qPCR testing is usually the method of choice when high sensitivity and specificity are required. For instance, in diagnostic and forensic applications, qPCR allows the crucial detection of minute amounts of target material. While sensitivity is a significant advantage of qPCR this same trait also makes it vulnerable to contamination, leading to inaccurate results.
In highly-regulated industries, the consequences of assay contamination can be very serious, including:
- False test results leading to delays, inappropriate treatment choices or undue patient stress.
- Wasted resources spent on retesting and identifying contamination sources.
- Reduced confidence in testing methods.
Controlling for contamination in qPCR assay development
Assay developers must take great care during optimisation and validation processes to account for potential qPCR contamination risks. One way to achieve this is by designing and implementing effective controls. During the initial stages of qPCR assay development controls are included to:
- Ensure that assay components do not generate a signal in the absence of the target sequence (from dimerisation of primer or probe and subsequent amplification, or contamination of reaction components such as enzymes or buffers);
- Verify that the design is functional and specific. For example, assays for pathogen detection need to distinguish the target sequence from any similar sequences (e.g. SARS-CoV-2 assays do not detect the common cold coronavirus template);
- Assess assay efficiency and the limits of quantification and detection to ensure adequate sensitivity.
| Control | Expected result | Result | Interpretation | Action |
|
No template control
|
Negative | Negative | No contamination or primer dimers evident | • Ensure positive control is correct. |
| Negative | Positive | Primer dimers or contamination evident |
• Check assay with an intercalating dye and compare product sizes using melt curve analysis. | |
|
Target template*
|
Positive | Positive | Assay functioning | • Ensure negative control is correct. Optimisation may still be required. |
| Positive | Negative | Failed reaction | • Repeat using intercalating dye to determine whether primers, probe or both have failed. • Repeat assay using a different template to identify alternative explanations for reaction failure. |
|
|
No reverse transcription
|
Negative | Negative | No DNA amplification | • Ensure positive control is positive and NTC is negative. |
| Negative | Positive | DNA amplification (or primer dimers) |
• Read in conjunction with NTC. Both samples being positive indicates primer dimers; negative NTC and positive minus RT indicates detection of contaminating DNA. • Redesign assay to span exon junctions, or repeat RNA extraction. |
|
|
Target template serial dilution to single copies of target/minimum three replicates
|
Estimate of assay efficiency and reproducibility |
Efficiency (95–105%) Replicates highly reproducible |
Assay optimised | • Validate in conjunction with negative controls. |
| Estimate of assay efficiency and reproducibility |
Efficiency <95% or >105% Replicates highly variable |
Assay improvements required (where possible) |
• Optimise assay conditions (oligo concentrations and/or annealing temperature). • Redesign assay |
|
| Nonspecific template control (SPUD) [Internal positive control]
|
Positive (of specified Cq) |
Negative or higher Cq than expected |
Presence of contaminants inhibiting reaction efficiency |
• Systematically explore the source of contamination. May occur at any stage from sample preparation to test set-up. |
| *Validation using control template material provides additional information around assay efficiency and limit of detection. Bustin et al.2, described an exemplary study for the development of a clinical diagnostic assay. They developed a multiplex qRT-PCR for detection of SARS-CoV-2 from clinical samples and included controls for both development and inclusion of quality control assessments when applied to patient material. The negative control was a simple “No Template Control” containing all assay components, with the exception of template. This was run when assays were being validated and when combined in multiplex. The assay description clearly states that no product was amplified in the absence of a template, neither for single nor multiplex assays. This confirmed that the primers of all assays did not interact and that specific template had not found its way into the reaction components. |
Once the assay has been developed, optimised and verified, laboratory technicians run positive and negative controls with each clinical sample plate to ensure reliable diagnostic results.
Controls are included to identify vulnerabilities in the experimental or diagnostic process and validate accurate data interpretation. In diagnostic applications, parallel controls ensure that false positives and negatives are correctly identified and reveal possible contamination events.
The usual method to monitor for contamination in a qPCR assay is to use “no template controls” (NTCs). NTC wells contain all the qPCR reaction components, such as primers, master mix and water, but lack the sample nucleic acid template. Amplification in the NTC wells indicates that there is possible contamination. Contamination may have originated from one of the reaction components or from environmental cross-contamination of reactions.
Contamination sources
The high sensitivity of qPCR assays makes them vulnerable to even the slightest contamination risks. Two of the most common sources of false positive results in a clinical diagnostic assay are sample or reaction template carryover and contaminated assay components.2
Sample or reaction template carryover
A positive target-material sample or specific template materials, including positive controls, can contaminate qPCR assays and produce false positive results. Typically, this type of contamination occurs in the laboratory. Strict workflows and best practices must be enforced to prevent contamination events. While qPCR enables automated systems that employ real-time amplification and simultaneous detection in a closed system, potential carryover events may occur if template is present within the vicinity of the reaction preparation area. Even the smallest amount of aerosolised amplified template can build up in laboratory spaces, eventually contaminating lab reagents, equipment and ventilation systems.3
Contaminated assay components Contamination may also be introduced during the qPCR assay component manufacturing processes. Many enzymes used for amplification technologies are manufactured using recombinant bacteria to express high enzyme levels. Although the enzymes are extensively purified as part of the production process, traces of bacterial sequences may remain in the enzyme preparations. Introducing bacterial nucleic acids into either sample preparation or downstream amplifications (or both) could compromise the validity of microbiome or metagenomics analyses.
Similarly, oligonucleotides may be contaminated by the solvents used in their HPLC purification. Some aqueous buffers can support bacterial growth, which can develop into biofilms in the liquid handling instrumentation used in oligo preparation. If these bacterial sequences are introduced late in the preparation method, they could carry through into the final oligonucleotide preparation. However, the use of bacteriostatic compositions in the HPLC buffers significantly reduces this form of bacterial DNA contamination.
Oligonucleotide probes and primers are also vulnerable to contamination when synthetic templates are produced in the same physical environment.4 In this situation, the template material could cross-contaminate the assay primers and/or probes during manufacture, resulting in potentially catastrophic consequences for diagnostic testing. In this event, new oligonucleotides would need to be produced. To avoid such eventualities, LGC Biosearch Technologies does not synthesise any oligonucleotide with a sequence from a known organism that may serve as a template.
Synthetic templates require extreme care when handling. If opened in an unprotected space, concentrated template solutions can easily contaminate entire facilities. If this happens, a new assay may need to be designed and optimised, or laboratories may need to move into new physical spaces.
| Contamination | Source | Result | Action |
| Positive sample to negative sample |
Cross-contamination during sample handling |
False positive | • May not be detected, indicated if negative control is also contaminated. |
|
Inhibitory materials
|
Carried over during sample preparation | False negative | • Detected by multiplexed Internal or Full Process Control, or separate positive control (for example, SPUD) of known Cq/concentration. Each sample must be checked. Repeat sample processing when inhibitors are evident. |
| Contaminated reagents | All reactions delayed (lower Cq than expected) |
• Verify Cq of positive control is correct for quantity. • Replace reagents. • Consider inhibition-resistant reagents |
|
| Specific template material | PCR product leakage from previous reactions |
False positive | • If identified as such, implement a deep cleaning regimen and ensure reactions are free from contaminants before proceeding |
| Synthetic oligonucleotides (used as positive controls) |
False positive | • It is difficult to remove template contamination from such highly concentrated source material and may be necessary to redesign and optimise a new assay. Some labs have had to resort to new physical spaces. | |
| Artificial control material | False positive | • It is difficult to remove template contamination from such highly concentrated source material and may be necessary to redesign and optimise a new assay. Some labs have had to resort to new physical spaces. | |
| Human DNA/RNA | End-use contamination | Potential false positive if the assay targets human sequences |
• Use new batches of reagent. |
| Contamination of buffers occurring during manufacture |
Potential false positive if the assay targets human sequences |
• Supplier must investigate and ensure reagents free from contaminants are provided. | |
| Bacterial DNA/RNA | Contamination of buffers occurring during manufacture | Potential false positive if the assay targets bacterial sequences |
• A well-recognised challenge since many enzymes are produced in bacterial systems. Specifically manufactured reagents may be required. |
Reducing contamination risks for qPCR assays
qPCR assay end users and assay component manufacturers must take steps to reduce contamination risks and maintain assay result integrity.
End-user responsibilities
Diagnostic laboratory teams must implement procedures to prevent sample cross-contamination. Mechanical barriers, such as employing separate physical spaces for reagent preparation and sample amplification, can reduce the risk of reaction template carryover.3 Thorough cleaning of laboratory areas with a bleach solution, followed by removal with ethanol, is also recommended to reduce contamination risks.
Laboratories may also incorporate pre- and post-amplification sterilisation techniques to reduce carryover contamination concerns. For example, when the same assay is repeated many times, using master mixes containing dUTP incorporates Uracil into subsequent PCR amplicons. Including a pre-PCR step of digestion by uracil N-glycosylase (UNG) or uracil DNA glycosylase (UDG) degrades potentially contaminating templates created by previous PCR amplification. Therefore, adding a master mix containing these enzymes to a reaction tube with target specimens allows for the hydrolysation and removal of potential contaminating amplification products before the next qPCR reaction.3 | Method | Mode of action | Advantages | Disadvantages |
| UV light | Thymidine dimmer | Inexpensive, requires no change in PCR protocol. | Ineffective against G+C-rich and short (>300 bp) amplification products. |
| UNG* | Enzymatic hydrolysis of the aerosolized amplicons. | Easy to incorporate, most active against T-rich amplicons. | Expensive, may reduce amplification efficiency. |
| Hydroxylamine | Chemically modifies C and prevents C+G pairing. | Inexpensive, effective on short and G+C-rich amplicons. | Carcinogenic, may interfere with amplicon analysis. |
| Isopsoralen (IP) | Modifies target by cyclobutane adduct. | Relatively inexpensive, requires minor modification of the PCR protocol. | Carcinogenic, inhibitory effect on PCR not very effective for controlling G+C-rich and short amplicons, requires added equipment. |
| Psoralen | Same as IP | same as IP | May interfere with amplicon analysis. |
| Primer hydrolysis | Post PCR hydrolysis of RNA residues of the amplicons by NaOH. | Equally effective on G+C-rich amplicons. | Variable efficacy, may generate aerosol during NaOH addition. |
*Uracial-N-glycosylase
Other methods of pre- and post-amplification sterilisation include UV light irradiation of preparation spaces and equipment, nucleic acid inactivation with furocoumarins, primer hydrolysis and addition of hydroxylamine hydrochloride to PCR reaction tubes after amplification.3
Manufacturer responsibilities
qPCR manufacturing facilities and kit suppliers need to take care to produce and source high-quality, reliable raw materials and reagents for their customers. This requires diligent attention to product segregation, line clearance and cleaning.
Oligonucleotide manufacturers must also have a streamlined process to identify requests for potentially contaminating oligos and an operating procedure addressing how such requests are managed.
It is paramount that companies supplying products to the scientific discovery and diagnostic processes have failsafe processes in place to ensure that the raw materials, reagents and protocols are reliable, robust and fit for purpose.
Download the LGC Biosearch Technologies application note for more information about controlling for contamination in qPCR assays.
Related content
References
1. Nolan T, Will S, McClelland L et al. (2020). Ensuring qPCR Data Reliability – Controlling for Contamination.
2. Huggett JF, Benes V, Bustin SA et al. (2020). Cautionary Note on Contamination of Reagents Used for Molecular Detection of SARS-CoV-2. Clin Chem. 66(11):1369–1372. https://doi.org/10.1093/clinchem/hvaa214
3. Aslanzadeh J. (2004). Preventing PCR Amplification Carryover Contamination in a Clinical Laboratory. Ann Clin Lab Sci. 34(4):389-96. https://pubmed.ncbi.nlm.nih.gov/15648778/
4. Sadowski I and Bogutz A. (2020). Management of Inadvertent Template Contamination in Production of Oligonucleotide qPCR Reagents. BioTechniques. 69(6). https://doi.org/10.2144/btn-2020-0129
