Detection and typing method for genetic types of fusobacterium

Abstract

The invention provides a group of specific DNA (deoxyribonucleic acid) sequences of L1 and L5 genetic fusobacterium, and designs corresponding detection primers to be applied to detection and typing of L1 / L5 genetic fusobacterium, and the detection and typing method is a visual and accurate method for determining the L1 / L5 genetic typing fusobacterium. The method can directly and rapidly qualitatively and quantitatively detect L1 and L5 fusobacterium, is simple to operate, short in time consumption and suitable for a single strain sample or a sample containing mixed bacteria, can directly interpret according to PCR and quantitative PCR results, does not need additional sequencing and data analysis work, can achieve the accuracy of 100% in currently tested strains, and has a good application prospect. The L1 / L5 genetic type can play different pathogenic roles in colorectal cancer (CRC), accurate detection and identification have profound significance in knowing the pathogenic mechanism of the colorectal cancer (CRC), and the L1 / L5 genetic type has potential clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a group of specific DNA sequences for detecting and typing L1 and L5 genotype fusobacteria and a screening method thereof, as well as an application of the specific sequences in detecting and typing fusobacteria genotypes. Background Art

[0002] Imbalance of intestinal flora is closely related to the occurrence and development of many human diseases. Among them, Fusobacterium in the intestinal flora is enriched in intestinal diseases such as colorectal cancer (CRC), promoting the occurrence and development of the disease. Among the members of the genus Fusobacterium, Fusobacterium nucleatum has been studied most in-depth in CRC. However, Fusobacterium has extensive genetic diversity, and the clinical distribution and pathogenicity of other members have rarely been reported.

[0003] In the previous study, the inventors have found through evolutionary analysis that Fusobacterium species constitute 9 major genetic branches (lineages), and defined them as L1-L9 genotypes (Dexi Bi, et al. "Profiling Fusobacterium infection at high taxonomic resolution reveals lineage-specific correlations in colorectal cancer", Nature Communications, 2022, 13: 3336). Through large-sample clinical studies, it was also found that L1, L4, L5, and L9 genotypes of Fusobacterium are the most common in the intestine, among which L1 and L5 genotypes (referred to as L1 type and L5 type, respectively) Fusobacterium may play different pathogenic roles in colorectal cancer (CRC). L1 type Fusobacterium, including closely related species such as Fusobacterium nucleatum, F. periodonticum, and F. hwasookii, is widely present and significantly enriched in the feces and tumor tissues of CRC patients, suggesting a close association with the occurrence of CRC; L5 type Fusobacterium, including Fusobacterium proteum and Fusobacterium ulcerans, did not show enrichment characteristics, but its abundance was significantly correlated with vascular infiltration.

[0004] The FrpoB-seq technology developed by the inventors in the early stage can realize the accurate detection of Fusobacterium species and subspecies. Based on the results, data analysis can indirectly realize more accurate qualitative and quantitative detection of L1 and L5 types of Fusobacterium in samples. However, this method relies on high-throughput sequencing and needs to be combined with amplicon high-throughput sequencing and real-time quantitative PCR to realize the estimation of target abundance in samples. Therefore, its operation is relatively cumbersome and the detection cycle is long.

[0005] However, there is currently no intuitive and accurate method to determine the genetic typing of Fusobacterium, especially the L1 and L5 types that are significantly associated with the disease, that is, there is no technology to directly detect L1 and L5 genetic types of Fusobacterium. Summary of the invention

[0006] In order to overcome at least one problem existing in the prior art, the present invention screened a group of specific DNA sequences for detecting L1 and L5 genotypes of Fusobacterium, and designed corresponding detection primers based on them for application in the detection and typing of Fusobacterium genotypes.

[0007] The present invention obtains L1 and L5 genotype (referred to as L1 type and L5 type, respectively) Fusobacterium-specific DNA sequences by comparative genomics. The specific method is to download the sequenced Fusobacterium genome from the NCBI public database, including the completed map and the draft map; according to the previously published genetic classification (Dexi Bi, et al. "Profiling Fusobacterium infection at high taxonomic resolution reveals lineage-specific correlations in colorectal cancer", Nature Communications, 2022, 13: 3336), the genome is divided into L1 type, L5 type and other types. Among them, the L1 type includes genomes of Fusobacterium nucleatum, F. periodonticum, F. pseudoperiodonticum, F. hwasookii, F. canifelinum, Fusobacterium sp. W1481, Fusobacterium sp. oral taxon370str. F0437, etc.; the L5 type includes genomes of F. varium, F. ulcerans, Fusobacterium sp. 81A6, Fusobacterium sp. Fv113-g1, etc.; other types include genomes of F. mortiferum, F. gonidiaformans, F. massiliense, F. necrogenes, F. necrophorum, F. perfoetens, F. russii, Fusobacterium sp. clos_1_1, F. naviforme, etc. According to the reported previous research results, these strains have covered the vast majority of known and common strains of the above genotypes and are sufficiently representative.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] The first aspect of the present invention is to provide a method for screening specific sequences for detecting and typing the genotype of Fusobacterium, which comprises the steps of: performing a pan-genome analysis on a specific genotype of Fusobacterium; screening out genes that are highly conserved at the nucleotide sequence level in the specific genotype of Fusobacterium from the obtained gene clusters by sequence comparison; then screening out genes that have no homology with other genotypes of Fusobacterium at the nucleotide and protein sequence levels by comparison; performing a nucleotide sequence comparison on the obtained gene sequence with non-Fusobacterium species in the NCBI nucleotide database, eliminating genes that have a certain sequence similarity with the non-Fusobacterium species, and excluding genes containing insertions, truncations, and inconsistent copy numbers between strains.

[0010] Furthermore, the highly conserved gene at the nucleotide sequence level exists in all L1 type genomes, and the nucleotide sequence identity of the gene among strains is not less than 87%.

[0011] Furthermore, the gene having a certain sequence similarity with a non-Fusobacterium species is a gene having a higher sequence similarity, specifically, the sequence similarity is >60%.

[0012] Furthermore, the specific genetic type of Fusobacterium is L1 genetic type (abbreviated as L1 type) Fusobacterium or L5 genetic type (abbreviated as L5 type) Fusobacterium.

[0013] It is understandable that the above screening method is also applicable to the screening of specific sequences of other genotypes of Fusobacterium, including L2-L4 and L6-L9 genotypes.

[0014] Furthermore, through the above screening method, 4 L1-type specific DNA fragments (L1-type specific sequences) were finally screened and obtained, which were named L1-125, L1-479, L1-746, and L1-820 respectively; using the same method, 6 L5-type specific DNA fragments (L5-type specific sequences) were screened and obtained, which were named L5-211, L5-962, L5-1251, L5-1277, L5-1430, and L5-1621 respectively.

[0015] Further, the specific sequence includes a specific sequence for detecting and typing L1 type Fusobacterium or a specific sequence for detecting and typing L5 type Fusobacterium; wherein the specific sequence for detecting and typing L1 type Fusobacterium includes at least one of the following sequences: a sequence (L1-125) as shown in SEQ ID No.1, a sequence (L1-479) as shown in SEQ ID No.2, a sequence (L1-746) as shown in SEQ ID No.3, a sequence (L1-820) as shown in SEQ ID No.4, or a homologous sequence having 85-99% similarity to the sequences shown in SEQ ID NO.1 to SEQ ID No.4; and the specific sequence for detecting and typing L5 type Fusobacterium includes at least one of the following sequences: a sequence (L5-211) as shown in SEQ ID No.5, a sequence (L5-962) as shown in SEQ ID No.6, a sequence (L5-1251) as shown in SEQ ID No.7, a sequence (L5-1277) as shown in SEQ ID No.8, a homologous sequence having 85-99% similarity to the sequences shown in SEQ ID No.1 to SEQ ID No.4. The sequence shown in SEQ ID No.9 (L5-1430), the sequence shown in SEQ ID No.10 (L5-1621), or a homologous sequence having 85-99% similarity to the sequence shown in SEQ ID NO.5 to SEQ ID No.10.

[0016] It is understandable that DNA sequences are inevitably polymorphic and cannot be enumerated exhaustively, but considering that its homologous fragments have not been found in other species and are highly specific to L1 / L5 types, the characteristic DNA sequences referred to here include the following sequences and unlisted fragments that have nucleotide sequence similarity with any of the following sequences.

[0017] The second aspect of the present invention is to provide a group of applications of specific sequences, which are selected from at least one of the following applications: application in detection and typing of Fusobacterium genotype, application in preparation of detection and typing products of Fusobacterium genotype, application in bioinformatics analysis, and application in determining the pathogenic mechanism and molecular epidemiological characteristics of colorectal cancer; wherein the specific sequence includes a specific sequence for detection and typing of L1 type Fusobacterium or a specific sequence for detection and typing of L5 type Fusobacterium; the specific sequence for detection and typing of L1 type Fusobacterium includes at least one of the following sequences: a sequence (L1-125) as shown in SEQ ID No.1, a sequence (L1-479) as shown in SEQ ID No.2, a sequence (L1-746) as shown in SEQ ID No.3, a sequence (L1-820) as shown in SEQ ID No.4, or a homologous sequence having 85-99% similarity to the sequences shown in SEQ ID NO.1 to SEQ ID No.4; and the specific sequence for detection and typing of L5 type Fusobacterium includes at least one of the following sequences: a sequence (L1-125) as shown in SEQ ID No.1, a sequence (L1-479) as shown in SEQ ID No.2, a sequence (L1-746) as shown in SEQ ID No.3, a sequence (L1-820) as shown in SEQ ID No.4, or a homologous sequence having 85-99% similarity to the sequences shown in SEQ ID NO.1 to SEQ ID No.4; and The sequence shown in SEQ ID No.5 (L5-211), the sequence shown in SEQ ID No.6 (L5-962), the sequence shown in SEQ ID No.7 (L5-1251), the sequence shown in SEQ ID No.8 (L5-1277), the sequence shown in SEQ ID No.9 (L5-1430), the sequence shown in SEQ ID No.10 (L5-1621), or a homologous sequence having 85-99% similarity to the sequences shown in SEQ ID NO.5 to SEQ ID No.10.

[0018] Furthermore, in the application, the bioinformatics analysis includes identifying the typing of the sequenced Fusobacterium strain by sequence alignment; or detecting and analyzing the presence of genetic Fusobacterium in the analysis of metagenomic sequencing data.

[0019] Furthermore, in the application, the detection typing product includes a detection kit, a detection microfluidic chip, etc.

[0020] The third aspect of the present invention is to provide a primer composition for amplifying any specific sequence described in the second aspect of the present invention.

[0021] Furthermore, based on the screened specific sequences, multiple sequence alignment is performed on all known sequences corresponding to each specific fragment, and universal amplification primers are designed in relatively conservative regions.

[0022] Further, the primer composition includes at least one of the following primer pairs: a primer pair shown in SEQ ID No.11 and SEQ ID No.12 (for amplifying L1-125), a primer pair shown in SEQ ID No.13 and SEQ ID No.14 (for amplifying L1-125), a primer pair shown in SEQ ID No.15 and SEQ ID No.16 (for amplifying L1-125), a primer pair shown in SEQ ID No.17 and SEQ ID No.18 (for amplifying L1-746), a primer pair shown in SEQ ID No.19 and SEQ ID No.20 (for amplifying L1-820), a primer pair shown in SEQ ID No.21 and SEQ ID No.22 (for amplifying L5-211), a primer pair shown in SEQ ID No.23 and SEQ ID No.24 (for amplifying L5-962), a primer pair shown in SEQ ID No.25 and SEQ ID No.26 (for amplifying L5-1251), a primer pair shown in SEQ ID No.27 and SEQ ID No.28 (for amplifying L5-1252), a primer pair shown in SEQ ID No.28 and SEQ ID No.29 (for amplifying L6-1264), a primer pair shown in SEQ ID No.29 and SEQ ID No.30 (for amplifying L6-1275), a primer pair shown in SEQ ID No.29 and SEQ ID No.31 (for amplifying L6-1276), a primer pair shown in SEQ ID No.29 and SEQ ID No.32 (for amplifying L6-1277). The primer pair shown in No.28 (used to amplify L5-1277), the primer pair shown in SEQ ID No.29 and SEQ ID No.30 (used to amplify L5-1430), and the primer pair shown in SEQ ID No.31 and SEQ ID No.32 (used to amplify L5-1621).

[0023] Further, the primer combination is a primer pair shown in SEQ ID No. 17 and SEQ ID No. 18 (for amplifying L1-746), or a primer pair shown in SEQ ID No. 31 and SEQ ID No. 32 (for amplifying L5-1621). It has been verified that L1-746-F / R and L5-1621-F / R are the optimal primers for detecting L1 and L5 types of Fusobacterium, respectively, and the specificity and sensitivity in the test strain can reach 100%.

[0024] During the primer design process, if different base possibilities appeared at the same site, the corresponding degenerate base was used as a replacement (R; A / G; Y: C / T; M: A / C; K: G / T; S: C / G; W: A / T; H: A / C / T; B: C / G / T; V: A / C / G; D: A / G / T; N: A / C / G / T).

[0025] It is understandable that the above primer pairs can be used for L1 / L5 type Fusobacterium detection by amplifying the corresponding DNA fragment. No conserved region suitable for primer design has been found in L1-479, so primers are not designed for this fragment (the design can be optimized later). The primer pairs listed above are only an application example. Primers or probes designed based on the sequence corresponding to the L1 / L5 type specific fragment described in the present invention and used for the purpose described in the present invention all belong to the protection scope of the present invention.

[0026] The above-mentioned specific sequences and their corresponding primer sequences are shown in the following table:

[0027] Table 1 - L1 / L5 type Fusobacterium specific sequences and primer sequences

[0028]

[0029]

[0030]

[0031]

[0032] The fourth aspect of the present invention is to provide a kit containing any primer combination described in the third aspect of the present invention.

[0033] Furthermore, the kit also includes a PCR amplification reagent.

[0034] Furthermore, the PCR amplification includes conventional PCR amplification and real-time quantitative PCR amplification.

[0035] The fifth aspect of the present invention is a method for detecting and typing the genotype of Fusobacterium using any of the primer combinations described in the third aspect of the present invention, comprising the steps of: using the primer combination to perform PCR amplification on the sample to be detected, analyzing and identifying the obtained amplification product to perform genetic typing of Fusobacterium. The above PCR amplifications all use commercial kits, such as the Premix Taq (Ex Taq Version 2.0 plus dye) kit for ordinary PCR and the TB Green Premix Ex Taq TMII (Tli RNaseH Plus) kit, according to the reaction system given in the manual, the total amount of DNA template is 10-100ng per reaction. The ordinary PCR reaction conditions are: 98℃10 seconds, 54℃20 seconds, 72℃30 seconds, 35 cycles; the amplified product is detected by agarose gel electrophoresis. The real-time quantitative PCR reaction conditions are: 50℃2 minutes, 95℃30 seconds, and then 40 cycles of 98℃5 seconds, 50℃34 seconds, 70℃30 seconds; quantitative analysis is based on Ct value.

[0036] Furthermore, the sample to be tested is a single strain sample or a sample containing mixed bacteria.

[0037] Furthermore, the sample includes feces, ex vivo tissue, whole blood, colorectal cancer (CRC) tissue, etc.

[0038] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0039] The detection typing method proposed in the present invention can realize direct and rapid qualitative and quantitative detection of L1 and L5 genotypes of Fusobacterium, with simple operation and short time consumption. The above detection typing method is applicable to single strain samples or samples containing mixed bacteria, and can be directly interpreted according to PCR and quantitative PCR results without the need for additional sequencing and data analysis. The accuracy can reach 100% in the strains currently tested. The above L1 / L5 genotypes may play different pathogenic roles in colorectal cancer (CRC), and accurate detection and identification are of far-reaching significance for understanding their pathogenic mechanisms and have potential clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0041] Figure 1 This is a schematic diagram of the results of using the L1 / L5 genotype Fusobacterium-specific fragment sequence for genome analysis in one embodiment of the present invention.

[0042] Figure 2 This is a schematic diagram of the results of using specific primers for the L1 / L5 genotype of Fusobacterium in a single strain sample in one embodiment of the present invention;

[0043] Figure 3 Schematic diagram of the results of using primers L1-746-F / R and L5-1621-F / R in a sample containing mixed bacteria in one embodiment of the present invention. DETAILED DESCRIPTION

[0044] The following will be combined with the accompanying drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0045] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0046] Example 1 - Screening of L1 / L5 type specific fragment sequences and their application in genome analysis

[0047] In this embodiment, the sequenced Fusobacterium genome, including the completed map and the draft map, is downloaded from the NCBI public database; the genome is divided into L1 type, L5 type and other types according to the genetic classification published previously, and the PGAP software is used to perform pan-genome analysis and comparison on L1 type and L5 type respectively, wherein the intermediate process is to take the intersection or union, and is implemented using a perl script or in Excel. For L1 type, from the obtained gene clusters, the genes that are highly conserved at the nucleotide sequence level in L1 type are screened out by sequence comparison, that is, they exist in all L1 type genomes, and the nucleotide sequence consistency of the gene between strains is not less than 87% (if there are too many genes, the threshold is increased to reduce the number of candidate genes); and then the genes that have no homology with L5 and other types at the nucleotide and protein sequence levels are screened out by comparison, that is, the genes that are conservative and specific to L1. The obtained gene sequence was compared with the non-Fusobacterium species in the NCBI nucleotide database, and genes with high sequence similarity (>60%) with other species were further eliminated, and genes with insertions, truncations, and inconsistent copy numbers between strains were excluded. Finally, 4 L1-type specific DNA fragments were screened and named L1-125, L1-479, L1-746, and L1-820. Using the same method, 6 L5-type specific DNA fragments were screened and named L5-211, L5-962, L5-1251, L5-1277, L5-1430, and L5-1621. The specific sequence information of the above-mentioned specific DNA fragments is shown in Table 1 above.

[0048] The nucleotide sequence of the genome and the specific fragment is compared, and the results are as follows Figure 1 As shown, it shows that each specific fragment only appears in the L1 or L5 type Fusobacterium genome (homologous sequences exist) and does not appear in other genotypes, indicating that the corresponding DNA fragment can be used to characterize the Fusobacterium L1 / L5 genotype.

[0049] Based on the L1 / L5 type specific fragments screened above, multiple sequence alignment was performed on all known sequences corresponding to each specific fragment, and universal amplification primers were designed in relatively conservative regions. The primer sequences are shown in Table 1 above.

[0050] Example 2 - Application of L1 / L5 type specific primers in single strain samples

[0051] This example verifies the application of all primer sequences in Table 1 to detect L1 / L5 type Fusobacterium in a single strain sample, which includes the steps of:

[0052] (1) Obtaining clearly typed Fusobacterium strains;

[0053] Fusobacterium strains were isolated from colorectal cancer tumor tissues, and their isolation, identification and culture have been reported in previous studies (Dexi Bi, et al. "Profiling Fusobacterium infection at high taxonomic resolution reveals lineage-specific correlations in colorectal cancer", Nature Communications, 2022, 13: 3336). Each strain was assigned to a different genotype according to the results of strain identification. In this example, Fusobacterium culture was performed using a commercial Columbia blood plate or thioglycollate liquid medium at 37°C.

[0054] (2) Extraction Figure 2 Total DNA of the Fusobacterium strains that have been clearly typed;

[0055] The Fusobacterium strains were inoculated into commercial Columbia blood agar plates or thioglycollate liquid medium and cultured anaerobically at 37°C for 2 days. The bacteria were collected and the total DNA of each strain was extracted using a commercial bacterial genomic DNA extraction kit according to the steps listed in the instructions.

[0056] (3) PCR amplification using the primer pairs in Table 1 to identify whether it is L1 or L5 genotype;

[0057] Premix Taq (Ex Taq Version 2.0 plus dye) or similar commercial PCR kits were used, according to the reaction system specified in the instructions, with a DNA template amount of 10 ng per reaction, and PCR amplification was performed using the primer pairs in Table 1. The reaction conditions were: 98°C for 10 seconds, 54°C for 20 seconds, and 72°C for 30 seconds, for 35 cycles. The amplified product was detected by agarose gel electrophoresis to detect whether a band appeared at the expected size position. The experimental results are shown in Figure 1. Figure 2 As shown in the figure, the designed primers all had positive amplification in the strains of the corresponding typing, and there was a single band at the expected position, indicating that the genotype of a single strain of Fusobacterium can be effectively identified. Among the tested strains, the sensitivity of all primers can reach 100%, but some primers have non-specific amplification in other typing; among them, L1-746-F / R and L5-1621-F / R are the optimal primers for L1 and L5 type detection, respectively, and the specificity and sensitivity can reach 100% in the included test strains.

[0058] Example 3-Application of L1 / L5-specific primers in samples containing mixed bacteria

[0059] This example verifies the application of the optimal primers for L1 and L5 type detection determined in Example 2, namely L1-746-F / R and L5-1621-F / R, in detecting L1 / L5 type Fusobacterium in mixed bacterial samples, which includes the steps of:

[0060] (1) Acquisition of samples and preparation of standards;

[0061] Whole blood samples were collected from healthy volunteers, stool samples were collected from healthy volunteers (HC) and colorectal cancer (CRC) patients, and fresh frozen CRC tumor tissue samples were obtained from surgically resected specimens; DNA in the samples was extracted using the corresponding commercial kits. The collection of human samples was approved by the Ethics Committee of Shanghai Tenth People's Hospital (approval number: No. SHSY-IEC-4.1 / 20-85 / 01). At the same time, different CFU (colony-forming unit) standards of a single strain were prepared (F. nucleatum ATCC 25586, i.e., Fn, was used for L1 type; F. varium THCT13E1, i.e., Fv, was used for L5 type). The strains were inoculated into commercial thioglycollate liquid medium, cultured anaerobically at 37°C until the logarithmic phase, and diluted to 1×10 8 CFU / ml, the bacterial concentration was confirmed by dilution and coating the plate, the total DNA was extracted using a kit, and the obtained DNA product was appropriately diluted to obtain the DNA standard corresponding to the specific bacterial amount (10CFU / μL-1×10 6 CFU / μL).

[0062] (2) PCR detection and result analysis;

[0063] 1) Testing of standard products:

[0064] Using TB Green Premix Ex Taq TM II (Tli RNaseH Plus) kit, according to the reaction system specified in the instructions, 1 μl of standard was added to each reaction as a template, that is, 10 CFU-1×10 6 The reaction conditions are: 50℃ for 2 minutes, 95℃ for 30 seconds, and then 40 cycles of 98℃ for 5 seconds, 50℃ for 34 seconds, and 70℃ for 30 seconds. The Ct value (the higher the bacterial count, the lower the Ct value) is taken and analyzed with the log10 value of the CFU corresponding to the DNA template in the system. The results are as follows Figure 3 As shown in part A of the results, the amplification results (ie, Ct values) of the two pairs of primers have a good linear relationship with the bacterial load, indicating that they have good quantitative ability.

[0065] 2) Sample testing:

[0066] The primer sequences (5'-3') used in the following verification experiments are shown in the following table:

[0067]

[0068] 2-1) First, in order to verify the specificity of the detection primers (L1-746-F / R and L5-1621-F / R) in samples with complex backgrounds (such as host background and other flora backgrounds), whole blood DNA samples were taken to characterize the human host DNA background, and feces and colorectal cancer (CRC) tissue samples that were verified to be free of Fusobacterium were taken to characterize the human flora DNA background and flora + host background, respectively, for PCR amplification; at the same time, the above-mentioned Fn / Fv strain DNA was used as a positive control, and the reaction system without adding DNA template was used as a negative control. Premix Taq (Ex Taq Version 2.0 plus dye) or similar commercial PCR kits were used, and the reaction system given in the instructions was used. The amount of human specimen DNA template was 100ng per reaction, and the amount of strain DNA template was 10ng per reaction. The reaction conditions were; 98℃ 10 seconds, 54℃ 20 seconds, 72℃ 30 seconds, and 35 cycles. The amplified product was detected by agarose gel electrophoresis to observe whether a band appeared at the expected size position. Among them, the presence of Fusobacterium in the samples was verified using Fusobacterium primers fuso-rsub-F2 and fuso-rsub-R2 (Dexi Bi, et al. "Profiling Fusobacterium infection at high taxonomic resolution reveals lineage-specific correlations in colorectal cancer", Nature Communications, 2022, 13: 3336). The absence of amplified bands indicated that Fusobacterium was not present. At the same time, the commonly used Fusobacterium nucleatum detection primers Fn-F and Fn-R (Castellarin, M. et al. "Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma", Genome Research, 2012, 22: 299–306) were used to support the amplification results. The primers showed positive amplification for Fn, and negative amplification for Fv and samples without Fusobacterium, and the results were consistent with expectations.

[0069] The above experimental results are as follows Figure 3As shown in part B of the figure, two pairs of primers (L1-746-F / R and L5-1621-F / R) were amplified in the corresponding positive controls, but not in human DNA (whole blood samples), as well as stool and colorectal cancer (CRC) tissue samples that were verified to be free of Fusobacterium, indicating that they are not affected by the host and other bacterial backgrounds.

[0070] 2-2) Furthermore, in this example, the detection primers L1-746-F / R and L5-1621-F / R were applied to real-time quantitative PCR to detect the abundance of L1 / L5 type Fusobacterium in stool samples, and the universal primers (a combination of 5 primers) in the V6 region of the 16S rRNA gene were used as internal references (Geller, LT et al. "Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine", Science, 2017, 357: 1156-1160), and TB Green Premix Ex Taq TM II (Tli RNaseH Plus) kit, according to the reaction system specified in the instructions, the amount of DNA template is 100ng per reaction. The reaction conditions are: 50℃ for 2 minutes, 95℃ for 30 seconds, and then 40 cycles of 98℃ for 5 seconds, 50℃ for 34 seconds, and 70℃ for 30 seconds, and the -ΔCt value is calculated.

[0071] The above experimental results are as follows Figure 3 As shown in part C, it was found that the abundance of L1 / L5 fusobacteria in different samples was heterogeneous; at the same time, the test results were compared with the FrpoB-seq results corresponding to the samples, and it was found that there was a high degree of consistency, and the Spearman correlation could reach about 0.9, indicating that L1-746-F / R and L5-1621-F / R have accurate quantitative detection capabilities in samples containing mixed bacteria.

[0072] It can be seen from the above embodiments that the present invention provides an intuitive and accurate method for determining the L1 / L5 genetic typing of Fusobacterium, which screens a group of L1 and L5 type Fusobacterium-specific DNA sequences, designs corresponding detection primers, and verifies the application of the detection primers in the detection of L1 and L5 type Fusobacterium, which helps to reveal the genetic typing composition of Fusobacterium in lesions, and its relationship with the disease and molecular epidemiological characteristics.

[0073] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A method for screening specific sequences for detecting and typing the genotype of Fusobacterium, characterized in that: The method comprises the following steps: performing a pan-genome analysis on a specific genetic type of Fusobacterium: screening out genes that are highly conserved at the nucleotide sequence level in the specific genetic type of Fusobacterium from the obtained gene clusters by sequence comparison; screening out genes that have no homology with other genetic types of Fusobacteria at both the nucleotide and protein sequence levels by comparison; performing a nucleotide sequence comparison on the obtained gene sequence with non-Fusobacterium species in the NCBI nucleotide database, eliminating genes that have certain sequence similarities with non-Fusobacterium species, and excluding genes that contain insertions, truncations, and inconsistent copy numbers among strains.

2. The screening method according to claim 1, characterized in that The specific genotype of Fusobacterium is L1 genotype of Fusobacterium or L5 genotype of Fusobacterium.

3. The screening method according to claim 2, characterized in that The specific sequence includes a specific sequence for detecting and typing the L1 genotype Fusobacterium or a specific sequence for detecting and typing the L5 genotype Fusobacterium; Wherein, the specific sequence for detecting and typing the L1 genotype Fusobacterium includes at least one of the following sequences: a sequence as shown in SEQ ID No.1, a sequence as shown in SEQ ID No.2, a sequence as shown in SEQ ID No.3, a sequence as shown in SEQ ID No.4, or a homologous sequence having 85-99% similarity to the sequences shown in SEQ ID NO.1 to SEQ ID No.4; Furthermore, the specific sequence for detecting and typing the L5 genotype Fusobacterium includes at least one of the following sequences: a sequence as shown in SEQ ID No.5, a sequence as shown in SEQ ID No.6, a sequence as shown in SEQ ID No.7, a sequence as shown in SEQ ID No.8, a sequence as shown in SEQ ID No.9, a sequence as shown in SEQ ID No.10, or a homologous sequence having 85-99% similarity to the sequences shown in SEQ ID NO.5 to SEQ ID No.

10.

4. The use of a set of specific sequences, characterized in that: The application is selected from at least one of the following applications: application in detection and typing of Fusobacterium genotype, application in preparation of detection and typing products of Fusobacterium genotype, application in bioinformatics analysis, and application in determining the pathogenic mechanism and molecular epidemiological characteristics of colorectal cancer; wherein the specific sequence includes a specific sequence for detection and typing of L1 genotype Fusobacterium or a specific sequence for detection and typing of L5 genotype Fusobacterium; The specific sequence for detecting and typing the L1 genotype Fusobacterium includes at least one of the following sequences: a sequence as shown in SEQ ID No. 1, a sequence as shown in SEQ ID No. 2, a sequence as shown in SEQ ID No. 3, a sequence as shown in SEQ ID No. 4, or a homologous sequence having 85-99% similarity to the sequences shown in SEQ ID NO. 1 to SEQ ID No. 4; Furthermore, the specific sequence for detecting and typing the L5 genotype Fusobacterium includes at least one of the following sequences: a sequence as shown in SEQ ID No.5, a sequence as shown in SEQ ID No.6, a sequence as shown in SEQ ID No.7, a sequence as shown in SEQ ID No.8, a sequence as shown in SEQ ID No.9, a sequence as shown in SEQ ID No.10, or a homologous sequence having 85-99% similarity to the sequences shown in SEQ ID NO.5 to SEQ ID No.

10.

5. The use according to claim 4, characterized in that: The bioinformatics analysis includes: identifying the typing of sequenced Fusobacterium by sequence comparison; or detecting and analyzing the presence of genetic Fusobacterium in the analysis of metagenomic sequencing data.

6. A primer composition for amplifying the specific sequence according to claim 4, characterized in that: The primer composition comprises at least one of the following primer pairs: The primer pair shown in SEQ ID No.11 and SEQ ID No.12, the primer pair shown in SEQ ID No.13 and SEQ ID No.14, the primer pair shown in SEQ ID No.15 and SEQ ID No.16, the primer pair shown in SEQ ID No.17 and SEQ ID No.18, the primer pair shown in SEQ ID No.19 and SEQ ID No.20, the primer pair shown in SEQ ID No.21 and SEQ ID No.22, the primer pair shown in SEQ ID No.23 and SEQ ID No.24, the primer pair shown in SEQ ID No.25 and SEQ ID No.26, the primer pair shown in SEQ ID No.27 and SEQ ID No.28, the primer pair shown in SEQ ID No.29 and SEQ ID No.30, and the primer pair shown in SEQ ID No.31 and SEQ ID No.

32.

7. The primer composition according to claim 6, characterized in that The primer combination is a primer pair shown by SEQ ID No.17 and SEQ ID No.18, or a primer pair shown by SEQ ID No.31 and SEQ ID No.

32.

8. A kit comprising the primer combination according to claim 6 or 7, characterized in that: The kit also includes PCR amplification reagents.

9. A method for detecting and typing the genotype of Fusobacterium using the primer combination of claim 6 or 7, characterized in that: The method comprises the steps of: using the primer combination to carry out PCR amplification on the sample to be detected, analyzing and identifying the obtained amplification product to perform typing of Fusobacterium.

10. The detection typing method according to claim 9, characterized in that: The sample to be tested is a single strain sample or a sample containing mixed bacteria.

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