A novel developed method based on single primer isothermal amplification for rapid detection of Alicyclobacillus acidoterrestris in apple juice

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

“…The SPIA for detection of A. acidoterrestris developed by our research team presented high sensitivity with the LOD as low as 4.8 CFU/mL . However, the design of the DNA/RNA chimeric primer and blocker is intricate and the expensive Bca DNA polymerase is indispensable in SPIA.…”

“…In order to monitor the quality of juice, the International Federation of Fruit Juice Producers (IFU), the Japan Fruit Juice Association (JFJA), and the Australian Fruit Juice Association (AFJA) have recommended conventional microbiological cultivation procedures that are reliable and accurate. , However, for products with short shelf-lives such as refrigerated juices, the procedures are unsuitable because they are labor intensive and require 3–5 days for the identification of target bacteria, which is too long to minimize the producer’s economic losses. ,, To realize sensitive and rapid detection of A. acidoterrestris, a variety of strategies have been described, such as use of commercially available kits (e.g., Vermicon AG, Germany), antibody-mediated immunological assay, ,, polymerase chain reaction (PCR)-based methods, and nucleic acid isothermal amplification approaches. , Although commercial kits have the merit of cutting down the detection time through DNA probes targeting specific genes of Alicyclobacillus spp., essential cultivation still takes several days . Since antibodies have high specificity and affinity, antibody-mediated immunological techniques could be used as alternative methods, including the enzyme-linked immunosorbent assay (ELISA), immunomagnetic separation-ELISA (IMS-ELISA), and double-antibody sandwich ELISA (DAS-ELISA) .…”

“…However, the usage of animals during antibody manufacturing is limited by directive 2010/63/EU for scientific purposes. , PCR-based approaches as rapid and robust alternatives have been widely used, and the reason is that thermostable DNA exhibits lower cross-reactivity and greater stability under harsh conditions in comparison with proteins. , However, expert assistance and advanced thermal cycle equipment are needed to complete the detection using the aforementioned methods. Isothermal amplification approaches such as loop-mediated isothermal amplification (LAMP), single primer isothermal amplification (SPIA), and rolling circle amplification (RCA) have been well studied in recent years for nucleic acid analysis, depending on their outstanding specificity, high efficiency, and rapidity.…”

“…3,6,9 To realize sensitive and rapid detection of A. acidoterrestris, a variety of strategies have been described, such as use of commercially available kits 10 (e.g., Vermicon AG, Germany), antibody-mediated immunological assay, 2,11,12 polymerase chain reaction (PCR)-based 13 methods, and nucleic acid isothermal amplification approaches. 14,15 Although commercial kits have the merit of cutting down the detection time through DNA probes targeting specific genes of Alicyclobacillus spp., essential cultivation still takes several days. 10 Since antibodies have high specificity and affinity, antibody-mediated immunological techniques could be used as alternative methods, including the enzyme-linked immunosorbent assay (ELISA), 2 immunomagnetic separation-ELISA (IMS-ELISA), 11 and double-antibody sandwich ELISA (DAS-ELISA).…”

Development and Application of a Simple “Easy To Operate” Propidium Monoazide-Crossing Priming Amplification on Detection of Viable and Viable But Non-culturable Cells of O157 Escherichia coli

“…Isothermal amplification is a novel method for DNA amplification at a constant temperature, providing simple, fast, independent of sophisticated instruments and cost-effective techniques to detect biological targets, especially for less well-equipped laboratories, as well as for filed detection. Isothermal technologies mainly include loop-mediated isothermal amplification, rolling circle amplification, single primer isothermal amplification, polymerase spiral reaction, strand displacement amplification, and recombinase polymerase amplification ( Walker et al, 1992 ; Notomi et al, 2000 ; Haible et al, 2006 ; Zhao et al, 2009 , 2010 , 2011 ; Wang et al, 2011 ; Xu Z. et al, 2012 ; Xu et al, 2020 ; Liu et al, 2015 ; Yang et al, 2017 ). Cross priming amplification (CPA) is a novel isothermal method that relies on five primers (2a/1s, 2a, 3a, 4s, and 5a) to amply the target nucleotide sequences ( Xu G. et al, 2012 ).…”