ABSTRACT
As to date, more than 49 million confirmed cases of Coronavirus Disease 19 (COVID-19) have been reported worldwide. Current diagnostic protocols use qRT-PCR for viral RNA detection, which is expensive and requires sophisticated equipment, trained personnel and previous RNA extraction. For this reason, we need a faster, direct and more versatile detection method for better epidemiological management of the COVID-19 outbreak. In this work, we propose a direct method without RNA extraction, based on the Loop-mediated isothermal amplification (LAMP) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein (CRISPR-Cas12) technique that allows the fast detection of SARS-CoV-2 from patient samples with high sensitivity and specificity. We obtained a limit of detection of 16 copies/µL with high specificity and at an affordable cost. The diagnostic test readout can be done with a real-time PCR thermocycler or with the naked eye in a blue-light transilluminator. Our method has been evaluated on a small set of clinical samples with promising results.
ABSTRACT
The development of a quantitative-competitive reverse transcription-PCR (RT-PCR) assay to quantify dengue virus (DEN) genome (vRNA) and its replicative intermediate RNA (vRI) is described. A highly conserved region located on the DEN capsid-premembrane genes was used to produce a competitor RNA molecule which contains an internal deletion of 70 nucleotides. The competitor provides a suitable internal control useful to quantify viral RNA from all four dengue virus (DEN 1-4) serotypes. The detection limit of the assay was found to be 100 copies per reaction. This is a rapid, simple, sensitive, inexpensive and easy method for quantitation of DEN RNA species.
Subject(s)
Dengue Virus/isolation & purification , Dengue/virology , Genome, Viral , RNA, Viral/analysis , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Animals , Cell Line , Dengue/diagnosis , Dengue Virus/classification , Dengue Virus/genetics , Humans , RNA, Viral/blood , Sensitivity and Specificity , SerotypingABSTRACT
Since its identification in April 2009, an A(H1N1) virus containing a unique combination of gene segments from both North American and Eurasian swine lineages has continued to circulate in humans. The lack of similarity between the 2009 A(H1N1) virus and its nearest relatives indicates that its gene segments have been circulating undetected for an extended period. Its low genetic diversity suggests that the introduction into humans was a single event or multiple events of similar viruses. Molecular markers predictive of adaptation to humans are not currently present in 2009 A(H1N1) viruses, suggesting that previously unrecognized molecular determinants could be responsible for the transmission among humans. Antigenically the viruses are homogeneous and similar to North American swine A(H1N1) viruses but distinct from seasonal human A(H1N1).
Subject(s)
Antigens, Viral/immunology , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/immunology , Influenza, Human/virology , Animals , Antibodies, Viral/immunology , Antigens, Viral/genetics , Disease Outbreaks , Evolution, Molecular , Genes, Viral , Genetic Variation , Genome, Viral , Hemagglutination Inhibition Tests , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Humans , Influenza A Virus, H1N1 Subtype/classification , Influenza A Virus, H1N1 Subtype/isolation & purification , Influenza A Virus, H3N2 Subtype/genetics , Influenza A virus/genetics , Influenza, Human/epidemiology , Influenza, Human/immunology , Mutation , Neuraminidase/genetics , Orthomyxoviridae Infections/veterinary , Orthomyxoviridae Infections/virology , Phylogeny , Reassortant Viruses/genetics , Swine , Swine Diseases/virology , Viral Matrix Proteins/genetics , Viral Nonstructural Proteins/geneticsABSTRACT
Dengue virus replication involves synthesis of a replicative intermediate RNA (RI-RNA), whose presence reveals an actual infection. We report on a simple and rapid reverse transcription-PCR for the detection of viral RI-RNA in infected cells. The product is demonstrated at 20 min postinfection. This method is useful for the study of virus-cell tropism.