CN111996270A - Reagent for detecting bifidobacterium in feces and application thereof - Google Patents
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Abstract
The application discloses a reagent for detecting bifidobacterium in excrement and application thereof. The reagent for detecting the bifidobacterium in the excrement comprises an upstream primer with a sequence shown in Seq ID No.1 and a downstream primer with a sequence shown in Seq ID No. 2. The reagent for detecting the bifidobacterium in the excrement can accurately and effectively detect the bifidobacterium in the excrement from nucleic acid extracted from an excrement sample, can better avoid the omission of the bifidobacterium in the excrement and has important significance for clinical examination guidance and medication.
Description
Technical Field
The application relates to the field of fecal microbe detection, in particular to a reagent for detecting bifidobacterium in feces and application thereof.
Background
A large number of different types of microorganisms live in the intestinal tract of human beings, and the microecological biomembrane barrier formed by the microorganisms plays an important role in maintaining the normal immune defense function of the human body. Intestinal microorganisms are the most bulky and complex micro-ecosystem in the human body. The intestinal microorganisms and metabolites thereof can regulate human health, and are important reference indexes of human health or diseases.
Among the intestinal microorganisms, Bifidobacterium (Bifidobacterium) accounts for about 3% of the normal adult intestinal flora, and the infant intestinal flora accounts for 75% -91%, is an important physiological bacterium for maintaining the intestinal microecological balance, and has the effects of preventing diarrhea, preventing intestinal infection, relieving constipation and regulating immunity. Therefore, how to rapidly and effectively detect the bifidobacteria in the human intestinal tract has important significance for analyzing the intestinal flora structure, clinically inspecting and guiding and taking medicines, keeping the dynamic balance of the intestinal flora, maintaining health, preventing diseases and the like.
At present, the conventional method for detecting the bifidobacteria in the intestinal tract of a human body is to detect the bifidobacteria in excrement, and the conventional method is to separate and count the faecal sample by a dilution seed-dropping method, so that the method is time-consuming and labor-consuming, has high technical dependence on operators and can better detect the bifidobacteria by experiential testers. In addition, research reports also adopt methods such as conventional PCR, etc. to carry out conventional PCR amplification detection on nucleic acid extracted from a stool sample; however, due to the inherent defects of the conventional PCR detection system, missed detection or false negative is easily generated.
Disclosure of Invention
The purpose of the present application is to provide a new reagent for the detection of bifidobacteria in feces and its use.
The following technical scheme is adopted in the application:
the first aspect of the application discloses a reagent for detecting bifidobacterium in excrement, which comprises an upstream primer with a sequence shown in Seq ID No.1 and a downstream primer with a sequence shown in Seq ID No.2,
Seq ID No.1：5’-ATACCCTGGTAGTCCACGC-3’
Seq ID No.2：5’-ACATCCAGCATCCACCGT-3’。
the reagent of the present application is capable of specifically detecting bifidobacterium faecalis in a faecal sample. The subject of the present application is nucleic acid extracted from a stool sample; in principle, genomic information from humans, as well as genomic information from various microorganisms in feces, is contained in these nucleic acids. However, the genomic information of which microorganisms are contained in a particular fecal nucleic acid sample is uncertain; the reagents of the present application can be used to determine or exclude the presence of faecal bifidobacteria in a faecal nucleic acid sample. In terms of detection principle, the present application relates to detection by PCR amplification using genus-specific detection primers of bifidobacterium faecalis, and related studies on PCR detection of bifidobacterium have been made in the prior art, but the existing PCR detection scheme for bifidobacterium cannot effectively detect bifidobacterium faecalis in a faecal nucleic acid sample. Therefore, the present application has developed a new reagent capable of specifically detecting fecal bifidobacteria among fecal microorganisms.
In one implementation of the present application, the reagents of the present application further comprise reagents for dye-based real-time fluorescent quantitative PCR amplification.
In one implementation of the present application, the reagent for real-time fluorescent quantitative PCR amplification by dye method comprises TB Green Premix Ex Taq reaction solution and ROX reference dye.
It is noted that the reagent for real-time fluorescence quantitative PCR amplification by dye method is combined together, so that the application is convenient to use, and the detection of the bifidobacterium in the feces is convenient; it is understood that reagents for dye-based real-time fluorescent quantitative PCR amplification are also commercially available.
In a second aspect of the present application, the use of the present reagent for the detection of bifidobacteria in stool for the detection of fecal microorganisms for non-diagnostic therapeutic purposes is disclosed.
In a third aspect of the present application, there is disclosed a method for detecting Bifidobacterium in feces for non-diagnostic therapeutic purposes, comprising subjecting a nucleic acid sample obtained from a fecal sample to dye-based real-time quantitative fluorescence PCR amplification using the reagent for detecting Bifidobacterium in feces of the present application, and determining whether the fecal sample contains Bifidobacterium in the feces based on the amplification result.
In an implementation manner of the present application, the detection method further includes performing melting curve analysis on the dye-based real-time fluorescence quantitative PCR amplification result, and if the melting curve has a single peak, determining that the detection result has strong specificity, and if the melting curve has no single peak, determining that the detection result has non-specific amplification or primer dimer.
It will be appreciated that the reagents of the present application are capable of specifically detecting faecal bifidobacteria and therefore, provided that an amplification curve is present, it is essentially possible to determine that a faecal sample contains faecal bifidobacteria; the determination is made by a melting curve, and may be selected and used as needed only for further determination of the specificity of the amplified product.
In an implementation manner of the present application, the detection method further includes sequencing the real-time fluorescence quantitative PCR amplification product by the dye method, and determining whether the amplification product is fecal bifidobacterium according to the sequencing result.
It can be understood that sequencing is the most intuitive judgment method, and the sequencing result is consistent with the fecal bifidobacterium, so that the fecal bifidobacterium in the fecal sample can be directly and accurately indicated. In principle, the faecal bifidobacteria can be accurately judged by the amplification curve judgment and the melting curve analysis of the application, and sequencing analysis can be selected according to requirements.
In one implementation of the present application, sequencing a dye-based real-time fluorescent quantitative PCR amplification product specifically includes connecting the dye-based real-time fluorescent quantitative PCR amplification product to a vector, and then sequencing a recombinant vector.
It should be noted that, the PCR amplification product is recombined into the vector for sequencing, mainly to ensure the stability of the PCR amplification product, so as to facilitate the accurate analysis and judgment of the subsequent sequencing; in theory, the PCR amplification product can also be directly sequenced, and is not particularly limited herein.
In one implementation of the present application, the vector is a T-vector. It will be appreciated that the T vector is a conventionally used plasmid vector, and it is not excluded that other vectors may also be employed.
In one implementation of the present application, determining whether the bifidobacterium is faecal bifidobacterium or not according to the sequencing result specifically includes performing BLAST comparison on the sequencing result by using NCBI and determining whether the bifidobacterium is faecal bifidobacterium or not according to the comparison result.
The beneficial effect of this application lies in:
the reagent for detecting the bifidobacterium in the excrement can accurately and effectively detect the bifidobacterium in the excrement from nucleic acid extracted from an excrement sample, can better avoid the omission of the bifidobacterium in the excrement and has important significance for clinical examination guidance and medication.
Drawings
FIG. 1 is a real-time fluorescent quantitative PCR amplification curve of a fecal nucleic acid sample by a dye method in an example of the present application;
FIG. 2 is a real-time fluorescence quantitative PCR amplification melting curve of a fecal nucleic acid sample by a dye method in the present example;
FIG. 3 is a diagram showing the sequence alignment of the sequencing results of the real-time fluorescent quantitative PCR amplification products of the fecal nucleic acid sample by the dye method in the present example;
FIG. 4 is a BLAST alignment chart of sequencing results of real-time fluorescent quantitative PCR amplification products of stool nucleic acid samples by dye method in the examples of the present application.
Detailed Description
The genus bifidobacterium is a gram-positive, immotile, strictly anaerobic bacterium with rod-shaped cells and sometimes bifurcated ends, and is widely present in the physiological environments of the digestive tract, vagina and oral cavity of humans and animals. Bacteria of the genus bifidobacterium are one of the important components of the human and animal intestinal flora, and some strains of bifidobacterium may be used as probiotics in the food, pharmaceutical and feed sectors. The bifidobacterium is an important beneficial microorganism in intestinal tracts, is used as a physiological beneficial bacterium, and has various important physiological functions of biological barrier, nutrition, anti-tumor, immunity enhancement, gastrointestinal tract function improvement, aging resistance and the like on human health. Studies have shown that bifidobacteria have some association with diabetes: the number of Bifidobacterium with the function of protecting intestinal barrier in diabetes mellitus patients is remarkably increased, so that the permeability of intestinal tracts is increased, free antigens in the intestinal tracts, such as Lipopolysaccharide (LPS), Free Fatty Acid (FFA) and the like enter blood, metabolic endotoxemia is caused, systemic chronic inflammation is caused, metabolic diseases such as obesity, diabetes mellitus and the like are finally caused, and bifidobacteria and glycosylated hemoglobin are in negative correlation. Therefore, the rapid and effective detection of the bifidobacterium in the feces is of great significance. However, the conventional detection method is liable to cause omission or false negatives, and cannot satisfy the use requirements such as clinical examination instructions and medication.
Based on the above research and knowledge, the present application has developed a novel reagent capable of specifically detecting Bifidobacterium faecalis in fecal microorganisms, comprising an upstream primer having a sequence shown in Seq ID No.1 and a downstream primer having a sequence shown in Seq ID No. 2.
The reagent can accurately and effectively detect the bifidobacterium in the excrement from the nucleic acid sample in the excrement, thereby providing an important reference basis for clinical examination guidance and medication.
The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application. In the following description, numerous details are set forth in order to provide a better understanding of the present application. However, one skilled in the art will readily recognize that some of the features may be omitted in different instances or may be replaced by other kits, materials, methods. In some instances, certain operations related to the present application have not been shown or described in detail in this specification in order to avoid obscuring the core of the present application from excessive description, and it is not necessary for those skilled in the art to describe these operations in detail, so that the related operations will be fully understood from the description in the specification and the general knowledge of the art. In the following examples, the reagents or instruments used are not indicated by manufacturers, but are all conventional products available on the market.
Examples
Materials and methods
1. Fecal nucleic acid extraction
In this example, the nucleic acid was extracted from feces using a kit for extracting genomic DNA from the rhizobacteria under the accession number DP302-02, and the detailed method steps were as described in the specification.
In this example, 8 nucleic acid samples from different stool sources were extracted for subsequent testing.
2. Primer design and Synthesis
In this example, specific detection primers were designed for Bifidobacterium faecalis in fecal microorganisms, and the primers were synthesized by Biotechnology engineering (Shanghai) Co., Ltd. The designed primer sequences are shown in Table 1.
TABLE 1 primers for the detection of Bifidobacterium faecalis specificity
| Primer name | Sequence (5 '→ 3') | SeqIDNo. | |
| Bifidobacterim- | ATACCCTGGTAGTCCACGC | 1 | |
| Bifidobacterim- | ACATCCAGCATCCACCGT | 2 |
3. Fecal nucleic acid sample detection
The specific detection primers of the bifidobacterium faecalis designed in the example are adopted to carry out dye-based real-time fluorescence quantitative PCR amplification (QPCR) detection on the extracted nucleic acid samples of the faeces, a water negative control is arranged, the QPCR reaction system is shown in the table 2, and the QPCR reaction conditions are shown in the table 3.
TABLE 2 QPCR reaction System
| Name of reactant | Dosage of |
| TB Green Premix Ex Taq(2×) | 5.0μL |
| ROX | 1.0μL |
| 10mM Bifidobacterim-F | 0.2μL |
| 10mM Bifidobacterim-R | 0.2μL |
| DNA template | 1.0μL |
| ddH2O | 2.6μL |
| Total of | 10μL |
TABLE 3 QPCR reaction conditions
In this example, QPCR products from all nucleic acid samples were purified, ligated to T-vector, sequenced by Kingzhi sequencing Inc., BLAST the sequencing results using NCBI, and analyzed for specific species of QPCR products. Wherein, the QPCR product is purified by adopting the DNA Clean-up Kit of Kangji century, the product has the code of CW2301M, and the specific method steps refer to the specification. T vector ligation Using the full-size gold pEASY-T5 Cloning Kit for ligation transformation, cat 501, the specific method steps are referred to the description.
Second, results and analysis
1. Real-time fluorescent quantitative PCR amplification result by dye method
The results of QPCR detection on 8 nucleic acid samples in this example are shown in FIG. 1, and show that Bifidobacterium faecalis was detected in all 8 nucleic acid samples. Furthermore, the QPCR melting curves of the 8 nucleic acid samples are shown in fig. 2, and it can be seen that the QPCR melting curves of all fecal nucleic acid samples have a single peak, indicating that the target sequence of bifidobacterium sp was obtained by amplification with the desired specificity.
2. Sequencing and sequence alignment results
The QPCR products from 8 nucleic acid samples were sequenced and BLAST aligned using NCBI, with the results shown in figures 3 and 4. FIG. 3 shows the alignment of partial sequences of the sequencing results, and FIG. 4 shows the alignment of BLAST. The results of the alignment showed that the QPCR products of the 8 nucleic acid samples were of bifidobacterium faecalis, consistent with the expectation, indicating that the primers designed in this example were successful in detecting bifidobacterium faecalis in faecal nucleic acid samples.
Therefore, the specific detection primer designed in this example can be used as a reagent for detecting Bifidobacterium faecalis in fecal microorganisms, and with the reagent of this example, Bifidobacterium faecalis can be accurately and efficiently detected from a nucleic acid sample extracted from a fecal sample.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.
SEQUENCE LISTING
<110> one Macro (Shenzhen) Gene Co Ltd
<120> reagent for detecting bifidobacterium in feces and application thereof
<130> 20I30394
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 19
<212> DNA
<213> Artificial sequence
<400> 1
ataccctggt agtccacgc 19
<210> 2
<211> 18
<212> DNA
<213> Artificial sequence
<400> 2
Claims (10)
1. A reagent for the detection of bifidobacteria in feces, characterized in that: comprises an upstream primer of the sequence shown in Seq ID No.1 and a downstream primer of the sequence shown in Seq ID No.2,
Seq ID No.1：5’-ATACCCTGGTAGTCCACGC-3’
Seq ID No.2：5’-ACATCCAGCATCCACCGT-3’。
2. the reagent for the detection of Bifidobacterium in feces according to claim 1, characterized in that: also comprises a reagent for real-time fluorescent quantitative PCR amplification by a dye method.
3. The reagent for the detection of Bifidobacterium in feces according to claim 2, characterized in that: the reagent for dye method real-time fluorescent quantitative PCR amplification comprises TB Green Premix Ex Taq reaction solution and ROX reference dye.
4. Use of a reagent for the detection of bifidobacterium in stool according to any one of claims 1 to 3 for the detection of stool microorganisms for non-diagnostic therapeutic purposes.
5. A method for the detection of faecal bifidobacteria for non-diagnostic therapeutic purposes, characterized in that: the method comprises the steps of carrying out dye-based real-time fluorescence quantitative PCR amplification on a nucleic acid sample obtained from a fecal sample by using the reagent for detecting Bifidobacterium in feces according to any one of claims 1 to 3, and judging whether the fecal sample contains the Bifidobacterium in feces or not according to the amplification result.
6. The detection method according to claim 5, characterized in that: and performing melting curve analysis on the dye method real-time fluorescence quantitative PCR amplification result, judging that the specificity of the detection result is strong if the melting curve has a single peak, and judging that the detection result has non-specific amplification or primer dimer if the melting curve has a single peak.
7. The detection method according to claim 5 or 6, characterized in that: and sequencing the real-time fluorescence quantitative PCR amplification product by a dye method, and judging whether the amplification product is the fecal bifidobacterium or not according to a sequencing result.
8. The detection method according to claim 7, characterized in that: the sequencing method of the dye-process real-time fluorescence quantitative PCR amplification product specifically comprises the steps of connecting the dye-process real-time fluorescence quantitative PCR amplification product with a vector, and then sequencing the recombinant vector.
9. The detection method according to claim 8, characterized in that: the vector is a T vector.
10. The detection method according to claim 7, characterized in that: and judging whether the bacillus is the faecal bifidobacterium or not according to the sequencing result, specifically, carrying out BLAST comparison on the sequencing result by using NCBI (national center for Biotechnology information) and judging whether the bacillus is the faecal bifidobacterium or not according to the comparison result.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040033547A1 (en) * | 2001-09-28 | 2004-02-19 | Field Katharine G | Detection of fecal contamination |
| JP2014068545A (en) * | 2012-09-27 | 2014-04-21 | Meiji Co Ltd | Method for detecting bifidobacterium |
| CN107653306A (en) * | 2017-11-13 | 2018-02-02 | 江南大学 | A kind of Bifidobacterium quick determination method and application based on high-flux sequence |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040033547A1 (en) * | 2001-09-28 | 2004-02-19 | Field Katharine G | Detection of fecal contamination |
| JP2014068545A (en) * | 2012-09-27 | 2014-04-21 | Meiji Co Ltd | Method for detecting bifidobacterium |
| CN107653306A (en) * | 2017-11-13 | 2018-02-02 | 江南大学 | A kind of Bifidobacterium quick determination method and application based on high-flux sequence |
Non-Patent Citations (2)
| Title |
|---|
| MARK J HOPKINS等: "Characterisation of intestinal bacteria in infant stools using real-time PCR and northern hybridisation analyses", 《FEMS MICROBIOL ECOL》, vol. 54, no. 1, 1 September 2005 (2005-09-01), pages 78 * |
| 郭世奎等: "SYBR GreenⅠ实时荧光定量PCR法分析结直肠癌患者肠道菌群变化", 《中国普外基础与临床杂志》, vol. 17, no. 5, 25 May 2010 (2010-05-25), pages 465 * |
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