TABLE 4.

List of issues to be considered and sources of possible error in context with assay setup for NAAT^a

Issue	PCR type(s)	Consider and indicate	Pitfalls and comments
Format	Conventional	Single, semi-nested, or nested PCR	Employing a unidirectional workflow, negative air pressure conditions, and/or dedicated safety cabinets are essential prerequisites for nested PCR reactions; dUTP/AmpErase (see below) cannot be used in nested assays, since the template for the second amplification would be destroyed
	Real-time	Platform: instrument and software version	Current state-of-the-art, closed, automated system, preferentially including amplicon inactivation; combines amplification, product detection, and quantification in a single step
		Chemistry: TaqMan probes, LightCycler (FRET) hybridization probes, Biprobes, molecular beacons, Scorpions, etc., SYBR green with/without probe, melting curve analysis	Various chemistries for different purposes with variable specificity are available, e.g., application of SYBR green without probe applied on clinical samples might be unspecific despite melting curve analysis, since SYBR green stain has a high affinity for any amplified double-stranded DNA; although yet not officially stipulated, “diagnostic” NAATs have to rely on sequence-specific analysis methods; detection by gel electrophoresis or SYBR green is thus unacceptable
Concn and conditions	Conventional and real-time	Quality and effective concn of oligonucleotide components	Quality of oligonucleotides can be assessed by MALDI-TOF analysis or analytical HPLC and concn can be determined spectrophotometrically (OD260); in practice, there are significant lot-to-lot or supplier-to-supplier variations; quality of oligonucleotides can also suffer from inadequate storage and handling; resulting problems could be complex
			Some well-balanced multiplex NAATs could heavily suffer from residual salts in the supplied primer or probe preparations
			Typical melting point of the real-time PCR hybridization probe may change
		dUTP/AmpEraseUNG	Amplicon generated by master mixes containing dUTP instead of dTTP is destroyed by the enzyme UNG during the first denaturation cycle in each newly set up PCR, allowing only genomic DNA (containing natural dTTP)—and not carried over amplicon—to be amplified
		Cycling conditions (times, temps, no. of cycles) for denaturation, annealing, and extension	To achieve a higher stringency concerning binding of primers, some conventional assays are based on the “touchdown” technique—a high annealing temperature during the first cycles to increase specificity is followed by decreasing annealing temperatures, e.g., every second cycle, for efficient amplicon amplification; if primer sequences are suboptimal or start temp too low, unspecific product may be generated
		State any changes made to a published assay	Inappropriate concn might lead to unspecific product formation or “destroying” the analytical sensitivity of well-balanced multiplex applications (e.g., Mg2+ concn)
Controls at PCR level and standards for quantification	Conventional and real-time	No., quality, and concn of TPC	Too many and/or too highly concentrated TPCs can be a contamination source, as can certain formulations
		No. and quality of NTP	NTPs contain master mix only and are used to exclude contamination at the PCR level; if too few NTPs are included, contamination might be missed
		Inhibition control: internal controls, genomic target DNA, target spiked into specimens’ DNA or amplification of human gene sequences, etc.	If amplification of a gene different from the target (or some other oligonucleotide spiked in) is used, the primers and/or probe should have attributes similar to those used for the target; otherwise, inhibition cannot be ruled out; has it been demonstrated that the presence of inhibition control does not negatively influence the efficiency or analytical sensitivity of the NAAT for the respective “native” target gene?
		Standards	Did each quantitative NAAT include a standard curve (prepared on how many replicates of how many dilutions?), or was the standard curve imported from a separate run? In the absence of codified standards, absolute quantification data may be highly variable!

^aFRET, fluorescent resonance energy transfer; MALDI-TOF, matrix-assisted laser desorption ionization-time of flight; HPLC, high-pressure liquid chromatography; OD₂₆₀, optical density at 260 nm; UNG, uracil N-glycosylase; TPC, template-positive control; NTP, no-template control.