Comparison of bacterial culture plus PCRs and SYBR Green real-time PCR methods in detecting Arcobacter butzleri in pork meat with different pre-enrichment periods
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Abstract
Objective: A speedy diagnosis of food poisoning bacteria is crucial. Although PCR can detect Arcobacter faster than bacterial culture-based diagnosis, sample pre-enrichment (PE) for 48 hours is usually needed to increase the test’s susceptibility. This study aimed to find Arcobacter butzleri in pork samples and figure out the shorter PE times of pork samples for real-time PCR versus the traditional diagnostic methods.
Materials and Methods: Sixty fresh pork samples were collected from retail meat shops in Mueang Khon Kaen district, Khon Kaen province, from October 2021 to March 2022. This study compared A. butzleri diagnosis between bacterial culture plus classical PCRs and SYBR Green real-time PCR. Both methods used the same samples of pork meat without (0 hour) or with pre-enriched samples (PE) for 24 and 48 hours to target species-specific 16S rRNA genes in the bacterium.
Results: Using 10-fold serial concentrations of the reference strain (A. butzleri DMST19680), the detection limit of SYBR Green real-time PCR was 2.8 x 10 CFU/mL in bacterial suspension and 2.8 x 104 CFU/mL in pork matrix. In sixty pork samples, real-time PCR did not directly detect A. butzleri (0 h PE) but did detect it at 90.0% in the 24 h and 48 h PE samples. A. butzleri was found at 3.3%, 63.3%, and 85.0% in the 0 h, 24 h, and 48 h PE samples, respectively, by bacterial culture-based methods. Considering 48 h PE as the gold reference, the prevalence of A. butzleri in the pork samples was 85.0% and 90.0% when using culture-based and real-time PCR approaches, respectively. Real-time PCR (24 h PE) had 100% sensitivity, 66.7% specificity (95%CI: 35.9–97.5), and 95.0% accuracy (95%CI: 89.5–100). The bacterial culture-based (48 h PE) and real-time PCR (24 h PE) methods had good agreement (kappa = 77.3%; 95%CI: 59.7–94.9).
Conclusions: A. butzleri was highly prevalent in pork samples sold in the Mueang district of Khon Kaen. The results proved that SYBR Green real-time PCR with 24 h PE was a reliable screening method for faster detection of A. butzleri in pork samples.
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