| 摘要 |
Current invention is directed for rapid sample pretreatment method that allows highly sensitive and specific detection of target nucleic acid (e.g. human genomic DNA, human pathogen genomic DNA, human non-pathogen genomic DNA) by amplification directly from crude unpurified biological samples lysates (e.g. human 5 urine, saliva, blood, urethra and cervical swabs and other samples containing biological material). Invention is focused on the description of the biological sample pretreatment method that enables fast release of the genomic material from human and pathogen cells, components of what are compatible with the following nucleic acid amplification method. As an example of the application, 10 invention also discloses protocols and primer sequences for isothermal nucleic acid amplification (recombinase polymerase amplification – RPA, loop-mediated isothermal amplification - LAMP), that enable highly specific and sensitive diagnostics of the genomic material from Homo sapiens, Chlamydia trachomatis and Mycoplasma genitalium from crude biological sample lysates and/or purified 15 total DNA. The example amplification can be combined with immunochromotographic product detection using lateral-flow strips and allows rapid (under 20 min) isothermal nucleic acid amplification based C. trachomatis and M. genitalium diagnostics from human urine samples, that does not require specific laboratory equipment nor qualified personnel, and is therefore well suited 20 for point-of-care settings applications. |
