Real-time Fluorescent PCR Techniques to Study Microbial-Host Interactions

Abstract

This chapter describes how real-time polymerase chain reaction (PCR) performs and how it may be used to detect microbial pathogens and the relationship they form with their host. Research and diagnostic microbiology laboratories contain a mix of traditional and leading-edge, in-house and commercial assays for the detection of microbes and the effects they impart upon target tissues, organs, and systems. The PCR has undergone significant change over the last decade, to the extent that only a small proportion of scientists have been able or willing to keep abreast of the latest offerings. The chapter reviews these changes. It discusses the second-generation of PCR technology-kinetic or real-time PCR, a tool gaining widespread acceptance in many scientific disciplines but especially in the microbiology laboratory.

Figure 10.1

Time versus temperature plot during a single PCR Cycle. The denaturation (D), primer and probe annealing (A) and primer extension (E) steps are shown. At the indicated optimal temperature ranges, dsDNA denatures (T_D) then oligoprobes anneal (T_M-PROBE) followed by the primers (T_M-PRIMER) as a precursor to their extension. The actual thermal cycler incubation temperature (dashed line) may overshoot the desired temperature to varying degrees, depending on the quality of the thermal cycler employed

Figure 10.2

Mechanisms of fluorescence resonance energy transfer (FRET). When the reporter (R) and quencher (Q, unfilled) of a nuclease oligoprobe are in close proximity and illuminated by an instrument's light source (where h is Planck's constant and ν is the frequency of the electromagnetic radiation); (a) the quencher “hijacks” the emissions from excitation of the reporter. The quencher then emits this energy. When the fluorophores are separated, as occurs upon oligoprobe hydrolysis as depicted in (b), the quencher can no longer influence the reporter which now fluoresces at a distinctive wavelength recorded by the instrument. In the reverse process using adjacent oligoprobes (c), the fluorophores begin the cycle as separated entities. Whilst the emission of the donor (D) is monitored, it is the signal from the acceptor (A) produced when in close proximity to the donor that indicates a positive reaction (d). In (e), another form of quenching is shown, caused by the intimate contact of labels attached to hairpin oligoprobes. The fluorophore (F) and an NFQ (Q, filled) interact more by collision than FRET, disrupting each other's electronic structure and directly passing on the excitation energy which is dissipated as heat (jagged, arrows). When the labels are separated, as is the case in (f), the fluorophore is free to fluoresce

Figure 10.3

Kinetic analysis of amplicon accumulation. A chart of the amplification of a template by real-time PCR (solid line) ideally appears as a sigmoidal curve when plotted as cycle number versus fluorescence emission intensity. Early exponential amplification cannot be viewed because the signal is below the sensitivity of the detector. However, when enough amplicon is present, the assay's exponential progress can be monitored as the rate of amplification enters a log-linear phase (LP). Under ideal conditions, the amount of amplicon increases at a rate of one log₁₀ every 3.32 cycles (i.e. it doubles, or increases by one log₂ every cycle). As primers and enzymes become limiting and products accumulate that are inhibitory to the PCR or compete for hybridisation with an oligoprobe, the reaction slows, entering a transition phase (TP). Eventually a plateau phase (PP) is reached in which there is little or no increase in fluorescent signal although amplicon may continue to accumulate. Some fluorescent detection chemistries display an overall reduction in fluorescence intensity after the plateau phase (Hook). The point at which fluorescence surpasses a pre-defined background noise (dashed horizontal line) is called the threshold cycle or crossing point (C_T or C_P; indicated by an arrow). These data are used for the calculation of template quantity when constructing a standard curve. Traditional PCR data collection is performed at the end of the assay (dashed vertical line). Also shown are curves representing a serial titration of template (dashed curves), consisting of decreasing starting template concentrations, which produce increasing numerical C_T or C_P values

Figure 10.4

Function of 5′ nuclease oligoprobes. Following primer hybridisation, the DNA polymerase (pol) progresses along the relevant strand during the extension step of the PCR, displacing and then hydrolysing the oligoprobe. Once the reporter (R) is removed from the extinguishing influence of the quencher (Q), it is able to release excitation energy at a wavelength that is monitored by the instrument and different from the emissions of the quencher. Inset shows the NFQ (Q; filled) and minor groove-binding molecule (grey diamonds) which make up the MGB nuclease oligoprobes. These bimolecular systems acquire data from the reporter's emissions: the opposite of the HybProbe chemistry. Data can be collected during the annealing or extension steps of the PCR

Figure 10.5

Function of the UT-oligoprobe. After the UT-primer is extended, the nascent strand acts as the template for the second primer. The polymerase encounters and hydrolyses the UT-oligoprobe whilst extending the second primer in the same fashion as a TaqMan oligoprobe. This trimolecular system can produce fluorescence data from the emissions of the released fluorophore during the annealing or extension steps

Figure 10.6

Function of the DzyNA primers. When the strand incorporating the primer is duplicated by a complementary strand (dashed line), a DNAzyme is created. A complementary, dual-labelled oligonucleotide substrate will be specifically cleaved by the DNAzyme releasing the fluorophore (F; circle) from its proximity to the quencher (Q; pentagon), releasing the labels and permitting fluorescence. Data can be collected during the annealing or extension steps of the PCR

Figure 10.7

Function of HybProbes. Adjacent hybridisation results in a FRET signal due to interaction between the donor (D) and acceptor (A) spectra detected during the annealing step of the PCR. This trimolecular system (two oligoprobes and a target) acquires its data from the acceptor's emissions: the opposite of the 5′ nuclease oligoprobe chemistry

Figure 10.8

Function of displacement oligoprobes. The shorter NFQ-labelled strand (Q; hexagon) is displaced when the fluorophore-labelled (F; circle) strand preferentially hybridises to the longer specific amplicon strand. Data is collected from this trimolecular system during the annealing step of the PCR

Figure 10.9

Function of the Q-PNA displacement primer. In the absence of the longer specific amplicon, quenching of the chemistry is achieved by a short NFQ-labelled PNA backbone (grey hexagons) designed to hybridise with the fluorophore-labelled primer (F; circle). Fluorescence data can be collected from this trimolecular system during the annealing and extension steps of the PCR

Figure 10.10

Function of the Light-Up Probe. These PNA (grey hexagons) oligoprobes fluoresce when their asymmetric thiazole orange fluorophore (T; open triangle) hybridises to the specific DNA strand. Data is collected from this bimolecular system during the annealing step of the PCR

Figure 10.11

Function of the HyBeacon Oligoprobe. The fluorophore (F; circle) emits fluorescence when in close proximity to DNA as occurs upon hybridisation with the specific amplicon strand. Data is collected from this bimolecular system during the annealing step of the PCR

Figure 10.12

Function of the Lightspeed probe. In aqueous solution the PNA (grey hexagons) probe forms a random coil conformation that is quenched due to the proximity of the fluorophore (F; circle) and quencher (Q; hexagon). Upon hybridisation this bimolecular system is stretched open and the fluorophore can emit fluorescence which is acquired during the annealing step of the PCR

Figure 10.13

Function of the molecular beacon. Hybridisation of the loop section of the beacon to the target separates the fluorophore (F; open circle) and NFQ (Q; filled hexagon) allowing fluorescence. Data from this bimolecular system is collected during the annealing step of the PCR. Inset shows a wavelength-shifting hairpin oligoprobe incorporating a harvester molecule (H; filled circle)

Figure 10.14

Function of the tripartite molecular beacon. The fluorophore (F; circle) is removed from the influence of the NFQ (Q; hexagon) upon binding to specific amplicon, which opens the hairpin and permits fluorescent emissions. Data are collected from this tetramolecular system during the annealing step of the PCR

Figure 10.15

Function of the sunrise primer. The fluorophore (F; circle) is separated from the NFQ (Q; hexagon) during disruption of the sunrise primer's hairpin structure and free to fluoresce. This disruption occurs during extension of a nascent complementary DNA strand and whenever dsDNA duplexes form during re-annealing. Data from this unimolecular system can be collected during the annealing step of the PCR

Figure 10.16

Function of the scorpion primer. The scorpion primer is blocked from being extended by a hexethylene glycol molecule (B; diamond) so that the hairpin can only be disrupted by specific hybridisation and not by the extension of a complementary amplicon strand as occurs for sunrise primers. The 5′ fluorophore (F; circle) is separated from a 3′ methyl red quencher (Q; hexagon) during self-hybridisation of the loop portion of the scorpion with a complementary region on the nascent amplicon strand. Inset shows a duplex scorpion. Data is collected from this unimolecular system during the annealing step of the PCR

Figure 10.17

Function of the LUX primer. The LUX primer is labelled with a single fluorophore (F; circle) positioned next to a guanine nucleotide when the hairpin portion of the primer is intact. The G naturally quenches the fluorophore. In the presence of the specific target strand the primer hybridises, disrupts the hairpin and is extended. The fluorophore is now free to fluorescence and data can be collected from this unimolecular signalling system during the annealing or extension step of the PCR

Figure 10.18

Fluorescence melting curve analysis (FMCA). At the completion of a real-time PCR using a fluorogenic chemistry, the reaction is rapidly heated and then cooled to a temperature below the expected T_D of the dsDNA or T_M of the oligoprobe(s). It is then heated to 85°C or more at a fraction of a degree per second (A). During heating, the raw data representing the emissions of a relevant fluorophore are constantly acquired (B). Software calculates the negative derivative of the fluorescence with temperature which is plotted against temperature to produce a melt peak indicative of the T_D of the dsDNA, or the T_M of the oligoprobe-target melting transition (dashed line and black peak; C). When sequence differences exist, the T_D or T_M is reduced (grey peak) due to the lower stability of heteroduplexes and the extent of the resulting temperature shift is used diagnostically to characterise an amplified template. Generally speaking, a ramp rate of 0.2°C/s permits clear discrimination between different genotypes. However, variation of the ramp rate can be helpful if unsatisfactory results are generated. Hybridisation can be enhanced by slowing the ramp rate whereas an accelerated ramp rate can help remove stable secondary structures at the oligoprobe hybridisation region resulting in sharper melt peaks

Figure 10.19

External standard curve for quantitation. Threshold cycle or crossing point data collected from an amplified titration of standard plotted against the concentration of each template. Through interpolation, C_T/C_P values for unknowns (Unk) permit the calculation of the starting template concentrations (dashed line). This is performed by rearranging the equation for the linear regression line (i), to solve for the x-axis value of interest, (ii). When the relevant values are substituted (iii), the concentration of template in each unknown can be calculated