Viability PCR
Viability PCR, also named v-PCR or vPCR, is an evolution of PCR. Through the use of a simple pre-treatment of the sample by the means of specific intercalating photo-reactive reagents it's possible to neutralize the DNA of dead cells. As a result, only DNA from live cells will be detected by PCR. This approach expands a lot the analytical scope of PCR procedures. The capability to detect only living cells become very important, because in key applications is more important to know the amount of live cells, than the total cell level. Examples of this are: food and water quality control, infectious diseases diagnostic, veterinary applications, ecological dynamics...
The first referenced work about this analytical approach was in 2003, Norwegian researchers[1] suggest the use of Ethidium Monoazide, an azide form of Ethidium Bromide, which was used in other analytical fields as Flow Cytometry as a candidate for viability PCR. However, the main important advances were done by Nocker and colleagues, which demonstrated in successive works[2][3][4][5] the potential of this technology and also suggested Propidium monoazide as a better reagent for vPCR.[6]
This field still is on development, from 2003 up to 2015, the scientific evidences about the applicability of vPCR are stacking, nowadays main efforts are focused in procedure optimization. Since a simple reagent mix with the sample, photo-activation and subsequent PCR not always shows expected results, each procedure needs some optimization. Up to now the main improvements has been :
- Improving the efficiency of photo activation: early procedures were based on high power halogen lamps which overheated the samples and don't ensured constant light dose, these home made solutions have been replaced by led based instruments.
- The use of long PCR aplicons as targets.[7]
- The increase of temperature during dark incubation.[8]
Through Combining different different optimizations strategies[9] and controlling the analytical bias, nowadays the vPCR becomes a powerful analytical tool.
References
- ↑ Nogva, Hege Karin; Drømtorp, Signe Marie; Nissen, Hilde; Rudi, Knut (2003-04-01). "Ethidium monoazide for DNA-based differentiation of viable and dead bacteria by 5'-nuclease PCR". BioTechniques 34 (4): 804–808, 810, 812–813. ISSN 0736-6205. PMID 12703305.
- ↑ Nocker, Andreas; Sossa, Katherine E.; Camper, Anne K. (2007-08-01). "Molecular monitoring of disinfection efficacy using propidium monoazide in combination with quantitative PCR". Journal of Microbiological Methods 70 (2): 252–260. doi:10.1016/j.mimet.2007.04.014. ISSN 0167-7012. PMID 17544161.
- ↑ Nocker, Andreas; Sossa-Fernandez, Priscilla; Burr, Mark D.; Camper, Anne K. (2007-08-01). "Use of propidium monoazide for live/dead distinction in microbial ecology". Applied and Environmental Microbiology 73 (16): 5111–5117. doi:10.1128/AEM.02987-06. ISSN 0099-2240. PMC 1951001. PMID 17586667.
- ↑ Nocker, Andreas; Camper, Anne K. (2009-02-01). "Novel approaches toward preferential detection of viable cells using nucleic acid amplification techniques". FEMS microbiology letters 291 (2): 137–142. doi:10.1111/j.1574-6968.2008.01429.x. ISSN 1574-6968. PMID 19054073.
- ↑ Nocker, Andreas; Mazza, Alberto; Masson, Luke; Camper, Anne K.; Brousseau, Roland (2009-03-01). "Selective detection of live bacteria combining propidium monoazide sample treatment with microarray technology". Journal of Microbiological Methods 76 (3): 253–261. doi:10.1016/j.mimet.2008.11.004. ISSN 0167-7012. PMID 19103234.
- ↑ Nocker, Andreas; Cheung, Ching-Ying; Camper, Anne K. (2006-11-01). "Comparison of propidium monoazide with ethidium monoazide for differentiation of live vs. dead bacteria by selective removal of DNA from dead cells". Journal of Microbiological Methods 67 (2): 310–320. doi:10.1016/j.mimet.2006.04.015. ISSN 0167-7012. PMID 16753236.
- ↑ Schnetzinger, Franz; Pan, Youwen; Nocker, Andreas (2013-03-01). "Use of propidium monoazide and increased amplicon length reduce false-positive signals in quantitative PCR for bioburden analysis". Applied Microbiology and Biotechnology 97 (5): 2153–2162. doi:10.1007/s00253-013-4711-6. ISSN 1432-0614. PMID 23354451.
- ↑ Nkuipou-Kenfack, Esther; Engel, Holger; Fakih, Sarah; Nocker, Andreas (2013-04-01). "Improving efficiency of viability-PCR for selective detection of live cells". Journal of Microbiological Methods 93 (1): 20–24. doi:10.1016/j.mimet.2013.01.018. ISSN 1872-8359. PMID 23389080.
- ↑ Fittipaldi, Mariana; Nocker, Andreas; Codony, Francesc (2012-11-01). "Progress in understanding preferential detection of live cells using viability dyes in combination with DNA amplification". Journal of Microbiological Methods 91 (2): 276–289. doi:10.1016/j.mimet.2012.08.007. ISSN 1872-8359. PMID 22940102.