Management of inadvertent template contamination in production of oligonucleotide qPCR reagents

摘要

The quantitative polymerase chain reaction, qPCR, is an important routine tool for diagnosis of microbial infections 1. This assay employs forward and reverse oligonucleotide primers for exponential amplification of pathogen-specific nucleic acids by thermostable DNA polymerases. Amplification of target sequences can be detected in each cycle by fluorescence produced by double stranded DNA-specific fluorescent dyes, or release of fluorescent dyes coupled to quenched oligonucleotide pathogen-specific probes 2. Double stranded DNA-specific dyes (e.g., SYBR, EVA Green) provide a sensitive and inexpensive option for monitoring amplification of specific sequences but are usually limited to detection of a single target sequence per reaction. In contrast, fluorescently labeled ‘TaqMan®’ oligonucleotide probes allow for ‘multiplexing’, enabling the detection of multiple specific sequences from samples in a single reaction 3. This capability is particularly important for clinical diagnosis, which may require internal positive controls or detection of multiple specific nucleic acid sequences within thousands or more routine samples 3. During the current COVID-19 pandemic, caused by the coronavirus SARS-CoV-2, diagnostic labs around the world are utilizing this technology to identify infections 4.Sensitive diagnostic detection of pathogens from clinical samples using qRT-PCR can detect as little as ten or fewer copies of template DNA or RNA 5. Consequently, these assays require that all reagents – including oligonucleotides, fluorescent probes, enzymes and nucleotides – be free of contamination by pathogen-specific nucleic acids. As production of detection reagents and control templates has scaled up during the COVID-19 pandemic, there have been multiple reports of reagents from commercial suppliers of oligonucleotide probes and primers that produced amplification in nontemplate (NT)/nonsample control reactions, indicating background contamination by viral sequences (pers. comm.). These inadvertent contaminations may have been introduced through manipulation of reagents by asymptomatic infected individuals, or more likely by close proximity to laboratory production of SARS-CoV-2 nucleic acids.During the present COVID-19 pandemic, many research groups have redirected some focus toward assisting with diagnosis, or the development of therapies or vaccines. To support local diagnostic efforts, we organized a collaboration to produce test reagents, including enzymes, oligonucleotides and TaqMan probes specific for SARS-CoV-2. For production of oligonucleotides and probes, we used an ABI394 synthesizer, on which we produced primers for amplification of nucleic acids specific for the SARS-CoV-2 RNA-dependent RNA polymerase (RdRP) and E genes to detect the virus, in addition to the human RNAseP positive control 4,5. Initial oligonucleotides from our instrument produced results for detection of viral sequences comparable to those obtained with oligos purchased from commercial suppliers.To enable routine quality control and analysis of our test reagents, we synthesized positive control template nucleic acids for the RdRP and E gene target sequences, which were cloned into pSP72. Both template fragments were synthesized as six overlapping 30- to 60-nt oligonucleotides on the same ABI394 instrument as was used for production of the diagnostic oligos. The full approximately 200-nucleotide fragments were produced using a single round of assembly PCR, digested with restriction enzymes for sites included at the 5′ and 3′ ends and cloned into pSP72. Clones of these constructs were grown in miniprep cultures, and DNA was prepared and analyzed in rooms adjacent to open space housing the DNA synthesizer.Unexpectedly, the oligo primer sets synthesized immediately following construction and growth of the template plasmids failed quality control, in that they produced amplification with a cycle threshold (Ct) value of approximately 31 in no-templ