Molecular detection assay using direct treatment with a bisulphite reagent
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Publication Number
US-9732375-B2
Publication Date
2017-08-15
Expiration Date
Abstract
A molecular detection assay including treating a biological sample directly with a bisulphite agent under conditions that allow cell disruption and nucleic acid treatment; removing the bisulphite agent from the treated sample; and detecting a target nucleic acid in the treated sample.
Core Innovation
The invention relates to a molecular detection assay that treats a biological sample directly with a treating agent consisting of sodium bisulphite or sodium metabisulphite at a concentration of about 2.5M to 3.5M and a pH of about 4.5 to 5.5, followed by heating at a temperature of about 75°C to 95°C for about 1 to 30 minutes to allow cell disruption and nucleic acid treatment. This combined treatment enables subsequent nucleic-acid detection in the treated sample.
After the direct sodium bisulphite or sodium metabisulphite treatment and heating, the assay removes the sodium bisulphite or sodium metabisulphite from the treated sample and then detects a target nucleic acid in the treated sample. The assay is carried out without pre-treatment of the biological sample prior to treating with the sodium bisulphite or sodium metabisulphite.
The invention is directed to molecular detection in a workflow that eliminates conventional pre-treatment/cleanup prior to bisulphite-based nucleic-acid processing, while still enabling downstream nucleic-acid detection modalities such as amplification or sequencing. The document further describes nucleic-acid targets including DNA and RNA, including infectious and genetic/epigenetic indicators.
Claims Coverage
The independent claim is clm-00001. The inventive features focus on direct bisulphite/metabisulphite treatment conditions, heating for cell disruption and nucleic-acid treatment, bisulphite removal, nucleic-acid detection, and carrying out the assay without pre-treatment prior to the treating step. Dependent claim sets further refine treatment/removal parameters and detection formats including primer/probe use, amplification methods, sequencing, and an elution-buffer pH constraint.
Direct bisulphite/metabisulphite treatment with defined conditions
Treating a biological sample directly with a treating agent consisting of sodium bisulphite or sodium metabisulphite at a concentration of about 2.5M to 3.5M and a pH of about 4.5 to 5.5.
Heating to disrupt cells and treat nucleic acids
Heating at a temperature of about 75°C to 95°C for about 1 to 30 minutes to allow cell disruption and nucleic acid treatment.
Bisulphite removal followed by target nucleic-acid detection
Removing the sodium bisulphite or sodium metabisulphite from the treated sample; and detecting a target nucleic acid in the treated sample.
No pre-treatment prior to the bisulphite treatment step
Carrying out the assay without pre-treatment of the biological sample prior to treating with the sodium bisulphite or sodium metabisulphite.
Overall, the claim coverage centers on a bisulphite-based molecular detection workflow that directly treats the biological sample under specified sodium bisulphite/sodium metabisulphite concentration and pH, applies defined heating to disrupt cells and treat nucleic acids, removes the treating agent, and detects a target nucleic acid, while explicitly omitting pre-treatment prior to the treating step.
Stated Advantages
Eliminates conventional pre-treatment/cleanup prior to bisulphite-based nucleic-acid processing.
Provides improved sensitivity and rapid turnaround as reported in the document.
Documented Applications
Molecular detection in feces for C. difficile detection (tcdA/B, tcdB) as compared with in-house and Qiagen.
Detection of parasites as compared with microscopy and EasyPlex.
Bacterial detection comparisons involving bacterial culture.
HCV RNA detection from serum at very low input as described in the document.
Simultaneous RNA and DNA virus lysis/conversion as described in the document.
Human cell processing in fecal material enabling β-globin amplification and suitability for colorectal cancer as described in the document.
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