Arora S, Agarwal R K, Bist B
Division of Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar, Bareilly (UP)-243 122, India.
Int J Food Microbiol. 2006 Feb 1;106(2):177-83. doi: 10.1016/j.ijfoodmicro.2005.06.019. Epub 2005 Oct 10.
The present study was conducted to assess the best method of the most commonly used methods for detection of aeromonads in foods of animal origin. With this objective an OMP based indirect plate ELISA and a duplex-PCR using primers targeting aerolysin gene and 16S rRNA gene and yielding amplicons of 252 bp and 599 bp, respectively, were standardized. The standardized protocols and the conventional cultural method were then compared for their respective sensitivities and specificities for detecting aeromonads from chicken and milk samples. Both the standardized assays were found to be highly specific for Aeromonas. The efficiency of the standardized indirect-ELISA and duplex-PCR protocols was assessed by artificial inoculation studies with varying concentrations of Aeromonas cells inoculated in chicken and milk samples followed by enrichment in Alkaline Peptone Water supplemented with 10 mg/ml cephalothin (APW-C) for 12 h. The results revealed that indirect-ELISA was able to detect a minimum of 10(3) cells/ml or g of Aeromonas cells in spiked milk and chicken samples, respectively. Whereas, duplex-PCR and cultural method were able to detect as low as 1 cell/ml or g of Aeromonas cells in spiked milk and chicken samples. The developed assays were also tested for their efficiency to detect Aeromonas spp. in naturally contaminated milk and chicken samples. Out of a total 50 milk samples screened for presence of Aeromonas by the three methods viz., indirect-ELISA, duplex-PCR and cultural method only 1 (2%) turned out to be positive showing positive results by all three methods. Similarly, 50 samples of chicken were tested by all three methods. Three samples (6%) turned out to be positive and here again by all the three methods.
本研究旨在评估动物源性食品中气单胞菌检测常用方法中的最佳方法。为此，基于外膜蛋白的间接平板酶联免疫吸附测定法（ELISA）以及使用靶向气溶素基因和16S rRNA基因的引物进行的双重聚合酶链反应（duplex-PCR）得以标准化，分别产生252 bp和599 bp的扩增子。然后比较标准化方案与传统培养方法对鸡肉和牛奶样品中气单胞菌检测的各自敏感性和特异性。发现这两种标准化检测方法对气单胞菌具有高度特异性。通过在鸡肉和牛奶样品中接种不同浓度的气单胞菌细胞进行人工接种研究，并随后在添加10 mg/ml头孢噻吩的碱性蛋白胨水（APW-C）中富集12小时，评估标准化间接ELISA和双重PCR方案的效率。结果显示，间接ELISA分别能够在加标牛奶和鸡肉样品中检测到最低10³个细胞/ml或g的气单胞菌细胞。而双重PCR和培养方法能够在加标牛奶和鸡肉样品中检测到低至1个细胞/ml或g的气单胞菌细胞。还测试了所开发的检测方法在检测天然污染的牛奶和鸡肉样品中气单胞菌属的效率。通过间接ELISA、双重PCR和培养方法这三种方法对总共50份牛奶样品进行气单胞菌检测筛选，只有1份（2%）呈阳性，且三种方法均显示阳性结果。同样，对50份鸡肉样品进行了所有三种方法的检测。三份样品（6%）呈阳性，同样也是三种方法均呈阳性。
