Molecular diagnostics amplification

Stepan J, Pantucek R, Ruzickova V, Rosypal S, Hajek V, Doskaf J (2001) Identification of Staphylococcus aureus based on PCR amplification of species specific genomic 826 bp sequence derived from a common 44-kb Snwl restriction fragment. Mol Cell Probes 15 249-257 Stepan J, Pantucek R, Doskaf J (2004) Molecular diagnostics of clinically important staphylococci. Folia Microbiol 49 353-386... 

Nucleic acid probe methods which rely on the specificity of nucleic acid hybridization reaction, combined with the powerful methods of nucleic acid amplification, are expected to spawn many new diagnostic tests which may be useful in the detection and treatment of infectious diseases, cancer, and inherited disorders. The impact of development and applications of diagnostic tests based on nucleic acid probes and on Mab technology wiU be felt increasingly as deeper understanding of diseases at a molecular level is gained. 

Enzyme immunoassay is widely used, both in competitive and non-competitive formats, for the bioanalysis of a broad range of low-molecular-weight compounds and macromolecules. Through the use of fluorogenic substrates and amplification systems such as avidin-biotin, the sensitivity of enzyme immunoassay has been developed to equal or exceed that of radioimmunoas-say.f ° The technique has found particularly wide applicability in the determination of new recombinant proteins, in demonstrating antibody responses to macromolecules, and in the measurement of biomarkers of disease, as well as in diagnostic medicine. 

Figure 37-28 Finding the cycle number at which fluorescence rises above background. Real-time fluorescence data (F) from the amplification reaction are shown with the first (F ) and second (F ") derivatives.The maximum of the second derivative corresponds to a defined point on the real-time data where the curve starts to rise. (Modified with permission of the pubiisber from Wittwer CT, Kusukawa N. Real-time PCR. In Persing DH, Tenover FC,Versalovic J,TangYW, Unger ER, Reiman DA, White TJ (eds.), Molecular Microbiology Diagnostic Principles and Practice, Washington, DC ASM Press, 2004 71-84.)...

We have exemplified BART applications in molecular IVDs and demonstrated its compatibility with various amplification methods, its ability to detect and quantify different targets, to cope with crude sample preparations and to provide rapid results under challenging conditions. Overall, BART is a universal reporter system for any molecular in vitro diagnostic tests based on isothermal nucleic acid amplification techniques. 

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