PCR assay

HBV DNA was measured by hybridization or branched chain DNA assays (lower detection limit 20,000-200,000 lU/mL) and PCR assays (lower detection limit approximately 50 lU/mL). For references see text... 

The second experiment was carried out with the same RNA isolated from mycelia growing on presence of glucose and apple pectin. cDNAs were obtained from those RNA by reverse trancription and used as template for PCR assays. Specific oligonucleotide primers for PG gene were used in the amplification reaction. The Fig-5 shows the results of this amplification experiment. 

La Rosa, G., Fontana, S., Di Grazia, A., laconelli, M., Pourshaban, M., and Muscillo, M. (2007). Molecular identification and genetic analysis of Norovirus genogroups I and II in water environments Comparative analysis of different reverse transcription-PCR assays. Appl. Environ. Microbiol. 73,4152M161. 

Making sure that the sample size does not exceed the maximum area or weight is important to minimize the amounts of interfering substances that are coextracted. If the leaf sample is larger than 2 mm, coextractive substances can inhibit the PCR assay. Regardless of which extraction method is used, it is important that the PCR assay is evaluated for coextractive interferences or inhibitors. 

Primer design is one of the most important aspects of a robust PCR assay. In general, primers should be designed such that they are not able to form secondary structures such as stemloop or hairpin configurations. A primer must not be complementary at the 3 end, as this will cause primer dimers to form. All primers should have similar melting temperatures and should not contain stretches of individual nucleotides. There are software programs available to assist in primer design, but it is crucial that primers are tested in the assay, especially in a multiplex system. 

Because the templates compete for amplification and, in the case of reverse transcription PCR (RT-PCR), also for reverse transcription, any variable affecting amplification has the same effect on both. Thus, the ratio of PCR products reflects the ratio of the initial amounts of the two templates as demonstrated by the function C/W=C (l+ )"/Wi(l+ )n, where Cand Ware the amounts of competitor and wild-type product, respectively, and C and W are the initial amounts of competitor and wild-type template, respectively, (Clementi etal., 1993). From this linear relationship, it could be concluded that a single concentration of competitor could be sufficient for quantitating unknown amounts of wild-type templates. However, in practice, the precise analysis of two template species in very different amounts has proved difficult and cPCRs using three to four competitor concentrations within the expected range of wild-type template concentrations are usually performed. In a recent study of different standardization concepts in quantitative RT-PCR assays, coamplification on a single concentration of a competitor with wild-type template was comparable to using multiple competitor concentrations and was much easier to perform (Haberhausen et al, 1998). 

The studies described here demonstrate that bDNA can be used to quantitate mRNA at physiologic levels and that the technology can also be used effectively in cell biology as well as infectious diseases. The bDNA assays do not have the sensitivity of RT-PCR assays that have been described for the same purposes, but bDNA may be better suited for truly quantitative mRNA measurements, since it does not require reverse transcription or amplification of the target sequences. 

Haberhausen, G., etal. (1998). Comparative study of different standardization concepts in quantitative competitive reverse transcription-PCR assays. J. Clin. Microbiol. 36,628-633. 

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. 

Fukushima, H. Tsunomori, Y. Seki, R. Duplex real-time SYBR Green PCR assays for detection of 17 species of food- or waterborne pathogens in stools. J. Clin. Microbiol. 2003,41,5134-5146. 

Other regions of the genome (s), such as repetitive elements (see later), also evolve rapidly and can be exploited as polymorphic markers in random or selective PCR assays. Characterization of such markers is of significance... 

Medhurst AD et al. The use of TaqMan RT-PCR assays for semiquantitative analysis of gene expression in CN S tissues and disease models. J Neurosci Methods 2000 98 9-20. 

Raymond, C. K., Roberts, B. S., Garrett-Engele, P., Lim, L. P., and Johnson, J. M. (2005). Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs. RNA 11, 1737-1744. 

K Withby, J Garson, A Baret. A microtiter format quantitative PCR assay for HCV RNA employing xanthine oxidase generated chemiluminescence. Proceedings of... 

M. Panning, M. Asper, S. Kramme, H. Schmitz and C. Drosten, Rapid detection and differentiation of human pathogenic orthopox viruses by a fluorescence resonance energy transfer real-time PCR assay, Clin. Chem., 50 (2004) 702-708. 

HIV-1 RNA in plasma can also be quantitated by a branched-DNA (bDNA) signal amplification assay which has a quantitation limit of 1 x 104 HIV-1 eq/ml. A novel internally controlled PCR assay (ICPCR) has been used to quantitate HIV-1 Gag DNA and RNA in peripheral blood mononuclear cells and plasma. The linear range of amplification for the ICPCR assay is between 10° and 103 copies for HIV-1 DNA, while for HIV-1 RNA the amplification range is from 101 to 104 copies. The ICPCR assay correlates with the bDNA signal amplification assay for the quantitation of HIV-1 RNA, although subtle differences between the two assays were noted (G2). Nevertheless, the fall in HIV-1 RNA levels in plasma in response to antiretroviral therapy was comparable with both the bDNA and ICPCR assays. 

A simple assay for the detection of the malarial parasite Plasmodium falciparum involves saponin lysis of blood sample and membrane filtration followed by amplification of the consensus sequence of eight 21-bp repeats. This procedure has been successfully used in the field (F2). A PCR assay targeting kinetoplast DNA sequences of Leishmania species was also successfully tested in the field (F2). Molecular methods for the detection of toxoplasma gondii and several other parasites have been reviewed (F2). 

C4. Coutlee, F., Provencher, D., and Voyer, H., Detection of human papillomavirus DNA in cervical lavage specimens by a nonisotopic consensus PCR assay. J. Clin. Microbiol. 33, 1973-1978 (1995). 

Fackler, M. J., McVeigh, M., Mehrotra, J., et al. (2004) Quantitative multiplex methylation-specific PCR assay for the detection of promoter hypermethylation in multiple genes in breast cancer. Cancer Res. 64, 4442-4452. 

Luchi N etal., A real-time quantitative PCR assay for the detection of Sphaeropsis sapinea from inoculated Pinus nigra shoots, JPhytopathol 153 37—42, 2005. 

Waage, A. S., Vardund, T., Lund, V., and Kapperud, G. (1999). Detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples by a seminested PCR assay. Appl. Environ. Microbiol. 65,1636-1643. 

Amagliani, G., Omiccioli, E., del Campo, A., Bruce, I. ]., Brandi, G., and Magnani, M. (2006). Development of a magnetic capture hybridization-PCR assay for Listeria monocytogenes direct detection in milk samples. /. Appl. Microbiol. 100, 375-383. 

Jenikova, G., Pazlarova, J., and Demnerova, K. (2000). Detection of Salmonella in food samples by the combination of immunomagnetic separation and PCR assay. Int. Microbiol. 3, 225-229. 

TABLE 3.1 Overview of PCR assays for the detection of mycotoxin-producing fungi and toxins produced by the target organisms... 

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