| Sensitive and rapid detection methods for Alicyclobacillus acidoterrestris are required. Alicyclobacillus acidoterrestris is a motile, spore-forming, rod-shaped organism with a central, subterminal, or terminal oval spore. At times, A. acidoterrestris is called acidophilic thermophilic bacteria (ATB). As the name implies, ATB grows well in acidic environments (such as fruit juice), surviving at pH levels as low as 2.5 and grows at temperatures of 25–70 oC. The prevention of spoilage by ATB is a current challenge for fruit juice and beverage industries worldwide due to the acidothermophilic growth capability, heat resistance, and spoilage potential of the bacterium. Fruit juices and fruit juice-containing drinks that are most susceptible to this bacterium are either fresh (not heat-treated) or pasteurized (but not UHT heat-treated). ATB causes a flat sour type of spoilage and produces an offensive-smelling compound, guaiacol, and other taint chemicals. Nowadays, available methods for identifying ATB are based on biochemical tests. However, these procedures are time-consuming and not always accurate.Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method that relies on an auto-cycling strand displacement DNA synthesis performed by the Bst DNA polymerase large fragment. LAMP assay was a specific, rapid and sensitive method of detecting viruses, fungi, bacteria and Genetically modified food. Therefore, the establishment of LAMP technology for the detection of Salmonella was very important.A loop-mediated isothermal amplification (LAMP) assay was developed for the rapid detection (within 2 h) of Alicyclobacillus acidoterrestris. One of the six primers recognizing eight distinct regions of the AB042057 gene were designed using RNA structure and the PrimerExplorer V3 software (Eiken Chemical Co. Ltd.; https://primerexplorer.jp/lamp3.0.0/index.html).The final LAMP reaction (total: 25μL) contained three primer pairs in the following concentrations: 18 pM each of inner primers (FIP and BIP), 3 pM each of outer primers (F3 and B3), and 9 pM each of loop primers (LoopF and LoopB). The reaction mixtures also contained 8U Bst DNA polymerase, 3 mM MgSO4, 5 mM dNTPs and 2μL of sample. Reaction mixtures were incubated at 63 oC for 20 min, followed by incubation at 80 oC for 10 min to completed the reaction. White pyrophosphate magnesium precipitation can be observed by the naked eye. Reaction products were analyzed by gel electrophoresis (5μL aliquots on 1.5% agarose gel).Using LAMP assay, the 8 strains of A. acidoterrestris were successfully amplified, but 6 strains of other bacillus Acidocaldarius and 13 bacterial species other than bacillus Acidocaldarius were not.The sensitivity of the LAMP assay was at 2.25 CFU/mL. This sensitivity is greater than that obtained by polymerase chain reaction (PCR) assay which is 225 CFU/mL. The LAMP assay was examined further for its ability to detect A. acidoterrestris in juice samples. The results were compared with those of conventional PCR detection. The detection limit of artificially contamination was 22.5 CFU/mL with LAMP detection for two hour. In contrast, the detection limit of artificially contaminated was 2250 CFU/mL with PCR detection for four hours.The effect of six methods of extracting A. acidoterrestris DNA in juice were compared, the method of extracting DNA, is no strict requirements.Results indicate that the proposed LAMP assay is a rapid, specific, and sensitive method for detecting A. acidoterrestris. As the amplification has been conducted under isothermal conditions, only water bath or heating block is needed to maintain the required temperature. Thus, the method can be generalized and popularized easily in the future. The development of an extremely rapid and highly efficient genetic detection system for ATB using the LAMP method is now under way. |
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