Quantitative PCR detects target DNA irrespective of cellular viability, leading to false positives when dead-cell templates persist. Propidium monoazide (PMA) has been widely used to suppress nonviable DNA, but protocol-dependent variability and concentration-driven failure modes motivate evaluation of alternative chemistries. Here we assess two reagents implemented under a single, standardized workflow (same culture, incubation, extraction, and uidA qPCR): a psoralen-based, UV-activated dye (MycoLight™ vPCR 350) and a light-independent, minor-groove–targeted dye (MycoLight™ vPCR Star).

With Escherichia coli, vPCR 350 at 50 μM plus 365-nm activation strongly reduced amplification from heat-killed cells while minimally affecting live cells. Relative to matched no-dye controls, dead samples shifted +12.79 cycles and live samples +1.19 cycles, yielding an 11.60-cycle separation under activation. Pre-activation effects were negligible. Under the same assay conditions, vPCR Star suppressed amplification from heat-killed cells (no detectable signal within 40 cycles), while allowing live cells to amplify (mean Cq = 33.86).

These results indicate that psoralen photochemistry can provide large dead-cell right-shifts with limited impact on viable templates, and that a light-independent, minor-groove–targeted approach can eliminate the need for photoactivation while preserving live-cell detection in this model system. Broader organism and matrix testing will define operating limits and application scope, but the present data support alternative dye chemistries as practical routes to improved live–dead discrimination in vPCR workflows.

The distinction between DNA from viable and nonviable organisms remains a significant analytical challenge in molecular diagnostics. Standard quantitative PCR amplifies target sequences regardless of cellular viability status, leading to false-positive results with practical implications across multiple sectors. In food safety applications, residual DNA from sanitized pathogens can trigger product recalls. Clinical laboratories encounter difficulties differentiating treatment success from persistent dead bacterial DNA. Water quality assessment programs report false contamination alerts from chlorine-killed organisms (Elizaquível et al., 2014; Randazzo et al., 2016; Kayigire et al., 2016).

Viability PCR addresses this challenge using DNA-binding dyes that selectively penetrate compromised cell membranes. Following photoactivation, these dyes covalently modify DNA to prevent amplification. Propidium monoazide (PMA) has served as the standard reagent, offering improved membrane selectivity compared to earlier compounds such as ethidium monoazide (Nocker et al., 2006). PMAxx™ formulation, based on patent US10570463B2 (Mao et al., 2020), incorporates polyethylene glycol (PEG) linkers to further limit entry into intact cells.

Despite widespread adoption, PMA-based methods show substantial interlaboratory and protocol-dependent variability, with DNA extraction often the dominant source and multi-lab studies reporting 10- to >100-fold differences across protocols (Jansriphibul et al., 2025). Recent characterization of the "hook effect" by Kaur et al. (2025), where excessive PMA concentrations reduce dead cell discrimination, identified a fundamental limitation of the chemistry. Their conclusion that PMA is "unreliable for quantitative live-dead assays" under many conditions highlighted the need for alternative approaches.

Phenanthridinium dyes such as propidium monoazide (PMA) and ethidium monoazide (EMA) rely on photoactivation of an aryl-azide to generate a highly reactive nitrene that should form covalent DNA adducts and block amplification (Fittipaldi et al., 2012; Kaur et al., 2025). While effective within narrow windows, this mechanism introduces several hard-to-control failure modes:

To address these constraints, we evaluated two complementary strategies under a single, standardized workflow: a psoralen-based, UV-activated reagent (MycoLight™ vPCR 350) and a light-independent, minor-groove–targeted reagent (MycoLight™ vPCR Star). Assays used a single incubation protocol (20 min, room temperature), uidA qPCR readout, and the same E. coli preparation; the only procedural difference between products was the UV-A activation step for vPCR 350 (365 nm via PhotoLyst™ 100).

The development of vPCR 350 employs psoralen as an alternative to the phenanthridium core of PMA. This approach addresses specific limitations of PMA through different molecular mechanisms.

vPCR 350 replaces the phenanthridinium–azide chemistry of PMA with a psoralen core. Psoralens are planar, polycyclic intercalators that, upon UV-A illumination (365 nm), undergo photoactivated covalent coupling to DNA from the intercalated state. The vPCR 350 scaffold intercalates reversibly; upon 365-nm activation, the engineered psoralen directs covalent modification to guanine bases in accessible regions of the target, converting those templates into non-amplifiable substrates. Molecules that do not react relax back to the ground state and remain inert. Unlike PMA's azide → nitrene radical pathway, psoralen activation in vPCR 350 does not generate nitrenes and therefore avoids radical–radical quenching and azo-dimer side reactions that constrain usable dye concentrations.

UV-A activation at 365 nm provides different optical properties compared to visible light activation. The PhotoLyst™ X100 device delivers standardized UV exposure through a single-chamber design accommodating 0.2 mL PCR tubes. The instrument features dual LED channels (365 nm and 465 nm) for compatibility with both psoralen-based and traditional PMA protocols.

With E. coli, vPCR 350 (50 µM) activated with UV-A (365 nm) substantially suppressed amplification from nonviable cells while imposing only a minor penalty on viable cells. Relative to no-dye controls, dead samples shifted +12.79 cycles (≈3.85 log10; ≈7.1×10³-fold), whereas live samples shifted +1.19 cycles (≈0.36 log10; ≈2.3-fold). The resulting selectivity (ΔΔCq = 12.79 - 1.19 = 11.60) corresponds to ≈3.49 log10 (≈3.1×10³-fold) stronger impact on dead than on live templates (n = 2 dead; n = 2 live). Pre-activation effects were minimal (dead +0.42 cycles; live -0.08 cycles).

For context, across pure-culture vPCR studies, PMA-based protocols often report reductions on the order of ~5–8 cycles are reported under favorable conditions, with larger shifts generally requiring long amplicons or harsher treatments (Fittipaldi 2012). Mechanistic analysis further indicates diminishing returns with increasing dye concentration or light dose (Codony 2023).

vPCR Star employs a chemical modification approach that eliminates photoactivation requirements entirely.

vPCR Star is a minor-groove–targeted reagent that blocks amplification without photoactivation. After sequence-selective binding in the minor groove, the dye forms covalent adducts that prevent strand separation and polymerase progression. Because the reaction is chemical (not photochemical), performance is independent of light dose, penetration, or lamp calibration.

Minor groove binding differs fundamentally from intercalation. The reagent associates with the DNA helix exterior without disrupting base pairing or causing significant structural distortion. This binding mode reaches saturation when preferred sites are occupied, potentially avoiding the over-binding effects observed with intercalating dyes. The resulting covalent modifications block polymerase progression without requiring extensive DNA structural changes.

The reaction proceeds at room temperature with a 20-minute incubation period in the dark. No specialized equipment or light exposure is required. This simplified protocol eliminates variables associated with photoactivation, including light penetration in turbid samples, exposure time optimization, and equipment calibration.

Dead-cell suppression.
vPCR Star suppressed dead-cell amplification in 2/2 wells (0/2 amplified). In the matched no-dye dead-cell controls, 1/2 wells amplified (Cq ≈ 37.0) and 1/2 was Undetermined.

Live-cell performance.
Live-cell wells amplified with both vPCR Star and no-dye controls. Mean Cq with vPCR Star was 33.86 vs 36.20 for no-dye (ΔCq = -2.34 cycles), indicating preserved—and slightly earlier—detection for live cells when using the dye/activation step.

The binary outcome—amplification for viable cells, no amplification for nonviable cells—contrasts with the partial suppression typically observed with PMA-based methods.

The higher discrimination achieved with vPCR 350 (ΔCq = 12.79) suits applications requiring maximum differentiation between viable and nonviable populations. Pharmaceutical sterility testing and validation of sterilization processes benefit from the reduced ambiguity in dead cell signal suppression. The 50 μM optimal concentration provides operational flexibility for samples with elevated dead cell loads compared to PMA's narrower working range.

Research laboratories with existing photoactivation infrastructure can implement vPCR 350 using standard UV exposure equipment or the PhotoLyst™ X100 device. The UV-A wavelength may offer improved penetration in certain sample types compared to visible light, though this requires case-specific validation.

The elimination of photoactivation requirements expands viability PCR accessibility to field applications and resource-limited settings. Environmental monitoring, on-site food safety testing, and point-of-care diagnostics become feasible without specialized equipment. The 10-minute protocol reduces total assay time, beneficial for time-sensitive applications.

Sample opacity and turbidity do not affect chemical activation, making vPCR Star suitable for complex matrices including blood, soil extracts, and biofilm suspensions where light-based methods show reduced performance. The binary discrimination pattern (complete suppression versus normal amplification) simplifies result interpretation and may improve reproducibility across operators.

MycoLight™ vPCR 350 and vPCR Star represent alternative chemical approaches to viability discrimination that address specific limitations of traditional phenanthridium-based dyes. The psoralen chemistry of vPCR 350 eliminates nitrene radical formation while achieving substantial improvements in dead cell signal suppression (ΔCq = 12.79 cycles). The minor groove binding mechanism of vPCR Star removes photoactivation requirements entirely, providing complete dead cell signal elimination in preliminary studies.

These technologies offer complementary solutions for different application requirements. vPCR 350 provides enhanced discrimination for controlled laboratory applications, while vPCR Star enables viability PCR in settings where photoactivation is impractical. Both approaches demonstrate performance improvements compared to standard PMA protocols in initial validation studies.

Further characterization across diverse organisms and sample matrices will establish the operational boundaries and optimal applications for these chemistries. The preliminary data suggest that alternative chemical mechanisms can address fundamental constraints of traditional viability dyes, potentially improving the reliability and accessibility of molecular viability assessment.

Bacterial strain and culture
Escherichia coli (ATCC 25922) was grown overnight in non-selective medium. No cell enumeration was performed prior to dye treatment. Heat-killed controls were prepared by incubating aliquots at 90 °C for 10 min.

Dye treatment
For each condition, 100 µL of culture (live or heat-killed) was aliquoted per tube and mixed with viability dye to 50 µM final concentration. Samples were incubated in the dark at room temperature for 20 min. Matched "no-dye" tubes were processed in parallel.

Photoactivation (vPCR 350 only)
After incubation, vPCR 350 tubes were exposed to UV-A (365 nm) for 20 minutes using the PhotoLyst™ 100 device. "No-UV" controls were kept in the dark.

DNA extraction
Tubes were centrifuged (live cells: 3000 rpm, 5 min; dead cells: 10,000 rpm, 5 min). Supernatants were discarded and genomic DNA was purified from pellets using a silica-membrane kit per the manufacturer's instructions. DNA was eluted in 200 µL and used directly without quantification.

qPCR
qPCR reactions (20 µL) contained 10 µL DNA template and 10 µL of a 2× SYBR Green master mix with low ROX, with uidA primers at 400 nM each (Forward 5'-TGGATCGCGAAAACTGTGGA-3'; Reverse 5'-CGGTGATATCGTCCACCCAG-3'). Thermal cycling on an ABI 7500 FAST used 95 °C for 30 s, followed by 40 cycles of 95 °C for 20 s and 60 °C for 30 s. A melt curve was collected for specificity assessment.

MycoLight™ vPCR 350
Catalog Number: 24210
Chemistry: Psoralen-based photocrosslinking
Activation: UV-A (365 nm)
Working Concentration: 50 μM

MycoLight™ vPCR Star
Catalog Number: 24211
Chemistry: Minor groove binding alkylator
Activation: Chemical (no light required)
Protocol Time: 10 minutes

PhotoLyst™ X100 Photoactivation Instrument
Catalog Number: PLX100
Wavelengths: 365 nm, 465 nm
Format: Single chamber, 0.2 mL tubes