CN111118187A - Primer group, kit and detection method for detecting esophageal squamous carcinoma tissue and paracancerous tissue differential flora - Google Patents
Primer group, kit and detection method for detecting esophageal squamous carcinoma tissue and paracancerous tissue differential flora Download PDFInfo
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Abstract
The invention provides a primer group, a kit and a detection method for detecting a flora different between esophageal squamous carcinoma tissue and paracancerous tissue, belonging to the technical field of microbial molecular biology detection; the primer set includes an upstream primer 341F and a downstream primer 806R. The genome DNA of the esophageal squamous carcinoma tissue and the genome DNA of the tissue beside the carcinoma are taken as templates, the primer group is utilized to detect the floras in the two tissues, and the differential floras of the esophageal squamous carcinoma tissue and the tissue beside the carcinoma can be detected through statistical analysis, so that the problems of poor specificity and insufficient representativeness of the esophageal squamous carcinoma differential bacteria detected by using the oral mouthwash sample in the existing scheme can be solved. In addition, the primer group can detect all bacterial flora in esophageal squamous carcinoma tissues and tissues beside the esophageal squamous carcinoma tissues, and can effectively solve the problem that the paraffin embedding method in the prior scheme only detects single flora and has insufficient representativeness.
Description
Technical Field
The invention relates to the technical field of microbial molecular biology detection, in particular to a primer group, a kit and a detection method for detecting a flora different between esophageal squamous carcinoma tissues and tissues beside carcinoma.
Background
Esophageal cancer is one of the most common malignant tumors seriously harming human health in China, 95 percent of which is esophageal squamous carcinoma, and has the characteristics of high malignancy, rapid metastasis, poor postoperative quality of life, obvious regional morbidity and the like.
The current research methods for analyzing the difference of esophageal squamous carcinoma tissue flora comprise paraffin embedding detection and oral cavity mouth wash detection; the detection sample for detecting the oral cavity mouth wash is from the oral cavity mouth wash, and has poor specificity and insufficient representativeness; the paraffin embedding detection method uses paraffin-embedded tissues, and related pathogenic bacteria are single, so that the possibility of secondary association cannot be eliminated, and the detection is not representative enough. At present, a method for detecting the different flora between the esophageal squamous carcinoma tissue and the tissue beside the carcinoma with good representativeness and reliable results is lacked.
Disclosure of Invention
The invention aims to provide a primer group, a kit and a detection method for detecting a flora different between esophageal squamous carcinoma tissues and paracarcinoma tissues, and the primer group, the kit and the detection method have the advantages of good representativeness and reliable results.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a primer group for detecting a bacterial flora difference between an esophageal squamous carcinoma tissue and a para-carcinoma tissue, which comprises an upstream primer 341F and a downstream primer 806R; the nucleotide sequence of the upstream primer 341F is shown as SEQ ID No. 1; the nucleotide sequence of the downstream primer 806R is shown as SEQ ID No. 2.
The invention provides various kits comprising the primer group in the scheme.
Preferably, the kit further comprises 2 × Phusion Master Mix and double distilled water.
The invention provides a method for detecting a differential flora between esophageal squamous carcinoma tissues and tissues beside carcinoma based on the primer group or the kit in the scheme, which is not the purpose of diagnosis, and comprises the following steps:
1) extracting flora genome DNA in cancer tissues and tissues beside the cancer of the esophageal squamous cell carcinoma patient respectively;
2) respectively using the flora genome DNA in cancer tissues and the flora genome DNA in tissues beside cancer as templates, and performing PCR amplification reaction by using the primer group of claim 1 to obtain PCR amplification products;
3) carrying out agarose gel electrophoresis detection on the PCR amplification product, selecting a band with a main band size of 400-450 bp, cutting the gel and recovering to obtain a target band;
4) constructing a library by using the target strip, and sequencing to obtain sequencing data of the flora of the cancer tissue and the tissue beside the cancer;
5) performing quality control on the flora sequencing data of the cancer tissues and the tissues beside the cancer to obtain the data after quality control;
6) introducing the quality-controlled data into Qiime2019.4 software, denoising by using a DADA2 algorithm to obtain specific amplicons, filtering the specific amplicons with the total sequence number lower than 10 or appearing in less than 5 samples, and applying
Comparing a Bayes classifier with a Greengenes13.8 database, and performing species annotation on the representative specific amplicon to obtain data after the species annotation;
7) analyzing the data after the species annotation by adopting Qiame2019.4 software, and respectively calculating Alpha diversity, Beta diversity and relative abundance of cancer tissues and paracancerous tissue floras to obtain a calculation result;
8) and counting the calculation result, and screening to obtain the differential flora of the esophageal squamous carcinoma tissue and the tissue beside the carcinoma.
Preferably, the concentration of the forward primer 341F and the concentration of the backward primer 806R are 10. mu. mol/L respectively.
Preferably, the procedure of the PCR amplification reaction in step 3) is: at 98 deg.C for 1 min; 30 cycles of 98 deg.C, 10s, 50 deg.C, 30s, 72 deg.C, 30 s; 72 deg.C, 5 min.
Preferably, the quality control of the flora sequencing data of the cancer tissues and the tissues beside the cancer in the step 6) comprises the following steps:
s1, removing the 3' end primer joints of the sequencing data of the cancer tissues and the cancer adjacent tissues and flora, and splicing paired sequences into a sequence according to the repeated relation between PE reads to obtain a first sample sequence;
s2, distinguishing the first sample sequence through the tag sequence, removing the tag sequence, generating two or more mismatched bases in an overlapping region, and obtaining a second sample sequence, wherein the error rate is higher than 1% and the fragment length is lower than 200 bp;
and S3, removing the chimera in the second sample sequence to obtain the sequencing data of the flora of the cancer tissue and the tissue beside the cancer to be analyzed.
The invention has the beneficial effects that: the invention provides a primer group for detecting a flora different between esophageal squamous carcinoma tissues and paracancerous tissues, which comprises an upstream primer 341F and a downstream primer 806R. The genome DNA of the esophageal squamous carcinoma tissue and the genome DNA of the tissue beside the carcinoma are taken as templates, the primer group is utilized to detect the flora in the two tissues, and the differential flora between the esophageal squamous carcinoma tissue and the tissue beside the carcinoma can be detected through statistical analysis, so that the problems of poor specificity and insufficient representativeness of the esophageal squamous carcinoma differential flora detected by using an oral mouthwash sample in the prior art can be solved. In addition, the primer group can detect all bacterial flora in esophageal squamous carcinoma tissues and tissues beside the esophageal squamous carcinoma tissues, and can effectively solve the problem that the paraffin embedding method in the prior scheme only detects single flora and has insufficient representativeness.
Drawings
FIG. 1 is a flow chart of an embodiment;
FIG. 2 is a diagram of genomic DNA detection of cancer tissues of an esophageal squamous carcinoma patient, wherein cancer tissues corresponding to lanes 3-8 are samples numbered from A25 to A30;
FIG. 3 is a genome DNA detection map of tissues beside cancer of an esophageal squamous carcinoma patient), wherein 3-7 lanes sequentially correspond to the tissues beside cancer, and the serial numbers of the tissues beside cancer are B1-B5 samples;
FIG. 4 is a partial diagram of the detection of cancer tissues 16S rRNA V3-V4 of patients with esophageal squamous carcinoma, wherein the cancer tissues in lanes 2-5 are numbered A37, A59, A58 and A99;
FIG. 5 is a partial diagram of the detection of 16S rRNA V3-V4 region of the tissue beside the esophageal squamous carcinoma patient, wherein the cancer tissues in lanes 2, 3, 4 and 6 are numbered as B31, B59, B32 and B43 samples;
FIG. 6 is a statistic of Alpha diversity of cancer and paracancer microbial communities;
FIG. 7 is a graph showing the spatial distribution of the microbial composition of cancer and paracarcinoma tissues;
FIG. 8 shows the statistical results of the microbial community Beta diversity between cancer and the tissue beside cancer.
Detailed Description
The invention provides a primer group for detecting a bacterial flora difference between an esophageal squamous carcinoma tissue and a para-carcinoma tissue, which comprises an upstream primer 341F and a downstream primer 806R; the nucleotide sequence of the upstream primer 341F is shown as SEQ ID No.1, and specifically comprises the following steps: CCTAYGGGRBGCASCAG; the nucleotide sequence of the downstream primer 806R is shown as SEQ ID No.2, and specifically comprises the following steps: GGACTACNNGGGTATCTAAT, respectively; the upstream primer 341F and the downstream primer 806R carry a tag sequence (Barcode).
In the invention, the primer group is used for amplifying the V3-V4 region of the 16S rRNA gene of bacteria, and can realize the amplification of all bacterial flora.
The invention provides various kits comprising the primer group in the scheme; the kit preferably further comprises 2 XPPhusion Master Mix and double distilled water.
The invention provides a method for detecting a differential flora between esophageal squamous carcinoma tissues and tissues beside carcinoma based on the primer group or the kit in the scheme, which is not the purpose of diagnosis, and comprises the following steps:
1) extracting flora genome DNA in cancer tissues and tissues beside the cancer of the esophageal squamous cell carcinoma patient respectively;
2) respectively using the flora genome DNA in cancer tissues and the flora genome DNA in tissues beside cancer as templates, and performing PCR amplification reaction by using the primer group of claim 1 to obtain PCR amplification products;
3) carrying out agarose gel electrophoresis detection on the PCR amplification product, selecting a band with a main band size of 400-450 bp, cutting the gel and recovering to obtain a target band;
4) constructing a library by using the target strip, and sequencing to obtain sequencing data of the flora of the cancer tissue and the tissue beside the cancer;
5) performing quality control on the flora sequencing data of the cancer tissues and the tissues beside the cancer to obtain the data after quality control;
6) introducing the quality-controlled data into Qiime2019.4 software, denoising by using a DADA2 algorithm to obtain specific amplicons, filtering the specific amplicons with the total sequence number lower than 10 or appearing in less than 5 samples, and applying
Comparing a Bayes classifier with a Greengenes13.8 database, and performing species annotation on the representative specific amplicon to obtain data after the species annotation;
7) analyzing the data after the species annotation by adopting Qiame2019.4 software, and respectively calculating Alpha diversity, Beta diversity and relative abundance of cancer tissues and paracancerous tissue floras to obtain a calculation result;
8) and counting the calculation result, and screening to obtain the differential flora of the esophageal squamous carcinoma tissue and the tissue beside the carcinoma.
Before extracting the flora genome DNA in the cancer tissue and the tissue beside the cancer of the esophageal squamous cell carcinoma patient, the invention preferably also comprises the steps of taking the cancer tissue and the tissue beside the cancer of the esophageal squamous cell carcinoma patient; the distance between the tissue beside the cancer and the cancer tissue is preferably 3-5 cm; in the invention, the cancer tissues are confirmed to be esophageal squamous cell carcinoma through pathological Hematoxylin-eosin staining (HE), and meanwhile, the existence of tumor cells is not detected in a tissue specimen beside the cancer.
After taking the cancer tissue and the tissue beside the cancer of the esophageal squamous carcinoma patient, the invention also comprises the steps of respectively cutting the two tissues into small pieces, respectively putting the small pieces into an autoclaved cryopreservation tube, storing the small pieces in liquid nitrogen within 1h, and then transferring the small pieces into a refrigerator at the temperature of minus 80 ℃ for storage. All consumables of the invention are sterilized by high pressure.
After obtaining the cancer tissue and the tissue beside the cancer, the invention respectively extracts the flora genome DNA in the cancer tissue and the tissue beside the cancer of the esophageal squamous cell carcinoma patient; the method for extracting the genome DNA of the two floras is not particularly limited by the invention, and the conventional extraction method in the field can be adopted. In the specific implementation process of the invention, the DNA of the flora in the cancer tissue and the tissue beside the cancer is extracted by adopting an SDS method.
After obtaining the flora genome DNA in the cancer tissue and the tissue beside the cancer, the invention respectively takes the flora genome DNA in the cancer tissue and the flora genome DNA in the tissue beside the cancer as templates, and utilizes the primer group in the scheme to carry out PCR amplification reaction to obtain PCR amplification products; the system of the PCR amplification reaction comprises the following components in 30 mu L: 2 XPHUSION Master Mix15 uL, template 10 uL, upstream primer 341F 1 uL, downstream primer 806R 1 uL and sterile double distilled water 3 uL; the concentrations of the upstream primer 341F and the downstream primer 806R are respectively preferably 10 mu mol/L; the concentration of the template is preferably 10-20 ng/mu L; in the present invention, use is made of
The High-Fidelity PCR Master Mix with GC Buffer enzyme is used for PCR, so that the amplification efficiency and accuracy can be ensured; the procedure for the PCR amplification reaction is preferably: at 98 deg.C for 1 min; 30 cycles of 98 deg.C, 10s, 50 deg.C, 30s, 72 deg.C, 30 s; 72 deg.C, 5min
After obtaining a PCR amplification product, carrying out agarose gel electrophoresis detection on the PCR amplification product, selecting a band with a main band size of 400-450 bp, cutting and recovering gel to obtain a target band; after obtaining the target band, the invention preferably further comprises purifying the product in the target band; the kit for purifying the product is preferably a GeneJET glue recovery kit of Thermoscientific company.
After obtaining the purified product, constructing a library by using the purified product, and sequencing to obtain sequencing data of the flora of the cancer tissue and the tissue beside the cancer; the Kit for constructing the Library is preferably a Library construction Kit of TruSeqDNA PCR-Free Library Preparation Kit of Illumina company; the constructed library is preferably subjected to Qubit quantification and library detection.
After obtaining the sequencing data of the flora of the cancer tissue and the tissue beside the cancer, the invention controls the quality of the sequencing data of the flora of the cancer tissue and the tissue beside the cancer to obtain the data after quality control; the method for controlling the quality of the flora sequencing data of the cancer tissues and the tissues beside the cancer comprises the following steps:
s1, removing the 3' end primer joints of the sequencing data of the cancer tissues and the cancer adjacent tissues and flora, and splicing paired sequences into a sequence according to the repeated relation between PE reads to obtain a first sample sequence; barcode is positioned at the beginning of reads and used for distinguishing which sample the sequence belongs to, and the Barcode is deleted after allocation is finished;
s2, distinguishing the first sample sequence through the tag sequence, removing the tag sequence, generating two or more mismatched bases in an overlapping region, and obtaining a second sample sequence, wherein the error rate is higher than 1% and the fragment length is lower than 200 bp;
and S3, removing the chimera in the second sample sequence to obtain the sequencing data of the flora of the cancer tissue and the tissue beside the cancer to be analyzed.
After the quality-controlled data are obtained, the quality-controlled data are introduced into Qiime2019.4 software, noise reduction is carried out by using a DADA2 algorithm to obtain specific amplicons, the specific amplicons with the total sequence number lower than 10 or appearing in less than 5 samples are filtered, and the specific amplicons are applied
Species annotation was performed on representative specific amplicons against the greengenes13.8 database using a Bayes classifier to obtain species annotated data.
After obtaining the data after species annotation, the method adopts Qiime2019.4 software to analyze the data after species annotation, and respectively calculates the Alpha diversity, Beta diversity and relative abundance of cancer tissues and tissues beside cancer flora (in a certain tissue, a specific bacterial sequence accounts for the percentage of all sequencing sequences) to obtain a calculation result; alpha diversity refers to the diversity within a particular area or ecosystem, measured by species abundance; beta diversity is a comparison of diversity between different species and is used to indicate the response of biological species to environmental heterogeneity.
In the implementation process of the invention, the Beta diversity of the species is evaluated by non-weighted and weighted principal axis analysis (PCoA). In screening differential flora between two groups, statistical differential analysis is performed by using a mixed linear model (R3.6 software), and the research result is corrected by False Discovery Rate (FDR).
After the calculation result is obtained, the calculation result is counted, and the differential flora of the esophageal squamous carcinoma tissue and the tissue beside the carcinoma is obtained by screening.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
The flowchart of this embodiment is shown in fig. 1.
1. Esophageal squamous carcinoma patient screening
120 patients with esophageal squamous carcinoma who are diagnosed in tumor hospitals in Fujian province and Zhangzhou hospitals in Fujian province from 2 months to 2017 months in 2013 are selected. Inclusion criteria were: receiving a radical surgical treatment for esophageal cancer; all subjects were diagnosed with esophageal squamous cell carcinoma by surgical pathology; before operation, the patient does not receive treatment such as radiotherapy or chemotherapy; no antibiotic use record 2 months before the operation; no other infectious diseases. Exclusion criteria: incomplete clinical pathological data and tissue specimens; metastatic tumor and esophageal cancer recurrence cases; there are patients with severe organic lesions of the brain, a history of psychiatric illness, etc.
2. Method of producing a composite material
2.1 tissue sample Collection
And taking cancer tissues and tissues beside the cancer (3-5 cm away from the cancer tissues) of the esophageal squamous carcinoma patient. Cutting the tissue into small pieces, respectively placing into autoclave sterilized freezing tubes, storing in liquid nitrogen within 1h, and transferring into-80 deg.C refrigerator for storage. All consumables were autoclaved. The cancer tissue specimens are confirmed to be esophageal squamous cell carcinoma through pathological Hematoxylin-eosin staining (HE), and meanwhile, the existence of tumor cells is not detected in the tissue specimens beside the cancer.
Observing the size, shape, depth, range, color and texture of the tissue, removing the tissue with putrefaction and erosion on the surface, and determining the existence of squamous cell carcinoma by pathological examination.
2.2 extraction and detection of genomic DNA
DNA of the flora in cancer tissues and tissues adjacent to the cancer is extracted by SDS method.
1) Taking out the sample from an ultralow-temperature refrigerator at minus 80 ℃, shearing 50-100 mg of tissues by using sterile scissors, putting the tissue into a 1.5 ml EP tube, adding 1ml of DNAzol, and treating the tissue for 5-10 times by using a homogenizer. And (3) placing the EP pipe treated by the homogenizer into a centrifuge, centrifuging for 10min at 10000g, and transferring the obtained supernatant into a new pipe.
2) EDTA (0.5M, pH8.0) and 50. mu.L lysozyme were added to the centrifuged EP tube, mixed well, then Protease K was added thereto, mixed well by inversion, and incubated at 55 ℃ for 2 hours.
3) Centrifuging at 12000rpm for 5min, sucking supernatant, adding protein precipitation solution, gently vortexing for 10s, standing at-20 deg.C for several minutes, and centrifuging at 12000rpm for 10 min.
4) The supernatant was pipetted into a fresh EP tube and centrifuged at 12000rpm for 5min, with possible precipitation of trace proteins at the bottom of the tube. The supernatant was aspirated into a new EP tube, isopropanol was added, mixed well and left at-20 ℃ for 20min, and then centrifuged at 12000rpm for 10 min.
5) The liquid was decanted, washed twice with 1ml of 75% ethanol and the small amount of liquid remaining was collected by centrifugation again and then aspirated off with a pipette tip. And drying the clean bench or airing the clean bench at room temperature.
6) Add 50. mu.L of ddH2O dissolving the DNA sample and the shaker aid the dissolution. Add 1. mu.L RNase A, reverse mix and incubate at 37 ℃ for 15 min.
7) And (4) quantitatively detecting the extracted DNA by using a Qubit instrument, wherein the detection results are qualified. Detection using 1% agarose gel electrophoresis: the voltage is 100V and 40 min. Photographing recording by a UVI gel imaging system: the DNA electrophoresis has no miscellaneous band or tailing, which indicates that the DNA fragment has good purity and no obvious degradation, and is shown in figure 2 (shown in a detection chart of cancer tissue genome DNA of an esophageal squamous carcinoma patient), wherein cancer tissues corresponding to 3-8 lanes are numbered as samples A25-A30; FIG. 3 (genome DNA detection map of tissues beside esophageal squamous carcinoma patient), wherein the corresponding tissues beside carcinoma in lanes 3-7 are samples numbered B1-B5. An appropriate amount of sample was taken in a centrifuge tube and the sample was diluted to 1 ng/. mu.L with sterile water. The DNA was stored in a freezer at-20 ℃ until use.
2.3PCR amplification
The V3-V4 region of the 16S rRNA gene of bacteria was amplified using bacterial universal primers 341F (SEQ ID No. 1: CCTAYGGGRBGCASCAG) and 806R (SEQ ID No. 2: GGACTACNNGGGTATCTAAT) using Barcode-carrying specific primers according to the selection of the sequencing region using diluted genomic DNA as a template. Use of
The High-Fidelity PCR Master Mix with GC Buffer enzyme is used for PCR, and the amplification efficiency and accuracy are ensured. A30 μ L PCR reaction system and procedure included: phusion Master Mix (2X) 15. mu.L, 1. mu.L each of upstream and downstream primers 341F and 806R (10uM/L), 10. mu.L of template DNA (equivalent to 20ng by quantification using a nucleic acid quantification apparatus), and 8. mu.L of double distilled water. The negative control was identical to the sample except that no template DNA was added. PCR reaction procedure: pre-denaturation at 98 ℃ for 1min, then denaturation at 98 ℃ for 10s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles, and finally extension at 72 ℃ for 5 min. The PCR product was detected by 2% agarose gel electrophoresis: voltage 80V, 40 min. Photographing recording by a UVI gel imaging system: no fragmentation, no primer dimer, and amplification of bright target fragment. FIG. 4 is a partial diagram of the detection of cancer tissues 16S rRNA V3-V4 of patients with esophageal squamous carcinoma, wherein the cancer tissues in lanes 2-5 are numbered A37, A59, A58 and A99;FIG. 5 is a partial diagram of the detection of 16SrRNA V3-V4 area of the cancer tissues beside the esophageal squamous carcinoma patient, wherein the cancer tissues in lanes 2, 3, 4 and 6 are numbered as B31, B59, B32 and B43 samples. The PCR product was stored at-20 ℃ until use.
2.4 PCR amplification product mixing and purification
And (3) performing equal-mass sample mixing according to the concentration of the PCR product, fully and uniformly mixing, purifying the PCR product by agarose gel electrophoresis with the concentration of 1 × TAE of 2%, selecting a sequence with the main band size of 400-450 bp, tapping and recovering a target band. The product purification kit used was a Thermo Scientific GeneJET gel recovery kit.
2.5 library construction and on-machine sequencing
Constructing a Library by using a Library construction Kit of TruSeq DNA PCR-Free Library Preparation Kit of Illumina company, carrying out Qubit quantification and Library detection on the constructed Library, and carrying out flora sequencing by using an IlluminaHiSeq2500 PE250 sequencing platform after the constructed Library is qualified.
2.6 Pre-processing of sequencing data
The quality control of the primary data for sequencing the esophageal flora of the esophageal squamous cell carcinoma patient comprises the following specific steps:
1) data splicing: removing the 3' end primer joint, and splicing the paired sequences into a sequence according to the repeated relation between the PE reads.
2) Cutting a warehouse: the sample sequences are distinguished through a tag sequence (Barcode), and sequences with two or more mismatched bases, the error rate higher than 1 percent and the fragment length lower than 200bp in the Barcode and the overlapping region are removed. The Barcode is located at the beginning of reads to distinguish to which sample this one sequence belongs. The Barcode is deleted after the allocation is completed.
3) Removing chimera: in the PCR reaction, some heterozygous DNA fragments are generated, and the accuracy of data analysis can be improved by removing these chimeras.
2.7 specific Amplicon (ASV) based data processing analysis: introducing quality control data into Qiime (2019.4) platform, denoising by using DADA2 algorithm to obtain specific Amplicon (ASV), filtering ASV with total sequence number lower than 10 or appearing in less than 5 samples, and applying
A Bayes classifier (NBC) was compared against greengeneses 13.8 database and species annotations were made on representative ASVs.
2.8 statistics
The Qiime (v2019.4) software is adopted to analyze the flora sequencing data in the cancer tissues and the para-cancer tissues, and the Alpha and Beta diversity of the flora and the relative abundance of the flora are calculated respectively.
3. Results of the study
1) Comparison of flora distribution of cancer tissue and tissue beside cancer of esophageal squamous carcinoma patient
In a study of flora diversity, the results of the Alpha diversity study showed that the signed rank sum test was used to compare Alpha diversity between cancer and paired paraneoplastic tissues, see figure 6, where the Observed OTUs (a in figure 6), Shannon index (B in figure 6) and Faith's PD index (C in figure 6) all differed (P < 0.05). The graphs show that there is a difference in Alpha diversity between cancer and paracancer microbial communities.
2) Species abundance was used to calculate curtis (bc), unweighted unifrac (uu) and weighted unifrac (wu) distance matrices in Bray between specimens, using principal axis analysis (PCoA) to find the most dominant axis in the distance matrices for presentation of differences in composition of cancerous and paracancerous tissue microorganisms. The results are shown in FIG. 7. The results show that cancer and paracancerous tissue microbial compositions differ spatially, regardless of the main axis of synthesis using BC (a in fig. 7), UA (B in fig. 7), and WU (C in fig. 7).
3) Differences in Beta diversity between cancer and tissue species surrounding cancer were analyzed using the PERMANOVA and PERMDISP tests. The results are shown in FIG. 8. The PERMDISP test suggested that the intragroup distance of the cancer and the paracarcinoma tissue did not differ, regardless of BC distance (a in fig. 8, P ═ 0.718), UU distance (B in fig. 8, P ═ 0.412), and WU distance (C in fig. 8, P ═ 0.496). The results of the PERMANOVA test showed that the distance between the groups of cancer and tissue species adjacent to cancer was greater than the distance between the groups (A, P ═ 0.001 in FIG. 8; B, P ═ 0.002 in FIG. 8; C, P ═ 0.028 in FIG. 8), suggesting that there was a difference in the microbial community Beta diversity between the two different tissues.
4) The flora can be classified into 7 levels according to the nature of the flora, and the application analyzes the distribution of the flora species levels in cancer tissues and tissues beside cancer of esophageal squamous carcinoma patients, the results of the species level research are shown in Table 1, and the results show that Clostridium butyricum (Clostridium butyricum), helicobacter pylori (helicobacter pylori), Oersonia (Olsenerella profusion), Porphyromonas pulposus (Porphyromonanodondylis), Bifidobacterium pseudolongum (Bifidobacterium pseudoolonggum), Brevundimonas dimia (Brevundimonas diminuta), Rosemophila viscosus (Rothia mulilaginosa), Clostridium novaeformis (Clostridium neoforma), Propionibacterium intervorans (Prevotella intermedia), and the distribution of the bacterial species levels in the cancer tissues and tissues around cancer (Prevotella tissue <0.05) of esophageal squamous carcinoma patients are analyzed, the total number of the different strains is 13.
TABLE 1 screening of differential bacteria between esophageal squamous carcinoma tissue and paracancerous tissue strains
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
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ggactacnng ggtatctaat 20
Claims (8)
1. A primer group for detecting a bacterium group different from a cancer-associated tissue of esophageal squamous carcinoma comprises an upstream primer 341F and a downstream primer 806R; the nucleotide sequence of the upstream primer 341F is shown as SEQ ID No. 1; the nucleotide sequence of the downstream primer 806R is shown as SEQ ID No. 2.
2. A kit comprising the primer set of claim 1.
3. The kit of claim 2, further comprising a2 x Phusion MasterMix and double distilled water.
4. A method for detecting a differentiated flora of esophageal squamous carcinoma tissues and tissues beside carcinoma based on the primer group of claim 1 or the kit of claim 2 or 3, which comprises the following steps:
1) extracting flora genome DNA in cancer tissues and tissues beside the cancer of the esophageal squamous cell carcinoma patient respectively;
2) respectively using the flora genome DNA in cancer tissues and the flora genome DNA in tissues beside cancer as templates, and performing PCR amplification reaction by using the primer group of claim 1 to obtain PCR amplification products;
3) carrying out agarose gel electrophoresis detection on the PCR amplification product, selecting a band with a main band size of 400-450 bp, cutting the gel and recovering to obtain a target band;
4) constructing a library by using the target strip, and sequencing to obtain sequencing data of the flora of the cancer tissue and the tissue beside the cancer;
5) performing quality control on the flora sequencing data of the cancer tissues and the tissues beside the cancer to obtain the data after quality control;
6) introducing the quality-controlled data into Qiime2019.4 software, denoising by using a DADA2 algorithm to obtain specific amplicons, filtering the specific amplicons with the total sequence number lower than 10 or appearing in less than 5 samples, and applying
Comparing the Greengenes13.8 database with the classifier, and performing species annotation on the representative specific amplicon to obtain data after the species annotation;
7) analyzing the data after the species annotation by adopting Qiame2019.4 software, and respectively calculating Alpha diversity, Beta diversity and relative abundance of cancer tissues and paracancerous tissue floras to obtain a calculation result;
8) and counting the calculation result, and screening to obtain the differential flora of the esophageal squamous carcinoma tissue and the tissue beside the carcinoma.
5. The method according to claim 4, wherein the PCR amplification reaction system in step 3) comprises, in 30 μ L: 2 XPisuion Master Mix15 uL, template 10 uL, upstream primer 341F 1 uL, downstream primer 806R 1 uL and sterile double distilled water 3 uL.
6. The method of claim 5, wherein the concentrations of the forward primer 341F and the backward primer 806R are 10 μmol/L, respectively.
7. The method of claim 4, wherein the PCR amplification reaction in step 3) is performed by: at 98 deg.C for 1 min; 30 cycles of 98 deg.C, 10s, 50 deg.C, 30s, 72 deg.C, 30 s; 72 deg.C, 5 min.
8. The method of claim 4, wherein the step 6) of performing quality control on the flora sequencing data of the cancer tissue and the para-carcinoma tissue comprises the following steps:
s1, removing the 3' end primer joints of the sequencing data of the cancer tissues and the cancer adjacent tissues and flora, and splicing paired sequences into a sequence according to the repeated relation between PE reads to obtain a first sample sequence;
s2, distinguishing the first sample sequence through the tag sequence, removing the tag sequence, generating two or more mismatched bases in an overlapping region, and obtaining a second sample sequence, wherein the error rate is higher than 1% and the fragment length is lower than 200 bp;
and S3, removing the chimera in the second sample sequence to obtain the sequencing data of the flora of the cancer tissue and the tissue beside the cancer to be analyzed.
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