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1.
JNMA J Nepal Med Assoc ; 59(235): 248-251, 2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-34506444

RESUMO

INTRODUCTION: The virus that causes COVID-19 is known as severe acute respiratory syndrome Coronavirus-2. This new variant of Corona Virus introduced in China has urged the massive health system resources to focus on its screening and management of sick patients worldwide. We aimed to find the prevalence of COVID-19 positive cases diagnosed by Real-time polymerase chain reaction in a tertiary care hospital of Nepal. METHODS: This is a descriptive cross-sectional study that was conducted from 11th of November to 15th December 2020. Nasopharyngeal and Oropharyngeal swabs were collected, and confirmation of cases of COVID-19 was done based on the detection of viral ribonucleic acid by nucleic acid amplification tests such as real-time reverse transcriptase-polymerase chain reactions. The viral genes targeted include the E, N, and ORF. RESULTS: A total of 15247 samples have been processed, of which s (14.81%) positive cases were included in this study. There were 1427 (63.19%) male and 831 (36.68%) females. The majority of the cases were asymptomatic 1386 (61.38%). The most common age group infected was between 15 to 40 years, 841 (58.93%) male and 542 (65.22%) females. The most common presenting symptoms were cough 315 (13.95%) and fever 306 (13.55%). CONCLUSIONS: Most of the individuals reported for real-time polymerase chain reaction were asymptomatic patients who might be contagious and have the potential to transmit infection. Among symptomatic cases, common symptoms were cough and fever.


Assuntos
COVID-19 , Adolescente , Adulto , Estudos Transversais , Feminino , Humanos , Masculino , Nepal/epidemiologia , Prevalência , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2 , Centros de Atenção Terciária , Adulto Jovem
2.
Zhongguo Zhong Yao Za Zhi ; 46(12): 3116-3122, 2021 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-34467703

RESUMO

Screening suitable reference genes is the premise of quantitative Real-time PCR(qRT-PCR)for gene expression analysis. To provide stable reference genes for expression analysis of genes in Aconitum vilmorinianum, this study selected 19 candidate re-ference genes(ACT1, ACT2, ACT3, aTUB1, aTUB2, bTUB, 18S rRNA, UBQ, eIF2, eIF3, eIF4, eIF5, CYP, GAPDH1, GAPDH2, PP2A1, PP2A2, ACP, and EF1α) based on the transcriptome data of A. vilmorinianum. qRT-PCR was conducted to profile the expression of these genes in the root, stem, leaf, and flower of A. vilmorinianum. The Ct values showed that 18S rRNA with high expression level and GAPDH2 with large expression difference among organs were not suitable as the reference genes. NormFinder and geNorm showed similar results of the expression stability of the other candidate reference genes and demonstrated PP2A1, EF1α, and CYP as the highly stable ones. However, BestKeeper suggested EF1α, ACT3, and PP2A1 as the top stable genes. In view of the different results from different softwares, the geometric mean method was employed to analyze the expression stability of the candidate re-ference genes, the results of which indicated that PP2A1, EF1α, and ACT3 were the most stable. Based on the comprehensive analysis results of geNorm, NormFinder, BestKeeper, and geometric mean method, PP2A1 and EF1α presented the most stable expression in different organs of A. vilmorinianum. PP2A1 and EF1α were the superior reference genes for gene expression profiling in different organs of A. vilmorinianum.


Assuntos
Aconitum , Perfilação da Expressão Gênica , Genes de Plantas/genética , Reação em Cadeia da Polimerase em Tempo Real , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa
3.
Zhongguo Zhong Yao Za Zhi ; 46(15): 3832-3837, 2021 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-34472256

RESUMO

Freshly collected seeds of Amomum tsaoko demonstrate obvious dormancy. Therefore, the selection of stable reference genes during seed dormancy release is very important for the subsequent functional research of related genes. In this study, ten commonly used reference genes(GAPDH, 40S, actin, tubulin, EIF4A-9, EIF2α, UBC, UBCE2, 60S, and UBQ) were selected as candidates for quantitative Real-time polymerase chain reaction(qRT-PCR) of the embryo samples of A. tsaoko at different dormancy release stages. Three kinds of software(BestKeeper, geNorm, and Normfinder) and the Delta CT method were used to evaluate the expression stability of the candidate reference genes, and the RefFinder online tool was employed to integrate the results and generate a comprehensive ranking. The results showed that the expression levels of the ten candidate reference genes differed greatly in different embryo samples. GAPDH and UBC had high expression levels, as manifested by the small Ct values. GeNorm identified 40S and UBCE2 as the most stable genes. NormFinder ranked EIF2α as the most stable gene and UBC as the least stable gene. UBCE2 was found to be the most stable gene and actin the least stable one by BestKeeper. Delta CT analysis suggested that the expression of 40S was most stable. UBCE2 was recommended as the most stably expressed gene by RefFinder. Thus, UBCE2 is the ideal reference gene for qRT-PCR analysis of A. tsaoko seeds at different dormancy release stages. The results may lay a foundation for analyzing the expression of related genes during seed dormancy release of A. tsaoko.


Assuntos
Amomum , Perfilação da Expressão Gênica , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sementes/genética
4.
Pan Afr Med J ; 39: 89, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34466191

RESUMO

Coronavirus disease 2019 (COVID-19), a severe acute respiratory syndrome caused by SARS-CoV-2 was declared a global pandemic by the World Health Organization (WHO) in March 2020. As of 21st April 2021, the disease had affected more than 143 million people with more than 3 million deaths worldwide. Urgent effective strategies are required to control the scourge of the pandemic. Rapid sample collection and effective testing of appropriate specimens from patients meeting the suspect case definition for COVID-19 is a priority for clinical management and outbreak control. The WHO recommends that suspected cases be screened for SARS-CoV-2 virus with nucleic acid amplification tests such as real-time Reverse Transcription-Polymerase Chain Reaction (rRT-PCR). Other COVID-19 screening techniques such as serological and antigen tests have been developed and are currently being used for testing at ports of entry and for general surveillance of population exposure in some countries. However, there are limited testing options, equipment, and trained personnel in many African countries. Previously, positive patients have been screened more than twice to determine viral clearance prior to discharge after treatment. In a new policy directive, the WHO now recommends direct discharge after treatment of all positive cases without repeated testing. In this review, we discuss COVID-19 testing capacity, various diagnostic methods, test accuracy, as well as logistical challenges in Africa with respect to the WHO early discharge policy.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Guias de Prática Clínica como Assunto , África , Humanos , Programas de Rastreamento/métodos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Manejo de Espécimes , Organização Mundial da Saúde
5.
Artigo em Inglês | MEDLINE | ID: mdl-34501904

RESUMO

Reliability, accuracy, and timeliness of diagnostic testing for SARS-CoV-2 infection have allowed adequate public health management of the disease, thus notably helping the timely mapping of viral spread within the community. Furthermore, the most vulnerable populations, such as people with intellectual disability and dementia, represent a high-risk group across multiple dimensions, including a higher prevalence of pre-existing conditions, lower health maintenance, and a propensity for rapid community spread. This led to an urgent need for reliable in-house rapid testing to be performed prior to hospital admission. In the present study, we describe a pooling procedure in which oropharyngeal and nasopharyngeal swabs for SARS-CoV-2 detection (performed prior to hospital admission using rapid RT-PCR assay) are pooled together at the time of sample collection. Sample pooling (groups of 2-4 samples per tube) allowed us to significantly reduce response times, consumables, and personnel costs while maintaining the same test sensitivity.


Assuntos
COVID-19 , Deficiência Intelectual , Hospitais , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/epidemiologia , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , SARS-CoV-2 , Sensibilidade e Especificidade
6.
Water Res ; 203: 117516, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34412018

RESUMO

Due to the coronavirus disease 2019 (COVID-19) pandemic, wastewater surveillance has become an important tool for monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within communities. In particular, reverse transcription-quantitative PCR (RT-qPCR) has been used to generate large datasets aimed at detecting and quantifying SARS-CoV-2 RNA in wastewater. Although RT-qPCR is rapid and sensitive, there is no standard method yet, there are no certified quantification standards, and experiments are conducted using different assays, reagents, instruments, and data analysis protocols. These variations can induce errors in quantitative data reports, thereby potentially misleading interpretations, and conclusions. We review the SARS-CoV-2 wastewater surveillance literature focusing on variability of RT-qPCR data as revealed by inconsistent standard curves and associated parameters. We find that variation in these parameters and deviations from best practices, as described in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines suggest a frequent lack of reproducibility and reliability in quantitative measurements of SARS-CoV-2 RNA in wastewater.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Transcrição Reversa , Águas Residuárias
7.
ACS Appl Mater Interfaces ; 13(35): 41445-41453, 2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34428374

RESUMO

Airborne transmission of exhaled virus can rapidly spread, thereby increasing disease progression from local incidents to pandemics. Due to the COVID-19 pandemic, states and local governments have enforced the use of protective masks in public and work areas to minimize the disease spread. Here, we have leveraged the function of protective face coverings toward COVID-19 diagnosis. We developed a user-friendly, affordable, and wearable collector. This noninvasive platform is integrated into protective masks toward collecting airborne virus in the exhaled breath over the wearing period. A viral sample was sprayed into the collector to model airborne dispersion, and then the enriched pathogen was extracted from the collector for further analytical evaluation. To validate this design, qualitative colorimetric loop-mediated isothermal amplification, quantitative reverse transcription polymerase chain reaction, and antibody-based dot blot assays were performed to detect the presence of SARS-CoV-2. We envision that this platform will facilitate sampling of current SARS-CoV-2 and is potentially broadly applicable to other airborne diseases for future emerging pandemics.


Assuntos
Testes Respiratórios/instrumentação , Teste para COVID-19/instrumentação , Máscaras , SARS-CoV-2/isolamento & purificação , Microbiologia do Ar , Anticorpos Antivirais/imunologia , Testes Respiratórios/métodos , Teste para COVID-19/métodos , Colódio/química , Colorimetria/métodos , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Cimento de Policarboxilato/química , Porosidade , Estudo de Prova de Conceito , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/química , Proteínas Virais/análise , Proteínas Virais/imunologia
8.
Mikrobiyol Bul ; 55(3): 435-444, 2021 Jul.
Artigo em Turco | MEDLINE | ID: mdl-34416808

RESUMO

Patients infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) show different clinical courses ranging from asymptomatic to severe infection requiring intensive care treatment and death. Real-time reverse transcription polymerase chain reaction (rRT-PCR), used in the diagnosis, screening and surveillance of coronavirus-2019 (COVID-19), provides the viral load as a cycle threshold (Ct) value. It has been reported that the Ct value may be related to the course of the infection and the clinical condition of the patient. In this study, it was aimed to compare the Ct and C reactive-protein (CRP) results of symptomatic and asymptomatic patients who were found to be positive with rRT-PCR. Between 14 April and 29 August 2020, a total of 355 patients aged 18 years and older with positive SARS-CoV-2 rRT-PCR test were included in the study. The COVID-19 rRT-PCR test was performed with Bio-speedy SARS-CoV-2 rRT-PCR kit (Bioeksen, Turkey) versions, the kit targeting the RdRp gene region, and the dual gene kit versions targeting the N and ORF1ab gene regions were used. Patients were classified as symptomatic and asymptomatic according to their clinical findings. Ct and CRP results of the patients were analyzed statistically. Of the 355 patients included in the study, 237 (66.7%) were symptomatic and 118 (33.2%) were asymptomatic patients. The mean age of symptomatic patients (46.68 ± 18.03) was observed significantly higher than asymptomatic patients (38.27 ± 13.82) (p<0.001). When the patients are evaluated according to the age groups, the rate of asymptomatic patients was significantly higher in the 21-39 age group, while the rate of symptomatic patients was significantly higher in 65 years and older group (p<0.05). The rate of comorbidity was significantly higher in symptomatic patients (n= 69, 29.1%) than in asymptomatic patients (n= 11, 9.3%) (p<0.001). Hypertension (12.2%), diabetes mellitus (9.7%), chronic respiratory disease (9.3%) and cardiovascular diseases (5.5%) were the most common diseases in symptomatic patients. However, among these, hypertension and chronic respiratory disease were found significantly higher in symptomatic patients (p<0.05). Increased CRP rate in symptomatic patients (64.6%) was found significantly higher than asymptomatic patients (27.3%) (p<0.001). The median of Ct value was found significantly higher in asymptomatic patients (26.34, IQR= 19.78-35.48), than in symptomatic patients (21.77, IQR= 17.81-26.51) (p<0.001). Regarding the medians of Ct values obtained from target genes; RdRp gene Ct value was found significantly higher in asymptomatic patients than in symptomatic patients (p<0.001). However, no statistical difference was found between symptomatic and asymptomatic patients in the ORF1ab and N genes Ct value medians (p> 0.05). As a result, it was observed that SARS-CoV-2 PCR positive patients were symptomatic in the presence of advanced age and comorbidity. Increased CRP value at the time of admission to the hospital was found significantly higher in symptomatic patients. Ct value has been shown to be lower in symptomatic patients, as expected. Although Ct and CRP values are thought to be useful in monitoring the clinical course and prognosis of patients with COVID-19, more detailed studies are needed to prove their clinical value.


Assuntos
COVID-19 , RNA Viral , Idoso , Humanos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2 , Carga Viral
9.
PLoS One ; 16(8): e0256352, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34403456

RESUMO

Rapid tests for SARS-COV-2 infection are important tools for pandemic control, but current rapid tests are based on proprietary designs and reagents. We report clinical validation results of an open-access lateral flow assay (OA-LFA) design using commercially available materials and reagents, along with RT-qPCR and commercially available comparators (BinaxNOW® and Sofia®). Adult patients with suspected COVID-19 based on clinical signs and symptoms, and with symptoms ≤7 days duration, underwent anterior nares (AN) sampling for the OA-LFA, Sofia®, BinaxNOW ™, and RT-qPCR, along with nasopharyngeal (NP) RT-qPCR. Results indicate a positive predictive agreement with NP sampling as 69% (60% -78%) OA-LFA, 74% (64% - 82%) Sofia®, and 82% (73% - 88%) BinaxNOW™. The implication for these results is that we provide an open-access LFA design that meets the minimum WHO target product profile for a rapid test, that virtually any diagnostic manufacturer could produce.


Assuntos
Antígenos Virais/análise , COVID-19/diagnóstico , Imunoensaio , SARS-CoV-2/metabolismo , Área Sob a Curva , COVID-19/virologia , Humanos , Nasofaringe/virologia , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/análise , RNA Viral/metabolismo , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade
10.
PLoS One ; 16(8): e0255807, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34407100

RESUMO

The use of saliva for the diagnosis of SARS-CoV-2 has shown to be a good alternative to nasopharyngeal swabs (NPS), since it permits self-collection, avoids the exposure of healthy persons to infected patients, reduces waiting times, eliminates the need of personal protective equipment and is non-invasive. Yet current saliva testing is still expensive due to the need of specialized tubes containing buffers to stabilize the RNA of SARS-CoV-2 and inactivate the virus. These tubes are expensive and not always accessible in sufficient quantities. We now developed an alternative saliva testing method, using TRIzol for extraction, viral inactivation, and storage of SARS-CoV-2 RNA, combined with RT-qPCR, which was comparable in its performance to NPS. Paired saliva samples and NPS were taken from 15 asymptomatic healthcare workers and one patient with SARS-CoV-2. Further 13 patients with SARS-CoV-2 were only saliva-tested. All the tests were performed according to CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel. Saliva (4 mL) was taken in sterile 50 mL tubes, 1.5 mL TRIzol were added and mixed. Our results show that 5 µL of saliva RNA extracted with TRIzol allow for an adequate detection of the virus in patients positive for SARS-CoV-2 and was equally sensitive to NPS in TRIzol. We conclude that saliva testing using TRIzol is a recommendable method for diagnosis of SARS-CoV-2 since it has several advantages over currently used saliva tests: it can be done with normal sterile tubes, does not need cold-chain handling, is stable at room temperature, is non-invasive and less costly, making it more accessible for low-income countries. Cheaper saliva testing using TRIzol is especially relevant for low-income countries to optimize diagnosis and help define quarantine durations for families, healthcare workers, schools, and other public workplaces, thus decreasing infections and mortality caused by SARS-CoV-2.


Assuntos
COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Manejo de Espécimes/instrumentação , Adulto , Idoso , Idoso de 80 Anos ou mais , Países em Desenvolvimento , Testes Diagnósticos de Rotina/economia , Diagnóstico Precoce , Guanidinas/química , Humanos , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Fenóis/química , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/genética , Sensibilidade e Especificidade , Fatores Socioeconômicos , Manejo de Espécimes/economia , Adulto Jovem
11.
PLoS One ; 16(8): e0256316, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34407126

RESUMO

Efficient and effective viral detection methodologies are a critical piece in the global response to COVID-19, with PCR-based nasopharyngeal and oropharyngeal swab testing serving as the current gold standard. With over 100 million confirmed cases globally, the supply chains supporting these PCR testing efforts are under a tremendous amount of stress, driving the need for innovative and accurate diagnostic solutions. Herein, the utility of a direct-to-PCR method of SARS-CoV-2 detection grounded in mechanical homogenization is examined for reducing resources needed for testing while maintaining a comparable sensitivity to the current gold standard workflow of nasopharyngeal and oropharyngeal swab testing. In a head-to-head comparison of 30 patient samples, this initial clinical validation study of the proposed homogenization-based workflow demonstrated significant agreeability with the current extraction-based method utilized while cutting the total resources needed in half.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Manejo de Espécimes/instrumentação , Teste de Ácido Nucleico para COVID-19/instrumentação , Estudos de Viabilidade , Humanos , Nasofaringe/virologia , Orofaringe/virologia , Estudos Prospectivos , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2/genética , Sensibilidade e Especificidade , Fluxo de Trabalho
12.
Mem Inst Oswaldo Cruz ; 116: e210085, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34406222

RESUMO

BACKGROUND: The high demand for adequate material for the gold standard reverse transcription real-time polymerase chain reaction (RT-qPCR)-based diagnosis imposed by the Coronavirus disease 2019 (COVID-19) pandemic, combined with the inherent contamination risks for healthcare workers during nasopharyngeal swab (NP) sample collection and the discomfort it causes patients, brought the need to identify alternative specimens suitable for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). OBJECTIVES: The aim of this work was to compare saliva and gingival fluid swabs to NP swabs as specimens for RT-qPCR-based SARS-CoV-2 diagnosis. METHODS: We compared gingival fluid swabs (n = 158) and saliva (n = 207) to the rayon-tipped NP swabs obtained from mild-symptomatic and asymptomatic subjects as specimens for RT-qPCR for SARS-CoV-2 detection. FINDINGS: When compared to NP swabs, gingival fluid swabs had a concordance rate of 15.4% among positive samples, zero among inconclusive, and 100% among negative ones. For saliva samples, the concordance rate was 67.6% among positive samples, 42.9% among inconclusive, and 96.8% among negative ones. However, the concordance rate between saliva and NP swabs was higher (96.9%) within samples with lower cycle threshold (Ct) values (Ct > 10 ≤ 25). MAIN CONCLUSIONS: Our data suggests that whereas gingival fluid swabs are not substitutes for NP swabs, saliva might be considered whenever NP swabs are not available or recommended.


Assuntos
Teste para COVID-19 , COVID-19 , Humanos , Nasofaringe , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2 , Saliva , Manejo de Espécimes
13.
J AOAC Int ; 104(4): 924-934, 2021 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-34416762

RESUMO

BACKGROUND: The SureFast® SARS-CoV-2 PLUS Test is a reverse transcription qPCR (RT-qPCR) assay for the direct, qualitative detection of novel coronavirus (SARS-CoV-2) RNA from stainless-steel environmental sample swabs. OBJECTIVE: To validate the SureFast SARS-CoV-2 PLUS Kit as part of the AOAC Research Institute's Emergency Response Validation Performance Tested Method(s)SM program. METHOD: The SureFast SARS-CoV-2 PLUS Kit was evaluated for specificity using in silico analysis of 15 764 SARS-CoV-2 sequences and 65 exclusivity organisms (both near neighbors and background organisms) using the ThermoBLAST program. The candidate method was evaluated in an unpaired study design for one environmental surface (stainless steel) and compared to the US Centers for Disease Control and Prevention 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel, Instructions for Use (Revision 4, Effective 6/12/2020). RESULTS: Results of the in silico analysis demonstrated 99.99% selectivity of the method in being able to detect target sequences of the known CoV-2 genomes and discriminate them from near neighbors. In the matrix study, the candidate method demonstrated statistically significant better recovery of the target analyte than the PCR detection reference method. CONCLUSIONS: The SureFast SARS-CoV-2 PLUS Kit is a rapid and accurate method that can be utilized by food producers to detect the causative agent of COVID-19 on stainless-steel contact surfaces. HIGHLIGHTS: SureFast SARS-CoV-2 PLUS test method is highly specific for primer/probe binding to the E target genome region for the SARS-CoV-2 virus, 99.99% binding specificity using in silico analysis.


Assuntos
COVID-19 , RNA Viral , Humanos , Reação em Cadeia da Polimerase em Tempo Real , SARS-CoV-2 , Sensibilidade e Especificidade , Aço Inoxidável , Estados Unidos
14.
Artigo em Inglês | MEDLINE | ID: mdl-34444305

RESUMO

The collection and storage of water-related matrices such as biofilm from collection to processing are critical for the detection of Legionella pneumophila by cultural and molecular tests. SRK™ is a liquid medium that acts both as an antimicrobial neutralizing agent and a transport medium for bacterial culture enumeration and is useful to maintain the stability of the sample from collection to analysis. The aims of this study were to evaluate Legionella pneumophila viability and bacterial nucleic acids' stability in SRK™ medium over time at different storage conditions. Artificial bacterial inoculates with an approximate concentration of 104, 103 and 102 CFU/mL were made using Legionella pneumophila certified reference material suspended in SRK™ medium. Bacteria recovery was analyzed by cultural and molecular methods at time 0, 24 and 48 h at room temperature and at 0, 24, 48 and 72 h at 2-8 °C, respectively. SRK™ medium supported Legionella pneumophila culture viability with CFU counts within the expected range. The recovery after 72 h at 2-8 °C was 83-100% and 75-95% after 48 h at room temperature. Real-time PCR appropriately detected Legionella pneumophila DNA at each temperature condition, dilution and time point. Results demonstrated a good performance of SRK™ medium for the reliable recovery of environmental Legionella.


Assuntos
Legionella pneumophila , Legionella , Meios de Cultura , Legionella/genética , Legionella pneumophila/genética , Reação em Cadeia da Polimerase em Tempo Real , Microbiologia da Água
15.
Nat Commun ; 12(1): 4811, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376648

RESUMO

Circulating microRNAs (miRNAs) could improve colorectal cancer (CRC) risk prediction. Here, we derive a blood-based miRNA panel and evaluate its ability to predict CRC occurrence in a population-based cohort of adults aged 50-75 years. Forty-one miRNAs are preselected from independent studies and measured by quantitative-real-time-polymerase-chain-reaction in serum collected at baseline of 198 participants who develop CRC during 14 years of follow-up and 178 randomly selected controls. A 7-miRNA score is derived by logistic regression. Its predictive ability, quantified by the optimism-corrected area-under-the-receiver-operating-characteristic-curve (AUC) using .632+ bootstrap is 0.794. Predictive ability is compared to that of an environmental risk score (ERS) based on known risk factors and a polygenic risk score (PRS) based on 140 previously identified single-nucleotide-polymorphisms. In participants with all scores available, optimism-corrected-AUC is 0.802 for the 7-miRNA score, while AUC (95% CI) is 0.557 (0.498-0.616) for the ERS and 0.622 (0.564-0.681) for the PRS.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Herança Multifatorial/genética , Polimorfismo de Nucleotídeo Único , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Neoplasias Colorretais/sangue , Neoplasias Colorretais/diagnóstico , Feminino , Humanos , Modelos Logísticos , Masculino , MicroRNAs/sangue , Pessoa de Meia-Idade , Estudos Prospectivos , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real/métodos , Fatores de Risco
16.
Methods Mol Biol ; 2351: 337-352, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382199

RESUMO

DNA methylation is thought to regulate accessibility of chromatin and binding of regulatory elements; however, it is difficult to determine if chromatin accessibility or transcription factor (TF) binding overlap with methylated or unmethylated DNA if the assays are performed separately. In order to examine accessibility or TF binding simultaneously with methylation on the same DNA molecule, we developed EpiMethylTag which combines ATAC-Seq or ChIP-Seq (M-ATAC or M-ChIP) with bisulfite conversion. Our approach provides a fast, low-input, low sequencing depth method to determine whether DNAme and accessibility/TF binding are mutually exclusive or can coexist in certain locations.


Assuntos
Montagem e Desmontagem da Cromatina , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Cromatina/genética , Cromatina/metabolismo , Metilação de DNA , Fatores de Transcrição/metabolismo , Sítios de Ligação , Ilhas de CpG , Elementos de DNA Transponíveis , Epigênese Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo Real
17.
Gene ; 802: 145864, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34352300

RESUMO

Milk fat is the most important energy substance in milk and contributes to its quality and health benefits. Water buffalo milk is well known for its high milk quality with higher fat contents compared with cattle milk. Dehong buffalo is a unique local swamp breed in Yunnan Province with higher milk fat and excellent milk quality which provides a good model for the investigation of the molecular mechanisms of milk fat deposition. In this study, we used RNA-seq to obtain mammary tissue transcriptomics of buffalo with different milk fat phenotypes including high(H), medium (M)and low (L) fat content groups. Comparative analyses of buffalo among three groups yielded differentially expressed genes (DEGs). Analyzing the number of different genes among H_VS_L, H_VS_M, and M_VS_L showed the same expression pattern between H_VS_M. The increasing expression levels of genes including CSN1S1, BTN1A1, LALBA, ALDH1L2, SCD and MUC15, and down-regulated expression levels of genes containing CCL2, CRABP2, ADTRP, CLU and C4A in H_VS_L and M_VS_L were found. GO and KEGG enriched pathways revealed these DEGs involved in milk protein and fat as well as immune response. The findings would enhance the understanding of the interplay between the milk composition and immune response, which suggests that the immune capacity should be considered when we tried to improve the milk quality.


Assuntos
Búfalos/metabolismo , Gorduras/metabolismo , Glândulas Mamárias Animais/metabolismo , Leite/metabolismo , Animais , Búfalos/genética , Feminino , RNA-Seq/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Transcriptoma
18.
Gene ; 802: 145863, 2021 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-34358628

RESUMO

Hydrophobins are small, secreted proteins with important physiological functions in mycelial growth and fungal development. Here, 1 nucleus-specific and 35 allelic hydrophobin genes were identified in the genome of a white rot fungus, Coriolopsis trogii. Among these, 22 were eight-cysteine class I hydrophobin genes and the other 14 were uncommon six-cysteine hydrophobin genes. The six-cysteine hydrophobins were speculated to have originated from a common ancestor. The hydrophobin genes favored a clustering distribution and two recent duplication pairs were identified. The genes had conserved gene structures with three exons and two introns. Cthyd18, Cthyd19, and Cthyd32 were constitutively highly expressed in all developmental stages. Cthyd20, Cthyd21, Cthyd22, Cthyd28, Cthyd30, Cthyd31, and Cthyd33 were highly expressed in mycelia, and Cthyd12 and Cthyd35 in the reproductive stages. Sixteen hydrophobin genes were regulated differently in the transition from mycelia to primordia; Cthyd35 showed maximal upregulation of 1922-fold, and Cthyd23 showed maximal downregulation of 552-fold. Most (32) hydrophobin genes showed significant differential expression between mycelia cultured in different media (potato dextrose agar or broth). Weighted gene co-expression network analysis and promoter analysis revealed that C2H2 zinc finger proteins may regulate hydrophobin genes. These results may support further research into the function and evolution of hydrophobins.


Assuntos
Proteínas Fúngicas/genética , Polyporaceae/genética , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Micélio/genética , Micélio/crescimento & desenvolvimento , Polyporaceae/crescimento & desenvolvimento , RNA-Seq , Reação em Cadeia da Polimerase em Tempo Real
19.
J Plant Physiol ; 264: 153483, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34371311

RESUMO

Tomato plants are susceptible to drought stress, but the mechanism involved in this process still remains poorly understood. In the present study, we demonstrated that SlNAC6, a nuclear-localized protein induced by exogenous abscisic acid (ABA) or polyethylene glycol (PEG) stress treatment, plays a positive role in tomato plant response to PEG stress. Down-regulation of SlNAC6 (SlNAC6-RNAi) resulted in a semidwarf phenotype, and the SlNAC6-RNAi lines showed reduced tolerance to PEG stress, exhibiting a higher water loss rate and degree of oxidative damage, as well as lower values of proline content and antioxidant enzyme activity, when compared with those in wild type (WT). In contrast, overexpression of SlNAC6 (SlNAC6-OE) leads to a significant delay of growth, and the SlNAC6-OE lines showed greatly enhanced tolerance to PEG stress concomitant with a lower water loss rate and degree of oxidative damage, as well as higher values of proline content and antioxidant enzyme activity. Further study showed that the transcription level of ABA signaling-related genes and the ABA content are respectively decreased or increased in SlNAC6-RNAi and SlNAC6-OE seedlings, as verified by multiple physiological parameters, such as stomatal conductance, water loss rate, seed germination, and root length. Moreover, overexpression of SlNAC6 can accelerate tomato fruit ripening. Collectively, this study demonstrates SlNAC6 exerts important roles in tomato development, drought stress response, and fruit ripening processes, some of them perhaps partly through modulating an ABA-mediated pathway, which implies SlNAC6 may hold the potential applications in improving agronomic traits of tomato or other Solanaceae crops.


Assuntos
Lycopersicon esculentum/metabolismo , Proteínas de Plantas/fisiologia , Fatores de Transcrição/fisiologia , Desidratação , Regulação da Expressão Gênica de Plantas , Lycopersicon esculentum/genética , Lycopersicon esculentum/fisiologia , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reprodução , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
BMC Plant Biol ; 21(1): 365, 2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34380415

RESUMO

BACKGROUND: Kiwifruit (Actinidia Lindl.) is considered an important fruit species worldwide. Due to its temperate origin, this species is highly vulnerable to freezing injury while under low-temperature stress. To obtain further knowledge of the mechanism underlying freezing tolerance, we carried out a hybrid transcriptome analysis of two A. arguta (Actinidi arguta) genotypes, KL and RB, whose freezing tolerance is high and low, respectively. Both genotypes were subjected to - 25 °C for 0 h, 1 h, and 4 h. RESULTS: SMRT (single-molecule real-time) RNA-seq data were assembled using the de novo method, producing 24,306 unigenes with an N50 value of 1834 bp. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs showed that they were involved in the 'starch and sucrose metabolism', the 'mitogen-activated protein kinase (MAPK) signaling pathway', the 'phosphatidylinositol signaling system', the 'inositol phosphate metabolism', and the 'plant hormone signal transduction'. In particular, for 'starch and sucrose metabolism', we identified 3 key genes involved in cellulose degradation, trehalose synthesis, and starch degradation processes. Moreover, the activities of beta-GC (beta-glucosidase), TPS (trehalose-6-phosphate synthase), and BAM (beta-amylase), encoded by the abovementioned 3 key genes, were enhanced by cold stress. Three transcription factors (TFs) belonging to the AP2/ERF, bHLH (basic helix-loop-helix), and MYB families were involved in the low-temperature response. Furthermore, weighted gene coexpression network analysis (WGCNA) indicated that beta-GC, TPS5, and BAM3.1 were the key genes involved in the cold response and were highly coexpressed together with the CBF3, MYC2, and MYB44 genes. CONCLUSIONS: Cold stress led various changes in kiwifruit, the 'phosphatidylinositol signaling system', 'inositol phosphate metabolism', 'MAPK signaling pathway', 'plant hormone signal transduction', and 'starch and sucrose metabolism' processes were significantly affected by low temperature. Moreover, starch and sucrose metabolism may be the key pathway for tolerant kiwifruit to resist low temperature damages. These results increase our understanding of the complex mechanisms involved in the freezing tolerance of kiwifruit under cold stress and reveal a series of candidate genes for use in breeding new cultivars with enhanced freezing tolerance.


Assuntos
Aclimatação/genética , Actinidia/genética , Actinidia/fisiologia , Congelamento , Regulação da Expressão Gênica de Plantas , Frutas/genética , Frutas/fisiologia , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Sistema de Sinalização das MAP Quinases , Anotação de Sequência Molecular , Fosfatidilinositóis/metabolismo , Melhoramento Vegetal , Reguladores de Crescimento de Plantas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Amido/metabolismo , Sacarose/metabolismo
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