Polymerase Chain Reaction Inhibitors

Year 2018, Volume: 1 Issue: 1, 31 - 36, 01.10.2018

Begüm Maşlak A Funda Bağcıgil

Abstract

Polymerase chain reaction (PCR) is a method which is based on the principle that the desired gene regions are copied and reproduced in-vitro conditions and that the obtained products are made visible. PCR has advantages such as rapidity and specificity related with selected primers and high sensitivity. However, it has some disadvantages. The most important disadvantages are; difficulties encountered in evaluating the results, false positives and false negatives. And the most important cause of false negatives is the PCR inhibitors. In veterinary microbiology, major samples sent for diagnostic purposes and molecular examinations are blood, necropsy materials and stool samples. In this review, the major inhibitory substances in the samples for molecular examinations, their action mechanisms and the methods recommended for the elimination of these substances will be explained

Keywords

Polymerase Chain Reaction, PCR, PCR inhibitors, elimination methods, veterinary microbiology

Polimeraz Zincir Reaksiyonu İnhibitörleri

Abstract

Polimeraz zincir reaksiyonu (PZR), istenilen gen bölgelerinin in-vitro koşullarda kopyalanması, çoğaltılması ve elde edilen ürünlerin gözle görülebilir bir hale getirilerek saptanması prensibine dayanan bir yöntemdir. PZR’ nın hızlı ve primerlerinin özelliğine göre spesifik olması, duyarlılığının yüksek olması gibi avantajlarının yanında bazı dezavantajları da bulunmaktadır. Bunlardan en önemlileri; sonuçların değerlendirilmesinde karşılaşılan güçlükler, yanlış pozitiflikler ve yanlış negatifliklerdir. Yanlış negatifliklerin en önemli sebebi de PZR inhibitörleridir. Veteriner hekimliğinde, başta tanı amaçlı olmak üzere moleküler incelemeler amacıyla sıklıkla incelenen örnekler dışkı, kan ve iç organlardır. Bu derlemede, bahsedilen örnekler içerisinde yer alan inhibitör maddeler, bu maddelerin etki mekanizmaları ve son olarak da bunların eliminasyonu için önerilen yöntemler açıklanacaktır.

Keywords

Polimeraz Zincir Reaksiyonu, PZR, PZR inhibitörleri, eliminasyon yöntemleri, veteriner mikrobiyolojisi

References

• Abbaszadegan M., Huber M.S., Gerba C.P., Pepper I.L. (1993): Detection of enteroviruses in groundwater with the polymerase chain reaction, Applied and Enviromen- tal Microbiology, 59: 1318–1324.
• Al-Soud W.A., Radstrom P. (1998): Capacity of nine thermostable DNA polymerases to mediate DNA ampli- fication in the presence of PCR-inhibiting samples, App- lied and Enviromental Microbiology, 64: 3748–3753.
• Al-Soud W.A., Radstrom, P. (2000): Effects of amplifi- cation facilitators on diagnostic PCR in the presence of blood, feces, and meat, Journal of Clinical Microbiology, 38: 4463–4470.
• Bickley J., Short J.K., McDowell D.G., Parkes H.C. (1996): Polymerase chain reaction (PCR) detection of Listeria monocytogenes in diluted milk and reversal of PCR inhibition caused by calcium ions, Letters in App- lied Microbiology, 22: 153–158.
• Burgess L.C., Hall J.O. (1999): UV light irradiation of plastic reaction tubes inhibits PCR, Biotechniques, 27(2): 252- 256.
• Burkardt H.J. (2000): Standardization and quality cont- rol of PCR analyses, Clinical Chemistry and Laboratory Medicine, 38(2): 87–91.
• Chaturvedi U., ve ark. (2008): Detection of canine ade- noviral infections in urine and faeces by the polymerase chain reaction, Journal of Virological Methods, 149(2): 260–263.
• Da Silva N., ve ark. (1995): Rapid and sensitive detecti- on of the bovine viral diarrhea virüs genome in semen, Journal of Virological Methods, 55: 209-218.
• Demeke T., Jenkins G.R. (2010): Influence of DNA ext- raction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-de- rived traits, Analytical and Bioanalytical Chemistry, 396: 1977–1990.
• Eckhart L., Bach J., Ban J., Tschachler E. (2000): Melanin binds reversibly to thermostable DNA polymerase and inhibits its activity, Biochemical and Biophysical Research Communications, 271: 726–730.
• Erlich H.A., Bugawan T.L. (1989): HLA class II gene polymorphism: DNA typing, evolution, and relationship to disease susceptibility. In: Erlich H.A. (ed.) PCR tech- nology-Principles and application for DNA amplifica- tion, Stockton Press, 193-208.
• Garibyan L., Avashia N. (2013): research tecniques made simple: polymerase chain reaction (PCR), Journal of In- vestigative Dermatology, 133(3): e6.
• Giese S.B., Ahrens P. (2000): Veterinary Microbiology, 77 (3-4), 291-297.
• Hale A.D., Green J., Brown D.W. (1996): Comparison of four RNA extraction methods for the detection of small round structured viruses in faecal specimens, Journal of Virological Methods, 57: 195-201.
• Katcher H.L., Schwartz I. (1994): A distinctive property of Tth DNA polymerase: enzymatic amplification in the presence of phenol, Biotechniques, 16: 84–92.
• Khan G., Kangro H.O., Coates P.J., Heath R.B. (1991): Inhibitory effects of urine on the polymerase chain re- action for cytomegalovirus DNA, Journal of Clinical Pa- thology, 44: 360–365.
• Ma T.S. (1995): Applications and Limitations of Polyme- rase Chain Reaction Amplification, CHEST, 108: 1393- 1404.
• Maher N., Dillon H.K., Vermund S.H., Unnasch T.R. (2001): Magnetic bead capture eliminates PCR inhibi- tors in samples collected from the airborne environment, permitting detection of Pneumocystis carinii DNA, App- lied and Environmental Microbiology, 67: 449–452.
• Monteiro L., ve ark. (1997): Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model, Jour- nal of Clinical Microbiology,35: 995–998.
• Mullis K.B. (1990): The unusual origin of the polymerase chain reaction, Scientific American, 264: 56-65.
• Opel K.L., Chung D., McCord B.R. (2010): A study of PCR inhibition mechanisms using real time PCR, Jour- nal of Forensic Science, 55: 25–33.
• Peist R., Honsel D., Twieling G., Loffert D. (2001): PCR inhibitors in plant DNA preparations, QIAGEN News, 3: 7–9.
• Powell H.A., ve ark. (1994): Protease inhibition of the detection of Listeria monocytogenes in milk using the polymerase chain reaction, Letters in Applied Microbio- logy, 18: 59–61.
• Radstrom P., ve ark. (2004): Pre-PCR processing: stra- tegies to generate PCR- compatible samples, Molecular Biotechnology, 26(2): 133-46.
• Rossen L., Norskov P., Holmstrom K., Rasmussen O.F. (1992): Inhibition of PCR by components of food samp- les, microbial diagnostic assays and DNA-extraction so- lutions, International Journal of Food Microbiology, 17: 37–45.
• Saulnier P., Andremont A. (1992): Detection of genes in feces by booster polymerase chain reaction, Journal of Clinical Microbiology, 30: 2080–2083.
• Schmidt B.L., ve ark. (1995): Detection of Borrelia burg- dorferi DNA by polymerase chain reaction in the urine and breast milk of patients with Lyme borreliosis, Diag- nostic Microbiology and Infectious Disease, 21: 121-128.
• Schrader C., Johne R., Schielke A., Ellerbroek, L. (2011): Food associated viruses and their detection – a review, Journal of Food Quality, 62: 36–51.
• Schrader C., Schielke A., Ellerbroek, L., Johne R. (2012): PCR inhibitors – occurence, properties and removal, Journal of Applied Microbiology, 113: 1014- 1026.
• Sipahioglu H.M., Usta M., Ocak M. (2006): Use of dried high-phenolic laden host leaves for virus and viroid pre- servation and detection by PCR methods, Journal of Vi- rological Methods, 137: 120–124.
• Smith C.J., Osborn A.M. (2009): Advantages and limita- tions of quantitative PCR (Q-PCR)-based approaches in microbial ecology. FEMS Microbiology Ecology, Volume 67, Issue 1, 6-20.
• Tamariz J., Voynarovska K., Prinz M., Caragine T. (2006): The application of ultraviolet irradiation to exogenous sources of DNA in plasticware and water for the amp- lification of low copy number DNA, Journal of Forensic Science, 51: 790–794.
• Wadowsky R.M., ve ark. (1994): Inhibition of PCR-based assay for Bordetella pertussis by using calcium alginate fi- ber and aluminum shaft components of a nasopharynge- al swab, Journal of Clinical Microbiology, 32: 1054–1057.
• Wan C.Y., Wilkins T.A. (1994): A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.), Analytical Biochemistry, 223: 7–12.
• Wilkins T.A., Smart L.B. (1996): Isolation of RNA from plant tissue. In A Laboratory Guide to RNA: Isolation, Analysis, and Synthesis, ed. Krieg, P.A. New York: Wi- ley-Liss, 21–42.
• Wilson I.G. (1997): Inhibition and facilitation of nucleic acid amplification, Applied and Environmental Microbio- logy, 63: 3741–3751.
• Yedidag E.N., ve ark. (1996): Acyclovir triphosphate in- hibits the diagnostic polymerase chain reaction for cyto- megalovirus, Transplantation, 62: 238–242.