Hielm S, Hyytiä E, Ridell J, Korkeala H
Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, Finland.
Int J Food Microbiol. 1996 Aug;31(1-3):357-65. doi: 10.1016/0168-1605(96)00984-1.
A test protocol for the detection and enumeration of Clostridium botulinum in fish and sediment samples with specific identification of neurotoxin types A, B, E and F was developed. Specific amplification products generated by polymerase chain reaction (PCR) formed the basis of identification of the toxin-producing organism, whereas quantification of the results was achieved with the most probable number (MPN) method. Twenty-six C. botulinum strains studied with PCR assays after enrichment in trypticase-peptone-glucose-yeast extract (TPGY) broth gave identical results as with the mouse bioassay. The suitability of the detection method for food and environmental surveys was assessed by running it on 32 samples of rainbow trout inoculated with spore loads ranging from 10(2) to 10(6) C. botulinum type E spores per kg. The organism was detected in all samples, and MPN estimates corresponded well to inoculum levels. In order to assess possible natural contamination, 16 fish and 16 visceral samples of rainbow trout, as well as ten aquatic sediment samples were tested. Of these, eight (80%) of the sediment samples were positive, with estimated spore counts of C. botulinum type E ranging from 95-2710 per kg sample.
开发了一种用于检测和计数鱼类及沉积物样本中肉毒梭菌，并对A、B、E和F型神经毒素进行特异性鉴定的检测方案。通过聚合酶链反应（PCR）产生的特异性扩增产物构成了产毒生物体鉴定的基础，而结果的定量则采用最大可能数（MPN）法。在胰蛋白酶-蛋白胨-葡萄糖-酵母提取物（TPGY）肉汤中富集后，用PCR检测法研究的26株肉毒梭菌菌株与小鼠生物测定法的结果相同。通过对32份每千克接种有10²至10⁶个E型肉毒梭菌孢子的虹鳟鱼样本进行检测，评估了该检测方法在食品和环境调查中的适用性。在所有样本中均检测到了该生物体，MPN估计值与接种水平相符。为了评估可能的自然污染情况，对16份虹鳟鱼的鱼体和16份内脏样本以及10份水生沉积物样本进行了检测。其中，8份（80%）沉积物样本呈阳性，E型肉毒梭菌的估计孢子数为每千克样本95 - 2710个。
