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《现代养猪生产技术》读者互动专区

《现代养猪生产技术》读者互动专区

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详解隐性霉菌毒素的检测方法(一)

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kk 发表于 2016-8-16 12:04:20 | 显示全部楼层 |阅读模式
有时,你看不到的东西才是你真正需要担心的东西。饲料中的霉菌毒素并不总是肉眼可见,现有的检测方法并不能检测出所有的霉菌毒素。

部分植物还可以改变其毒性物征,不再称之为“霉菌毒素”。饲料生产者该如何处理这些“隐藏的”霉菌毒素?

当检测结果显示,并没有什么可担心时,此时对于危险霉菌毒素的判断则是较难的。

霉菌毒素,霉菌产生的破坏性的复合物,动物生产者和饲料加工厂应密切注视原料质量。渗透入饲料和储存仓的霉菌毒素,甚至在快速样品测试宣布毒素水平低于检测范围时,还可继续隐藏,导致养猪者误认为他们所用的饲料并没有污染。
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常规检测方法

当处理饲料时,第一步需认识到,没有霉菌毒素也可能是一个问题。因为,通过肉眼霉菌计数法来观察霉菌与霉菌毒素的存在两者间的相关性很弱。即使对霉菌基因型的毒性分析也不能担保霉菌毒素的存在或缺乏,这是因为它们的合成机制受多种环境因素诱导。目前所用的鉴定霉菌毒素污染的众多方法中,部分操作快速、容易,但部分则需要更深入的实验室分析。

在大多数情况下,可用快速检测法或确认检测法来检测霉菌毒素。快速检测法可用于定性检测,作为拒收/接收原料的第一步检测。

但是,快速检测法检测范围狭窄,结果的准确度低、巨大的成本是其重大的缺点。不管怎样,快速检测法可用于判断样品是否存在某些霉菌毒素。如果存在,则需要确认检测法来定量其中的含量。

快速检测法

霉菌毒素的快速检测法有免疫测定法或ELISA(抗体检测法)、薄层色谱法、TLC等。这些检测方法常用于商业和公共的实验室,服务于兽医、营养家、农场,有时还使用于饲料加工厂、兽医工作室、部分农场。这些方法可快速出结果,一天仅需花几分钟即可。

依靠于较快速的检测速度和较大的样品数量,ELISA检测法是最常见的选择。它可以作为不同水平、不同范围的霉菌毒素的定性、或半定量分析法。商业ELISA快速试剂盒可适用于多数重要毒素,如黄曲霉毒素、赭曲霉毒素A、T-2毒素、DON、HT-2毒素、玉米赤霉烯酮、伏马毒素。

作为一种筛查工具,薄层色谱法或TLC也可像ELISA 法一样使用,且具备更好的重复性和更少的交叉反应,但是这些方法也需要进一步的样品清洁处理,和花费更多的时间。

其它的检测方法,如免疫检测试纸条、免疫渗滤法都使用了表面分布抗体的横向流装置。化学、生物传感器也可用于抗体、酶、细菌、受体、DNA、表面等离子共振体、红外线光谱等检测方法中。这些方法都可用于主要的、常规的毒素检测,但需注意交叉污染和假阳性。

确认检测法

定量确认检测法使用了?高效液相色谱法(HPLC)、气相色谱法,及UV或荧光检测法(FLD)。它们一般用于快速检测法结果为强阳性时,可进一步确认检测结果和为存在的霉菌毒素提供更准确的信息。

HPLC-UV 或 HPLC-FLD可根据霉素的化学特性来分离、鉴定、定量霉菌毒素,因为其可靠性、较低的检测低限、可行的定量法,目前仍然是霉菌毒素检测的“黄金法则”。但是这种方法也存在较多的限制性,如复杂性、耗时的增加、成本的增加、每次只能集中于结构类似的毒素。因此,用这些方法检测多种霉菌毒素时,所用的时间和分析也会随之增加。

虽然快速检测法、确认检测法可以用于判断、鉴定污染物,但研究者应知道,不仅自然界存在的霉菌毒素远多于我们目前可检测出的毒素数量,而且还有很多的因素可以帮助我们已知的某些霉菌毒素逃避我们的检测。

附原文:

Testing for hidden mycotoxins

Sometimes it's what you can't see that you really need to worry about. Mycotoxins are not always visible in feed and current testing methods don't always give the complete picture.

Some plants are able to change the character of toxins so they don't register as mycotoxins. What are feed producers to do about these “hidden” mycotoxins?

It's difficult to take action against dangerous mycotoxins when a test result might show that there is nothing to fear.

When it comes to mycotoxins—destructive compounds created by molds—producers and feed mill operators should keep a wary eye on their feedstuffs. Many toxins lurking in feed or storage bins continue to hide themselves even after rapid sample tests proclaim mycotoxin masses below range of detection, which can leave farmers with the assumption that they have been spared from contamination.

Common testing methods?

When dealing with feed, the first step towards recognizing that there may be a problem may be noting the presence of mold. However, there is poor correlation between the observations of mold, both visually and through mold count analysis, and mycotoxin presence. Even testing for toxigenic characteristics of the mold through genotyping cannot guarantee the presence or the absence of mycotoxins because their synthesis is induced by multi-factorial environmental stress.

Various methods of testing to identify mycotoxin contamination currently exist, some of which can be conducted quickly and easily and others requiring more in-depth laboratory analysis.

In most cases, mycotoxins are analyzed by either quick tests or confirmatory tests. Quick tests are rapid solutions that can be useful for in-field analysis and as a first set of measures for rejection/acceptance of feed material.

However, the narrow range of mycotoxins targeted, problems with result accuracy and their significant costs are key limitations. Nevertheless, quick tests are used to determine whether samples are positive for some mycotoxins. If they are positive, quantification generally involves confirmatory tests.

Quick tests?

Quick tests include immunoassays, or ELISA, which use antibodies to detect mycotoxins, and thin layer chromatography, TLC, testing. These tests are available from many commercial and public laboratories serving veterinarians, nutritionists and farmers and are sometimes run at feed mills, veterinary offices and on some farms. They provide fast results and may take only a few minutes to one day.

ELISA is commonly chosen due to its speed and the significant number of samples that can be analyzed, and is available as tests with a yes/no answer, or as semi-quantitative tests for various concentration levels and ranges. Commercial ELISA quick test kits are available for most of the key mycotoxins, including aflatoxins, ochratoxin A, T-2 toxin, DON, HT-2 toxin, zearalenone and fumonisins.

As a screening tool, thin-layer chromatography, or TLC, can be applied in the same way as ELISA is, with better repeatability and less cross-reactivity, but it requires further sample clean-up and a consequent increase in the amount of time needed to obtain a precise ratio.

Other strategies include using lateral flow devices such as immunostrips, immunodipsticks and immunofiltration with immobilized antibodies on their surface. Sensors and biosensors are also available using antibodies, enzymes, bacteria, receptors, DNA, surface plasmon resonance or infrared spectroscopy. These techniques are able to rapidly test for the main groups of regulated mycotoxins, but need to be used with caution due to cross-reactivity and false-positives.

Confirmatory tests?

Quantitative confirmatory tests use high-performance liquid chromatography, HPLC, or gas chromatography coupled with UV or fluorescent detection, FLD. They are used when quick tests are strongly positive. They can confirm results and provide more accurate information about the amount of mycotoxin present.

HPLC-UV or HPLC-FLD is used to separate, identify and quantify compounds according to their chemical properties and still represents the “gold standard” in the detection of mycotoxins because of its reliability and the low limits of detection and quantification achievable. However, although more definitive, such methods involve complicated, time-consuming extraction and a consequent rise in cost, and only focus on groups of toxins of similar chemical structure at one time. Thus, with these methods, testing for a range of toxins requires significant time and investment.

While the quick tests and confirmatory tests can be helpful in identifying contaminants, researchers are learning that not only are there more mycotoxins present than we can currently test for, but there are factors that may allow some well-known toxins to escape detection.

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