CN114317783B - Caries marker microorganisms and uses thereof - Google Patents
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- CN114317783B CN114317783B CN202111492646.2A CN202111492646A CN114317783B CN 114317783 B CN114317783 B CN 114317783B CN 202111492646 A CN202111492646 A CN 202111492646A CN 114317783 B CN114317783 B CN 114317783B
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Abstract
The invention provides caries marker microorganisms and uses thereof, the caries marker microorganisms comprise a first microorganism set, and further provides a kit comprising reagents suitable for detecting at least one species in the first microorganism set, the first microorganism set consisting of the following species: miracle bacillus (Cryptobacterium curtum), euglena (olsen ella uli), parasilvered juxta-karl-vered bacteria (Parascardovia denticolens), prasugrel denticola (Prevotella denticola), prasugrel melanosis (Prevotella nigrescens), streptococcus mutans (Streptococcus mutans), prasuvorexa saccharophila (Prevotella multisaccharivorax), sarteus (Shuttleworthia satelles). Compared with the healthy population, the microorganism provided by the invention is remarkably enriched in the caries patient population, can be used as a distinguishing mark of the healthy population and the caries patient population, and can be used as a mark for detecting and/or treating caries.
Description
Technical Field
The present invention relates to the field of biotechnology, in particular, to a caries marker microorganism and its use, more particularly, to a kit, use of a reagent in the preparation of a kit, a pharmaceutical composition or food composition for preventing or treating caries, a method of determining whether an individual has caries, a device for determining whether an individual has caries, a device, a method of screening for a drug.
Background
Dental caries, also called tooth decay and dental caries, is a bacterial disease, can cause secondary pulpitis and periapical periodontitis, can cause alveolar bone and jawbone inflammation when serious, is more likely to generate lesions to form caries holes, damage dental crowns and finally lead to tooth loss, is a very common oral disease, seriously affects the life quality of people, has high incidence rate and wide distribution, and can appear in any age group, especially in teenagers. The research result of the Chinese oral health epidemiology in 2018 shows that the prevalence of caries in children is obviously increased by 5.8% compared with that before 10 years.
Chronic caries progresses slowly, generally it takes about 1-2 years for caries to develop, and early symptoms are less obvious, and its cause is complex, and it is related to bacteria, food, host, time and other factors, if a patient is affected by bad oral environment for a long time, such as long-term eating of confectionery, no oral cleaning, bacterial growth, or influence of salivary flow and flow velocity, teeth are damaged to some extent, and caries is caused.
Because early symptoms of caries are not obvious, teeth may be mistaken for sensitive teeth, so that attention is not paid, and the teeth are frequently found on tooth surfaces which are not easy to keep clean, such as point gaps, cracks, adjacent surfaces and the like of teeth, and are not easy to find. Caries can be determined by taking X-ray films, where black shadows are visible. Or optical fiber transillumination, electrical impedance, ultrasonic wave, elastic mold separation, staining and other techniques can be used to improve the accuracy and sensitivity of early diagnosis of caries, but the examination process is more complicated.
With the completion of human genome sequencing and the rapid development of high-throughput sequencing technology, gene screening is another direction of caries diagnosis, and whether caries is caused or not can be determined by a saliva sample. There has been a great deal of research on caries, such as the use of streptococci, actinomycetes and fluoride, but there is still a need to study at deeper levels to reveal caries microbial markers.
Disclosure of Invention
The present application is made based on the discovery and recognition by the inventors of the following facts and problems:
the applicant of the present application has unexpectedly found, through extensive prior studies, that some microorganisms can be used as marker microorganisms for detecting caries, providing a non-invasive method for early caries detection; the method can reasonably and effectively apply the marking microorganism, support the growth of beneficial bacteria in the oral cavity, inhibit the potential pathogenic bacteria in the oral cavity, and treat or alleviate the clinical symptoms of dental caries.
For this purpose, in a first aspect of the invention, the invention proposes a kit. According to an embodiment of the invention, the kit comprises reagents suitable for detecting at least one species of a first set of microorganisms consisting of the following species: miracle bacillus (Cryptobacterium curtum), euglena (olsen ella uli), parasilvered juxta-karl-vered bacteria (Parascardovia denticolens), prasugrel denticola (Prevotella denticola), prasugrel melanosis (Prevotella nigrescens), streptococcus mutans (Streptococcus mutans), prasuvorexa saccharophila (Prevotella multisaccharivorax), sarteus (Shuttleworthia satelles). The marker microorganism of the embodiment of the invention is determined by the inventor through difference comparison analysis and verification of the abundance of microorganisms in saliva samples of a large number of individuals suffering from dental caries and a large number of healthy individuals, so that the microbial marker related to the dental caries is defined, and the kit comprising the reagent for detecting at least one strain of the first microorganism set in the marker microorganism can be used for noninvasively discovering or assisting in detecting the dental caries at an early stage and accurately determining the probability of the individuals suffering from the dental caries or the probability of the individuals in a healthy state.
In a second aspect of the invention, the invention proposes the use of a reagent in the preparation of a kit, said reagent being suitable for detecting at least one species of a first set of microorganisms. According to an embodiment of the invention, the kit is for diagnosing lipid caries or detecting the therapeutic effect of caries, the first set of microorganisms consisting of the following species: miracle bacillus (Cryptobacterium curtum), euglena (olsen ella uli), parasilvered juxta-karl-vered bacteria (Parascardovia denticolens), prasugrel denticola (Prevotella denticola), prasugrel melanosis (Prevotella nigrescens), streptococcus mutans (Streptococcus mutans), prasuvorexa saccharophila (Prevotella multisaccharivorax), sarteus (Shuttleworthia satelles). According to the kit prepared by the reagent of the specific embodiment of the invention, at least one strain in the first microorganism set can be accurately detected, and caries patients and healthy individuals can be extremely accurately distinguished, so that caries diagnosis can be effectively carried out in early stages, or caries change in the treatment process can be detected.
In a third aspect of the invention, the invention provides a pharmaceutical or food composition for preventing or treating dental caries. According to an embodiment of the invention, neisseria lactose (Neisseria lactamica) is included. The neisseria lactose (Neisseria lactamica) in the caries marker micro-organism according to the embodiment of the invention can be used for noninvasively discovering or assisting in detecting caries at an early stage, and determining the probability of suffering from caries or the probability of being in a healthy state; at the same time, increasing the lactoneisseria (Neisseria lactamica) in the oral cavity of a caries-prone population or a caries-prone patient can reduce the probability of caries or slow down and cure caries, and therefore, the pharmaceutical or food composition comprising the lactoneisseria (Neisseria lactamica) can be used to balance the oral flora and effectively prevent or treat caries.
In a fourth aspect of the invention, the invention features a method of determining whether an individual has caries. According to an embodiment of the invention, it comprises: (1) Determining an abundance of a marker microorganism in a saliva sample of the individual, the marker microorganism comprising at least one species of a first set of microorganisms and neisseria lactose (Neisseria lactamica); (2) Comparing the abundance obtained in step (1) to a predetermined threshold to determine if the individual has caries; wherein the first set of microorganisms consists of the following species: miracle bacillus (Cryptobacterium curtum), euglena (olsen ella uli), parasilvered juxta-karl-vered bacteria (Parascardovia denticolens), prasugrel denticola (Prevotella denticola), prasugrel melanosis (Prevotella nigrescens), streptococcus mutans (Streptococcus mutans), prasuvorexa saccharophila (Prevotella multisaccharivorax), sarteus (Shuttleworthia satelles). The method according to the embodiment of the invention can determine whether the individual has caries according to the abundance of various strains in the marker microorganism in the saliva sample of the individual, wherein the marker microorganism is determined by verifying a plurality of saliva samples in known states and analyzing the abundance of various oral microorganisms through difference comparison.
In a fifth aspect of the invention, the invention features a device for determining whether an individual has caries. According to an embodiment of the invention, it comprises: an abundance determination unit for determining an abundance of a marker microorganism in a saliva sample of the individual, the marker microorganism comprising at least one species of a first set of microorganisms and neisseria lactose (Neisseria lactamica); a comparison unit for comparing the abundance obtained with a predetermined threshold in order to determine whether the individual has caries; wherein the first set of microorganisms consists of the following species: miracle bacillus (Cryptobacterium curtum), euglena (olsen ella uli), parasilvered juxta-karl-vered bacteria (Parascardovia denticolens), prasugrel denticola (Prevotella denticola), prasugrel melanosis (Prevotella nigrescens), streptococcus mutans (Streptococcus mutans), prasuvorexa saccharophila (Prevotella multisaccharivorax), sarteus (Shuttleworthia satelles). The marker microorganisms are determined by analyzing the abundance of various oral microorganisms in saliva samples of caries patients and healthy people through difference comparison and verifying a large number of saliva samples in known states, and the device according to the embodiment of the invention can accurately determine whether the individuals are high-risk people with caries or caries patients.
In a sixth aspect of the invention, the invention provides an apparatus. According to an embodiment of the invention, it comprises: a computer-readable storage medium having stored thereon a computer program for use in the method of the fourth aspect; and one or more processors configured to execute the program in the computer-readable storage medium. The device according to the embodiment of the invention can accurately determine whether an individual is a high-risk group of caries or a caries patient.
In a seventh aspect of the invention, the invention provides a method of screening for a drug. According to an embodiment of the present invention, the medicament is for treating or preventing dental caries, the method comprising: administering a candidate drug to a subject, detecting abundance of a marker microorganism in saliva of the subject before and after administration, the marker microorganism comprising at least one species of a first microorganism set and neisseria lactose (Neisseria lactamica), wherein the candidate drug meeting at least one of the following conditions is suitable for use in treating or preventing caries: (1) After said administering, said abundance of at least one species of said first microorganism set is decreased; and (2) said increase in said abundance of neisseria lactose (Neisseria lactamica) following said administration; wherein the first set of microorganisms consists of the following species: miracle bacillus (Cryptobacterium curtum), euglena (olsen ella uli), parasilvered juxta-karl-vered bacteria (Parascardovia denticolens), prasugrel denticola (Prevotella denticola), prasugrel melanosis (Prevotella nigrescens), streptococcus mutans (Streptococcus mutans), prasuvorexa saccharophila (Prevotella multisaccharivorax), sarteus (Shuttleworthia satelles). The methods according to embodiments of the present invention can produce or screen for agents that inhibit the growth of various species in the first set of microorganisms in the marker microorganism, and/or promote the growth of neisseria lactose (Neisseria lactamica) in the oral marker microorganism, which are of great significance in aiding in the reduction of clinical symptoms of caries.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
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The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a schematic flow chart of an experimental analysis for screening caries marker micro-organisms according to an embodiment of the present invention; and
FIG. 2 is a graph showing the results of evaluation of the AUC of a marker microorganism according to an embodiment of the present invention, wherein:
2-a is a graph of AUC values and confidence interval results under ROC curve of 29 sample data at the first stage;
2-B is a graph of AUC values and confidence interval results under the ROC curve for 15 sample data from the second phase.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present invention, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise.
The term "optionally" is used for descriptive purposes only and is not to be construed as indicating or implying relative importance. Thus, a feature defined as "optional" may explicitly or implicitly include or exclude that feature.
Biological markers are cell/biochemical or molecular changes that can be detected from biological media. Biological agents include various body fluids, tissues, cells, saliva, hair, breath, and the like.
The abundance of a microorganism refers to the abundance of that microorganism in a population of microorganisms, e.g., the level of that microorganism in a population of oral microorganisms, can be expressed as the level of that microorganism in the population.
According to one embodiment of the present invention, a kit is provided, comprising reagents suitable for detecting at least one species of a first set of microorganisms consisting of: miracle bacillus (Cryptobacterium curtum), euglena (olsen ella uli), parasilvered juxta-karl-vered bacteria (Parascardovia denticolens), prasugrel denticola (Prevotella denticola), prasugrel melanosis (Prevotella nigrescens), streptococcus mutans (Streptococcus mutans), prasuvorexa saccharophila (Prevotella multisaccharivorax), sarteus (Shuttleworthia satelles).
According to a specific embodiment of the invention, the kit further comprises reagents suitable for detecting neisseria lactose (Neisseria lactamica).
According to a specific embodiment of the invention, the kit comprises reagents suitable for detecting all of the species in the first set of microorganisms.
According to a specific embodiment of the present invention, the marker microorganism is a microorganism marker related to dental caries in oral microorganisms, which has been determined by the inventors by comparative analysis and verification of the differences in abundance of microorganisms in saliva samples of a large number of individuals suffering from dental caries and a large number of healthy control individuals. The use of a kit comprising reagents for detecting the marker microorganism can determine whether an individual is at risk for caries or healthy, and can be used for noninvasive early detection or auxiliary detection of caries.
According to a specific embodiment of the present invention, the reagent suitable for detecting the first microorganism collection and/or neisseria lactose (Neisseria lactamica) is not particularly limited, and reagents which can detect the microorganism strain are included in the scope of the present invention, such as reagents for detecting the microorganism strain by morphological characteristics, physiological and biochemical characteristics, ecological characteristics and serological reactions, sensitivity to phage, molecular biology, etc., in particular, such as antibodies, enzymes, nucleic acid molecules.
Herein, the morphological characteristics of the microorganism refer to: the shape, size, arrangement, etc. of the microorganism, the cell structure, the gram stain reaction, the movement, the site and number of the flagellum, the presence or absence of spores and capsules, the size and position of spores, the shape, structure, the number, shape, size, color, surface characteristics, etc. of the actinomycetes and the reproductive organs of fungi are observed under a microscope.
Herein, the microbial physiological biochemical reaction characteristics refer to: the ability of the microorganism to utilize the substance, the specificity of the metabolite, such as whether H is produced 2 S, indole and CO 2 Alcohol, organic acid, whether nitrate can be reduced, whether milk can be coagulated, frozen, etc., the growth environment (suitable for growth temperature, humidity, concentration of oxygen and carbon dioxide, pH, high permeability resistance, halophilicity, etc.), and other biological relationships (such as symbiotic, parasitic, host range)Enclose and cause disease conditions), etc.
Herein, the microbiological serological reaction refers to: the highly sensitive specific reaction of antigen and antibody is used to identify similar species or to identify microorganisms of the same species, such as antisera made with known species, types or strains, with the presence or absence of specific serological reactions with the microorganism to be identified.
Herein, the molecular biological method for detecting microorganisms mainly includes: PCR technology, high throughput sequencing and other methods are utilized.
The use of a reagent according to the invention for the preparation of a kit adapted for detecting at least one species of a first set of microorganisms for diagnosing dental caries or for detecting the therapeutic effect of dental caries, said first set of microorganisms consisting of: miracle bacillus (Cryptobacterium curtum), euglena (olsen ella uli), parasilvered juxta-karl-vered bacteria (Parascardovia denticolens), prasugrel denticola (Prevotella denticola), prasugrel melanosis (Prevotella nigrescens), streptococcus mutans (Streptococcus mutans), prasuvorexa saccharophila (Prevotella multisaccharivorax), sarteus (Shuttleworthia satelles).
According to a specific embodiment of the present invention, the marker microorganism is a microorganism marker related to dental caries in oral microorganisms, which has been determined by the inventors by comparative analysis and verification of the differences in abundance of microorganisms in saliva samples of a large number of individuals suffering from dental caries and a large number of healthy control individuals. The use of the agent for detecting the marker microorganism can determine the probability of an individual suffering from caries or in a healthy state, and can be used for noninvasive early detection or auxiliary detection of caries.
According to some specific embodiments of the invention, the reagent is further suitable for detecting neisseria lactose (Neisseria lactamica).
According to a specific embodiment of the present invention, the reagent suitable for detecting the first microorganism group or neisseria lactose (Neisseria lactamica) is not particularly limited, and reagents which can detect the microorganism strain are included in the scope of the present invention, such as reagents for detecting the microorganism strain by morphological characteristics, physiological and biochemical characteristics, ecological characteristics, and serological reactions, sensitivity to phage, molecular biology, and the like, in particular, such as antibodies, enzymes, nucleic acid molecules.
Herein, the morphological characteristics of the microorganism refer to: the shape, size, arrangement, etc. of the microorganism, the cell structure, the gram stain reaction, the movement, the site and number of the flagellum, the presence or absence of spores and capsules, the size and position of spores, the shape, structure, the number, shape, size, color, surface characteristics, etc. of the actinomycetes and the reproductive organs of fungi are observed under a microscope.
Herein, the microbial physiological biochemical reaction characteristics refer to: the ability of the microorganism to utilize the substance, the specificity of the metabolite, such as whether H is produced 2 S, indole and CO 2 Alcohol, organic acid, whether nitrate can be reduced, whether milk can be coagulated, frozen, etc., the growth environment (temperature, humidity, concentration of gases such as oxygen and carbon dioxide, pH, whether high permeability resistance, salt tolerance, etc.), the relationship with other organisms (such as symbiosis, parasitism, host range and pathogenic condition), etc.
Herein, the microbiological serological reaction refers to: the highly sensitive specific reaction of antigen and antibody is used to identify similar species or to identify microorganisms of the same species, such as antisera made with known species, types or strains, with the presence or absence of specific serological reactions with the microorganism to be identified.
Herein, the molecular biological method for detecting microorganisms mainly includes: PCR technology, high throughput sequencing and other methods are utilized.
According to the present invention there is provided a method of determining whether an individual has caries comprising steps (1) and (2).
(1) Determining the abundance of a marker microorganism in a saliva sample of the individual.
The marker microorganism includes at least one species of a first microorganism group and neisseria lactose (Neisserialactamica). Wherein the first set of microorganisms consists of the following species: mystery bacillus (Cryptobacterium curtum), euglena (olsen ella uli), paradentitis kavaliensis (paraascadellica), prasuvorexa (prevotella dinetica), prasugrel melanosis (prevotella nigrescens), streptococcus mutans (stretococcus), prasuvorexa saccharophila (Prevotella multisaccharivorax), sarteus (Shuttleworthia satelles).
According to some specific embodiments of the invention, step (1) further comprises: obtaining nucleic acid sequencing data in a saliva sample of the individual; comparing the sequencing data to a reference genome; based on the results of the alignment, the abundance of the marker microorganism is determined.
According to a specific embodiment of the present invention, in step (1), the abundance of the marker microorganism is determined according to the following formula: ab (S) =ab (US) +ab (MS), wherein S represents the number of the marker microorganism, ab (S) represents the abundance of the marker microorganism S, ab (U) S )＝U S /l S ，U S For the number of reads in the sequencing data that are uniquely compared to the reference genome of the marker microorganism S, l S For the total length of the reference genome of the marker microorganism S,M S for the number of reads in the sequencing data that are non-uniquely aligned with the reference genome of the marker microorganism S, i represents the number of the non-uniquely aligned reads, co i For the corresponding abundance ratio of the ith read,Co i,s representing the abundance ratio of the non-uniquely aligned reads i for the marker microorganism S, N being the total number of microorganisms to which the non-uniquely aligned reads i can be aligned, j representing the number of microorganisms to which the non-uniquely aligned reads i can be aligned.
The sequencing data is obtained by sequencing the nucleic acid sequence in the sample, and the sequencing can be selected from, but is not limited to, semiconductor sequencing technology platforms such as PGM, ion Proton, BGISEQ-100 platform, synthetic edge sequencing technology platforms such as Hiseq, miseq sequence platform of Illumina company and single molecule real-time sequencing platform such as PacBio sequence platform. The sequencing mode can be single-ended sequencing or double-ended sequencing, and the obtained off-machine data is the fragment which is read out and called reads.
The alignment can be performed by using known alignment software, such as SOAP, BWA, teraMap, etc., and in the alignment process, the alignment parameters are generally set, one or a pair of reads (reads) is set to allow at most s base mismatches (mismatch), for example, s.ltoreq.2, and if more than s bases in the reads are mismatched, the reads are considered to be unable to align to (align with) the assembled fragment. The obtained comparison results include the comparison of each read with the reference genome of each species, including information as to whether the read is capable of comparing to the reference genome of a certain species or species, to compare to only one species or to reference genomes of multiple species, to compare to the location of the reference genome of a species, to compare to the unique location of the reference genome of a species, or to multiple locations.
The reference genome of the strain/microorganism refers to a predetermined sequence of the microorganism species, and may be any reference template of a biological class to which a pre-obtained sample to be tested belongs or is included, for example, the target is a microorganism in the sample to be tested, the reference sequence may be selected from the reference genome of various microorganisms in the NCBI database and/or the DACC oral genome disclosed in the HMP and MetaHIT projects, further, a resource library including more reference sequences may be pre-configured, for example, a sequence which is more similar to the reference sequence may be selected or determined and assembled according to factors such as the state, region, and the like of an individual from which the sample to be tested is derived. According to one embodiment of the invention, the reference genomes of various microorganisms are obtained from a public database, typically, one microorganism has multiple versions of the reference genome, i.e., one microorganism has multiple public reference genomes.
reads can be aligned with a reference genome of a species, which can be divided into two parts: a) Unique reads (U): uniquely comparing the genome of the previous species; these reads are called unique reads. That is, reads are defined as unique reads if the genomes on the reads are all from the same species. b) Multiplex reads (M): the genome of more than one species is aligned and defined as multiple reads. That is, reads are defined as multiple reads if the genome on which they are aligned is from at least two species.
(2) Abundance comparisons to determine if an individual has caries.
According to one embodiment of the invention, the abundance obtained in step (1) is compared to a predetermined threshold in order to determine whether the individual has caries.
According to some embodiments of the invention, the threshold is preset. Comparing the abundance of the marker microorganism in the sample of the individual to be detected with the threshold value, and determining the state of the individual to be detected based on whether the abundance in the sample of the individual to be detected reaches the threshold value. The threshold may be a value or range of values, for example, based on the mean of the abundance of a marker microorganism in an individual of known diseased or healthy status, the corresponding threshold for that microorganism may be set to a confidence interval (Confidence interval) of 95% of the mean of the abundance.
The confidence interval refers to an estimated interval of the overall parameters constructed by the sample statistics. In statistics, the confidence interval of a probability sample is an interval estimate of some overall parameter of the sample. The confidence interval reveals the extent to which the true value of this parameter falls around the measurement with a certain probability. The confidence interval gives the degree of confidence in the measured value of the measured parameter, i.e. the "certain probability" as required before, this probability being referred to as the confidence level.
According to some embodiments of the invention, the individual is determined to have caries when the abundance of the marker microorganism determined in step (1) reaches the caries-causing abundance threshold and does not reach the non-caries-causing abundance threshold, and the individual is determined to not have caries when the abundance of the marker microorganism determined in step (1) reaches the non-caries-causing abundance threshold and does not reach the caries-causing abundance threshold.
It should be noted that, depending on the purpose or requirement, there may be different requirements for determining the confidence level of the individual status result, and those skilled in the art may select different significance levels or thresholds.
The method is based on detecting the abundance of various strains in a marker microorganism in a saliva sample of an individual, comparing the abundance of various strains determined by detection with a threshold value thereof, and determining the probability that the individual is a decayed tooth individual or a healthy individual according to the obtained comparison result. A non-invasive method of aiding detection or aiding in the treatment of caries in an early stage is provided.
All or part of the steps of the method of determining whether an individual has caries using a marker microorganism in any of the above embodiments may be performed using an apparatus/system comprising detachable corresponding unit functional modules, or the method may be programmed, stored on a machine-readable medium, which is executed by a machine.
In accordance with the present invention there is provided an apparatus for determining whether an individual has caries, the apparatus comprising: an abundance determination unit for determining an abundance of the marker microorganism in the saliva sample of the individual, the marker microorganism comprising a first set of microorganisms and neisseria lactose (Neisseria lactamica); a comparison unit for comparing the abundance obtained with a predetermined threshold in order to determine whether the individual has caries; wherein the first set of microorganisms consists of the following species: miracle bacillus (Cryptobacterium curtum), euglena (olsen ella uli), parasilvered juxta-karl-vered bacteria (Parascardovia denticolens), prasugrel denticola (Prevotella denticola), prasugrel melanosis (Prevotella nigrescens), streptococcus mutans (Streptococcus mutans), prasuvorexa saccharophila (Prevotella multisaccharivorax), sarteus (Shuttleworthia satelles). The above description of the technical features and advantages of the method for determining whether an individual has caries using a marker microorganism according to any of the embodiments of the present invention applies equally to the apparatus according to this aspect of the present invention, and will not be described in detail here.
According to an embodiment of the invention, the abundance determination unit is adapted to determine the abundance by: obtaining nucleic acid sequencing data in a saliva sample of the individual; comparing the sequencing data to a reference genome; based on the results of the alignment, the abundance of the marker microorganism is determined.
The sequencing data is obtained by sequencing the nucleic acid sequence in the sample, and the sequencing can be selected from, but is not limited to, semiconductor sequencing technology platforms such as PGM, ion Proton, BGISEQ-100 platform, synthetic edge sequencing technology platforms such as Hiseq, miseq sequence platform of Illumina company and single molecule real-time sequencing platform such as PacBio sequence platform. The sequencing mode can be single-ended sequencing or double-ended sequencing, and the obtained off-machine data is the fragment which is read out and called reads.
The alignment can be performed by using known alignment software, such as SOAP, BWA, teraMap, etc., and in the alignment process, the alignment parameters are generally set, one or a pair of reads (reads) is set to allow at most s base mismatches (mismatch), for example, s.ltoreq.2, and if more than s bases in the reads are mismatched, the reads are considered to be unable to align to (align with) the assembled fragment. The obtained comparison results include the comparison of each read with the genome referenced by each species, including information as to whether the read is capable of comparing to the reference genome of a certain species or species, to only one species or to reference genomes of multiple species, to the location of the reference genome of a species, to the unique location of the reference genome of a species, or to multiple locations.
The reference genome of the microorganism refers to a predetermined sequence of the microorganism species, and may be any reference template of a biological class to which a pre-obtained sample to be tested belongs or is included, for example, the target is the microorganism in the sample to be tested, the reference sequence may be selected from the reference genome of various microorganisms in the NCBI database and/or the genome disclosed in HMP and MetaHIT projects, further, a resource library including more reference sequences may be pre-configured, for example, a sequence which is closer to the reference sequence may be selected or determined according to factors such as the state, region, etc. of the individual from which the sample to be tested is derived. According to one embodiment of the invention, the reference genomes of various microorganisms are obtained from a public database, typically, one microorganism has multiple versions of the reference genome, i.e., one microorganism has multiple public reference genomes.
reads can be aligned with a reference genome of a species, which can be divided into two parts: a) Uniquereads (U): uniquely comparing the reference genome of the previous species; these reads are called uniquereads. That is, reads are defined as unquerads if the reference genomes on the reads are all from the same species. b) Multiplereads (M): the reference genome of more than one species is aligned and defined as multiplexing. That is, reads are defined as multiplexing if the reference genome on which they are aligned is from at least two species.
According to one embodiment of the invention, the abundance of the marker microorganism is determined according to the following formula:
Ab(S)＝Ab(U S )+Ab(M S ) Wherein S represents the number of the marker microorganism, ab (S) represents the abundance of the marker microorganism S, ab (U) S )＝U S /l S ，U S For the number of reads in the sequencing data that are uniquely compared to the reference genome of the marker microorganism S, l S For the total length of the reference genome of the marker microorganism S,M S for the number of reads in the sequencing data that are non-uniquely aligned with the reference genome of the marker microorganism S, i represents the number of the non-uniquely aligned reads, co i For the corresponding abundance coefficient of the ith read,/->Co i,s Representing the non-unique ratio for the marker microorganism SAnd the abundance coefficient of the paired reads i, wherein N is the total number of the microorganisms which can be aligned by the non-uniquely aligned reads i, and j represents the number of the microorganisms which can be aligned by the non-uniquely aligned reads i. The above description of the technical features and advantages of the method for determining whether an individual has caries using a marker microorganism according to any of the embodiments of the present invention applies equally to the apparatus according to this aspect of the present invention, and will not be described in detail here.
According to the present invention there is provided an apparatus comprising: a computer readable storage medium having stored thereon a computer program for performing a method of determining whether an individual has caries as described above; and one or more processors configured to execute the program in the computer-readable storage medium.
According to the present invention there is provided a method of screening for a drug for the treatment or prevention of dental caries, the method comprising: administering a drug candidate to a subject, detecting the abundance of a marker microorganism in saliva of the subject before and after administration, the marker microorganism comprising neisseria lactose (Neisseria lactamica), wherein the drug candidate that meets at least one of the following conditions is suitable for treating or preventing caries: (1) After said administering, said abundance of at least one species of said first microorganism set is decreased; and (2) said increase in said abundance of neisseria lactose (Neisseria lactamica) following said administration; wherein the first set of microorganisms consists of the following species: miracle bacillus (Cryptobacterium curtum), euglena (olsen ella uli), parasilvered juxta-karl-vered bacteria (Parascardovia denticolens), prasugrel denticola (Prevotella denticola), prasugrel melanosis (Prevotella nigrescens), streptococcus mutans (Streptococcus mutans), prasuvorexa saccharophila (Prevotella multisaccharivorax), sarteus (Shuttleworthia satelles). The method according to embodiments of the present invention may produce or screen for agents that inhibit the growth of various species in the first set of microorganisms in the marker microorganism and/or promote the growth of neisseria lactose (Neisseria lactamica) in the oral marker microorganism, which are of great significance in aiding in the alleviation of clinical symptoms of caries.
By utilizing the method for producing or screening the medicine for treating the dental caries, which is provided by the invention, the medicine capable of supporting the growth of beneficial oral bacteria and/or inhibiting potential pathogenic bacteria of the oral cavity can be obtained by reasonably and effectively applying the determined dental caries biomarker for screening, and the method has important significance for curing and/or relieving clinical symptoms of the dental caries.
The embodiments will be described in detail below. The reagents, sequences, software and instrumentation referred to in the examples below, which are not specifically addressed, are all conventional commercial products.
Example 1 identification of biomarkers
In this example, the inventors studied saliva samples of 16 caries patients and 13 healthy controls to obtain oral microbiota and functional component characteristics. The inventor constructs a caries reference gene set by sequencing and quality control to obtain about 36.7Gb high-quality healthy human data and 47.3Gb high-quality caries patient sequencing data, and constructs a more complete gene set with the HOMD gene set. Metagenomic analysis showed that 25 species of microorganisms are closely related to caries, of which 4 are enriched in oral microorganisms in healthy humans and 21 are enriched in oral microorganisms in caries patients.
1. Sample collection and DNA extraction
All samples were from the university of Zhejiang medical college affiliated oral hospitals, samples were aged between 45-73 months, no antibiotics were used for 3 months prior to sampling, 16 caries patients and 13 healthy persons were all tested for saliva samples, subjects did not eat or drink water 2 hours prior to sampling, and saliva was required to be held in the oral cavity for 3 minutes. The subjects were flushed into sterile cryovials, and each saliva sample was then transferred into a sterile 1.5ml Eppendorf tube, snap frozen in liquid nitrogen, and stored at 80 ℃. Total DNA from 29 individual saliva samples was extracted using a QIAamp DNAmini kit kit (Qiagen, hilden, germany) and NEBNextMicrobiome DNAEnrichmentKit was used to isolate microbial DNA from host DNA, the specific experimental procedure being performed according to the kit instructions. The DNA was purified by ethanol precipitation, and the concentration was measured and stored at-20 ℃.
2. Construction of DNA library and sequencing
DNA banking was performed according to the instructions of the instrument manufacturer (Illumina). The library was PE100 bp sequenced and the library of 29 samples was sequenced on an Illumina Hiseq2000 (Illumina, san Diego, calif.) platform. Each sample produced on average 4.3Gb (sd. ±0.68 Gb) sequencing results, totaling 125Gb sequencing data volume.
Referring to the experimental procedure of fig. 1, the relevant biomarkers of caries are identified, wherein omitted steps or details are well known to those skilled in the art, and several important steps are described below.
3. Microbial species abundance analysis
3.1 sequence optimization statistics
Firstly, carrying out first-stage sequencing, acquiring data of 29 samples in the first-stage sequencing, filtering the data after the sequencing data of the 29 samples in the first stage are obtained, and carrying out quality control according to the following standard: a) Removing reads greater than 5N bases; b) Removing more than 50% of reads of low quality bases (Q20); c) Tail low mass (Q20) and N bases were removed. Using the SOAPAligner2.21 default parameters to remove reads of the human genome (HG 19), missing paired reads sequences is considered as a single reads for assembly.
Wherein the HOMD gene set is downloaded from http:// www.homd.org/ftp/all_oral_genome/20160329.
3.2 species abundance analysis
SOAPAlign2.21 was used to match the paired-endclearreads for the redundant genome, with parameters of-r 2-m200-x 1000. The comparison result of Reads and redundant genome can be divided into two parts: a) Uniquereads (U): reads aligned only to the genome of the last species; these reads are defined as uniquerads. That is, if the genomes are from the same species, the inventors define these reads as unique reads. b) Multiplereads (M): if reads align the genomes of two or more species, it is defined as multiplexing. That is, if the genomes on the alignment are from different species, the inventors define these reads as multiplexing.
For species S, if the abundance is Ab (S), it is likely that it is associated with U-specific reads and M-shared reads, the abundance is calculated as follows:
Ab(S)＝Ab(U S )+Ab(M S )，
wherein S represents the number of the marker microorganism,
ab (S) represents the abundance of the marker microorganism S,
Ab(U S )＝U S /l S ，
U S for the number of reads in the sequencing data that are uniquely compared to the reference genome of the marker microorganism S,
l S for the total length of the reference genome of the marker microorganism S,
M S for the number of reads in the sequencing data that are not uniquely aligned with the reference genome of the marker microorganism S,
Co i for the corresponding abundance ratio of the ith read,
i represents the number of the non-uniquely aligned reads,
Co i,s representing the abundance ratio of the non-uniquely aligned reads i for the marker microorganism S,
n is the total number of microorganisms to which the non-uniquely aligned reads i can be aligned,
j represents the number of microorganisms to which the non-uniquely aligned reads i can be aligned.
And (3) obtaining a normalized species abundance table after dividing all the calculated species abundance values in each sample by the total abundance of each sample.
Example 2 screening of microbial species markers
In order to obtain oral microorganism species markers closely related to caries disease, the inventors made a study on the species level using two sets of oral microorganism species abundance data of caries patient group (16 cases) and normal person group (13 cases). Based on the species abundance table obtained in example 1, the inventors set the criteria as follows: (1) The median of species abundance in the caries patient group or healthy person group must be greater than 0.00001; (2) Checking through Wilcoxon rank sum check to obtain the correlation p value of each species and dental caries; (3) Screening was performed using the above parameters using a relatively stringent threshold (p_values < 0.02). The inventors obtained 25 oral microbial species closely related to Caries disease, data analysis is shown in table 1, where 21 species of microorganisms are enriched in Caries (Caries) patient's oral cavity and 4 species are enriched in healthy people (Health), and these 25 microbial species markers are shown in table 1.
Table 1:
| species (Taxonomy) | p value | Enriched populations |
| Prevotella_amanii (Prevotella) | 3.11E-04 | Caries |
| Streptococcus mutans (streptococcus_mutans) | 3.11E-04 | Caries |
| Paralobacter (Dialister_invsu) | 3.84E-04 | Caries |
| Lactobacillus gasseri (Lactobacillus gasseri) | 6.16E-04 | Caries |
| Gum Euler's bacteria (Olsenella_ uli) | 1.23E-03 | Caries |
| Prevotella_oris (Prevotella_oris) | 2.12E-03 | Caries |
| Oenomonas (Selenomonas_sp. _oral_taxon_137) | 2.12E-03 | Caries |
| Prevotella_multi-formis (Prevotella) | 4.11E-03 | Caries |
| Prevotella multisaccharidovorax | 4.11E-03 | Caries |
| Werong fungus (Anaeroglobus_geminatus) | 4.81E-03 | Caries |
| Mystery bacillus (Cryptobacterium_curtum) | 5.16E-03 | Caries |
| Serratia (Shuttleworthia_proteins) | 6.52E-03 | Caries |
| Prevotella-buccae (Prevotella-buccae) | 8.73E-03 | Caries |
| Golden bacillus (Oribacterium_sp. _oral_tax_078) | 1.15E-02 | Caries |
| Prevotella-dentritica (Prevotella-dentritica) | 1.15E-02 | Caries |
| Wei Gesi card multidimensional bacteria (Scardovia_wiggsiae) | 1.15E-02 | Caries |
| Legionella parabacilis (Parascadovia_denticolens) | 1.51E-02 | Caries |
| Prevotella nigrescens (Prevotella nigrescens) | 1.51E-02 | Caries |
| Eubacterium (eubacterium_bacterium_AC19a) | 1.72E-02 | Caries |
| Proprietas gingivitis (Prevotella_oulrum) | 1.72E-02 | Caries |
| Oenomonas (Selenomonas_sp. _oral_taxon_138) | 1.95E-02 | Caries |
| Bacillus (Bacillus_bataviensis) | 1.84E-02 | Health |
| Neisseria lactis (Neisseria_lactamica) | 1.95E-02 | Health |
| Streptococcus (Streptomyces sp. M334) | 1.95E-02 | Health |
| Salmonella enterica (Salmonella_enterica) | 1.01E-03 | Health |
Example 3 validation of microbial species markers
To verify the findings in example 2, the inventors further analyzed the abundance of 25 bacteria shown in table 1 in saliva samples of 9 healthy persons and 6 caries patients in the verified population, and deleted the microorganism species markers shown in table 1 according to the enrichment of each species in healthy and disease groups, and analysis of DNA extraction, sequencing and species abundance of the verified population was performed with reference to examples 1 and 2.
The verification result is as follows: of the 4 species enriched in healthy humans, 1 gave high quality validation (p-value < 0.05) in validation sets, with p-values for markers of microbial species enriched in healthy humans in validation sets as shown in table 2 below. The microbial species is neisseria lactose (Neisseria lactamica). The bacterium is not pathogenic, can be parasitic in the nasopharynx of infants and children to become a bacterial strain with bacterium, is a long resident bacterium of human pharynx, and can generate cross antibodies against A group meningococcus, thus having the potential of resisting meningococcal infection.
Table 2:
| species (Taxonomy) | p value | Enriched populations |
| Neisseria lactose (Neisseria lactamica) | 1.75E-02 | Health |
For 21 species enriched in caries patients in Table 1, 8 of them gave high quality verification in the verification set (p-value < 0.05), p-value cases of the microbiota enriched in caries patients in the verification population are shown in Table 3 as mysterious bacteria (Cryptobacterium curtum), gum European bacteria (Olsenella uli), parschel Vibrio denticola (Parascardovia denticolens), prevotella denticola (Prevotella denticola), prevotella saccharophila (Prevotella multisaccharivorax), prevotella melanosis (Prevotella nigrescens), serratia (Shuttleworthia satelles), streptococcus mutans (Streptococcus mutans), respectively.
Miracle bacillus (Cryptobacterium curtum), euglena (olsen eli), paradentitis kai vickers (Parascardovia denticolens) are oral bacteria, were originally obtained from pulp infected or caries patients, are oral pathogenic bacteria, and are saccharide-consuming by Prevolella (Prevolella), salmonella typhimurium (Streptomyces_mutans) and actinomycetes (Streptomyces_mutans), and have acid-producing and acid-resistant cariogenic properties.
Table 3:
the inventor believes that 1 microorganism species marker enriched from healthy people can be used as a reverse index of caries disease, or as a component of a microbial preparation drug flora for developing and treating caries, or as an index for detecting caries and monitoring caries progress; 8 microorganism species markers enriched by caries patients are used as positive indicators of caries diseases and are used for caries simple and non-invasive detection and diagnosis.
The inventor utilizes the 9 microorganism species markers to construct a comprehensive index, estimates the area under ROC (Receiver-operating characteristic) curve AUC, and the greater the AUC value is, the higher the diagnostic ability is, and the diagnostic ability of caries is reflected by the AUC value. The evaluation was performed on 29 samples of the first phase (first phase) and 15 samples of the second phase (second phase), respectively, both of which show a very good diagnostic capacity as shown in fig. 2, with auc=97.6% obtained in the first phase, with a confidence interval of 92.6% -100% as shown in fig. 2 a; auc=94.4% was obtained in stage two, with confidence intervals of 82.3% -100% as shown in fig. 2 b.
Example 4 detection of individual State
The abundances of the species in mystery bacillus (Cryptobacterium curtum), euglena (olsen ella uli), paradentitis carlsberg (Parascardovia denticolens), prasuvorexa denticola (Prevotella denticola), prasuvorexa melanogaster (Prevotella nigrescens), and streptococcus mutans (Streptococcus mutans) 6 shown in table 3 in each saliva sample were determined by the method of example 3, and whether or not the abundances of these 6 strains in each sample fall within a confidence interval of 95% of the abundances of the disease control group or the healthy control group, and the state of the individual corresponding to the sample in which the abundances of the 6 bacteria fall within the disease group corresponding interval was determined as a caries patient, and the state of the individual corresponding to the sample in which the abundances of the 6 bacteria fall within the healthy group corresponding interval was determined as a non-caries patient.
The experimental results show that the method is used for judging the individual states of 7 disease samples and 3 healthy samples in known states, wherein the judgment of the states of 8 sample individuals is consistent with the recorded states of the sample individuals.
In addition, the inventors have found that the combined detection of the species in Table 2 and Table 3, e.g., detection of the enrichment of the species markers in Table 3, while the absence of the enrichment of the species markers in Table 2, allows a more accurate determination of caries patients or susceptible populations.
In the case of using the markers for caries treatment, the inventors found that the species markers in table 3 are suppressed or cleared, and at the same time, the species markers in table 2 are enriched, resulting in a better treatment effect.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (6)
1. A kit for detecting caries comprising reagents suitable for detecting a first set of microorganisms, said first set of microorganisms consisting of: mystery bacillusCryptobacterium curtum）Euler gumOlsenella uli）Leuconostoc spParascardovia denticolens）Prevotella denticola @Prevotella denticola）Prevotella melanosis @Prevotella nigrescens）Streptococcus mutansStreptococcus mutans）、Prevotella saccharophila @Prevotella multisaccharivorax）Buddha's bacteriaShuttleworthia satelles）。
2. The kit of claim 1, further comprising a reagent suitable for detecting neisseria lactoseNeisseria lactamica) Is a reagent of (a).
3. Use of a reagent for detecting a species of a first set of microorganisms for diagnosing caries or detecting a therapeutic effect of caries in the preparation of a kit, said first set of microorganisms consisting of: mystery bacillusCryptobacterium curtum）Euler gumOlsenella uli）Leuconostoc spParascardovia denticolens）Prevotella denticola @Prevotella denticola）Prevotella melanosis @Prevotella nigrescens）Streptococcus mutansStreptococcus mutans）、Prevotella saccharophila @Prevotella multisaccharivorax）Buddha's bacteriaShuttleworthia satelles）。
4. The use according to claim 3, wherein the reagent is further suitable for detecting neisseria lactose @Neisseria lactamica）。
5. A device for determining whether an individual has caries, comprising:
An abundance determination unit for determining the abundance of a marker microorganism in a saliva sample of the individual, the marker microorganism being a first set of microorganisms and neisseria lactose @Neisseria lactamica）；
A comparison unit for comparing the abundance obtained with a predetermined threshold in order to determine whether the individual has caries;
wherein the first set of microorganisms consists of the following species: mystery bacillusCryptobacterium curtum）Euler gumOlsenella uli）Leuconostoc spParascardovia denticolens）Prevotella denticola @Prevotella denticola）Prevotella melanosis @Prevotella nigrescens）Streptococcus mutansStreptococcus mutans）、Prevotella saccharophila @Prevotella multisaccharivorax）Buddha's bacteriaShuttleworthia satelles）。
6. The apparatus of claim 5, wherein the abundance determination unit is adapted to determine the abundance by:
obtaining nucleic acid sequencing data in a saliva sample of the individual;
comparing the sequencing data to a reference genome;
based on the results of the alignment, the abundance of the marker microorganism is determined.
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