Optimisation of diagnostic PCR a study of PCR inhibitors in blood and sample pretreatment

• PCR is widely employed as a rapid, sensitive and specific molecular diagnostic technique in clinical diagnosis, environmental investigations and for monitoring biotechnical processes. However, the full potential of diagnostic PCR is limited, in part, by the presence of PCR-inhibitory substances in biological samples. Among the mechanisms by which these inhibitors may act are through interfering with the DNA polymerase, with target nucleic acids and with lysis of cells. Therefore characterisation of PCR inhibitors is an important step for the development of more efficient sample preparation methods, which are used to reduce the effect of inhibitors and/or to concentrate target DNA. The aim of this work was to identify PCR-inhibitory components in blood and to develop pre-PCR treatment methods to eliminate the effect of inhibitors. Three major inhibitors were identified in blood namely, immunoglobulin G in plasma, haemoglobin in erythrocytes and lactoferrin in leukocytes. In addition, the quantitative effects of inhibitors on DNA synthesis were also investigated. Pre-PCR treatment methods based on selection of DNA polymerases more resistant to PCR-inhibitory components and the addition of amplification facilitators were tested. This approach was very efficient in reducing inhibition of PCR by blood and other complex biological samples. For example, when rTth was used instead of Taq DNA polymerase it became possible to amplify DNA in the presence of 20% (vol/vol) blood without reduced amplification sensitivity, whereas Taq DNA polymerase was totally inhibited in the presence of 0.004% (vol/vol) blood in the PCR mixture. Furthermore, the addition of bovine serum albumin to rTth the reaction mixtures allowed DNA amplification in the presence of 20% instead of 2% (vol/vol) meat and 4% instead of 0.4% (vol/vol) faeces. Thus, the PCR-inhibitory effect of various components in biological samples can, to some extent, be eliminated by the use of an appropriate thermostable DNA polymerase and amplification facilitators. This approach will allow direct detection of pathogens in biological samples, or facilitate the development of simple and rapid sample preparation methods more suitable for automation. Blood culture is an important step for the detection of blood pathogens, due to the low number of pathogens in the blood (often less than one per ml) and the small sample volume of blood obtainable from neonates and young infants. Therefore, a pre-PCR treatment was developed to remove the effect of PCR inhibitors in blood cultures, and to concentrate the bacterial cells prior to PCR. This method was based on a combination of dilution, centrifugation, washing with NaOH, and the addition of bovine serum albumin. Using this pre-PCR treatment allowed detection of 1 CFU of bacteria per ml of blood culture medium.

Ämnesord

TEKNIK OCH TEKNOLOGIER  -- Industriell bioteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Industrial Biotechnology (hsv//eng)

Nyckelord

mycology

virology

bacteriology

Microbiology

AmpliTaq Gold

Amplification facilitators

Betaine

Blood culture medium

Blood

BSA

cheese

faeces

gp32

Haemoglobin

Ions

Immunoglobulin G

Mikrobiologi

bakteriologi

virologi

mykologi

Publikations- och innehållstyp

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