Methods for Identifying Microorganisms in Clinical and Non-Clinical Settings

Abstract

The present invention relates to a method for identifying microorganisms in a biological sample by polymerase chain reaction (PCR), comprising the following steps: a) providing a biological sample suspected of containing microorganisms, and optionally isolating nucleic acid sequences from the biological sample; b) using a broad taxonomic range amplification primer set to PCR amplify at least one microbial ribosomal RNA internal transcribed spacer (ITS) region contained in the optionally isolated nucleic acid sequences, thereby generating PCR amplicons from nucleic acid sequences of microbial origin; c) recording the high-resolution melting curve of the PCR amplicons and recording the length of the PCR amplicons; d) comparing the high-resolution melting curve with a database of high-resolution melting curves of reference amplicons containing known microbial species or strains, thereby obtaining a first identification index; e) comparing the length of each PCR amplicon having a different length with a database of PCR amplicon lengths of reference amplicons containing known microbial species or strains, thereby obtaining a second identification index; and f) when the first identification index matches the second identification index, identifying the microorganisms present in the sample at the species or strain level.

Description

Technical Field

[0001] The present invention belongs to the field of microbial diagnostics, and in particular relates to the rapid identification of microorganisms in a sample at the species or strain level, especially bacteria in clinical and non-clinical samples. The present invention relates to a rapid microbial identification method based on DNA analysis and a system for performing this method. Background Art

[0002] Population growth, urbanization, and global connectivity have greatly increased the transmission efficiency of infectious disease pathogens. The recent and very rapid global spread of highly virulent multi-drug resistant bacterial clones clearly illustrates this, and these clones have now become an important public health threat. Therefore, there is a greater need for early detection programs for harmful bacteria. In addition, according to the World Health Organization's "One Health" approach, laboratory diagnosis of livestock infections or microbial contamination in non-clinical settings, such as microbial detection of sterile products in the food sector or the pharmaceutical industry, should include improved programs for accurately detecting and identifying microbial pathogens or contaminants while reducing turnaround time and cost.

[0003] Since traditional culture techniques are not sensitive enough and are time-consuming (>24 hours), the focus in recent years has shifted to culture-independent methods, including DNA-based molecular methods, to detect, characterize, and study the epidemiology and clinical impact of such microorganisms. There are many DNA-based diagnostic methods available currently. Although this progress is encouraging, no single diagnostic platform can fully meet the demand for actionable data in all settings with a short turnaround time.

[0004] Although microbial detection has been revolutionized by the development and application of species-specific and strain-specific polymerase chain reaction (PCR) procedures (for highly specific amplification and detection of even the smallest amounts of microbial DNA in a sample), this method requires knowledge of where the presence of microorganisms in clinical samples can be expected. Since only the absence of one or a few specific species is shown, the negative test results provide limited information. In addition, in cases where the pathogenic pathogen is difficult to predict, evades detection through mutation, or is not actually a single species or microbial type, the applicability of DNA-based diagnostic methods is limited. Clearly, the high specificity of PCR amplification has both advantages and disadvantages when performing clinical and non-clinical diagnoses.

[0005] Moreover, despite their high specificity, the identification of amplicons still inevitably needs to be verified in these methods to exclude the amplification of non-target sequences. Amplicon verification can include the use of hybridization probes, detection of restriction sites in the amplicon, confirmation of the expected length (in terms of the number of nucleotides) of the amplicon, or sequencing of the amplicon.

[0006] Currently, methods for bacterial identification by next-generation DNA sequencing are not suitable for clinical applications because these sequencing methods take at least approximately 24 hours to obtain results, while treatment decisions need to be made within the shortest possible time frame. In addition, common sequencing methods only allow the analysis of relatively short DNA fragments (e.g., about 300 bp), which is too short to provide sufficient resolution at the species or strain level, and sequencing technologies that allow the sequencing of longer fragments are either too expensive, require large batch samples to be affordable, or are too inaccurate to be implemented in clinical routine.

[0007] Summarizing the situation of clinical diagnosis, there are five key issues that determine the applicability of any test: (1) scope, (2) speed, (3) sensitivity, (4) specificity, and (5) scalability. Classical culture-based microbial diagnosis scores highly in three of these five items: scope (many different microorganisms can be detected), sensitivity (single bacterial cells can be detected at most), and specificity (the determination of bacterial species / strains is accurate). However, culture does not score highly in terms of speed and scalability (high throughput).

[0008] Current qPCR-based diagnostics score highly in four of the five items: speed, sensitivity, specificity, and scalability. However, the scope of these assays is extremely limited: a single qPCR can only detect a single or a few target microorganisms, which means that an endless array of qPCRs is required to achieve a scope slightly comparable to that of culture.

[0009] In addition, even in such an environment, negative results (the absence of any species of microorganisms) cannot be given. Negative results are extremely important in clinical microbiology because some of the most important clinical decisions are made based on negative results, and examples include searching for other possible causes of disease besides infection, stopping the use of antibiotics, discharging the patient, removing indwelling devices (postoperative drainage tubes and iv catheters), and deciding to retain very important foreign bodies (prosthetic joints, bone plates, etc.) in the patient. Due to the importance of negative results and the inability of qPCR to accurately provide these results, culture still remains the mainstay of microbial diagnosis despite its obvious limitations.

[0010] Obviously, there is a high desire to provide a cost-effective and efficient PCR-based microbial detection system that scores highly in all five key issues, especially with clinical applicability. Summary of the Invention

[0011] The present inventors have now unexpectedly discovered a method for the broad identification of microorganisms that can also be applied clinically. The inventors have realized that developing more species- and strain-specific PCR amplification tests to add to the existing array of available diagnostic tests for specific pathogens does not solve the problem. The inventors have adopted a method that combines a less specific amplification protocol as a first part with a highly specific detection / validation procedure as a second part, where both parts can be run in high throughput.

[0012] The method of the present invention uses a more "generic" amplification of the internal transcribed spacer (ITS) region of microbial rRNA from genomic DNA, rather than amplifying species-specific or strain-specific sequences. The identification of the microorganism from which the ITS region was amplified is then determined by combining information on the amplicon length with the DNA melting curve of the amplicon. Surprisingly, this method provides very high resolution at the species and strain levels, can be applied generically to a large number of microorganisms, and has enabled the establishment of a database that has data on the amplicon lengths and melting curves annotated with species and strains, improving the accuracy of microorganism identification in subsequent samples. Most surprisingly, this relatively simple method is able to provide clinically relevant information based on which clinicians can make treatment decisions, even for very important negative samples (where no microorganisms are detected).

[0013] In the method of the present invention, the amplification of the ribosomal RNA internal transcribed spacer (ITS) region of microbial genomic DNA is essentially not species-specific or strain-specific. Although the highest resolution at the strain level can be obtained by using species-specific amplification protocols, in the method of the present invention, the amplification procedure preferably targets the rRNA ITS regions of a large number of microbial species (covering a wider range of multiple taxonomic genera, families, orders, classes, phyla or even kingdoms), so that the amplicons are derived from a potentially large group of microorganisms. Therefore, "universal" DNA amplification is preferably carried out by using higher taxonomic level or group-specific primers (such as genus-specific, family-specific, order-specific, class-specific, phylum-specific, kingdom-specific and / or domain-specific primers) for amplifying the rRNA ITS region of microbial genomic DNA suspected to be present in a sample. By selecting a universal amplification method, all bacterial species can be detected, thus eliminating the need for many separate qPCRs to achieve a wide range. In addition, since all bacterial species should be amplified, a negative result means that there are no bacteria in the sample and can indeed be used as a solid basis for clinical decisions. So far, such a universal PCR method has not been applied to clinical microbial identification because such extensive PCR amplification will result in the amplification of multiple PCR amplicons from multiple species, each having a different length and a different nucleotide sequence, which does not lead to practical identification in all cases because the specificity level is considered too low for clinical purposes. In addition, the nature of clinical samples is usually that human DNA is present at a much higher abundance than bacterial DNA. Since these concentration differences can be very high (it is not uncommon for human DNA to be 10 10 times more than potential bacterial DNA), even when using very specific bacterial amplification primers, cross-reactions are common, resulting in non-specific amplification and false positive results.

[0014] Surprisingly, despite the above problems, the present inventors have found a way to make the universal PCR method applicable to clinical use by combining two techniques that provide clinically sufficient sensitivity and specificity in the said universal PCR method, and the above two techniques are (i) the technique of high-resolution melting curve analysis (hrMCA) of the amplified PCR products and (ii) PCR amplicon length analysis, such as by capillary electrophoresis separation and subsequent fragment length analysis of the amplified PCR products (i.e., amplicons).

[0015] The present invention now provides a method for detecting and identifying microorganisms in a biological sample at the species or strain level by polymerase chain reaction (PCR), said method comprising the following steps:

[0016] a) providing a biological sample suspected of containing microorganisms and isolating a nucleic acid sequence from said biological sample;

[0017] b) PCR amplifying at least one microbial ribosomal RNA internal transcribed spacer (ITS) region contained in the isolated nucleic acid sequences using a set of broad-taxonomic range amplification primers to generate PCR amplicons;

[0018] c) Recording the high-resolution melting curve of the PCR amplicons generated in step b), and recording the length of the PCR amplicons generated in step b) by capillary electrophoresis or sequencing;

[0019] d) Comparing the high-resolution melting curve recorded in step c) with a database of high-resolution melting curves of reference amplicons to obtain a first identification index of the microorganisms present in the sample, wherein the reference amplicons are generated from reference microbial species or strains of known taxonomic identification using the same primer set;

[0020] e) Comparing the length of each PCR amplicon of different lengths recorded in step c) with a database of PCR amplicon lengths including reference amplicons to obtain a second identification index of the microorganisms present in the sample, wherein the reference amplicons are generated from reference microbial species or strains of known taxonomic identification using the same primer set;

[0021] f) When the first identification index matches the second identification index, the microorganisms present in the sample are identified at the species or strain level.

[0022] The method of the present invention can be used to identify the microorganisms present in a sample at the species or strain level, where the microorganisms can be, for example, microorganisms capable of causing diseases or microorganisms corresponding to products that should be sterile but are contaminated, such as bacteria, viruses, protozoa, or fungi.

[0023] If no signal is detected in any of the steps of the method of the present invention, it can be concluded that the biological sample is negative for the presence of microorganisms.

[0024] The method claimed in the present application provides an assay with a broad range (i.e., capable of detecting many different microorganisms; e.g., general bacteria), which can potentially be performed very rapidly, is scalable, and has clinically relevant (i.e., very high) sensitivity and specificity. This is because a combination of high-resolution melting curves (hrMC) differentiated according to DNA sequences and PCR amplicon length analysis differentiated according to amplicon lengths is used, and based on other results, the confidence in the accuracy of each result (hrMC features and amplicon length) is interpreted. Although each individual result alone may often not be sufficient to derive a definite species name, or may misidentify contaminated human DNA as microbial DNA, uniquely finding an hrMC result that combines with or matches the amplicon length measurement provides an extremely accurate identification of the microbial origin of the amplicon, and thus the microorganisms present in the sample. Each result is compared with a database that includes hrMC data annotated to microbial strains or species, as well as amplicon length data annotated to microbial strains or species. Only when both results are annotated to the same species or strain in the database will the microbial species or strain be positively identified. The high-resolution melting curve depends on the length and sequence of the amplicon, but there may be overlap between different amplicons because different combinations of length and sequence may still result in comparable melting curves. Combining mrMC with the length measurement of the amplicon solves this problem. When amplicons have the same melting curve and the same length, they must come from the same microorganism because amplicons of the same length with the same melting curve must be almost identical in sequence, and thus from the same species (the sequences of the rRNA ITS region vary highly between different species, so only minor variations can be found within the same species). Therefore, amplicons that do not originate from the same microorganism but show comparable melting curves must have different lengths, and conversely, amplicons that do not originate from the same species but show comparable lengths must have different melting curves. Thus, differentiating rRNA ITS amplicons based on melting curve data and amplicon length provides a highly robust clinical method for microbial identification.

[0025] One of the current problems in clinical microbial identification is that when using microbial amplification primers, human DNA is often co-amplified. It is well known that the proportion of human DNA in clinical samples can exceed bacterial DNA by up to 10 10 times. Although the primer pairs used for amplifying microbial DNA are specific to microbial DNA, due to the existence of regions showing homology with some bacterial primers, there is always the possibility that non-target human DNA is amplified. Even if the probability of such non-specific amplification is very small (e.g., a probability of 1 in 10 9 ), but when the number of true targets exceeds 10 10In an environment with a high proportion of human DNA in clinical samples, the possibility of such events also becomes very real. This means that in principle, in every PCR-based microbial detection assay using samples obtained from humans, false positive amplification products are very likely to form. When using classical qPCR methods, in addition to amplification primers, highly specific probes can be used to mitigate this problem. These probes can only bind to very specific target regions within the amplicon. They do not bind to non-specific products and thus alleviate the problem of non-specific amplification of human DNA. However, this method also precludes the possibility of developing assays with a broad range. When aiming to develop assays with a broad range, this non-specific DNA needs to be addressed in an alternative way. When only amplicon length analysis is used, since the length of non-specific (human) amplicons may overlap, be similar or even identical to that of the microorganism, such false positives cannot be appropriately identified with sufficient clinical certainty. This problem also remains when using hrMC alone: non-specific amplicons may produce hrMC curves similar to those of certain microbial strains or species. Once again, by combining hrMC with length analysis, this problem can be solved. The combination of hrMC and amplicon length is so specific that an assignment can be made with certainty (e.g., for specific microbial amplicons or non-specific amplicons). When the length and hrMC are consistent with the microbial characteristics, the amplicon must be from that microorganism (since the length and hrMC are the same, the sequences must be almost the same). When this combination is inconsistent with the microbial characteristics, the amplicon is not from that microbial source. Since non-specific human amplicons are usually similar among different patients, a library of hrMC and human amplicon lengths can be constructed, enabling them to be directly and positively identified as non-specific. By combining broad microbial detection with accurate identification of false positives, an assay can be performed that can accurately give positive and negative results for clinical samples, and both positive and negative results can be used to make clinical decisions.

[0026] In some aspects of the present invention, a database of high-resolution melting curves and PCR amplicon lengths including reference amplicons generated by reference microbial species or strains identified by known classification as described herein may further include high-resolution melting curves and PCR amplicon lengths of reference amplicons generated from human sequences using the broad classification range amplification primer set as a control for non-specific amplicons. The term "non-specific amplicon generation" must be understood herein to refer to non-microorganisms where non-specific binding of primers to non-target DNA templates occurs.

[0027] Finally, combining hrMC with amplicon length detection opens up the possibility of miniaturizing detection devices, paving the way for Point Of Care (POC) applications of a wide range of microbial detection assays. Currently, the machines required to measure hrMC or amplicon length are typically large and expensive. However, it is envisioned that these measurements could also be performed on very small and relatively inexpensive devices, such as lab-on-a-chip (LOC) devices. The current problem with these devices is that they lack the accuracy of their larger counterparts. The accuracy of measuring amplicon length may not be 1 nucleotide, but for example 5 nucleotides. High resolution is essential for determining bacterial species using either hrMC alone or length measurement (even then, specificity is lacking). However, by combining hrMC information with length measurement, the resolution requirements for each of these measurements may not be as stringent. For example, if the 16S-23S ITS amplicon lengths of three different bacterial species are separated by only two nucleotide lengths, then only very accurate measurements can distinguish between them. However, the fact that these amplicons are from different bacterial species means that the sequences of the 16S-23S ITS region will necessarily be different (the sequence of ribosomal DNA and its spacer are unique to each bacterial species). If the lengths are similar, the sequence differences will necessarily result in very different hrMC curves, since the melting curve depends only on length and sequence. Thus, even if three different bacterial species cannot be separated by amplicon length alone, they can always be separated by hrMC. As previously mentioned, this is not an accidental event, because different bacterial species have different 16S-23S ITS amplicon sequences. If the lengths are similar, then different sequences will necessarily result in different hrMC curves.

[0028] It has now been found that by combining high-resolution melting curve analysis with amplicon length analysis, a wide range of microbial detection and identification for clinical diagnosis has become feasible. The method described in this paper can uniquely meet all five basic requirements for optimal clinical diagnosis: a very wide range, for example covering all bacterial species; speed is guaranteed because it is a molecular method and thus does not require culturing; high sensitivity because it is a PCR-based method; by combining hrMC with amplicon length analysis, the specificity of bacterial identification is extremely good (also, from a clinical perspective, the specificity, that is, the ability to correctly identify true negative samples and identify contaminated human DNA, is also very good); finally, the scalability of current machines is already very good and will be further improved in the future through miniaturization.

[0029] In a preferred embodiment of the present invention, the biological sample is a sample from a human body, an animal body, a plant, a food product, a pharmaceutical product or a chemical product, a laboratory culture or an environmental sample (such as a sample from soil or water). In other preferred embodiments, the biological sample is a bodily sample of a patient, selected from body fluids or exudates, including but not limited to uterine fluid, whole blood, serum, plasma, lymph fluid, mucus, saliva, sputum, stool / feces, sweat, wound fluid, pus / purulence, gastric contents, ascites / ascetic fluid, bile, urine, semen, cerebrospinal fluid / liquor and breast milk; or a bodily sample of a subject selected in the form of a swab, a biopsy, a lavage fluid or a paper point sample, including but not limited to samples from the skin, organs, tissues, oral cavity, urogenital tract, vagina, gastrointestinal tract, respiratory tract or pulmonary system, and the cardiovascular system.

[0030] In a preferred embodiment of the present invention, the microorganism is selected from archaea, bacteria, viruses, protozoa and fungi. Preferably, the microorganism is a pathogenic microorganism capable of causing diseases, preferably selected from pathogenic bacteria, viruses, protozoa or fungi, and most preferably bacteria.

[0031] It should be known that the microorganism or microorganism nucleic acid sequence in some aspects of the present invention can be prokaryotic or eukaryotic. It is a fact known to those skilled in the art that the internal transcribed spacer (ITS) in the genome of these microorganisms is the spacer DNA located between the small subunit ribosomal RNA (rRNA) and the large subunit rRNA gene in the chromosome or the corresponding transcribed region in the polycistronic rRNA precursor transcript. Bacteria and archaea must have two ITS regions. The first ITS is located between the 16S and 23S rRNA genes, herein referred to as the 16S-23S rRNA ITS region, and the second is located between the 23S and 5S rRNA genes, herein referred to as the 23S-5S rRNA ITS region. There are also two ITSs in eukaryotes; ITS1 is located between the 18S and 5.8S rRNA genes, herein referred to as the 18S-5.8S rRNA ITS region, and ITS2 is located between the 5.8S and 26S (plants) or 28S (other eukaryotes) rRNA genes, herein referred to as the 5.8S-26S / 28S rRNA ITS region.

[0032] In a preferred embodiment of the present invention, at least one rRNA ITS region may thus be selected from the 16S-23S rRNA ITS region, the 23S-5S rRNA ITS region, the 18S-5.8S rRNA ITS region, and the 5.8S-26S / 28S rRNA ITS region. In a most preferred embodiment of the present invention, at least one rRNA ITS region is the 16S-23S rRNA ITS region.

[0033] In another preferred embodiment of the present invention, the reference amplicons in the database include amplicons generated by in vivo and / or in silico PCR amplification reactions for amplifying the ribosomal RNA internal transcribed spacer (ITS) of a reference microbial species or strain with known taxonomic identification. Preferably, the reference microbial species or strain belongs to the same microbial phylum, more preferably to the same microbial kingdom, and most preferably is from the bacterial kingdom.

[0034] In another preferred embodiment of the present invention, the databases in steps d) and e) are combined into a single database, and wherein the database includes data on bacteria.

[0035] In yet another preferred embodiment of the present invention, broad taxonomic range amplification primers are used to amplify the ribosomal RNA ITS regions of multiple, preferably substantially all, strains or species of microorganisms from a microbial genus, family, order, class, phylum, kingdom, and / or domain, preferably for amplifying the ribosomal RNA ITS regions of substantially all strains or species from a microbial phylum, more preferably for amplifying the ribosomal RNA ITS regions of substantially all strains or species from a microbial kingdom, and most preferably for amplifying the ribosomal RNA ITS regions of bacteria.

[0036] As used herein, the term "substantially all strains or species" preferably refers to the case where more than 50%, preferably more than 60%, 70%, 80%, 90% or more of the microbial strains or species included in the genus, family, order, class, phylum, kingdom, and / or domain are amplified by the broad taxonomic range amplification primers.

[0037] In yet another preferred embodiment of the present invention, the broad taxonomic range amplification primers for amplifying at least one ribosomal RNA internal transcribed spacer (ITS) region include forward and reverse primers for amplifying the 16S-23S rRNA ITS region, the 23S-5S rRNA ITS region, the 18S-5.8S rRNA ITS region, or the 5.8S-26S / 28S rRNA ITS region. In a most preferred embodiment of the present invention, the broad taxonomic range amplification primers for amplifying at least one ribosomal RNA internal transcribed spacer (ITS) region include forward and reverse primers for amplifying the 16S-23S rRNA ITS region.

[0038] As used herein, the term "at least one microbial rRNA ITS region" refers to an embodiment in which the microbial rRNA ITS region is amplified over its entire length. Those skilled in the art will readily understand that the amplification of a wide taxonomic range of microbial rRNA ITS regions requires the presence of the same sequence (or sequences having a high level of sequence similarity over the entire sequence length, such as more than 90%, preferably more than 95%, 96%, 97%, 98% or 99% sequence similarity) in the genomic DNA of a large number of taxonomically related strains or species to serve as primer annealing sites. Such identical sequences or sequences with high sequence similarity are typically present in the conserved regions encoding the rRNA sequences, rather than in the ITS regions. Thus, amplifying at least one microbial rRNA ITS region by amplification of a wide taxonomic range of microbial rRNA ITS regions generally requires the identification of conserved regions in the opposite coding rRNA regions of the rRNA ITS of interest. Thus, the ITS region is typically amplified as a whole. Those skilled in the art will understand that subsequent annotation of taxonomic identification can be performed by considering only a portion of the microbial rRNA ITS of interest or a portion of the PCR amplicons generated in the methods of the present invention, although it is contemplated that this preferably occurs by treating the microbial rRNA ITS as amplified as a whole.

[0039] In yet another preferred embodiment of the aspects of the present invention, the wide taxonomic range amplification primer set comprises each amplification primer of SEQ ID NOs: 1 and 3 - 5, or each amplification primer of SEQ ID NOs: 2 - 5, or each amplification primer of SEQ ID NOs: 1 - 5.

[0040] In yet another preferred embodiment of the aspects of the present invention, the wide taxonomic range amplification primer set comprises each amplification primer of SEQ ID NOs: 6 and 7 - 13.

[0041] In yet another preferred embodiment of the aspects of the present invention, the wide taxonomic range amplification primer set is a universal bacterial amplification primer set, which preferably comprises each amplification primer of SEQ ID NOs: 14 - 15.

[0042] In yet another preferred embodiment of the aspects of the present invention, the step of PCR amplification comprises qPCR.

[0043] In yet another preferred embodiment of the aspects of the present invention, the length of the PCR amplicon is recorded by capillary electrophoresis.

[0044] In yet another preferred embodiment of the present invention, the PCR amplification reaction further comprises a PCR calibration system, the PCR calibration system comprising: a set of PCR amplification primers, wherein at least one primer comprises a tag; and a set of at least two PCR calibration subunits, each PCR calibration subunit consisting of a spacer-containing DNA fragment of a given length, the spacer having a given length and flanked by upstream and downstream adapter DNA sequences, the adapter DNA sequences comprising primer binding sites for binding to the PCR amplification primers, wherein the set of PCR amplification primers is used for PCR amplification of the spacer DNA sequences of all the PCR calibration subunits in the set of at least two PCR calibration subunits, wherein the spacer DNA sequences comprised in each of the at least two PCR calibration subunits in the set have different lengths, and wherein each of the at least two PCR calibration subunits in the set is present in an equal or known amount relative to the other PCR calibration subunits in the set; and wherein step b) of the PCR amplification further comprises PCR amplifying the at least two PCR calibration subunits using the PCR amplification primers of the PCR calibration system.

[0045] In yet another preferred embodiment of the present invention, a broad taxonomic range amplification primer set for amplifying at least one rRNA internal transcribed spacer (ITS) region comprises a tagged forward and / or a tagged reverse primer, preferably a tagged forward primer, more preferably a fluorescently tagged forward primer. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Is a cycle time schedule, including melt curve analysis performed on a LightCycler480 machine. Fluorescence acquisition was performed at the end of the primer extension phase at 72 °C (point) and throughout the melt indicated by the temperature gradient starting at 1:43:17.

[0047] Figure 2 Is an example of melt curve analysis. Upper side: As the temperature increases, a decrease in fluorescence can be observed because the double-stranded DNA increasingly becomes single-stranded and EvaGreen loses its fluorescence. Lower side: The first derivative of the top melt curve clearly highlights the region where the fluorescence suddenly decreases: the melting temperature of the target amplicon.

[0048] Figure 3 Are melt curves (left column) and fragment length analyses of 16S-23S fragments of eight different bacterial species from three different phyla.

[0049] Figure 4The length of the 16S-23S segment for Streptococcus pyogenes and Corynebacterium jeikeium. Both species exhibit the same length profile. However, the melting curve for each species is unique. By combining the length data with the melting curve data, the two species can be clearly identified.

[0050] Figure 5 Combined fragment length analysis and melting curve analysis of the 16S-23S IS profiles for Klebsiella pneumoniae and Enterobacter cloacae. Although the melting curves are barely distinguishable, each species can be identified by its unique fragment length characteristics. Detailed implementation

[0051] Definitions

[0052] The term "nucleic acid sequence" or "nucleotide sequence" refers to the base sequence of a DNA or RNA molecule in single-stranded or double-stranded form, especially a DNA sequence encoding the ITS region of a ribosomal RNA gene, and these terms are used interchangeably herein.

[0053] "Isolated nucleic acid sequence" refers to a nucleic acid sequence that is no longer in its natural environment from which it was isolated. This term particularly refers to a nucleic acid molecule that has been isolated from at least about 50%, 75%, 90% or more of the proteins, lipids, carbohydrates or other materials naturally associated therewith, such as a microbial host cell.

[0054] As used herein, the term "microbial" refers to a subject derived from a microorganism / microbe, which generally refers to a microscopic organism, meaning it is too small to be seen with the naked eye.

[0055] As used herein, the term "target microbial nucleic acid sequence" refers to a nucleic acid fragment targeted for replication (or amplification) and subsequent detection, and is a diagnosis of the specific microorganism whose presence is to be determined.

[0056] As used herein, the term "polymerase chain reaction (PCR)" refers to a well-known in vitro technique for making a large number of copies of a specific fragment of target DNA from template DNA (i.e., DNA containing the target region to be replicated). During the reaction, a mixture containing the target DNA, primers, dNTPs, and a thermostable DNA polymerase is heated to 90°C to 95°C to denature the strands of the target DNA. The solution is cooled to a temperature that allows the primers (single-stranded DNA molecules approximately 18 to 30 nucleotides in length) to anneal to the complementary sequences on the target DNA and provide the 3'-OH required for DNA synthesis. Subsequently, the DNA polymerase synthesizes a new DNA strand complementary to the target by extending the primers, which typically occurs at a temperature of approximately 72°C. The thermal cycling protocol of denaturation / primer annealing / primer extension is repeated multiple times, where the DNA synthesized in the previous cycles serves as a template for each subsequent cycle. As a result, the amount of target DNA present in each cycle doubles, and the target DNA sequence accumulates exponentially over 20 to 40 cycles. A heating block with an automated thermal cycler is used to precisely control the temperature. A preferred method for use in the present invention is qPCR amplification (also known as real-time PCR), where the amplification of the target DNA molecules is typically monitored during PCR (i.e., in real-time) using a non-specific fluorescent dye that intercalates into any double-stranded DNA or a sequence-specific DNA probe composed of oligonucleotides, where these oligonucleotides are labeled with a fluorescent reporter for the real-time detection of PCR products.

[0057] As used herein, the term "template" refers to the nucleic acid from which a target sequence is amplified in a nucleic acid amplification reaction. As used herein, the term "amplifiable template" refers to a template that produces a single amplicon upon amplification. An amplifiable template contains primer binding sites for hybridization with amplification primers.

[0058] As used herein in the context of isolating nucleic acid sequences from a biological sample, the term "isolate" refers to an in vitro process of extracting nucleic acids, preferably genomic DNA, from a sample of interest. The process typically may involve, but is not limited to, lysing the biological sample (in cells) using a guanidine detergent lysis solution that allows for the selective precipitation of DNA from the (cell) lysate, and precipitating genomic DNA from the lysate with ethanol. After ethanol washing, the precipitated DNA can be dissolved in water or 8 mM NaOH and used as a template in a PCR reaction. Analysis of genomic DNA samples for diagnostic purposes can be obtained by using DNA isolation techniques that are generally known. Total genomic DNA can be purified by using, for example, a combination of physical and chemical methods. Very suitable commercially available DNA isolation systems can be used, such as A nucleic acid extraction system (bioMérieux, Marcy l′Etoile, France) or a MagNA Pure 96 system (Roche Diagnostics GmbH, Mannheim, Germany).

[0059] As used herein, the term "PCR mixture" refers to a small amount of biochemical reactants in an aqueous liquid used to perform a PCR reaction, including: (genomic) template DNA containing the target DNA sequence; a set of at least two oligonucleotide primers that hybridize to opposite strands of the target DNA sequence and flank the region to be amplified; a thermostable DNA polymerase; four deoxynucleoside triphosphates (dNTPs) and Mg 2+ ions.

[0060] As used herein, the term "amplification primer" refers to an oligonucleotide primer that hybridizes to the opposite strand of the target DNA sequence and flanks the region to be amplified.

[0061] The terms "amplification product" and "amplicon", which are used interchangeably herein, refer to nucleic acid fragments that are products of nucleic acid amplification or replication events, such as those formed in polymerase chain reaction (PCR). As used herein, the term "PCR amplicon" refers to the PCR product or the amplified target DNA.

[0062] As used herein, the term "high-resolution melting curve (hrMC) analysis" refers to a post-PCR analysis method for identifying variations in nucleic acid sequences. This method is based on detecting small differences in the PCR melting (dissociation) curve. The temperature-dependent dissociation between two DNA strands can be exploited using DNA intercalating fluorophores such as SYBR Green, EvaGreen or as I. The "saturation dye" of LCGreen Plus or Cyto9 (a dye that does not inhibit PCR even when producing maximum fluorescence (saturation)) is measured in combination with a real-time PCR instrument with precise temperature ramp control and advanced (fluorescence) data acquisition capabilities. Software specifically designed for hrMC analysis is used to analyze and process the data. High-resolution melting curves are generated by slow ramping through a temperature gradient with high precision (e.g., 0.1 °C or lower), and the fluorescence level of the intercalating dye is measured at each step. The melting temperature of a DNA molecule is determined by the nucleic acid sequence and length, and differences in these nucleotide sequences between samples result in species-unique melting curves, even when using universal primers to isolate amplicons. Details of the hrMC analysis procedure are well known to those skilled in the art and are described, for example, in Reed GH, Kent JO, Wittwer CT (2007) High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics, 8, 597-608; US7,297,484; US7,387,887; US7,524,632; US20090117553 and US20100041044, the contents of which are incorporated herein by reference.

[0063] As used herein, the term "high-resolution melting curve (hrMC)" refers to a dissociation curve that describes the temperature-dependent dissociation between two DNA strands measured using a fluorophore intercalated into the DNA. The high-resolution melting curve can be referenced to a negative first derivative plot of the melting curve, which makes it easier to precisely determine (pin-point) the dissociation temperature (defined as 50% dissociation) based on the peaks formed thereby.

[0064] As used herein, the term "electrophoretic separation and amplicon length analysis" refers to a technique in which a mixture of charged molecules, preferably nucleic acids, especially PCR-amplified DNA fragments, loaded on a gel matrix, migrates from the negative electrode (cathode) towards the positive electrode (anode) according to size, charge, and structure when the gel is placed in an electric field, such that shorter nucleic acid fragments migrate faster than longer ones, thereby separating based on size. Electrophoretic separation and amplicon length analysis is preferably performed by capillary electrophoresis, whereby DNA is detected by ultraviolet absorption or fluorescence labeling. In the presence of appropriate standards, the fragment size can be accurately determined based on the relative electrophoretic mobility, for example, using an ABI Prism 3500 genetic analyzer (Applied Biosystems) or a similar analyzer.

[0065] "Electrophoretic separation and amplicon length analysis" as defined herein can also be performed by DNA sequencing of the amplicons. DNA sequencing can provide accurate information about the length of the amplicons.

[0066] As used herein, the term "negative control reaction" refers to a post-PCR mixture that does not contain PCR amplicons due to the deliberate absence of the target nucleic acid sequence or template DNA in the pre-PCR mixture.

[0067] As used herein, the term "primer dimer (PD)" refers to a potential by-product in PCR, which consists of primer molecules that anneal (hybridize) to each other due to complementary base strings in the primers. Thus, DNA polymerase amplifies the PD, initiating competition with the PCR reagents and potentially inhibiting the amplification of the DNA sequence targeted by the PCR. In quantitative PCR, the PD may interfere with accurate quantification.

[0068] As used herein, the term "non-specific PCR amplicon" refers to a potential by-product in PCR, which consists of amplified DNA that is not the target DNA, usually resulting from non-specific annealing (hybridization) of primer molecules to other nucleic acid sequences (such as human DNA) in the template DNA. The hrMCs generated by non-specific PCR amplicons are different from the PCR amplicons generated by the target microbial DNA sequences.

[0069] As used herein, the term "human PCR amplicon" refers to a non-specific PCR amplicon in which the primer molecules anneal to human nucleic acid sequences in the template DNA rather than microbial DNA sequences. The hrMCs generated by human PCR amplicons are different from the PCR amplicons generated by (microbial) target DNA.

[0070] As used herein, the term "quantification cycle" or "Cq" includes the measurement performed in a real-time PCR assay or qPCR assay, whereby a positive reaction is detected by the accumulation of a signal (such as a fluorescence signal). The Cq (quantification cycle) can be defined as the number of cycles required for the signal to cross a threshold (i.e., exceed the background level). The Cq level is inversely proportional to the amount of target nucleic acid in the sample (i.e., the lower the Cq level, the higher the amount of target nucleic acid in the sample).

[0071] As used herein, the term "sample" or "biological sample" includes a sample from a human body, an animal body, a plant, a laboratory culture, an environmental sample, or a food item, a drug product, or a chemical product, preferably where the food item, drug, or chemical product is intended to be free of microorganisms or microbial DNA.

[0072] As used herein, the term "subject" is intended to denote any individual or patient upon whom the methods described herein are performed. Generally, the subject is a human, although as will be appreciated by those skilled in the art, the subject can be an animal. Thus, other animals including mammals and birds are also included within the definition of subject.

[0073] Performing PCR in the method of the present invention

[0074] In the method of the present invention, the step of amplifying DNA is carried out by polymerase chain reaction (PCR).

[0075] Preferably, in the method of the present invention, the PCR is qPCR or real-time PCR. The present invention relates to the broad-range classification amplification or universal amplification of at least one rRNA ITS region of microbial genomic DNA in a sample, i.e., the taxon-specific amplification of genomic DNA of entire microbial genera, families, orders, classes, phyla or kingdoms.

[0076] Methods and means for establishing such PCR reactions are well known to those skilled in the art. Preferably, the method of the present invention is used for detecting or identifying bacteria or bacterial DNA in a sample. Exemplary primer sets for amplifying the 16S-23S rRNA ITS region of genomic DNA of one or more of the phyla Firmicutes, Bacteriodetes or Proteobacteria are provided as SEQ ID NO: 1-13. Exemplary primer sets for the universal amplification of the 16S-23S rRNA ITS region of bacterial genomic DNA are provided as SEQ ID NO: 14-15. Designing further primer sets is within the routine capabilities of those skilled in the art, and these primer sets allow for broad-range classification amplification of microbial, preferably bacterial DNA.

[0077] High-resolution melting curve (hrMC) analysis in the method of the present invention

[0078] Characterizing nucleic acids by high-resolution melting curve (hrMC) analysis is a powerful technique for identifying sequence variations in a sample during or after PCR. By measuring the fluorescence of a saturated intercalating dye as the DNA fragment amplified by PCR is heated and dissociates, a sequence-defined melting curve with single-nucleotide resolution can be generated.

[0079] In a preferred embodiment of the aspect of the present invention, the hrMC dye is selected from, but not limited to, the group consisting of LC Green, SYTO9, EvaGreen, Chromofy, BEBO or SYBR Green, preferably Eva Green. Preferably, the saturated intercalating dye should not inhibit PCR.

[0080] In the method of the present invention, the hrMC curve of the PCR amplicon can be generated during or after the PCR amplification step by measuring the fluorescence of a saturating intercalating dye of the amplicon present in the post-PCR sample. As used herein, a "melting curve" refers to a curve generated by hrMC analysis. Those skilled in the art are well aware of how to perform hrMC. Ample guidance can be found, for example, in Reed et al. 2007 Pharmacogenomics 8(6):597-608 and Wittwer et al. 2003 Clin Chem. 49:853-860.

[0081] High-resolution melting curve analysis in qPCR (i.e., real-time PCR) is not equivalent to the analysis performed by digital PCR. In digital PCR, each partition contains only a single sample template, which means that all amplicons in each partition are derived from a single template. This homogeneity makes it easier to interpret the hrMC curve and enables direct comparison with other partitions. In real-time PCR, a heterogeneous sample of multiple targets after amplification will still remain heterogeneous, and thus the melting curve will reflect this heterogeneity. In the case of multiple amplicons present in a sample, this makes it difficult to distinguish multiple targets by hrMC analysis.

[0082] In the method of the present invention, an hrMC curve of the post-PCR sample is generated. Preferably, the PCR is qPCR or real-time PCR. The generated hrMC curve is compared with one or more corresponding (predetermined) reference hrMC curves of 16S-23S rRNA ITS amplicons of known (i.e., taxonomically identified) microbial strains or species. Preferably, the reference hrMC curves of one or more corresponding 16S-23S rRNA ITS amplicons of the known strain or species are included in a library or database of reference hrMC curves. Such a library or database can be established by: (i) generating in vitro 16S-23S rRNA ITS PCR amplicons of individual strains and / or species of microorganisms, (ii) performing hrMC analysis on the amplicons, and (iii) storing the reference hrMC curves in a library or database of reference hrMC curves, where the species or strain identity is annotated to the hrMC curves. Alternatively, a database or library of hrMC curves can be generated by computer predicting the hrMC curves of one or more 16S-23S rRNA ITS PCR amplicons of individual strains and / or species of microorganisms.

[0083] From a clinical perspective, if the hrMC curve of the 16S-23S rRNA ITS PCR amplicon in a PCR sample indicates the presence of a microorganism, such as a bacterium, in the clinical sample, the clinician can already make an important first treatment decision: if the microorganism present is sensitive to an antimicrobial agent, initiate administration of a therapeutic amount of the antimicrobial agent. For example, if it is determined based on hrMC analysis that bacteria are present, the clinician can make a treatment decision to initiate administration of an antibiotic.

[0084] However, in many cases, qPCR or real-time PCR does not ultimately identify the microorganism present. In the case where only a single microorganism is present in the sample, if it matches the reference hrMC curve, hrMC analysis of the PCR product can provide a definitive answer regarding the identification of the microorganism. In other cases, such as when more than one microorganism is present in the sample, hrMC analysis generally does not provide the clinician with actionable information regarding the identification of the microorganism.

[0085] The inventors have found that combining hrMC analysis of a discrimination sequence with PCR fragment length analysis based on fragment length in a clinical assay is highly beneficial in increasing the sensitivity and specificity of a PCR-based microorganism identification assay. This combined approach enables the interpretation of the resulting hrMC curve based on the PCR fragment length profile and vice versa, which significantly improves specificity. This has been hitherto unknown in the art. The combination of these two techniques provides the clinician with actionable information regarding the identification of the microorganism. Preferably, the specificity (of microorganism identification) provided by the method of the present invention is preferably at least 90%, more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%.

[0086] PCR fragment length analysis in the method of the present invention

[0087] The method of the present invention relates to the step of performing amplicon fragment length analysis on the amplicons in a PCR sample to generate a fragment length profile of the amplicons present therein.

[0088] Methods and means for determining (PCR) fragment length profiles are generally known in the art. An exemplary and highly useful method for PCR fragment length analysis is capillary gel electrophoresis. Preferably, the capillary gel electrophoresis method, such as the capillary gel electrophoresis method called the IS-pro method described in Budding et al., FASEB J., 24, 4556-4564 (2010), WO2008 / 125365 and WO2015 / 170979, and in a preferred embodiment, may involve differentiating bacterial species based on the length of the 16S-23S rDNA ITS region with taxonomic classification by gate-specific fluorescent labeling of PCR primers.

[0089] The IS-pro method described in the above publications is expressly incorporated herein by reference. For example, the method for analyzing microbial populations described on page 4, line 11 and further in WO2008 / 125365, including the primer sets described therein, are incorporated herein by reference. In addition, for example, the PCR calibration system and its uses described in WO 2015 / 170979 (see, for example, page 44 and further), the IS-pro analysis described in WO 2015 / 170979 (see, for example, page 53 and further), the primer sets and probes described in WO 2015 / 170979 (see, for example, pages 65 and 66 of WO 2015 / 170979), and the types of patient samples described in WO 2015 / 170979 (see, for example, page 11, line 18 and further) are incorporated herein by reference.

[0090] In short, for the IS-pro method, the sequences of conserved DNA regions in the 16S and 23S rRNA gene sequences flanking the intergenic region contained in the genomic DNA of the microorganism are used as primer binding sites for amplifying the (polymorphic) ITS DNA region.

[0091] The taxonomic diversity analysis of IS-pro is based on the fact that prokaryotic microorganisms, including bacteria and archaea, include one or more copies of rrn operons in their genomes, and the rrn operon copies include genes for 5S, 16S, and 23S ribosomal RNAs. In most prokaryotes, the order of the ribosomal genes in the operon is 16S-23S-5S and is co-transcribed in a single polycistronic RNA, which is processed to provide the RNA species present in mature ribosomes. The spacer region between the 16S and 23S genes contains regions with secondary structure and sometimes tRNA genes. The variation in the spacer regions of rRNA operons found in relatively closely related taxa is very high. The great differences in the size and sequence of spacer regions between different prokaryotic groups make them very suitable as taxonomic markers.

[0092] In the IS-pro method, primer pairs targeting conserved regions in ribosomal gene sequences are used to amplify the 16S-23S rRNA gene internal transcribed spacer (ITS) region. More preferably, the conserved DNA regions are those closest to the 3′ end of the 16S rRNA gene and closest to the 5′ end of the 23S rRNA gene.

[0093] Depending on the microbiome being studied, phylum-specific primer sets can be used. Suitable phylum-specific primer sets in IS-pro include those capable of simultaneously amplifying multiple sequences in a single reaction in a process called multiplex PCR.

[0094] To amplify the intergenic spacer regions of bacterial DNA of the phyla Firmicuta and Actinobacteria, suitable primer sets include:

[0095] FirISf: 5′-CTGGATCACCTCCTTTCTAWG-3′ (SEQ ID NO: 1) as the forward primer, and one of DUISrI: 5′-AGGCATCCACCGTGCGCCCT-3′ (SEQ ID NO: 3), DUISr2: 5′-AGGCATTCACCRTGCGCCCT-3′ (SEQ ID NO: 4), and DUISr3: 5′-AGGCATCCRCCATGCGCCCT-3′ (SEQ ID NO: 5) as the reverse primer. Preferably, in this article, the FirISf primer is tagged with a fluorescent label. Preferably, in this article, the reverse primer is not tagged.

[0096] Genera within the phylum Firmicutes include: Bacilli, the order Bacillales (genus Bacillus, genus Listeria, genus Staphylococcus); Bacilli, the order Lactobacillales (genus Enterococcus, genus Lactobacillus, genus Lactococcus, genus Leuconostoc, genus Pectinatus, genus Pediococcus, genus Streptococcus); Clostridia (genus Acetobacterium, genus Clostridium, genus Eubacterium, genus Heliobacterium, Heliospirillum, Sporomusa); Mollicutes (genus Mycoplasma, genus Spiroplasma, Ureaplasma (genus Ureaplasma), genus Erysipelothrix).

[0097] To amplify the intergenic spacer regions of genes from bacteria of the phylum Bacteroidetes, suitable primer sets include:

[0098] BacISf: 5′-CTGGAACACCTCCTTTCTGGA-3′ (SEQ ID NO: 2) as the forward primer, and one or more, preferably all, of DUISrI: 5′-AGGCATCCACCGTGCGCCCT-3′ (SEQ ID NO: 3), DUISr2: 5′-AGGCATTCACCRTGCGCCCT-3′ (SEQ ID NO: 4), and DUISr3: 5′-AGGCATCCRCCATGCGCCCT-3′ as the reverse primer. Herein, preferably, the BacISf primer bears a fluorescent label. Herein, preferably, the reverse primer is unlabeled.

[0099] In a broad community analysis, the intergenic spacer regions of bacteria from the phyla Firmicutes and Actinobacteria can be amplified in a single multiplex reaction with the intergenic spacer regions of bacteria from the phylum Bacteroidetes. In such a multiplex reaction, the PCR mixture includes the forward primers FirISf and BacISf and all three reverse primers. Preferably herein, the FirISf primer bears a first fluorescent label, such as FAM. And the BacISf primer bears a second fluorescent label, such as HEX. Preferably herein, the reverse primers are unlabeled.

[0100] For a broader or alternative community analysis, the intergenic spacer regions of bacteria from the phylum Proteobacteria can be amplified, and suitable primer sets include:

[0101] ProtISf: 5′-CCGCCCGTCACACCATGG-3′ (SEQ ID NO: 6) as the forward primer, and one or more of DPISr1: 5′-AATCTCGGTTGATTTCTTTTCCT-3′ (SEQ ID NO: 7), DPISr2: 5′-AATCTCGGTTGATTTCTTCTCCT-3′ (SEQ ID NO: 8), DPISr3: 5′-AATCTCTTTTGATTTCTTTTCCTCG-3′ (SEQ ID NO: 9), DPISr4: 5′-AATCTCATTTGATGTCTTTTCCTCG-3′ (SEQ ID NO: 10), DPISr5: 5′-AATCTCTTTTGATTTCTTTTCCTTCG-3′ (SEQ ID NO: 11), DPISr6: 5′-AATCTCTCTTGATTTCTTTTCCTTCG-3′ (SEQ ID NO: 12), and DPISr7: 5′-AATCTCAATTGATTTCTTTTCCTAAGG-3′ (SEQ ID NO: 13), preferably all as reverse primers. Preferably herein, the ProtISf primer is labeled with a fluorescent label. Preferably herein, the reverse primers are unlabeled.

[0102] Genera within the phylum Bacteroidetes include: Bacteroides (a prevalent organism in the feces of warm-blooded animals including humans; including, for example, B. acidifaciens, B. distasonis, B. gracilis, B. fragilis, B. oris, B. ovatus, B. putredinis, B. pyogenes, B. stercoris, B. suis, B. tectus, B. thetaiotaomicron, B. vulgatus) and Porphyromonas (a group of organisms that inhabit the human oral cavity).

[0103] Very advantageously, amplification of the phylum Proteobacteria is carried out by multiplex PCR in order to provide sufficient taxonomic resolution within the phylum Proteobacteria (see WO 2015 / 170979).

[0104] Preferred microorganisms in the context of the present invention belong to bacterial phyla selected from the group consisting of: Firmicutes, Fusobacterium, Deferribacteres, Spirochaetes, Cyanobacteria, Acidobacteria, Nitrospina, Nitrospirae, Caldithrix, Haloanaerobiales, Verrrucomicrobia, Chlamydiae, Planctomycetes, Gemmimonas, Fibrobacteres, Chlorobi, Bacteroidetes, Proteobacteria, Thermotogae, Corprothermobacter, Synergites, Thermodesulfobacteria, Desulfurobacterium, Aquificae, Deinococcus-Thermus, Chloroflexi, Tenericutes, and Actinobacteria. More preferred phyla targeted in the method of the present invention include Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, Fusobacterium, and Verrrucomicrobia. Highly preferred are Bacteroidetes, Firmicutes, Actinobacteria, and Proteobacteria.

[0105] These phyla are known to the person skilled in the art and have been described, inter alia, in Schloss 2004 (Microb. Mol. Biol. Rev. 6(4): 686-691) or in Bergey manual of systematics of archaea and bacteria (2015).

[0106] In the method of the present invention, universal amplification of the bacterial 16S-23S ribosomal RNA internal transcribed spacer (ITS) region of genomic DNA can also be employed. For such universal amplification of bacterial DNA, a universal bacterial primer set can be used that includes: a forward primer ITS1FD having the sequence 5′-CGGTGAATACGTTCCCGGIIIIIGTACAC-3′ (SEQ ID NO: 14) in combination with a reverse primer ITS2RD having the sequence 5’-CGTCCTTCDTCGVCTBIIIIIGCCARG-3’ (SEQ ID NO: 15).

[0107] Samples to which the method of the present invention can be applied can be from any environment containing microorganisms, but are preferably samples from mammals (such as humans). Suitable sample materials include, for example, samples from humans, plants, animals, water, food (such as dairy products), yeast cultures (for example for industry), or soil.

[0108] In the IS-pro technology, microbial DNA is suitable and is usually isolated by an automated separation procedure (such as EasyMag, Biomerieux, Marcy 1'Etoile, France) according to the manufacturer's instructions. In the method of the present invention, microbial DNA is preferably isolated from the sample in the form of genomic DNA. Methods for isolating genomic DNA available in the art are well known to those skilled in the art.

[0109] Then, according to the protocol provided by the manufacturer, or using any of the primer sets described above, the 16S-23S rRNA ITS region was amplified from the isolated microbial DNA by IS-pro assay (inBiome, Amsterdam, the Netherlands) using phylum-specific fluorescently labeled PCR primers. IS-pro involves the amplification of the 16S-23S rRNA ITS region and subsequent identification of microbial species based on the length of the amplicons as the source of the DNA template that produced the amplicons. Briefly, the appropriately diluted isolated DNA can be amplified in a standardized PCR amplification. An example of a standardized PCR amplification includes using a phylum-specific primer set to amplify the 16S-23S rRNA ITS region of the combined phyla Firmicutes, Actinobacteria, Fusobacteria, and Verrucomicrobia (FAFV). Another example of a standardized PCR amplification includes using a phylum-specific primer set to amplify the 16S-23S rRNA ITS region of Bacteroidetes. Additionally, the 16S-23S rRNA ITS regions of Firmicutes and Bacteroidetes can be amplified in a single reaction to provide amplicons from any bacterial species from these phyla from a DNA sample (e.g., using SEQ ID NOs: 1-5 in a single multiplex reaction). Another example of a standardized PCR amplification includes using a phylum-specific primer set to amplify the 16S-23S rRNA ITS region of Proteobacteria (e.g., using the primers of SEQ ID NOs: 6 and 7-13). Another example of a standardized PCR amplification includes using a kingdom-specific primer set to amplify the 16S-23S rRNA ITS region of microorganisms belonging to the kingdom Bacteria (e.g., using the primers of SEQ ID NOs: 14 and 15).

[0110] After PCR amplification and generation of the amplicons, the PCR products were then divided into small aliquots and analyzed by capillary electrophoresis to reveal the 16S-23S rRNA ITS amplicon lengths, for example, in an ABI Prism 3500 Genetic Analyzer (Applied Biosystems, Carlsbad, California, USA).

[0111] In IS-pro, when the 16S-23S rRNA ITS region is amplified from the microbial genomic DNA template in a sample using a broad taxonomic range or universal amplification primers, different bacterial species with different 16S-23S ITS DNA sequences produce amplicons of different lengths, which are easily separated by capillary gel electrophoresis, providing a profile of different DNA fragments, each representing a separate 16S-23S ITS DNA sequence with a characteristic length (in number of nucleotides or base pairs).

[0112] Typically, each fragment is considered to represent a single ITS region from a different bacterial species or operational taxonomic unit (OTU). However, different bacterial species may produce 16S-23S ITS amplicons of the same length. In addition, since the genome may contain multiple rrn operons with different ITS region sequences, not every fragment necessarily represents a different microbial species. Thus, different peaks (amplicons of different lengths) may originate from a single species or different species. Finally, minor biological length variations in the ITS sequence may complicate simple species identification based solely on length measurements.

[0113] To improve resolution and species identification and better classify and assign amplicons from a microbial DNA sample to a specific species or strain, the present inventors have now found that adding an hrMC analysis step allows individual species that produce amplicons of the same length (i.e., produce a single electrophoretic peak) to be separately identified. hrMC analysis is capable of identifying sequence differences between amplicons of the same length, thus enabling the species under the same CE peak to be separately identified. In addition, by adding the hrMC step, multiple amplicons of different lengths derived from a single species can be unambiguously assigned to the correct species. Further, fragments in combined form derived from a single species or (more) different species can be classified and assigned as being from a specific species or strain, or from multiple species or strains. This can be done in both complex and uncomplex microbial populations.

[0114] These improvements are extremely important for the reliability of assays used in clinical practice.

[0115] In addition, it has now been found that certain amplicon peaks in the amplicon length profile can now be attributed to human DNA (false positives), which greatly improves the specificity of the assay in clinical settings, because if hrMC analysis confirms the presence of human DNA, then ambiguous length peaks in the amplicon length profile that could not be excluded as "non-microbial" by the clinician based on the amplicon length profile can now be excluded.

[0116] Since the hrMCs of different microbial species are generally easily distinguishable, the specificity of the test is improved, thus enabling individual species that produce the same amplicon length in PCR (e.g., Corynebacterium jeikeium / Streptococcus pyogenes) to be clearly detected based on differences in their hrMCs.

[0117] The method of the present invention comprises the steps of adding hrMC staining before, during, or after PCR amplification and performing hrMC analysis on the post-PCR mixture, particularly the PCR amplicons.

[0118] Another advantage of using hrMC analysis in a diagnostic method is the amplification reaction involving the use of primers with a wide classification range and subsequent electrophoretic separation and amplicon length analysis, where hrMC analysis can increase the speed of the procedure. Once the hrMC of the reaction product is known, electrophoretic separation and amplicon length analysis can be performed only on fragments within a predetermined length range based on the hrMC data (e.g., only fragments with lengths between 300 and 500 bp are separated and analyzed). For example, if the hrMC indicates the presence of one of three possible species, the known amplicon lengths of these species can be immediately "amplified" to resolve the problem.

[0119] The method of the present invention can be used for genotyping, diagnosing, investigating, analyzing, and monitoring various microbial hiding samples such as those associated with the intestinal or gastrointestinal tract, skin, urogenital tract, oral cavity, and lung system, and for diagnosing, monitoring, or predicting diseases. The method can be used for diagnostic purposes, such as for diagnosing, monitoring, or predicting (including early detection) microbial infections, and can even be used for environmental diagnosis, such as for determining the microbial status of a sample source, where the sample source is of environmental, plant, animal, or food origin, or a sample of a drug or chemical product intended to be free of microorganisms or microbial DNA, and where the specific profile of the ITS region (or its absence) indicates the sterility of the sample, the quality of the environment, the microbial safety of food and drugs, the quality of chemical products, or the health of plants or animals.

[0120] An alternative method for determining the length of a PCR amplicon is by the step of DNA sequencing of the amplicon in the post-PCR sample. Preferably, the DNA sequencing step is performed by a next-generation sequencing method capable of determining the sequence length of amplicons with base pair lengths in the range of 100 to 1200 bps, which length range generally resembles the overall variation in the lengths of 16S-23S ITS DNA sequences between different microbial species. Suitable next-generation sequencing methods for determining amplicon length include: nanopore-based methods (e.g., devices manufactured by Oxford Nanopore) or Pacific-Biosciences or single-molecule real-time sequencing methods (e.g., devices manufactured by Pacific Biosciences).

[0121] When performing the step of determining the length of PCR amplicons in the method of the present invention, an amplicon length profile can be generated. The generated amplicon length profile can be compared with one or more reference or control amplicon length profiles of the corresponding 16S-23S rRNA internal transcribed spacer (ITS) regions of known microbial strains or species. Preferably, the reference amplicon length profiles of the corresponding 16S-23S rRNA internal transcribed spacer (ITS) regions of the known microbial strains or species are included in a library or database of reference amplicon length profiles. Such a library or database can be established by: (i) in vitro generating PCR amplicons of the target 16S-23S rRNA internal transcribed spacer (ITS) regions of the genomic DNA of known individual microbial strains and / or species; (ii) performing fragment length analysis on the amplicons; and (iii) storing the reference fragment length profiles in a library or database of reference fragment length profiles. Alternatively, a database or library of reference fragment length profiles can be generated by computationally predicting the fragment length profiles of PCR amplicons of the target 16S-23S rRNA internal transcribed spacer (ITS) regions of the genomic DNA of individual microbial strains and / or species.

[0122] It is within the routine experimentation of those skilled in the art to establish (predetermined) reference or control amplicon length profiles suitable for comparison with the amplicon length profiles generated from test samples. For example, the method of the present invention can include the step of analyzing the amplification products or amplicons (after PCR) based on length differences in the amplification products, thereby providing an amplicon length profile of the microbial population composition in the microbiome or sample as disclosed herein; and comparing the fragment length profile with at least one reference fragment length profile of known microorganisms.

[0123] For purposes of clear and concise description, features are described herein as part of the same or separate embodiments, however, it should be understood that the present disclosure includes embodiments having combinations of all or some of the described features.

[0124] The content of the documents cited herein is incorporated herein by reference.

[0125] Examples

[0126] Example 1 Combination of melting curve and fragment length data for unambiguous species identification

[0127] To detect and identify different microorganisms, DNA regions that are widely conserved across taxa can be used when they possess specific characteristics. Important characteristics are the presence of conserved regions in the DNA that can be used to amplify the DNA of many different species using a wide range of primers; and the presence of variable regions flanked by conserved regions that can be used to distinguish between different species. There are different methods for analyzing the variable regions, which have different applicability based on the specific characteristics of the variable regions. The DNA region that is present in all life forms and harbors both conserved and variable regions scattered throughout is ribosomal DNA (rDNA). In bacterial ribosomal DNA, the 16S-23S intergenic spacer (IS) region is of particular interest because it is present in almost all bacteria, exhibits high variability in sequence composition and length, and is flanked by highly conserved regions, making it highly suitable for targeting by a wide range of primers.

[0128] In this paper, we will demonstrate a very effective and highly accurate method for detecting and identifying bacteria that is based on a combination of melting curve and fragment length analysis of the 16S-23S IS region.

[0129] The melting curve of the 16S-23S intergenic spacer (IS) fragment in bacteria can be used to distinguish between different bacterial species. Additionally, length measurements can also distinguish between different bacterial species. While melting curve analysis or fragment analysis can be used alone to identify bacterial species, this is not possible in many cases. In these cases, the fragment profiles between species are the same, or the melting curves do not provide sufficient discrimination between species. When fragment length analysis is combined with melting curve analysis and the results are compared to a database, unambiguous species identification can be performed. Here, we will show that the combination of fragment length analysis and melting curve analysis can clearly distinguish bacteria down to the species level, whereas neither fragment length analysis nor melting curve analysis alone can achieve this.

[0130] Materials and Methods

[0131] Strain Selection and Cultivation

[0132] Twelve different clinical isolates were used in this analysis: Bacteroides fragilis, Bacteroides thetaiotaomicron, Enterococcus faecalis, Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus cristatus, Streptococcus pyogenes, Corynebacterium jeikeium, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Enterobacter cloacae. Aerobic bacteria were cultured on sheep blood agar (BioMerieux) at 37 °C for 2 days of aerobic incubation, and anaerobic bacteria were cultured on Schaedler agar (Oxoid) for 3 days of anaerobic incubation. Bacterial colonies were identified by MALDI-TOF (VITEK MS system, Biomérieux).

[0133] DNA Isolation

[0134] DNA was isolated by adding 200 μl of suspended bacteria (0.1 McFarland) to 400 μl of AL buffer (Qiagen, Hilden, Germany) and 40 μl of proteinase K in an Eppendorf container. The mixture was centrifuged at 9000 g for 10 seconds, then vortexed and incubated at 56 °C while shaking at 1400 rpm for 1 hour. Further DNA extraction was performed using an easyMAG automated DNA isolation machine (Biomérieux).

[0135] The sample mixture (640 μl) was transferred to an 8-well easyMAG container and suspended in 2 ml of the nucliSENS lysis buffer provided by the manufacturer. After incubation at room temperature for 1 hour, 70 μl of magnetic silica beads, as provided by the easyMAG machine, were added. Subsequently, the mixture was inserted into the easyMAG machine, the "Specific A" protocol was selected, the off-site workflow was chosen, and the DNA was eluted in 110 μl of NucliSens easyMAG extraction buffer 3 provided by the manufacturer (Biomérieux). All DNA was stored at 4 °C.

[0136] PCR

[0137] Three different PCRs were used for various analyses: the first two PCRs were used for phylum-specific amplification and fragment analysis of the 16S-23S region, and the third PCR was used for general amplification, fragment analysis, and melting curve analysis of the 16S-23S region. The first PCR contained two different fluorescently labeled forward primers (SEQ ID NO: 1 and 2) targeting different bacterial groups and three reverse primers (SEQ ID NO: 3-5) providing general coverage for these bacterial groups. The first forward primer (SEQ ID NO: 1) was specific for the phyla Firmicutes, Actinobacteria, Fusobacteria, and Verrucomicrobia (FAFV), while the second tagged forward primer (SEQ ID NO: 2) was specific for the phylum Bacteroidetes. The second PCR used a tagged forward primer (SEQ ID NO: 6) in combination with seven reverse primers (SEQ ID NO: 7-13) and was specific for the phylum Proteobacteria (see Table 1). The third PCR reaction mixture contained a primer set that recognized a broad collection of bacteria: FOR: 5’CGGTGAATACGTTCCCGGIIIIIGTACAC 3’ (SEQ ID NO: 14) and REV 5’CGTCCTTCDTCGVCTBIIIIIGCCARG-3’ (SEQ ID NO: 15). Inosine binds to all 4 DNA bases, ensuring broad reactivity. The FOR primer contained the ATTO550 fluorescent moiety. The PCR reaction mixture contained EvaGreen, an intercalating dye that fluoresces after binding to double-stranded DNA.

[0138] Amplification of the first two PCRs was performed on a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA).

[0139] Melting curve analysis

[0140] The third PCR included melting curve analysis and was performed in a LightCycler 480 (Roche) using the Figure 1 cycle schedule shown (the cycle schedule was the same for all three PCRs, and only the melting curve analysis was added on the lightCycler machine).

[0141] The program ended with a melting curve in which all PCR amplicons were slowly heated. The temperature of the DNA strand segments depended on the sequence and length. The derivative of the fluorescence was then converted to 1 of the melting temperature of the PCR product. For example, see Figure 2 .

[0142] Species identification could be performed by melting curve analysis by comparison with a proprietary melting curve database (inBiome).

[0143] Fragment length analysis

[0144] After PCR, 5 μl of the PCR product was mixed with 20 μl of eMix (inBiome). DNA fragment analysis was performed on an ABI Prism 3500 Genetic Analyzer (Applied Biosystems). The data was analyzed using the inBiome proprietary software suite (inBiome, Amsterdam, the Netherlands), and the results were presented in the form of a microbial profile. Automated species calling of the fragment length profile was performed using a dedicated software suite (inBiome), where the peaks were associated with a database containing IS-profile information of >500 microbial species.

[0145] Peaks below 128 RFU were considered background noise and discarded from further analysis. The entire process from DNA isolation to analysis data could be completed within four hours.

[0146] Results

[0147] Melting curves and fragment analyses were performed on the 16S-23S IS regions of eight different species from three different bacterial phyla to demonstrate the discriminatory potential of the combination of melting curve and fragment length data. The results are as Figure 3 shown. Here, it can be clearly seen that the melting curves and fragment length distributions are highly variable between species, even between different species within the same genus. By comparing these combined melting curve and fragment length data with a database (inBiome) containing reference profiles of known species, each species can be unambiguously identified at the species level in this analysis.

[0148] Example 2 Accurate identification in the case of the same fragment length

[0149] There may be cases where the melting curve or fragment length profile of one species is similar to that of another species. By combining the melting curve data with the fragment length data, unambiguous identification can still be performed. In this example, an exemplary real situation with similar fragment length distributions is given.

[0150] Although the lengths of DNA fragments from the 16S-23S intergenic region are highly specific to each species, in some cases, different species have the same fragment length characteristics. In these cases, although the fragment lengths may be the same, the nucleotide sequences in these fragments are necessarily different: modern taxonomy is mainly based on the sequences of ribosomal DNA, and different species always have different nucleotide sequences in this region, especially in the intergenic (IS) region, which is the most variable region.

[0151] The melting temperature of a fragment is determined by two factors: the length and the sequence of the fragment. Thus, when two fragments from different species have the same length, their sequences must be different, and thus their melting curve characteristics must be different. This is the case as Figure 4 shown for two clinically highly relevant bacterial species, Streptococcus pyogenes and Corynebacterium jeikeium, where species-level identification is crucial for the correct treatment of patients.

[0152] It is shown that the method of the present invention can accurately distinguish and identify these two species.

[0153] Example 3 Accurate identification in the case of identical melting curves

[0154] Since the melting curve depends on the length and sequence of the fragment, although the length and sequence are different, the mutual influence between the length and sequence may still result in similar melting curves.

[0155] In addition, the similarity of the melting curves may also be caused by the presence of multiple (PCR) fragments, where the melting curves of the individual fragments are superimposed on each other. This results in a complex melting curve, which will exhibit reduced resolution. This phenomenon is particularly problematic in closely related species, where each species harbors multiple rRNA operons and thus harbors 16S-23S IS PCR fragments. In these cases, combining the melting curve data with fragment length analysis can still uniquely identify each bacterial species. This exemplary real situation is illustrated in the analysis of the 16S-23S IS profiles of closely related species in the Enterobacteriaceae, which is a highly relevant bacterial family in clinical microbiology and contains many human pathogens. The following figure shows the melting curve and fragment length analysis of Enterobacter cloacae and Klebsiella pneumoniae. Although it is difficult to distinguish the melting curves due to the many similar IS fragments in these closely related species, the unique length distribution can still distinguish these species (see Figure 5 ).

[0156]

Claims

1. A method for identifying microorganisms in a biological sample at the species or strain level by polymerase chain reaction (PCR), the method comprising the following steps: a) Providing a biological sample suspected of containing microorganisms and containing microbial nucleic acid sequences; b) Using a broad taxonomic range amplification primer set to PCR amplify at least one microbial ribosomal RNA internal transcribed spacer (ITS) region contained in the microbial nucleic acid sequences, thereby generating PCR amplicons, wherein the broad taxonomic range amplification primer set is used to amplify the at least one microbial ribosomal RNA ITS; c) Recording the high-resolution melting curve of the PCR amplicons generated in step b) and recording the length of the PCR amplicons generated in step b) by capillary electrophoresis or sequencing; d) Comparing the high-resolution melting curve recorded in step c) with a database of high-resolution melting curves of reference amplicons, thereby obtaining a first taxonomic identification index of the microorganisms present in the sample, wherein the reference amplicons are generated from reference microbial species or strains with known taxonomic identification using the same amplification primer set; e) Comparing the length of each PCR amplicon with different lengths recorded in step c) with a database of PCR amplicon lengths of reference amplicons, thereby obtaining a second taxonomic identification index of the microorganisms present in the sample, wherein the reference amplicons are generated from reference microbial species or strains with known taxonomic identification using the same amplification primer set; f) When the first taxonomic identification index matches the second taxonomic identification index, then the microorganisms present in the sample are identified at the species or strain level; The method is for non-diagnostic purposes.

2. The method according to claim 1, wherein, The method includes the step of isolating the microbial nucleic acid sequences contained in the biological sample.

3. The method according to claim 1 or 2, wherein The biological sample suspected of containing microorganisms and containing microbial nucleic acid sequences is a biological sample suspected of containing archaea, bacteria, viruses, protozoa or fungi, or a combination thereof.

4. The method according to claim 1 or 2, wherein, The reference amplicons in the database include amplicons generated by in vivo and / or in silico PCR amplification reactions for amplifying the corresponding ribosomal RNA ITS regions of reference microbial species or strains with known taxonomic identification using the same broad taxonomic range amplification primer set for amplifying the at least one microbial ribosomal RNA ITS region.

5. The method according to claim 1 or 2, wherein Combining the databases in steps d) and e) into a single database.

6. The method according to claim 1 or 2, wherein The database includes ribosomal RNA ITS sequences of bacteria and corresponding taxonomic identification data.

7. The method according to claim 1 or 2, wherein The broad taxonomic range amplification primers are used to amplify the ribosomal RNA ITS regions of microorganisms from multiple strains or species of the genus, family, order, class, phylum, kingdom and / or domain of microorganisms.

8. The method according to claim 1 or 2, wherein The broad taxonomic range amplification primers are used to amplify the ribosomal RNA ITS regions of microorganisms from more than 50% or more of the strains or species of the genus, family, order, class, phylum, kingdom and / or domain of microorganisms.

9. The method according to claim 1 or 2, wherein, The broad taxonomic range amplification primers are used to amplify the ribosomal RNA ITS regions of microorganisms from more than 50% or more of the strains or species of the phylum of microorganisms.

10. The method according to claim 1 or 2, wherein The broad taxonomic range amplification primers are used to amplify the microbial rRNA ITS region, and the microorganisms are from more than 50% or more strains or species of the microbial kingdom.

11. The method according to claim 1 or 2, wherein, The broad taxonomic range amplification primers are used to amplify the microbial rRNA ITS region of bacteria.

12. The method according to claim 1 or 2, wherein, The broad taxonomic range amplification primers for amplifying the at least one microbial rRNA ITS region include forward and reverse primers for amplifying the 16S-23S rRNA ITS region, 23S-5S rRNA ITS region, 18S-5.8S rRNA ITS region of microorganisms, or 5.8S-26S / 28S rRNA ITS region of microorganisms.

13. The method according to claim 1 or 2, wherein The broad taxonomic range amplification primers for amplifying the at least one microbial rRNA ITS region include forward and reverse primers for amplifying the 16S-23S rRNA ITS region.

14. The method according to claim 1 or 2, wherein, The broad taxonomic range amplification primer set is an amplification primer set for amplifying at least one rRNA ITS region of bacteria of the phylum Bacteriodetes and / or Firmicutes.

15. The method according to claim 1 or 2, wherein The broad taxonomic range amplification primer set includes each amplification primer of SEQ ID NO: 1 and 3-5, or each amplification primer of SEQ ID NO: 2-5, or each amplification primer of SEQ ID NO: 1-5.

16. The method according to claim 1 or 2, wherein The broad taxonomic range amplification primer set includes each amplification primer of SEQ ID NO: 6 and 7-13.

17. The method according to claim 1 or 2, wherein The broad taxonomic range amplification primer set is a universal bacterial amplification primer set.

18. The method according to claim 1 or 2, wherein The broad taxonomic range amplification primer set includes each amplification primer of SEQ ID NO: 14-15.

19. The method according to claim 1 or 2, wherein, The step of PCR amplification includes qPCR.

20. The method according to claim 1 or 2, wherein, In step c), the length of the PCR amplicon is recorded by capillary electrophoresis or sequencing.

21. The method according to claim 1 or 2, wherein Step c) and optionally step b) are carried out in a miniaturized device.

22. The method according to claim 1 or 2, wherein Step c) and optionally step b) are carried out on a lab-on-a-chip device.

23. The method according to claim 1 or 2, wherein The database containing the high-resolution melting curves and PCR amplicon lengths of reference amplicons generated by known taxonomically identified reference microbial species or strains further includes: high-resolution melting curves and PCR amplicon lengths of reference amplicons generated by human sequences used as controls, and the human sequences are used to generate non-specific amplicons using the broad taxonomic range amplification primer set.

24. The method according to claim 1 or 2, wherein, The PCR amplification reaction further includes the use of a PCR calibration system, comprising: a set of PCR amplification primers, wherein at least one amplification primer includes a tag; and a set of at least two PCR calibration sub-units, each PCR calibration sub-unit consisting of a DNA fragment of a given length, the DNA fragment flanked by upstream and downstream adaptor DNA sequences, the adaptor DNA sequences including primer binding sites for binding to the PCR amplification primers, wherein the set of PCR amplification primers is used for PCR amplification of the DNA sequences of all the PCR calibration sub-units in the set of at least two PCR calibration sub-units, wherein the spacer DNA sequences contained in each of the at least two PCR calibration sub-units in the set have different lengths, and wherein each of the at least two PCR calibration sub-units in the set is present in an equal amount or a known amount relative to the other PCR calibration sub-units in the set; And wherein step b) of the PCR amplification further includes PCR amplifying the at least two PCR calibration sub-units using the PCR amplification primers of the PCR calibration system.

25. The method according to claim 1 or 2, wherein A broad taxonomic range amplification primer set for amplifying at least one microbial rRNA ITS region includes a tagged forward and / or a tagged reverse primer.

26. The method according to claim 1 or 2, wherein A broad taxonomic range amplification primer set for amplifying at least one microbial rRNA ITS region includes a tagged forward primer.

27. The method according to claim 1 or 2, wherein A broad taxonomic range amplification primer set for amplifying at least one microbial rRNA ITS region includes a fluorescently tagged forward primer.

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