A plasma circulating microbial marker, reagent, kit for colorectal cancer and its application

Abstract

The application discloses a kind of colorectal cancer plasma circulating microbial markers, reagent, kit and its application, belong to biomedical detection technical field.The colorectal cancer plasma circulating microbial markers of the present application include alternaria alternata, xanthomonas campestris, clostridium perfringens, human rumblus, pseudomonas syringae, alternaria alternata, parabacillus licheniformis and pseudomonas szechuanensis.The colorectal cancer plasma circulating microbial markers of the present application can be used for early screening of colorectal cancer, with high sensitivity and specificity, significantly improve the accuracy and reliability of detection;Break through the limitation of traditional ctDNA detection, improve the detection rate of early colorectal cancer.Through low-depth whole genome sequencing, the colorectal cancer plasma circulating microbial markers in colorectal cancer plasma sample are detected, the requirement for depth in experiment is reduced, while ensuring high sensitivity and specificity, cost can be greatly reduced, more suitable for large-scale screening.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, and in particular relates to a plasma circulating microbial marker, reagent, kit for colorectal cancer, and their applications. Background Technology

[0002] In the field of early colorectal cancer screening, existing technologies mainly rely on colonoscopy, fecal occult blood tests, fecal DNA testing (such as Cologuard), and liquid biopsy (ctDNA testing). Colonoscopy, as the "gold standard" for colorectal cancer diagnosis, allows direct observation of intestinal lesions and biopsy, boasting high accuracy and specificity. However, due to its invasiveness, the need for bowel preparation, complex operation, and high medical costs, patient acceptance is low, hindering its widespread adoption as a screening method. Fecal occult blood tests are a non-invasive screening method that detects occult blood in stool to determine the presence of bleeding lesions in the intestines. While less expensive, its sensitivity and specificity are limited, especially for early colorectal cancer and precancerous lesions, with a low detection rate and a tendency for false negatives or false positives. Fecal DNA testing improves sensitivity by analyzing tumor exfoliated cell DNA (such as KRAS and APC gene mutations) and methylation markers (such as NDRG4 and BMP3) in stool. Although its sensitivity is improved compared to fecal occult blood tests, it is more expensive, still relies on stool samples, is inconvenient to operate, and has poor patient compliance. In recent years, liquid biopsy technology based on circulating tumor DNA (ctDNA) in blood has attracted attention in early cancer screening. This method screens for cancer by detecting mutated DNA fragments released by tumors. However, the content of ctDNA in early-stage colorectal cancer patients is extremely low, resulting in limited detection sensitivity (especially in stage I / II colorectal cancer). Furthermore, whole-genome or deep sequencing in liquid biopsy is costly and difficult to popularize. Additionally, tumor heterogeneity leads to insufficient specificity of some mutational biomarkers, potentially resulting in overlap with other cancers or benign lesions, limiting its application in large-scale screening. Therefore, there is an urgent need to find an efficient, low-cost, and non-invasive solution to overcome the shortcomings of existing technologies. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a plasma circulating microbial marker for colorectal cancer that is highly sensitive and specific, non-invasive, and has low detection cost.

[0004] Another object of the present invention is to provide a reagent.

[0005] Another object of the present invention is to provide a reagent kit.

[0006] Another object of the present invention is to provide the application of the aforementioned colorectal cancer plasma circulating microbial marker, reagent, or kit in the preparation of colorectal cancer screening and diagnostic products.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a circulating microbial marker in the plasma of colorectal cancer, comprising Alternaria incomplexa, Ogataeaphilodendri, Clostridium perfringens, Romboutsia hominis, Ralstonia solanacearum, Alternaria conjuncta, Bacillus paralicheniformis, and Pseudomonas sichuanensis.

[0009] Preferably, the circulating microbial markers in the plasma of colorectal cancer include a colorectal cancer group and a healthy group; the colorectal cancer group includes Alternaria, Ogatoria philodendron, Clostridium perfringens, Ralstonia solanacearum, and Ralstonia solanacearum; the healthy group includes Alternaria spp., Bacillus paralichrysogenum, and Pseudomonas schistosporum.

[0010] The present invention also provides a reagent comprising a reagent for detecting the content or abundance of eight microorganisms in the circulating microbial markers of colorectal cancer plasma.

[0011] Preferably, the test sample for the reagent includes peripheral blood of the subject.

[0012] The present invention also provides a kit comprising the reagents.

[0013] The present invention also provides the application of the aforementioned colorectal cancer plasma circulating microbial marker, reagent, or kit in the preparation of colorectal cancer screening and diagnostic products.

[0014] Preferably, the criteria for colorectal cancer screening and diagnosis include: standardizing the original abundance data of 8 microorganisms so that the sum of their relative abundances is 100%, and recording 0% for no detection, calculating the risk score = Σ(relative abundance of each microorganism × corresponding feature weight), and determining the colorectal cancer positive when the risk score is ≥15.

[0015] Preferably, the method for screening and diagnosing colorectal cancer includes: extracting plasma DNA from peripheral blood and sequencing it; identifying the content of circulating microbial markers in the plasma of colorectal cancer; and determining whether the peripheral blood comes from a colorectal cancer patient based on the criteria for screening and diagnosing colorectal cancer.

[0016] Preferably, the sequencing method includes low-depth whole-genome sequencing.

[0017] Preferably, the colorectal cancer includes stage I or stage II colorectal cancer.

[0018] The beneficial effects of this invention are:

[0019] This invention screens out eight circulating microorganisms in plasma and combines these microorganisms to form a plasma circulating microbial biomarker for colorectal cancer. This biomarker can be used for early screening of colorectal cancer, exhibiting high sensitivity and specificity, and significantly improving the accuracy and reliability of detection. Applying this invention's plasma circulating microbial biomarker for colorectal cancer screening and diagnosis can overcome the limitations of traditional ctDNA detection and improve the detection rate of early colorectal cancer. By using low-pass whole-genome sequencing (low-pass WGS) to detect the plasma circulating microbial biomarker in colorectal cancer plasma samples, the depth requirements in the experiment are reduced, and the cost can be significantly reduced while ensuring high sensitivity and specificity, making it more suitable for large-scale screening. Attached Figure Description

[0020] Figure 1 This is a flowchart of the screening and validation process for circulating microbial biomarkers in colorectal cancer plasma in Example 1;

[0021] Figure 2 The microorganisms that showed significant differences between the colorectal cancer group and the healthy group in Example 1;

[0022] Figure 3 The weight values ​​of the eight circulating microbial markers in the plasma of colorectal cancer in Example 1;

[0023] Figure 4 The performance of the circulating microbial markers in the plasma of colorectal cancer in Example 1 in the colorectal cancer training set;

[0024] Figure 5 The performance of the circulating microbial markers in the plasma of colorectal cancer in Example 1 in the colorectal cancer validation set. Detailed Implementation

[0025] This invention provides a circulating microbial marker in the plasma of colorectal cancer, comprising Alternaria incomplexa, Ogataeaphilodendri, Clostridium perfringens, Romboutsia hominis, Ralstonia solanacearum, Alternaria conjuncta, Bacillus paralicheniformis, and Pseudomonas sichuanensis.

[0026] In this invention, the circulating microbial markers in the plasma of colorectal cancer preferably include a colorectal cancer group and a healthy group; the colorectal cancer group preferably includes Alternaria, Ogatoria philodendron, Clostridium perfringens, Ralstonia solanacearum, and Ralstonia solanacearum; the healthy group preferably includes Alternaria spp., Bacillus paralichrysogenum, and Pseudomonas schistosii.

[0027] This invention extracted nucleic acids, constructed libraries, and sequenced plasma samples from 206 colorectal cancer patients and 33 healthy individuals. Through MaAsLin differential analysis and feature screening using the random forest algorithm, eight marker microorganisms with optimal classification performance suitable for screening and diagnosing colorectal cancer were obtained. Among them, *Alternaria incomplexa*, *Ogataeaphilodendri*, *Clostridium perfringens*, *Romboutsia hominis*, and *Ralstonia solanacearum* were enriched in colorectal cancer patients; while *Alternaria conjuncta*, *Bacillus paralicheniformis*, and *Pseudomonas sichuanensis* were enriched in the healthy group. The circulating microbial biomarkers for colorectal cancer in plasma of this invention were validated in 87 colorectal cancer patients and 14 healthy individuals. The AUC value was 0.9635, the sensitivity was 91.95%, and the specificity was 78.57%, indicating that the eight circulating microbial biomarkers for colorectal cancer screened by this invention can effectively distinguish between colorectal cancer patients and healthy individuals (P<0.001), and have significant clinical screening application value.

[0028] The present invention also provides a reagent comprising a reagent for detecting the content or abundance of eight microorganisms in the circulating microbial markers of colorectal cancer plasma.

[0029] In this invention, the reagent preferably includes primers, probes, aptamers, or antibodies that are specific to the eight microorganisms in the circulating microbial markers of colorectal cancer plasma.

[0030] In this invention, the detection sample of the reagent preferably includes peripheral blood of the subject, and more preferably includes plasma DNA in the peripheral blood of the subject.

[0031] The present invention also provides a kit comprising the reagents.

[0032] The present invention also provides the application of the aforementioned colorectal cancer plasma circulating microbial marker, reagent, or kit in the preparation of colorectal cancer screening and diagnostic products.

[0033] In this invention, the preferred criteria for colorectal cancer screening and diagnosis include: standardizing the original abundance data of 8 microorganisms so that the sum of their relative abundances is 100%, and recording 0% for no detection; calculating the risk score = Σ(relative abundance of each microorganism × corresponding feature weight); and determining the colorectal cancer positive when the risk score is ≥15. The corresponding feature weights can be evaluated using a random forest algorithm to assess the classification contribution of each microbial marker and quantify their feature weight values. In some embodiments, the feature weight of *Alternaria incomplexa* is preferably 1.63, the feature weight of *Ogataea philodendri* is preferably 1.24, the feature weight of *Clostridium perfringens* is preferably 2.17, the feature weight of *Romboutsia hominis* is preferably 0.85, the feature weight of *Ralstonia solanacearum* is preferably 1.08, the feature weight of *Alternaria conjuncta* is preferably -1.12, the feature weight of *Bacillus paralicheniformis* is preferably -1.59, and the feature weight of *Pseudomonas sichuanensis* is preferably -0.83.

[0034] In this invention, the preferred method for colorectal cancer screening and diagnosis includes: extracting plasma DNA from peripheral blood and sequencing it; identifying the content of circulating microbial markers in the plasma of colorectal cancer; and determining whether the peripheral blood comes from a colorectal cancer patient based on the criteria for colorectal cancer screening and diagnosis.

[0035] In this invention, the sequencing method preferably includes low-pass whole-genome sequencing. This invention uses low-pass whole-genome sequencing (WGS) to detect circulating microbial markers in colorectal cancer plasma samples, reducing the depth requirements in the experiment. While ensuring high sensitivity and specificity, it can significantly reduce costs and is more suitable for large-scale screening.

[0036] In this invention, the colorectal cancer screening and diagnosis preferably includes early colorectal cancer screening and diagnosis, and the early colorectal cancer preferably includes stage I or stage II colorectal cancer. Validation experiments of this invention show that, among all included colorectal cancer samples, early stage (stage I / II) samples accounted for 74.40%, indicating that the plasma circulating microbial markers for colorectal cancer of this invention can serve as reliable tumor markers and are suitable for non-invasive early screening of colorectal cancer.

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] Unless otherwise specified, the following embodiments are all conventional methods.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0040] Example 1

[0041] 1. Sample selection

[0042] Plasma samples were collected from 293 pathologically confirmed colorectal cancer patients (who had not received preoperative treatment) and 47 healthy controls (collected using cfDNA anticoagulant tubes). All cancer cases were confirmed by imaging, laboratory testing, and pathology, while healthy controls were from routine physical examinations. All samples underwent cfDNA extraction and low-pass whole-genome sequencing (Low Pass WGS). Samples were randomly assigned to a training set (206 cancer patients / 33 healthy individuals) and a validation set (87 cancer patients / 14 healthy individuals), with no significant differences in age or sex between the two groups. Cancer patients were staged as follows:

[0043] Training set: 87 cases in stage I (42.23%), 64 cases in stage II (31.07%), and 55 cases in stage III and above (26.70%).

[0044] Validation set: 39 cases in stage I (44.83%), 28 cases in stage II (32.18%), and 20 cases in stage III and above (22.99%).

[0045] 2. Screening and validation of plasma circulating microbial markers for colorectal cancer

[0046] Screening for combinations of plasma circulating microbial biomarkers for colorectal cancer, and constructing a colorectal cancer screening and diagnostic model, the process is as follows: Figure 1 As shown.

[0047] Based on high-throughput sequencing technology, plasma nucleic acid extraction and library construction were performed on plasma samples isolated from peripheral blood. The process included plasma separation, nucleic acid extraction, end repair, adapter ligation, library amplification, circularization, and DNB preparation. The obtained library samples were sequenced using an MGISEQ-2000 sequencer (manufactured by BGI Genomics) with a PE100 sequencing strategy.

[0048] After sequencing data was processed on the MGISEQ-2000 platform, the data underwent several steps. First, FASTP was used for quality control of the raw data, filtering out low-quality sequences, short sequences, and adapter contamination to obtain high-quality clean data. Then, Bowtie2 alignment to the human reference genome (GRCh38 / hg38) was used to remove human sequences. To reduce interference, BBmap was used to filter low-complexity sequences. Microbial species were identified through alignment with database reference sequences, then classified using Kraken2, and relative abundance was calculated using Bracken. Finally, a decontamination process was implemented to remove potential endogenous and exogenous contaminants, and differentially expressed genera were screened to construct a classification model.

[0049] For the training set, after processing using the constructed bioinformatics analysis pipeline, MaAsLin differential analysis (corrected for sex and age) revealed significant differences in 37 microorganisms between the two groups (P<0.05). Figure 2The study included 20 microorganisms significantly enriched in the colorectal cancer group and 17 microorganisms enriched in the healthy group. Feature selection based on the random forest algorithm showed that the feature combination consisting of 8 microorganisms had the best classification performance. The finally identified colorectal cancer screening biomarkers include *Alternaria incomplexa* (fungus), *Ogataeaphilodendri* (fungus), *Clostridium perfringens* (bacteria), *Romboutsia hominis* (bacteria), *Ralstonia solanacearum* (bacteria), *Alternaria conjuncta* (fungus), *Bacillus paralicheniformis* (bacteria), and *Pseudomonas sichuanensis* (bacteria). Five microorganisms, namely Alternaria incomplexa, Ogataea philodendri, Clostridium perfringens, Romboutsia hominis, and Ralstonia solanacearum, were enriched in colorectal cancer, while the other three were enriched in the healthy group.

[0050] We evaluated the classification contribution of each microbial biomarker using the random forest algorithm and quantified their feature importance: 5 colorectal cancer-enriched bacteria received positive weights (+), and 3 healthy-enriched bacteria received negative weights (-). The absolute value of the weight was positively correlated with its discriminative importance in the model. Figure 3 The feature weight values ​​of the eight microbial biomarkers are shown in Table 1.

[0051] Table 18 Characteristic Weights of Microbial Biomarkers

[0052] Microbial name Feature Importance Alternariaincomplexa 1.63 Ogataeaphilodendri 1.24 Clostridium perfringens 2.17 Romboutsiahominis 0.85 Ralstoniasolanacearum 1.08 Alternaria conjuncta -1.12 Bacillus paralicheniformis -1.59 Pseudomonas sichuanensis -0.83

[0053] Based on the eight colorectal cancer microbial biomarkers obtained through screening, this invention establishes a colorectal cancer diagnostic model. In the training set, the model constructed using the random forest algorithm exhibits excellent discriminative ability. The discriminative criterion is as follows: the original abundance data of the eight microorganisms are standardized so that their relative abundance sum is 100% (0% for non-detection), and a risk score is calculated as Risk Score = Σ(relative abundance of each microorganism × corresponding feature weight). A Risk Score ≥ 15 indicates a positive colorectal cancer diagnosis. The detection sensitivity and specificity in the training set both reached 100% and 100% respectively. Figure 4In the independent validation set, the model performed as follows: AUC 0.9635, sensitivity 91.95%, and specificity 78.57%. Figure 5 Experimental data confirmed that the eight circulating microbial markers for colorectal cancer selected in this invention can effectively distinguish between colorectal cancer patients and healthy individuals (P<0.001), demonstrating significant clinical screening application value.

[0054] In this invention, among 293 colorectal cancer samples, the proportion of early stage (stage I / II) colorectal cancer samples reached 74.40%, which proves that the plasma circulating microbial markers of colorectal cancer of this invention can be used as reliable tumor markers and can be used in non-invasive early screening of colorectal cancer.

[0055] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A colorectal cancer plasma circulating microbial marker characterized in that, The colorectal cancer plasma circulating microbial markers are Alternaria alternata Alternaria incomplexa , Ogataea siamensis Ogataea philodendri , Clostridium perfringens Clostridium perfringens , Lachnospira pectinosciurae Romboutsia hominis , Ralstonia pickettii Ralstonia solanacearum , Sympodiomycopsis scabiosae Alternaria conjuncta , Bacteroides caccae Bacillus paralicheniformis , and Pseudomonas szechuanensis Pseudomonas sichuanensis .

2. The plasma circulating microbial marker of colorectal cancer according to claim 1, characterized in that, The colorectal cancer plasma circulating microbial markers include a colorectal cancer group and a healthy group; the colorectal cancer group is Alternaria incomplexa, Ogataea philodendri, Clostridium perfringens, Romboutsia hominis and Ralstonia solanacearum; the healthy group is Alternaria conjuncta, Bacillus paralicheniformis and Pseudomonas sichuanensis.

3. An agent, characterized in that, The reagent includes a reagent for detecting the content or abundance of 8 microorganisms in the colorectal cancer plasma circulating microbial markers of claim 1 or 2.

4. The agent of claim 3, wherein The detection sample of the reagent includes peripheral blood of a subject.

5. A kit characterized in that, The kit includes the reagent of claim 3 or 4.

6. Use of the reagent of claim 3 or 4 or the kit of claim 5 in the preparation of a colorectal cancer screening diagnostic product.

7. Use according to claim 6, characterized in that, The colorectal cancer screening diagnostic standard includes: normalizing the original abundance data of 8 microorganisms so that the relative abundance sum is 100%, and recording 0% for undetected, calculating the risk score = Σ (relative abundance of each microorganism × corresponding characteristic weight), and determining colorectal cancer positive when the risk score is greater than or equal to 15.

8. Use according to claim 6, characterized in that, The method for colorectal cancer screening diagnosis includes: extracting plasma DNA in peripheral blood and sequencing; identifying the content of the colorectal cancer plasma circulating microbial markers of claim 1 or 2; and determining whether the peripheral blood is from a colorectal cancer patient according to the colorectal cancer screening diagnostic standard.

9. Use according to claim 8, characterized in that, The sequencing method includes low-depth whole genome sequencing.

10. Use according to claim 6, characterized in that, The colorectal cancer includes colorectal cancer stage I or colorectal cancer stage II.

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