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Publication numberUS20030143531 A1
Publication typeApplication
Application numberUS 10/321,784
Publication dateJul 31, 2003
Filing dateDec 16, 2002
Priority dateMar 1, 1999
Also published asCA2367587A1, DE60025906D1, DE60025906T2, EP1155148A1, EP1155148B1, EP1605062A1, US6495325, WO2000052206A1
Publication number10321784, 321784, US 2003/0143531 A1, US 2003/143531 A1, US 20030143531 A1, US 20030143531A1, US 2003143531 A1, US 2003143531A1, US-A1-20030143531, US-A1-2003143531, US2003/0143531A1, US2003/143531A1, US20030143531 A1, US20030143531A1, US2003143531 A1, US2003143531A1
InventorsHendrik van Haeringen, Willem Van Haeringen
Original AssigneeVan Haeringen Hendrik, Van Haeringen Willem Anne
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Detection and quantification of microorganisms using amplification and restriction enzyme analysis
US 20030143531 A1
Abstract
In livestock, a number of pathological conditions and/or syndromes have been noticed that do not seem to be attributable to, for example, an infection with a single microorganism. These syndromes however do seem to have an effect on the equilibrium which normally exists within the different bacteria constituting the bacterial flora. A number of these syndromes are referred to as dysbacteriosis and bacterial overgrowth. To enable at least in part the evaluation of the “general” health of the livestock the invention provides means and methods for analyzing the composition of a microbiological flora. In one aspect a method of the invention comprises providing a sample of the flora, selectively amplifying nucleic acid present in the sample, subjecting the amplificate to restriction digestion and analyzing the resulting pattern of restriction fragments.
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Claims(20)
What is claimed is:
1. A method of analyzing a microbiological flora containing more than one species of microorganism, said method comprising:
providing a sample of said microbiological flora;
amplifying nucleic acid from the more than one species of microorganism presenting in said sample to produce an amplificate;
subjecting the amplificate to at least one restriction digestion to produce restriction fragments from the more than one species of microorganism; and
preparing a resulting pattern based on the restriction fragments of the more than one species of microorganism in the sample.
2. The method according to claim 1, wherein said microbiological flora is selected from the group consisting of bacterial flora, yeast flora, fungal flora, viral flora, and mixtures of any thereof
3. The method according to claim 1, wherein said sample is taken from a source selected from the group consisting of digestive tract, organ, tissue, water, and food product.
4. The method according to claim 1 further comprising:
comparing the resulting pattern of the sample with at least one reference pattern.
5. The method according to claim 5, wherein said reference pattern is derived from another source.
6. The method according to claim 1, further comprising analyzing the resulting pattern of restriction fragments to determine the relative abundance of micro-organisms included in flora.
7. The method according to claim 1 wherein the sample is analyzed to determine hygienic status.
8. The method according to claim 4, wherein said reference value is obtained from a database.
9. A method of determining the relative ratios of microorganisms present in a microbiological flora, said method comprising:
providing a sample of said microbiological flora;
amplifying nucleic acid from the more than one species of microorganism present in said sample to produce an amplificate;
subjecting the amplificate to at least one restriction digestion to produce restriction fragments from the more than one species of microorganism;
preparing a resulting pattern based on the restriction fragments of the more than one species of microorganism in the sample; and
analyzing the resulting pattern of restriction fragments to determine the relative ratios of microorganisms present in said flora.
10. A test kit for analyzing a composition of microbiological flora having more than one species of microorganism, the test kit comprising:
two primers for amplification of nucleic acids in a the composition of microbiological flora to produce an amplificate;
at least one restriction enzyme for digesting an amplificate of a microbiological flora sample to produce restriction fragments from more than one species of microorganism; and at least one reference pattern for comparative analysis of a resulting pattern of restriction fragments from digestion of a microbiological flora sample.
11. The method according to claim 1, wherein analyzing the resulting pattern of restriction fragments comprises determining presence or absence of equilibrium in intestinal bacterial flora.
12. The method according to claim 1, wherein providing a sample comprises obtaining a sample from feces of a subject.
13. The method according to claim 1, wherein said providing a sample comprises obtaining the sample from a broiler or human subject.
14. The method according to claim 1, wherein said providing a sample comprises obtaining a sample from a tissue or organ of a subject, and further comprising analyzing the total bacterial content of said tissue or organ.
15. The method of claim 4, wherein said reference pattern is a standardized baseline pattern derived from a plurality of subjects with healthy flora.
16. The method of claim 1, wherein said subjecting the amplificate to restriction digestion comprises subjecting the amplificate to restriction enzymes recognizing nucleotide sequences of about 4 to about 8 nucleotides.
17. A method of analyzing the composition of a bacterial flora, comprising: obtaining a sample of bacterial flora taken from the digestive tract of a subject;
amplifying nucleic acid from more than one species of bacteria present in said sample with at least one primer of about 20 to 30 nucleotides directed to a nucleic acid believed to be present in said sample to produce an amplificate; subjecting the amplificate to restriction digestion;
analyzing a resulting pattern of restriction fragments by comparing the restriction pattern of the sample with at least one reference restriction pattern; and determining relative abundance and relative ratios of said bacterial flora.
18. The method according to claim 17, wherein analyzing the resulting pattern of restriction fragments comprises determining presence or absence of equilibrium in intestinal bacterial flora.
19. A method of analyzing a sample of microbiological flora containing more than one species of microorganism, said method comprising:
providing the sample of said microbiological flora;
subjecting the sample to at least one restriction digestion to produce restriction fragments from the more than one species of microorganism to produce a restriction digestion product;
amplifying the restriction digestion product to produce an amplificate; and
preparing a resulting pattern based on the restriction fragments of the more than one species of microorganism in the sample.
20. The method according to claim 19 further comprising:
comparing the resulting pattern of the sample with at least one reference pattern.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser. No. 09/702,561, filed on Oct. 31, 2000, U.S. Pat. No. 6,495,325 (Dec. 17, 2002), which is a continuation of International Application No. PCT/NL00/00125, filed Mar. 1, 2000, designating the United States of America, (published Sep. 8, 2000 in English as WO 00/55206) the contents of the entirety of all of which are incorporated by this reference.

TECHNICAL FIELD

[0002] The present invention relates to methods and means for diagnosis of pathological or other detrimental conditions or the general health of a group of individuals, in particular vertebrates, more typically mammals and/or birds, such as livestock. The invention further relates to methods and means for microbiological typing of substances comprising a variety of microbiological organisms such as but not limited to food, food additives, waste material, soils etc.

BACKGROUND

[0003] In livestock, a number of pathological conditions and/or syndromes have been noticed that do not seem attributable to, for example, an infection with a single microorganism. These syndromes, however, do seem to have an effect on the equilibrium which normally exists within the different bacteria constituting the bacterial flora. A number of these syndromes are referred to as dysbacteriosis and bacterial overgrowth.

[0004] Although not wishing to be bound by theory a possible explanation for the presence of these syndromes, that these diseases may occur due to

[0005] 1. The feed intake/feed type. The feed interferes with the digestion, resulting in changes in the bacteria: some particular bacteria or groups of bacteria are abundant and overwhelming, some particular bacteria or groups of bacteria are inhibited and cannot survive.

[0006] 2. Invasion of one (or more) bacterial species. The bacteria do not need to be pathogenic themselves, but disturb the balance within the flora.

[0007] 3. Invasion of one (or more) viral or fungal species.

[0008] 4. Immune depression.

[0009] 5. Environmental effects such as temperature and humidity.

[0010] Typically, these syndromes influence the general health and performance of an animal and often even on the whole of the group of animals. In poultry, there are even a number of syndromes which show clinical symptoms directly related to the condition of the intestinal bacterial flora.

[0011] Therefore, it is of importance to be capable to check the general health of a population of livestock and/or to have a means of at least determining the presence of such an ill-defined pathological condition in a group (herd, flock) of, for example, livestock..

[0012] Examples of syndromes related to bacterial disequilibrium are, for example, steathorrea in calves, ileitis in pigs, and also diarrhoea at weaning, diarrhoea in poultry.

DISCLOSURE OF THE INVENTION

[0013] The present invention provides novel means (e.g., test kits) and methods for determining (relative) abundance as well as estimates of actual amounts of different bacteria present in intestinal bacterial flora, in order to determine, for example, the presence or absence of equilibrium in the intestinal bacterial flora, especially when compared to a set of normal compositions of intestinal bacterial flora until the present invention analyses of bacterial flora occurred with methods used in classical bacteriology. Classical bacteriology is based on cultivation and identification of different bacterial strains. Various methods are used, including aerobic and anaerobic growth, pre-enrichment, etc.

[0014] Furthermore, counting the number of colonies has generally been used to obtain information about the number of bacteria present in samples.

[0015] Using these methods, only an estimated 20% of the bacteria have been isolated and characterized until now. Nearly axiomatic in these classical bacterial techniques (as well as with techniques finding other microorganisms, such as viruses, yeast or fungi) is the thinking that one disease has one causal agent, or at least that one or a distinct few agents can be considered instrumental in causing the distinct disease. However, these classical insights do not provide any practical manner of obtaining insight into the presence or absence of a “healthy” equilibrium in intestinal tracts.

[0016] Thus, the present invention provides a method for analyzing the composition of microbiological (e.g., viral, fungal, yeast or bacterial) flora, especially in an intestinal tract. The method includes providing a sample of the flora, selectively amplifying nucleic acid in the sample, subjecting the amplificate to restriction digestion, and analyzing the resulting pattern of restriction fragments, providing detection of interrelationships between flora constituents such as known and unknown bacteria, fungi, viruses and the like, for example, under distinct disease conditions without the need to identify an organism. The nucleic acid is selectively amplified, albeit usually from as many, or the greater part of, microorganism species as possible present in the sample, optionally supplemented with specific amplification of known organism species or parts thereof possibly present in the sample to provide optional identification, when so desired. Typical results include the detection of a cluster or cluster of amplified fragments (which may have been subjected to restriction), the fragments localized or detected in a pattern (governed, for example, by molecular mass) allowing the recognition of specific (cluster) patterns. These may be identical in various samples, me be different, or may be supplemented with additional clusters (typically nearly equally sized fragments). Typically, when the sample is representative of a “normal” population, a well balanced cluster pattern may be seen, whereas, when for example dysbacteriosis or overgrowth has occurred, one cluster (commonly representing one microorganism) may be over-represented, typically to the detriment of other clusters (representing others). The sample may typically be derived from feces, or a biopsy and in many cases can be taken post-mortem.

BRIEF DESCRIPTION OF THE FIGURES

[0017]FIGS. 1 and 2 represent the DNA-patterns of three microbiological populations obtained from three individuals. FIG. 1 shows DNA-fragments with sizes ranging from 100 to 900 base pairs, whereas FIG. 2 shows DNA-fragments between 300 and 600 base pairs. Each peak in the figures represents a specific DNA-fragment.

[0018] The DNA-fragments for three individual chickens were based on samples form the duodenum. Two samples were derived from clinically ‘healthy, individuals (A and B), whereas one sample represents a non-healthy, individual (C). The non-healthy individual showed one major additional DNA-fragment(marked ‘Additional,) which was not present in two ‘healthy individuals.

[0019]FIGS. 3 and 4 illustrate part of a procedure to test for bacterial overgrowth in broilers. Illustrations show that under distinct disease conditions samples exhibit the same clusters, whereas bacterial variation can easily be identified with the technology. Bacterial clusters, or species are identified through DNA-fragment size, using base pairs.

[0020]FIG. 3. A total of eleven samples are shown. Each sample is based on a specimen from the intestinal tract from clinically healthy chicken at 3.5 weeks of age. At least four major bacterial clusters are present, whereas a few other bacterial species are present in some of the samples as well. Two issues are herewith illustrated. A. Major clusters are present in all samples. B. The relative number of bacteria present is different in the samples.

[0021]FIG. 4. A total of four samples are shown. Each sample is known to exhibit large differences at common bacterial/germ counts. Two issues are illustrated here. A. The procedure is clearly capable of identifying similar, large, differences as detected in the germ counts. Samples with high counts for specific groups in the germ counts show identical DNA-fragments, whereas these fragments are not present in the samples without these specific bacteria. B. This new technology is identifying additional information compared to the ‘classical, germ counts, because our methods are including all bacterial species in one analysis. Even unknown bacteria are included.

DETAILED DESCRIPTION OF THE INVENTION

[0022] Preferably, samples are taken under conditions which can be repeated, so that the differences in the flora are attributable to the conditions to be diagnosed and/or analyzed and not to temporal, dietary or other variations. Post-mortem samples and biopsies should be taken from the same area in the intestinal tract. When using this technique as provided by the invention, it is not per se the mere identification of a tentative causal microorganism that matters, as well as the recognition of distinct patterns in the analysis's results that allow demonstrating equilibrium with respect to a distinct or fitting condition (diseased or non-diseased) in the flora, and allow identification of similar patterns in animals from the same or different flock. Clearly, classical techniques have not paid attention to patterns in flora that for example relate to multifactorial disease patterns, but have commonly tried to identify one or more distinct causal agents, which in general are sought after with predetermined and specific detection means, such as nucleic acid primers or probes, antibodies, and so on, for the detection of specific microorganisms or specific components thereof. For example, in U.S. Pat. No. 5,543,294; JP 05 317096; Ratcliff et al., Path. (1994) 26:477-479; Wood et al., Appl. Env. Microb. (1998) 64:3683-3689; and U.S. Pat. No. 5,571,674 all give specific and direct instructions on how to identify a specific bacterium, or a bacterium belonging to a specific genus or family, but do not pay attention to other microorganisms that are or may be present in the test samples, clearly demonstrating that they are not interested in the whole content of the flora but only in specific microorganisms therein. They will, therefore, never be able to detect interrelationships between flora constituents such as known and unknown bacteria, fungi, viruses and the like.

[0023] In the field of bacteriology, the invention provides, for example, means for quality control, identification and quantification of bacterial species of interest, and detection of presence/absence of highly pathogenic and lethal bacterial species, or the detection of new, previously unknown, bacteria. The invention provides, for example, information on the total of bacteria in any given sample. Because of the nature of the technology, many unidentified bacteria are amplified Thus, when testing clinical samples, new bacterial species are found which could not be identified until now from unknown bacterial populations.

[0024] Furthermore, the invention provides analysis of bacterial populations to provides a method for quality control. For example, in sewage treatment plants, bacteria are used as a part of the process to clean waste water. The nature of the bacteria present in these systems needs to be verified for many reasons. The quality and composition of the bacteria present in these systems need to be checked on a regular basis. Further, the resulting water needs to be verified as well. Both checks are currently performed using classical, methods, amongst which are culturing under different conditions. The invention provides a rapid, better, technology for “quality control” of the whole process. A further advantage of the invention is, that known pathological bacteria (e.g., legionella, salmonella spp.) can be identified in the same process of testing the sample with a method according to the invention.

[0025] In one embodiment, the invention provides a method for testing disease or health status of livestock such as broilers. The bacterial flora in the intestinal tract of broilers is influenced by the composition of feed.

[0026] During the growth of broilers, generally at two or more occasions differences are provided in the composition of the feed. These changes are done to optimize the ratio between feed-intake and growth. Due to the change in feed, the bacterial populations in the intestinal tract are influenced as well. Analysis of the bacterial patterns according to the invention will show the differences due to the changes in feeding.

[0027] In another example, the invention provides a method involving germ counts. On many occasions, culturing of bacterial populations is performed for many purposes and in many research projects. However, it is known that only particular groups of bacteria are visualized or identified (e.g., all coliform bacteria in one group). The present invention is quantitative, when so desired, and can replace the existing use of culturing bacteria. Also, on many occasions, diseases in the intestinal tract are hard to identify. Analysis of the total bacterial contents in any organ, or tissue can be performed in a rapid, reliable fashion, and be helpful in diagnosis of disease of man and animal alike.

[0028] Yet another example comprises food technology. In the process of preparing food, or the quality control of (raw or fermented) products, such as sausages or cheese (it is essential that the same product is manufactured at a constant level. The invention provides a method enabling the detection of bacterial, viral or fungal species, allowing also to verify manufacturing conditions in the same procedure.

[0029] Generally, the sample will have to be pre-treated, for example, homogenized in order to make the nucleic acid accessible for amplification. The sample may be taken from any vertebrate having an intestinal tract and a bacterial flora therein.

[0030] However, mammals and birds are preferred subjects of the present invention, especially humans and domestic animals, in particular cattle, poultry and other livestock. Amplification of nucleic acid should be selective in that the choice of primers should be such that a representative amplificate of all or at least a representative number of microorganisms present in the intestinal flora is obtained. If the primers are chosen well the further details of the amplification technique employed are less critical, so that any amplification method can be used, although PCR is preferred.

[0031] The length of the primers and the amplification conditions can be determined by the person skilled in the art, depending on the sample, the kind of animal, the need for specificity, etc. Typically, primers will vary in length between 20 and 30 nucleotides and typically they will he directed to a nucleic acid sequence which is present in the selected microorganisms present in the intestinal flora of the subjects to be sampled, such as 16S rRNA and 23S rRNA. In some cases, a nested PCR may be useful.

[0032] The amplificate itself typically will not give sufficient specific fragments so that a good analysis of the frequency presence/absence/etc. of the respective microorganisms can be obtained. Therefor it is important to further differentiate the nucleic acids obtained from the respective microorganisms by subjecting them to a treatment with a number of restriction enzymes. In that manner patterns will be obtained which contain sufficient information to enable doing a good analysis on richness, evenness, relative abundance, amounts, in short the state of the intestinal flora. This will certainly be very easy in the case where it is not necessary to identify particular microorganisms, and where it suffices to compare obtained patterns with known patterns of healthy individuals, preferably an average over many healthy individuals with certainty intervals also given.

[0033] The restriction enzymes should be selected such that fragments giving relevant information are obtained. The skilled person is capable of designing a set of restriction enzymes capable of doing just that. Typically the enzymes should recognize sequences of 4-8 base pairs, which enables selective digestion to enhance the information present in the amplificate. Typically, at least 10 different enzymes are used, but usually no more than 4 enzymes are necessary.

[0034] Examples of useful restriction enzymes include, but are not limited to CfoI and HaeIII.

[0035] The analysis of the restricted amplificate can be done in any manner capable of distinguishing between sizes of nucleic acids, in particular DNA fragments. The person skilled in the art is capable of selecting a method best fitted for his purposes. In the case of many samples having to be analyzed some kind of automated system is of course preferred. Good examples of methods for automated analysis systems require possibilities to separate and quantify DNA fragments. For instance, for the present invention, the ABI Prism (Perkin Elmer) is suitable. In order to be able to analyze the pattern in the way of the invention it is preferred to compare the result of amplification and restriction of the sample with at least one reference sample. Some reference should be available for comparing a present state (equilibrium) of a certain animal with a healthy state(equilibrium) of the same animal, another animal or preferably a group of animals known to be generally healthy.

[0036] The more information is present about the healthy state, the easier it becomes to see differences between healthy intestinal floras and floras related to pathological conditions or related to general health problems. Of course, the comparison is most reliable when samples are taken in the same manner under similar, if not the same, circumstances. It will often not be necessary to make direct comparisons between samples, it will generally suffice to compare some kind of test result (numbers, bar codes, graphs, patterns and the like).

[0037] Standardizing the picture of bacterial flora can be done, for example, in the following manner.

[0038] To enable interpretation of samples for various purposes, a baseline is established of the intestinal tract of poultry and fecal material of pigs.

[0039] The choice of animals used for the baseline is depending on a) age, b) housing, c) feed (intake), d) breeding line, e) clinical health, and f) sex.

[0040] An important aspect is the comparability of the samples constituting the baseline and the sample to be tested. Thus, calibration is needed for at least some of the parameters given above.

[0041] An easy way of obtaining a number of references is subjecting pure bacteria cultures (of a bacterium of which it is known that it is present in the intestinal flora) to amplification and restriction in the same manner as sample will be treated.

[0042] The presence and/or abundance of these bacteria in intestinal tracts can be determined based on the presence or absence of (a number of) characteristic peaks.

[0043] The invention also provides a method, wherein the analysis involves determining the relative abundance of microorganisms included in the intestinal flora or even determining simply whether a pattern is within a healthy variation of patterns without even knowing much about the constituting bacteria. This can be done once a collection of healthy patterns is available. Thus, one merely measures the relative abundance and presence/absence of peaks known to be present in the floras of healthy individuals, without knowing from which organisms the peaks originate.

[0044] Of course, the earlier mentioned pure bacteria cultures give the possibility of identifying which peaks belong to which microorganism if so desired. Of such peaks, the height and/or the area under the peak, especially in comparison with other known peaks is a measure for the amount of a certain microorganism present in a sample. Thus, the invention also provides a method according to the invention, wherein the analysis involves determining the amount of at least one microorganism present in the intestinal flora. In another embodiment the invention provides a diagnostic test kit for performing a method according to the invention comprising primers for amplification, restriction enzymes for digestion and optionally at least one reference sample comprising material derived from sat least one pure bacteria culture. Preferably, such a test kit comprises at least one reference pattern as described herein before.

[0045] Suitable primers to include in a test kit according to the invention are primers derived from aligned sequences. This sequence information enables the selection of homologues sequences from which primers can be developed. Preferably, primers are chosen from ribosomal RNA sequences. Preferably, at least one primer comprises the sequence 5′-AGAGTTTGATCCTGGCTCAG-3′ (SEQ ID NO: 1) or a functional fragment and/or derivative thereof. A functional derivative or fragment is a primer having a sequence which base pairs with the sequence with which the original sequence base pairs or with a sequence very closely in the vicinity of the original sequence so that the same or similar nucleic acids are obtained after amplification.

[0046] Another preferred primer comprises the sequence 5′-CCGTCAATTCCTTTRAGTTT-3′ (SEQ ID NO:2) or a functional fragment and/or derivative thereof.

[0047] The invention also provides the use of a test kit according to the invention for determining presence or absence of equilibrium in intestinal bacterial flora as explained hereinbefore. The invention also provides the use of a method according to the invention for determining presence or absence of equilibrium in intestinal bacterial flora.

[0048] The term “intestinal flora” as used herein means a flora present in the digestive tract such as the intestinal tract, the stomach, the oesophagus the mouth or beak or other parts of, or close to, the digestive tract. Thus, intestinal flora samples maybe taken from any part of, or close to, the digestive tract.

[0049] The invention is exemplified by using feces as a source of microbiological populations. However, the methods and means of the invention are equally applicable to microbiological populations from other sources.

[0050] The means and methods of the invention are in general suited for the analysis of a microbiological sample suspected of containing a variety of different microbiological organisms. An example of a microbiological organism is any kind of bacterium, phage, virus, fungus or yeast. A sample of microbiological flora can be obtained from any source suspected of containing a variety of microorganisms, preferably the sample is obtained from the digestive tract, most preferably the intestinal tract. It will also be clear to the person skilled in the art that the means and methods of the invention may also be used to determine and/or trace the kind of plant and mammal material in nucleic acid containing foods. It is clear to the person skilled in the art that the disclosed methods and means are not limited to intestinal tract bacterial flora but are indeed suited for the typing of microbiological flora from many different sources, such as food samples, food additives, waste material, soils etc.

[0051] With the term “flora” is meant a collection of microbiological organisms.

[0052] The invention will be explained in more detail by the following illustrative examples.

EXAMPLES

[0053] Material and Methods

[0054] Sampling: Parts of the intestinal tract were isolated/obtained during post mortem examination. In poultry, for example, a well-defined part of the ileum between the exit of the pancreatic duct and the Mickel's Diverticulum, which was used.

[0055] Endoscopial investigation is used to collect samples from particular parts of the intestinal tract during life. Feces were obtained from living subjects. Conservation and transport: Samples were put and maintained on dry ice immediately after collection and transported to the laboratory.

[0056] At arrival at the laboratory, samples were stored at −20 ° C.

[0057] Materials:

[0058] Lysis Buffer DNA-Extraction (pH 6.4)

[0059] 142.5 g GuSCN

[0060] 4.8 g TRIS

[0061] 1.63 g EDTA

[0062] Aqua ad 200 ml

[0063] Wash-Solution DNA-Extraction (pH 6.4)

[0064] 142.5 g GuSCN

[0065] 4.8 g TRIS

[0066] Aqua ad 200 ml

[0067] DE-Solution DNA-Extraction

[0068] 0.75 ml HCl 35%

[0069] 10 g Diatomaceous Earth

[0070] Aqua ad 50 ml

[0071] Sample Preparation

[0072] 1. Sample handing

[0073] a. intestinal tract

[0074] Approximately, 2 cm of the specified location was sliced lengthwise using a scalpel. The entire contents was transferred into a sterile tube.

[0075] b. endoscopial biopsies and fecal material.

[0076] Samples were transferred into sterile tubes.

[0077] 2. Homogenization

[0078] Mixing by manually shaking was performed until the substance was visually homogeneous.

[0079] 3. DNA-extraction

[0080] a. Quantity

[0081] Approximately 0.2 g of homogeneous material was used. In cases of watery samples, a volume of approximately 250 μl was used.

[0082] b. DNA-extraction

[0083] The DNA-extraction was performed generally according to Boom's method.

[0084] One ml of lysis buffer was added to the homogeneous sample.

[0085] The suspension was vortexed for 30 seconds, and stored at room temperature for one hour.

[0086] The suspension was centrifuged at 15000-17000 g for 20 seconds.

[0087] The supernatant was transferred to a sterile tube containing 50 μl of DE-solution. The suspension was vortexed for 30 seconds, and centrifuged at 15000-17000 g for 20 seconds.

[0088] After removal of the supernatant, five washing steps were performed.

[0089] 1. 200 μl wash-solution,

[0090] 2. 200 μl wash-solution,

[0091] 3. 200 μl 70% Ethanol,

[0092] 4. 200 μl 70% Ethanol,

[0093] 5. 200 μl Acetone p.a.

[0094] The pellet was air-dried for 15 minutes at 56° C.

[0095] The pellet was resuspended in 75 μl ddH20 (overnight 37° C.).

[0096] Finally, the supernatant was transferred to a new Eppendorf cup and stored at 4° C.

[0097] PCR

[0098] Polymerase Chain Reaction was performed using 1.5 ul DNA in a total reaction volume of 15 μl, containing amplitaq DNA polymerase (0.07 unit/μl), primers (1.3 mM of forward and reverse primers), dNTPs (200 mM) and 60 mM KCl, 12 mM HCl pH 8.3 and 1.8 mM MgCl2.

[0099] Primer Sequences.

(SEQ ID NO:1) Label FAM
Forward 5′-AGAGTTTGATCCTGGCTCAG-3′
(SEQ ID NO:2) Label TET
Reverse 51-CCGTCAATTCCTTTRAGTTT-31

[0100] PCR Program.

1 cycle of 5 min 95° C.
35 cycles of 30 sec 95° C.
45 sec 56° C.
2 min 72° C.
1 cycle of 5 min 72° C.

[0101] Both primers were labeled with fluorescent probes to enable automated analysis.

[0102] The purpose of this reaction is an amplification of as many bacterial organisms as possible.

[0103] This generally results in about 50 fragments.

[0104] Restriction:

[0105] Restriction digestion was performed on the PCR-products with different restriction enzymes, such as

HaeIII GG′CC,
CfoI (=HhaI) GCG′C,
TaqI TICGA,
HinfI G′ANTC,
MspI C′CGG,
PstI CTGCA′G,
HinDIII A′AGCTT.

[0106] Analysis

[0107] Size analysis of the DNA-fragments was performed on an ABI5 377 DNA Sequencer using fluorescently labeled primers. The conditions of the tun on the ABI377 Prism Sequencing system were as follows: ABI Collection software version 2.1 with well-to-read distance 36 cm; running conditions 3000 V, 60 mA, 200 W, collection time 5 hours.

[0108] Size-analysis was performed using ABI-software. Lanetracking, Genotyper, Data analysis was performed using GeneScan 3.1 and Genotyper 2.0 software according to the manufacturer's instructions.

[0109] Pure Culture Bacteria

[0110] Several bacterial strains such as, for example, Clostridium perfringens, Staphylococcus aureus, Mycoplasma gallisepticum, Mycoplasma synovium, Ornitobacter rhinotracheale, Escherichia coli, Lactobacilliae, Enterococcae and Pseudomonas aeruginosa were used as references.

[0111] Polymerase Chain Reaction as described before is carried out on pure culture bacteria. The size of the PCR-product is specific for each bacterium.

[0112] Restriction enzymes can be used to identify a number of fragments.

[0113] The overall picture of fragments after the PCR and after the use of restriction enzymes results in a “pattern” of a pure culture. This pattern can be identified in any intestinal sample depending on the amount of bacteria present.

[0114] Interpretation

[0115] 1. Richness

[0116] The results of the analysis are visualized in a figure, showing peaks corresponding to the size of the DNA-fragments.

[0117] These peaks generally are related to bacterial species. The number of peaks identified is an indication for the total number of bacterial species. Using information from pure cultured bacteria, this also provides information on the presence or absence of specific bacteria.

[0118] 2. Evenness.

[0119] Furthermore, the peaks provide information on the relative quantity of each DNA-fragment. The ratio of the DNA-fragments based on peak-height and peak-area can be used to estimate the number of certain bacteria present in the sample.

[0120] Polymerase chain reaction as described before is carried out after DNA extraction form pure culture bacteria. Thus, samples can be interpreted to see whether some bacteria are overwhelmingly present or almost absent.

1 2 1 20 DNA Unknown Primer 1 agagtttgat cctggctcag 20 2 20 DNA Unknown Primer 2 ccgtcaattc ctttragttt 20

Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US8563295Jan 12, 2010Oct 22, 2013Dupont Nutrition Biosciences ApsLactic acid bacteria and their use in swine direct-fed microbials
Classifications
U.S. Classification435/5, 435/91.2, 435/6.16
International ClassificationC12N15/09, A01K67/00, C12Q1/68, C12Q1/70, C12P19/34
Cooperative ClassificationC12Q1/689, C12Q1/683
European ClassificationC12Q1/68B6A, C12Q1/68M10B