Detection of Alicyclobacillus spp. in Fruit Juice by Combination of Immunomagnetic Separation and a SYBR Green I Real-Time PCR Assay

Abstract

An approach based on immunomagnetic separation (IMS) and SYBR Green I real-time PCR (real-time PCR) with species-specific primers and melting curve analysis was proposed as a rapid and effective method for detecting Alicyclobacillus spp. in fruit juices. Specific primers targeting the 16S rDNA sequences of Alicyclobacillus spp. were designed and then confirmed by the amplification of DNA extracted from standard strains and isolates. Spiked samples containing known amounts of target bacteria were used to obtain standard curves; the correlation coefficient was greater than 0.986 and the real-time PCR amplification efficiencies were 98.9%- 101.8%. The detection limit of the testing system was 2.8×101 CFU/mL. The coefficient of variation for intra-assay and inter-assay variability were all within the acceptable limit of 5%. Besides, the performance of the IMS-real-time PCR assay was further investigated by detecting naturally contaminated kiwi fruit juice; the sensitivity, specificity and accuracy were 91.7%, 95.9% and 95.3%, respectively. The established IMS-real-time PCR procedure provides a new method for identification and quantitative detection of Alicyclobacillus spp. in fruit juice.

Development of the Saltatory Rolling Circle Amplification Assay for Rapid and Visual Detection of Alicyclobacillus acidoterrestris in Apple Juice

“…Cai et al could detect Alicyclobacillus spp. as low as 2.8 × 10 1 CFU/mL using IMS coupled with real-time PCR . The LOD was no more than 10 CFU/mL for A.…”

“…as low as 2.8 × 10 1 CFU/mL using IMS coupled with real-time PCR. 33 The LOD was no more than 10 CFU/mL for A. acidoterrestris detection with IMS-real-time PCR, and 2−3 h was needed to accomplish the test. 3 However, PCR-based methods required a costly thermocycle instrument and tedious electrophoresis.…”

Fruit Juice Spoilage by Alicyclobacillus: Detection and Control Methods—A Comprehensive Review

“…Furthermore, the 5′-end hyper-variable region of the gene varies among Alicyclobacillus species and makes it sufficient for discrimination among species [ 201 ]. Moreover, the immunomagnetic separation method (IMS), which is based on magnetic beads that capture the microorganism cells improved the sensitivity of PCR and RT-PCR when cooperating with the 16 S rRNA gene for the detection of Alicyclobacillus [ 164 , 165 , 196 ].…”

Immunomagnetic separation: An effective pretreatment technology for isolation and enrichment in food microorganisms detection

“…detection in fruit juices (Figure 5). With the treatment of IMS, the interference of juice ingredients was eliminated and the PCR detection limitation is enhanced one order of magnitude (28 CFU/mL) (Cai et al., 2015).…”