Real-time PCR as a Diagnostic Tool for Bacterial Diseases

Max Maurin


Expert Rev Mol Diagn. 2012;12(7):731-754. 

In This Article

Real-time PCR Technology

Although extraction of bacterial nucleic acids from clinical samples remains of variable sensitivity, either using manual or automated methods, currently available methods are not discussed here because they have been presented in recent publications.[1,2] qPCR comprises both amplification and fluorescent detection of amplified DNA target in the same step. As compared with conventional PCR, a post-PCR step is unnecessary, which reduces the turn-around time of the analytical process and the risk of contamination with previously amplified nucleic acids, because there is no need for manipulation of the amplified products after the reaction. There are two main methods for detection of amplified qPCR products:[1,2] the first using a fluorescent dye, such as SYBR® Green, which binds nonspecifically to double stranded DNA, where the resulting DNA–dye complex absorbs blue light (lmax = 497 nm) and emits green light (lmax = 520 nm) detected by the qPCR instrument; and the second using fluorescent resonance energy transfer (FRET) probes, which bind specifically to the amplified DNA. The term FRET probe refers to a generic mechanism in which emission/quenching relies on the interaction between the electron-excitation states of two fluorescent dye molecules.[4] Different FRET probes exist, including 5′-nuclease probes (also named hydrolysis or TaqMan® probes), dual-hybridization probes (also named LightCycler® probes), molecular beacons and scorpion probes (Figure 1). A TaqMan probe carries both a fluorophore and a dark quencher (which absorbs excitation energy from the fluorescent dye) at its extremities; hydrolysis of the probe due to 5′-nuclease activity of the DNA polymerase separates the fluorophore from the dark quencher at the time of elongation, resulting in increased fluorescence. A dual hybridization probe consists of two DNA probes (upstream and downstream probes), each carrying a fluorophore at the 3′ end and 5′ end, respectively. At the time of probe hybridization, the fluorescence emitted by the upstream probe is absorbed by the downstream probe, which emits light with a specific wavelength detected by the qPCR instrument. A molecular beacon is a hairpin-shaped molecule with an internally quenched fluorophore whose fluorescence is restored when the probe binds to its target DNA. A scorpion probe consists of a hairpin-shaped single-stranded DNA, labelled at its 5′ and 3′ ends by a fluorophore and a quencher, respectively. The probe is directly linked to the 5′ end of a PCR primer by a blocker, which prevents the polymerase from extending the PCR primer at the 5′ end. The DNA polymerase extends the primer 3′ end, which allows the probe to hybridize to the newly synthesized DNA. The distance between the fluorophore and the quencher increases, allowing fluorescence emission detected by the real-time PCR instrument.

Figure 1.

Real-time PCR probes. (A) 5′ nuclease probe (TaqMan) probe. Fluorescence occurs when the fluorophore and the quencher are separated from each other by the nuclease activity of the DNA polymerase. (B) Dual hybridization probes. The two probes carry a fluorophore, respectively at their 3′ and 5′ ends. At the time of probe hybridization, energy transfer occurs between the two fluorophores 3′ ends because they are close to each other. (C) Molecular beacon. This hairpin-shaped molecule has an internally quenched fluorophore whose fluorescence is restored when the probe binds to its target DNA. (D) Scorpion probe. The primer (attached to the probe by a blocker) binds to the target DNA. After extension of the primer by DNA polymerase and its separation from the target DNA by heating, the probe binds to the newly synthesized DNA strand. The distance between the fluorophore and the quencher increases, allowing fluorescence emission.

SYBR Green and dual-hybridization probes are often used for melting point analysis at the end of the amplification. qPCR presents a number of advantages over conventional PCR, including a faster turn-around time, a lower risk of DNA cross contaminations and the possibility to quantify target DNA in clinical samples. The use of qPCR and FRET probes can sometimes lead to better sensitivity and/or specificity compared with conventional PCR, especially when a large number of target sequences are available, allowing the design of appropriate primers and probes. qPCR shares the same limitations as PCR methods, including false-negative results due to inhibition of DNA polymerase or variations in the target nucleic acid sequence among strains of a same bacterial species, false-positive results especially because of clinical sample contamination, and difficulties in differentiating commensa-lism from pathogenicity for opportunistic pathogens. These limitations can be partially controlled by proper use of negative, positive and internal controls. A positive control (i.e., containing target DNA) will check that the DNA amplification reaction has occurred. A negative control (DNA-free sample) will check that no external DNA contamination has occurred. An internal process control will test the presence of inhibitors of DNA amplification (especially DNA polymerase). A negative qPCR result will be interpreted as unreliable in the presence of inhibitors, and will necessitate specific procedures to remove these inhibitors.