Herrmann J E, Cliver D O
Appl Microbiol. 1968 Apr;16(4):595-602. doi: 10.1128/am.16.4.595-602.1968.
The purpose of this study was to develop methods for detection and quantitation of food-borne virus. Samples (25 g) of cottage cheese, contaminated with various quantities of coxsackievirus type A9, comprised the model system. Two of the methods presented have at least a 50% probability of detecting virus at levels below 5 plaque-forming units/25-g sample. Noteworthy aspects of these methods include use of a glycine-NaOH buffer (pH 8.8) containing approximately 1 m MgCl(2) as the diluent in which the sample is slurried, treatment of the slurry with Freon TF and bentonite to facilitate centrifuge clarification, and concentration of the clarified sample extract by a two-stage process employing polyethylene glycol followed by ultracentrifugation. Virus in the final 0.5-ml sample concentrate was detected and quantitated by the plaque technique in rhesus monkey kidney cell cultures. Processing of the sample requires approximately 2 days, and the inoculated cultures may have to be observed for as long as 7 days thereafter. If these levels of sensitivity are desired, and if 12 samples per day are tested on a routine basis, the cost savings achieved by employing these methods rather than testing sample extracts without concentration may range from 75 to 90%.
本研究的目的是开发检测和定量食源性病毒的方法。用含有不同数量A9型柯萨奇病毒污染的农家干酪样品(25克)构成模型系统。所提出的两种方法在病毒含量低于5个空斑形成单位/25克样品时,至少有50%的概率能检测到病毒。这些方法的值得注意之处包括使用含有约1 mM氯化镁的甘氨酸 - 氢氧化钠缓冲液(pH 8.8)作为稀释剂来匀浆样品,用氟利昂TF和膨润土处理匀浆以利于离心澄清,以及通过聚乙二醇然后超速离心的两步法浓缩澄清的样品提取物。在恒河猴肾细胞培养物中,通过空斑技术检测和定量最终0.5毫升样品浓缩物中的病毒。样品处理大约需要2天,此后接种的培养物可能需要观察长达7天。如果需要这样的灵敏度水平,并且如果每天常规检测12个样品,采用这些方法而不是不浓缩就检测样品提取物所节省的成本可能在75%到90%之间。