/11 (27%) patients tested positive on all sequential swabs using culture, and 5/11 (45%) tested positive using PCR (Figures 2-4). On average, samples collected from nares/hands swabs had lower Ct values (mean=27) compared to axilla/groin swabs (mean=31) (p-value=< 0.001) (Figure 5). Discussion: Identifying the swab site with most consistent C. auris detection is important for surveillance purposes. In our study, there were more positives and consistent positivity for nares/hands by both culture and PCR-based methods, as well as lower Ct values, suggesting that these swabs provide more reliable detection of C. auris colonization. Alternative screening methods deserve consideration as CDC continues to explore whether swabbing of other body sites (e.g., nares, hands) would improve accuracy and consistency when identifying colonized patients.

A Rapid PCR-Based Diagnostic Method for Skin Infection with Mycobacterium marinum.

The increasing incidence of chronic skin infections caused by Mycobacterium marinum, coupled with the time-consuming and low detection rates nature of traditional culture and histological-based diagnostic methods, underscores the need for an expedited approach. The study aims to develop a rapid and efficient method for detecting M. marinum with PCR technology. We designed four pairs of primers based on DNA sequences from GeneBank and prior studies, we utilized both PCR and Real-time PCR to identify M. marinum. Specificity and sensitivity assessments were conducted in vitro by DNAs extracted from M. marinum and other bacterial or fungal cultures. Further validation was performed through the implementation of a mouse skin infection model to optimize and confirm the efficacy of the detection method in both fresh and paraffin-embedded skin tissues. The same PCR testing system was further confirmed with paraffin-embedded skin tissues samples from patients as well. The results of the study indicate promising outcomes for the four-pair primers system. It demonstrated 100% sensitivity in detecting M. marinum from purified cultures, including typical strains and nine clinical isolates, while achieving a specificity of 100%. This specificity was evidenced by the absence of PCR products from 12 bacterial species, 12 fungi species, and six other non-tuberculous mycobacterium (NTM) species. In the animal model, the PCR assay exhibited high detection efficacy for both infected fresh tissues and paraffin-embedded tissues, with a slight superiority observed in fresh tissues. However, the PCR assay exhibited high detection efficacy for clinical paraffin-embedded tissues. These findings collectively underscore the robust detection capabilities of our four-pair primers in both in vitro and in vivo settings. A sensitive and highly specific rapid detection system has been successfully developed that can be used to detect M. marinum in both infected fresh tissues and paraffin-embedded tissues.

The effect of genetic alterations detected by the circulating tumor DNA-based next-generation sequencing technique on prognosis and survival in metastatic colorectal cancer

Purpose: Studies conducted to date showed that circulating tumor DNA (ctDNA)-based next generation sequencing (NGS) panels are beneficial in the treatment strategies of patients with metastatic colorectal cancer (mCRC). In this study; we planned to determine the frequencies of various genetic alterations in patients with mCRC by ctDNA-based NGS analyses, evaluate the concordance rates by comparing these results with the results in standard polymerase chain reaction (PCR) analyses, and investigate the effect of the detected alterations on overall survival and progression-free survival. Materials and methods: The study was conducted by retrospective screening and analysis of the data on 48 patients, who were followed up with a diagnosis of mCRC and who received chemotherapy and/or biological agents. The data were analyzed using SPSS 25.0 [IBM SPSS Statistics 25 software (Armonk, NY: IBM Corp.)] package program. Results: In this study, ctDNA-based NGS analyses, compared to the quantitative PCR-based gold standard method, were found to have a sensitivity rate of 64.7%, specificity rate of 55.6% and concordance rate of 59.1% for KRAS mutation; a sensitivity rate of 100%, specificity rate of 86.7% and concordance rate of 87.1% for NRAS mutation; a sensitivity rate of 50%, specificity rate of 96.4% and concordance rate of 90.6% for BRAF mutation. In addition, concordance rates were evaluated based on to the time elapsed between the time of taking the liquid biopsy and tissue biopsy samples. As a result, concordance rates for KRAS, NRAS, and BRAF mutations were found to be 60.9%, 100%, and 100% respectively, in cases where this elapsed time was less than 6 months; and were found to be 57.1%, 78.9%, and 85% respectively, in cases where this elapsed time was more than 6 months. Furthermore, the comprehensive analyzes revealed that the frequency of many molecular changes in mCRC as well as the relationship of these changes with clinicopathological features and survival times. Conclusion: Our study demonstrates the clinical benefit of ctDNA-based NGS analyzes in patients with mCRC.

A large cohort of Hb H disease in northeast Thailand: A molecular revisited, diverse genetic interactions and identification of a novel mutation

Background and aimsTo update the molecular characteristics of α-thalassemia in northeast Thailand, the molecular basis and genetic interactions of Hb H disease were examined in a large cohort of patients. Materials and methodsA study was done on 1,170 subjects with Hb H disease and various genetic interactions encountered during 2009–2023. Hb and DNA analyses were carried out. ResultsAs many as 40 genotypes with several known, previously undescribed, and novel mutations were observed. These included 698 subjects (59.8 %) of Hb H disease, 357 (30.6 %) with EABart’s disease, 63 (5.4 %) with EEBart’s disease, 18 (1.7 %) with abnormal Hbs, 17 (1.5 %) with β-thalassemia, and 4 (0.4 %) with EFBart’s or EFABart’s disease. The molecular basis of 13 subjects (1.1 %) remains unknown. The α0-thalassemia included --SEA (n = 1,139, 97.4 %) and --THAI (n = 21, 1.8 %). Two rare mutations were identified in 3 subjects (0.3 %) with --SA and --CR deletions. For α+-thalassemia, −α3.7 kb del (n = 626, 53.5 %), Hb Constant Spring (n = 415, 35.5 %), −α4.2 kb del (n = 44, 3.8 %), Hb Paksé (n = 36, 3.1 %), and Hb Q-Thailand (n = 19, 1.6 %), were detected. Ten rarer α+-thalassemia were identified, including a novel mutation, namely the Hb Chumphae (HBA2:c.32T>A). The Hb H-Lansing-Ramathibodi, Hb H-Jax, and Hb H-Chumphae are hitherto undescribed in this region. PCR-based diagnostic methods for these α-thalassemia defects were described. ConclusionsThis study confirms the diverse heterogeneity and genetic interactions causing Hb H disease in northeast Thailand. The results should prove useful for laboratory diagnosis and genetic counseling of this genetic disorder in the region.

Hematodinium perezi (Dinophyceae: Syndiniales) in Morocco: The First Record on the African Atlantic Coast and the First Country Record of a Parasite of the Invasive Non-Native Blue Crab Callinectes sapidus

Dinoflagellates belonging to the genus Hematodinium are key parasites of marine crustaceans, primarily decapods. In this study, we document the first report of H. perezi Chatton & Poisson, 1930 on the African Atlantic coast. This is also the first parasite record in the invasive non-native Atlantic blue crab Callinectes sapidus Rathbun, 1896 in Morocco. Specimens of C. sapidus were sampled in winter 2023 from two Ramsar sites on the Moroccan Atlantic, namely Merja Zerga and Oualidia Lagoons, and were screened to detect the presence of parasites in their hemolymph. Based on staining fresh hemolymph smears, we did not detect Hematodinium in any of the 36 investigated individuals (20 and 16 from Merja Zerga and Oualidia Lagoons, respectively), probably due to methodological artifacts. The PCR-based method was revealed to be more accurate in diagnosing the Hematodinium parasite. It showed that at Merja Zerga Lagoon, 13 individuals of C. sapidus were infected by the parasite (prevalence: 65%) in comparison to four at Oualidia Lagoon (25%). Genetic analysis, based on the ITS1 rDNA gene from Hematodinium, confirmed the sequences as being those of Hematodinium perezi.

Routine use of MSI testing in colorectal cancer using a proposed algorithm.

Fifteen percent of all colorectal cancers have detectable defects in the mismatch repair system (dMMR). MMR status is used to identify possible Lynch Syndrome (LS) and to determine prognosis and choice of treatment. Two standard techniques for determining MMR status are immunohistochemistry (IHC) and analysis for microsatellite instability (MSI) by PCR. Recently, our department introduced Idylla™ MSI assay as an alternative option to IHC, and as part of this, we introduced a decision algorithm. The purpose of this study was to review the use of the new method and our algorithm and to assess possible false-positive results. Retrospectively, we identified 629 cases of colorectal cancer in which either IHC (336 cases) or Idylla™ MSI (293 cases) was performed. Similar results were obtained by the two methods. IHC detected dMMR in 55 cases (16%) and Idylla™ MSI in 52 cases (18%). In all 52 cases of MSI, subsequent IHC was performed. One case was not confirmed by IHC, but was confirmed by another PCR-based method. Overall, we found that the Idylla™ MSI works well as a screening method for dMMR with no false-positive cases detected. The proposed algorithm was useful and easily applicable.

Multiplexing LAMP Assays: A Methodological Review and Diagnostic Application.

The loop-mediated isothermal amplification (LAMP) technique is a great alternative to PCR-based methods, as it is fast, easy to use and works with high sensitivity and specificity without the need for expensive instruments. However, one of the limitations of LAMP is difficulty in achieving the simultaneous detection of several targets in a single tube, as the methodologies that allow this rely on fluorogenic probes containing specific target sequences, complicating their adaptation and the optimization of assays. Here, we summarize different methods for the development of multiplex LAMP assays based on sequence-specific detection, illustrated with a schematic representation of the technique, and evaluate their practical application based on the real-time detection and quantification of results, the possibility to visualize the results at a glance, the prior stabilization of reaction components, promoting the point-of-care use, the maximum number of specific targets amplified, and the validation of the technique in clinical samples. The various LAMP multiplexing methodologies differ in their operating conditions and mechanism. Each methodology has its advantages and disadvantages, and the choice among them will depend on specific application interests.

Fast and Simple Molecular Test for Sex Determination of the Monomorphic Eudromia elegans Individuals.

Sex determination based just on morphological traits such as plumage dichromatism, sexual size dimorphism, behavior, or vocalizations is really challenging because of the sexual monomorphism present in more than half of avian species. Currently, a lot of them can be tested through DNA-based procedures, but they do not fit all the avian species, such as Eudromia elegans. The aim of this study was to design a new molecular method suitable for routine sex determination for that species that is fast, simple, and cost- and time- effective. DNA was isolated from dry blood stain and/or chest feather samples of E. elegans species. We used two sets of sex-specific primers (ZF/ZR and WF/WR) to amplify the expected fragments localized on the highly conserved CHD1 gene to distinguish between sexes due to the W-specific DNA sequence present only in females. We confirmed the accuracy and consistency of the PCR-based method based on length differences to distinguish between the sexes of E. elegans, which amplified two fragments in females and one fragment in males.

Analysis of genetic variants for different horn phenotypes and their inheritance in Icelandic sheep

Abstract. Icelandic sheep are characterized by a great diversity in horn phenotypes. Within their breed, they show a variability in terms of this trait to an extent rarely observed elsewhere. Previously, several genetic variants were published as markers for horn status (in terms of absence or presence of horns, including scurs) and horn traits (e.g., oval horns, horn length and polyceraty). The aim of this study was to genotype, for the first time, five of these genetic variants in Icelandic sheep with different horn phenotypes, as well as to analyze their inheritance. Phenotypic and pedigree data, as well as DNA samples from two Icelandic sheep farms, were used. Genetic variants were genotyped by published PCR-based methods in all samples (n=94) or in subsets. As in other sheep breeds with variable horn status, the inheritance of the presence or absence of horns was shown to be complex in Icelandic sheep, especially when sheep carry anything other than regularly formed horns. The 1.78 kb sized RXFP2 insertion on ovine chromosome 10 previously described to be associated with polledness in several sheep breeds was also found to be present in Icelandic sheep and showed some association but not a perfect segregation with the individuals' horn statuses. Missing associations were especially seen in sheep with scurs and oval horns. Regarding horn shape, there was no agreement with the studied variants described in Chinese breeds having comparable horn traits. However, matching tendencies were seen for the horn size variant that was found in the same study. All sheep with four or more horns carried the already published 4 bp deletion in HOXD1, as previously described for three other sheep breeds. Interestingly, for the first time, the deletion was also detected in phenotypically polled animals originating from multi-horned families. According to the results from animals genotyped simultaneously for the RXFP2 and the HOXD1 variants, polledness in sheep with a genetic disposition for polyceraty seems not to be controlled by the RXFP2 insertion. However, this and all other findings in Icelandic sheep need to be confirmed by analyzing a higher number of well-phenotyped animals.

Occurrence, Impact, and Multilocus Sequence Analysis of Alder Yellows Phytoplasma Infecting Common Alder and Italian Alder in Southern Italy.

Alder yellows (ALY) phytoplasma (16SrV-C) is associated with ALY, a disease of several Alnus (alder) species in Europe and A. rubra in North America. In all affected species, the symptoms are similar. However, latent infections are common. ALY phytoplasma includes different strains which may be occasionally transmitted to grapevines leading to some grapevine yellows diseases. In the current study, visual symptom assessment and PCR-based methods using universal and group-specific phytoplasma primers were used to update and extend knowledge on the occurrence, impact, and genetic diversity of ALY phytoplasma in declining and non-symptomatic A. glutinosa and A. cordata trees in the Basilicata and Campania regions of southern Italy. ALY phytoplasma was detected in 80% of alder trees examined. In symptomatic trees, no other cause of disease was observed. More than half of alder trees that tested phytoplasma-positive proved to be latently infected. A considerable genetic variability was observed among the newly recorded ALY phytoplasma strains in southern Italy in almost of the genes examined. These included 16S rRNA, 16S/23S rDNA spacer region, ribosomal protein rpsV (rpl22) and rpsC (rps3), map, imp, and groEL genes. Eleven new genotypes were identified at map gene sequence level. However, the genetic differences observed were not related to plant host species, geographical origin, and symptoms shown by infected alder trees. Also, this study indicates that ALY phytoplasma is more widespread than previously thought.

Development and validation of a ddPCR assay to detect and quantify tobacco DNA in smoke and smokeless tobacco and tobacco-free products

The last decade, smoke and smokeless products claiming to be tobacco-free, including herbal cigarettes and herbal shisha, became available on the European market and gained popularity. This study proposes a new digital droplet PCR (ddPCR) method, designed based on a previously developed real-time PCR (qPCR) method being currently used by the U.S. Food and Drug Administration (FDA) to specifically detect the presence of tobacco DNA in targeting a sequence from the Nicotiana tabacum nia-1 gene. To ensure a harmonized and reliable control by enforcement laboratories, both of these qPCR and ddPCR methods were then evaluated and validated for their compliance to an international standard. First, the performance of these PCR-based methods was successfully assessed as specific and sensitive, and in line with minimum performance requirements from international standard. Secondly, the transferability to external laboratory was confirmed for these PCR-based methods. Finally, the applicability of these PCR-based methods was demonstrated using 7 ground tobacco reference materials from the Tobacco Research Center (TRC) Toronto University as well as 6 commercial smokeless and tobacco-free smoke and smokeless products. Based on this study, the previously developed qPCR method was confirmed as complying with international standard, ensuring a efficient and harmonize use by enforcement laboratories for tobacco control on the European market. Moreover, this study proposed to enforcement laboratories the possibility to use a ddPCR method, enabling the simultaneous detection and absolute quantification of tobacco DNA as well as a limited impact of PCR inhibitors.

Development and validation of a PCR-based method to differentiate human sex in blood-fed Aedes aegypti (Diptera: Culicidae).

Monitoring mosquito host choice to identify high-risk groups for different vector-borne diseases is important to devise vector control strategies and disease management. The present study was conducted to develop and validate a PCR-based method to identify human sex in blood-fed Aedes aegypti mosquitoes. Several human genes present in both the X and Y chromosomes were screened and diagnostic PCR primers were successfully designed and amplified for the human STS gene. The limit of detection of this PCR assay was carried out on Ae. aegypti fed with human blood up to 5 days (120 hours) post blood-meal under laboratory condition. The efficiency of this PCR assay was evaluated in field-collected Ae. aegypti mosquitoes and compared with other existing methods. The developed PCR primers can successfully amplify and distinguish human sex in mosquitoes up to 72 hours after a blood meal, with an amplified product of 627bp and 298bp for male (XY) and 627bp for female (XX) blood-fed mosquitoes. Further, validation of this assay in field-collected Ae. aegypti mosquitoes revealed that this assay could detect human sex in mosquito blood meal substantially more efficiently (c2 = 4.5, p = 0.034) than other PCR based assay. The newly developed PCR assay highly specific to human DNA and can distinguish male and female DNA for up to 72 hours. This assay can be is used for identifying highrisk groups and extended to other medically important hematophagous insects to assess their role in disease transmission and epidemic preparedness.

Association of TaqI (rs731236) Polymorphism of Vitamin D Receptor Gene with Lumbar Degenerative Disc Disease

Lumbar degenerative disc disease (LDDD) significantly contributes to low back pain, with a complicated etiology involving genetic and environmental facts. The aim of study was to investigate the association between the TaqI (rs731236) polymorphism of the vitamin D receptor (VDR) gene with LDDD. In total, 248 patients with symptomatic LDDD and 146 control subjects were examined. The evaluation of clinical features of patients with LDDD comprised radiodiagnostic magnetic resonance imaging, neurologic examinations, pain scores including the visual analog scale (VAS), and disability investigation with Oswestry Disability Index (ODI). Genotyping of the VDR gene polymorphism was conducted using polymerase chain reaction-based methods. Individuals of the LDDD group who were VDR TaqI AA genotype carriers were significantly greater than the other group (P= 0.014), whereas those with GG genotype were significantly lower (P= 0.028) in the patient group. In addition, VAS and ODI scores were significantly lower in the GG genotype carrier group, whereas AA genotype carriers had the greatest scores (P= 0.004). Carrying the G allele decreased the risk of LDDD 1.7 times (P= 0.014) and carrying the A allele enhanced the risk 1.8 times (P= 0.028). Moreover, G-allele carriers had significantly lower VAS (P= 0.002) and ODI scores (P < 0.0001). VDR TaqI (rs731236) GG genotype and G allele have protective potential, whereas the AA genotype and A allele are risk factors for LDDD. The findings reveal a statistically significant association of the TaqI (rs731236) polymorphism of VDR gene polymorphism with LDDD. This result highlights the potential role of genetic factors in developing LDDD and suggests avenues for future research in genetic screening and personalized treatment strategies.

Comprehensive Investigation of Salmonella spp.: Isolation, Characterization and PCR Primer Set Evaluation for Enhanced Detection and Identification

Background: Salmonellosis is a serious health concern for both animals and humans. According to a report of the Centers for Disease Control and Prevention, 40,000 human infections occur annually; however, the number of unconfirmed cases is as high as approximately 800,000-4000,000. These organisms gain entry into the gastrointestinal cells and cause serious diseases, resulting in intestinal damage and even death. Methods: In this research, 115 Salmonella isolates from different sources were tested. Using morphological, cultural, and biochemical methods, they were classified into 30 serovars. Plasmid profiling was used for quick identification, which showed similarities among the isolates.PCR primer sets targeting specific genes, such as invA, hilA, iroB, oriC, fimA, hitJ and stn, demonstrated reliability, accuracy and specificity for identifying S. enterica serovars. Result: This study recommends the use of specific primer sets in PCR-based methods for rapid and reliable identification and detection of locally isolated Salmonella serovars/strains. This research adds to the continuing efforts for improving the diagnostic techniques to tackle the challenges presented by Salmonella spp.

Distribution of toxigenic cyanobacteria in Alpine lakes and rivers as revealed by molecular screening

The increasing frequency of cyanobacteria blooms in waterbodies caused by ecosystem eutrophication could endanger human health. This risk can be mitigated by effective monitoring incorporating molecular methods. To date, most molecular studies on toxigenic cyanobacteria have been limited to microcystins (MCs), disregarding other cyanotoxins, to freshwater planktic habitats while ignoring benthic habitats, and to limited geographic areas (usually one or a few specific waterbodies). In this study, we used PCR-based methods including PCR product sequencing and chemical-analytical methods (LC-MS/MS) to screen many plankton (n = 123) and biofilm samples (n = 113) originating from 29 Alpine lakes and 18 rivers for their cyanotoxin production potential. Both mcyE (indicating MC synthesis) and anaC (indicating anatoxin (ATX) synthesis) gene fragments were able to qualitatively predict MC or ATX occurrence. The abundance of mcyE gene fragments was significantly related to MC concentrations in plankton samples (R2 = 0.61). mcyE gene fragments indicative of MC synthesis were most abundant in planktic samples (65 %) and were assigned to the genera Planktothrix and Microcystis. However, mcyE rarely occurred in biofilms of lakes and rivers, i.e., 4 % and 5 %, respectively, and were assigned to Microcystis, Planktothrix, and Nostoc. In contrast, anaC gene fragments occurred frequently in planktic samples (14 % assigned to Tychonema, Phormidium (Microcoleus), and Oscillatoria), but also in biofilms of lakes (49 %) and rivers (18 %) and were assigned to the genera Phormidium, Oscillatoria, and Nostocales. The cyrJ gene fragment indicating cylindrospermopsin synthesis occurred only once in plankton (assigned to Dolichospermum), while saxitoxin synthesis potential was not detected. For plankton samples, monomictic and less eutrophic conditions were positively related to mcyE/MC occurrence frequency, while oligomictic conditions were related to anaC/ATX frequency. The anaC/ATX frequency in biofilm was related to the lake habitats generally showing higher biodiversity as revealed from metabarcoding in a parallel study.

PCR-based genome walking methods (review)

The review discusses a range of classical and modern methods used to determine the nucleotide sequence of unknown DNA regions flanking known ones. These methods are applied to decipher the regulatory regions of genes, identify integration sites of T-DNA or viruses, and so on, in cases where the use of whole-genome sequencing is not justified. To amplify a DNA segment, a binding site for a primer must be added to the end of the unknown sequence. This can be achieved either by ligating an adapter or by annealing a degenerate primer under gentle conditions, or by looping the DNA fragment so that the target region is surrounded by known sequences. The second important task is to eliminate the inevitable products of nonspecific binding of adapters or degenerate primers, which is often resolved through multiple rounds of nested PCR. Different methods vary significantly in terms of complexity, prevalence, and the availability of required reagents.

Limited availability of methods for the detection of xenotransplantation-relevant viruses in veterinary laboratories.

The German Xenotransplantation Consortium is in the process to prepare a clinical trial application (CTA) on xenotransplantation of genetically modified pig hearts. In the CTA documents to the central and national regulatory authorities, that is, the European Medicines Agency (EMA) and the Paul Ehrlich Institute (PEI), respectively, it is required to list the potential zoonotic or xenozoonotic porcine microorganisms including porcine viruses as well as to describe methods of detection in order to prevent their transmission. The donor animals should be tested using highly sensitive detection systems. I would like to define a detection system as the complex including the actual detection methods, either PCR-based, cell-based, or immunological methods and their sensitivity, as well as sample generation, sample preparation, sample origin, time of sampling, and the necessary negative and positive controls. Lessons learned from the identification of porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) in the xenotransplanted heart in the recipient in the Baltimore study underline how important such systems are. The question is whether veterinary laboratories can supply such assays. A total of 35 veterinary laboratories in Germany were surveyed for their ability to test for selected xenotransplantation-relevant viruses, including PCMV/PRV, hepatitis E virus, and porcine endogenous retrovirus-C (PERV-C). As comparison, data from Swiss laboratories and a laboratory in the USA were analyzed. Furthermore, we assessed which viruses were screened for in clinical and preclinical trials performed until now and during screening of pig populations. Of the nine laboratories that provided viral diagnostics, none of these included all potential viruses of concern, indeed, the most important assays confirmed in recent human trials, antibody detection of PCMV/PRV and screening for PERV-C were not available at all. The situation was similar in Swiss and US laboratories. Different viruses have been tested for in first clinical and preclinical trials performed in various countries. Based on these results it is necessary to establish special virological laboratories able to test for all xenotransplantation-relevant viruses using validated assays, optimally in the xenotransplantation centers.

Considerations and Opportunities for Probe Capture Enrichment Sequencing of Emerging Viruses from Wastewater.

Until recently, wastewater-based monitoring for pathogens of public health concern primarily used PCR-based quantification methods and targeted sequencing for specific pathogens (e.g., SARS-CoV-2). In the past three years, researchers have expanded sequencing to monitor a broad range of pathogens, applying probe capture enrichment to wastewater. The goals of those studies included (1) monitoring and expanding fundamental knowledge of disease dynamics for known pathogens and (2) evaluating the potential for early detection of emerging diseases resulting from zoonotic spillover or novel viral variants. Several studies using off-the-shelf probe panels designed for clinical and environmental surveillance reported that enrichment increased virus relative abundance but did not recover complete genomes for most nonenteric viruses. Based on our experience and recent results reported by others using these panels for wastewater, clinical, and synthetic samples, we discuss challenges and technical factors that affect the rates of false positive and false negative results. We identify trade-offs and opportunities throughout the workflow, including in wastewater sample processing, probe panel design, and bioinformatic analysis. We suggest tailored methods of virus concentration and background removal, carefully designed probe panels, and multithresholded bioinformatics analysis.