5-gene uniplex probe-based qPCR for antibiotic resistance genes SOP

Authors: Condon, Justine; Durso, Lisa

Submitted to: Protocols.io
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/1/2025
Publication Date: 4/25/2025
Citation: Condon, J.C., Durso, L.M. 2025. 5-gene uniplex probe-based qPCR for antibiotic resistance genes SOP. Protocols.io. https://doi.org/10.17504/protocols.io.ewov1dw1yvr2/v1.
DOI: https://doi.org/10.17504/protocols.io.ewov1dw1yvr2/v1

Interpretive Summary: There is growing concern that antibiotic resistance from agriculture and the environment can be transferred to bacteria that make people and animals sick. One way to do this is to track antibiotic resistance genes – the DNA instructions carried by bacteria that code for the resistance.  However, before risk can be calculated it is essential to be able to know now only what is present, but also how much is there.  One tool that allows us to measure how many antibiotic resistance genes are present in a sample is quantitative Polymerase Chain Reaction (qPCR). This protocol describes the qPCR procedures for detecting and measuring three important types of antibiotic resistance, and one gene that helps antibiotic resistance genes move between bacteria. 

Technical Abstract: Purpose To describe the correct procedures on how to set-up quantitative polymerase chain reaction (qPCR), for 16S ribosomal RNA, and antibiotic resistance determinants for sulfonamide (sul1), erythromycin (erm(B)), extended spectrum ß-lactamase (blaCTX-M-1), and integrase (intI1). Scope In conventional PCR, the amplified DNA product, or amplicon, is detected in an end-point analysis. In real-time PCR, the accumulation of amplification product is measured as the reaction progresses, in real time, with product quantification after each cycle. Real-time detection of PCR products is enabled by the inclusion of a fluorescent reporter molecule in each reaction well that yields increased fluorescence with an increasing amount of product DNA. The fluorescence chemistries employed for this purpose include DNA-binding dyes and fluorescently labeled sequence-specific primers or probes. Specialized thermal cyclers equipped with fluorescence detection modules are used to monitor the fluorescence signal as amplification occurs. The measured fluorescence is proportional to the total amount of amplicon; the change in fluorescence over time is used to calculate the amount of amplicon produced in each cycle. (What is Real-Time PCR (qPCR)? | Bio-Rad) A probe is a sequence specific oligonucleotide with a reporter dye and a quencher. The chemical reaction behind its mechanism is hydrolysis of a probe (cleavage of the reporter from the quencher), causing the emission of a fluorescent signal. When the probe is intact, the short distance between the reporter and the quencher permits the excitation energy transfer from the reporter to the quencher or fluorescence resonance energy transfer (FRET). As a result, the quencher absorbs the light emitted by the reporter. During DNA amplification, the probe binds to the template and Taq DNA Polymerase with its 5’-->3’ exonuclease activity cleaves the probe. Therefore, a light signal is emitted, and qPCR instruments are used to detect the light emitted by the reporter. The light emitted by the reporter corresponds with the amplified DNA. (All about Probe-Based Real-Time qPCR Assays | GoldBio) This assay is for the detection of 16S rRNA, sul1, ermB, blaCTX-M-1, and intI1. These target genes utilize primer/probe pairs based on Dungan 2018 and were validated in accordance with the NORMAN (2015) and MIQE guidelines. The use of real time PCR is done primarily for determining expression of these antibiotic resistant genes (ARG) commonly present in environmental samples.