Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90226
標題: 建立奈米金球之側層流檢測平臺以同時偵測仙人掌桿菌的致嘔吐及腹瀉基因
Development of a gold-nanoparticle based lateral flow assay for simultaneously detecting Bacillus cereus emetic and diarrheal toxin genes
作者: 許琇婷
Hsiou-Ting Hsu
關鍵字: multiplex PCR
Bacillus cereus
gold nanoparticles
lateral flow assay
多重聚合酶連鎖反應
仙人掌桿菌
奈米金球
側層流檢測試紙
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摘要: Bacillus cereus can be commonly found in environment and causes emetic and diarrheal food illness. Acute nausea and vomiting caused by the emetic type of B. cereus is due to the cereulide that is an acidic and thermal stable cyclic peptide. The diarrheal type of B. cereus is associated with several enterotoxins, such as haemolysin BL (Hbl), non-haemolytic enterotoxin (Nhe), cytotoxin K (CytK), enterotoxin T (BceT) and enterotoxin FM (Ent FM). A multiplex polymerase chain reaction (mPCR) was combined with a multiplex lateral flow assay (mLFA) to simultaneously detect the nheA and ces genes of B. cereus. A mPCR was used to amplify nheA and ces genes by using tag-labeled primers, and the double-labeled amplicons (FITC-& Biotin-labeled for the nheA amplicon and DIG-& Biotin-labeled for the ces amplicon) were captured by anti-FITC or anti-DIG antibodies immobilized on the strips. The complexes were detected by avidin-tagged gold nanoparticle (avidin-tagged AuNPs) and the remaining nanoparticles were captured by BSA-Biotin immobilized on the control line. After optimization, the best conditions for the mPCR included: the annealing temperature as 59oC, 0.5 U Taq DNA polymerase, 0.2 mM of each dNTP, 0.2 μM of each nheA primers, 0.1 μM of each ces primers and 36 reaction cycles. The optimal conditions for the mLFA included: using the HFB07502 membrane, the coating density for anti-FITC antibody and anti-DIG antibody as 3.5 μg/cm and 0.2 μg/cm, and the volume ratio of AuNP to amplicon as 8:1. The detection limit of this mPCR-mLFA system was 104 CFU/mL in buffer, and could detect 101 CFU/mL in foods after a 4- to 5-hour pre-enrichment. Hence, we have developed a rapid, sensitive and specific mPCR-mLFA assay for simultaneously detecting the nheA and ces gene of B. cereus.
URI: http://hdl.handle.net/11455/90226
文章公開時間: 2017-08-31
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