The Real Time Polymerase Chain Reaction (PCR) or Quantitative PCR is a molecular diagnostic tool used to detect nucleic acids, and in this case Brucella DNA (Bounaadja et al., 2009). This method is applied on DNA extracted from specimens (lymph nodes, genital swab, fluids, organs...) or from calibrated suspension of an isolated strain.

 

Principle

This method consists of an exponential amplification of DNA, mediated by a Polymerase Chain Reaction. As a quantitative method, it allows to estimate the initial amount of DNA in a sample.

Samples are previously inactivated and DNA is extracted using appropriate methods: manual or automated, silica column or magnetic beads, …

This PCR is based on Taqman technology, with the hydrolysis of oligonucleotide probe marked by fluorochromes. Fluorescence is released during each cycle of Taq Polymerase proportionally to targeted DNA amplification  and is measured at each cycle. This increase of fluorescence is translated into an amplification curve which makes possible to determine a Cycle Threshold (Ct) value.

To detect Brucella with this PCR, different genes are targeted: IS711 (multicopy), bcsp31 and per (monocopy) using specific primers (forward and reverse) and specific probes.

 

To validate the PCR run, several controls should be included in each test:

- An Internal Positive Control (IPC) = an exogenous DNA introduced in each sample before the extraction step, which can indicate potential inhibition during the extraction steps; its presence validates the extraction

- A negative extraction control = negative control (as Phosphate Buffer Saline Solution) extracted at the same time as samples, which allows to check the presence of possible contamination during the extraction steps;

- A positive extraction control = well-characterised sample that contains target DNA, which confirms the correct extraction of DNA. (This control must be calibrated around 35 (Ct value) to limit the risk of contamination);

- A PCR positive control = known target DNA previously extracted and included to ensure the good running of PCR;

- A PCR negative control = ultrapure water instead of sample to validate the no-contamination of the PCR.

 

Interpretation:

- An IPC amplification curve should be found in each well, except for the PCR positive control. Homogeneity of IPC curves should be checked to check potential inhibitions. For PCR on DNA from specimens, samples are tested pure and at a dilution of 1/10. The expected difference in IPC curves in terms of C_t is approximately 3 (1 log). If the difference is greater than 3 C_t, an inhibition is probable.

- Extraction negative control should be negative for the target(s) but positive for IPC

- Extraction positive control should be positive for the target(s) and positive for IPC

- PCR positive control should be positive for the target(s) and negative for IPC

- PCR negative control should be negative (no amplification curve)

- Sample should be positive for IPC and are considered as:

- Positive if there is an amplification curve for IS711 only or for IS711 and at least one other target (bcsp31 or per) with a C_t value lower than 38

- Doubtful if there is an amplification curve for IS711 and at least one other target (bcsp31 or per) with a C_t value between 38 and 40. Samples should be retested.

- Negative if the C_t value is higher than 40 or if there is no amplification curve for all targets.

- Inconclusive if there is an amplification curve for bcsp31 or per only. Samples should be retested.