ABSTRACT
Recent efforts in biological engineering have made detection of nucleic acids in samples more rapid, inexpensive and sensitive using CRISPR-based approaches. We expand one of these Cas13a-based methods to detect small molecules in a one-batch assay. Using SHERLOCK-based profiling of in vitrotranscription (SPRINT), in vitro transcribed RNA sequence-specifically triggers the RNase activity of Cas13a. This event activates its non-specific RNase activity, which enables cleavage of an RNA oligonucleotide labeled with a quencher/fluorophore pair and thereby de-quenches the fluorophore. This fluorogenic output can be measured to assess transcriptional output. The use of riboswitches or proteins to regulate transcription via specific effector molecules is leveraged as a coupled assay that transforms effector concentration into fluorescence intensity. In this way, we quantified eight different compounds, including cofactors, nucleotides, metabolites of amino acids, tetracycline and monatomic ions in samples. In this manner, hundreds of reactions can be easily quantified in a few hours. This increased throughput also enables detailed characterization of transcriptional regulators, synthetic compounds that inhibit transcription, or other coupled enzymatic reactions. These SPRINT reactions are easily adaptable to portable formats and could therefore be used for the detection of analytes in the field or at point-of-care situations.
Subject(s)
Bacterial Proteins/metabolism , Biosensing Techniques/methods , CRISPR-Associated Proteins/metabolism , Endodeoxyribonucleases/metabolism , Enzyme Assays/methods , Nucleic Acid Synthesis Inhibitors/analysis , Bacterial Proteins/genetics , CRISPR-Associated Proteins/genetics , CRISPR-Cas Systems , Endodeoxyribonucleases/genetics , Fluorescent Dyes/chemistry , Leptotrichia , Ligands , Nucleic Acid Synthesis Inhibitors/pharmacology , Riboswitch , Rifampin/analysis , Transcription Factors/metabolism , Transcription, Genetic/drug effectsABSTRACT
Frozen berries have been repeatedly identified as vehicles for norovirus (NoV) transmission causing large gastroenteritis outbreaks. However, virus detection in berries is often hampered by the presence of RT-PCR-inhibiting substances. Here, several virus extraction methods for subsequent real-time RT-PCR-based NoV-RNA detection in strawberries were compared and optimized. NoV recovery rates (RRs) between 0.21 ± 0.13% and 10.29 ± 6.03% were found when five different artificially contaminated strawberry batches were analyzed by the ISO/TS15216-2 method indicating the presence of different amounts of RT-PCR inhibitors. A comparison of five different virus extraction methods using artificially contaminated strawberries containing high amounts of RT-PCR inhibitors revealed the best NoV RRs for the ISO/TS15216 method. Further improvement of NoV RRs from 2.83 ± 2.92% to 15.28 ± 9.73% was achieved by the additional use of Sephacryl(®)-based columns for RNA purification. Testing of 22 frozen strawberry samples from a batch involved in a gastroenteritis outbreak resulted in 5 vs. 13 NoV GI-positive and in 9 vs. 20 NoV GII-positive samples using the original ISO/TS15216 method vs. the extended protocol, respectively. It can be concluded that the inclusion of an additional RNA purification step can increase NoV detection by the ISO/TS15216-2 method in frozen berries containing high amounts of RT-PCR inhibitors.
Subject(s)
Fragaria/chemistry , Fragaria/virology , Frozen Foods/virology , Norovirus/isolation & purification , RNA, Viral/isolation & purification , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Food Microbiology/methods , Limit of Detection , Norovirus/genetics , Nucleic Acid Synthesis Inhibitors/analysis , RNA, Viral/geneticsABSTRACT
This chapter presents methods and protocols suitable for the identification and characterization of inhibitors of the prokaryotic and/or eukaryotic translational apparatus as a whole or targeting specific, underexploited targets of the bacterial protein synthetic machinery such as translation initiation and aminoacylation. Some of the methods described have been used successfully for the high-throughput screening of libraries of natural or synthetic compounds and make use of model "universal" mRNAs that can be translated with similar efficiency by cellfree extracts of bacterial, yeast, and HeLa cells. Other methods presented here are suitable for secondary screening tests aimed at identifying a specific target of an antibiotic within the translational pathway of prokaryotic cells.
Subject(s)
Drug Evaluation, Preclinical/methods , Nucleic Acid Synthesis Inhibitors/isolation & purification , Protein Biosynthesis/drug effects , Animals , Bacteria/drug effects , Bacteria/genetics , Bacteria/metabolism , Cell-Free System/metabolism , Cells, Cultured , Clinical Laboratory Techniques , Humans , Luciferases, Renilla/genetics , Luciferases, Renilla/metabolism , Nucleic Acid Synthesis Inhibitors/analysis , Prokaryotic Initiation Factor-2/antagonists & inhibitors , Prokaryotic Initiation Factor-2/physiology , RNA Cap-Binding Proteins/physiology , RNA, Messenger/isolation & purification , RNA, Messenger/metabolism , Transfer RNA Aminoacylation/drug effects , Yeasts/genetics , Yeasts/metabolismABSTRACT
A capillary zone electrophoretic method with indirect UV-detection for determination of rimantadine, an antiviral drug against influenza A, in tablets was validated. Instrumental precision, the method precision, accuracy, calibration curve linearity, selectivity, robustness, and time stability of the sample and the standard were tested. The method was also applied to monitor dissolution tests of the tablets. The possibility of addition of an internal standard for improvement of the method precision was discussed.
Subject(s)
Electrophoresis, Capillary/methods , Nucleic Acid Synthesis Inhibitors/analysis , Rimantadine/analysis , Nucleic Acid Synthesis Inhibitors/chemistry , Rimantadine/chemistry , Solubility , TabletsABSTRACT
Deoxycytidine kinase (dCyd kinase) is important for the phosphorylation of several different nucleoside antimetabolites. To understand the significance of dCyd kinase levels in chemotherapy, dCyd kinase mRNA levels were evaluated in several cells with a quantitative competitive polymerase chain reaction (PCR) assay. dCyd kinase catalytic activity and intracellular ara-CTP production were also compared with the levels of dCyd kinase mRNA. The assay was able to show: (i) that dCyd kinase catalytic activity and dCyd kinase mRNA levels were correlated in cells; (ii) that dCyd kinase mRNA levels were more sensitive in lower levels of 10 amol/micrograms of total RNA; and (iii) in cytosine arabinoside (ara-C)-resistant cells, both dCyd kinase mRNA levels and intracellular ara-CTP levels were lower compared with levels in sensitive cells. The PCR assay for dCyd kinase mRNA could be useful in the selection and monitoring of patients treated with nucleosides that are activated by this enzyme.
