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1.
Zhongguo Zhong Yao Za Zhi ; 46(15): 3832-3837, 2021 Aug.
Artículo en Chino | MEDLINE | ID: mdl-34472256

RESUMEN

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.


Asunto(s)
Amomum , Perfilación de la Expresión Génica , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Semillas/genética
2.
Pan Afr Med J ; 39: 89, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34466191

RESUMEN

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.


Asunto(s)
Prueba de COVID-19/métodos , COVID-19/diagnóstico , Guías de Práctica Clínica como Asunto , África , Humanos , Tamizaje Masivo/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Manejo de Especímenes , Organización Mundial de la Salud
3.
Artículo en Inglés | MEDLINE | ID: mdl-34501904

RESUMEN

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.


Asunto(s)
COVID-19 , Discapacidad Intelectual , Hospitales , Humanos , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/epidemiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , SARS-CoV-2 , Sensibilidad y Especificidad
4.
JNMA J Nepal Med Assoc ; 59(235): 248-251, 2021 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-34506444

RESUMEN

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.


Asunto(s)
COVID-19 , Adolescente , Adulto , Estudios Transversales , Femenino , Humanos , Masculino , Nepal/epidemiología , Prevalencia , Reacción en Cadena en Tiempo Real de la Polimerasa , SARS-CoV-2 , Centros de Atención Terciaria , Adulto Joven
5.
Zhongguo Zhong Yao Za Zhi ; 46(12): 3116-3122, 2021 Jun.
Artículo en Chino | MEDLINE | ID: mdl-34467703

RESUMEN

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.


Asunto(s)
Aconitum , Perfilación de la Expresión Génica , Genes de Plantas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Estándares de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
6.
Vet Res ; 52(1): 121, 2021 Sep 16.
Artículo en Inglés | MEDLINE | ID: mdl-34530902

RESUMEN

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is causing a global crisis. It is still unresolved. Although many therapies and vaccines are being studied, they are still in their infancy. As this pandemic continues, rapid and accurate research for the development of therapies and vaccines is needed. Therefore, it is necessary to understand characteristics of diseases caused by SARS-CoV-2 through animal models. Syrian hamsters are known to be susceptible to SARS-CoV-2. They were intranasally inoculated with SARS-CoV-2. At 2, 4, 8, 12, and 16 days post-infection (dpi), these hamsters were euthanized, and tissues were collected for ultrastructural and microstructural examinations. Microscopic lesions were prominent in the upper and lower respiratory tracts from 2 and 4 dpi groups, respectively. The respiratory epithelium in the trachea, bronchiole, and alveolar showed pathological changes. Inflammatory cells including neutrophils, lymphocytes, macrophages, and eosinophils were infiltrated in/around tracheal lamina propria, pulmonary vessels, alveoli, and bronchiole. In pulmonary lesions, alveolar wall was thickened with infiltrated inflammatory cells, mainly neutrophils and macrophages. In the trachea, epithelial damages started from 2 dpi and recovered from 8 dpi, consistent with microscopic results, High levels of SARS-CoV-2 nucleoprotein were detected at 2 dpi and 4 dpi. In the lung, lesions were most severe at 8 dpi. Meanwhile, high levels of SARS-CoV-2 were detected at 4 dpi. Electron microscopic examinations revealed cellular changes in the trachea epithelium and alveolar epithelium such as vacuolation, sparse micro-organelle, and poor cellular margin. In the trachea epithelium, the number of cytoplasmic organelles was diminished, and small vesicles were prominent from 2 dpi. Some of these electron-lucent vesicles were filled with virion particles. From 8 dpi, the trachea epithelium started to recover. Because of shrunken nucleus and swollen cytoplasm, the N/C ratio of type 2 pneumocyte decreased at 8 and 12 dpi. From 8 dpi, lamellar bodies on type 2 pneumocyte cytoplasm were increasingly observed. Their number then decreased from 16 dpi. However, there was no significant change in type 1 pneumocyte. Viral vesicles were only observed in the cytoplasm of type 2 pneumocyte. In conclusion, ultra- and micro-structural changes presented in this study may provide useful information for SARS-CoV-2 studies in various fields.


Asunto(s)
COVID-19/patología , Sistema Respiratorio/patología , SARS-CoV-2/patogenicidad , Animales , Cricetinae , Inmunohistoquímica/veterinaria , Masculino , Mesocricetus , Proyectos Piloto , ARN Viral/química , ARN Viral/aislamiento & purificación , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Sistema Respiratorio/química , Sistema Respiratorio/ultraestructura , Sistema Respiratorio/virología , Factores de Tiempo , Tráquea/patología , Tráquea/ultraestructura , Tráquea/virología , Pérdida de Peso
7.
J Med Microbiol ; 70(9)2021 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-34516365

RESUMEN

Introduction. The identification of enteropathogens is critical for the clinical management of patients with suspected gastrointestinal infection. The FLOW multiplex PCR system (FMPS) is a semi-automated platform (FLOW System, Roche) for multiplex real-time PCR analysis.Hypothesis/Gap Statement. FMPS has greater sensitivity for the detection of enteric pathogens than standard methods such as culture, biochemical identification, immunochromatography or microscopic examination.Aim.The diagnostic performance of the FMPS was evaluated and compared to that of traditional microbiological procedures.Methodology. A total of 10 659 samples were collected and analysed over a period of 7 years. From 2013 to 2018 (every July to September), samples were processed using standard microbiological culture methods. In 2019, the FMPS was implemented using real-time PCR to detect the following enteropathogens: Shigella spp., Salmonella spp., Campylobacter spp., Giardia intestinalis, Entamoeba histolytica, Blastocystis hominis, Cryptosporidum spp., Dientamoeba fragilis, adenovirus, norovirus and rotavirus. Standard microbiological culture methods (2013-2018) included stool culture, microscopy and immunochromatography.Results. A total of 1078 stool samples were analysed prospectively using the FMPS from July to September (2019): bacterial, parasitic and viral pathogens were identified in 15.3, 9.71 and 5.29 % of cases, respectively. During the same period of 6 years (2013-2018), the proportion of positive identifications using standard microbiological methods from 2013 to 2018 was significantly lower. A major significant recovery improvement was observed for all bacteria species tested: Shigella spp./enteroinvasive Escherichia coli (EIEC) (P <0.05), Salmonella spp. (P <0.05) and Campylobacter spp. (P <0.05). Marked differences were also observed for the parasites G. intestinalis, Cryptosporidium spp. and D. fragilis.Conclusion. These results support the value of multiplex real-time PCR analysis for the detection of enteric pathogens in laboratory diagnosis with outstanding performance in identifying labile micro-organisms. The identification of unsuspected micro-organisms for less specific clinical presentations may also impact on clinical practice and help optimize patient management.


Asunto(s)
Gastroenteritis/diagnóstico , Reacción en Cadena de la Polimerasa Multiplex , Reacción en Cadena en Tiempo Real de la Polimerasa , Adenoviridae/aislamiento & purificación , Blastocystis hominis/aislamiento & purificación , Campylobacter/aislamiento & purificación , Cryptosporidium/aislamiento & purificación , Dientamoeba/aislamiento & purificación , Entamoeba histolytica/aislamiento & purificación , Heces/microbiología , Heces/parasitología , Heces/virología , Gastroenteritis/microbiología , Gastroenteritis/parasitología , Giardia lamblia/aislamiento & purificación , Humanos , Norovirus/aislamiento & purificación , Rotavirus/aislamiento & purificación , Salmonella/aislamiento & purificación , Shigella/aislamiento & purificación
8.
J Hazard Mater ; 416: 126137, 2021 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-34492926

RESUMEN

Fomites are known to spread infectious diseases, but their role in determining transmission risk remains unclear. The association of surface touch networks (STNs), proposed to explain this risk, with real-life surface contamination has not yet been demonstrated. To construct STNs, we collected surface touch data from 23 to 26 scholars through 2 independent experiments conducted in office spaces for 13 h each. In parallel, a tracer bacterium (Lactobacillus bulgaricus) was spread by a designated carrier in each experiment during normal activities; the subsequent extent of surface contamination was assessed using qPCR. The touch data were also analyzed using an agent-based model that predicted the observed contamination. Touching public (door handles) and hidden public (desks, chair seatbacks) surfaces that connected occupants, sparse hand-to-hand contact, and active carriers contributed significantly to contamination spread, which was also correlated with the size of the social group containing carriers. The natural and unsupervised experiments reflected realistic exposure levels of mouths (1-10 ppm of total contamination spread by one root carrier), nostrils (~1 ppm), and eyes (~0.1 ppm). We conclude that the contamination degree of known and hidden public surfaces can indicate fomite exposure risk. The social group effect could trigger superspreading events through fomite transmission.


Asunto(s)
Fómites , Tacto , Mano , Reacción en Cadena en Tiempo Real de la Polimerasa
9.
Mikrobiyol Bul ; 55(3): 435-444, 2021 Jul.
Artículo en Turco | MEDLINE | ID: mdl-34416808

RESUMEN

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.


Asunto(s)
COVID-19 , ARN Viral , Anciano , Humanos , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , SARS-CoV-2 , Carga Viral
10.
Artículo en Inglés | MEDLINE | ID: mdl-34444305

RESUMEN

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.


Asunto(s)
Legionella pneumophila , Legionella , Medios de Cultivo , Legionella/genética , Legionella pneumophila/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Microbiología del Agua
11.
Gene ; 802: 145864, 2021 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-34352300

RESUMEN

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.


Asunto(s)
Búfalos/metabolismo , Grasas/metabolismo , Glándulas Mamarias Animales/metabolismo , Leche/metabolismo , Animales , Búfalos/genética , Femenino , RNA-Seq/veterinaria , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Transcriptoma
12.
Gene ; 802: 145863, 2021 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-34358628

RESUMEN

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.


Asunto(s)
Proteínas Fúngicas/genética , Polyporaceae/genética , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Micelio/genética , Micelio/crecimiento & desarrollo , Polyporaceae/crecimiento & desarrollo , RNA-Seq , Reacción en Cadena en Tiempo Real de la Polimerasa
13.
Viruses ; 13(7)2021 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-34372527

RESUMEN

Real-time reverse transcription polymerase chain reaction (RT-qPCR) is the most sensitive and specific assay and, therefore, is the "gold standard" diagnostic method for the diagnosis of SARS-CoV-2 infection. The aim of this study was to compare and analyze the detection performance of three different commercially available SARS-CoV-2 nucleic acid detection kits: Sansure Biotech, GeneFinderTM, and TaqPathTM on 354 randomly selected samples from hospitalized COVID-19 patients. All PCR reactions were performed using the same RNA isolates and one real-time PCR machine. The final result of the three evaluated kits was not statistically different (p = 0.107), and also had a strong positive association and high Cohen's κ coefficient. In contrast, the average Ct values that refer to the ORF1ab and N gene amplification were significantly different (p < 0.001 and p < 0.001, respectively), with the lowest obtained by the TaqPathTM for the ORF1ab and by the Sansure Biotech for the N gene. The results show a high similarity in the analytical sensitivities for SARS-CoV-2 detection, which indicates that the diagnostic accuracy of the three assays is comparable. However, the SanSure Biotech kit showed a bit better diagnostic performance. Our findings suggest that the imperative for improvement should address the determination of cut-off Ct values and rapid modification of the primer sets along with the appearance of new variants.


Asunto(s)
Prueba de Ácido Nucleico para COVID-19/métodos , COVID-19/virología , Juego de Reactivos para Diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , SARS-CoV-2/aislamiento & purificación , COVID-19/diagnóstico , COVID-19/epidemiología , Estudios Transversales , Humanos , ARN Viral/genética , ARN Viral/aislamiento & purificación , SARS-CoV-2/genética , Sensibilidad y Especificidad , Serbia/epidemiología
14.
PLoS One ; 16(8): e0255999, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34379690

RESUMEN

BACKGROUND: The primary goal of the presented cross-sectional observational study was to determine the clinical and demographic risk factors for adverse coronavirus disease 2019 (COVID-19) outcomes in the Pakistani population. METHODS: We examined the individuals (n = 6331) that consulted two private diagnostic centers in Lahore, Pakistan, for COVID-19 testing between May 1, 2020, and November 30, 2020. The attending nurse collected clinical and demographic information. A confirmed case of COVID-19 was defined as having a positive result through real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay of nasopharyngeal swab specimens. RESULTS: RT-PCR testing was positive in 1094 cases. Out of which, 5.2% had severe, and 20.8% had mild symptoms. We observed a strong association of COVID-19 severity with the number and type of comorbidities. The severity of the disease intensified as the number of comorbidities increased. The most vulnerable groups for the poor outcome are patients with diabetes and hypertension. Increasing age was also associated with PCR positivity and the severity of the disease. CONCLUSIONS: Most cases of COVID-19 included in this study developed mild symptoms or were asymptomatic. Risk factors for adverse outcomes included older age and the simultaneous presence of comorbidities.


Asunto(s)
COVID-19/diagnóstico , COVID-19/epidemiología , COVID-19/patología , COVID-19/virología , Comorbilidad , Demografía , Complicaciones de la Diabetes/patología , Humanos , Hipertensión/complicaciones , Pakistán/epidemiología , ARN Viral/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Riesgo , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Índice de Severidad de la Enfermedad
15.
Talanta ; 234: 122672, 2021 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-34364473

RESUMEN

An Enzyme Linked ImmunoMagnetic Electrochemical assay (ELIME) was developed for the detection of the hepatitis A virus (HAV). This system is based on the use of new polydopamine-modified magnetic nanobeads as solid support for the immunochemical chain, and an array of 8 screen-printed electrodes as a sensing platform. Enzymatic-by-product is quickly measured by differential pulse voltammetry. For this purpose, all analytical parameters were optimized; in particular, different blocking reagents were evaluated in order to minimize the nonspecific interaction of bioreagents. Using the ELIME assays, a quantitative determination of HAV can be achieved with a detection limit of 1·10-11 IU mL-1 and a working range between 10-10 - 5 × 10-7 IU mL-1. The cross-reactivity of the commercial monoclonal antibodies against HAV used in ELIME assays was tested for Coxsackie B4, resulting very low. The sensitivity was also investigated and compared with spectrophotometric sandwich ELISA. The average relative standard deviation (RSD) of the ELIME method was less than 5% for the assays performed on the same day, and 7% for the measurements made on different days. The proposed system was applied to the cell culture of HAV, which title was quantified by Real-Time Quantitative Reverse Transcription PCR (RT¬qPCR). To compare the results, a correlation between the units used in ELIME (IU mL-1) and those used in RT¬qPCR (genome mL-1) was established using a HAV-positive sample, resulting in 1 IU mL-1-10-4 gen mL-1 (R2 = 0.978). The ELIME tool exhibits good stability and high biological selectivity for HAV antigen detection and was successfully applied for the determination of HAV in tap water.


Asunto(s)
Virus de la Hepatitis A , Bioensayo , Virus de la Hepatitis A/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad
16.
Mem Inst Oswaldo Cruz ; 116: e210085, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34406222

RESUMEN

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.


Asunto(s)
Prueba de COVID-19 , COVID-19 , Humanos , Nasofaringe , Reacción en Cadena en Tiempo Real de la Polimerasa , SARS-CoV-2 , Saliva , Manejo de Especímenes
17.
BMC Bioinformatics ; 22(1): 398, 2021 Aug 24.
Artículo en Inglés | MEDLINE | ID: mdl-34433408

RESUMEN

BACKGROUND: The analyses of amplification and melting curves have been shown to provide valuable information on the quality of the individual reactions in quantitative PCR (qPCR) experiments and to result in more reliable and reproducible quantitative results. IMPLEMENTATION: The main steps in the amplification curve analysis are (1) a unique baseline subtraction, not using the ground phase cycles, (2) PCR efficiency determination from the exponential phase of the individual reactions, (3) setting a common quantification threshold and (4) calculation of the efficiency-corrected target quantity with the common threshold, efficiency per assay and Cq per reaction. The melting curve analysis encompasses smoothing of the observed fluorescence data, normalization to remove product-independent fluorescence loss, peak calling and assessment of the correct peak by comparing its melting temperature with the known melting temperature of the intended amplification product. RESULTS: The LinRegPCR web application provides visualization and analysis of a single qPCR run. The user interface displays the analysis results on the amplification curve analysis and melting curve analysis in tables and graphs in which deviant reactions are highlighted. The annotated results in the tables can be exported for calculation of gene-expression ratios, fold-change between experimental conditions and further statistical analysis. Web-based LinRegPCR addresses two types of users, wet-lab scientists analyzing the amplification and melting curves of their own qPCR experiments and bioinformaticians creating pipelines for analysis of series of qPCR experiments by splitting its functionality into a stand-alone back-end RDML (Real-time PCR Data Markup Language) Python library and several companion applications for data visualization, analysis and interactive access. The use of the RDML data standard enables machine independent storage and exchange of qPCR data and the RDML-Tools assist with the import of qPCR data from the files exported by the qPCR instrument. CONCLUSIONS: The combined implementation of these analyses in the newly developed web-based LinRegPCR ( https://www.gear-genomics.com/rdml-tools/ ) is platform independent and much faster than the original Windows-based versions of the LinRegPCR program. Moreover, web-based LinRegPCR includes a novel statistical outlier detection and the combination of amplification and melting curve analyses allows direct validation of the amplification product and reporting of reactions that amplify artefacts.


Asunto(s)
Artefactos , Internet , Reacción en Cadena en Tiempo Real de la Polimerasa
18.
J Plant Physiol ; 264: 153483, 2021 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-34371311

RESUMEN

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.


Asunto(s)
Lycopersicon esculentum/metabolismo , Proteínas de Plantas/fisiología , Factores de Transcripción/fisiología , Deshidratación , Regulación de la Expresión Génica de las Plantas , Lycopersicon esculentum/genética , Lycopersicon esculentum/fisiología , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducción , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
19.
BMC Plant Biol ; 21(1): 365, 2021 Aug 11.
Artículo en Inglés | MEDLINE | ID: mdl-34380415

RESUMEN

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.


Asunto(s)
Aclimatación/genética , Actinidia/genética , Actinidia/fisiología , Congelación , Regulación de la Expresión Génica de las Plantas , Frutas/genética , Frutas/fisiología , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Sistema de Señalización de MAP Quinasas , Anotación de Secuencia Molecular , Fosfatidilinositoles/metabolismo , Fitomejoramiento , Reguladores del Crecimiento de las Plantas/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Almidón/metabolismo , Sacarosa/metabolismo
20.
PLoS One ; 16(8): e0256352, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34403456

RESUMEN

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.


Asunto(s)
Antígenos Virales/análisis , COVID-19/diagnóstico , Inmunoensayo , SARS-CoV-2/metabolismo , Área Bajo la Curva , COVID-19/virología , Humanos , Nasofaringe/virología , Sistemas de Atención de Punto , ARN Viral/análisis , ARN Viral/metabolismo , Curva ROC , Reacción en Cadena en Tiempo Real de la Polimerasa , SARS-CoV-2/aislamiento & purificación , Sensibilidad y Especificidad
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