Polymerase chain reaction quantification

Vanlandingham, D.L., Schneider, B.S., Klingler, K., Fair, J., Beasley, D., Huang, J., Hamilton, P. and Higgs, S. (2004) Real-time reverse transcriptase-polymerase chain reaction quantification of West Nile virus transmitted by Culex pipiens quinquefasciatus. Am. J. Prop. Med. Hyg. 71, 120-123. 

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 (PGR), 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 PGR where the quantity of amplified product is compared to a control PGR 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. PGR methods can be very sensitive however. 

Becker-Andre M, Hahlbrock K. 1989. Absolute quantification using the polymerase chain reaction (PCR). Nucleic Acid Res 17 9347-9446. 

HCV and HIV-1). The bDNA assay is being much employed for the quantification of messenger RNA. Moreover, for the detection of viral and pathogenic disorders based on alkahne-phosphatase-sensitive dioxetanes, several assay methods are available these include the Polymerase-Chain-Reaction (PCR) amphfication, probe ligation, strand-displacement amplification and the ligase chain reaction. ... 

Fig. 8.2.2 Principle of genomic quantification by multiplex ligation-dependent probe amplification (MLPA). Synthetic oligonuleotides are designed to bind to exon 1 (oligo 1a and 1b) and 2 (2a and 2b). After specific hybridisation, the oligos are ligated. The ligation products undergo quantitative polymerase chain reaction using universal primers X and Y...

Eig. 5. Several endpoint detection methods were compared for the detection of immuno-polymerase chain reaction (IPCR) amplificate from a direct IPCR (Fig. 3A) of mouse-IgG. Although all IPCR/DNA-detection combinations were able to improve the detection limit of a comparable enzyme-linked immunosorbent assays (ELISA) of approximately 10 amol IgG in a 30-fL sample volume, several differences were observed in actual detection limit, and the linearity of the concentration/signal ratio dependent on the DNA quantification was applied. Best results were obtained for PCR-ELISA (see also Fig. 6) in combination with fluorescence- or chemiluminescence-generating substrates (b, c). With photometric substrates (d) or gel electrophoresis and subsequent spot densitometry (a), a 10-fold decrease in sensitivity was observed. In addition to the more sigmoid curve in gel electrophoresis, an enhanced overall error of 20% compared to 13% in PCR-ELISA was observed for two independent assays. The simple addition of a double-strand sensitive intercalation marker to the PCR-amplificate and measurement in a fluorescence spectrometer further decreased sensitivity (e) and appears therefore to be unsuited for IPCR amplificate quantification. (Figure modified according to references 37 and 65.)... 

Niemeyer CM, Adler M, Blohm D. Fluorometric polymerase chain reaction (PCR) enzyme-linked immunosorbent assay for quantification of immuno-PCR products in microplates. Anal Biochem 1997 246(1) 140-145. 

Endrizzi K, Fischer J, Klein K, Schwab M, Nussler A, Neuhaus P, Eichelbaum M, Zanger UM. Discriminative quantification of cytochrom P4502D6 and 2D7/8 psuedogene expression by TaqMan real-time reverse transcriptase polymerase chain reaction. Anal Biochem 2002 15 121-131. 

Wang T, Brown MJ (1999) mRNA quantification by real time TaqMan polymerase chain reaction validation and comparison with RNase protection. Anal Biochem 269 198-201... 

Since alterations in global DNA methylation are implicated in various pathobio-logical processes, a gradient IPC-ESI-MS/MS method with a volatile IPR was used to determine cytosine and 5-methylcytosine in DNA quantification relied on stable isotope dilution [58], Muscular dystrophies caused by various mutations in the dystrophin gene are amenable to easier prenatal diagnosis via a multiplex polymerase chain reaction (PCR)/IPC assay [59]. Some guidelines for the analysis of genomic DNA by IPC-ESl-MS can be found in Reference 60. 

Kreiner G, Sanak M, Zelek-Molik A, Nalepa I. Using reverse transcription and a competitive polymerase chain reaction for quantification of a1B-adrenoceptor mRNA. Pol J Pharmacol 2002 54 401 105. 

Hochhaus A, Lin F, Reiter A, Skladny H, Mason PJ, van Rhee F, et al. Quantification of residual disease in chronic myelogenous leukemia patients on interferon-alpha therapy by competitive polymerase chain reaction. Blood 1996 87 1549-55. 

An introduction to single gene transcript quantification methods. A number of methods are now available for the detection of gene activity via messenger RNA (mRNA). In all protocols, the first step is to isolate total RNA. This can be probed directly or used for reverse transcriptase polymerase chain reaction (RT-PCR)... 

Polymerase chain reaction (PCR)-based assays amplify a specific target DNA which can be used in a variety of analytical applications. Quantitative PCR has been used for lot release testing, the detection of viral contaminants (both in raw material testing as well as in-process control), the quantification of endogenous retroviruses in cell cultures, host cell DNA quantification, and the determination of genetic stability in cell lines [13-15]. 

Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 2000 25 169-193. 

Weber, M., Hagege, H., Lutfalla, G., et al. (2003) A real-time polymerase chain reaction assay for quantification of allele ratios and correction of amplification bias. Anal. Biochem. 320, 252-258. 

Lopez 1, Pardo MA (2005). Application of relative quantification TaqMan real-time polymerase chain reaction technology for the identification and quantification of Thunnus alalunga and Thunnus albacares. J. Agric. Food Chem., 53 4554-4560. 

A DNA molecule can be amplified by the polymerase chain reaction (PCR) (section 6.2), if part of its sequence is known. One DNA molecule is sufficient to generate millions of identical copies in a controlled amplification reaction. With real-time PCR, the DNA quantity can be measured during the amplification reaction (section 6.2.4). Other methods of DNA quantification include DNA arrays (section 5.3) and, if available, biosensors (section 5.2). 

M7. Miyake, Y., Fujiwara, Y., Ohue, M., Yamamoto, H., Sugita, Y., Tomita, N., et al.. Quantification of micrometastases in lymph nodes of colorectal cancer using real-time fluorescence polymerase chain reaction. Int. J. Oncol 16, 289-293 (2000). 

Related mRNAs encoding various proteins can be detected by different types of in situ hybridization. For this method, the number of specific mRNAs detectable per tumor sample is limited. However, the advantage of in situ hybridization is the same as in immunohistochemistry where the morphology of the tumor is still visible. Specific mRNA-species can be detected by northern blot, nuclease protection assay or reverse transcription (RT) combined with polymerase chain reaction (PCR). Using the modern real-time PCR protocols, reliable quantification of PCR targets is possible. A more complex approach is possible by using the micro-array technology, where hundreds or even more of mRNAs can be detected simultaneously in a semi-quantitative fashion. 

Ornstein, K. and Barbour, A.G. (2006) A reverse transcriptase-polymerase chain reaction assay of Borrelia burgdorferi 16S rRNA for highly sensitive quantification of pathogen load in a vector. Vector Borne Zoonotic Dis. 6, 103-112. 

Leung KT, Watt A, Lee H, Trevors JT (1997) Quantification detection of penta-chlorophenol-degrading Sphingomonas sp. UG30 in soil by a most-probable-number polymerase chain reaction protocol. J Microbiol Method 31 59-66... 

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