scholarly journals In-House Development, Standardization, and Validation of a RT-qPCR Assay for the Detection of SARS-CoV-2 Virus in Ecuador.

2020 ◽  
Author(s):  
David De la Torre ◽  
Ronny Pibaque ◽  
Tatiana Veloz ◽  
Paúl Beltrán ◽  
Lucy Baldeón

Abstract The World Health Organization (WHO) reported about 30 million cases of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and around 1 million deaths worldwide. Ecuador is the country with the highest mortality rate of confirmed cases in South America where both the poor ability to identify SARS-CoV-2 carriers and shortages in reagent supply have contributed the high infection rate observed. Hence, there is an urgent need to develop, standardize and validate an in-house protocol that cannot only reduces testing costs, but also increase the ability to screen widely the population. Primer-probe sets for the SARS-CoV-2 envelope protein E and the human ribonuclease P (RP) were validated for a duplex quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) for the Coronavirus Disease-19 (COVID-19) detection. Optimal E primers concentration was 400 nM and TaqMan probe 200 nM. The primer efficiency was set at 94.9% and R2 value at 0.99, which was comparable to commercial kits. The lower detection limit was found at 15 copies/ μL (50 copies/rx). In comparison to a Loop-mediated Isothermal Amplification (LAMP) commercial kit, there was a higher detection rate (30%) and results were highly reproducible (95%). We were able to develop a highly sensitive and low-cost duplex in-house RT-qPCR test for COVID-19 detection comparable to other commercially available kits.

2020 ◽  
Vol 10 (2) ◽  
pp. 90-92
Author(s):  
Rano Mal Piryani ◽  
Suneel Piryani ◽  
Shomeeta Piryani ◽  
Ganesh Dangal ◽  
Muzaherul Huq ◽  
...  

COVID-19 is mainly transmitted through droplet infection and spread very fast compared to SARS-CoV and MERS-CoV. For the countries, it is important to know at what stage the COVID-19 epidem­ic is? So, as to take appropriate steps to contain the epidemic. This will only be known by testing the suspects and contacts of confirmed cases. If there is poor testing, then most of the infected people may remain undetected, however they could spread the virus to hundreds of other people and potential contacts, which could not be known and quarantined in time continuing the spread. If there is quality assured, highly sensitive and specific testing along with adequate isolation and quarantine, then the spread will be limited. There are two types of tests available for COVID-19: the tests directly detecting the viral ribonucleic acid (RNA) collected in nasopharyngeal or throat swabs, and tests detecting antibodies from the blood sample. At this point in time, the polymerase chain reaction (PCR) tests are used for confirmation of the disease while antibodies tests may provide information regarding the prevalence of infection. World Health Organization advices the countries to increase the testing and get to know the level of epidemic and act accordingly for containment of infection.


2020 ◽  
Vol 17 ◽  
Author(s):  
Ajoy Basak ◽  
Sarmistha Basak

: The current global pandemic outbreak of a novel type of corona virus termed by World Health Organization as COVID-19 became an grave concern and worry to human health and world economy. Intense research efforts are now underway worldwide to combat and prevent the spread of this deadly disease. This zoonotic virus, a native to bat population is most likely transmitted to human via a host reservoir. Due to its close similarity to previously known SARS CoV (Severe Acute Respiratory Syndrome Corona Virus) of 2002 and related MERS CoV (Middle East Respiratory Syndrome Corona Virus) of 2012, it is also known as SARS CoV2. But unlike them it is far too infectious, virulent and lethal. Among its various proteins, the surface spike glycoprotein “S” has drawn significant attention because of its implication in viral recognition and host-virus fusion process. A detail comparative analysis of “S” proteins of SARS CoV (now called SARS CoV1), SARS CoV2 (COVID-19) and MERS CoV based on structure, sequence alignment, host cleavage sites, receptor binding domains, potential glycosylation and Cys-disulphide bridge locations has been performed. It revealed some key features and variations that may elucidate the high infection and virulence character of COVID-19. Moreover this crucial information may become useful in our quest for COVID-19 therapeutics and vaccines.


2021 ◽  
Vol 11 (8) ◽  
pp. 3495
Author(s):  
Shabir Hussain ◽  
Yang Yu ◽  
Muhammad Ayoub ◽  
Akmal Khan ◽  
Rukhshanda Rehman ◽  
...  

The spread of COVID-19 has been taken on pandemic magnitudes and has already spread over 200 countries in a few months. In this time of emergency of COVID-19, especially when there is still a need to follow the precautions and developed vaccines are not available to all the developing countries in the first phase of vaccine distribution, the virus is spreading rapidly through direct and indirect contacts. The World Health Organization (WHO) provides the standard recommendations on preventing the spread of COVID-19 and the importance of face masks for protection from the virus. The excessive use of manual disinfection systems has also become a source of infection. That is why this research aims to design and develop a low-cost, rapid, scalable, and effective virus spread control and screening system to minimize the chances and risk of spread of COVID-19. We proposed an IoT-based Smart Screening and Disinfection Walkthrough Gate (SSDWG) for all public places entrance. The SSDWG is designed to do rapid screening, including temperature measuring using a contact-free sensor and storing the record of the suspected individual for further control and monitoring. Our proposed IoT-based screening system also implemented real-time deep learning models for face mask detection and classification. This module classified individuals who wear the face mask properly, improperly, and without a face mask using VGG-16, MobileNetV2, Inception v3, ResNet-50, and CNN using a transfer learning approach. We achieved the highest accuracy of 99.81% while using VGG-16 and the second highest accuracy of 99.6% using MobileNetV2 in the mask detection and classification module. We also implemented classification to classify the types of face masks worn by the individuals, either N-95 or surgical masks. We also compared the results of our proposed system with state-of-the-art methods, and we highly suggested that our system could be used to prevent the spread of local transmission and reduce the chances of human carriers of COVID-19.


Sensors ◽  
2018 ◽  
Vol 18 (10) ◽  
pp. 3423 ◽  
Author(s):  
Shrikant Warkad ◽  
Satish Nimse ◽  
Keum-Soo Song ◽  
Taisun Kim

According to the World Health Organization (WHO), 71 million people were living with Hepatitis C virus (HCV) infection worldwide in 2015. Each year, about 399,000 HCV-infected people succumb to cirrhosis, hepatocellular carcinoma, and liver failure. Therefore, screening of HCV infection with simple, rapid, but highly sensitive and specific methods can help to curb the global burden on HCV healthcare. Apart from the determination of viral load/viral clearance, the identification of specific HCV genotype is also critical for successful treatment of hepatitis C. This critical review focuses on the technologies used for the detection, discrimination, and genotyping of HCV in clinical samples. This article also focuses on advantages and disadvantages of the reported methods used for HCV detection, quantification, and genotyping.


2021 ◽  
Vol 24 (5-esp.) ◽  
pp. 650-654
Author(s):  
Gabriela Paschoalini Romagni ◽  
Paula Marino Costa ◽  
Sandra Mara Maciel ◽  
Maria Paula Jacobucci ◽  
Regina Célia Poli-Frederico

A doença cárie é considerada, atualmente, como biofilme sacarose dependente, entretanto, estudos recentes apontam que fatores genéticos também podem influenciar seu desenvolvimento. Variantes nos gene amelogenina (AMELX) e enamelina (ENAM), responsáveis pela formação do esmalte, têm sido propostas como potencialmente envolvidos na doença. O objetivo deste estudo foi avaliar se a ocorrência de cárie dentária em adolescentes está relacionado às variantes nos genes AMELX e ENAM. Para a avaliação da prevalência de cárie foi utilizado o índice de dentes cariados, perdidos e obturados (CPO-D), segundo critérios da Organização Mundial de Saúde. As amostras de DNA foram extraídas das células da mucosa oral. Para a análise dos polimorfismos de nucleotídeo único (SNPs) dos genes AMELX (rs17878486) e ENAM (rs7671281) foi utilizada  a técnica de amplificação de fragmentos de DNA pela reação em cadeia da polimerase foi realizada (PCR) em tempo real pelo sistema TaqMan (Applied Biosystems, Foster City, EUA). Para a análise estatística, foi utilizado o teste exato de Fisher e qui-quadrado com nível de significância de 5%. Apenas os fatores socioeconômicos influenciaram a experiência de cárie. Concluiu-se que o componente genético, na população deste estudo, não influenciou o desenvolvimento da cárie.   Palavras-chave: Polimorfismo genético. Adolescentes. Esmalte.   Abstract Caries disease is currently considered a sucrose-dependent biofilm, however recent studies indicate that a genetic component can also influence its development. Variants in the amelogenin (AMELX) and enamelin (ENAM) genes, responsible for the enamel formation, have been proposed as potentially involved in the disease. The purpose of this study was to evaluate whether the occurrence of dental caries in adolescents is related to variants in the AMELX and ENAM genes. To assess the caries prevalence, the index of decayed, missing and filled teeth (DMFT) were used, according to World Health Organization criteria. DNA samples were extracted from oral mucosa cells. For the analysis of single nucleotide polymorphisms (SNPs) of the AMELX (rs17878486) and ENAM (rs7671281) genes, the amplifying DNA fragments technique  by the polymerase chain reaction was performed (PCR) in real time by the TaqMan system (Applied Biosystems, Foster City, USA). For the statistical analysis, Fisher's exact test and chi-square were used with a 5% significance level. Only socioeconomic factors influenced the caries experience. It was concluded that the genetic component in the population of this study, did not influence the development of caries.   Keywords: Genetic polymorphism. Adolescents. Enamel.


2021 ◽  
Vol 29 (3) ◽  
pp. 299-306
Author(s):  
Simona Roxana Gheorghe ◽  
Cătălin Marian ◽  
Ligia Gabriela Tătăranu ◽  
Anica Dricu ◽  
Cees Vermeer ◽  
...  

Abstract Meningiomas are classified by the World Health Organization (WHO) in three grades, based on morphological features. Independent of this grading, the presence of calcification in meningiomas influences their growth rate. The messenger RNA of matrix Gla protein (MGP), an extra-hepatic protein with different conformations involved in the homeostasis of ectopic calcification has been found in meningiomas and was shown to be regulated in breast cancer by miR-155-5p, a specific micro RNA. Therefore, we investigated the expression of miR-155-5p and its relationship with local MGP conformations in different grade meningiomas. According to the WHO classification, our 41 samples of meningiomas were stratified in groups WHO I and WHO II. Using real time polymerase chain reaction, we observed a higher miR-155-5p expression in group WHO I versus group WHO II [with a fold change (FC) of 3.83, p=0.027)]. Moreover, the expression of miR-155-5p was higher in calcified tumors compared to non-calcified tumors in all samples (FC=3.01, p=0.047) and in group WHO I (FC=3.65, p=0.048). Utilizing immunohistochemistry, we determined the concurrent presence of all MGP conformations in calcified meningiomas. This study was the first to establish higher miR-155-5p expression in grade WHO I and calcified meningiomas, which could improve molecular classification and targeted therapy and also the presence of all MGP conformations in calcified meningiomas, confirming the existence of an anti-calcification mechanism in meningiomas..


2016 ◽  
Vol 36 (3) ◽  
pp. 239-243
Author(s):  
Grisel Marrero ◽  
Lilian P. Delgado ◽  
Humberto Caroll ◽  
Neise Ortiz ◽  
Alexis Musacchio ◽  
...  

Abstract A rapid, specific and sensible polystyrene latex-based reagent has been developed to detect rheumatoid factor (RF) in human sera. Detection of RF is one of the criteria for rheumatoid arthritis (RA) diagnosis. RF includes immunoglobulins IgG, IgM or IgA targeting human-IgG Fc region, found in sera of 70–90% of patients with RA. Spherical, clean polystyrene particles of 480±80 nm with a Z-potential of -28 mV were synthesized. Purified gamma-globulin human blood fraction, used as IgG source, was adsorbed on particles in glycine-buffered saline with a fivefold excess of the immunoglobulins needed to saturate the calculated total surface area of the particles. The reagent was calibrated against the World Health Organization international serum reference preparation and tested with commercial positive and negative RF controls. Reactivity of the developed diagnostic reagent with human sera, either positive or negative for RF, was evaluated. The sera were tested both intact and following complement inactivation. When complement-inactivated sera were used, our reagent showed 100% coincidence of results with a commercial diagnostic kit of specificity of 80% and sensibility of 93%. The in-house reagent developed in the present work is easy to prepare and at relatively low cost, making it ideal for RF detection in low-income settings.


2020 ◽  
Author(s):  
Michael G. Ison

Coronaviruses (CoVs) are a group of viral pathogens that infect mammals and birds. The presentation in humans is typically that of a mild upper respiratory tract infection, similar to the common cold. However, in recent years, dramatic attention has arisen for more lethal members of this viral family (e.g., severe acute respiratory syndrome [SARS-CoV], Middle East respiratory syndrome [MERS-CoV], and coronavirus disease 2019 [COVID-19]). The epidemiology, clinical presentation, diagnosis, and management of these viruses are discussed in this review. Importantly, new guideline tables from the Centers for Disease Control and Prevention, as well as the World Health Organization are provided at the conclusion of the review. This review contains 12 tables, 3 figure and 48 references. Keywords: Coronavirus, severe acute respiratory distress syndrome (SARS), Middle East respiratory syndrome (MERS), COVID-19, respiratory infection, antiviral, real-time polymerase chain reaction


2020 ◽  
Author(s):  
Michael G. Ison

Coronaviruses (CoVs) are a group of viral pathogens that infect mammals and birds. The presentation in humans is typically that of a mild upper respiratory tract infection, similar to the common cold. However, in recent years, dramatic attention has arisen for more lethal members of this viral family (e.g., severe acute respiratory syndrome [SARS-CoV], Middle East respiratory syndrome [MERS-CoV], and coronavirus disease 2019 [COVID-19]). The epidemiology, clinical presentation, diagnosis, and management of these viruses are discussed in this review. Importantly, new guideline tables from the Centers for Disease Control and Prevention, as well as the World Health Organization are provided at the conclusion of the review. This review contains 12 tables, 3 figure and 48 references. Keywords: Coronavirus, severe acute respiratory distress syndrome (SARS), Middle East respiratory syndrome (MERS), COVID-19, respiratory infection, antiviral, real-time polymerase chain reaction


2020 ◽  
Author(s):  
Michael G. Ison

Coronaviruses (CoVs) are a group of viral pathogens that infect mammals and birds. The presentation in humans is typically that of a mild upper respiratory tract infection, similar to the common cold. However, in recent years, dramatic attention has arisen for more lethal members of this viral family (e.g., severe acute respiratory syndrome [SARS-CoV], Middle East respiratory syndrome [MERS-CoV], and coronavirus disease 2019 [COVID-19]). The epidemiology, clinical presentation, diagnosis, and management of these viruses are discussed in this review. Importantly, new guideline tables from the Centers for Disease Control and Prevention, as well as the World Health Organization are provided at the conclusion of the review. This review contains 12 tables, 3 figure and 48 references. Keywords: Coronavirus, severe acute respiratory distress syndrome (SARS), Middle East respiratory syndrome (MERS), COVID-19, respiratory infection, antiviral, real-time polymerase chain reaction


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