1. Introduction
Nucleic acids, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), are hereditary macromolecule biopolymers composed of nucleotide subunits. Each nucleotide contains a five-carbon sugar coordinated with a 5′ phosphate group, a 3′ hydroxyl group, and a 1′ pyrimidine or purine nitrogenous base. Strands of nucleotides have a 5′ phosphate group at one end and a 3′ hydroxyl group at the other, to which new nucleotides are added, resulting in 5′ to 3′ sense or 3′ to 5′ antisense directionality. DNA consists of two antiparallel nucleotide strands in a double helix structure joined together by hydrogen bonds that link complementary nitrogenous bases: guanine with cytosine and adenine with thymine [
1,
2]. This complementary DNA base pairing is highly specific and utilised in molecular diagnostics for infectious disease pathogen detection [
3]. Nucleic acid amplification diagnostics, such as polymerase chain reaction (PCR), are used to target pathogen specific genomic nucleotide sequences and amplify these biomarkers to detectable levels [
4]. Although PCR was first conceived in the 1980s [
5,
6,
7,
8], it remains the most commonly used nucleic acid amplification technology and has been the primary diagnostic tool of choice during the global COVID-19 pandemic [
9,
10,
11].
PCR is a temperature-mediated technology that mimics aspects of DNA replication by rapidly cycling high-temperature DNA denaturing phases with specific lower-temperature annealing phases. This process causes separation of the target DNA to be amplified, enabling sense and antisense, or forward and reverse, single-stranded oligonucleotide primers to complement bind at opposing ends of a short nucleotide sequence located in the biomarker target. The free 3′ hydroxyl group of each hybridised primer is targeted by a thermostable DNA polymerase leading to the addition of nucleotide subunits, generating new DNA strands. Each denaturation and annealing temperature cycle doubles the biomarker target, leading to exponential amplification that can be monitored in real-time, typically using thermocycling fluorometers in combination with hydrolysis probes or DNA-binding dyes. Hydrolysis probes are single-stranded oligonucleotides, with a 5′ fluorophore and 3′ quencher, located between PCR primers and designed with higher melting temperatures to ensure earlier target hybridisation. The 3′ quencher absorbs fluorophore fluorescence and acts as an extension blocker preventing non-specific signal generation. Extension from the flanking primers during each PCR cycle dissociates the hydrolysis probe fluorophore and quencher producing fluorescence which is relative to the amplified product, enabling both target detection and quantification [
6,
12]. Real-time PCR monitoring using the two-oligonucleotide system of a forward and reverse primer with nucleic acid dyes facilitates simpler assay design and lower cost compared to the three-oligonucleotide hydrolysis probe system [
13]. However, the dye-binding two-oligonucleotide system is less specific and more prone to primer-dimer formation or non-target amplification, leading to false-positive results [
14].
Various PCR applications or performance properties such as multiplex target detection and single-base specificity are essential for effective infectious disease diagnostics. Multiplex PCR involves the simultaneous detection of multiple targets in a single reaction, enabling reduced analysis time and reagent cost, conservation of clinical specimen, and incorporation of assay validating internal controls [
15,
16]. Typically, multiplex PCR is achieved using the three-oligonucleotide hydrolysis probe system with differentially coloured fluorophores and multichannel thermocycling fluorometers. The dye-binding two-oligonucleotide PCR system also enables multiplex detection; however, this approach requires post-amplification melt analysis and is more prone to assay cross-reactivity [
12,
17]. Single-base specificity in PCR is an important performance property as it can enable effective differentiation between closely related targets as well as the identification of specific single-nucleotide polymorphisms (SNPs). SNPs are genomic point mutations or single nucleotide differences found in at least 1% of a population. Particular SNPs can be associated with various diseases, pathogenic microorganisms, or antimicrobial resistance, and as a result are commonly targeted in molecular diagnostics. SNP variations at a particular genome location are referred to as alleles, with the most commonly found variation in a population known as the wild-type allele and alterations from this referred to as mutant alleles [
18,
19,
20,
21]. SNP detection using single-base-specific PCR has been demonstrated with both allele-biased amplification [
22,
23] and allele-biased signal generation methods [
24,
25]. Allele-biased amplification, such as allele-specific PCR, biases amplification of a particular allele by locating SNP mismatches at primer 3′-ends to inhibit polymerisation and enable differentiation between variant alleles. This approach, however, produces variable results and generally requires extensive optimisation to successfully differentiate targets [
26]. Another major limitation to this method is that once a non-specific priming event of the SNP template occurs, incorporating primers into the amplicon, no further target discrimination can be achieved. Allele-biased signal generation methods, such as the three-oligonucleotide hydrolysis probe system, differentiate between unbiased allele amplification by designing probes to coordinate with a particular allele SNP. For effective SNP differentiation using this method, shorter hydrolysis probes are used, requiring the incorporation of modified bases such as locked nucleic acids (LNAs) to maintain appropriate probe melting temperature values [
27,
28].
This article introduces 3′
Tth endonuclease cleavage PCR (3TEC-PCR) technology, a novel two-oligonucleotide PCR system that enables real-time multiplex target detection with single-base specificity. The 3TEC-PCR method uses standard PCR conditions with a modified primer/probe and a thermostable endonuclease,
Tth endonuclease IV. The modified primer/probe incorporates a 5′ fluorophore, an internal abasic site and a 3′ quencher (
Figure 1). In single-stranded form, the modified primer/probe is quenched and blocked from polymerase extension by the 3′ quencher. In target-bound duplex form, the modified primer/probe abasic site is cleaved by the
Tth endonuclease IV enzyme, enabling extension and fluorescence production via dissociation of the fluorophore and quencher. The presence of an SNP immediately 3′ of the abasic site causes cleavage inhibition, which facilitates the 3TEC-PCR single-base specificity. In this study, we have demonstrated 3TEC-PCR real-time detection, analytical specificity and sensitivity, SNP differentiation, and multiplex detection using bacterial meningitis pathogens
Haemophilus influenzae,
Neisseria meningitidis, and
Streptococcus pneumoniae as model target organisms.
3. Discussion
Real-time PCR, typically performed using a two-oligonucleotide dye-binding method or a three-oligonucleotide hydrolysis probe system, is the most widely used nucleic acid diagnostic approach for the identification of infectious disease pathogens [
9,
12]. Multiplex target detection and single-base specificity are essential diagnostic assay performance properties for effective PCR application. Multiplex PCR enables simultaneous detection of multiple targets in a single reaction, reducing assay costs and facilitating internal control incorporation [
15,
16]. Single-base specificity enables highly specific PCR and the identification of single-nucleotide polymorphisms (SNPs) using either allele biased amplification or allele biased signal generation methods [
22,
24]. In this study, we introduce a novel two-oligonucleotide PCR system that enables real-time multiplex target detection with single-base specificity, 3′
Tth endonuclease cleavage PCR (3TEC-PCR). Bacterial-meningitis-associated pathogens,
H. influenzae,
N. meningitidis, and
S. pneumoniae, were used to demonstrate the real-time detection, analytical specificity and sensitivity, SNP differentiation, and multiplex detection capabilities of the 3TEC-PCR method.
Singleplex 3TEC-PCR real-time target detection was exemplified in this study using the
H. influenzae 3TEC-PCR wild-type assay (
Figure 2). Standard real-time PCR typically produces sigmoidal shaped fluorescence amplification curves with lag, log, and stationary phases, representing target concentration as a function of the PCR cycle on a linear scale [
29]. Efficient real-time PCR reactions are exponential and cause the doubling of target product at each cycle, typically producing Ct value differences of approximately 3.3 cycles between 10-fold serially diluted target concentrations [
30]. Ct values higher than 40 cycles, indicating low-level detection, are usually disregarded or considered questionable [
31]. The resulting amplification curves from the
H. influenzae 3TEC-PCR wild-type assay (
Figure 2) are sigmoidal in shape with an approximate Ct value difference of 3.5–4 between each of the 10-fold serially diluted concentrations of
H. influenzae DNA tested, typical of standard real-time PCR. Each DNA concentration tested, apart from the single genome copy concentration, was successfully detected in duplicate, producing similar amplification curves for each replicate. This result illustrates the reproducibility of the 3TEC-PCR assay, as well as sensitive low-level detection considering the single genome copy detection was observed before the 40-cycle threshold. As signal generation was not observed in the negative control reaction, this indicates that no non-specific interactions occurred using the 3TEC-PCR method. Complete analytical specificity was displayed using the 3TEC-PCR method as only the inclusivity panel strains were detected during specificity testing of the
H. influenzae 3TEC-PCR wild-type assay (
Table S1). Additionally, sensitive low-level detection using the 3TEC-PCR method was highlighted, as the
H. influenzae 3TEC-PCR wild-type assay produced an LOD with 95% confidence of 4.1 genome copies per reaction (
Table S3). To obtain this value, 12 replicates of serially diluted
H. influenzae genomic DNA concentrations were tested. The minimum number of replicates required to give statistically valid LOD results is six; however, testing a higher number of replicates increases the confidence of the sensitivity testing [
32]. The lowest target concentration at which 95% of positive samples are detected is referred to as the LOD, and the theoretical LOD for PCR is limited to 3 genome copies per reaction according to Poisson distribution [
31].
The single-base specificity of 3TEC-PCR was demonstrated using the
H. influenzae 3TEC-PCR wild-type and mutant allele assays in combination with synthetic DNA templates, SNP0 and SNP1 (
Figure 3). Each assay only detected the complementary SNP-free target and did not detect the non-complementary SNP-containing target. This result indicates that an SNP present on the 3′ side of the forward primer/probe abasic site inhibits
Tth endonuclease IV cleavage by preventing duplex DNA structure formation, highlighting the utility of 3TEC-PCR for SNP detection. Additionally, this result indicates that the blocked 3TEC-PCR forward primer/probe requires hybridisation to an absolute complementary target match to enable cleavage, extension, and signal production. This 3TEC-PCR property ensures increased assay specificity compared to standard allele-specific PCR methods by preventing the priming of partially homologous target sequences and reducing the occurrence of non-specific template-independent interactions such as primer-dimer formation. The synthetic DNA templates, SNP0 and SNP1, tested at 10
4 copies to highlight 3TEC-PCR single-base specificity (
Figure 3), produced similar amplification curves and Ct values compared to the
H. influenzae genomic DNA tested at 10
4 genome copies (
Figure 1).
Multiplex 3TEC-PCR real-time target detection was successfully exhibited using the
H. influenzae,
N. meningitidis, and
S. pneumoniae 3TEC-PCR multiplex assay (
Figure 4). All three bacterial targets, tested at 10
4 genome copies, were each detected in separate target reactions and in one combined reaction with all three targets, producing similar approximate Ct values in both formats: 26 for
H. influenzae, 28 for
N. meningitidis, and 29 for
S. pneumoniae. However, it can be observed that there is a reduction in the fluorescence production between the single-target reactions and the combined multiple-target reaction. This is a result of the expected reaction inhibition from the simultaneous co-amplification of three bacterial targets in one reaction. Although bacterial pathogen co-infection can be rare, particularly with leading meningitis-associated organisms [
33], this demonstration of pathogen co-amplification in a single reaction highlights the robustness of the 3TEC-PCR method. There is a slight reduction in single-target detection efficiency between the singleplex and multiplex 3TEC-PCR assays. The singleplex
H. influenzae 3TEC-PCR wild-type assay produced at Ct value of 23.5 for
H. influenzae at 10
4 copies (
Figure 2), compared to a Ct value of 26 in the multiplex assay (
Figure 4). This reduced efficiency of target detection is a result of reaction inhibition due to the presence of additional primer sets in the multiplex reaction and is an expected limitation of multiplex PCR compared to singleplex PCR [
34,
35]. In terms of assay sensitivity, initial LOD analysis for the
H. influenzae,
N. meningitidis, and
S. pneumoniae 3TEC-PCR multiplex assay demonstrated low-level detection of 10 genome copies per reaction for each target when tested separately. Multiplex PCR typically requires assay optimisation involving biasing primer sets based on assay performance to ensure that each set produces similar Ct values and satisfactory LODs [
34]. The multiplex 3TEC-PCR assay in this study used unbiased, balanced primer set concentrations, without assay optimisation. However, resulting Ct values between each target were relatively similar, with low limits of detection observed, highlighting the compatibility of 3TEC-PCR with multiplexing applications.
Numerous PCR technologies with two or three-oligonucleotide systems facilitate real-time multiplex detection and single-base specificity; however, these approaches have various limitations compared to 3TEC-PCR. Three-oligonucleotide PCR systems incorporating forward and reverse primers flanking fluorescently labelled probes, such as hydrolysis probes, fluorescence resonance energy transfer hybridisation probes, or molecular beacons, enable multiplex detection and SNP identification using LNA modifications [
12,
27]. However, the design of three-oligonucleotide PCR systems can be restrictive when targeting biomarkers with limited suitable target sequence regions. Two-oligonucleotide PCR systems using novel primer/probe modifications with fluorescent labels, such as Scorpion [
36], Amplifluor [
37], LUX [
38], Cyclicon [
39], Angler [
40], and Plexor [
41], also enable multiplex detection and SNP identification. These methods, however, involve complicated probe design and are subject to the same previously outlined limitations for SNP detection as standard allele-specific PCR, with some approaches requiring additional nucleic acid analogues to enable single-base specificity [
14]. Dual priming oligonucleotide (DPO) technology is a two-oligonucleotide PCR system that facilitates SNP detection via internal primer polydeoxyinosine linkers; however, for multiplex detection this method requires the incorporation of fluorescently labelled hydrolysis probes [
42]. RNase H2-dependant PCR (rhPCR) is another two-oligonucleotide real-time PCR system that enables SNP detection using blocked primers with RNA residues that are cleaved upon target hybridisation by a thermostable RNase H2 enzyme [
43]. The rhPCR method, however, has only been demonstrated in a singleplex format, with real-time multiplex detection requiring the inclusion of hydrolysis probes. Cycling probe technology (CPT) is a pre-cursor to rhPCR, with initial isothermal diagnostic applications involving RNase H cleavage of chimeric DNA-RNA-DNA probes [
44], and subsequent applications utilising LNA hydrolysis probes for SNP detection [
45]. However, the universal application of diagnostic methods utilising RNase H enzymes with RNA residue primers or probes is limited as the occurrence of non-specific cleavage by RNases other than RNase H, or inhibition due to human genomic DNA, has been reported [
46]. Cairns and colleagues previously reported a two-oligonucleotide real-time PCR system using an enzyme-cleavable primer/probe with a 5′-end restriction enzyme sequence flanked by a fluorophore and quencher [
47]. This method enables multiplex detection; however, as the restriction enzyme sequence is not target-specific, and due to reverse-primer activity, this system is subject to the same SNP detection limitations as standard allele-specific PCR.
3TEC-PCR technology, derived from our previously reported isothermal diagnostic methods incorporating cleavable primer/probe systems [
48,
49], requires minor alterations to standard real-time PCR assay design and performance. In addition to the inclusion of a modified primer/probe and
Tth endonuclease IV, zinc chloride is also required as a cleavage enzyme stabilising agent. Primer/probe Tm values should be compatible with the 3TEC-PCR 65 °C annealing temperature, the optimal incubation temperate for
Tth endonuclease IV. The thermostability of
Tth endonuclease IV in combination with the blocked primer/probe facilitates hot-start PCR reactions. Placement of the primer/probe abasic site can be altered; however, the cleaved oligonucleotide should be of a sufficient Tm value to enable efficient post-cleavage primer activity. Alternate positioning of the fluorophore and quencher labels can be used, and cytosine residues as well as thymine residues can be utilised for internal oligonucleotide labelling. Positioning of the primer/probe quencher label is ideally located on a guanine residue to enable increased quenching activity [
50]; however, this is not essential. Compared to standard three-oligonucleotide hydrolysis probe PCR systems, 3TEC-PCR does not need to accommodate an oligonucleotide probe between the forward and reverse primers, enabling improved variability of reverse primer positioning, increased design flexibility especially when targeting restrictive diagnostic biomarkers, and reduced assay oligonucleotide requirements. This study details the first report of a two-oligonucleotide real-time multiplex PCR method with single-base specificity using
Tth endonuclease IV, providing effective transferable diagnostics technology for infectious disease pathogen detection and SNP identification.