3.2. Quantitative (real-time) PCR

3.2. Quantitative (real-time) PCR STUART E. DENMAN and CHRISTOPHER S. MCSWEENEY CSIRO Livestock Industries, 306 Carmody Rd, St Lucia, Queensland 4067, Australia

Introduction Many nucleic acid-based probe and PCR assays have been developed for the detection tracking of specific microbes within the rumen ecosystem [4–6, 8–11, 14, 15]. Conventional PCR assays detect PCR products at the end stage of each PCR reaction, where exponential amplification is no longer being achieved. This approach can result in different end product (amplicon) quantities being generated [3]. In contrast, using quantitative, or real-time PCR, quantification of the amplicon is performed not at the end of the reaction, but rather during exponential amplification, where theoretically each cycle will result in a doubling of product being created w [13]. For real-time PCR, the cycle at which fluorescence is deemed to be detectable above the background during the exponential phase is termed the cycle threshold (Ct). The Ct values obtained are then used for quantitation, which will be discussed later. Quantitative PCR allows the researcher to view the entire reaction and product being generated throughout all stages of the reaction. In its simplest and cheapest form, real-time PCR employs the DNA-binding dye, SYBR Green. SYBR Green binds to the minor groove of double-stranded DNA and fluoresces at a much higher intensity when bound to double-strand DNA when compared with the dye in free solution. As the amplification reaction proceeds and more double-stranded amplicons are produced, the SYBR Green dye fluorescence signal will increase and can be detected. As SYBR Green will bind indiscriminately to any double-stranded piece of DNA, it is important that the assay is completely optimized so that only the specific target of interest is amplified and that non-specific products or primer dimers are excluded. Dissociation curve analysis is performed at the completion of the amplification cycles to reveal the purity of the amplicon produced for each reaction. The dissociation curve is produced by monitoring the loss of fluorescence signal, as the temperature is slowly raised from 60◦ C to 95◦ C causing the double-stranded amplicon to dissociate and the SYBR Green to be released. A single specific amplicon product will dissociate at a given melting temperature, producing a single sharp dissociation curve. Any nonspecific products or primer dimers will be detected during this analysis as multiple or broader dissociation peaks. 105 H.P.S. Makkar and C.S. McSweeney (eds.), Methods in Gut Microbial Ecology for Ruminants, 105–115. © 2005 IAEA. Printed in the Netherlands.

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Other detection assay systems have evolved that show greater specificity than the SYBR Green assay by combining a fluorescence reporter dye with a quencher dye. The most common of these, the 5 Nuclease (Taqman) assay, utilizes target specific amplification primers and a probe that contains both the reporter and quencher chemistry. The probe is designed in such a way, that while intact, the quencher is in close proximity to the fluorescence reporter and quenches any signal. The probe will bind to its target site on the template DNA between the amplification primers. As the Taq polymerase extends along the template, the 5 -nuclease activity of the polymerase will degrade the probe, resulting in an increase in detectable fluorescence, as the quencher is separated from the reporter flurophore. This type of detection does not suffer from the presence of non-specific product amplification or primer dimers, as long as the probe is sequence specific. Regardless of which method is employed, several common requirements should be addressed so as to produce an accurate and robust assay. (1) The amplicon product size should be restricted to a size range of 50–200 bp, as amplification efficiencies of 2 (a doubling of product at each cycle) are more easily obtainable when the amplicon size is kept to a minimum (