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Molecular fingerprinting methods to detect and genotype dna targets through polymerase chain reaction

A chain reaction and genotyping technology, applied in the field of multiplex polymerase chain reaction (PCR) analysis, can solve the problems of increasing the test cost and affecting the stability of the test, and achieves the effect of being cheap in implementation.

Inactive Publication Date: 2020-03-31
ULISSE BIOMED
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  • Claims
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Problems solved by technology

However, fluorescently labeled probes can greatly increase assay cost, affect assay stability, and require more than one fluorescent channel; this means that assays can only be performed using dedicated instruments with specific specificities

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  • Molecular fingerprinting methods to detect and genotype dna targets through polymerase chain reaction
  • Molecular fingerprinting methods to detect and genotype dna targets through polymerase chain reaction
  • Molecular fingerprinting methods to detect and genotype dna targets through polymerase chain reaction

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specific Embodiment approach

[0143] 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:

[0144] a) dNTPs (final concentration range: 0.05 mM to 0.3 mM)

[0145] b) MgCl 2 (Final concentration range: 0.3 mM to 4 mM)

[0146] c) TrisHCl buffer solution (final concentration range: 10 mM to 50 mM; pH: 6.00 to 10.00)

[0147] d) KCl (final concentration range: 10 mM to 50 mM)

[0148] e) BSA (final concentration range: 0.005 to 0.05 mg / ml)

[0149] f) Hot start polymerase

[0150] g) SYTO-9 (final concentration range: 1 μM to 8 μM).

[0151] 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 molecular fingerprinting method is disclosed to detect and genotype pathogens DNA targets in a sample through polymerase chain reaction (PCR). The method comprises performing PCR amplification and then a High Resolution Melting (HRM) analysis. The PCR reaction mixture used comprises two or more pairs of amplification primers designed in order to generate amplicons with a different melting temperature each other in order to discriminate, in the HRM analysis, each amplicon by observing the specific melting temperature of each amplicon. 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 DNAs, due to the release of the intercalating molecule or compound. Discrimination and genotyping of different strains of the same pathogen, different pathogens belonging to separated genus and genetic variations in the sample can be determined through a reader analysing the signal variation; the result of the analysis is obtained through a graphic interface connected to the reader.

Description

technical field [0001] Embodiments of the present disclosure relate to methods for multiplex polymerase chain reaction (PCR) analysis capable of distinguishing and genotyping different strains of the same pathogen, different pathogens belonging to different genera, and genetic variations in a single reaction. Primers, oligonucleotides, chemical components and methods. In particular, embodiments of the present disclosure relate to the detection and characterization of pathogens associated with sexually transmitted diseases (such as, but not limited to, high-risk human papillomavirus ( Primers, oligonucleotides, methods and conditions for different strains of HPV). Background technique [0002] 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6848C12Q1/6858C12Q1/70
CPCC12Q1/6848C12Q1/6858C12Q1/708C12Q2527/107C12Q2537/143C12Q2563/173C12Q2525/119C12Q2525/301G01N2800/50C12Q2565/10C12Q1/6853C12Q2537/163
Inventor 鲁迪·伊波迪里诺布鲁纳·马里尼爱丽丝·阿维安马尔科·莫塞尼戈尼古拉·福斯基伊丽莎白·莫罗米歇尔·蒙特龙
Owner ULISSE BIOMED
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