Microbial species marker for continuously increasing colorectal cancer and application thereof

Abstract

The invention discloses a continuous increasing colorectal cancer microbial species marker. The composition comprises at least two of a fusobacterium oral classification strain, fusobacterium horngii, fusobacterium wardsii, leuconostoc, fusobacterium, bloom bacteria, fusobacterium, streptomyces expansum, mycobacterium jatrovii, microsporidium parvum, fusobacterium nucleatum, prevotella nivalis, fusobacterium, prevotella aureus, mycobacterium, alcanivorans and microbacterium. The microbial marker is continuously and progressively increased in a healthy individual group, a colorectal adenoma patient group and a colorectal cancer patient group, so that the microorganism as the marker can be used for distinguishing healthy individuals, colorectal adenoma patients and colorectal cancer patients, especially can be used for diagnosing colorectal adenoma and colorectal cancer, and can be used for diagnosing colorectal cancer. And susceptible people suffering from colorectal adenoma or colorectal cancer can be effectively monitored, early patients suffering from colorectal adenoma or colorectal cancer can be found, or the treatment effect of colorectal adenoma or colorectal cancer can be monitored.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically, the present invention relates to continuously increasing colorectal cancer microbial species markers and applications, and more specifically, the present invention relates to a group of markers, the use of microorganisms as markers for diagnosing colorectal adenoma or colorectal cancer, a kit, a method for determining the source of a sample, an apparatus for determining the source of a sample, a drug, and a method for producing or screening a drug. Background Art

[0002] Colorectal adenoma refers to a benign tumor originating from the glandular epithelium of the colorectal mucosa. It is generally divided into tubular adenomas, villous adenomas, and tubular-villous adenomas, which are mixed types with some tubular and some villous adenomas. Adenomas are usually mixed types, but the proportion of tubular and villous is different. If most of them are mainly composed of tubular adenomas, they are called tubular adenomas. If most of them are mainly composed of villous adenomas, they are villous adenomas. Colorectal adenomas are closely related to colorectal cancer. Studies have shown that at least 80% of colorectal cancers evolve from colorectal adenomas, which takes about 5 years or more, with an average of 10 to 15 years. Colorectal adenomas are precancerous lesions of colorectal cancer. Active diagnosis and treatment of colorectal adenomas is an important way to control and reduce colorectal cancer. Colorectal cancer is a common malignant tumor, including colon cancer and rectal cancer. The incidence of colorectal cancer is from high to low in the order of rectum, sigmoid colon, cecum, ascending colon, descending colon and transverse colon. In recent years, there has been a trend of developing towards the proximal end (right colon). Its incidence is closely related to lifestyle, genetics, colorectal adenoma, etc. The age of onset tends to be older, with a male-to-female ratio of 1.65:1. There are no symptoms in the early stages of colorectal cancer, or the symptoms are not obvious, only discomfort, indigestion, occult blood in the stool, etc. As the tumor develops, symptoms gradually appear, manifested as changes in bowel habits, abdominal pain, blood in the stool, abdominal mass, intestinal obstruction, etc., with or without systemic symptoms such as anemia, fever and weight loss. Tumors can cause changes in the affected organs due to metastasis and infiltration. Colorectal cancer exhibits different clinical symptoms and signs due to its different sites of occurrence. Actively exploring markers related to colorectal adenoma or colorectal cancer is of great significance for the treatment and prevention of colorectal adenoma or colorectal cancer.

[0003] Therefore, there is an urgent need to develop a relevant marker for diagnosing colorectal adenoma or colorectal cancer. Summary of the invention

[0004] The present invention aims to solve one of the technical problems existing in the prior art to at least a certain extent. To this end, the present invention provides a microbial marker for diagnosing colorectal adenoma and colorectal cancer, which can diagnose colorectal adenoma and colorectal cancer.

[0005] In one aspect of the present invention, the present invention proposes a group of markers. According to an embodiment of the present invention, the markers include Fusobacterium sp. oral taxon 370, Fusobacterium gonidiaformans, Fusobacterium hwasookii, Leuconostoc sp. DORA_2, Fusobacterium sp. CM22, Blumeria graminis, Fusobacterium sp. OBRC1, Streptomyces turgidiscabies, Mycobacterium leprae, Parvimonas micra, Fusobacterium nucleatum, Prevotella nigrescens, Fusobacterium sp. CM1, Prevotella aurantiaca, Mycobacterium sp.1554424_7, Alcanivorax hongdengensis and Dialister sp.CAG:588. According to an embodiment of the present invention, the above-mentioned microbial markers are continuously increased in the healthy individual group, the colorectal adenoma patient group and the colorectal cancer patient group. Thus, the above-mentioned microorganisms are used as markers to distinguish healthy individuals, colorectal adenoma patients and colorectal cancer patients, especially to diagnose colorectal adenoma and colorectal cancer, and to provide a non-invasive method for discovering colorectal adenoma and colorectal cancer diseases. In addition, it is also possible to effectively monitor the susceptible population of colorectal adenoma or colorectal cancer (including family inheritance and external factors), discover early patients with colorectal adenoma or colorectal cancer, or monitor the treatment effect of colorectal adenoma or colorectal cancer.

[0006] In another aspect of the present invention, the present invention proposes a use of a microorganism as a marker for diagnosing colorectal adenoma and / or colorectal cancer, wherein the microorganism includes Fusobacterium sp. oraltaxon 370, Fusobacterium gonidiaformans, Fusobacterium hwasookii, Leuconostoc sp. DORA_2, Fusobacterium sp. CM22, Blumeria graminis, Fusobacterium sp. OBRC1, Streptomyces turgidiscabies, Mycobacterium leprae, Parvimonas micra, Fusobacterium nucleatum, Prevotella nigrescens, Fusobacterium sp. CM1, Prevotella aurea aurantiaca, Mycobacterium sp.1554424_7, Alcanivorax hongdengensis and Dialister sp.CAG: 588. According to an embodiment of the present invention, the above-mentioned microbial markers are continuously increased in the healthy individual group, the colorectal adenoma patient group and the colorectal cancer patient group. Thus, the above-mentioned microorganisms are used as markers to distinguish healthy individuals, colorectal adenoma patients and colorectal cancer patients, especially to diagnose colorectal adenoma and colorectal cancer, and to effectively monitor the susceptible population of colorectal adenoma or colorectal cancer (including family inheritance and external factors), find early patients of colorectal adenoma or colorectal cancer, or monitor the treatment effect of colorectal adenoma or colorectal cancer.

[0007] In another aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit includes a reagent, and the reagent is used to detect the aforementioned marker. The kit according to the embodiment of the present invention can accurately detect the aforementioned microbial marker, can determine whether the biological sample contains the aforementioned microbial marker, and further use the kit to accurately distinguish or diagnose healthy individuals, colorectal adenoma patients and colorectal cancer patients, and has a high diagnostic accuracy.

[0008] In yet another aspect of the present invention, the present invention provides a method for determining the source of a sample. According to an embodiment of the present invention, the method includes: detecting microorganisms in a sample to be tested; and determining the source of the organism based on the detection result of the microorganism; wherein the microorganisms include Fusobacterium sp. oral taxon 370, Fusobacterium gonidiaformans, Fusobacterium hwasookii, Leuconostoc sp. DORA_2, Fusobacterium sp. CM22, Blumeria graminis, Fusobacterium sp. OBRC1, Streptomyces turgidiscabies, Mycobacterium leprae, Parvimonas micra, Fusobacterium nucleatum, Prevotella nigrescens, Fusobacterium sp. CM1, Prevotella aurea aurantiaca, Mycobacterium sp.1554424_7, Alcanivorax hongdengensis and Dialister sp.CAG: 588. As can be seen from the above, the above microbial markers increase continuously in the healthy individual group, the colorectal adenoma patient group and the colorectal cancer patient group, and can be used as markers for distinguishing or diagnosing healthy individuals, colorectal adenoma patients and colorectal cancer patients. Therefore, the above method can accurately detect the source of the sample by detecting the microbial markers.

[0009] In another aspect of the present invention, the present invention proposes a device for determining the source of a sample. According to an embodiment of the present invention, the device includes: a processor for executing an executable program, and executing the executable program includes completing the aforementioned method; a storage unit connected to the processor for storing data, including the executable program. As can be seen from the foregoing, the above-mentioned microbial markers continuously increase in the healthy individual group, the colorectal adenoma patient group and the colorectal cancer patient group, and can be used as markers for distinguishing or diagnosing healthy individuals, colorectal adenoma patients and colorectal cancer patients. Therefore, the above-mentioned device can accurately detect the source of the sample by detecting the microbial markers.

[0010] In another aspect of the present invention, the present invention provides a drug for treating colorectal adenoma or colorectal cancer, wherein the drug inhibits or eliminates microorganisms, and the microorganisms include Fusobacterium sp. oral taxon 370, Fusobacterium gonidiaformans, Fusobacterium hwasookii, Leuconostoc sp. DORA_2, Fusobacterium sp. CM22, Blumeria graminis, Fusobacterium sp. OBRC1, Streptomyces turgidiscabies, Mycobacterium leprae, Parvimonas micra, Fusobacterium nucleatum, Prevotellanigrescens, Fusobacterium sp.CM1, Prevotella aurantiaca, Mycobacterium sp.1554424_7, Alcanivorax hongdengensis, Dialister sp.CAG: 588. The drug according to the embodiment of the present invention can effectively treat colorectal adenoma or colorectal cancer, and has important significance for assisting in alleviating the clinical symptoms of colorectal adenoma and colorectal cancer.

[0011] In another aspect of the present invention, the present invention provides a method for producing or screening the aforementioned drug. According to an embodiment of the present invention, the method includes: a step of screening a substance that inhibits the relative abundance of the microorganism as the drug. The method according to an embodiment of the present invention can effectively screen out drugs for treating colorectal adenoma or colorectal cancer.

[0012] Beneficial effects of the present invention:

[0013] The microbial marker of the present invention can diagnose colorectal adenoma and colorectal cancer, can effectively monitor the susceptible population of colorectal adenoma or colorectal cancer (including those caused by family inheritance and external factors), discover early patients of colorectal adenoma or colorectal cancer, or monitor the treatment effect of colorectal adenoma or colorectal cancer; the microbial marker is used in drugs for treating colorectal adenoma or colorectal cancer, inhibiting potential intestinal pathogenic bacteria (such as Fusobacterium sp. oral taxon 370), etc., which can improve and restore the intestinal microecological structure and alleviate the clinical symptoms of colorectal adenoma or colorectal cancer, which is of great significance; or the microbial marker can be used for producing or screening drugs for treating colorectal adenoma or colorectal cancer; a kit using the microbial marker is provided, etc.

[0014] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0016] Figure 1 This is an experimental flow chart for identifying relevant microbial markers for colorectal adenoma or colorectal cancer in Example 1 of the present invention;

[0017] Figure 2 The abundance results of 9 microbial species markers among the 23 microbial species markers in the healthy control group, the colorectal adenoma group and the colorectal cancer group in Example 1 of the present invention;

[0018] Figure 3 The abundance results of 14 microbial species markers among the 23 microbial species markers in the healthy control group, the colorectal adenoma group and the colorectal cancer group in Example 1 of the present invention;

[0019] Figure 4 The abundance results of 9 microbial species markers among the 17 microbial species markers in the healthy control group, the colorectal adenoma group and the colorectal cancer group in Example 2 of the present invention;

[0020] Figure 5 The abundance results of 8 microbial species markers among the 17 microbial species markers in the healthy control group, the colorectal adenoma group and the colorectal cancer group in Example 2 of the present invention;

[0021] Figure 6 This is an AUC evaluation graph of the comprehensive indexes of 23 microbial species markers in Example 3 of the present invention; wherein, Figure 6a shows the AUC values ​​and confidence intervals of 23 microbial species markers under the ROC curve of 11 healthy control groups and 37 colorectal adenoma group sample data; Figure 6 b shows the AUC values ​​and confidence intervals of 23 microbial species markers under the ROC curve of 11 healthy control groups and 54 colorectal cancer group sample data;

[0022] Figure 7 This is an AUC evaluation graph of the comprehensive indicators of 23 microbial species markers in the validation set data in Example 3 of the present invention; wherein, Figure 7 a shows the AUC values ​​and confidence intervals of 23 microbial species markers under the ROC curve of 30 healthy control group and 30 colorectal adenoma group samples in the validation set data; Figure 7 b shows the AUC values ​​and confidence intervals of 23 microbial species markers under the ROC curve of 30 healthy control group and 30 colorectal cancer group sample data in the validation set data. DETAILED DESCRIPTION

[0023] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0024] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0025] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0026] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention but not excluding other contents.

[0027] As used herein, the terms "optionally", "optional" or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0028] In this article, the term "marker" or "biomarker" should be understood in a broad sense, including any detectable biological indicator that can reflect an abnormal state, and can include gene markers, species markers (species markers / genus markers) and functional markers. Among them, the meaning of gene markers is not limited to existing genes that can be expressed as proteins with biological activity, but also includes any nucleic acid fragments, which can be DNA or RNA, which can be modified DNA or RNA, or unmodified DNA or RNA. In particular, the biomarker of the present invention is a microbial marker.

[0029] In this article, the term "abundance of a microorganism" or "abundance value of a microorganism" refers to the abundance of the microorganism in a certain microbial population, such as the abundance of the microorganism in the intestinal microbial population, which can be expressed as the content of the microorganism in the population.

[0030] The present invention proposes a group of markers, the use of microorganisms as markers for diagnosing colorectal adenoma or colorectal cancer, a kit, a method for determining the source of a sample, an apparatus for determining the source of a sample, a drug, and a method for producing or screening a drug, which will be described in detail below.

[0031] Markers

[0032] In one aspect of the present invention, the present invention proposes a group of markers. According to an embodiment of the present invention, the markers include Fusobacterium sp. oral taxon 370, Fusobacterium gonidiaformans, Fusobacterium hwasookii, Leuconostoc sp. DORA_2, Fusobacterium sp. CM22, Blumeria graminis, Fusobacterium sp. OBRC1, Streptomyces turgidiscabies, Mycobacterium leprae, Parvimonas micra, Fusobacterium nucleatum, Prevotella nigrescens, Fusobacterium sp. CM1, Prevotella aurantiaca, Mycobacterium sp.1554424_7, Alcanivorax hongdengensis and Dialister sp.CAG:588.

[0033] According to an embodiment of the present invention, the above-mentioned microbial markers continuously increase in the healthy individual group, the colorectal adenoma patient group and the colorectal cancer patient group. Therefore, the above-mentioned microorganisms can be used as markers to distinguish healthy individuals, colorectal adenoma patients and colorectal cancer patients, especially to diagnose colorectal adenoma and colorectal cancer, and to effectively monitor the susceptible population of colorectal adenoma or colorectal cancer (including those caused by family inheritance and external factors), discover early patients with colorectal adenoma or colorectal cancer, or monitor the treatment effect of colorectal adenoma or colorectal cancer.

[0034] According to an embodiment of the present invention, the marker further includes at least one of Alistipes sp. CAG: 435, Prevotella sp. CAG: 5226, Prevotella intermedia, Prevotella veroralis, Prevotella pleuritidis and Enterococcus casseliflavus. Thus, the accuracy of diagnosis of colorectal adenoma and colorectal cancer can be further improved.

[0035] use

[0036] In another aspect of the present invention, the present invention proposes a use of a microorganism as a marker for diagnosing colorectal adenoma and / or colorectal cancer, wherein the microorganism includes Fusobacterium sp. oraltaxon 370, Fusobacterium gonidiaformans, Fusobacterium hwasookii, Leuconostoc sp. DORA_2, Fusobacterium sp. CM22, Blumeria graminis, Fusobacterium sp. OBRC1, Streptomyces turgidiscabies, Mycobacterium leprae, Parvimonas micra, Fusobacterium nucleatum, Prevotella nigrescens, Fusobacterium sp. CM1, Prevotella aurea aurantiaca, Mycobacterium sp.1554424_7, Alcanivorax hongdengensis and Dialister sp.CAG:588.

[0037] According to an embodiment of the present invention, the above-mentioned microbial markers continuously increase in the healthy individual group, the colorectal adenoma patient group and the colorectal cancer patient group. Therefore, the above-mentioned microorganisms can be used as markers to distinguish healthy individuals, colorectal adenoma patients and colorectal cancer patients, especially to diagnose colorectal adenoma and colorectal cancer, and to effectively monitor the susceptible population of colorectal adenoma or colorectal cancer (including those caused by family inheritance and external factors), discover early patients with colorectal adenoma or colorectal cancer, or monitor the treatment effect of colorectal adenoma or colorectal cancer.

[0038] According to an embodiment of the present invention, the microorganism further includes at least one of Alistipes sp.CAG:435, Prevotella sp.CAG:5226, Prevotella intermedia, Prevotella veroralis, Prevotella pleuritidis and Enterococcus casseliflavus. Thus, the accuracy of diagnosis of colorectal adenoma and colorectal cancer can be further improved.

[0039] Reagent test kit

[0040] In another aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit includes a reagent, and the reagent is used to detect the aforementioned marker. The kit according to the embodiment of the present invention can accurately detect the aforementioned microbial marker, can determine whether the biological sample contains the aforementioned microbial marker, and further use the kit to accurately distinguish or diagnose healthy individuals, colorectal adenoma patients and colorectal cancer patients, and has a high diagnostic accuracy.

[0041] Method and device for determining the origin of a sample

[0042] In yet another aspect of the present invention, the present invention provides a method for determining the source of a sample. According to an embodiment of the present invention, the method includes: detecting microorganisms in a sample to be tested; and determining the source of the organism based on the detection result of the microorganism; wherein the microorganisms include Fusobacterium sp. oral taxon 370, Fusobacterium gonidiaformans, Fusobacterium hwasookii, Leuconostoc sp. DORA_2, Fusobacterium sp. CM22, Blumeria graminis, Fusobacterium sp. OBRC1, Streptomyces turgidiscabies, Mycobacterium leprae, Parvimonas micra, Fusobacterium nucleatum, Prevotella nigrescens, Fusobacterium sp. CM1, Prevotella aurea aurantiaca, Mycobacterium sp.1554424_7, Alcanivorax hongdengensis and Dialister sp.CAG: 588. As can be seen from the above, the above microbial markers increase continuously in the healthy individual group, the colorectal adenoma patient group and the colorectal cancer patient group, and can be used as markers for distinguishing or diagnosing healthy individuals, colorectal adenoma patients and colorectal cancer patients. Therefore, the above method can accurately detect the source of the sample by detecting the microbial markers.

[0043] According to an embodiment of the present invention, the microorganism further includes at least one of Alistipes sp.CAG:435, Prevotella sp.CAG:5226, Prevotella intermedia, Prevotella veroralis, Prevotella pleuritidis and Enterococcus casseliflavus. Thus, the accuracy of sample source detection can be further improved.

[0044] According to an embodiment of the present invention, the detection result is a relative abundance value of the microorganism.

[0045] According to an embodiment of the present invention, the relative abundance value is obtained in the following manner: extracting a nucleic acid sample from the biological sample, constructing a sequencing library for the nucleic acid sample; performing high-throughput sequencing on the sequencing library, and obtaining the relative abundance value of the microorganism based on the sequencing result.

[0046] According to an embodiment of the present invention, the sample to be tested is a stool sample.

[0047] According to an embodiment of the present invention, the organism is derived from a patient with colorectal cancer, a patient with colorectal adenoma or a healthy individual.

[0048] According to an embodiment of the present invention, it further includes: comparing the relative abundance value with a predetermined threshold.

[0049] In this article, the "predetermined threshold value" can be obtained by detecting microbial markers in a large number of samples to be tested of known sample sources, and statistically analyzing the abundance values ​​of microbial markers obtained from a large number of samples to be tested. Among them, the "predetermined threshold value" can be the abundance values ​​of multiple microbial markers directly obtained, or it can be a weighted value based on the abundance values ​​of multiple microbial markers, and the specific type is not limited.

[0050] According to an embodiment of the present invention, the predetermined threshold includes a first threshold and a second threshold.

[0051] It should be noted that the "first threshold" can be obtained by detecting the microbial markers of a large number of healthy individuals and patients with colorectal adenoma, and statistically analyzing the abundance values ​​of the microbial markers in the two groups. The "second threshold" can be obtained by detecting the microbial markers of a large number of patients with colorectal adenoma and colorectal cancer, and statistically analyzing the abundance values ​​of the microbial markers in the two groups.

[0052] According to an embodiment of the present invention, the relative abundance value is not lower than the second threshold, which is an indication that the sample to be tested is derived from a patient with colorectal cancer; or, the relative abundance value is greater than or equal to the first threshold and less than or equal to the second threshold, which is an indication that the sample to be tested is derived from a patient with colorectal adenoma; or, the relative abundance value is lower than the first threshold, which is an indication that the sample to be tested is derived from a healthy individual.

[0053] In another aspect of the present invention, the present invention proposes a device for determining the source of a sample. According to an embodiment of the present invention, the device includes: a processor for executing an executable program, and executing the executable program includes completing the aforementioned method; a storage unit connected to the processor for storing data, including the executable program. As can be seen from the foregoing, the above-mentioned microbial markers continuously increase in the healthy individual group, the colorectal adenoma patient group and the colorectal cancer patient group, and can be used as markers for distinguishing or diagnosing healthy individuals, colorectal adenoma patients and colorectal cancer patients. Therefore, the above-mentioned device can accurately detect the source of the sample by detecting the microbial markers.

[0054] Drugs and methods for producing or screening them

[0055] In another aspect of the present invention, the present invention provides a drug for treating colorectal adenoma or colorectal cancer, wherein the drug inhibits or eliminates microorganisms, and the microorganisms include Fusobacterium sp. oral taxon 370, Fusobacterium gonidiaformans, Fusobacterium hwasookii, Leuconostoc sp. DORA_2, Fusobacterium sp. CM22, Blumeria graminis, Fusobacterium sp. OBRC1, Streptomyces turgidiscabies, Mycobacterium leprae, Parvimonas micra, Fusobacterium nucleatum, Prevotellanigrescens, Fusobacterium sp.CM1, Prevotella aurantiaca, Mycobacterium sp.1554424_7, Alcanivorax hongdengensis, Dialister sp.CAG: 588. The drug according to the embodiment of the present invention can effectively treat colorectal adenoma or colorectal cancer, and has important significance for assisting in alleviating the clinical symptoms of colorectal adenoma and colorectal cancer.

[0056] According to an embodiment of the present invention, the microorganism further comprises at least one of Alistipes sp.CAG:435, Prevotella sp.CAG:5226, Prevotella intermedia, Prevotella veroralis, Prevotella pleuritidis and Enterococcus casseliflavus. Thus, the therapeutic effect on colorectal adenoma or colorectal cancer can be further improved.

[0057] In another aspect of the present invention, the present invention provides a method for producing or screening the aforementioned drug. According to an embodiment of the present invention, the method includes: a step of screening a substance that inhibits the relative abundance of the microorganism as the drug. The method according to an embodiment of the present invention can effectively screen out drugs for treating colorectal adenoma or colorectal cancer.

[0058] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0059] Embodiment 1:

[0060] In this example, DNA sequencing analysis was performed by extracting stool samples from 54 colorectal cancer patients, 37 colorectal adenoma patients, and 11 healthy controls to conduct a study on the continuous increase of intestinal flora microorganisms from the healthy control group to the colorectal adenoma group and then to the colorectal cancer group, and about 626.307Gb of high-quality sequencing data was obtained. Metagenomic analysis showed that 23 intestinal flora microorganism species markers met the conditions for continuous increase from the healthy control group to the colorectal adenoma group and then to the colorectal cancer group. The specific steps are as follows:

[0061] 1. Sample collection and DNA extraction

[0062] The samples of healthy controls, colorectal adenoma patients and colorectal cancer patients were all from Shanghai Changhai Hospital. The experiment collected stool samples from 54 colorectal cancer patients, 37 colorectal adenoma patients and 11 healthy controls. The fresh stool samples of each individual were divided into 200 mg / portion, a total of 5 portions, and immediately frozen in a -80℃ refrigerator. DNA was extracted using the phenol chloroform treatment DNA extraction method.

[0063] 2. Library construction and sequencing

[0064] The DNA extracted in step 1 was used to construct a library. The DNA library was constructed according to the operating instructions of the instrument manufacturer (Illumina). The library was sequenced by PE 2*150bp. The Illumina NovaSeq platform sequenced the libraries of 102 samples. Each sample generated an average of 6.14Gb of high-quality sequencing results, with a total of 626.307Gb of sequencing data.

[0065] Reference Figure 1 The experimental process for identifying the microbial markers related to colorectal adenoma or colorectal cancer, wherein the omitted steps or details are well known to those skilled in the art, several important steps are as follows:

[0066] 1) Basic processing of sequencing data

[0067] After obtaining the sequencing data of the sample, it is filtered and quality controlled according to the following standards: a) remove reads greater than 3 N bases; b) remove low-quality (Q20) N50 reads; c) remove more than 10 low-quality (Q2) bases or a specified number of tail N bases. Sequences with missing paired reads are considered as single reads for assembly.

[0068] 2) Species abundance analysis

[0069] SOAPalign 2.21 was used to match paired-end clean reads against redundant genomes with parameters –r 2–m200–x 1000. Reads aligned to redundant genomes may be divided into two parts: a) Unique reads (U): reads aligned to only one genome; these are defined as unique reads. b) Multiple reads (M): reads aligned to more than one genome if these genomes are from the same species; these reads are defined as unique reads. If they are from different species, they are defined as multiple reads.

[0070] For species S, if the abundance is Ab(S), it may be associated with U-specific reads and M-shared reads, and the evaluation method is as follows:

[0071] Ab(S)=Ab(U)+Ab(M)

[0072] Ab(U)=U / l

[0073]

[0074] Ab(U) and Ab(M) are the abundances of unique and multiple reads, respectively, and l represents the genome length. For each multiple read, there is a species-specific coefficient Co; assuming that {M} has a related N in different species, then CO is calculated as follows:

[0075]

[0076] For these reads, the unique abundance with N added was used as the standard.

[0077] The species abundance values ​​calculated in each sample were divided by the total abundance of each sample to obtain the normalized species abundance table.

[0078] 3) Screening for continuously increasing intestinal microbial species markers

[0079] In order to obtain intestinal microbial species markers that accompany the development of colorectal cancer, we first used the intestinal microbial species abundance data of three groups, namely the healthy control group (11 cases), the colorectal adenoma group (37 cases) and the colorectal cancer group (54 cases), to study the continuous increase of markers from the healthy control group to the colorectal adenoma group and then to the colorectal cancer group at the species level. Based on the species abundance table obtained by the above calculation method, the following standards were set: (1) The p value and FDR value of each species were obtained by Kruskal-Wallis test to determine whether there was a difference between the three groups; (2) A strict threshold (FDR<0.01) was used to screen out species markers with significant differences between the three groups; (3) By comparing the medians of the three groups, the median abundance of the colorectal cancer group was greater than 1.5 times the median abundance of the colorectal adenoma group, and the median abundance of the colorectal adenoma group was greater than 1.5 times the median abundance of the healthy control group.

[0080] Finally, 23 intestinal microbial species markers were obtained, which increased continuously from the healthy control group to the colorectal adenoma group and then to the colorectal cancer group. These 23 microbial species markers are shown in Table 1. The abundance increases continuously as shown in Figure 2 and Figure 3 shown.

[0081] Table 1: 23 microbial species markers

[0082] Species Markers NC_median A_median CRC_median p value(KW test) FDR(KW test) Fusobacterium_sp_oral_taxon_370 2.53E-07 1.01E-06 1.23E-05 9.70E-09 3.98E-07 Alistipes_sp_CAG_435 0 7.63E-08 2.49E-06 1.85E-07 3.80E-06 Alcanivorax_hongdengensis 0 2.01E-08 3.32E-07 3.82E-06 2.41E-05 Dialister_sp_CAG_588 9.20E-07 1.44E-06 1.71E-05 1.85E-06 2.41E-05 Fusobacterium_gonidiaformans 2.78E-08 5.16E-08 1.10E-06 2.99E-06 2.41E-05 Fusobacterium_hwasookii 3.94E-08 2.83E-07 3.18E-06 3.33E-06 2.41E-05 Leuconostoc_sp_DORA_2 0 1.11E-06 4.47E-06 4.12E-06 2.41E-05 Fusobacterium_sp_CM22 4.02E-09 3.56E-07 2.93E-06 6.00E-06 3.07E-05 Blumeria_graminis 3.52E-09 1.09E-08 1.16E-07 1.12E-05 5.08E-05 Fusobacterium_sp_OBRC1 1.78E-07 9.69E-07 5.45E-06 2.84E-05 0.000116515 Streptomyces_turgidiscabies 0 7.31E-09 9.19E-08 5.05E-05 0.000188078 Mycobacterium_leprae 1.59E-07 2.59E-07 2.74E-06 9.69E-05 0.000331024 Parvimonas_micra 2.12E-06 4.09E-06 1.82E-05 0.00011066 0.000349004 Fusobacterium_nucleatum 2.03E-06 5.37E-06 3.33E-05 0.000151067 0.00044241 Prevotella_nigrescens 4.20E-07 1.06E-06 4.31E-06 0.000163377 0.000446563 Fusobacterium_sp_CM1 3.94E-07 6.47E-07 4.26E-06 0.000203367 0.000512606 Prevotella_intermedia 4.68E-07 9.99E-07 1.41E-05 0.000212544 0.000512606 Prevotella_aurantiaca 1.65E-08 1.93E-07 8.65E-07 0.000310111 0.000706364 Prevotella_sp_CAG_5226 9.64E-07 2.13E-06 5.54E-06 0.000832879 0.001797265 Prevotella_veroralis 2.42E-07 3.64E-07 2.14E-06 0.001370576 0.002809681 Prevotella_pleuritidis 8.26E-06 1.87E-05 3.48E-05 0.003029049 0.005913858 Mycobacterium_sp_1554424_7 1.09E-07 1.71E-07 5.03E-07 0.004513505 0.008411532 Enterococcus_casseliflavus 1.34E-09 1.58E-08 1.94E-07 0.005029294 0.008965263

[0083] Example 2: Validation of continuously increasing gut microbial species markers in 90 independent validation set data

[0084] In order to verify the intestinal microbial species markers associated with the development of colorectal cancer, 90 additional independent cohort data were collected, including a healthy control group (30 cases), a colorectal adenoma group (30 cases) and a colorectal cancer group (30 cases). Based on the species abundance table obtained by the same method as in Example 1, the same screening criteria were set as follows: (1) The p value and FDR value of each species in the three groups were obtained by Kruskal-Wallis test; (2) A strict threshold (FDR<0.01) was used to screen out species markers with significant differences in the three groups; (3) By comparing the medians of the three groups, the median abundance of the colorectal cancer group was greater than 1.5 times the median abundance of the colorectal adenoma group, and the median abundance of the colorectal adenoma group was greater than 1.5 times the median abundance of the healthy control group.

[0085] Finally, among the 23 continuously increasing intestinal microbial species markers found previously, 17 microbial species markers that met the above conditions were obtained again (as shown in Table 2), and the abundance increased continuously as follows Figure 4 and Figure 5 shown.

[0086] Table 2: 17 microbial species markers

[0087] Species Markers NC_median A_median CRC_median p value(KW test) FDR(KW test) Fusobacterium_sp_oral_taxon_370 1.15E-08 9.80E-06 0.000136082 3.89E-12 6.62E-11 Parvimonas_micra 1.30E-06 7.59E-06 0.000286734 1.31E-11 1.11E-10 Dialister_sp_CAG_588 7.80E-07 5.46E-06 0.000169283 3.26E-11 1.85E-10 Fusobacterium_nucleatum 1.66E-06 1.14E-05 4.88E-05 1.18E-07 4.99E-07 Fusobacterium_sp_CM1 3.06E-07 2.14E-06 7.08E-06 3.55E-07 1.21E-06 Fusobacterium_gonidiaformans 2.05E-08 1.30E-07 2.82E-06 8.64E-07 2.45E-06 Fusobacterium_sp_OBRC1 1.64E-07 1.85E-06 6.92E-06 1.23E-06 2.86E-06 Fusobacterium_hwasookii 2.66E-07 1.50E-06 1.00E-05 1.35E-06 2.86E-06 Fusobacterium_sp_CM22 9.03E-08 1.39E-06 5.26E-06 2.86E-06 5.41E-06 Prevotella_nigrescens 6.27E-07 5.35E-06 1.60E-05 1.09E-05 1.85E-05 Alcanivorax_hongdengensis 1.13E-08 1.16E-07 5.76E-07 2.89E-05 4.46E-05 Prevotella_aurantiaca 1.13E-07 3.44E-07 2.95E-06 5.16E-05 7.31E-05 Streptomyces_turgidiscabies 0 4.93E-08 1.72E-07 0.000183047 0.000239369 Leuconostoc_sp_DORA_2 5.80E-07 1.43E-06 5.35E-06 0.00020553 0.000249572 Mycobacterium_leprae 3.17E-07 5.05E-07 4.17E-06 0.000607743 0.000688776 Mycobacterium_sp_1554424_7 1.08E-07 2.63E-07 8.36E-07 0.000747206 0.000793906 Blumeria_graminis 8.65E-09 1.89E-08 8.91E-08 0.002089654 0.002089654

[0088] Example 3: ROC evaluation of continuously increasing microbial markers for disease classification

[0089] 1. In order to evaluate the diagnostic effect of continuously increasing microbial markers on the classification of colorectal adenoma and healthy people, and the diagnostic effect of classifying colorectal cancer and healthy people, the 23 microbial species markers that continuously increase from the healthy control group to the colorectal adenoma group and then to the colorectal cancer group obtained by screening in Example 1 were further used to carry out receiver operating characteristic ROC (receiver-operating characteristic) curve analysis (the larger the area under the curve AUC, the higher the diagnostic ability), and evaluate the diagnostic ability of the 23 markers for colorectal adenoma and colorectal cancer, respectively. By evaluating 11 healthy control groups and 37 colorectal adenoma group samples, and by evaluating 11 healthy control groups and 54 colorectal cancer group samples, specifically: collecting stool samples according to the method of Example 1, extracting DNA from the stool samples, sequencing, and performing species abundance analysis of 23 microbial markers on the sequencing results, a comprehensive index was constructed, and the area under the ROC (receiver operating characteristic) curve AUC was estimated (the larger the AUC, the higher the diagnostic ability), and the diagnostic ability of the comprehensive score for colorectal adenoma and colorectal cancer was evaluated.

[0090] The results showed that AUC = 78.87% was obtained in 11 healthy control groups and 37 colorectal adenoma samples, showing good diagnostic ability (see Figure 6 a); AUC = 91.92% was obtained in 11 healthy control group and 54 colorectal cancer group samples, which also showed good diagnostic ability (see Figure 6 b).

[0091] 2. In the validation set data, the receiver operating characteristic (ROC) curve analysis was also further performed to evaluate the diagnostic ability of the 23 markers for colorectal adenoma and colorectal cancer. Using the method in step 1, 30 healthy control group samples and 30 colorectal adenoma group samples were evaluated. AUC = 81% was obtained in the 30 healthy control group samples and 30 colorectal adenoma group samples in the validation set data, showing good diagnostic ability (see Figure 7 a); Using the method in step 1, 30 healthy control group samples and 30 colorectal cancer group samples were evaluated, and the AUC of 30 healthy control group samples and 30 colorectal cancer group samples in the validation set data was 94.33%, which also showed good diagnostic ability (see Figure 7 b).

[0092] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0093] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A set of markers, characterized in that Including Fusobacterium sp.oraltaxon370, Fusobacterium gonidiaformans, Fusobacteriumhwasookii, Leuconostoc sp.DORA_2, Fusobacterium sp.CM22, Blumeria graminis, Fusobacterium sp.OBRC1, Streptomycesturgidiscabies, Mycobacterium leprae, Parvimonas micra, Fusobacterium nucleatum, Prevotella nigrescens, Fusobacterium sp.CM1, Prevotella aurantiaca, Mycobacterium sp.1554424_7, Alcanivorax hongdengensis and Dialister sp.CAG:

588.

2. The marker according to claim 1, characterized in that The present invention further comprises at least one of Alistipes sp. CAG:435, Prevotella sp. CAG:5226, Prevotella intermedia, Prevotella veroralis, Prevotella pleuritidis and Enterococcus casseliflavus.

3. Use of a microorganism as a marker for diagnosing colorectal adenoma and / or colorectal cancer, the microorganism comprising Fusobacterium sp. oral taxon 370, Fusobacterium gonidiaformans, Fusobacterium hwasookii, Leuconostoc sp. DORA_2, Fusobacterium sp. CM22, Blumeria graminis, Fusobacterium sp. OBRC1, Streptomyces turgidiscabies, Mycobacterium leprae, Parvimonas micra, Fusobacterium nucleatum, Prevotella nigrescens, Fusobacterium sp. CM1, Prevotella aurea aurantiaca, Mycobacterium sp.1554424_7, Alcanivorax hongdengensis, and Dialister sp.CAG:588; Optionally, the microorganism further comprises at least one of Alistipes sp. CAG:435, Prevotella sp. CAG:5226, Prevotella intermedia, Prevotella veroralis, Prevotella pleuritidis and Enterococcus casseliflavus.

4. A kit, characterized in that: Comprising a reagent for detecting the marker according to claim 1 or 2.

5. A method for determining the source of a sample, characterized in that: include: Detecting microorganisms in the sample to be tested; determining the source of the organism based on the detection result of the microorganism; The microorganisms include Fusobacterium sp. oral taxon 370, Fusobacterium gonidiaformans, Fusobacterium hwasookii, Leuconostoc sp. DORA_2, Fusobacterium sp. CM22, Blumeria graminis, Fusobacterium sp. OBRC1, Streptomyces turgidiscabies, Mycobacterium leprae, Parvimonas micra, Fusobacterium nucleatum, Prevotella nigrescens, Fusobacterium sp. CM1, Prevotella aurantiaca, Mycobacterium sp.1554424_7, Alcanivorax hongdengensis and Dialister sp.CAG:588; Optionally, the microorganism further comprises at least one of Alistipes sp. CAG:435, Prevotella sp. CAG:5226, Prevotella intermedia, Prevotella veroralis, Prevotella pleuritidis and Enterococcus casseliflavus.

6. The method according to claim 5, characterized in that The detection result is the relative abundance value of the microorganism; Optionally, the relative abundance value is obtained by: Extracting a nucleic acid sample from the biological sample, and constructing a sequencing library for the nucleic acid sample; Performing high-throughput sequencing on the sequencing library, and obtaining a relative abundance value of the microorganism based on the sequencing result; Optionally, the sample to be tested is a stool sample.

7. The method according to claim 6, characterized in that The organism is derived from a patient with colorectal cancer, a patient with colorectal adenoma, or a healthy individual; Optionally, further comprising: comparing the relative abundance value with a predetermined threshold; Optionally, the predetermined threshold includes a first threshold and a second threshold; Optionally, the relative abundance value is not less than the second threshold value, which is an indication that the sample to be tested is derived from a patient with colorectal cancer; or The relative abundance value is greater than or equal to the first threshold value and less than or equal to the second threshold value, which indicates that the sample to be tested is derived from a patient with colorectal adenoma; or The relative abundance value being lower than the first threshold is an indication that the sample to be tested is derived from a healthy individual.

8. A device for determining the source of a sample, characterized in that: include: A processor, configured to execute an executable program, wherein executing the executable program includes performing the method according to any one of claims 5 to 7; A storage unit is connected to the processor and is used to store data, including the executable program.

9. A drug for treating colorectal adenoma or colorectal cancer, characterized in that: The drug inhibits or eliminates microorganisms, and the microorganisms include Fusobacterium sp. oral taxon 370, Fusobacterium gonidiaformans, Fusobacterium hwasookii, Leuconostoc sp. DORA_2, Fusobacterium sp. CM22, Blumeria graminis, Fusobacterium sp. OBRC1, Streptomyces turgidiscabies, Mycobacterium leprae, Parvimonas micra, Fusobacterium nucleatum, Prevotella nigrescens, Fusobacterium sp. CM1, Prevotella aurantiaca, Mycobacterium sp.1554424_7, Alcanivorax hongdengensis, Dialister sp.CAG:588; Optionally, the microorganism further comprises at least one of Alistipes sp. CAG:435, Prevotella sp. CAG:5226, Prevotella intermedia, Prevotella veroralis, Prevotella pleuritidis and Enterococcus casseliflavus.

10. A method for producing or screening the drug according to claim 9, characterized in that: include: The step of screening a substance that inhibits the relative abundance of the microorganism as the drug.

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