Real-time PCR

From Poultry Hub

The real-time polymerase chain reaction (Real-time PCR) is a powerful analytical tool used in poultry research and molecular biology and is based on the principles of a standard PCR assay. The difference to a standard PCR is that real-time PCR continuously measures fluorescent signals from the PCR throughout the cycles. Thus, it offers the ability to monitor the accumulating PCR product (amplicon) in real time which has been made possible by the labelling of primers, probes or amplicons with fluorogenic molecules. The real-time PCR technique has emerged as an important technique for detection and analysis of nucleic acids due to its improved rapidity, sensitivity, reproducibility and lower risk of cross-contamination compared to standard PCR.

Contents 1 Real-time PCR and poultry research 2 Detection chemistries 3 Threshold cycle 4 Further information 5 See also 6 External links

Real-time PCR and poultry research

The real-time PCR technique is being used to detect and quantify viral or bacterial load in many various tissues, e.g. blood, immune or intestinal organs, feathers or feather dander and even faeces. Figure 1 shows an example of the application of real-time PCR assays to determine the daily shedding of Marek's disease viruses in single and mixed infections.

Real-time PCR protocols for many avian diseases have been developed which include:

• Avian influenza
• Chicken anaemia virus
• Chlamydiosis
• Coccidiosis
• Infectious bronchitis
• Marek's disease virus or MDV

Apart from numerous applications for poultry research purposes, real-time analysis is a powerful and increasingly inexpensive tool for routine monitoring of poultry diseases under field conditions. Data from real-time PCR assays thus provide valuable information to the farmer such as whether vaccinations have been successful and enable an early diagnosis of diseases.

Detection chemistries

There are currently five main chemistries used for the detection of the PCR product during real-time PCR and they can be classified into amplicon sequence specific or non-specific methods of real-time PCR detection. The simplest method uses fluorescent dyes, e.g.SYBR® Green I, that bind specifically to double-stranded DNA. Molecular Beacons, adjacent linear oligoprobes, Scorpions and the 5’-nuclease TaqMan®assay, rely on the hybridisation of fluorescent-labelled oligonucleotides to the correct amplicon.

Threshold cycle

The threshold cycle (Ct) is defined as the PCR cycle in which the gain in fluorescence generated by the accumulating amplicon exceeds a chosen threshold above background fluorescence. The threshold cycle is when the system begins to detect the increase in the signal associated with an exponential growth of PCR product during a log-linear phase. This phase provides the most useful information about the reaction. At the beginning of the exponential phase, all reagents are still in excess. This means the low amount of product will not compete with the primers’ annealing capabilities and the DNA polymerase is still highly efficient, making the data more accurate. Figure 2 shows a typical amplification plot of a tenfold serial dilution of DNA run in duplicate.

Further information

• Chousalkar, K.K., Cheetham, B.F. and Roberts, J.R. (2008): LNA probe-based real-time RT-PCR for the detection of infectious bronchitis virus from the oviduct of unvaccinated and vaccinated laying hens . J Virol Methods. 155, 67-71.
• Islam, A. and Walkden-Brown, S.W. (2007): Quantitative profiling of the shedding rate of the three Marek’s disease virus (MDV) serotypes reveals that challenge with virulent MDV markedly increases shedding of vaccinal virus. J. Gen. Virol. 88, 2121-2128.
• Mackay, I.M., Arden, K.E. and Nitsche, A. (2002): Real-time PCR in virology. Nucleic Acid Research 30, 1292-1305.
• Mullis, K., Fallona, F., Scharf, R., Saiki, R., Horn, G. and Ehrlich, H. (1986): Specific enzymatic amplification of DNA in vitro: The Polymerase Chain Reaction. Cold Spring Harb. Symp. Quant. Biol. 51, 263-273.
• Mullis, K.B. and Fallona, F.A. (1986): Specific synthesis of DNA in vitro via a polymerase- catalysed chain reaction. Methods Enzymol. 155, 335-350.
• Reece, R.J. (2004): Analysis of genes and genomes. John Wiley and Sons Ltd., England.
• Whitcombe, D., Theaker, J., Guy, S., Brown, T. and Little, S. (1999): Detection of PCR products using self-probing amplicons and fluorescence. Nat. Biotechnol. 17, 804-807.

See also

• Anatomy of the Chicken Learning Resource
• Microarrays

External links

The publications of real-time PCR articles below are just examples and are NOT exhaustive: http://www.poultryhub.org/index.php?title=Real-time_PCR&action=edit&section=6

• Avian influenza real-time PCR assay Journal of Medical Microbiology.
• Chicken anaemia virus real-time PCR assay British Society for Immunology.
• Chlamydia real-time PCR assayin Journal of Medical Microbiology.
• Coccidiosis real-time PCR assay in Avian Pathology.
• Infectious bronchitis virus in BioOne.
• Marek's disease real-time PCR assayin: Journal of Virological Methods