CN1320060A - Method and apparatus for enrichment of selected groups of microorganisms - Google Patents

Abstract

Translated fromChinese

本发明描述了从微生物混合物中浓集目标微生物的方法和装置。由诸如尼龙、聚苯乙烯或玻璃等材料制成的珠粒(1)包被了针对特定微生物的抗体。珠粒(1)容纳在栅格材料(4)围成的围栏(2)中。栅格的孔径小于珠粒体积以确保珠粒留在栅格材料内,并且大于微生物的体积以使微生物与珠粒相互作用。棒(5)与围栏(2)的上部相连,以便能在含有目标微生物的生长培养基中搅动该装置。

The present invention describes methods and devices for concentrating target microorganisms from a mixture of microorganisms. Beads (1) made of materials such as nylon, polystyrene or glass are coated with antibodies against specific microorganisms. The beads (1) are housed in a fence (2) surrounded by grid material (4). The grid has a pore size smaller than the volume of the beads to ensure that the beads remain within the grid material, and larger than the volume of the microbes to allow the microbes to interact with the beads. A rod (5) is attached to the upper part of the enclosure (2) in order to agitate the device in the growth medium containing the target microorganism.

Description

Translated fromChinese

浓集所选微生物群的方法和装置Method and apparatus for enrichment of selected groups of microorganisms

背景-发明领域Background - Field of Invention

本申请基于1998年8月24日提交的临时专利申请No.60/097,627。This application is based on Provisional Patent Application No. 60/097,627, filed August 24,1998.

本发明涉及用来检测样品中微生物的产品和方法。更具体地说，本发明提供了有助于检测食物样品、临床样品和其它产品中特定的微生物污染是否存在的方法和装置。The present invention relates to products and methods for detecting microorganisms in samples. More specifically, the present invention provides methods and devices that facilitate the detection of the presence of specific microbial contamination in food samples, clinical samples, and other products.

背景-现有技术Background - prior art

测试各种物质(如食物、饮料、药物、化妆品、水以及体液)中微生物污染，尤其是某些病原性细菌的污染，是非常有必要的。最近爆发的食物引起的疾病暗示各种食物已经被诸如大肠杆菌0157:H7、沙门氏菌属、李斯特氏菌属、空肠弯曲杆菌空肠亚种和圆孢子(Cyclospora)等病原性细菌污染，因此增强了对迅速分析微生物方法的需求。微生物分析对于评价安全性和质量、确定生产效率和与遵守规章条例是关键的。It is very necessary to test for microbial contamination in various substances (such as food, beverages, drugs, cosmetics, water and body fluids), especially for certain pathogenic bacteria. Recent outbreaks of food-borne illness suggest that various foods have been contaminated with pathogenic bacteria such as Escherichia coli 0157:H7, Salmonella spp., Listeria spp., Campylobacter jejuni subsp. The need for rapid analytical microbiological methods. Microbiological analysis is critical for evaluating safety and quality, determining production efficiency, and complying with regulations.

微生物测试范围、意义和需求的增加起了揭示常规方法有限性和缺点的作用。确定样品中病原性细菌存在的经典方法通常要花数天时间来进行。希望能迅速检测出引起疾病的具体病原菌。The increase in scope, significance, and demand for microbiological testing has acted to reveal the limitations and shortcomings of conventional methods. Classical methods to determine the presence of pathogenic bacteria in a sample usually take days to perform. It is hoped that the specific pathogen causing the disease can be quickly detected.

由于大多数病原菌试验所需的灵敏度低于每25克产品一个微生物，因此大多数测试方法依赖于首先的富集步骤。通常大量存在于许多食物中的固有的微生物菌丛通常会干扰病原菌的选择性分离和鉴定。诸如加热、冷却、干燥、冷冻、加入防腐剂等食物加工以及其它原因可能会亚致死性地损伤细菌细胞。这些受损的细胞对用于选择性微生物培养基中的组分极其敏感。因此，在许多试验中，方法从预富集开始，将样品培育在营养性非选择性培养基中，以使受损或受应激的细菌复苏。该步骤后是选择性富集步骤，使感兴趣的细菌生长而抑制固有的微生物菌丛。富集步骤后是常规的接种方法或各种更先进、迅速的方法如DNA扩增或免疫试验。Since the required sensitivity for most pathogen tests is less than one organism per 25 grams of product, most test methods rely on an enrichment step first. Intrinsic microbial flora, often present in abundance in many foods, often interferes with the selective isolation and identification of pathogenic bacteria. Food processing such as heating, cooling, drying, freezing, adding preservatives, and other causes can damage bacterial cells sublethally. These damaged cells are extremely sensitive to components used in selective microbial media. Therefore, in many assays, methods begin with pre-enrichment, incubating samples in nutrient non-selective media to allow recovery of damaged or stressed bacteria. This step is followed by a selective enrichment step, allowing the bacteria of interest to grow while suppressing the resident microbial flora. The enrichment step is followed by conventional vaccination methods or various more advanced and rapid methods such as DNA amplification or immunoassays.

因此希望在早期阶段将目标生物体从产品中其它菌群分离出来。一种方法是用免疫-磁性分离技术，该技术涉及采用对目标微生物有特异性的免疫磁性颗粒。将表面附有抗体的磁性珠粒与含有目标微生物的样品混合。该微生物将通过抗体与珠粒表面结合。用磁铁从溶液中吸出微生物-磁珠复合物，以浓集微生物。It is therefore desirable to separate the target organism from other flora in the product at an early stage. One approach is the immuno-magnetic separation technique, which involves the use of immunomagnetic particles specific for the target microorganism. Magnetic beads with antibodies attached to their surfaces are mixed with a sample containing the microorganism of interest. The microorganism will bind to the surface of the bead via the antibody. Concentrate the microorganisms by aspirating the microorganism-magnetic bead complexes from the solution with a magnet.

美国专利4,230,685描述了外表面结合有蛋白A的磁性反应性的微球。微球与对细胞、细菌或病毒有选择性的抗体反应，以便将其从混合群体中分离出来。微生物将附着于抗体，从而与微球相连，然后将微球用于磁性分离步骤中。较佳的微球由白蛋白、蛋白A和磁性颗粒的混合物制得。制备微球，使蛋白A在抗体结合的外表面上。美国专利4,695,393描述了一种制备这种可用来分离微生物的磁性珠粒的方法。US Patent 4,230,685 describes magnetically reactive microspheres with Protein A bound to the outer surface. The microspheres react with antibodies that are selective for cells, bacteria or viruses in order to separate them from mixed populations. The microbes will attach to the antibody, thereby linking to the microspheres, which are then used in the magnetic separation step. Preferred microspheres are made from a mixture of albumin, protein A and magnetic particles. Microspheres were prepared so that Protein A was on the outer surface for antibody binding. US Patent 4,695,393 describes a method for preparing such magnetic beads that can be used to isolate microorganisms.

美国专利5,491,068和5,695,946描述了一种方法，该方法的特征是利用专门的磁性珠粒来抗体捕获感兴趣的微生物。该方法需要将捕获细胞培育形成菌落；用菌落提升膜取出菌落中的物质；和用标记的抗体、PCR或核酸探针检测膜片上的菌落物质。该方法的主要问题是灵敏度低，只能测出1个微生物/克。该低灵敏度是该方法所固有的，它比检测大多数食物病原体所需的灵敏度低50-100倍。US Patents 5,491,068 and 5,695,946 describe a method that features the use of specialized magnetic beads for antibody capture of microorganisms of interest. The method needs to cultivate the captured cells to form a colony; use a colony lifting membrane to take out the substance in the colony; and use a labeled antibody, PCR or nucleic acid probe to detect the colony substance on the membrane. The main problem of this method is the low sensitivity, only 1 microorganism/gram can be detected. This low sensitivity is inherent to the method, which is 50-100 times less sensitive than is required to detect most food pathogens.

美国专利4,677,055描述了一种采用与抗特异性抗原决定簇抗体偶联的磁性凝胶来浓集细菌的方法。该方法涉及获得含有具有特异性抗原决定簇的微生物的培养基以及使培养基与磁性凝胶颗粒接触的步骤。该步骤后是用磁性方式分离培养基中的凝胶，并接种到新的培养基中。US Patent 4,677,055 describes a method for concentrating bacteria using magnetic gels coupled with antibodies against specific epitopes. The method involves the steps of obtaining a culture medium containing microorganisms with specific antigenic determinants and contacting the culture medium with magnetic gel particles. This step is followed by magnetic separation of the gel from the medium and inoculation into new medium.

总之，磁性珠粒存在很多问题。一个问题是由这些珠粒的小体积(3-10微米)和培养基的大体积(25-3000)毫升)引起的。结果，不可能从如此大的体积中取出磁性珠粒。因此，许多步骤或是使用较少体积的样品(从而降低了试验的灵敏度)，或在取出1-5毫升溶液后预富集一段时间(8-18小时)以便浓集目标微生物。与磁性珠粒有关的另一个问题是，它们被脂肪和蛋白质包覆，因此难以用磁铁来收集。从培养基中分离出珠粒并洗去未吸附的细菌要使用大量劳力，并且产生实验室表面和珠粒受污染的危害。In summary, there are many problems with magnetic beads. One problem arises from the small volume of these beads (3-10 microns) and the large volume of culture medium (25-3000 ml). As a result, it is impossible to remove magnetic beads from such a large volume. Therefore, many steps either use smaller sample volumes (thereby reducing the sensitivity of the assay), or pre-enrichment for a period of time (8-18 hours) after withdrawal of 1-5 ml of solution in order to concentrate the target microorganisms. Another problem with magnetic beads is that they are coated with fats and proteins, making them difficult to collect with magnets. Separating the beads from the culture medium and washing away unadsorbed bacteria is labor intensive and creates the risk of contamination of laboratory surfaces and beads.

目的和优点purpose and advantages

因此，本发明的目的是提供一种可使用大体积培养基浓集目标微生物的方法和装置。本发明的另一目的是提供耗劳力较少的、更迅速的且本身能自动化的方法。Accordingly, it is an object of the present invention to provide a method and a device which allow the concentration of target microorganisms using large volumes of culture medium. Another object of the present invention is to provide a method that is less labour-intensive, faster and itself automatable.

还有其它目的和优点可在阅读了随后的说明和附图后明白。Still other objects and advantages will become apparent upon reading the ensuing description and drawings.

附图简述Brief description of the drawings

图1显示了用来浓集目标微生物的较佳装置的侧视图。Figure 1 shows a side view of a preferred apparatus for concentrating target microorganisms.

图2显示了用来浓集目标微生物的装置的另一种设计的侧视图。Figure 2 shows a side view of another design of the device used to concentrate the microorganisms of interest.

较佳的实施方案-说明Preferred Embodiment - Description

图1显示了用来从含有微生物混合物的悬浮液中分离出目标微生物的装置的较佳实施方案。珠粒1由诸如尼龙、聚苯乙烯或玻璃等材料制成。珠粒上包被了针对诸如沙门氏菌属、大肠杆菌0157:H7和李斯特氏菌属等具体微生物的抗体。设计圆筒形围栏2来包含这些珠粒。围栏由框架3支持的覆盖该框架的栅格4构成。栅格的孔径小于珠粒体积，以确保珠粒留在围栏2中。然而，孔径大得足以使细菌自由进入围栏。围栏上部与棒5相连。棒5能使围栏2在溶液中移动以及随后将该装置取出溶液。Figure 1 shows a preferred embodiment of an apparatus for isolating microorganisms of interest from a suspension containing a mixture of microorganisms. The beads 1 are made of materials such as nylon, polystyrene or glass. The beads are coated with antibodies against specific microorganisms such as Salmonella, E. coli 0157:H7, and Listeria. Cylindrical enclosure 2 is designed to contain these beads. The fence consists of a frame 3 supported by a grid 4 covering the frame. The pore size of the grid is smaller than the volume of the beads to ensure that the beads stay in the enclosure 2. However, the pore size is large enough to allow bacteria to freely enter the enclosure. The upper part of the fence links to each other with the rod 5. The rod 5 enables the movement of the fence 2 in the solution and the subsequent removal of the device from the solution.

图2显示了该装置的不同设计。包被了抗体的珠粒11被容纳在栅格13制得的茶叶袋状的围栏12内。非灯芯材料的线14与围栏12上部相连，从而能使围栏12在溶液中移动，同时不会使线吸收含有细菌的溶液。栅格13的孔径小于珠粒体积，以确保珠粒留在围栏内。然而，该孔径大得足以使细菌自由进入围栏。Figure 2 shows different designs of the device. Antibody-coated beads 11 are accommodated in tea bag-shaped enclosures 12 made of grids 13 . A thread 14 of non-wicking material is attached to the upper portion of the fence 12 to allow the fence 12 to move through the solution without the thread absorbing the solution containing bacteria. The pore size of the grid 13 is smaller than the volume of the beads to ensure that the beads stay within the enclosure. However, the pore size is large enough to allow bacteria to freely enter the enclosure.

将待测试目标微生物是否存在的食物样品与合适的预先富集用的肉汤混合。将预先富集用的肉汤培育在合适的温度下。在培育期开始时，或在培育数小时后，将围栏2浸入含有样品的肉汤中，从而使其上固定了单克隆或多克隆抗体的珠粒与选择的感兴趣的细菌接触。通过将装置2降低到溶液中并搅动溶液至少30分钟至多达数小时，而实现这个目的。该步骤能使珠粒捕获细胞，并产生珠粒-目标微生物细胞的复合物。下一步骤是从含有食物颗粒和其它混合菌群的悬浮液中分离出结合有目标细胞的珠粒。这可通过用棒5从溶液中抽出整个装置来实现。随后在无菌盐水或缓冲溶液中洗涤该装置数次。每次洗涤后更换洗液，以除去未结合的微生物。在培育肉汤混合液中加入诸如吐温20(0.51-0.1％w/v)或鱼精蛋白等洗涤剂通常会减少非特异性吸附。吐温-20还可用于洗涤步骤中，用来除去非特异性结合的细胞。在洗涤步骤后，可用许多方法来检测目标微生物的存在。A food sample to be tested for the presence of the target microorganism is mixed with a suitable pre-enrichment broth. The pre-enrichment broth is incubated at a suitable temperature. At the beginning of the incubation period, or after several hours of incubation, pen 2 is dipped into the broth containing the sample, thereby bringing the beads immobilized with monoclonal or polyclonal antibodies into contact with the selected bacteria of interest. This is accomplished by lowering the device 2 into the solution and agitating the solution for at least 30 minutes up to several hours. This step enables the beads to capture the cells and create bead-target microbial cell complexes. The next step is to isolate the beads bound to the cells of interest from the suspension containing food particles and other mixed flora. This is accomplished by pulling the entire device out of solution with the rod 5 . The device is then washed several times in sterile saline or buffered solution. Change the wash solution after each wash to remove unbound microorganisms. Addition of detergents such as Tween 20 (0.51-0.1% w/v) or protamine to the incubation broth mix will usually reduce non-specific adsorption. Tween-20 can also be used in the wash step to remove non-specifically bound cells. Following the washing step, a number of methods can be used to detect the presence of target microorganisms.

一些检测程序可和本发明结合使用，以检测感兴趣微生物的存在。例如，可将该装置插入新的生长肉汤中，该肉汤含有染料指示剂，可用染料特征的变化来确定目标微生物的存在与否。无需将微生物从珠粒上分离下来，因为附着于珠粒对其生长没有影响。因此，细胞能继续在合适的培养基中增殖。或者，可从围栏中取出珠粒，接种到合适的选择性或差异性琼脂表面上。另一种方法是用免疫试验。大多数免疫试验要求每毫升有10³-10⁵个细胞，因此珠粒应当含有足量的细胞来进行直接免疫试验。类似地，该方法可以和DNA杂交以及诸如PCR等扩增技术组合。Several detection procedures can be used in conjunction with the present invention to detect the presence of microorganisms of interest. For example, the device can be inserted into new growth broth that contains a dye indicator, and a change in the signature of the dye can be used to determine the presence or absence of the target microorganism. There is no need to detach the microorganisms from the beads, as attachment to the beads has no effect on their growth. Thus, the cells can continue to proliferate in the appropriate medium. Alternatively, beads can be removed from the pens and plated onto a suitable selective or differential agar surface. Another way is to use immunoassays. Most immunoassays require 10³ -10⁵ cells per milliliter, so beads should contain sufficient cells for direct immunoassays. Similarly, this method can be combined with DNA hybridization and amplification techniques such as PCR.

从上文公开的内容看出，本发明方法的特征在于使用容纳在围栏中的免疫珠粒来从样品中选出目标微生物。该珠粒必需能从测试样品中有效地捕获目标微生物，同时不会捕获可能以多得多的数量存在的其它微生物。然而，所用的抗体无需对目标微生物具有完全的特异性，因为在试验最后还可进行额外的选择步骤。抗体结合部位的取向必须朝外，以使抗体的结合部分与目标微生物之间接触。珠粒大小必须大于微生物的体积，以便将珠粒留在围栏内，同时使目标微生物进入围栏附着于珠粒。珠粒与目标微生物之间的接触时间必须足够长，以便发生强的相互作用。发现数小时的相互作用才能产生最佳的结果，即产生强相互作用，获得高捕获效率。培育步骤完成后，通过取出装有珠粒的围栏，可从溶液中取出珠粒。洗涤围栏和珠粒数次，并将珠粒转移到检测系统中。From the above disclosure, the method of the present invention is characterized by the use of immunobeads contained in enclosures for the selection of target microorganisms from a sample. The beads must be effective at capturing the target microorganisms from the test sample while not capturing other microorganisms that may be present in much greater numbers. However, the antibodies used need not be completely specific for the target microorganism, since an additional selection step can be performed at the end of the assay. The orientation of the antibody binding site must be outward so that there is contact between the binding portion of the antibody and the target microorganism. The bead size must be greater than the volume of the microorganisms in order to keep the beads inside the enclosure while allowing the target microorganisms to enter the enclosure and attach to the beads. The contact time between the beads and the target microorganism must be long enough for a strong interaction to occur. Interactions of several hours were found to yield the best results, i.e. strong interactions resulting in high capture efficiencies. After the incubation step is complete, the beads can be removed from the solution by removing the enclosure containing the beads. Wash the pens and beads several times and transfer the beads to the detection system.

结论、分支和范围Conclusion, branches and scope

因此，可以看出该新的装置和方法可以利用大体积的培养基来浓集目标微生物，无需仅用磁性珠粒所需的一部分预先富集用的肉汤或是少量富集肉汤。本发明提供了劳动强度低、更快速且本身能自动化的方法和装置。在试验各步骤期间用于容纳珠粒的许多不同的设计均可利用。Thus, it can be seen that the new device and method can utilize large volumes of culture medium to concentrate target microorganisms without requiring only a fraction of the pre-enrichment broth or small amounts of enrichment broth required for magnetic beads. The present invention provides methods and apparatus that are less labor intensive, faster and inherently automatable. Many different designs are available for housing the beads during the various steps of the assay.

显然，在参看了上述技术后能对本发明作许多改进和变化。虽然上文的描述包含了许多具体特征，但是它们不应被理解成对本发明范围的限制，它们只是为本发明的一些较佳实施方案提供了说明。本发明可以本文具体描述以外的方式来实施。Obviously many modifications and variations of the present invention are possible in light of the above teachings. While the description above contains many specificities, these should not be construed as limitations on the scope of the invention but as illustrations of some of the preferred embodiments of this invention. The invention may be practiced otherwise than as specifically described herein.

Claims (7)

Translated fromChinese

1．一种从含有微生物混合群体的悬浮液中分离特定的目标微生物的装置，该装置包含：1. A device for isolating specific target microorganisms from a suspension containing a mixed population of microorganisms, the device comprising:多个包被了至少一种用来捕获目标微生物的抗体材料的珠粒；和a plurality of beads coated with at least one antibody material for capturing target microorganisms; and由包围所述珠粒的栅格材料制成的围栏，栅格的孔径小于所述珠粒的体积并大于微生物的体积。An enclosure made of a grid material surrounding the bead, the grid having a pore size smaller than the volume of the bead and larger than the volume of the microorganism.2．根据权利要求1所述的装置，其中所述珠粒用树脂材料制成。2. The device according to claim 1, wherein said beads are made of a resin material.3．根据权利要求1所述的装置，其中所述珠粒用非树脂材料制成。3. The device of claim 1, wherein said beads are made of a non-resin material.4．根据权利要求1所述的装置，它还包含在悬浮液中搅动所述围栏的手段。4. 2. The apparatus of claim 1, further comprising means for agitating said enclosure in suspension.5．一种从含有微生物混合群体的悬浮液中分离目标微生物的方法，该方法包括：5. A method of isolating a target microorganism from a suspension containing a mixed population of microorganisms, the method comprising:将多个包被了至少一种抗体材料的珠粒浸在该悬浮液中，所述珠粒被栅格制成的围栏所截留，栅格的孔径小于所述珠粒的体积并大于微生物的体积，从而使目标微生物被所述珠粒捕获；和Submerging in the suspension a plurality of beads coated with at least one antibody material, said beads being retained by a fence made of a grid having a pore size smaller than the volume of said beads and larger than that of the microorganism volume, so that target microorganisms are captured by the beads; and在将所述围栏从悬浮液中拉出后，洗涤所述珠粒以除去未与所述珠粒结合的微生物。After the pens are pulled out of suspension, the beads are washed to remove microorganisms not bound to the beads.6．根据权利要求5所述的方法，其中在所述洗涤中应用至少一种洗涤剂。6. The method according to claim 5, wherein at least one detergent is used in said washing.7．根据权利要求5所述的方法，该方法还包括在悬浮液中搅动容纳了所述珠粒的所述围栏。7. The method of claim 5, further comprising agitating said enclosure containing said beads in suspension.