CN118006815A - Specific DNA sequence of Fusobacterium nucleatum and its application and detection method - Google Patents

Abstract

The invention provides a section of specific DNA sequence of Fusobacterium nucleatum, the specific sequence of which is shown as SEQ ID No.1 or is a homologous sequence with 85-99% similarity with the sequence shown as SEQ ID No. 1; on the basis of the specific DNA sequences, primer compositions for detection are designed, and the primer compositions comprise the sequences shown in SEQ ID No. 2-SEQ ID No. 5. The specific DNA sequence of the fusobacterium nucleatum and the primer designed by the specific DNA sequence can realize the direct and rapid qualitative and quantitative detection of the fusobacterium nucleatum, and the detection method is applicable to single strain samples or mixed thallus-containing samples, and the detection accuracy is far higher than that of the prior art.

Description

Fusobacterium nucleatum specific DNA sequence and application and detection method thereof

Technical Field

The invention relates to the technical field of biological medicine, in particular to a segment of fusobacterium nucleatum specific DNA sequence, application and a detection method thereof.

Background

Intestinal flora plays an important role in human diseases. The imbalance of fusobacterium (Fusobacterium) in the intestinal flora is closely related to the occurrence and development of a variety of diseases. Clinical studies have shown that enrichment of Fusobacterium nucleatum (Fusobacterium nucleatum) in the gut is highly correlated with the development of colorectal cancer (colorectal cancer, CRC). Recent studies have further shown that fusobacterium nucleatum is an important CRC pathogen that has important regulatory effects on key pro-cancerous molecules in CRC tumor cells, promoting tumor invasion and migration; also can activate autophagy of tumor cells, resulting in CRC chemotherapy resistance; it also has important regulating effect on immune microenvironment, and can promote tumor metastasis by inducing macrophage to express chemotactic factor. In tumor tissues and feces of patients with CRC, the significantly elevated abundance of F.nucleatum is correlated with poor prognosis and recurrence. In addition, fusobacterium nucleatum can secrete outer membrane vesicles in inflammatory bowel disease to promote intestinal inflammation.

The primer relied by the quantitative PCR detection of the clostridium nucleatum in the intestinal tract is not specific, and the primer has non-specific amplification to related relatively-close strains such as F.periodontics, F.pseudobacteriovoricosum, F.hwasokii and the like, and influences the accuracy of results. Thus, there is still a lack of a more accurate method for PCR detection of F.nucleatum, and accurate determination of the abundance of F.nucleatum in the intestinal tract is crucial for understanding its distribution and the exact association with the disease.

The most widely used fusobacterium nucleatum detection primers at present are Fn-F/R (Genome Res2012, 22:299-306) targeting nusG genes, and in addition, primers targeting 16S rRNA genes and fadA genes (Ann Clin Biochem 2022,59:396-403;J Med Microbiol 2016,65:760-769) have been reported. However, these primers lack specificity and their targets are ubiquitous in F.periodontium, F.pseudobacteriophadiobium, F.hwasokiii, etc., e.g., the inventors have previously demonstrated that Fn-F/R primers can amplify bands in F.putida (Microb Biotechnol2021, 14:2176-2186); in addition, in the detection technology based on high-throughput sequencing, 16S rRNA gene amplicon sequencing cannot reach strain resolution, and thus detection of the level of Fusobacterium nucleatum strain cannot be achieved. Metagenome and FrpoB-seq technologies can be used for detecting the clostridium nucleatum more accurately at present, wherein FrpoB-seq technology is the most accurate clostridium high-resolution detection technology developed by the inventor in advance, but the two methods are complex in operation, long in detection period and complex in analysis, and cannot be applied to rapid detection.

Therefore, there is still a lack of a precise PCR detection method for Fusobacterium nucleatum, and there is an urgent need to develop a set of primers capable of precisely detecting Fusobacterium nucleatum.

Disclosure of Invention

In order to overcome at least one problem in the prior art, the invention screens a section of DNA sequence with high specificity to the clostridium nucleatum, designs corresponding detection primers, and describes the accuracy of the application of the detection primers to the clostridium nucleatum detection. The invention is helpful for precisely revealing the abundance of Fusobacterium nucleatum in the focus and the exact relation between the Fusobacterium nucleatum and the disease.

It is noted that F.nucleatum has a high degree of genetic diversity, 4 subspecies of which have been redefined as independent species and named F.animalis (primordium subspecies), F.polymorpha (primordium subspecies), F.venturi (primordium subspecies) and F.nucleatum (primordium subspecies). However, in keeping with previous studies, the term "Fusobacterium nucleatum" as used hereinafter in the present invention is intended to refer broadly to a collection of these 4 newly defined species (original 4 subspecies) rather than to the newly defined Fusobacterium nucleatum species (original "subspecies").

In order to achieve the above purpose, the invention adopts the following technical scheme:

In a first aspect, the present invention provides a DNA sequence specific for Fusobacterium nucleatum, which is shown as SEQ ID No.1 or a homologous sequence having 85-99% similarity with the sequence shown as SEQ ID No. 1.

It will be appreciated that the presence of unavoidable polymorphisms in the DNA sequence is not exhaustive, but that it is considered that homologous fragments thereof are not found in other species and exhibit a high degree of specificity for F.nucleatum, and that the DNA characteristic sequences referred to herein include the sequence shown in SEQ ID NO.1 and the unlisted fragments having nucleotide sequence similarity to the sequence shown in SEQ ID NO. 1.

Further, the screening method of the specific DNA sequence comprises the steps of: performing ubiquity genome analysis on the clostridium nucleatum, and screening genes highly conserved at the nucleotide sequence level in the clostridium nucleatum through sequence alignment; then screening genes which have no homology with other fusobacterium and other species at the nucleotide level (namely genes which are conserved and specific for the fusobacterium nucleatum) from the genes through alignment; the obtained gene sequence is subjected to nucleotide sequence alignment with non-fusobacterium species, genes with a certain sequence similarity with other species are further removed, and genes containing insertion, truncation and inconsistency of copy numbers among strains are removed, so that the specific DNA sequence (named Fn-SR) is obtained through screening.

Further, the genes highly conserved at the nucleotide sequence level are present in more than 98% of the genome of F.nucleatum, and the nucleotide sequence identity of the genes among strains is not less than 85%.

Further, the gene having a certain sequence similarity with other species is a gene having a higher sequence similarity, specifically, the sequence similarity is >60%.

In a second aspect of the present invention, there is provided a primer composition for amplifying the fusobacterium nucleatum specific DNA sequence of any one of the first aspect. Specifically, on the basis of the specific DNA sequences screened, all the known sequences corresponding to the specific DNA sequences are subjected to multi-sequence comparison, and a group of universal amplification primers are designed in a relatively conserved region.

Further, the primer composition includes: a forward primer with a sequence shown as SEQ ID No.2 and a reverse primer with a sequence shown as SEQ ID No. 3-SEQ ID No. 5.

It is understood that the above-listed primer composition is only one application example, and that primers or probes designed for the purposes described herein based on the sequences corresponding to the specific fragment of F.nucleatum described herein are all within the scope of the present invention.

Further, because of the unavoidable sequence polymorphism in the target region, the designed primers include 1 forward primer containing the degenerate base and 3 reverse primers containing the degenerate base, and the four primers need to be mixed simultaneously as one set of primers. Degenerate bases are expressed as: 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.

Further, in specific verification examples, the specificity and sensitivity of the primer composition in the test strain can reach 100%.

In a third aspect, the present invention provides a detection reagent for fusobacterium nucleatum, comprising the primer composition of any one of the second aspects and a PCR amplification reagent. Specifically, the PCR amplification reagents include a general PCR amplification reagent or a real-time quantitative PCR amplification reagent.

The specific sequences and the primer sequences corresponding to the specific sequences are shown in the following table:

TABLE 1 Fusobacterium nucleatum specific DNA sequence and detection primer thereof

In a fourth aspect, the present invention provides the use of a fusobacterium nucleatum specific DNA sequence according to any one of the first aspect, a primer composition according to any one of the second aspect, or a detection reagent according to any one of the third aspect, selected from at least one of the following applications: the application of the kit in detecting the clostridium nucleatum based on non-diagnostic purposes, the application in preparing a kit for detecting the clostridium nucleatum, the application in preparing a kit for early detecting colorectal cancer or inflammatory bowel disease, the application in bioinformatic analysis and the application in evaluating poor prognosis and recurrence of the colorectal cancer.

Further, the bioinformatics analysis includes: identifying whether the sequenced fusobacterium strain is fusobacterium nucleatum by sequence alignment; or detecting and analyzing the existence of the clostridium nucleatum in metagenome sequencing data analysis.

Further, the kit is a common PCR kit or a real-time quantitative PCR kit containing the primer composition. It is understood that the above-described kit also covers the form of a detection microfluidic chip or the like. The kit also comprises PCR amplification reagents.

In a fifth aspect, the present invention provides a method for detecting a DNA sequence specific for F.nucleatum according to any one of the first aspect, comprising the steps of: PCR amplification of a sample to be detected using the primer composition according to any one of the second aspects, and analysis and identification of the resulting amplified product to thereby perform detection of the specific DNA sequence.

The PCR amplification can be carried out by adopting a commercial kit, such as a common PCR kit adopting a Premix Taq (Ex Taq Version 2.0 Plus dye) kit, a real-time quantitative PCR kit adopting a TB Green Premix Ex Taq ^TM II (TLI RNASEH Plus) kit, and the total amount of the DNA template is 10-100ng per reaction according to a reaction system given by a specification. The common PCR reaction conditions are as follows; 98℃for 10 seconds, 55℃for 20 seconds, 72℃for 30 seconds, with 35 cycles; the amplified product was detected by agarose gel electrophoresis. The real-time quantitative PCR reaction conditions are as follows; 50 ℃ for 2 minutes, 95 ℃ for 30 seconds, then 40 cycles of 98 ℃ for 5 seconds, 55 ℃ for 34 seconds, 70 ℃ for 30 seconds; quantitative analysis was performed based on Ct values.

Further, the sample to be detected is a single strain sample or a mixed thallus sample.

Further, the side sample to be tested includes a whole blood sample, a fecal sample, a tissue sample (including an ex vivo tissue sample, a colorectal tissue sample, etc.).

Compared with the prior art, the invention has the following beneficial effects by adopting the technical scheme:

The screened specific DNA sequence of the fusobacterium nucleatum and the primer designed by the specific DNA sequence can realize the direct and rapid qualitative and quantitative detection of the fusobacterium nucleatum, the operation is simple, the time consumption is short, the detection method is applicable to single strain samples or mixed thallus-containing samples, the identification can be directly carried out according to PCR and quantitative PCR results, no additional sequencing and data analysis work is needed, and the accuracy in the tested strain can reach 100%; in the specific example, it is proved that the Spearman correlation between the qPCR result of Fn-F/R and the abundance of Fusobacterium nucleatum obtained by FrpoB-seq in the sample in the prior art is only about 0.6, and the Spearman correlation coefficient between the PCR result of the primer detection of the invention and the abundance of Fusobacterium nucleatum obtained by FrpoB-seq is close to 0.9, which indicates that the detection accuracy of the invention is far higher than that of the existing detection method.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and do not constitute a limitation on the invention. In the drawings:

FIG. 1 is a schematic diagram showing the results of genome analysis using the specific fragment sequences of F.nucleatum of FIG. 1 according to an embodiment of the present invention.

FIG. 2 is a schematic diagram showing the results of the application of the Fusobacterium nucleatum specific primer in a single strain sample according to an embodiment of the present invention;

FIG. 3 is a schematic diagram showing the results of the application of the Fusobacterium nucleatum specific primer in a mixed bacterial cell-containing sample according to an embodiment of the present invention.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The experimental procedures, which are not specified in the following examples, are generally determined according to national standards. The experimental materials not shown in the examples below are all commercially available. The equipment used in each step in the following examples is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standard, the conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise indicated, all parts are parts by weight and all percentages are percentages by mass. Unless defined or otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, any method and material similar or equivalent to those described may be used in the methods of the present invention.

It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other. The invention is further described below with reference to the drawings and specific examples, which are not intended to be limiting.

Example 1-selection of Fusobacterium nucleatum specific fragment Fn-SR and design of detection primer therefor

In this example, a specific DNA sequence (Fn-SR) of F.nucleatum is obtained by comparative genomics, and the detection primer is designed based on the specific fragment selected, and the specific method comprises the following steps:

(1) Screening of specific DNA sequences Fn-SR

The sequenced fusobacterium genome is downloaded from the NCBI public database, including the completion map and the sketch. The genome analysis was performed on F.nucleatum respectively using PGAP software. From the obtained gene cluster (gene cluster), genes highly conserved at the nucleotide sequence level in F.nucleatum are selected by sequence alignment, namely, the genes exist in more than 98% of F.nucleatum genome, and the nucleotide sequence consistency among strains is not lower than 85%. And then genes which have no homology with other fusobacterium and other species at the nucleotide level are screened out by comparison, namely genes which are conserved and specific for the fusobacterium nucleatum. The obtained gene sequence was subjected to nucleotide sequence alignment with non-fusobacterium species in NCBI nucleotide database, genes having higher sequence similarity (60%) with other species were further deleted, and genes containing insertions, truncations, and copy number inconsistencies among strains were excluded. Finally, 1 gene, defined as a specific DNA fragment of Fusobacterium nucleatum type, was obtained by screening and named Fn-SR. The above analysis incorporated 70 F.nucleatum genomes altogether, with this fragment being present in 69 (98.6%) genomes; specific fragments which are present in all F.nucleatum genomes and meet the above requirements have not been obtained. The sequence information of the Fn-SR is specifically as follows:

TTGATATCAACTTTAAGTTTTTCTTATCAAGTTAATTATGATGATGTAGTTGATATAGT

TTTAAGAAATTATCCTCAATCAAGAGTTACAAAAATAGAGATATTTAATTATAAAGA

TAAAACTGTATATGAGGGAGAAGCCTTTGATAAAGGTCAAAAAATAGAATTTATAAT

AGATGTAAATACTGGTGAAGTTTTTAAAATAGCACCCGATTATGATGATAAATACAA

TCCTAATTATAATTTACCAATTACTTTTGAACAGGCAAGTAGAATAGGCTTGGATAAT

TCATTCAATGGAAAAGTAAAAAGTATTGAGTTAAAAAATATAAATAAAAGAGCTTAT

TATATAGTTGAAGTTAAGGAAGATAAATCTGAAAAAGAAATAAGAATTGATGCTAATAGTGGGAAAGTTATTGGTATAAAAGAGGAGGAGTAA(SEQ ID NO.1)

The nucleotide sequence alignment of the genome of the F.nucleatum strain sequenced from NCBI with the specific fragment Fn-SR, as shown in FIG. 1, shows that the specific fragment is present in 98.6% (69/70) of F.nucleatum ('Fn') genome (homologous sequence is present) and not in other F.nucleatum species. Nucleotide sequence conservation was over 85%. The above results demonstrate that the specific fragment Fn-SR can be used to characterize F.nucleatum.

(2) Design of detection primers

On the basis of the screened specific fragment Fn-SR, all the corresponding known sequences are subjected to multiple sequence comparison, a group of universal amplification primers are designed in a relatively conserved region, and the designed primers comprise 1 forward primer containing a facultative base and 3 reverse primers containing a facultative base as a group of primers because of unavoidable sequence polymorphism in a target region, and the four primers need to be mixed simultaneously. Specific sequence information of the primers is as follows:

YTATCCTCAATCAAGAGTTACAA(Fn-SR-F，SEQ ID NO.1)；

AGRATTTTAYCRGTATTAGCATCRAT(Fn-SR-Ra，SEQ ID NO.2)；

AARATTTTWCCASTATTAGCATCAAT(Fn-SR-Rb，SEQ ID NO.3)；

ATAACTTTCCCACTATTAGCATCAAT(Fn-SR-Rc，SEQ ID NO.4)。

example 2 use of Fusobacterium nucleatum specific primers in Single Strain samples

This example demonstrates the detection of F.nucleatum in a single strain sample using the primer set designed in example 1, comprising the steps of:

Total DNA of Fusobacterium strains listed in FIG. 2, which have been clearly classified into species, was extracted. The Fusobacterium strains were inoculated to commercial Columbia blood plates or thioglycolate liquid medium, anaerobically cultured at 37℃for 2 days, and the bacterial cells were collected, and total DNA of each strain was extracted by using a commercial bacterial genome DNA extraction kit according to the procedures listed in the specification.

PCR amplification was performed using the primers designed in example 1 (Fn-SR-F, fn-SR-Ra, fn-SR-Rb, fn-SR-Rc of the sequences shown in SEQ ID No.1 to SEQ ID No. 4) to identify whether it was F.nucleatum. Specifically, PCR amplification was performed using primer pairs in Table 1 using a reaction system of 10ng of DNA template per reaction as specified in the instruction using Premix Taq (Ex Taq Version 2.0plus dye) or a similar commercial PCR kit. The reaction conditions are as follows; 98℃for 10 seconds, 55℃for 20 seconds, 72℃for 30 seconds, thereby cycling through 35. The amplified product was detected by agarose gel electrophoresis to detect the presence or absence of a band at the expected size position.

The results of the above experiments are shown in fig. 2: the primers were amplified in both F.nucleatum ('Fn') strains, but not in other F.nucleatum strains, indicating that the primers were effective in identifying F.nucleatum and that the specificity and sensitivity of the primers were 100% in the incorporated strains.

EXAMPLE 3 use of Fusobacterium nucleatum specific primers in mixed thallus containing samples

This example shows that the primers designed in example 1 (Fn-SR-F, fn-SR-Ra, fn-SR-Rb, fn-SR-Rc, shown in SEQ ID NO.1 to SEQ ID NO. 4) were not amplified in human DNA (whole blood sample) and in faeces and colorectal cancer (CRC) tissue samples without F.sub.bacteria (shown in part A of FIG. 3), indicating that they are not affected by the background of the host and other flora. Specifically, taking a whole blood DNA sample to represent a human host DNA background, taking feces and CRC tissue sample DNA without clostridium to represent a human flora DNA background and a flora plus host background respectively, and carrying out PCR amplification; meanwhile, the F.nucleic ATCC 25586 and F.varium THCT E1 strain DNA are adopted as positive control and negative control respectively, and a reaction system is adopted without adding a DNA template. The amount of DNA template of human sample is 100ng per reaction, and the amount of DNA template of strain is 10ng per reaction by adopting Premix Taq (Ex Taq Version 2.0 plus dye) or similar commercial PCR kit according to the reaction system given by the instruction. The reaction conditions are as follows; 98℃for 10 seconds, 55℃for 20 seconds, 72℃for 30 seconds, thereby cycling through 35. The amplified product was detected by agarose gel electrophoresis to see if a band appeared at the expected size position. The stool samples in part a of fig. 3 used healthy Human (HC) and CRC patient stools.

Further, the present example applies the primer to real-time quantitative PCR to detect abundance of fusobacterium nucleatum in stool and tissue samples; wherein the stool sample comprises stool samples of healthy people and CRC patients, and the tissue sample comprises tumor tissue of CRC patients and paired normal colorectal tissue samples. The detection was performed using the general primer of the V6 region of the 16S rRNA gene as an internal control (Geller,L.T.et al."Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine",Science,2017,357:1156–1160)., and also using the widely used F.nucleatum 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). A TB Green Premix Ex Taq ^TM II (TLI RNASEH Plus) kit was used, and the amount of DNA template per reaction was 100ng according to the reaction system given in the specification. The reaction conditions are as follows; the-DeltaCt values were calculated at 50℃for 2 minutes, 95℃for 30 seconds, then at 98℃for 5 seconds, 55℃for 34 seconds, and 70℃for 30 seconds for 40 cycles. Correlation analysis of the detection results with the FrpoB-seq results of the samples (methods and results the inventors have previously published in Dexi Bi,et al."Profiling Fusobacteriuminfection at high taxonomic resolution reveals lineage-specific correlations in colorectal cancer",Nature Communications,2022,13:3336), frpoB-seq is the most accurate fusobacterium high resolution detection technique so far, which can provide more accurate fusobacterium nucleatum abundance data.

The specific sequences of the above-used internal reference primers (general primers for the V6 region of the 16S rRNA gene) and Fn-F/R primers are as follows:

The correlation results are shown in section B of FIG. 3, and the experimental results show that the detection results of the primers of the embodiment have high consistency with the abundance of Fusobacterium nucleatum (Fn) obtained by FrpoB-seq, and the Spearman correlation coefficient is close to 0.9. However, the Spearman correlation coefficient of the Fn-F/R detection result in the prior art only reaches about 0.6. The above results demonstrate that the fusobacterium nucleatum specific primers employed in this example have accurate quantitative detection capability in mixed cell-containing samples.

As can be seen from the above examples, the present invention employs a specific screening method to determine a DNA sequence highly specific to Fusobacterium nucleatum, and on the basis of the sequence, a set of reference compositions are designed, which have excellent specificity and sensitivity, can realize direct and rapid qualitative and quantitative detection of Fusobacterium nucleatum, and the detection accuracy is significantly better than that of the existing detection method.

The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for the present invention will occur to those skilled in the art, and are also within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.

Claims (10)

1. A segment of specific DNA sequence of clostridium nucleatum is characterized in that the specific DNA sequence is shown as SEQ ID No.1 or is a homologous sequence with 85-99% similarity with the sequence shown as SEQ ID No. 1.

2. The fusobacterium nucleatum specific DNA sequence according to claim 1, wherein the method of screening the specific DNA sequence comprises the steps of: performing ubiquity genome analysis on the clostridium nucleatum, and screening genes highly conserved at the nucleotide sequence level in the clostridium nucleatum through sequence alignment; then screening genes which have no homology with other fusobacterium and other species at the nucleotide level from the genes by comparison; and (3) comparing the obtained gene sequence with nucleotide sequences of non-fusobacterium species, further removing genes with certain sequence similarity with other species, and removing genes containing insertion, truncation and inconsistency of copy numbers among strains, thereby screening and obtaining the specific DNA sequence.

3. A primer composition for amplifying the fusobacterium nucleatum specific DNA sequence of claim 1.

4. The primer composition of claim 3, wherein the primer composition comprises: a forward primer with a sequence shown as SEQ ID No.2 and a reverse primer with a sequence shown as SEQ ID No. 3-SEQ ID No. 5.

5. A detection reagent for F.nucleatum, wherein the detection reagent comprises the primer composition according to claim 3 or 4 and a PCR amplification reagent.

6. Use of the fusobacterium nucleatum specific DNA sequence of claim 1, the primer composition of claim 3, or the detection reagent of claim 4, wherein the use is selected from at least one of the following: the application of the kit in detecting the clostridium nucleatum based on non-diagnostic purposes, the application in preparing a kit for detecting the clostridium nucleatum, the application in preparing a kit for early detecting colorectal cancer or inflammatory bowel disease, the application in bioinformatic analysis and the application in evaluating poor prognosis and recurrence of the colorectal cancer.

7. The use according to claim 6, wherein the bioinformatics analysis comprises: identifying whether the sequenced fusobacterium strain is fusobacterium nucleatum by sequence alignment; or detecting and analyzing the existence of the clostridium nucleatum in metagenome sequencing data analysis.

8. The use according to claim 6, wherein the kit is a normal PCR kit or a real-time quantitative PCR kit containing the primer composition.

9. A method for detecting a DNA sequence specific for fusobacterium nucleatum according to claim 1, comprising the steps of: PCR amplification of a sample to be detected using the primer composition according to claim 3, and analysis and identification of the amplified product to detect the specific DNA sequence.

10. The method according to claim 9, wherein the sample to be tested is a single strain sample or a mixed bacterial cell-containing sample, and the sample to be tested includes a whole blood sample, a fecal sample, and a tissue sample.