Competitive PCR

Competitive PCR (cPCR) has emerged as the best strategy for controlling the sam-ple-to-sample variability of PCR. In cPCR different templates of similar lengths and with the same primer binding sequences are coamplified in the same tube. This ensures identical thermodynamics and amplification efficiency for both templates. The amount of one of the templates must be known and, after amplification, products of both templates must be distinguishable and separately quantifiable. 

The quantitation of HCV RNA in serum may be important in predicting and monitoring response to antiviral therapy (Davis, 1994). A variety of methods have been used to quantitate HCV RNA, including endpoint dilution RT-PCR, competitive PCR, multicyclic RT-PCR, nucleic acid sequence-based amplification, RT-PCR with a single internal quantitation standard, and bDNA (Chazouilleres et al, 1994 Detmer et al., 1996 Ishiyama et al, 1992 Kaneko et al., 1992 Klevits et al., 1991 Kobayashi etal., 1993 Miskovsky etal., 1996). 

Miskovsky, E. P et al. (1996). Clinical characterization of a competitive PCR assay for quantitative testing of hepatitis C virus. J. Clin. Microbiol. 34,1975-1979. 

Edwards S G, Pirgozliev S R, Hare M C and Jenkinson P (2001), Quantification of trichothecene-producing Fusarium species in harvested grain by competitive PCR to determine efficacies of fungicides against Fusarium head blight of winter wheat , Appl. Environ. Microbiol., 67, 1575-1580. 

Gilliand G, Perrin S, Bunn HF. 1990a. Competitive PCR quantitation of mRNA in PCR Protocols. Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. San Diego Academic Press pp. 60-69. 

The plaque assay is desirable because it is very sensitive and only detects infectious viral particles. However, there are viral agents which cannot be supported by cell lines. In these cases other methods must be used. The polymerase chain reaction (PCR), which amplifies DNA or RNA from viral agents, can be used to detect the presence and quantity of viral agents. The amount of RNA or DNA target in the initial sample can be determined by competitive PCR where the quantity of amplified product is compared to a control PCR product where the initial amount of target is known. Quantification is also possible by an end-point dilution method similar to that used to determine a tissue culture infections dose. PCR methods can be very sensitive however,... 

Waterkeyn, J.G., Cowman, A.F. and Lightowlers, M.W. (1997b) Taenia ovis copy number of genes encoding host-protective antigens determined by competitive PCR. Experimental Parasitology 86, 84-88. 

Vona, G., Caldini, A., Sestini, R., Rapi, S., Bianchi, S., Fanelli, A., Pazzagli, M., and Orlando, C. 1999. The c-erbB2 oncogene amplification by competitive PCR in aneuploid cell clones stored from breast cancer samples. Clin. Chem. Lab. Med. 37 649-654. 

Figure 10 Procedure for multiplexed quantitative gene expression analysis using competitive PCR coupled with MALDI-TOF MS (MassARRAY system). Primers for three transcripts (CXCR4, HMBS, and GAPDH) to be measured simultaneously are depicted. Figure 11 illustrates the results for such an experiment.

Ding C, Cantor CR. A high-throughput gene expression analysis technique using competitive PCR and matrix-assisted laser desorption ionization time-of-flight MS. Proc Natl Acad Sci USA 2003 100 3059-3064. 

Quantitation of a PCR product is possible through the generation of a standard curve showing linearity between amount of DNA template present prior to amplification and the corrected peak area of resulting PCR product. To develop this standard curve, a serial dilution of template is performed. Alternatively, a ratio of the unknown template s peak area relative to that of an internal standard can be determined (e.g., for competitive PCR). A linear polyacrylamide sieving buffer was used to determine viral burden. The buffer, which contained the intercalating dye EnhanCE, permitted visualization by LIF and quantitation of the products of reverse transcriptase (RT) PCR... 

Figure 7.14 (A) CE-LIF electropherogram generated during competitive PCR of cytochrome P450-1Al 1, competitor 2, target IS, internal standard CF, 5-carboxyflu-orescein. (B) Competitive PCR of GAPDH 3, competitor 4, target. [Reproduced with permission from Fasco et al., Anal Biochem 224 140 (1995).]...

Piatak M Jr, Saag MS, Yang LC, Clark SJ, Kappes JC, Luk KC, Hahn BH, Shaw GM, Lifson JD (1993a) High levels of HIV-1 in plasma during all stages of infection determined by competitive PCR. Science 259 1749-1754. 

Phillips, C. J., Pard, E. A., and Prosser, J. I. (2000). Quantitative analysis of ammonia oxidising bacteria using competitive PCR. FEMS Microbiology Ecology 32, 167—175. 

A commonly used approach, known as the competitive PCR method, exploits the competition and plateau features of PCR to quantitate the expression of the mRNA of interest (15,16). The reaction incorporates an RNA or DNA PCR template which is included with the sample and amplified along with the RNA of interest. The most frequently used alternate methods include the use of competitive RNA or DNA PCR templates that are different in size, contain a recognizable mutation detectable because of gel electrophoresis separation properties, or contain a restriction site to allow digestion following PCR. Typically, in these approaches, serial dilutions of a known concentration of the competitor RNA or DNA are added to tubes containing equal, but unknown quantities of the mRNA of interest. Subsequently, RT is performed, followed by PCR amplification. The differing PCR products are separated by gel electrophoresis, and the concentration in the unknown sample is obtained at the dilution where the product of the unknown sample is equal to the product of the competitor. 

McCulloch RK, Choong CS, Hurley DM (1995). An evaluation of competitor type and size for use in the determination of mRNA by competitive PCR. PCR Methods AppL, 4(4) 219-226. 

Wheat Barley Rye Buckwheat Quantitative Competitive PCR PCR, RT-PCR TrnL (chloroplast rRNA) ITSl 5.8S rRNA 201 (rye and wheat) 196 (harley) 146... 

Dahinden I, von Buren M, Liithy J (2001). A quantitative competitive PCR system to detect contamination of wheat, barley or rye in gluten-free food for coeUac patients. Fur. Food Res. TechnoL, 212 228-233. 

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