Molecular diagnostic labs must maintain rigorous experimental design to ensure a patient's test results are not false negatives or false positives. Either error could lead to unnecessary testing or potentially exacerbate a condition due to delay in treatment. Eliminating false positives or false negatives is even more important now that diagnostic testing has increased dramatically due to the emergence of novel viral diseases and the expanded use of molecular testing for flu strains [1, 2].
Basic molecular assay design to identify viral genomic DNA or RNA
Assays that use real-time or quantitative PCR (qPCR) are based on identifying viral DNA or RNA from a respiratory samples. qPCR assays quantify the amount of the viral nucleic acid by amplifying specific segment(s) of the viral genome in the presence of a fluorescent probe. The amount of fluorescence released during amplification corresponds to the amount of viral DNA found in the sample. When RNA viral genomes are evaluated, the RNA must be converted to cDNA with a reverse transcriptase (RT) reaction prior to amplification and quantification, referred to as RT-qPCR or real-time RT-PCR. DNA viral genomes do not require this additional step .
Several key controls must be performed in order to verify the accuracy of qPCR or RT‑qPCR (Table 1).
Table 1. Essential negative and positive qPCR controls
|Negative controls||No template control (NTC)||Omit gDNA or cDNA to assess for unwanted nucleic acid contamination or primer-dimer formation|
|No reverse transcriptase control (No RT or –RT)||Only for RT-qPCR assays: omit reverse transcriptase step to assess for genomic DNA contamination|
|No amplification control||Omit DNA polymerase from reaction in order to assess for background fluorescence|
|Positive controls||Exogenous positive control||An external piece of DNA or RNA with the target sequence such as a custom gene, gBlocks™ Gene Fragments, or Ultramer™ DNA or RNA oligos|
|Endogenous positive control||A native target sequence present in the sample that serves as a reference gene among the samples|
|Multiple primer/probe sets for the same viral or other pathogenic genome||Assess multiple regions (2–3) of the target viral RNA or DNA genome with individual qPCR reactions (or multiplex qPCR) to ensure that the amplification curve is due to the actual target. The results should be consistent across each pathogenic genome|
Fluorescence in the no template control can be due to primer-dimers
A false-positive reading in the no template control (NTC) reaction signals that either the primer/probe sets are forming dimers that are being amplified, or there is unwanted contaminating DNA in the sample. Primer-dimer amplification can be identified by using a different primer set to amplify the target . Negative results in a NTC from a second primer/probe set indicates the original primers were either annealing to a non-specific site or to each other. If the NTC is positive for each of the primer/probe sets, there is the potential of template contamination in your assay.
Template contamination in the NTC
Identifying the origin of the template contamination can be a time-consuming endeavor. In sensitive molecular assays such as qPCR, even the slightest amount of contamination can be detected if it is complementary to the primers and probes being used in the assay.
Sources of contamination can vary but can occur in the laboratory procedures themselves. These include cross‑contamination from a positive sample or reagent to the NTC during sampling, processing, and/or during analysis. This will vary from one experiment to the next or one location to another [4, 5]. Labs can reduce cross‑contamination by using skilled professionals with the required training and perform regularly scheduled cleaning and decontamination procedures for laboratory equipment. Other important safeguards include:
- Always perform control reactions.
- Aliquot reagents such as nuclease-free water, master mix, primers, and probes for single use.
- Physically separate qPCR set-up areas from sample collection and processing areas.
- Use separate equipment for sample collection and processing than used for qPCR reactions.
- Retest samples that are adjacent to a positive sample with a strong fluorescent signal in the testing plates.
Another source of cross-contamination that many researchers overlook can occur during the manufacturing process for kit components. Contamination can occur if the manufacturer does not provide adequate safeguards to prevent the cross-contamination. Identifying contamination in kit components is more elusive and time-consuming, so it is important to choose your reagent supplier carefully.
Preventing contamination during primer and probe manufacturing
IDT uses safeguards to minimize the potential of contamination from its manufacturing process. Some safeguards include:
- IDT offers customers the option to have verification that their primer and probes are template-free to cycle 45 in digital PCR NTC analysis. IDT performs quality controls for our oligos and probes. Ensuring that the probes and primers release no fluorescence in an actual digital PCR NTC test provides rigorous proof that no contamination has been introduced during the manufacturing process (Table 2).
- IDT separates the production of positive control sequences from the primer and probe sequences in order to reduce the chance of template contamination. We have several facilities dedicated to manufacturing oligonucleotide sequences in an environment under GMP and ISO 13285:2016 standards.
- IDT positive control plasmid DNA is produced in a separate manufacturing facility and is verified by NGS (next generation sequencing). The complete separation of manufacturing is important for studies on viruses for emergent diseases or other pathogenic viral strains.
- Rigorous decontamination procedures in every manufacturing facility. IDT implements rigorous cleaning procedures that decontaminate equipment, benches, tube racks, and any other surfaces touched through the manufacturing process of any nucleic acid sequences. Manufacturing equipment is also decontaminated to prevent any residual DNA from spreading around the facility. In addition, IDT has a group tasked to ensure the cleaning procedures are done properly and are effectively removing any potential contamination.
- IDT engineered their own proprietary DNA synthesizers designed for addition of complex modifications such as fluorophores, quenchers, linkers, spacers, and modified bases. In addition to DNA synthesizers, IDT makes almost everything needed to produce oligos themselves, which allows us to monitor performance and purity of each key reagent and make quick alterations if necessary. This results in an extremely high coupling efficiency, meaning IDT can manufacture longer oligos with higher percentages of full-length products (Figure 1).
Table 2. Custom no-template control testing results.
|Assays tested||Assays with positive NTCs||Total pass|
|Percent of tested assays||1.60%||98.4%|
Nucleic acid contamination is a serious problem. Raw qPCR data obtained from the lab that published the article demonstrating a link between autism and the measles, mumps, and rubella (MMR) vaccine had clear contamination in the NTC reactions. The authors disregarded the contamination and published the data even though it was obviously tainted . The source of the contamination cannot be established for these experiments, but the potential for contamination in real-time RT-PCR and PCR assays is a well-known risk [7, 8].
Accuracy of testing for viral pathogens, whether they are new or have been around for hundreds of years, begins with ensuring your reagents are clean of contamination. Making sure your supplier for primers and probes has adequate safeguards in place to reduce and/or eliminate contamination from your products will help you develop an accurate and reliable diagnostic test.