CN116426660A - Specific novel molecular targets of bacteroides fragilis and bacteroides fragilis BFS17 and rapid detection method thereof - Google Patents
Specific novel molecular targets of bacteroides fragilis and bacteroides fragilis BFS17 and rapid detection method thereof Download PDFInfo
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Abstract
The invention discloses a specific new molecular target of bacteroides fragilis and bacteroides fragilis BFS17 and a rapid detection method thereof. The molecular target nucleotide sequence of the bacteroides fragilis (Bacteroides fragilis) is shown as SEQ ID NO.1, and the molecular target nucleotide sequence of the bacteroides fragilis BFS17 is shown as SEQ ID NO. 2. The invention adopts a genome-wide method to obtain specific core genes of the bacteroides fragilis and the probiotics strain bacteroides fragilis BFS17, designs and obtains characteristic primers capable of sensitively detecting the bacteroides fragilis and the probiotics strain bacteroides fragilis BFS17, and simultaneously can rapidly and simply distinguish the bacteroides fragilis, the probiotics strain bacteroides fragilis BFS17 and other microbial strains. The invention has the advantages of simple and quick operation, high specificity, quantification and low detection cost.
Description
Technical Field
The invention belongs to the technical field of microorganisms and the technical field of medicines, and particularly relates to a specific novel molecular target of bacteroides fragilis and bacteroides fragilis BFS17 and a rapid detection method thereof.
Background
Bacteroides are the most common symbiotic flora of human bodies, and are widely colonized in intestinal tracts, oral cavities, respiratory tracts and reproductive tracts of human bodies, and are closely related to human health. Among 78 species and 5 subspecies of Bacteroides, bacteroides fragilis (Bacteroides fragilis) has the greatest influence on human health. Bacteroides fragilis is a gram-negative, short-rod-shaped, capsular, spore-free and obligate anaerobic microorganism which can decompose peptone and glucose to produce various substances such as succinic acid, acetic acid, formic acid, lactic acid and propionic acid, and the like, and is involved in carbohydrate fermentation, nitrogen-containing substance utilization and bile acid and steroid bioconversion in human bodies. On one hand, the bacteroides fragilis can assist a human body to generate microorganism K, regulate immunity and promote body health; on the other hand, part of bacteroides fragilis may carry a toxin-encoding gene, producing bacteroides fragilis toxin (Bacteroides fragilis toxin, BFT), causing the host to produce symptoms such as diarrhea. The inventor obtains a strain of bacteroides fragilis BFS17 (GDMCC 63172) through early separation in the intestinal tracts of healthy human bodies in China, and the strain can be used for high-yielding GABA, high-efficiency relieving intestinal inflammation, enhancing the barrier function of the intestinal tracts and improving the intestinal anxiety depression and has good application prospect. Therefore, the method has important application significance in accurately and sensitively detecting the existence of the bacteroides fragilis, in particular to the bacteroides fragilis BFS17 and carrying out quantitative analysis of the bacteroides fragilis and the bacteroides fragilis BFS17.
At present, the detection of the bacteroides fragilis mainly comprises morphological detection, biochemical test and the like based on the isolated culture. The methods need to enrich and selectively culture samples under anaerobic conditions, and then inspect under a microscope or carry out serial biochemical identification after obtaining single bacterial colonies, so that the method has the advantages of long experiment time consumption, complex operation and high cost, is not beneficial to early identification and identification of bacteroides fragilis, and can not carry out accurate quantitative analysis. In recent years, the molecular biological detection based on polymerase chain reaction PCR is characterized by rapidness, simplicity, accuracy, low cost and the like, and gradually replaces the traditional culture and biochemical detection, thereby becoming one of the most potential novel detection technologies of microorganisms. The key of the novel molecular detection method based on PCR is to search the special nucleotide sequence of the microorganism of the species for detection design. At present, although some patents relate to a molecular detection scheme of bacteroides fragilis, primer design is mainly carried out based on specific sequences in general genomes of microorganisms such as 16S rRNA genes of the bacteroides fragilis. In the case of Bacteroides fragilis, these approaches, while being able to distinguish the difference between Bacteroides fragilis and common microorganisms such as Escherichia coli well, are more difficult to distinguish between Bacteroides fragilis and its proximal microorganisms such as Bacteroides simplex, bacteroides thetaiotaomicron, and the like. At present, a specific identification or rapid quantitative detection mode does not exist for the bacteroides fragilis BFS17 with good probiotics.
In 2005, tettelin et al proposed the concept of the general genome of microorganisms (pangenome), which refers to the general term for all genes of a species. In the flood genome of a species, the genes of that species can be divided into three categories, core genes, meaning genes that exist conservatively in all strains; optional genome (dispensable genome), meaning genes present in a portion of the individual, which genes are involved in differentiation of the species, conferring a competitive advantage to the individual in the community; a strain-specific gene (strain-specific genes) refers to a gene that is specifically present in a certain individual. Researchers adopt high-performance calculators to collect all genome information of a certain species from a global public database and a self-built database of the researchers, and the comprehensive genome and core genome information of the species can be obtained efficiently by calculation and analysis through software such as Prokka, roary and the like. By using a core gene of a certain species, a core gene specific for that species can be obtained compared to other species in the database. The design of detection primers based on the species-specific core gene has become a more accurate way of microorganism detection.
Therefore, the core genes in the bacteroides fragilis and the strain specific genes of the bacteroides fragilis BFS17 are excavated based on the genome-wide analysis technology, and primer design and detection condition optimization are carried out according to the specific genes, so that the method has important application significance for efficiently and accurately identifying the bacteroides fragilis and the bacteroides fragilis BFS17 in a sample.
The invention comprises the following steps:
the invention aims to overcome the defects of the prior art and provide a specific new target for detecting and identifying bacteroides fragilis BFS17 and a probiotic bacterial strain and a corresponding detection scheme thereof. The invention obtains the core genome information of bacteroides fragilis according to the MP scheme in the prokaryotic universal genome automatic analysis software (Pan-Genomics Analysis Pipeline, PGAP). Extracting protein sequences of specific genes of the bacteroides fragilis according to NCBI database and all genome information of the bacteroides fragilis detected by the inventor, comparing non-redundant protein databases in the NCBI database through Blast analysis, removing protein sequences which can be compared with genes of other species to obtain core genes of the bacteroides fragilis, and comparing the NCBI database to obtain specific molecular targets SEQ ID NO.1 and SEQ ID NO.2 of the bacteroides fragilis BFS17 for identifying whether the bacteroides fragilis and the probiotic bacterial strain bacteroides fragilis BFS17 exist in a sample.
The invention provides a group of molecular targets for detecting bacteroides fragilis and bacteroides fragilis BFS17, wherein the nucleotide sequence of the molecular target of the bacteroides fragilis (Bacteroides fragilis) is shown as SEQ ID NO.1, and the nucleotide sequence of the molecular target of the probiotic bacterial strain bacteroides fragilis BFS17 is shown as SEQ ID NO. 2.
The invention also provides a primer group for detecting the bacteroides fragilis and the bacteroides fragilis BFS17, wherein the primer group comprises the following components: the primers for detecting the bacteroides fragilis comprise primers shown as SEQ ID NO.3 and SEQ ID NO. 4; the primers for detecting the bacteroides fragilis BFS17 comprise primers shown in SEQ ID NO.5 and SEQ ID NO. 6.
The invention also provides a method for detecting the bacteroides fragilis and the bacteroides fragilis BFS17, extracting DNA of a sample, and then carrying out PCR amplification by using the primer group for detecting the bacteroides fragilis, wherein if the obtained band size is 204bp, the bacteroides fragilis contains bacteroides; if the obtained band size is 203bp, the Bacteroides fragilis is contained.
Preferably, the PCR reaction system is 2X PrimeSTAR Max Premix 12.5.5 mu L, the forward primer is 10 mu mol/L1 mu L, the reverse primer is 10 mu mol/L1 mu L, the template DNA2.5 ng/mu L1 mu L, and ddH 2 O 9.5μL。
Preferably, the PCR reaction procedure is a pre-denaturation at 98℃for 3min; denaturation at 98℃for 10s, annealing at 60℃for 5s, elongation at 72℃for 5s, and a total of 35 cycles; extending at 72℃for 10min.
The invention also provides a qPCR detection method for quantifying the bacteroides fragilis BFS17 of the bacteroides fragilis and probiotic strain, which comprises the following steps:
(1) Respectively drawing standard curves of the bacteroides fragilis and the bacteroides fragilis BFS17 by using the primers for detecting the bacteroides fragilis and the bacteroides fragilis BFS17;
(2) And performing qPCR amplification on the sample by using the primer group for detecting the bacteroides fragilis and the bacteroides fragilis BFS17 to obtain a ct value of the sample, and calculating the content of the bacteroides fragilis BFS17 containing the bacteroides fragilis and the probiotic strain in the sample according to a standard curve.
Preferably, the qPCR amplification system is: 2X SYBR Green Premix reaction solution 5. Mu.L, template DNA to be detected 1. Mu.L, forward and reverse primers (10. Mu. Mol/L) each 0.5. Mu.L, sterilized double distilled water 3. Mu.L.
Preferably, the qPCR reaction procedure is a two-step PCR reaction procedure: preheating at 95 ℃ for 30s; the two-step method is carried out for amplifying at 95 ℃ for 5s and annealing at 60 ℃ for 30s, and 40 cycles are carried out.
The invention also provides a kit for detecting the bacteroides fragilis BFS17 of the bacteroides fragilis and probiotic strains, which comprises the primer group for detecting the bacteroides fragilis and the bacteroides fragilis BFS17.
Preferably, the kit further comprises: 2 XPCR Mix, template DNA and DEPC water.
The invention also provides the molecular targets for detecting the bacteroides fragilis and the probiotics bacteroides fragilis BFS17 and the application of the primer group for detecting the bacteroides fragilis and the probiotics bacteroides fragilis BFS17 in detecting the bacteroides fragilis and the probiotics bacteroides fragilis BFS17.
The invention has the beneficial effects that:
compared with the prior art, the detection method has the advantages of short detection time, simple and convenient operation, high accuracy and low cost, can detect the existence of the bacteroides fragilis and the probiotic bacterial strain bacteroides fragilis BFS17 in a sample without experiments such as microbial culture and the like, and has high practicability; the specific detection target has single specificity, the detection result is specific, and the result judgment is simple; the invention can qualitatively and quantitatively judge the existence of the bacteroides fragilis BFS17 of the bacteroides fragilis and the probiotics strain in the sample, and has wide application range.
The invention adopts a genome-wide method to obtain specific core genes of the bacteroides fragilis and the probiotics strain bacteroides fragilis BFS17, designs and obtains characteristic primers capable of sensitively detecting the bacteroides fragilis and the probiotics strain bacteroides fragilis BFS17, and simultaneously can rapidly and simply distinguish the bacteroides fragilis, the probiotics strain bacteroides fragilis BFS17 and other microbial strains. The invention has the advantages of simple and quick operation, high specificity, quantification and low detection cost.
Bacteroides fragilis BFS17 it was deposited at the Cantonese microorganism strain collection (GDMCC) at month 16 of 2023, accession number: building 5, building 59, guangzhou City, guangdong, first, china, qinghai, china: 510070 with accession number GDMCC No:63172.
description of the drawings:
FIG. 1 shows that the results of comparison of the 16S rRNA gene sequence of Bacteroides fragilis BFS17 on NCBI indicate that the 16S rRNA gene sequence of BFS17 has the highest similarity with the 16S rRNA gene sequences of Bacteroides fragilis ATCC 25285 and Bacteroides fragilis NCTC 9343 in NCBI database, and the gene coverage is 100% and the similarity is 99.43%.
FIG. 2 is a diagram showing specific probiotic excavation of Bacteroides fragilis BFS17
A. In order to compare the amino acid metabolism synthesis genes of different bacteroides fragilis by genome analysis, it is seen that although all the bacteroides fragilis isolates carry the glutamate decarboxylase synthesis gene gadB, the BFS17 and BFS22 with good anti-inflammatory effects specifically contain the amino acid synthesis gene gadC compared with other bacteroides fragilis with poor effect; B. dynamic observation of the levels of gamma-aminobutyric acid (GABA) in the supernatants of Bacteroides fragilis BFS17 and BFS22 fermentation broths increased continuously with the increase of culture time and bacterial load, and under the same culture conditions, the GABA production level of BFS17 is obviously higher than that of BFS22.
FIG. 3 is a heat map of gene expression in a Brevibacterium fragilis strain obtained from a ubiquity analysis: the left side is a genetic evolutionary tree constructed from the flood genome sequence of a representative strain of bacteroides fragilis; the right heat map shows the presence (black) or absence (white) of various genes of each representative Bacteroides fragilis. The analysis shows that the bacteroides fragilis has 739 core genes.
FIG. 4 shows the rapid detection of Bacteroides fragilis BFS17 as a probiotic strain in accordance with the present invention: A. detection of 5 Bacteroides fragilis isolates and 19 non-Bacteroides fragilis strains by using specific primers of Bacteroides fragilis: as can be seen, the specific primers are used for detecting 5 strains of different species of bacteroides fragilis (BF 1-BF 5), bright bands appear at 204bp, and the specific primers are used for detecting 19 strains of non-bacteroides fragilis (BF 6-BF 24), and no bands are detected at 204 bp; B. detection of the specific primer of Bacteroides fragilis BS17 on the probiotic bacterial strain, namely the BFS17 and 23 non-BFS 17 bacterial strains: as can be seen, the specific primers are used for detecting the Propionibacterium fragilis BFS17, bright bands appear at 203bp, while the specific primers are used for detecting other 5 Propionibacterium fragilis isolates (1-5), 1 Bacteroides thetaiotaomicron (6), 1 Bacteroides simplex (7), 1 Bacteroides oval (8) and 15 common human body and food-derived microorganism strains (9-23), no bands are detected at 203 bp.
FIG. 5 is a quantitative detection of a sample containing Bacteroides fragilis and probiotic bacterial strain Bacteroides fragilis BFS17 according to the present invention, and a quantitative detection of a sample containing Bacteroides fragilis and probiotic bacterial strain Bacteroides fragilis BFS17 according to the qPCR detection scheme provided by the present invention: the result of A (1) shows that when the colony concentration of the bacteroides fragilis in the sample is more than or equal to 1 multiplied by 10 2 When CFU/mL, the sample can detect fluorescent signals, A (2) the inventionThe quantitative detection effect on the bacterial colony samples containing the bacteroides fragilis with different concentrations is good, and the fitting degree of the standard curve is high; b (1) results show that when the colony concentration of the probiotic bacterial strain bacteroides fragilis BFS17 in the sample is more than or equal to 1 multiplied by 10 2 When CFU/mL is carried out, a fluorescent signal can be detected by the sample, and B (2) the quantitative detection effect of the invention on bacterial colony samples containing the probiotics strain BFS17 with different concentrations is good, and the fitting degree of a standard curve is high.
The specific embodiment is as follows:
in order to more clearly demonstrate the technical scheme, objects and advantages of the present invention, the present invention is described in further detail below with reference to the specific embodiments and the accompanying drawings.
Example 1 detection target mining of Bacteroides fragilis based on ubiquitination
1. Separation and preservation of bacteroides
The intestinal tract samples of healthy young people in China are collected for 30 parts. Under the aseptic environment, 0.1g of intestinal tract sample is taken and resuspended in 10mL of physiological saline, and 0.5mL of bacterial liquid is sucked for gradient dilution. Adding physiological saline to make into 10 -1 To 10 -9 Diluting gradient bacterial suspension, selecting 10 -7 、10 -8 、10 -9 Three gradient bacterial suspensions were respectively aspirated 100. Mu.L to Bacteroides separation medium, smeared uniformly with a spreading bar, and cultured at 37℃under anaerobic conditions for 48h. Picking a typical colony on a flat plate to a bacteroides separation culture medium for streak purification, picking a single colony after purification, inoculating the single colony to a bacteroides liquid culture medium for expansion culture, carrying out anaerobic culture at 37 ℃ for 48 hours, and then preserving 30% glycerol in an ultralow temperature refrigerator at-80 ℃. 82 probiotics were obtained from 30 healthy young intestinal samples.
2. Identification of Bacteroides
Bacterial DNA extraction was performed using a bacterial DNA extraction kit (Cracker, china), and then PCR amplification was performed using 2 XPCR mix (Cracker). The PCR amplification primers used were the 16S rRNA gene universal primers (27F and 1492R) (Garity GM, et al 2016.). The PCR reaction conditions were: pre-denaturation at 95℃for 5min; for 35 cycles of 95 ℃ 30s,56 ℃ 30s and 72 ℃ 1min30s, and annealing and extending for 10min at 72 ℃. And performing gel cutting recovery on the PCR product, and then performing first-generation sequencing. The obtained 16S rRNA gene sequences were aligned to NCBI database (https:// blast. NCBI. Lm. Nih. Gov), and the results showed the highest homology to Bacteroides (FIG. 1). The bacterial strains with similarity of Identity and Coverage to the known bacteroides of more than 99% in the comparison result can be determined to be bacteroides.
After identification, 42 bacteroides (BFS 17 and BFS 22) exist in 82 probiotics. Wherein the strain 16S rRNA gene sequence claimed in the patent is aligned with NCBI database (https:// blast. NCBI. Lm. Nih. Gov), and the result shows that the homology with the Lactobacillus fragilis is highest, and the strain is named as Bacteroides fragilis (Bacteroides fragilis) BFS17. Bacteroides fragilis (Bacteroides fragilis) BFS17 was deposited at the Guangdong province microbiological bacterial collection center (GDMCC) at 2023, 2 months and 16 days, accession number: building 5, building 59, guangzhou City, guangdong, first, china, qinghai, china: 510070 with accession number GDMCC No:63172.
3. digging and verifying beneficial metabolites of bacteroides fragilis
1. Genome level bacteroides fragilis BFS17 beneficial metabolite genome
Comparative genomic analysis of BFS17, BFS22 and other Bacteroides fragilis isolates without IBS inflammatory improvement was performed using the Prokka software and the Roary software, and the results are shown in FIG. 2A. Comparative genomic analysis revealed that all bacteroides fragilis in this study carried the gadB gene, whereas only bacteroides fragilis BFS17 and BFS22 carried the gadB and gadC genes at the same time, whereas the strain without ameliorating inflammatory effects did not carry the gadC gene. The current study indicates that the gadB gene is responsible for encoding glutamate decarboxylase in bacteria, which catalyzes the decarboxylation of glutamate in bacterial cells, consuming one proton to produce gamma-aminobutyric acid (gamma-aminobutyric acid, GABA) (De Biase D, mol Microbiol, 2012). In 2012, a professor team at Qinghai university found that GABA synthesized by bacteria could not be transported directly to the outside of the cell, and it was necessary to rely on an antiport protein GadC (responsible for the coding synthesis by the gadC gene) for the transport of GABA from the inside to the outside of the cell (Dan Ma, nature.2012). GadC can increase the GABA yield of the strain fermentation supernatant on the one hand, and can assist the strain to remove intracellular acidic substances such as GABA on the other hand, so that the inhibition effect of the acidic substances on the strain growth is relieved.
Recent studies indicate that GABA, especially GABA produced by microorganisms, which contains lipopeptides, is closely related to symptoms of IBS such as visceral hypersensitivity, abdominal pain, depression and anxiety (larute V, et al elife,2022;Petitfils C,et al.Gut.2022.). Thus, BFS17 and BFS22 may improve IBS symptoms, alleviate visceral hypersensitivity, and alleviate depressive anxiety levels by synthesizing and secreting GABA.
2. Metabolome level bacteroides BFS17 beneficial metabolite validation
BFS17 monoclonal colony is picked to BHIych culture medium, anaerobic fermentation is carried out in an anaerobic workstation at 37 ℃, and the GABA content in the BFS17 fermentation metabolite is quantitatively analyzed by using a GABA micro-method detection kit (enzyme-linked biology, china) during fermentation, and the result is shown in figure 2B. The results show that as the fermentation time is prolonged, the bacterial load of BFS17 and BFS22 is gradually increased, and the GABA content in the fermentation supernatant is also gradually increased, while the GABA of the supernatant of BFS17 is higher than that of BFS22 in 24 hours (521.34 mg/L vs 185.85mg/L, P < 0.001); and the gap is gradually increased with time to 48 hours, and GABA in the fermentation supernatant of BFS17 is further increased to 1089.36mg/L which is higher than that of the fermentation supernatant of BFS22 (515.50 mg/L); by 72 hours, GABA in the BFS17 fermentation supernatant was as high as 1221.64mg/L, higher than that of BFS22 fermentation supernatant (546.43 mg/L). The result suggests that BFS17 has stronger GABA synthesis function than BFS22.
4. Downloading the whole genome database of Bacteroides in the GenBank database in NCBI and established earlier by the inventor team, covers 455 genome sequences of Bacteroides fragilis. After annotating all the bacteroides fragilis genome sequences by using Prokka software (v 1.13), functional genes with similarity of more than or equal to 85% are clustered into the same gene cluster by using a universal genome analysis software Roary, and a matrix list of all the analyzed genomes is generated, as shown in figure 3. The genes common to all bacteroides fragilis in the matrix list are considered to be core genes of bacteroides fragilis. Extracting the nucleotide sequence of the core gene of the bacteroides fragilis, comparing the NCBI non-redundant protein databases one by one, removing the sequences existing in other species at the same time, and the rest is the potential specific molecular target of the bacteroides fragilis. Meanwhile, the specific nucleotide sequences of the BFS17 species of the probiotic bacterial strain are extracted, NCBI non-redundant protein databases are compared, sequences which exist in other species simultaneously are removed, and the rest is the potential specific molecular target of the BFS17 species of the probiotic bacterial strain. And (3) carrying out Primer design on the potential specific molecular targets by using NCBI database Primer-BLAST to obtain corresponding detection primers and detection target fragments. And (3) comparing the detection target fragment with the NCBI non-redundant gene database again, and removing sequences with similarity of more than 85% in other species to obtain the strain-specific molecular target suitable for molecular detection. And subsequently, applying the screened targets to detection in an actual sample, and selecting targets still having good detection specificity and sensitivity in the actual sample as specific molecular targets for detecting the bacteroides fragilis and the probiotic bacterial strain bacteroides fragilis BFS17.
According to the above flow, further screening and verification analysis are carried out to obtain specific molecular target sequences SEQ ID NO. 1-SEQ ID NO.2 with specific identification of Bacteroides fragilis and Bacteroides fragilis BFS17, namely, the specific identification nucleotide sequence of Bacteroides fragilis is shown as SEQ ID NO.1, and the specific identification nucleotide sequence of the probiotic bacterial strain Bacteroides fragilis BFS17 is shown as SEQ ID NO. 2.
Example 2 establishment of novel detection target Rapid detection method of Bacteroides fragilis BFS17
(1) The embodiment provides a method for detecting bacteroides fragilis and bacteroides fragilis BFS17, which comprises the following specific operations:
the primers SEQ ID NO. 1-SEQ ID NO.2, which can specifically amplify the target and obtain the target fragment, were designed by using Primer-BLAST software according to the specific detection targets SEQ ID NO. 1-SEQ ID NO.2 of Bacteroides fragilis and Bacteroides fragilis BFS17 obtained in example 1, and the rapid detection method was obtained as shown in Table 1, comprising the steps of:
TABLE 1 Bacteroides and Bacteroides fragilis specific PCR detection primer sequences
PCR detection system and amplification procedure:
extracting the genome DNA of the microorganism to be detected by adopting a microorganism genome DNA extraction kit (CHARY, china), and then adding the microorganism genome DNA into a PCR detection reaction system of the bacteroides fragilis and the bacteroides fragilis BFS17. The PCR reaction system of Bacteroides fragilis BFS17 is configured as follows:
the following PCR conditions were as follows:
after the PCR was completed, 5. Mu.LPCR was taken and subjected to 1.5% agarose electrophoresis. If the electrophoresis result shows that the single amplification band appears at the target band of the amplification product, the sample contains the corresponding bacteroides fragilis or probiotic bacterial strain bacteroides fragilis BFS17; if no corresponding single amplified band appears, the sample does not contain bacteroides fragilis or the probiotic strain bacteroides fragilis BFS17. In the invention, the size of a target band of the bacteroides fragilis is detected to be 204bp, and a nucleotide sequence shown as an underlined section sequence of SEQ ID NO.1 can be obtained by Sanger sequencing; the target band size of the bacteroides fragilis BFS17 is detected to be 203bp, and the nucleotide sequence shown as the underlined sequence of SEQ ID NO.2 can be obtained by Sanger sequencing.
(2) Sensitivity and specificity evaluation of novel specific detection target rapid detection method of bacteroides fragilis
The scheme (primers BF_F and BF_R) in the step (1) is adopted to detect 5 isolates of Bacteroides fragilis from different sources and 19 other common non-Bacteroides fragilis, the electrophoresis result is shown in figure 4A, and the result is shown in Table 2.
TABLE 2 detection results of novel specific detection targets of Bacteroides fragilis
(3) Sensitivity and specificity evaluation of novel specific detection target rapid detection method of bacteroides fragilis BFS17
The probiotic bacterial strain Bacteroides fragilis BFS17, other 5 Bacteroides fragilis isolates (1-5), bacteroides thetaiotaomicron 1 (6), bacteroides simplex 1 (7), bacteroides ovale 1 (8) and 15 common human and food-derived microbial strains (9-23) were detected by the scheme (primers S17_F and S17_R) in step (1), the electrophoresis results are shown in FIG. 4B, and the results are shown in Table 3.
TABLE 3 detection results of novel specific detection targets of Bacteroides fragilis
Example 3 Bacteroides fragilis BFS17 specific novel quantitative detection method for detecting target
(1) The embodiment provides a method for quantitatively detecting bacteroides fragilis BFS17 of bacteroides fragilis and probiotic bacterial strains in a sample, which adopts SEQ ID NO. 3-SEQ ID NO.6 as a primer of a qPCR amplification reaction and comprises the following specific operations:
primers for identifying specific nucleotide by bacteroides fragilis
Forward primer sequence SEQ ID NO.3
5′-GAGCTTTTGTAGCTGTCCGC-3′
Reverse primer sequence SEQ ID NO.4
5′-CGCCTGCCTGTTTGTCTTTG-3′
Primers for identifying specific nucleotide by bacteroides fragilis BFS17
Forward primer sequence SEQ ID No.5
5′-GTTAATCATCGCTGCGGCAC-3′
Reverse primer sequence SEQ ID No.6
5′-CAACTTCACGTCGCACGAAT-3′
(1) Template DNA preparation: extracting microorganism DNA in a BFS17 sample containing bacteroides fragilis or bacteroides fragilis by using a microorganism DNA extraction kit to serve as a template to be detected;
(2) qPCR detection system and amplification procedure:
the DNA template preparation method required for qPCR detection was performed using a tissue microorganism DNA extraction protocol. The qPCR reaction system for detecting the bacteroides is configured as follows:
the following is a qPCR amplification procedure:
(3) qPCR result reading:
by Roche
The system is amplified and detected on a 96 fluorescent quantitative amplification instrument, and software is utilized
96SW 1.1 reads the amplification result. On the premise that a blank control does not have a fluorescent signal, if the fluorescent signal is generated, the sample contains the bacteroides fragilis or probiotic bacterial strain bacteroides fragilis BFS17 corresponding to the detection primer; if the fluorescent signal is not generated, the sample does not contain the bacteroides fragilis or the probiotic strain bacteroides fragilis BFS17 corresponding to the detection primer.
(2) Evaluation of detection sensitivity of novel detection scheme on Bacteroides fragilis or Bacteroides fragilis BFS17
The concentration was 1X 10 by using physiological saline 9 Diluting the CFU/mL of Bacteroides fragilis or Bacteroides fragilis BFS17 according to a gradient of 10 times to obtain a concentration of 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1 The bacterial strain pure culture of CFU/mL is subjected to quantitative detection of the bacteroides fragilis or the bacteroides fragilis BFS17 in the sample according to the qPCR scheme after bacterial strain DNA is extracted, and each concentration sample is subjected to three parallel experiments.
Drawing a standard curve: taking the logarithm of the concentration of the bacteroides fragilis or the bacteroides fragilis BFS17 in the sample as an abscissa, taking the real-time Ct value of the corresponding qPCR as an ordinate, and fitting the obtained curve to obtain a standard curve for quantitatively detecting the bacteroides fragilis or the bacteroides fragilis BFS17. The standard curve is shown in fig. 5A (2) and fig. 5B (2), and the detection limit of the primer pair in the sample is 1×10, namely the bacteroides fragilis or bacteroides fragilis BFS17 2 CFU/mL. The result shows that the fitting standard curve of the bacteroides fragilis detection is y= -2.3394x+32.215, and the correlation coefficient R 2 0.9265; the fitting standard curve of the bacteroides fragilis detection is y= -2.3886x+33.607, and the correlation coefficient R 2 0.9194.
(3) qPCR quantitative detection of bacteroides fragilis or bacteroides fragilis BFS17 in actual sample
1g of human feces sample was collected, and microbial DNA in feces was extracted using a feces microbial DNA extraction kit, followed by qPCR detection as in example 3 (1). qPCR detection is carried out by adopting specific primers of bacteroides fragilis, ct value of a detection sample is found to be 22.30+/-0.13, and the amount of bacteroides fragilis bacteria contained in the sample is found to be 1 multiplied by 10 according to standard curve calculation 4.24 CFU/mL; qPCR detection is carried out by adopting specific primers of the bacteroides fragilis BFS17, the Ct value of a detection sample is found to be 24.72+/-0.04, and the bacterial load of the bacteroides fragilis BFS17 in the sample is found to be 1 multiplied by 10 according to standard curve calculation 3.72 CFU/mL。
1. Specific recognition nucleotide sequence of bacteroides
SEQ.ID NO.1
ATGCGAGCTTTTGTAGCTGTCCGCCAATTGGCCTCGGCTCTTCCGGTGAATGACATCACCAGGCTGCAA GATGAAATAAAAGAGTTAAAAGAATATGTAGAAGCGGCATTTGCCGATTACAATGATATTAATGAAGATACGAGGAT GCAACTTGAATTAATTAATCAGGCAATTGCTGAATTGCAGGCCAAAGACAAACAGGCAGGCGGAAAAACTCGTAATCCGATCGGTTTTATATC
TTATTAA
2. Specific recognition nucleotide sequence of bacteroides fragilis
SEQ.ID NO.2
ATGAACAAGAACATCAGACAAAAGTTAATCATCGCTGCGGCACTTATGATTGCCGCAAC
CGCCTCCGCTTTCGCACAGGGAAACGGTCTGGCTGGCATCAACGAAGCCACCTCTATGG
TGAGTTCGTATTTCGACCCCGGCACGAAATTAATCTACGCCATCGGCGCGGTAGTCGGGC
TTATCGGTGGCGTGAAAGTGTACGGAAAATTCGTGCGACGTGAAGTTGCGTAA
3. Primers for identifying specific nucleotide by bacteroides fragilis
Forward primer sequence SEQ ID NO.3
5′-GAGCTTTTGTAGCTGTCCGC-3′
Reverse primer sequence SEQ ID NO.4
5′-CGCCTGCCTGTTTGTCTTTG-3′
4. Primers for identifying specific nucleotide by bacteroides fragilis BFS17
Forward primer sequence SEQ ID No.5
5′-GTTAATCATCGCTGCGGCAC-3′
Reverse primer sequence SEQ ID NO.6
5′-CAACTTCACGTCGCACGAAT-3′
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. The molecular targets for detecting the bacteroides fragilis and the bacteroides fragilis BFS17 are characterized in that the molecular target nucleotide sequence of the bacteroides fragilis (Bacteroides fragilis) is shown as SEQ ID NO.1, and the molecular target nucleotide sequence of the bacteroides fragilis BFS17 is shown as SEQ ID NO. 2.
2. A primer set for detecting bacteroides fragilis and bacteroides fragilis BFS17, wherein the primer set comprises: the primers for detecting the bacteroides fragilis comprise primers shown as SEQ ID NO.3 and SEQ ID NO. 4; the primers for detecting the bacteroides fragilis BFS17 comprise primers shown in SEQ ID NO.5 and SEQ ID NO. 6.
3. Use of the molecular target of claim 1 or the primer set of claim 2 for detecting bacteroides fragilis, BFS17.
4. A method for detecting Bacteroides fragilis and Bacteroides fragilis BFS17 by a non-disease diagnosis and treatment method is characterized in that DNA of a sample is extracted, and then PCR amplification is respectively carried out by using the primer set in claim 2, if the obtained band size is 204bp, the DNA contains Bacteroides fragilis; if the obtained band size is 203bp, the strain contains bacteroides fragilis BFS17.
5. The method according to claim 4, wherein the PCR amplification is performed in a reaction system of 2X PrimeSTARMax Premix 12.5.5. Mu.L, 10. Mu. Mol/L of forward primer 1. Mu.L, 10. Mu. Mol/L of reverse primer 1. Mu.L, 2.5 ng/. Mu.L of template DNA 1. Mu.L, ddH 2 O 9.5μL。
6. The method of claim 4, wherein the PCR amplification is performed at 98℃for 3min; denaturation at 98℃for 10s, annealing at 60℃for 5s, elongation at 72℃for 5s, and a total of 35 cycles; extending at 72℃for 10min.
7. The qPCR detection method for quantifying the bacteroides fragilis BFS17 of the bacteroides fragilis and probiotic strain is characterized by comprising the following steps:
(1) Drawing standard curves of bacteroides fragilis and bacteroides fragilis BFS17 by using the primers for detecting bacteroides fragilis and bacteroides fragilis BFS17 according to claim 2;
(2) The primer group for detecting the bacteroides fragilis and the bacteroides fragilis BFS17 is used for qPCR amplification of a sample to obtain a ct value of the sample, and the content of the bacteroides fragilis BFS17 containing the bacteroides fragilis and the probiotic bacterial strain in the sample is calculated according to a standard curve.
8. The qPCR detection method according to claim 7, wherein the qPCR amplification system is: 5. Mu.L of 2 XSBRGreen Premix reaction solution, 1. Mu.L of template DNA to be detected, 0.5. Mu.L of forward and reverse primers each, and 3. Mu.L of sterilized double distilled water.
9. The qPCR detection method according to claim 7, wherein the qPCR reaction procedure is a two-step PCR reaction procedure: preheating at 95 ℃ for 30s; the two-step method is carried out for amplifying at 95 ℃ for 5s and annealing at 60 ℃ for 30s, and 40 cycles are carried out.
10. A kit for detecting Bacteroides fragilis and Bacteroides fragilis BFS17, which is characterized by comprising the primer group for detecting Bacteroides fragilis and Bacteroides fragilis BFS17 as set forth in claim 2.
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