Immunomagnetic Separation Combined with Polymerase Chain Reaction for the Detection of Alicyclobacillus acidoterrestris in Apple Juice

Zhouli Wang,Yahong Yuan,Rui Cai,Chen Niu,Tianli Yue,Jun Wang,Richard C Willson

doi:10.1371/journal.pone.0082376

Abstract

A combination of immunomagnetic separation (IMS) and polymerase chain reaction (PCR) was used to detect Alicyclobacillus acidoterrestris (A. acidoterrestris) in apple juice. The optimum technological parameters of the IMS system were investigated. The results indicated that the immunocapture reactions could be finished in 60 min and the quantity of IMPs used for IMS was 2.5 mg/mL. Then the combined IMS-PCR procedure was assessed by detecting A. acidoterrestris in apple juice samples. The agarose gel electrophoresis results of 20 different strains showed that the IMS-PCR procedure presented high specificity to the A. acidoterrestris. The sensitivity of the IMS-PCR was 2×101 CFU/mL and the total detection time was 3 to 4 h. Of the 78 naturally contaminated apple juice samples examined, the sensitivity, specificity and accuracy of IMS-PCR compared with the standardized pour plate method were 90.9%, 97.0% and 96.2%, respectively. The results exhibited that the developed IMS-PCR method will be a valuable tool for detecting A. acidoterrestris and improving food quality in juice samples.

Highlights

Alicyclobacillus acidoterrestris (A. acidoterrestris), which contains ω-fatty acids as the major membrane fatty acid component, is a thermophilic, aciduric, gram-positive, rodshaped, spore-forming and nonpathogenic bacterium [1,2,3]
The immunocapture time and the quantity of immunomagnetic nanoparticles (IMPs) used for the separation of A. acidoterrestris in apple juice samples were optimized for maximal immunocapture efficiency
As the coupling time continuously increased to 120 min, there was no significant change in the percentage of recovery

Summary

Introduction

Alicyclobacillus acidoterrestris (A. acidoterrestris), which contains ω-fatty acids as the major membrane fatty acid component, is a thermophilic, aciduric, gram-positive, rodshaped, spore-forming and nonpathogenic bacterium [1,2,3]. The performance of the developed IMS-PCR was further evaluated by detecting of A. acidoterrestris in naturally contaminated apple juice samples. The immunocapture time and the quantity of IMPs used for the separation of A. acidoterrestris in apple juice samples were optimized for maximal immunocapture efficiency. In order to determine the detection limit of the IMS-PCR assay, 1 mL ten-fold dilutions of A. acidoterrestris (from 2×100 CFU/mL to 2×105 CFU/mL) was added into 9 mL sterilized water and reconstituted apple juice (15 °Brix), respectively.

Results

Conclusion