阅读说明：本技术 分析遗传信息的方法 (Method for analyzing genetic information ) 是由 F·M·*** 于 2018-05-14 设计创作，主要内容包括：无需以纯化形式进行分离以及随后纵向株系跟踪的株系分型的系统和方法。该方法包括定位目标中存在的一个或多个遗传区域的步骤。遗传区域含有在目标的两个或多个变体(即株系)间变异的遗传位点。设备检测每个基因位点的独特序列。在取得具有遗传位点的生物材料样本后,该设备为目标中存在的遗传区域生成扩增子。扩增子与互补探针杂交,从而产生用于检测的杂交探针和非杂交探针。检测到的杂交探针分配以标识符。设备将标识符转换为模式。记录该模式并与记录的一个或多个其他模式进行比较,以确定该模式是否与一个或多个其他模式不同。(Systems and methods for strain typing without isolation in a purified form and subsequent longitudinal strain tracking. The method comprises the step of locating one or more genetic regions present in the target. The genetic region contains genetic loci that are mutated between two or more variants (i.e., strains) of interest. The device detects unique sequences at each gene locus. After taking a sample of biological material having a genetic locus, the apparatus generates amplicons for the genetic region present in the target. The amplicons are hybridized to complementary probes, thereby generating hybridized and non-hybridized probes for detection. The detected hybridization probes are assigned identifiers. The device converts the identifier into a pattern. The pattern is recorded and compared to one or more other patterns recorded to determine if the pattern is different from the one or more other patterns.)

1. A method of analyzing genetic information, comprising the steps of:

a. locating one or more genetic regions present in the target, wherein the genetic region comprises a genetic locus of variation between two or more variants of the target;

b. providing a device configured to detect a unique sequence for each genetic locus;

c. obtaining a sample of biological material having a genetic locus;

d. generating amplicons of one or more genetic regions present in the target;

e. hybridizing the amplicons to one or more probes of the genetic locus, wherein the one or more probes will hybridize to variants of the genetic locus and the one or more probes will not hybridize to variants of the genetic locus;

f. detecting each probe hybridized to the amplicon by the device;

g. assigning an identifier to each hybridization probe, the identifier being different from identifiers assigned to non-hybridization probes;

h. converting the identifier assigned to each probe into an identifier mode of a hybridization probe by the equipment and recording;

i. comparing the recorded pattern with one or more other recorded patterns;

j. it is determined whether the pattern of the recording is different from the pattern of one or more other recordings.

2. The method of claim 1, wherein at least six genetic loci are present in the target.

3. The method of claim 1, wherein the biological material is a purified culture.

4. The method of claim 1, wherein the biological material is a complex mixture.

5. The method of claim 1, wherein the pattern and one or more other patterns are stored in a database, the database being connected to the device by at least one of a wired connection or a wireless connection.

6. The method of claim 1, further comprising the step of comparing the pattern stored in the database with previous patterns stored in the database, wherein the previous patterns were obtained from previously analyzed samples.

7. The method of claim 1, further comprising the step of reporting a pattern, wherein the pattern represents a set of genetic characteristics defining a biological material.

8. The method of claim 1, wherein the target is listeria.

9. The method of claim 8, wherein the two or more variants of interest are listeria strains.

10. The method of claim 1, wherein the pattern is a series of two or more binary digits.

11. The method of claim 1, wherein the sample is a biological material from an environment.

12. The method of claim 1, wherein the step of hybridizing the amplicons to one or more probes of a genetic locus is performed on a hybridization array.

13. The method of claim 1, wherein the step of hybridizing the amplicons to one or more probes of a genetic locus is performed on microbeads.

14. A method for strain typing a target organism in a composite biomaterial comprising the steps of:

a. amplifying, by a device, a nucleic acid sequence containing a variable genetic locus from a composite biological material to produce an amplicon;

b. hybridizing the amplicons to one or more probes of the genetic locus on at least one of the first hybridization array and the first microbeads, wherein the one or more probes will hybridize to variants of the genetic locus and the one or more probes will not hybridize to variants of the genetic locus;

c. detecting one or more hybridization probes and one or more non-hybridization probes on at least one of the first hybridization array and the first microbeads;

d. assigning an identifier to each hybridization probe and non-hybridization probe on at least one of the first hybridization array and the first microbead;

e. generating a first pattern of one or more identifiers on at least one of the first hybridization array and the first microbeads;

f. comparing a first pattern of at least one of the first hybridization array and the first microbeads to a second pattern of at least one of the second hybridization array and the second microbeads;

g. it is determined whether the first mode is different from the second mode.

15. The method of claim 14, wherein the first schema and the second schema are stored in a database operatively connected with a device.

16. The method of claim 14, wherein the identifier is a binary number.

17. The method of claim 14, further comprising the step of reporting the first pattern and the second pattern, wherein the first pattern and the second pattern represent a set of genetic characteristics defining a composite biomaterial.

18. The method of claim 14, wherein at least six genetic loci are present in the target.

19. The method of claim 14, further comprising the step of capturing an image of the first hybridization array with a camera.

20. The method of claim 14, wherein the target is listeria.

Background of the invention

1.Invention of the inventionIn the field of

The present invention relates generally to systems and methods for nucleic acid analysis, and more particularly to systems and methods for analyzing nucleic acid sequences from complex biological samples and using the analysis to monitor the appearance of these sequences.

Disclosure of Invention

In particular, the present invention relates to systems and methods for analyzing nucleic acid sequences from complex biological samples and using the analysis to monitor the presence of these sequences. In one embodiment, a method for analyzing genetic information comprises the steps of: (i) locating one or more genetic regions present in the target, wherein the genetic region comprises a genetic locus that varies between two or more variants of the target; (ii) providing a device configured to detect a unique sequence for each genetic locus; (iii) obtaining a sample of biological material having a genetic locus; (iv) generating amplicons of one or more genetic regions present in the target; (v) hybridizing the amplicons to one or more probes of the genetic locus, wherein the one or more probes will hybridize to variants of the genetic locus and the one or more probes will not hybridize to variants of the genetic locus; (vi) detecting each probe hybridized to the amplicon by the device; (vii) assigning to each hybridization probe an identifier that is different from the identifiers assigned to non-hybridization probes; (viii) converting the identifier assigned to each probe into an identifier mode of a hybridization probe by the equipment and recording; (ix) comparing the recorded pattern with one or more other recorded patterns; and (x) determining whether the pattern of the recording is different from the pattern of the one or more other recordings.

In another embodiment, a method for strain typing a target organism in a composite biomaterial comprises the steps of: (i) amplifying, by a device, a nucleic acid sequence containing a variable genetic locus from a composite biological material to generate an amplicon; (ii) hybridizing the amplicons to one or more probes of the genetic locus on at least one of the first hybridization array and the first microbeads, wherein the one or more probes will hybridize to variants of the genetic locus and the one or more probes will not hybridize to variants of the genetic locus; (iii) detecting one or more hybridization probes and one or more non-hybridization probes on at least one of the first hybridization array and the first microbeads; (iv) assigning an identifier to each hybridization probe and non-hybridization probe on at least one of the first hybridization array and the first microbead; (v) generating a first pattern of one or more identifiers on at least one of the first hybridization array and the first microbeads; (vi) comparing a first pattern of at least one of the first hybridization array and the first microbeads to a second pattern of at least one of the second hybridization array and the second microbeads; and (vii) determining whether the first mode is different from the second mode.

Drawings

One or more aspects of the present invention are particularly pointed out and distinctly claimed as examples in the claims at the conclusion of the specification. The above and other objects, features and advantages of the present invention will become apparent from the following description taken in conjunction with the accompanying drawings, in which:

FIG. 1 is a top view of an exemplary embodiment of a consumable device used by a prior art system workstation;

FIG. 2 is a flow diagram of an exemplary embodiment of a method of analyzing nucleic acid sequences from a complex biological sample and using the analysis to monitor the occurrence of those sequences;

FIG. 3 is a diagram of an exemplary embodiment of an alignment between nucleic acid sequences that can be used to select targets for preparing hybridization arrays or microbeads and primers required for generating amplicons on the arrays or microbeads that bind to the specific nucleic acid sequences;

FIG. 4 is a diagram of an exemplary embodiment of a refinement protocol for strain characterization using microbeads;

FIG. 5A is a diagram of an exemplary embodiment of a simplified three-site refinement scheme for strain characterization using a hybridization array;

FIG. 5B is a diagram of another exemplary embodiment of a simplified three-site refinement scheme using hybridization arrays for strain characterization;

FIG. 6A is a top view of an exemplary embodiment of a 9X 3 hybridization array in which all available variant genetic locus probe spots are hybridized to amplicons and three probe spots are used for camera positioning to record the pattern of spots;

FIG. 6B is a top view of an exemplary embodiment of a 9X 3 hybridization array of biological material showing that only 7 variant genetic site probe spots hybridize to amplicons and three probe spots are available for camera positioning to record a pattern of spots, and a table representing the conversion of spot positions to binary codes representing the pattern of hybridization events for amplicons hybridizing to their complementary probes at known positions on the array;

FIG. 7 is a top view of an exemplary embodiment of a pair of hybridization arrays having the same probe position arrangement and camera positioning as in FIGS. 6A and 6B, produced from two separate biological materials, showing the same amplicon hybridization pattern on both arrays;

FIG. 8 is a top view of an exemplary embodiment of a pair of hybridization arrays with the same probe position arrangement and camera positioning as in FIGS. 6A and 6B, produced from two additional separate biological materials, showing different amplicon hybridization patterns on the two arrays;

FIG. 9 is a timeline of a current representative method of typing Listeria strains used by food safety regulatory agencies and epidemiologists, further including simplified reports comparing generated patterns for comparing pattern result repeatability or uniqueness as compared to the timeline of an exemplary embodiment of Listeria strain typing methods that do not require isolation in a purification culture;

FIG. 10 is a diagram of an exemplary embodiment of a method for strain typing six Listeria strains, including a filter key detailing probe location and probe type on a 9X 3 exemplary array, and another pattern encoding program;

FIG. 11 is a top view of a hybridization array generated from 6 different Listeria strains, which were first analyzed from a purified culture and then incorporated into a complex environmental enrichment culture; and

FIG. 12 is a top view of a hybridization array generated from 12 different Listeria strains, each strain being analyzed from incorporation into a negative environment enrichment culture.

Detailed Description

The various aspects of the invention and certain features, advantages and details thereof are explained more fully with reference to the non-limiting examples that are illustrated in the accompanying drawings. Descriptions of well-known structures are omitted so as not to unnecessarily obscure the details of the present invention. It should be understood, however, that the detailed description and the specific non-limiting examples, while indicating various aspects of the invention, are given by way of illustration only, not by way of limitation. Various substitutions, modifications, additions and/or arrangements within the spirit and/or scope of the basic inventive concept will become apparent to those skilled in the art from this disclosure.

The present invention is the analysis of nucleic acid sequence patterns occurring from complex biological samples and the use of the analysis to monitor the repetition or variation of patterns generated by these sequence analyses. The system may comprise a conventional workstation (including consumables, such as the Rheonix Optimum shown in FIG. 1)^TMCartridges (cartridges) used in workstations), and kits (not shown) containing reagents thereof. Exemplary structural and functional aspects of embodiments of the present invention are similar to or include elements of the workstation and its consumable components described and illustrated in U.S. patent No. 8,383,039. These similarities should be understood by one of ordinary skill in the art in conjunction with review of the present invention and the drawings and the patent disclosure, and are not discussed in detail herein. FIG. 1 is a top view of a consumable device (prior art, e.g., cartridge) interfaced with a workstation, which is an exemplary embodimentWhere the experiments were performed. The apparatus has a liquid reservoir 17, and the liquid reservoir 17 has a reservoir connected to a cut-off passage 16 formed at the bottom of the liquid reservoir 17. Some of the liquid reservoirs 17 contain an array 47 of low density nucleic acid probes for the hybridization of amplicons to the array probes. RheonixOptimum^TMThe workstation is coupled to an ink cartridge (as shown in fig. 1) to perform the steps of the methods described herein in an automated fashion. The system and method may also be performed using other means instead of hybridization arrays, such as microbeads each containing a unique identifier and coupling each microbead to their own oligonucleotide probe to complete the analysis of hybridization events by analyzing each microbead and generating a pattern corresponding to the pattern generated herein.

Turning now to fig. 2, a flow diagram of a method 100 for analyzing nucleic acid sequences of a complex biological sample and using the analysis to monitor for duplication or variation of patterns generated by these sequence analyses is shown. In a first step 102, a target monitored organism is identified. Such target organisms should have at least two genetic regions which are characteristic for the target organism and which vary between strains of the target organism. In order to perform the method directly from a sample containing a large background of non-target flora, it is crucial to select a genetic region that is unique to the target organism, since even in a background where there is a surplus of non-target genetic material, this unique region allows the assay to amplify only the genetic region of the target organism.

In a next step 104, genetic loci within the identified genetic region of the target organism are identified. In the example shown in FIG. 3, within the indicated genetic region, two genetic locus variants are shown at variant position 1 and variant position 2 of 6 strains of the target organism (i.e., base pairs of variant position 1 and variant position 2 differ in nucleic acid sequence between 6 strains). As shown in FIG. 3, each potential outcome of a hybridization event is assigned an identifier, such as a binary number. As shown in FIG. 3, the base pair at the first position of each of the 6 variants can be AA, GC or GT. In the example, the base pair AA is assigned the binary digit "1", while any other base pair referred to as a "non-AA" is assigned the binary digit "0". Similarly, theThe second-position base pair of each of the 6 variants may be either GT or AA. In the example, the base pair GT is assigned the binary digit "1", while any other so-called "non-GT" base pair is assigned the binary digit "0". Thus, the two sites exhibit variation by a binary system, with a total of four possible binary code combinations ("0, 0" or "0, 1" or "1, 0" or "1, 1") for each nucleic acid sequence. In the embodiment shown in FIG. 3, there are six target strains generating three of four possible binary code combinations. For example, a first strain has both an AA base pair at a first position and a GT base pair at a second position; thus, the binary code for the first strain is "1, 1". In another aspect, the sixth strain has a GT base pair at the first position and an AA base pair at the second position; thus, the binary code for the sixth strain is "0, 0". The examples further show that 3 patterns were generated in 6 lines. There are two "1, 1" modes, one "0, 1" mode, three "0, 0" modes and zero "1, 0" modes. Importantly, the patterns generated did not distinguish lines 1 and 2 from each other, but they did distinguish lines 1 and 2 from the other 4 lines. Lines 4, 5 and 6 are also distinguished from lines 1, 2 and 3, although they are also not distinguished from each other. In fact, n sites exhibiting such variant selection will provide up to 2ⁿ(where n is the number of positions) potential modes. For example, 2 sites in this example produce 4 patterns, 3 sites produce 8 patterns, 4 sites produce 16 patterns, 5 sites produce 32 patterns, 6 sites provide 64 potential hybridization patterns, and so on for multiple sites for which probes are prepared. The particular mutation site and the probes chosen must ensure that the probes classify the variants into sufficiently small groups (each group representing a pattern). In one embodiment, a sufficiently small grouping classifies up to 35% of the target organisms into any one group. However, the groupings must also be large enough so that each variant (i.e., strain) is not classified into its own group. In other words, the genetic locus and probes must be selected such that a single pattern represents at most 35% of the target organism, rather than any one particular strain of the target organism.

After selecting the variant sites, nucleic acid primers are generated to perform an amplification reaction to produce amplicons comprising sequences of the variant sites, as shown in step 106. In a next step 108 nucleic acid probes are generated and designed to hybridize with some amplicons and not others in the manner shown in fig. 3 to generate binary results for each position. Detection of a target nucleic acid sequence requires the use of a nucleic acid probe having a nucleotide base sequence that is sufficiently complementary to the target sequence or an amplicon thereof. Under selective assay conditions, the probe will hybridize to the target sequence or its amplicon, allowing the operator to detect the presence of the target sequence when it is present in the sample. Efficient probes are designed to prevent non-specific hybridization with any nucleic acid sequence that would interfere with the detection of the presence of the target sequence. The probe and/or amplicon may include a detectable label, wherein the label is, for example, a radioactive label, a fluorescent dye, biotin, an enzyme, an electrochemical or chemiluminescent compound. In some embodiments, hybridization may be to probes immobilized on microbeads, in which case the microbeads may also have specific labels to identify the microbeads themselves, or use identification of binding of the microbeads and oligonucleotide tags. In embodiments of the methods described herein, the presence or absence of the target sequence is detected by a camera of the workstation using reverse dot hybridization (RDB). The camera on the workstation captures an image of the composite hybridization array and under software control running a grayscale image processing program, selects hybridization points that are dark enough to represent a successful hybridization event or not dark enough to represent a successful hybridization event. The gray values are preset using data generated during the assay development process. Successful hybridization is then assigned an identifier, which may be a "1" or some other unique identifier for a location on the array. Unsuccessful hybridization is assigned an identifier, which may be "0".

In a next step 110, an assay is performed on the source of biological material. Such biological material may be in a native state such that the organisms contained therein are not separated from each other. In one embodiment, the biological material is from a purified culture. In another embodiment, the biological material is from a complex enrichment culture. In performing nucleic acid-based assays, sample preparation is the first and most critical step in releasing and stabilizing nucleic acids that may be present in a sample. Sample preparation may also help to eliminate nuclease activity and remove or inactivate potential inhibitors of nucleic acid amplification or nucleic acid detection. The system workstation performs all sample preparation steps in an automated manner, while only one technician is required to perform (i.e., the user performs) the pipetting steps. Using the test kit and workstation, the user can prepare the sample by performing cell lysis and nucleic acid purification (i.e., DNA isolation). In another embodiment of pattern typing, sample preparation involves preliminary immunomagnetic separation (IMS) performed on a workstation or off-line to remove cross-reactive species without the need for complete isolation and purification of the target organism in a purification culture. For example, a preliminary IMS may be required for a particular target organism (e.g., Salmonella).

In a next step 112, after the nucleic acid (e.g., DNA) is isolated, the workstation transfers the purified nucleic acid to a reaction cell where amplification of the specific nucleic acid sequence occurs without any additional input from the user. Amplifying a specific genetic sequence from the biological material to obtain a nucleic acid sequence of the biological material. In particular, nucleic acid amplification is the enzymatic synthesis of nucleic acid amplicons (i.e., copies) that comprise sequences homologous to the nucleic acid sequences being amplified. Examples of nucleic acid amplification procedures employed in the art include Polymerase Chain Reaction (PCR), Strand Displacement Amplification (SDA), Ligase Chain Reaction (LCR), nucleic acid sequence amplification (NASBA), transcription-related amplification (TAA), cold PCR and non-enzymatic amplification techniques (NEAT), among others.

Nucleic acid amplification is particularly beneficial when the amount of target sequence in the sample is low. By amplifying the target sequence and detecting the resultant amplicon, the sensitivity of the assay can be greatly improved, since the fewer target sequences required at the start of the assay, the better will be to ensure the detection of nucleic acids in a sample belonging to the target organism or virus. In the examples of the methods described herein, specific sequences of target organisms with inter-strain polymorphisms are amplified. In other words, sequences specific to the target organism are amplified, but also contain enough differences to enable detection and strain characterization. That is, the selected sequence is specific for the target organism (not present in other genera or species), but not present in 100% of the strains of the target genus or species. Sufficient of these sequences are selected and amplified to enable differentiation of strains of the target organism.

Referring now to fig. 4, a microbead-based format is shown. In the bead-based format of the embodiments, each variant of the probe may be added to a different fluorescently labeled bead. The beads are used to detect the binding event by illuminating the beads and analyzing them using a detector that detects the fluorescent properties of the beads and the properties of the nucleic acid probes linked to the fluorophore to determine which amplicon variants are present. In this case, fluorescent microbead 1 will provide a signal for its microbead and probe fluorophore (positive result), while fluorescent microbead 2 will provide a signal for only the microbead, not for its probe fluorophore (negative result), because no complementary amplicon is present. The binding types of many beads/probes can be highly multiplexed to form a pattern-type based protocol as described herein.

Finally, the amplicon hybridization produced by the above-described biological material is analyzed using a system having sufficient multiplexing capability such that at least 6 hybridization probes (i.e., at least 64 patterns) can be analyzed to determine the presence or absence of specific hybridization of amplicons produced by the biological material. As shown in the above examples, the presence of hybridization of the amplicons to the predetermined probe sequences can be known using colorimetric, fluorometric, radiographic, electrophoretic, mass spectrometry, or any other such identification analysis, which can determine the absence/presence of hybridization of each probe sequence.

For example, in the next step 114 of embodiments of the methods described herein, the amplicons are captured (i.e., hybridized) by their complementary probes. Following probe capture, the hybridized probes (and non-hybridized probes) are detected at a subsequent step 116. In one embodiment, a camera on the workstation detects bound DNA by imaging darkening of reporter molecules deposited as a result of enzyme activity binding to amplicons on the array. If no amplicon product (i.e., non-hybridizing probe) is made, the given spot is not darkened. In another embodiment, microbeads with hybridized probes may be detected and analyzed using a suitable system. In a next step 118, the system assigns an identifier to each probe location based on the gray scale threshold of the imaging software as described above. In a next step 120, the identifier is converted into a pattern of identifiers, and the pattern is recorded and stored.

Turning now to fig. 5A and 5B, there is shown a diagram of an exemplary embodiment of a simplified three-site refinement scheme for strain characterization. In the example, three sites (or 1 base pair more positions than shown in FIG. 3, providing up to 8 possible probe hybridization patterns) are used to differentiate strains of the target organism. As described above, when the probe hybridizes to the amplicon, the binary digit "1" is assigned. Similarly, when there is no hybridization, a binary digit "0" is assigned. For example, as shown in FIG. 5A, the binary code pattern is "1, 0, 1" because the assay shows two dark spots at the first probe position and the third probe position, and no spots at the second probe position (and thus no probe hybridization). In another example, as shown in FIG. 5B, the binary code pattern is "0, 0, 1" because the assay shows a dark spot at the third probe position, and no spots (and therefore no probes hybridized) at the first and second probe positions.

Referring now to FIGS. 6A and 6B, top views of exemplary embodiments of 9X 3 hybridization arrays are shown. The hybridization array of FIG. 6A shows all probes available for hybridization to amplicons and contains an arrangement of reference points between dark spots (shown in a circular grid pattern). The reference point is used for positioning of the workstation camera to correct image acquisition and to help verify that the assay is performed correctly. The camera used is a conventional camera mounted in a workstation, similar to the camera described and illustrated in U.S. patent No. 8,383,039. These similarities should be understood by one of ordinary skill in the art in conjunction with review of the present invention and the drawings and the patent disclosure, and are not discussed in detail herein. The array on the hybridization membrane was arranged in 3 columns and 9 rows so that the specific point was always at the same position relative to the camera reference point. The images captured by the camera are processed by a software program running on the workstation (or on a device connected to the workstation) and designed to characterize the gray scale at each particular point. Providing a specific gray value for the software, and when the gray value is higher than the gray value, the software assigns an identifier (such as '1') to indicate that the hybridization is successful; below this gray level, the software assigns a different identifier (e.g., "0") to indicate a hybridization failure. All spots in FIG. 6A have hybridized and will be assigned the identifier "1".

Turning now to fig. 6B, a representative hybridization membrane derived from a sample of biological material is shown. In step 120 of the method, the identifier assigned to the probe position of successful or unsuccessful hybridization is converted into a pattern (i.e. code) of identifiers of the biological material, and the pattern is recorded and stored. FIG. 6B illustrates this mode, which shows the same camera positioning as shown in FIG. 6A; however, there are fewer dark positive spots that represent hybridization (or binding) events. The probes were arranged in the same rows and columns as used in FIG. 6A. Binding events (i.e., dark dots) are assigned a binary digit "1" while non-binding events (i.e., no dots) are assigned a binary digit "0". The assigned "1" and "0" generate binary codes when each row and array is read. The generated binary codes represent all hybridization and non-hybridization events in a particular sample. In the embodiment shown in FIG. 6B, the first row has the binary code "1, 0, 1", where "1" represents a control point. The binary code of the second row has the binary code "0, 1, 1", where "1" indicates a positive dark spot in the second and third columns. Thus, the pattern of biological material (i.e., the code) can be read from left to right and top to bottom. Fig. 6A-6B show an array with 24 available probes, as it is an array of 3 columns and 9 rows, providing 27 probes, minus 3 probes for positioning. Thus, up to 2 are provided in the arrays of FIGS. 6A-6B²⁴Or 16777216 possible patterns.

Referring now to FIGS. 7 and 8, top views of exemplary embodiments of hybridization arrays produced from biological material are shown. At step 122 below the method, the patterns (i.e., codes) of one or more hybridization arrays are compared. FIG. 7 shows a pair of hybridization arrays generated from two separate biomaterials. The binary code (i.e., pattern) generated for each sample is displayed under the corresponding hybridization array for each sample. The pattern for each biomaterial in fig. 7 is a positive spot in the array, representing a hybridization event. The same binary code between two separate biological materials indicates that the sample contains the same set of variants. The sample may be the same strain or from two different strains reporting the same pattern (e.g., see strains 1 and 2 of fig. 3). The specific pattern that persists from two or more samples may indicate that one or more strains are in an invariant population. In a food production environment sampling program, pattern recognition test results developed using the present invention can be used to notify modifications to the health standards operating program (SSOP) to reduce the risk of continued potential contamination of the organism by the finished product. These organisms can be either pathogenic bacteria causing outbreaks of epidemic situations or quality organisms causing economic losses associated with food spoilage.

Turning now to fig. 8, this is another pair of individual biomaterials (also different from those shown in fig. 7). The binary codes (i.e., patterns) of the individual biomaterials shown in fig. 8 are not identical. Differences in the pattern are evidenced by differences in the position of the positive dark spots on the array indicating hybridization events. The different patterns generated between the two individual biomaterials indicate the presence of different sets of variants and the temporal nature of one or more strains or populations in the individual samples. Thus, the next step in the method, step 124, is to determine whether the patterns match or differ, thereby characterizing the biological material as persistent or transient. In other words, by comparing the patterns, it can be determined whether a strain or population of one or more target organisms (e.g., listeria) is present in the one or more biological materials.

As shown in FIGS. 6A-8, after determining (step 124) the pattern of each biological material analyzed, a final step 126 includes generating a useful report, which may be stored in a suitable computing system, database, or any other suitable storage medium for later comparison with the analysis of another biological material. Comparison between a previously stored pattern library and a newly obtained pattern library provides a means to identify similarities and differences between biological material samples. This comparison is of great significance for the detection of pathogens in many fields. For example, if a repeating pattern is found in a longitudinally collected food manufacturing facility environment, primary production, or food sample (e.g., a repeating pattern as shown in fig. 7), the target organism is likely to be a persistent population. On the other hand, if a different or transient pattern is found to exist from the longitudinally collected samples (as shown in FIG. 8), the target organisms are likely to be of a different population and from a different community.

Turning now to fig. 9, a timeline of the current listeria strain typing method is shown, in contrast to the exemplary embodiment flow diagram of a method of strain-typing listeria in a complex enrichment culture without isolation in a purification culture. In summary, current methods for typing listeria strains require approximately 1-2 days to begin with the enrichment step. Next, it takes several hours to perform a molecular diagnostic screening test. Then, culture separation is carried out, which takes about 3 to 4 days. Finally, the cultures were molecularly strain typed over the course of 1-7 days. Thus, the overall timeline for the current Listeria strain typing process is about 5-13 days.

Still referring to fig. 9, the current method of typing listeria strains is compared to the exemplary embodiments of the present invention. The method for typing the listeria strains from the complex enrichment culture greatly shortens the time required for completing the typing of the strains without the need of separation in the purification culture. Similar to current methods, the exemplary embodiment requires enrichment of the sample, which takes approximately 1-2 days. The molecular diagnostic screening assay is performed within 1-4 hours thereafter. However, the present invention is superior to current strain typing methods in the last step. According to the exemplary embodiment shown in FIG. 9, strain typing can be performed directly from enrichment culture in up to 5 hours. Thus, the present invention is used to compress the 4-11 days required for current isolation culture and typing methods from the midmolecular strains to 5 hours. Finally, an exemplary embodiment of the method of performing strain typing from complex enrichment culture without isolation culture takes about 2-3 days, compared to the current method requiring 5-13 days. Fig. 9 also shows a three sample reduction report indicating that sample 1(S1) and sample 2(S2) are new unique patterns, while sample 3(S3) is a previously generated pattern.

Referring now to fig. 10, a schematic representation of an exemplary embodiment of a method for strain typing six listeria strains is shown. Two different species of listeria of a total of six different strains are shown. The serotype line represents the results of the traditional strain typing method. As shown in the strain line, the present invention can distinguish two different strains, which are considered to be the same when serotype-wise (mode 7). Hybridization assays using the systems and methods of the invention are shown in the descending row of serotype results.

As shown in FIG. 10, the hybridization assay results from the systems and methods of the present invention show four patterns (6, 7, 10, and 19). The two patterns (7 and 19) are the same for the isolated strains. The mode is switched using the "filter key" shown. The filter key shows the camera location point (or reference point "RS"), two assay control points (MM1 and MM2), two variety identification points (Lm-ctr and L-spp-ctr) and assigns values to other potential point locations on the hybridization array. The array is converted to digital code by comparing the hybridized array to the screening key (typically performed by a camera and image processing software). For example, for strain 4, the array is converted to the pattern code 6, 12, 18, 20 and then further to the pattern number 10. The 20-dot array shown in this example can produce up to 2²⁰Or 1048576 possible modes.

The user of the system receives the digital code (i.e., the pattern) generated by each biological material being tested or a report thereof. From these patterns, the user can determine whether the biological material has the same listeria population or a different listeria population. If the user continues to observe the same pattern when testing multiple biological materials from the same or different locations, the user knows that the repetitive pattern represents the same listeria flora. With only the numeric code, the user has sufficient knowledge to make rapid scientific modifications to his health Standard Operating Program (SOP) to help produce a safe finished product.

This is advantageous for the user because the receive-only mode reduces the burden the user is responsible for using other subtype methods. In particular, the U.S. Food and Drug Administration (FDA) requires the reporting of specific listeria strains. The FDA will then post the presence of the reported listeria, stop production and other activities at the site of the reporter strain, and take various compliance measures on behalf of the user. Thus, the digital code provides the user with sufficient information to know whether a resident flora or transient flora is present without knowing the specific strain, thereby limiting the user's exposure to enhanced FDA regulations.

Turning now to fig. 11, a top view of a hybridization array resulting from comparing the performance of an assay between a purified cultured listeria strain and a composite environmentally enriched listeria strain is shown. The two sets of hybridization arrays shown in FIG. 11 have the same pattern for each specific strain shown in purified culture or complex enrichment. For complex enrichment, listeria is artificially introduced into a negative environment enrichment of pre-enriched listeria. Specifically, the same listeria replicate in the purified culture sample is inoculated into an environmental sample that is pre-enriched and negative for the presence of the target organism. The system and method of the present invention is used to analyze the environmental enrichment of incorporation. As a result, the hybridization array detects Listeria in an environment where different organisms are mixed and performs strain typing. FIG. 11 demonstrates that the system and method of the present invention produces the same pattern when testing isolates used in purification cultures and when extracting biological material from the environment. Thus, fig. 11 demonstrates that culture isolation and molecular strain typing, as currently and routinely performed by current methods, is no longer required, significantly reducing the time required to type biological material strains.

Turning now to fig. 12, a top view of a hybridization array generated from 12 listeria strains incorporating pre-enrichment listeria negative environmental enrichment is shown. These arrays demonstrate that the methods herein can classify 12 strains into 10 separate patterns. Note that the strain used in fig. 11 was again reused in fig. 12 with six additional strains added. These arrays indicate that the determination of a careful selection of genetic regions from a particular set of variable sites provides a robust method for classifying listeria populations residing in the population as actionable information based on the analysis of the pattern of site generation using the methods herein. Since listeria present in the enriched sample does not need to be isolated in a purification culture prior to molecular typing, this result can be used to make decisions about hygiene procedures much faster than currently available methods. This approach will also greatly reduce the cost of performing molecular typing as part of an environmental monitoring program, thereby enabling a wider range of food manufacturers to employ advanced molecular strain characterization.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Unless otherwise indicated, the terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to"). The term "coupled" should be interpreted as partially or wholly contained within, attached to, or combined with, even if intervening elements are present.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate embodiments of the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. It is not intended to limit the invention to the particular form or forms disclosed, but on the contrary, the intention is to cover all modifications, alternative constructions, and equivalents falling within the spirit and scope of the invention as defined by the appended claims. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.