A real-time polymerase chain reaction-based method for rapid and specific detection of spoilage Alicyclobacillus spp. in apple juice

Abstract

Aims: To develop a real-time PCR-based rapid detection method for spoilage Alicyclobacillus spp. in juice products. Methods and Results:The squalene-hopene cyclase-encoding gene was targeted for primer-and-probe development. Gene fragments from representative strains were cloned, and PCR primers and probe were designed by DNA sequence comparison. Selected bacteria were examined for cross-reactivity by the new method. Cells were serially diluted in apple juice and saline, and examined by the new method to establish detection sensitivity. Using the newly developed Taqman Ò real-time PCR-based method, strains of Alicyclobacillus acidocaldarius and A. acidoterrestris were detected without cross reactivity with other common food-borne micro-organisms. Detection of <10 cells per PCR reaction from juice samples was accomplished within 3-5 h. Conclusion: This is the first reported real-time PCR-based detection method for Alicyclobacillus spp. and its application in juice products is demonstrated. Significance and Impact of the Study: As a favourable alternative for the laborious and time-consuming culture-or biochemical characterization-based techniques, the system has great potential for industrial applications from raw material screening to final product quality control.

Real-Time PCR Detection of Paenibacillus spp. in Raw Milk To Predict Shelf Life Performance of Pasteurized Fluid Milk Products

“…Few rapid, molecular-based detection methods targeting spore-forming bacteria responsible for food spoilage have been developed (9,22,24), and of these, none have focused on Paenibacillus, the psychrotolerant spore-forming genus associated with dairy spoilage. The absence of appropriate tools may reflect, in part, the fact that the bacterial ecology of pasteurized fluid milk has only recently been characterized at the molecular level, which led to identification of Paenibacillus as the predominant fluid milk spore-forming spoilage genera (12,19,21,34).…”

Fruit juice and puree characteristics influence enrichment requirements for real-time PCR detection of Alicyclobacillus acidoterrestris

“…Ten out of 13 samples (77%) showed background contamination of the genus Alicyclobacillus, while 7 out of the 13 samples (54%) showed the specific presence of A. acidoterrestris background contamination (Table 1). A number of reports have shown that A. acidoterrestris can be present in both fresh juices and juice concentrates (Pettipher et al 1997;Luo et al, 2004;Groenewald et al 2009;Groenewald et al 2013;McKnight et al, 2010;Wang et al, 2013). The presence of background Alicyclobacillus spp.…”

“…spoilage is a global problem in the fruit juice industry (Cagnasso et al 2010;Groenewald et al 2009, McKnight et al 2010, Walls & Chuyate 2000. Most instances of contamination have been reported in apple and orange juices (McKnight et al, 2010;Groenewald et al, 2009;Luo et al 2004). In the US juice industry alone, Alicyclobacillus spp.…”

“…and other organisms have have been reported in the literature (Al-Qadiri et al, 2006;Luo et al 2004, Jasson et al, 2009, Cagnasso et al, 2010, Connor et al, 2005, Chang and Kang 2004. Many of the published reports have, however, focused only on limited types of samples (Luo et al 2004;Connor et al, 2005). Real-time PCR-based kits which have been tested for their specificity and sensitivity are currently commercially available.…”

“…The International Federation of Fruit Juice Producers (IFU) recommended method of conventional plating requires a minimum of 72 hours of enrichment prior to plating. Thus, conventional plating methods to detect this organism are generally very time consuming and ineffective as a quality control strategy (Luo et al, 2004;Lin et al, 2005). Considering the high product value of juices, storing fruit juices for an extended period of time until obtaining conventional plating results is not economically feasible (Lin et al, 2005).…”