Academic literature on the topic 'Kit qPCR'

The first Ukrainian real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) based test kit for the differential diagnosis of African (AFS) and Classical swine fever (CSF) has been developed in the Institute of Veterinary Medicine of NAAS. The proposed test kit allows simultaneous detection of three targets: ASFV DNA, CSFV cDNA and an internal control sample. The goal of this work was to provide an expert evaluation of the RT-qPCR kit for differential diagnosis of ASF and CSF according to appearance, analytical sensitivity, specificity and repeatability. Interdepartmental evaluation of the kit was conducted in the State Scientific and Research Institute of Laboratory Diagnostics and Veterinary and Sanitary Expertise (DNDILDEVSE) in accordance with the approved methodology. The RT-qPCR kit sensitivity was determined by testing 10-fold serial dilutions of the ASFV DNA and CSFV cDNA (concentration range was 103–100 copies/μl). For specificity determination reference samples of ASFV DNA different genotypes, ASF and CSF positive and negative field samples, as well as pathogens which cause similar to ASF and CSF clinical syndromes were used. Sample preparation and amplification were performed according to the test kit instructions. The amplification was accomplished on QuantStudio™ 5 System (Applied Biosystems). As a result of accomplished interdepartmental evaluation high sensitivity, specificity and repeatability of RT-qPCR kit were confirmed. In particular, it was determined that the limit of detection of the RT-qPCR kit was 5 copies of the ASFV and CSFV genomes per one reaction. The high specificity of the assay to ASFV (I, II, V, VIII, IX and X genotypes) and CSFV was confirmed. It was showп no cross-reactions with closely related pigs viruses (porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and virus of Aujeszky's disease). The high enough repeatability of results was also confirmed. In conclusion, the obtained results are in compliance with the requirements of the RT-qPCR kit normative and technical documentation. This RT-qPCR kit will be recommended for use in veterinary medicine laboratories after its registration would be done.

ABSTRACT Methods for detecting microorganisms on surfaces are needed to locate biocontamination sources and to relate surface and airborne concentrations. Research was conducted in an experimental room to evaluate surface sampling methods and quantitative PCR (QPCR) for enhanced detection of a target biocontaminant present on flooring materials. QPCR and culture analyses were used to quantitateBacillus subtilis (Bacillus globigii) endospores on vinyl tile, commercial carpet, and new and soiled residential carpet with samples obtained by four surface sampling methods: a swab kit, a sponge swipe, a cotton swab, and a bulk method. The initial data showed that greater overall sensitivity was obtained with the QPCR than with culture analysis; however, the QPCR results for bulk samples from residential carpet were negative. The swab kit and the sponge swipe methods were then tested with two levels of background biological contamination consisting of Penicillium chrysogenum spores. The B. subtilis values obtained by the QPCR method were greater than those obtained by culture analysis. The differences between the QPCR and culture data were significant for the samples obtained with the swab kit for all flooring materials except soiled residential carpet and with the sponge swipe for commercial carpet. The QPCR data showed that there were no significant differences between the swab kit and sponge swipe sampling methods for any of the flooring materials. Inhibition of QPCR due solely to biological contamination of flooring materials was not evident. However, some degree of inhibition was observed with the soiled residential carpet, which may have been caused by the presence of abiotic contaminants, alone or in combination with biological contaminants. The results of this research demonstrate the ability of QPCR to enhance detection and enumeration of biocontaminants on surface materials and provide information concerning the comparability of currently available surface sampling methods.

Abstract A reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is regarded as the most sensitive method available and is being used for screening procedure for all incoming passengers to Northern Cyprus for SARS-CoV-2. This study investigated the compatibility of two different RT-qPCR methodologies Diagnovital® and Bio-Speedy® by re-analyzing the previously confirmed positive samples. A total of 43 previously confirmed positive samples were re-analyzed by two different commercially available SARS-CoV-2 RT-qPCR kits. Only 23.5% of positive samples detected by Diagnovital® RT-qPCR kit were detected by Bio-Speedy® detection kit. In conclusion, adoption of Diagnovital® RT-qPCR kit detecting two regions of SARS-CoV-2 genome in our laboratories enabled the detection of SARS-CoV-2 in asymptomatic cases with higher sensitivity and contributed to the prevention of viral transmission within the country. The timely detection of infection in asymptomatic individuals may be the key to a successful fight against the COVID- 19 pandemic.

ABSTRACT BACKGROUND: The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) has prompted a need for mass testing to identify patients with viral infection. The high demand has created a global bottleneck in testing capacity, which prompted us to modify available resources to extract viral RNA and perform reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to detect SARS-COV-2. OBJECTIVES: Report on the use of a DNA extraction kit, after modifications, to extract viral RNA that could then be detected using an FDA-approved SARS-COV-2 RT-qPCR assay. MATERIALS AND METHODS: Initially, automated RNA extraction was performed using a modified DNA kit on samples from control subjects, a bacteriophage, and an RNA virus. We then verified the automated extraction using the modified kit to detect in-lab propagated SARSCOV-2 titrations using an FDA approved commercial kit (S, N, and ORF1b genes) and an in-house primer-probe based assay (E, RdRp2 and RdRp4 genes). RESULTS: Automated RNA extraction on serial dilutions SARS-COV-2 achieved successful one-step RT-qPCR detection down to 60 copies using the commercial kit assay and less than 30 copies using the in-house primer-probe assay. Moreover, RT-qPCR detection was successful after automated RNA extraction using this modified protocol on 12 patient samples of SARS-COV-2 collected by nasopharyngeal swabs and stored in viral transport media. CONCLUSIONS: We demonstrated the capacity of a modified DNA extraction kit for automated viral RNA extraction and detection using a platform that is suitable for mass testing. LIMITATIONS: Small patient sample size. CONFLICT OF INTEREST: None.

ABSTRACTRapid and reliable detection and identification of Francisella tularensis (a tier 1 select agent) are of primary interest for both medical and biological threat surveillance purposes. The Biotoxis qPCR detection kit is a real-time quantitative PCR (qPCR) assay designed for the detection of Bacillus anthracis, Yersinia pestis, and F. tularensis in environmental or biological samples. Here, we evaluated its performance for detecting F. tularensis in comparison to previously validated qPCR assays. The Biotoxis qPCR was positive for 87/87 F. tularensis subsp. holarctica (type B) strains but also for F. tularensis subsp. novicida. It was negative for Francisella philomiragia and 24/24 strains belonging to other bacterial species. For 31 tularemia clinical specimens, the Biotoxis qPCR displayed a sensitivity between 90.32% and 96.55%, compared to qPCR tests targeting ISFtu2 or a type B-specific DNA sequence, respectively. All 30 nontularemia clinical specimens were Biotoxis qPCR negative. For water samples, the Biotoxis qPCR limit of detection was 1,000 CFU/liter of F. tularensis. For 57 environmental water samples collected in France, the Biotoxis qPCR was positive for 6/15 samples positive for ISFtu2 qPCR and 4/4 positive for type B qPCR. In conclusion, the Biotoxis qPCR detection kit demonstrated good performances for F. tularensis detection in various biological and environmental samples, although cross-amplification of F. tularensis subsp. novicida must be considered. This plate format assay could be useful to test a large number of clinical or environmental specimens, especially in the context of natural or intentional tularemia outbreaks.

ABSTRACT Inhibited reactions have occasionally been observed when cilantro samples were processed for the detection of Cyclospora cayetanensis using quantitative real-time PCR (qPCR). Partial or total inhibition of PCR reactions, including qPCR, can occur, leading to decreased sensitivity or false-negative results. If inhibition occurs, this implies the need for additional purification or cleanup treatments of the extracted DNA to remove inhibitors prior to molecular detection. Our objective was to evaluate the performance of five commercial DNA cleanup kits (QIAquick purification kit from Qiagen [kit 1], OneStep PCR inhibitor removal by Zymo Research [kit 2], NucleoSpin genomic DNA cleanup XS from Macherey-Nagel [kit 3], DNA IQ system by Promega [kit 4], and DNeasy PowerPlant pro kit from Qiagen [5]) to minimize qPCR inhibition using the U.S. Food and Drug Administration–validated Bacteriological Analytical Manual (BAM) Chapter 19b method for detection of C. cayetanensis in cilantro samples containing soil. Each of the five commercial DNA cleanup kits evaluated was able to reduce the qPCR internal amplification control cycle threshold values to those considered to be normal for noninhibited samples, allowing unambiguous interpretation of results in cilantro samples seeded at both a high oocyst level (200 oocysts) and a low oocyst level (10 oocysts). Of the five kits compared, kits 1, 2, and 3 did not show significant differences in the detection of C. cayetanensis, while significantly higher cycle threshold values, indicating lower recovery of the target DNA, were observed from kits 4 and/or 5 in samples seeded with 200 and 10 oocysts (P < 0.05). This comparative study provides recommendations on the use of commercial cleanup kits which could be implemented when inhibition is observed in the detection of C. cayetanensis in cilantro samples using the BAM Chapter 19b method. HIGHLIGHTS

The global pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is having a tremendous impact on the global economy, health care systems and the lives of almost all people in the world. The Central European country of Slovakia reached one of the highest daily mortality rates per 100,000 inhabitants in the first 3 months of 2021, despite implementing strong prophylactic measures, lockdowns and repeated nationwide antigen testing. The present study reports a comparison of the performance of the Standard Q COVID-19 antigen test (SD Biosensor) with three commercial RT-qPCR kits (vDetect COVID-19-MultiplexDX, gb SARS-CoV-2 Multiplex-GENERI BIOTECH Ltd. and Genvinset COVID-19 [E]-BDR Diagnostics) in the detection of infected individuals among employees of the Martin University Hospital in Slovakia. Health care providers, such as doctors and nurses, are classified as “critical infrastructure”, and there is no doubt about the huge impact that incorrect results could have on patients. Out of 1231 samples, 14 were evaluated as positive for SARS-CoV-2 antigen presence, and all of them were confirmed by RT-qPCR kit 1 and kit 2. As another 26 samples had a signal in the E gene, these 40 samples were re-isolated and subsequently re-analysed using the three kits, which detected the virus in 22, 23 and 12 cases, respectively. The results point to a divergence not only between antigen and RT-qPCR tests, but also within the “gold standard” RT-qPCR testing. Performance analysis of the diagnostic antigen test showed the positive predictive value (PPV) to be 100% and negative predictive value (NPV) to be 98.10%, indicating that 1.90% of individuals with a negative result were, in fact, positive. If these data are extrapolated to the national level, where the mean daily number of antigen tests was 250,000 in April 2021, it points to over 4700 people per day being misinterpreted and posing a risk of virus shedding. While mean Ct values of the samples that were both antigen and RT-qPCR positive were about 20 (kit 1: 20.47 and 20.16 for Sarbeco E and RdRP, kit 2: 19.37 and 19.99 for Sarbeco E and RdRP and kit 3: 17.47 for ORF1b/RdRP), mean Ct values of the samples that were antigen-negative but RT-qPCR-positive were about 30 (kit 1: 30.67 and 30.00 for Sarbeco E and RdRP, kit 2: 29.86 and 31.01 for Sarbeco E and RdRP and kit 3: 27.47 for ORF1b/RdRP). It confirms the advantage of antigen test in detecting the most infectious individuals with a higher viral load. However, the reporting of Ct values is still a matter of ongoing debates and should not be conducted without normalisation to standardised controls of known concentration.

We evaluated a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS-CoV-2 kit) based on reverse transcription, isothermal amplification, and CRISPR-Cas12 reaction. From a total of 210 RNA samples extracted from nasopharyngeal swabs using spin columns, the Lyo-CRISPR SARS-CoV-2 kit detected 105/105 (100%; 95% confidence interval (CI): 96.55–100) positive samples and 104/105 (99.05%; 95% CI: 94.81–99.97) negative samples that were previously tested using commercial RT-qPCR. The estimated overall Kappa index was 0.991, reflecting an almost perfect concordance level between the two diagnostic tests. An initial validation test was also performed on 30 nasopharyngeal samples collected in lysis buffer, in which the Lyo-CRISPR SARS-CoV-2 kit detected 20/21 (95.24%; 95% CI: 76.18–99.88) positive samples and 9/9 (100%; 95% CI: 66.37–100) negative samples. The estimated Kappa index was 0.923, indicating a strong concordance between the test procedures. The Lyo-CRISPR SARS-CoV-2 kit was suitable for detecting a wide range of RT-qPCR-positive samples (cycle threshold range: 11.45–36.90) and dilutions of heat-inactivated virus (range: 2.5–100 copies/µL); no cross-reaction was observed with the other respiratory pathogens tested. We demonstrated that the performance of the Lyo-CRISPR SARS-CoV-2 kit was similar to that of commercial RT-qPCR, as the former was highly sensitive and specific, timesaving (1.5 h), inexpensive, and did not require sophisticated equipment. The use of this kit would reduce the time taken for diagnosis and facilitate molecular diagnosis in low-resource laboratories.

Arboviruses have been emerging and reemerging worldwide, predominantly in tropical and subtropical areas. As many arbovirus infections, including dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV), have similar signs and symptoms, clinical diagnosis of arbovirus infections is challenging. Therefore, reliable laboratory tests are necessary to improve the clinical management of patients with suspected arbovirus infections. Real-time reverse-transcription PCR (RT-qPCR) is among the more effective methods to distinguish these viruses. The aim of this study was to construct a unique positive external control derived from a unique plasmid using genetic engineering for specific use in RT-qPCR assays to detect Zika, dengue (1–4), and chikungunya. An external control derived from the MS2 bacteriophage was constructed using sequences from arbovirus and human genomes. Laboratories were asked to test the control in the ZDC Biomol kit, a RT-qPCR kit which is able to detect Zika, dengue serotypes 1–4, chikungunya, and an internal human control. RNA extracted from the external control was able to be amplified and detected in RT-qPCR assays for each virus detected by using the ZDC Biomol kit. The external control, samples from viral culture, and infected patient samples display similar amplification using this assay. The pET47b(+)MS2-ZDC vector is a viable expression system for the production of external control viral-like particles (MS2-ZDC). The RNA from the recombinant particles can be easily extracted and can function as a tool to validate all steps of process from the extraction to the amplification of all targets in specific reaction. Thus, the MS2-ZDC particles are laboratory-safe in order to avoid risk for operators, and the phages are effective as positive control for use in the ZDC Biomol kit amplifying all kit targets making them effective for commercial profile.

Les lymphocytes T régulateurs (Treg) sont une sous-population de cellules immunitaires connues pour leur rôle prépondérant dans la modulation de la réponse immunitaire innée et adaptative. Ils ont un rôle majeur dans la physiologie de l’organisme, mais également dans la mise en place des pathologies comme les inflammations chroniques, les maladies auto-immunes et les cancers. L’intérêt de mieux caractériser et suivre l’évolution des Tregs n’est plus à démontrer, qui plus est à l’ère des immunothérapies. En ce sens, notre équipe a établi une signature moléculaire unique des Tregs, basée sur l’expression de 81 gènes permettant de les caractériser et d’évaluer leur modulation. Ainsi, la validation de la signature moléculaire aboutira à l’établissement d’un kit compagnon couplé à un algorithme de lecture. Cela permettra de prévenir de façon fiable et rapide les effets indésirables de nouvelles molécules, de la recherche jusqu'à la clinique, vis-à-vis du système immunitaire.La première partie des résultats de qPCR sur les Tregs isolés d’individus sains a permis de conclure à la stabilité d’expression pour 84% des gènes de la signature. Ce ratio monte jusqu’à 92% pour l’évaluation de la stabilité d’expression des gènes dans les PBMC des mêmes donneurs. Ces résultats, très prometteurs, nécessitent d’être confirmés sur une plus grande population. Ainsi, l’étude d’une corrélation mathématique entre les niveaux d’expressions géniques des Tregs isolés et des PBMC autologues a été entrepris. Cette étude a permis de montrer que l’expression de 11 gènes est corrélée de manière linéaire à plus de 70% entre Tregs isolés et PBMC autologues.Dans un second temps, ce travail a permis d’évaluer la réponse de la signature face à des agents modulateurs décrits pour activer ou inhiber les Tregs. Cette étape a validé la bonne réactivité de certains gènes face aux agents modulateurs. C’est le cas de 4 gènes, qui diminuent significativement quand l’activité suppressive des Tregs est inhibée par un anticorps monoclonal anti-galectine-9. Néanmoins, l’analyse de variance multivariée n’a pas permis de montrer que les variations d’expression observées sont exclusivement dues au traitement sur la base des 10 donneurs, il semble que la variabilité interindividuelle entre en jeu dans l’analyse de certains groupes de gènes.Enfin, le comportement de la signature a été évalué chez 4 patients, inclus dans un essai clinique pour le traitement de différents types de cancer par radiothérapie stéréotaxique hypo fractionnée à haute dose. Encore une fois, les résultats obtenus sont très prometteurs puisque l’évolution biologique et clinique des patients est corrélée avec l’évolution d’expression des gènes de la signature. Ces données constituent une première confirmation de l’utilité d’une telle signature dans l’immuno-monitoring des patients.Ces études sont très encourageantes pour le développement d’un algorithme de lecture automatisé des résultats. Ainsi, le kit pourra être positionnable sur le marché de la recherche fondamentale jusqu’en clinique, pour l’évaluation des Tregs et leurs microenvironnements dans tous types d’études<br>Regulatory T cells (Treg) are an immune cell subpopulation known for their important role in innate and adaptive immune response contraction. They have a major role in physiology, but also in pathologies outcome such as chronic inflammations, autoimmune diseases and cancers. A better characterization and follow of Treg evolution are clearly needed, especially in the era of immunotherapies. Our team has established a unique Treg molecular signature based on the expression of 81 genes, allowing to characterize and to evaluate Treg modulation. Thus, the molecular signature validation will lead to the establishment of a companion kit coupled with a reading algorithm. This will allow to reliably and rapidly prevent the adverse effects of new molecules, from research to the clinic, regarding the immune system.The first part of the RT-PCR results on healthy donors isolated Tregs allowed to conclude that 84% of the genes have stable expression. This ratio rises to 92% for the stability evaluation of gene expression in PBMC from the same donors. These results are very promising, but need to be confirmed on a larger population. To go further the presence of a mathematical correlation between isolated Tregs and autologous PBMC gene expression levels, have been researched. This study showed that the expression of 11 gene are linearly correlated beyond 70%. Afterward, the signature response to modulating agents described to activate or inhibit Tregs have been asses. Here, the genes good response to modulating agents is proved: 4 gene expression decrease significantly when Tregs suppressive activity is inhibited by an anti-galectin-9 monoclonal antibody. Nevertheless, multivariate analysis of variance failed to show that the expression variations are exclusively due to treatment with 10 donors, it seems that interindividual variability has a role in some gene response to modulator. Finally, the signature behavior was evaluated in 4 patients, included in a clinical trial for cancer treatment by high dose hypo fractionated stereotactic radiotherapy. Once again, the results obtained are very promising since the biological and clinical patients’ evolution is correlated with the gene expression modulation. These data constitute a first confirmation of the usefulness of such a signature in the immuno-monitoring of patients.These outcomes are very encouraging for the development of an automated reading algorithm. Thus, the kit could be positioned on the market from basic research to the clinic, for the evaluation of Tregs and their micro environments in all types of studies

A utilização de Inibidores da Tirosino Quinase (ITQ) alterou drasticamente a expectativa de vida do paciente com Leucemia Mielóide Crônica (LMC) e o monitoramento da expressão do oncogene BCR-ABL1 tornou-se um fator prognóstico fundamental para avaliação da resposta ao tratamento. Atualmente, a necessidade de desenvolvimento de metodologias moleculares que facilitem a quantificação rápida, barata e sensível, associada à detecção precoce de baixos níveis de BCR-ABL1, tem proporcionado o surgimento de diversos ensaios comerciais para monitoramento molecular. Entretanto, estes kits possuem uma variabilidade na sua composição, execução e parâmetros analíticos, principalmente com relação à sensibilidade, o que torna os resultados, muitas vezes, não comparáveis. Esse trabalho teve como objetivo revisar a literatura buscando identificar as diferentes opções comerciais disponíveis para o monitoramento do BCR-ABL1, além de comparar os resultados de dois destes ensaios com a metodologia de referência. A partir da revisão realizada, identificamos cinco kits comerciais como principais opções disponíveis para monitoramento de BCR-ABL1 na LMC: GeneXpert® BCR-ABL Assay (Cepheid), Ipsogen® BCR-ABL1 Mbcr Fusion Quant Kit (QIAGEN), BCR-ABL1 Quant RUO™ Assay (Asuragen), LightCycler® t(9;22) Quantification Kit (Roche Molecular Biochemicals) e ODK-1201 (Otsuka Pharmaceutical Co. Ltd.). Posteriormente, comparamos os resultados e avaliamos o desempenho dos ensaios GeneXpert® BCR-ABL e do BCR-ABL1 Quant RUO™ com a metodologia de referência a partir de amostras de 60 pacientes com LMC em uso de ITQ. Identificamos uma concordância global ótima, com coeficientes de correlação de 0,97 (GeneXpert® BCR-ABL Assay) e 0,84 (BCR-ABL1 Quant RUO™ Assay). No entanto, na avaliação da concordância relacionada ao alcance ou não de uma Resposta Molecular Maior (RMM), o ensaio BCR-ABL1 Quant RUO™ apresentou melhores resultados, com uma menor discrepância para respostas moleculares profundas. A análise estratificada por subtipos de transcritos de BCR-ABL1 não mostrou diferença de desempenho entre os dois ensaios. A partir das análises comparativas realizadas e respectivas vantagens de cada teste, aliados aos dados obtidos a partir da revisão da literatura, sugere-se que o GeneXpert® BCR-ABL poderia ser utilizado como um teste primário, devido à rapidez do ensaio, enquanto o BCR-ABL1 Quant RUO™, por apresentar resultados associados a uma maior sensibilidade, poderia ser um teste secundário, a fim de confirmar resultados abaixo de uma RMM ou resultados não detectáveis. Fica evidente que a escolha de um ensaio comercial deve atender às necessidades de cada laboratório, mas que, fundamentalmente, esteja alinhada às recomendações internacionais de quantificação.<br>The use of tyrosine kinase inhibitors (TKIs) has drastically changed the life expectancy of patients with chronic myeloid leukemia (CML) and monitoring the expression of the BCR-ABL1 oncogene has become a key prognostic factor for assessing treatment response. The need to development molecular methodologies that facilitate fast, cheap and sensitive quantification associated with the early detection of low levels of BCR-ABL1 has led to the emergence of several commercial assays for molecular monitoring. However, these kits have variability in their composition, performance and analytical parameters, mainly in relation to the sensitivity, which makes the results often not comparable. This work aimed to review the literature in order to identify the different commercial options available for the monitoring of BCR-ABL1, in addition to comparing the results of two of these tests with the reference methodology. From the review, we identified five commercial kits as the main options available for monitoring BCR-ABL1 in the LMC: GeneXpert® BCR-ABL Assay (Cepheid), Ipsogen® BCR-ABL1 Mbcr Fusion Quant Kit (QIAGEN), BCR-ABL1 Quant RUO™ Assay (Asuragen), LightCycler® t (9; 22) Quantification Kit (Roche Molecular Biochemicals) and ODK-1201 (Otsuka Pharmaceutical Co. Ltd.). Subsequently, we compared the results and evaluated the performance of the GeneXpert® BCR-ABL and BCR-ABL1 Quant RUO™ with reference methodology from samples of 60 patients with CML using TKI. We identified an optimal overall agreement for the two trials, with correlation coefficients of 0.97 and 0.84, respectively. However, in the evaluation of the agreement related to the reach of a Major Molecular Response (MMR), the BCR-ABL1 Quant RUO™ assay presented better results, with a smaller discrepancy for deep molecular responses. Analysis stratified by subtypes of BCR-ABL1 transcripts showed no difference in performance between the two assays. From the comparative analyzes performed and the respective advantages of each test, allied to the data obtained from the literature review, it is suggested that GeneXpert® BCR-ABL assay could be used as a primary test, due to the rapidity of the assay, while the BCR-ABL1 Quant RUO™, for presenting results associated with increased sensitivity, could be a secondary test in order to confirm results below an MMR or undetected results. It is clear that the choice of a commercial assay should meet the needs of each laboratory, but that it is fundamentally in line with international quantification recommendations.

Introdução: O sistema RANK-RANKL-OPG é constituído pelo receptor RANK, o ligante RANKL e o receptor solúvel Osteoprotegerina (OPG). Esse sistema tem importante papel na regulação de processos fundamentais ao tecido ósseo, como a remodelação óssea. A literatura descreve o papel da via de sinalização RANK-RANKL na regulação da atividade de retículos sarcoplasmáticos no tecido muscular, bem como a participação de OPG na redução de fraqueza muscular e restauração de fibras em casos de distrofia muscular em camundongos. Dessa forma, destaca-se a relevância de estudos sobre a unidade osso-músculo e a atividade de proteínas solúveis na comunicação e homeostase desses tecidos. Objetivo: Verificar a expressão de RANK-RANKL-OPG ao longo do processo de diferenciação de miotubos in vitro. Materiais e Métodos: A linhagem de mioblastos C2C12 foi cultivada em meio DMEM suplementado com 10% de soro fetal bovino, 100U/ml de penicilina e 100 μg/ml de estreptomicina em estufa a 37ºC e 5% de CO2. A diferenciação em miotubos foi realizada durante cinco dias em DMEM suplementado com 2% soro equino. Estímulos com RANKL foram realizados ao longo da diferenciação, renovados a cada 48h. A análise da expressão gênica e proteica de RANK, RANKL e OPG foram realizadas por RT-qPCR utilizando o kit QuantiFast SYBR® Green (Qiagen®) e western blot, respectivamente. A dosagem de RANKL e OPG no sobrenadante foi realizada pelo kit ELISA Kit (R&amp;D Systems®). Resultados: A expressão proteica de RANK e OPG aumentou, destacando-se a maior expressão de OPG no terceiro dia de diferenciação. Observamos um aumento significativo de OPG no sobrenadante de células no 3º dia de diferenciação, enquanto RANKL foi indetectado. Além do aumento na expressão gênica de OPG, houve aumento na expressão de MyoD e MF20, fundamentais para a diferenciação de miotubos. Conclusão: Os resultados sugerem a ação de RANKL na indução da diferenciação de miotubos e também apontam que RANKL pode atuar como uma osteocina na regulação do músculo esquelético e este, por sua vez, pode regular este estímulo por meio da expressão de OPG. Assim, estes dados preliminares indicam um papel inédito da sinalização RANK-RANKL-OPG em processos fisiológicos da fibra muscular esquelética.

One step reverse-transcription polymerase chain reaction (RT-PCR) assays are an attractive option for further automating gene detection assays. One-step assays can reduce hands–on-time and the risk of sample crossover and contamination. The one-step chemistries are showing increasing use in virus detection and have been reported, in some cases, to be more appropriate than their two-step counterparts [1, 2]. Previous work presented by the Stokes Institute research group outlined a micro fluidic based continuous flow instrument which performed high throughput qPCR in nanolitre sized droplets [3]. This instrument had advantages over commercially available instruments in that it could process far more than the traditional 96 or 384 reaction setup in a single run and the reaction volume was reduced from 20–50 μl down to 30–100 nl sized droplets. Combining one-step chemistry with the technology offered by the devices being developed would lead to a high-throughput RNA-to-signal system capable of reverse transcribing and performing PCR on thousands of nanolitre sized reactions every day. It is envisaged that this technology will also lead to gene expression from single cells contained in nanolitre sized droplets. In this paper, a study was conducted in which an extra thermal region, manufactured from aluminium, was added to the existing continuous flow instruments. This region was maintained at a temperature suitable for reverse transcription, which was 48°C for the one-step kit tested. The thermal region was also a suitable length to maintain the sample at the required temperature for 15 minutes. Using a commercially available one step RT-PCR kit (TaqMan® RNA-to-CT™ 1-Step Kit, 4392653), the device was evaluated for its potential to perform one-step RT-PCR in continuously flowing nanolitre sized droplets. Electrophoresis gels were initially used in assessing specific amplification before an end-point detection method was utilized. RNA was extracted from the leukemic REH cell line with the housekeeping gene, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as the gene of interest. To investigate the possibility of further reducing sample preparation and facilitating further automation, amplification from cell lysates without nucleic acid extraction was carried out on the device. Cell lysates were prepared using the cell lysis buffer from the TaqMan® Gene Expression Cells-to-CT™ Kit (Cat #AM1728). It was found that the device was successful in one-step RT-PCR from extracted RNA samples and samples from cell lysates without nucleic acid extraction.

Introdução: A pandemia causada pelo vírus SARS-CoV-2, destacou no Brasil suas fragilidades sociais, econômicas e sistemáticas. O Sistema único de Saúde (SUS) durante o enfrentamento à pandemia, experimentou um cenário epidemiológico critico, sendo necessário medidas estratégicas que buscaram a contenção de óbitos e disseminação da COVID-19. Os profissionais da força de trabalho em saúde foram expostos de maneira acentuada, a demanda de acompanhar e assistir essa população foi urgente e necessária, permitindo a elaboração do planejamento e apoio, medidas restritivas com segurança e melhor conhecimento sobre a doença. Objetivos: Avaliar prevalência e incidência da infecção e reinfecção por SARS-CoV-2 entre vacinados e não vacinados contra a COVID-19. Material e métodos: O estudo consiste em uma coorte aberta prospectiva em trabalhadores da Unidade Básica de Saúde da Estrutural. Os dados primários foram coletados por questionário estruturado utilizando o software RedCap. A coleta de sangue para sorologia foi feita antes e depois da segunda dose, quando possível, a cada 30 dias. Se o trabalhador apresentasse sintomas gripais, procuraria a equipe de pesquisa é realizadaria coleta de esfregaço de nasofaringe para diagnóstico por RT-qPCR. Os resultados de SWAB, foram devolvidos entre 12 e 48 horas, com isso conseguimos monitorar os casos de infecção e reinfecção. Resultados: Entre 8 de fevereiro e 30 de setembro de 2021, 128 participantes foram entrevistados e cederam amostras. A adesão inicial ao projeto foi alta, provavelmente pela ausência de monitoramento prévio. No entanto, devido ao atraso no recebimento de kits para sorologia, houve atraso na devolutiva destes resultados e consequentemente, desistências. Houveram sete baixas (por desistência, remoção da unidade ou licença prolongada) e 12 novos trabalhadores aderiram. Neste momento, está sendo iniciado o quarto momento de coleta. Foram coletadas 43 amostras de SWAB de nasofaringe que reportaram sintomas gripais. Conclusão: O monitoramento permite entender o comportamento do vírus, impedir cadeias de transmissão e reduzir a ocorrência de desfechos desfavoráveis. Trabalhadores da saúde são exemplo de fonte primária de dados etiológicos de doenças transmissíveis, podendo funcionar como termômetro da casuística e se refletir em respostas precoces para a população. Monitorá-los é, portanto, estratégico para o controle da pandemia.