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ARS Home » Plains Area » Lincoln, Nebraska » Agroecosystem Management Research » Research » Publications at this Location » Publication #424476
Research Project: Managing Manure as a Soil Resource for Improved Biosecurity, Nutrient Availability, and Soil Sustainability

Location: Agroecosystem Management Research

Title: Campylobacter qPCR SOP

Author
item Condon, Justine
item Durso, Lisa

Submitted to: Protocols.io
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/1/2025
Publication Date: 4/18/2025
Citation: Condon, J.C., Durso, L.M. 2025. Campylobacter qPCR SOP. Protocols.io. https://doi.org/10.17504/protocols.io.j8nlk9jywv5r/v1.
DOI: https://doi.org/10.17504/protocols.io.j8nlk9jywv5r/v1

Interpretive Summary: Campylobacter bacteria are a common cause of foodborne illness.  In the U.S., it is the top foodborne cause of diarrhea.  It is commonly associated with poultry and raw milk, but also common in untreated water, and has also been spread through pets.  This protocol describes a probe-based method to detect and quantify Campylobacter in soil, water, and fecal samples by detecting Campylobacter-specific DNA. 

Technical Abstract: Purpose To describe the correct procedures on how to set-up quantitative polymerase chain reaction (qPCR) for Campylobacter. 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 protocol describes the procedures used to quantify Campylobacter in a variety of animal and environmental substrates using qPCR.