Methods for DNA targets detection directly in crude samples through polymerase chain reaction and genotyping via high resolution melting analysis
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A high-resolution melting, raw sample technology, used in the detection of several pathogens that cause sexually transmitted diseases
Inactive Publication Date: 2020-03-31
ULISSE BIOMED
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However, fluorescently labeled probes are more expensive, affect assay robustness, and analysis requires more than one fluorescent detector
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[0178] In one embodiment, the processing buffer for diagnostic purposes comprises proteinase K and TrisHCl buffered solution. For example, a specific embodiment of the processing buffer comprises:
[0179] a) Proteinase K (between 1 mg / mL and 1 μg / mL);
[0180] b) TrisHCl buffer solution (between 1 M and 10 mM; pH between 7.0 and 10.0).
specific Embodiment approach
[0181] In one embodiment, possible first amplification buffers for diagnostic purposes comprise dNTPs, sources of monovalent or divalent cations, buffer solutions, BSA, hot-start DNA polymerases, intercalating molecules or compounds. For example, one embodiment of the first amplification buffer comprises:
[0182] a) dNTPs (final concentration range: 0.05 mM to 0.3 mM)
[0183] b) MgCl 2 (Final concentration range: 0.3 mM to 4 mM)
[0184] c) TrisHCl buffer solution (final concentration range: 10 mM to 50 mM; pH: 6.00 to 10.00)
[0185] d) KCl (final concentration range: 10 mM to 50 mM)
[0189] In another possible embodiment, a possible alternative second amplification buffer for diagnostic purposes is provided, comprising dNTPs, a source of monovalent or divalent cations, a buffer solution, BSA, a hot-start DNA ...
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Abstract
A method id disclosed for DNA targets detection directly in crude samples through Polymerase Chain Reaction (PCR) and genotyping via High Resolution Melting (HRM) analysis. The method comprises diluting the crude sample, treating the diluted crude sample by performing a ramp of increasing and then decreasing temperature, performing PCR amplification and then an HRM analysis. The method further comprises monitoring, during the HRM analysis, the change in the signal emission resulting from the temperature-induced denaturation of the double-stranded amplicons into two single- stranded DNA, due tothe release of an intercalating molecule or compound. The detection of DNA targets in the crude sample is performed through a reader analysing the signal variation, obtaining the result of the analysis through a graphic interface connected to the reader.
Description
technical field [0001] Embodiments of the present disclosure relate to methods for detecting target DNA directly in raw samples by PCR and genotyping by HRM analysis. In particular, embodiments of the present disclosure describe methods for detecting several pathogens that cause sexually transmitted diseases, such as but not limited to HR-HPV (High Risk Human Papillomavirus). [0002] In more detail, the present disclosure allows for the detection of pathogen DNA directly from raw biological samples (including, for example, vaginal or cervical mucus) in one multiplex assay, without the need for a DNA extraction step. Background technique [0003] Polymerase chain reaction (PCR) is a reliable technique used in diagnostic applications worldwide. PCR consists of a primer extension reaction that amplifies specific nucleic acids in vitro. The reaction utilizes a thermostable DNA polymerase and is based on several cycles involving different temperature steps that allow for DNA d...
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