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Background: porcine circovirus type 2 (PCV2) is associated with reproductive disease in newly populated herds and in replacement breeding stock from new sources and is almost exclusively reported in gilts. Objective: the main purpose of this study was to assess the dynamics of porcine circovirus type 2 infection and neutralizing antibodies in subclinically infected gilts and the effect on their piglets. Methods: the study was conducted with 40 gilts selected at random from four breeding herds. Blood samples, nasal and vaginal swabs were obtained from the gilts at arrival, acclimatization, farrowing, and one day after farrowing. Colostrum samples were collected immediately after parturition and one day after farrowing. Blood, nasal swab, or tissue samples were collected from four piglets prior to suckling. All serums were analyzed by virus neutralization test (VNT) to establish the presence of antibodies. All samples were subjected to SYBER Green real-time PCR assay to detect PCV2 DNA. Results: high levels of viremia and viral load of PCV2 in nasal and vaginal swabs were found in healthy gilts at arriving, confirming the introduction of infected animals into the farms. In addition, most gilts were positive for PCV2 DNA in serum, nasal and vaginal swabs at farrowing. PCV2 shedding was also observed in nasal and vaginal fluids and colostrum even in presence of serum neutralizing antibodies (NA). Subclinically infected dams had detectable viremia, developed anti-PCV2 antibodies, and there was PCV2 DNA in tissue samples of their born alive and healthy piglets. PCV2a and PCV2b genotypes were confirmed in PCV2 subclinical infection in both dams and piglets in utero. Conclusion: replacement gilts can be infected with PCV2 before entering the farm and continuous exposure seems to occur horizontally in acclimatization and gestation units or before farrowing. Exposure and infection during gestation may result in infected but apparently healthy piglets. Antecedentes: o circovírus suíno tipo 2 (PCV2) está associado a casos de falha reprodutiva em granjas recém-assentadas e fazendas de criação de marrãs e afeta principalmente a porcas nulíparas. Clinicamente observam-se aumento dos fetos abortados no segundo e terceiro estágios da gravidez, fetos mumificados, natimortos e nascimento de leitões inviáveis. Objetivo: o principal objetivo deste estudo foi avaliar a dinâmica da infeção pelo circovirus porcino tipo 2 e os títulos de anticorpos neutralizantes em porcas nulíparas com infecção subclínica e o efeito em sua leitegada. Métodos: este estudo foi realizado com 40 porcas em quatro granjas selecionadas aleatoriamente. Foram coletados de cada animal amostras de sangue, esfregaços nasais e vaginais ao entrar na fazenda, durante a quarentena, no parto e um dia pós-parto. Também foram coletadas amostras de colostro no parto e um dia pós-parto. Amostras de sangue, esfregaços nasal e tecido foram tomadas de quatro leitões antes de consumir colostro. As amostras foram analisadas pelo PCR Sybr Green para detectar e quantificar o PCV2. A detecção de anticorpos contra o vírus do PCV2 em soro foi realizada pelo teste de soroneutralização e todas as amostras foram analisadas através da técnica de SYBR Green PCR para a detecção do ADN viral. Resultados: a detecção de um nível elevado de viremia e a demonstração da excreção viral em esfregaços nasais e vaginais nas fêmeas permitiram demonstrar a introdução de porcas nulíparas aparentemente saudávels. Igualmente, o soro e as secreções vaginais e nasais foram positivos por PCR SYBR Green em tempo real na maioria das porcas no parto. Observou-se excreção viral em secreções vaginais e nasais e colostro na presença de anticorpos neutralizantes. A infecção das porcas manifestou-se no desenvolvimento de anticorpos neutralizantes e detecção de infecção fetal em leitões recém-nascidos aparentemente saudáveis , confirmando a transmissão vertical do PCV2. Os genótipos PCV2a e PCV2b foram detectados na infecção in utero. Conclusões: as porcas nulíparas podem estar infectadas com PCV2 antes de entrar nas granjas de criação e pode ser infectadas por transmissão horizontal durante a quarentena e gravidez. Exposição e infecção viral durante a gestação pode resultar em infecção subclínica de leitões recém-nascidos. Antecedentes: el circovirus porcino tipo 2 (PCV2) es asociado con casos de falla reproductivas en granjas recién pobladas, en granjas de cría para cerdas jóvenes y casi exclusivamente en cerdas de reemplazo. Los signos clínicos descritos son: incremento en los abortos durante la segunda y tercera etapa de la gestación, fetos momificados, mortinatos y el nacimiento de lechones débiles no viables. Objetivo: el principal propósito de este estudio fue evaluar la dinámica de la infección por el circovirus porcino tipo 2 y títulos de anticuerpos neutralizantes en las cerdas de reemplazo subclinicamente infectadas y el efecto en su camada. Métodos: este estudio se realizó con 40 cerdas de reemplazo seleccionadas al azar en cuatro granjas porcinas de cría. De cada animal se colectaron muestras de sangre, hisopados nasales y vaginales al ingresar a la explotación, durante la cuarentena, en el momento del parto y un día post-parto. Igualmente, se colectaron muestras de calostro al terminar el parto y un día post-parto. De cuatro lechones neonatos, se colectaron muestras de sangre, hisopado nasal y tejidos antes de consumir calostro. Todos los sueros fueron analizados mediante la técnica de sero-neutralización para detectar anticuerpos anti-PCV2 y todas las muestras se analizaron por una técnica SYBER Green en tiempo real para detectar el ADN viral. Resultados: la detección de un alto nivel de viremia y la demostración de la eliminación viral en hisopados nasales y vaginales permitió demostrar la introducción a las granjas de cerdas de reemplazo infectadas, aparentemente sanas. Igualmente, el suero y los hisopados nasales y vaginales fueron positivos por PCR SYBER Green en la mayoría de las hembras al parto. Se demostró eliminación viral en fluidos nasales, vaginales y en calostro en presencia de anticuerpos séricos neutralizantes. La infección de las cerdas se manifestó en viremia, en el desarrollo de anticuerpos frente al PCV2 y en la presencia del ADN viral en los tejidos de lechones neonatos aparentemente sanos. Los genotipos PCV2a y PCV2b fueron detectados en la infección in utero. Conclusiones: las cerdas de reemplazo pueden estar infectadas con el PCV2 antes de ingresar a las explotaciones de cría o pueden infectarse por transmisión horizontal durante la cuarentena y gestación. La exposición e infección viral de las cerdas durante la gestación puede resultar en infección subclínica de los lechones neonatos.

Haloferaxmediterranei is a haloarchaeon of high interest in biotechnology because it produces and mobilizes intracellular polyhydroxyalkanoate (PHA) granules during growth under stress conditions (limitation of phosphorous in the culture media), among other interesting metabolites (enzymes, carotenoids, etc.). The capability of PHA production by microbes can be monitored with the use of staining-based methods. However, the staining of haloarchaea cells is a challenging task; firstly, due to the high ionic strength of the medium, which is inappropriate for most of dyes, and secondly, due to the low permeability of the haloarchaea S-layer to macromolecules. In this work, Haloferax mediterranei is used as a halophilic archaeon model to describe an optimized protocol for the visualization and analysis of intracellular PHA granules in living cells. The method is based on double-fluorescence staining using Nile red and SYBR Green by confocal fluorescence microscopy. Thanks to this method, the capability of PHA production by new haloarchaea isolates could be easily monitored.

Background Telomere shortening and mitochondrial dysfunction are well-known independent contributors to many diseases, but emerging evidence suggests a reciprocal relationship between the two processes. The role of the so-called telomere-mitochondrial axis in colorectal cancer (CRC) remains largely unknown. Methods This prospective cohort study screened CRC patients who underwent surgery, from whom peripheral blood, intestinal mucosa, and tumor samples were collected. Colonoscopically confirmed cancer- and adenoma-free healthy individuals were screened as controls, from whom peripheral blood and intestinal mucosa samples were obtained. Relative mitochondrial DNA copy number (mtDNA-CN) and relative telomere length (RTL) were measured in all samples by real-time quantitative polymerase chain reaction and were further compared and correlated considering clinical data. Relative mtDNA-CN was quantified using both TaqMan probes and SYBR Green to compare both methods. Finally, multivariable analyses were conducted to investigate the association between both biomarkers and the risk of tumor recurrence and mortality. Results A total of 166 CRC patients and 61 healthy individuals were included in the study. In TNM stage I patients, relative mtDNA-CN and RTL were negatively correlated with each other in intestinal mucosa (ρ = -0.77, p  < 0.0001), tumor tissue (ρ = -0.41, p  = 0.032), and the tumor-to-intestinal mucosa ratio (ρ = -0.39, p  = 0.046). However, these associations disappeared with increasing TNM stage, suggesting a dysregulation of the telomere-mitochondrial axis in advanced disease. Higher relative mtDNA-CN in blood was associated with a lower risk of disease recurrence even after adjusting for multiple covariates (HR = 0.43, 95% CI 0.20–0.97, p  = 0.041), highlighting its potential use as a prognostic tool. The quantification of mtDNA-CN performed by both methods -TaqMan probes and SYBR Green- was shown to be positively correlated ( p  < 0.01). Relative mtDNA-CN and RTL were found to be tissue-dependent in both CRC patients and healthy controls. Conclusions This study provides a novel contribution to the understanding of the almost unexplored telomere-mitochondrial axis in CRC, highlighting its potential role in disease progression and prognosis. Graphical Abstract Created with https://www.BioRender.com .

Background As an alternative to PCR methods, LAMP is increasingly being used in the field of molecular diagnostics. Under isothermal conditions at 65 °C, the entire procedure takes approximately 30 min to complete. In this study, we establish a sensitive and visualized LAMP method in a closed-tube system for the detection of Plasmodium knowlesi . Methods A total of 71 malaria microscopy positive blood samples collected in blood spots were obtained from the Sarawak State Health Department. Using 18s rRNA as the target gene, nested PCR and SYBR green I LAMP assay were performed following the DNA extraction. The colour changes of LAMP end products were observed by naked eyes. Results LAMP assay demonstrated a detection limit of 10 copies/µL in comparison with 100 copies/µL nested PCR. Of 71 P. knowlesi blood samples collected, LAMP detected 69 microscopy-positive samples. LAMP exhibited higher sensitivity than nested PCR assay. The SYBR green I LAMP assay was 97.1% sensitive (95% CI 90.2–99.7%) and 100% specific (95% CI 83.2–100%). Without opening the cap, incorporation of SYBR green I into the inner cap of the tube enabled the direct visualization of results upon completion of amplification. The positives instantaneously turned green while the negatives remained orange. Conclusions These results indicate that SYBR green I LAMP assay is a convenient diagnosis tool for the detection of P. knowlesi in remote settings.

Quantitative polymerase chain reaction (qPCR) is a widespread method to quantify RNA or DNA. The results are reported as cycle of quantification (Cq), scaled to absolute numbers of copies or relative to reference genes. The reported Cq values of the same reaction vary between different machines and cannot be compared between different laboratories. This study shows that the third derivative zero (TD0) method is machine independent and more reproducible than the classic Cq calculations. Together with the mean PCR efficiency it allows the calculation of the number of copies initially present (Ncopy), a parameter easy to interpret. A large dataset was created for the evaluation of this method including amplicons with different length, primer concentrations, reaction mixes, and fluorescence reporter systems. Furthermore, the calculated Ncopy values can be corrected at the same time using known concentrations of a standard and for the expression of reference genes and combining absolute and relative quantification. The algorithms were implemented in the open-source program RDML-Tools, which can perform all steps of a qPCR analysis using the raw fluorescence amplification data and is available on the internet. We conclude that qPCR analysis today should widen its focus and include the three essential parameters, TD0, mean PCR efficiency and Ncopy.

Glyphosate (GLYP) is a broad-spectrum, nonselective, organic phosphine postemergence herbicide registered for many food and nonfood fields. Herein, we developed a biosensor (Mbs@dsDNA) based on carboxylated modified magnetic beads incubated with NH2-polyA and then hybridized with polyT-glyphosate aptamer and complementary DNA. Afterwards, a quantitative detection method based on qPCR was established. When the glyphosate aptamer on Mbs@dsDNA specifically recognizes glyphosate, complementary DNA is released and then enters the qPCR signal amplification process. The linear range of the method was 0.6 μmol/L–30 mmol/L and the detection limit was set at 0.6 μmol/L. The recoveries in tap water ranged from 103.4 to 104.9% and the relative standard deviations (RSDs) were <1%. The aptamer proposed in this study has good potential for recognizing glyphosate. The detection method combined with qPCR might have good application prospects in detecting and supervising other pesticide residues.

Microscopic detection and enumeration of microbial biomass provide essential primary information about the biosphere. Unlike aquatic samples, the application of specific microbial detection methods, such as fluorescence staining, to particle-rich samples such as marine sediments has been challenging because of the abundance of non-cellular particles. Even with recently developed fluorescence-based techniques for distinguishing microbial cells from sediment particles, reliable detection remains hindered by particle interference, often necessitating the expertise of trained specialists. In this study, we developed a deep learning-based image recognition method for identifying microbial cells in sediment samples, aiming to increase the accuracy of microbial cell detection and enumeration in microscopic images while eliminating the need for labor-intensive expert training. Our program first detects and identifies “cell-like particles” based on their green fluorescence and then classifies them via a trained classifier. The classifier successfully distinguished cell-like particles in pre-annotated images with accuracies of 94.1% for two-class classification and 88.8% for four-class classification. The accuracy was further improved to 96.6% by setting the confidence index cutoff to 0.7 and pre-screening focused images. The cell recognition program developed in this study will facilitate accurate and reliable detection of microbes in particle-rich environmental samples, reducing the reliance on intensive expert training.

The pandemic of coronavirus disease 2019 (COVID-19) continues to threaten public health. For developing countries where vaccines are still in shortage, cheaper alternative molecular methods for SARS-CoV-2 identification can be crucial to prevent the next wave. Therefore, 14 primer sets recommended by the World Health Organization (WHO) was evaluated on testing both clinical patient and environmental samples with the gold standard diagnosis method, TaqMan-based RT-qPCR, and a cheaper alternative method, SYBR Green-based RT-qPCR. Using suitable primer sets, such as ORF1ab, 2019_nCoV_N1 and 2019_nCoV_N3, the performance of the SYBR Green approach was comparable or better than the TaqMan approach, even when considering the newly dominating or emerging variants, including Delta, Eta, Kappa, Lambda, Mu, and Omicron. ORF1ab and 2019_nCoV_N3 were the best combination for sensitive and reliable SARS-CoV-2 molecular diagnostics due to their high sensitivity, specificity, and broad accessibility. Key points • With suitable primer sets, the SYBR Green method performs better than the TaqMan one. • With suitable primer sets, both methods should still detect the new variants well. • ORF1ab and 2019_nCoV_N3 were the best combination for SARS-CoV-2 detection.

Introduction. Yersinia enterocolitica (Ye) species is divided into 6 biotypes (BT), 1A, 1B, 2, 3, 4, 5 classified based on biochemical reactions and about 70 serotypes, classified based on the structure of the lipopolysaccharide O-antigen. The BT1A is considered non-pathogenic, while the BT 1B-5 are considered pathogenic.Methods. Evaluate the distribution of eleven chromosomal and plasmid virulence genes, ail, ystA, ystB, myfA, hreP, fes, fepD, ymoA, sat, virF and yadA, in 87 Ye strains isolated from food, animals and humans, using two SYBR Green real-time PCR platforms.Results. The main results showed the presence of the ail and ystA genes in all the pathogenic bioserotypes analyzed. The ystB, on the other hand, was identified in all non-pathogenic strains biotype 1A. The target fes, fepD, sat and hreP were found in both pathogenic biotypes and in BT1A strains. The myfA gene was found in all pathogenic biotype and in some Ye BT1A strains. The virF and yadA plasmid genes were mainly detected in bioserotype 4/O:3 and 2/O:9, while ymoA was identified in all strains.Conclusions. The two molecular platforms could be used to better define some specific molecular targets for the characterization and rapid detection of Ye in different sources which important implications for food safety and animal and human health.