Nested PCR

Another PCR variation used for detecting bacteria is nested PCR, which uses two successive rounds of amplification with different sets of primers.16,96,97 After amplification of one DNA fragment in the first round, the amplicon is used as a template for a second round of amplification, together with another primer set that amplifies an internal segment of the first amplicon.96,97 Nested PCR is especially useful when the concentration of the target bacterium is too... 

Bertolini, E. Penyalver, R. Garcia, A. Olmos, A. Quesada, J. M. Cambra, M. Lopez, M. M. Highly sensitive detection of Pseudomonas savastanoi pv. savas-tanoi in asymptomatic olive plants by nested-PCR in a single closed tube./. Microbiol. Meth. 2003,52, 261-266. 

Sironi et al, 1995). In surveys of the human parasite B. malayi, Wolbachia was detected by PCR in all females and in 25% of males (Taylor et al, 1999). Re-analysis of male worms using a nested PCR revealed that all individual male worms were infected (Taylor et al, 2000). 

Mathis, A., Weber, R., Kuster, H. and Speich, R. (1996) Reliable one-tube nested PCR for detection and SSCP-typing of Pneumocystis carinii. Journal of Eukaryotic Microbiology 43, 7S. 

Lahr et al.59 Microdissected thyroid tissues PUREscript kit (BlOzym, Germany) Nested PCR, RET oncogene with amplicon size of 141-383 bp Expressed genes can be analyzed from routine FFPE tissue slides or pooled single cells. 

Another possibility to enhance the specific DNA-fragment is to perform a nested PCR. With this PCR method, sequences of very low abundancy can be amplified, but the risk to enhance the background at the same time is very high. For a nested PCR, a small aliquot of the first PCR reaction is used together with gene-specific primers which bind within the initial PCR product. 

ABCBl 1236C>T PCR amplification Nested PCR Sequencing PCR ABCBl 2677G>T/A (A893S/T)... 

Kanbe, T., Yamaki, K., and Kikuchi, A. (2002). Identification of the pathogenic Aspergillus species by nested PCR using a mixture of specific primers to DNA topoisomerase 11. Microbiol. Immunol. 46, 841-848. 

Klemsdal, S. S., and Elen, O. (2006). Development of a highly sensitive nested-PCR method using a single closed tube for detection of Fusarium culmorum in cereal samples. Lett. Appl. Microbiol. 42, 544—548. 

Difficulties in determining the optimal annealing temperature can often be overcome by performing touchdown PCR [10] or nested PCR [11]. 

Helguera, P. R., Taborda, R., Docampo, D. M., and Ducasse, D. A. (2001) Immunocapture reverse transcription-polymerase chain reaction combined with nested PCR greatly increases the detection of Prunus necrotic ring spot virus in the peach. J. Virol. Methods 95, 93-100. 

Ibeas, J. I., Lozano, I., Perdigones, F., and Jimenez, J. (1996). Detection of Dekkera-Brettano-myces strains in sherry by a nested PCR method. Appl. Environ. Microbiol. 62, 998-1003. 

Several strategies can be used to enhance amplification specificity. The most commonly used method is the nested PCR.7 The potential drawbacks with nested PCRs are 2-fold. First, successful amplification requires conservation of four primer binding regions and may compromise the range of variants detected. Second, the nested PCR, as commonly performed, lends itself to contamination as products from the first amplification are openly transferred to a new tube for the second amplification. 

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