Ready-To-Use qPCR for Detection of DNA from Trypanosoma cruzi or Other Pathogenic Organisms. 2022

Alexandre Dias Tavares Costa, and Steffanie Skau Amadei, and Amanda Bertão-Santos, and Tuany Rodrigues
Laboratório de Ciências e Tecnologias Aplicadas em Saúde (LaCTAS), Instituto Carlos Chagas (ICC), Fundação Oswaldo Cruz (Fiocruz); Pós-graduação em Engenharia de Bioprocessos e Biotecnologia, Universidade Federal do Paraná (UFPR); Pós-graduação em Biociências e Biotecnologia, Instituto Carlos Chagas (ICC), Fundação Oswaldo Cruz (Fiocruz); alexandre.costa@fiocruz.br.

Real-time PCR (qPCR) is a remarkably sensitive and precise technique that allows for amplifying minute amounts of nucleic acid targets from a multitude of samples. It has been extensively used in many research areas and achieved industrial application in fields such as human diagnostics and trait selection in crops of genetically modified organisms (GMO) crops. However, qPCR is not an error-proof technique. Mixing all reagents into a single master mix subsequently distributed onto 96 wells of a regular qPCR plate might lead to operator mistakes such as incorrect mixing of reagents or inaccurate dispensing into the wells. Here, a technique called gelification is presented, whereby most of the water present in the master mix is substituted by reagents that form a sol-gel mixture when submitted to a vacuum. As a result, qPCR reagents are effectively preserved for a few weeks at room temperature or a few months at 2-8 oC. Details of preparing each solution are shown here along with the expected aspect of a gelified reaction designed to detect T. cruzi satellite DNA (satDNA). A similar procedure can be applied to detect other organisms. Starting a gelified qPCR run is as simple as removing the plate from the refrigerator, adding the samples to their respective wells, and starting the run, thus decreasing the setup time of a full-plate reaction to the time it takes to load the samples. Additionally, gelified PCR reactions can be produced and controlled for quality in batches, saving time and avoiding common operator mistakes while running routine PCR reactions.

UI MeSH Term Description Entries
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D014349 Trypanosoma cruzi The agent of South American trypanosomiasis or CHAGAS DISEASE. Its vertebrate hosts are man and various domestic and wild animals. Insects of several species are vectors. Trypanosoma cruzus,cruzi, Trypanosoma
D014355 Chagas Disease Infection with the protozoan parasite TRYPANOSOMA CRUZI, a form of TRYPANOSOMIASIS endemic in Central and South America. It is named after the Brazilian physician Carlos Chagas, who discovered the parasite. Infection by the parasite (positive serologic result only) is distinguished from the clinical manifestations that develop years later, such as destruction of PARASYMPATHETIC GANGLIA; CHAGAS CARDIOMYOPATHY; and dysfunction of the ESOPHAGUS or COLON. Trypanosomiasis, South American,American Trypanosomiasis,Chagas' Disease,Trypanosoma cruzi Infection,Infection, Trypanosoma cruzi,Infections, Trypanosoma cruzi,South American Trypanosomiasis,Trypanosoma cruzi Infections,Trypanosomiasis, American
D016054 DNA, Protozoan Deoxyribonucleic acid that makes up the genetic material of protozoa. Protozoan DNA
D060888 Real-Time Polymerase Chain Reaction Methods used for detecting the amplified DNA products from the polymerase chain reaction as they accumulate instead of at the end of the reaction. Kinetic Polymerase Chain Reaction,Quantitative Real-Time PCR,Quantitative Real-Time Polymerase Chain Reaction,Real-Time PCR,PCR, Quantitative Real-Time,PCR, Real-Time,PCRs, Quantitative Real-Time,PCRs, Real-Time,Quantitative Real Time PCR,Quantitative Real Time Polymerase Chain Reaction,Quantitative Real-Time PCRs,Real Time PCR,Real Time Polymerase Chain Reaction,Real-Time PCR, Quantitative,Real-Time PCRs,Real-Time PCRs, Quantitative
D021141 Nucleic Acid Amplification Techniques Laboratory techniques that involve the in-vitro synthesis of many copies of DNA or RNA from one original template. DNA Amplification Technic,DNA Amplification Technique,DNA Amplification Techniques,Nucleic Acid Amplification Technic,Nucleic Acid Amplification Technique,RNA Amplification Technic,RNA Amplification Technique,RNA Amplification Techniques,Amplification Technics, Nucleic Acid,Amplification Techniques, Nucleic Acid,DNA Amplification Technics,Nucleic Acid Amplification Technics,Nucleic Acid Amplification Test,Nucleic Acid Amplification Tests,RNA Amplification Technics,Technics, Nucleic Acid Amplification,Techniques, Nucleic Acid Amplification,Amplification Technic, DNA,Amplification Technic, RNA,Amplification Technics, DNA,Amplification Technics, RNA,Amplification Technique, DNA,Amplification Technique, RNA,Amplification Techniques, DNA,Amplification Techniques, RNA,Technic, DNA Amplification,Technic, RNA Amplification,Technics, DNA Amplification,Technics, RNA Amplification,Technique, DNA Amplification,Technique, RNA Amplification,Techniques, DNA Amplification,Techniques, RNA Amplification

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