CN117701742A - Species-specific molecular target of bacteroides dorsalis and rapid detection method thereof - Google Patents
Species-specific molecular target of bacteroides dorsalis and rapid detection method thereof Download PDFInfo
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Abstract
The invention discloses a species-specific molecular target of bacteroides dorsalis and a rapid detection method thereof, wherein the sequence of the molecular target of bacteroides dorsalis is shown as SEQ ID NO. 1. The invention designs and obtains a special primer capable of sensitively detecting the bacteroides dorsalis based on a molecular target sequence: the forward primer F30 has a sequence shown as SEQ ID NO.2, the reverse primer R30 has a sequence shown as SEQ ID NO.3, and can be used for establishing a PCR rapid detection method of the bacteroides dorsalis and a qPCR quantitative detection method of the bacteroides dorsalis, effectively, rapidly and simply distinguishing bacteroides dorsalis and other microbial strains, has the advantages of strong specificity, simplicity in operation and capability of realizing quantification, can be applied to identification and quantification of the bacteroides dorsalis in samples such as food, excrement and the like, and provides technical support for detection and industrial application of the bacteroides dorsalis.
Description
Technical Field
The invention belongs to the technical field of microorganism detection, and particularly relates to a bacteroides dorsalis strain specific molecular target and a rapid detection method thereof.
Background
Bacteroides are a class of rod-shaped, sporefree, bile-tolerant, gram-negative strict anaerobes whose major byproducts of anaerobic respiration are acetic acid, isovaleric acid and succinic acid. As the important basic stone fungus of human intestinal canal, the bacteroides accounts for about 25 percent of the total number of human intestinal canal microorganisms, can establish stable symbiotic relation with human beings, can utilize carbohydrate fermentation to produce short chain fatty acid beneficial to human health, and is beneficial to regulating intestinal canal microenvironment and maintaining physiological functions of intestinal canal. Bacteroides are involved in many important metabolic activities in the human colon, and in addition to fermenting carbohydrates, nitrogen-containing substances can also be utilized, as well as to complete bioconversion of bile acids and other steroids. Bacteroides are closely related to human health, play an important role in human immune system regulation, increase in bone mineral density, and production of the inhibitory neurotransmitter gamma-aminobutyric acid, and decrease thereof is related to various diseases such as coronary artery disease, inflammatory bowel disease, and the like. Notably, part of bacteroides may carry a toxin-encoding gene, produce toxins, cause diarrhea and other symptoms in the host; in addition, when Bacteroides reaches a body part other than the intestinal tract, infection of the central nervous system, skin, soft tissues and the like may be caused. Bacteroides multocida (Bacteroides dorei) was originally isolated from human feces by Bakir et al and was reported to be effective in converting cholesterol to stigmasterol, thereby preventing cardiovascular disease caused by high-fat diet. Studies have shown that the abundance of Bacteroides dorsalis in patients with coronary artery disease is significantly reduced; in addition, bacteroides dorsalis has been shown to exert anti-influenza effects by promoting early interferon expression and down-regulating local and systemic inflammatory responses, and it is seen that bacteroides dorsalis is of interest for human health benefits. In recent years, the field of probiotics research has been unprecedented, and first generation probiotics such as bifidobacteria and lactobacillus have been widely used for the prevention and adjuvant treatment of human diseases. The bacteroides represented by the strains such as bacteroides fragilis and bacteroides dorsalis beneficial to human health are very likely to develop into the dominant strain of the second generation probiotics by virtue of the unique physiological activity and the advantages derived from human intestinal tracts, thereby playing a role in preventing and relieving diseases. Therefore, the presence of the bacteroides dorsalis is accurately and sensitively detected, the quantitative analysis of the bacteroides dorsalis is realized, and the method has important significance in the fields of probiotic product development and the like.
At present, the detection of the bacteroides is mainly as follows: based on morphological detection after separation and culture, biochemical test, mass spectrometry detection, etc. The method needs to perform enrichment and selective culture on the sample under anaerobic conditions, obtain single bacterial colonies, and then perform microscopic observation, biochemical identification or on-machine detection, so that the experimental operation is complicated, the time consumption is long, the cost is high, and quantitative analysis cannot be performed. In recent years, molecular biology has rapidly progressed, and detection methods based on nucleic acid amplification are established successively, and such methods have remarkable advantages in terms of detection time, specificity and sensitivity. The molecular biological detection method based on PCR gradually replaces the traditional culture and biochemical detection method, and becomes one of the most potential novel technologies in microorganism detection. The key of the nucleic acid detection method is the selection of target genes or target sequences, namely searching the nucleotide sequences specific to target species for detection design. Thanks to the development of the whole genome sequencing technology of the microorganism, the specific molecular detection target is obtained based on the whole genome sequence comparison analysis, and the PCR identification technology with simplicity, rapidness, economy, high efficiency and high sensitivity can be developed.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a species-specific molecular target for detecting and identifying bacteroides dorsalis and a corresponding detection method thereof, which can distinguish bacteroides dorsalis from bacteroides and other microorganisms.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a specific detection molecular target for detecting bacteroides dorsalis, wherein the bacteroides dorsalis molecular target has a nucleotide sequence shown as SEQ ID NO. 1.
The invention also provides a group of primers for detecting the specific molecular target of the bacteroides dorsalis, wherein the primers comprise a forward primer with a nucleotide sequence shown as SEQ ID NO.2 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 3.
The invention also provides a kit for detecting the bacteroides dorsalis, which comprises the primer for detecting the specific molecular target of the bacteroides dorsalis.
The invention also provides application of the specific molecular target of the bacteroides dorsalis, the primer or the kit for detecting the bacteroides dorsalis in detecting the bacteroides dorsalis.
The invention also provides a method for detecting the bacteroides dorsalis for the purposes of non-disease diagnosis and treatment, which comprises the following steps:
extracting DNA of microorganism of a sample to be detected, carrying out PCR amplification on the microorganism by adopting the primer for detecting the bacteroides dorsalis, and carrying out gel electrophoresis on an amplified product, if a band with the size of 193bp is obtained, judging that the bacteroides dorsalis is detected in the sample, and if the band with the size of 193bp is not obtained, judging that the bacteroides dorsalis is not detected in the sample.
Preferably, the PCR amplification system is 25. Mu.L, which comprises: 2X Dream Taq Green PCR Master Mix12.5. Mu.L, 10. Mu. Mol/L forward primer and reverse primer each 0.5. Mu.L, 1. Mu.L DNA template, 10.5. Mu.L sterilized double distilled water.
Preferably, the PCR reaction program is as follows: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 68℃for 30s, extension at 72℃for 30s, and a total of 34 cycles; extending at 72℃for 5min.
The invention also provides a qPCR detection method for quantifying the bacteroides dorsalis for non-disease diagnosis and treatment purposes, which comprises the following steps:
(1) Drawing a standard curve by using the primer for detecting the bacteroides dorsalis;
(2) The primer for detecting the bacteroides dorsalis is used for carrying out qPCR amplification on the sample to obtain a Ct value of the sample, and the content of the bacteroides dorsalis in the sample is calculated according to a standard curve.
Preferably, the qPCR amplification system is 20 μl, which comprises: 2X TB Green Premix EX Taq II 10. Mu.L, 10. Mu. Mol/L forward and reverse primers each 0.2. Mu.L, sterilized double distilled water 7.6. Mu.L, and DNA solution to be measured 2. Mu.L.
Preferably, the qPCR reaction procedure is a two-step PCR reaction procedure: preheating at 95 ℃ for 30s; denaturation at 95℃for 5s and annealing at 68℃for 30s were performed for 45 cycles in total.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) The invention digs out the specific molecular target of the bacteroides dorsalis strain by the pan genome analysis based on the whole genome sequencing information of the bacteroides dorsalis, and adopts the PCR amplification technology to verify, thereby having accurate and reliable result.
(2) The method for detecting the bacteroides dorsalis provided by the invention does not need to be subjected to steps such as microbial cultivation, has the characteristics of simplicity in operation, economy, rapidness, high efficiency and sensitivity, and can be suitable for various complex samples.
(3) The bacteroides dorsalis detection method provided by the invention can be qualitative and quantitative, and has a wide application range.
Drawings
FIG. 1 shows the rapid detection of Bacteroides multocida and 24 other microbial strains using the specific molecular targets of Bacteroides multocida species of the present invention, and shows that the detection of Bacteroides multocida (strain No. 1) using the specific primers showed a bright band at 193bp, while the detection of 24 strains other than Bacteroides multocida using the specific primers did not show a band at 193 bp. In the figure, the lane labeled M is DL2000 DNA Marker, and the lane labeled C is blank.
FIG. 2 shows the rapid detection of Bacteroides multocida in a detection model using the specific molecular target of Bacteroides multocida strain of the present invention as a substrate, A, B, C corresponds to three food systems: mineral water, milk and 10% bean dreg suspension. The results suggested that the positive group amplification products all showed a bright band at 193bp, and the negative group did not show the band.
FIG. 3 shows the quantitative detection of a sample containing Bacteroides thetaiotaomicron by the qPCR detection method provided by the invention: the numbers 1, 2, 3, 4, 5, 6, 7, 8 in the A legend respectively represent a concentration of 10 1 、10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 Amplification curve of bacterial liquid of CFU/mL by extracting DNA with bacterial DNA extraction kit and then carrying out qPCR detection, A result shows that when the concentration of bacterial colony of Bacteroides multocida in sample is more than or equal to 1X 10 1 When CFU/mL, the sample can detect stable fluorescent signals; the result B shows that the method has good quantitative detection effect on bacterial liquid samples containing the bacteroides dorsalis at different concentrations, and the fitting degree of a standard curve is high.
Detailed Description
The following examples are further illustrative of the invention and are not intended to be limiting thereof.
EXAMPLE 1 excavation of Bacteroides multocida species-specific molecular targets
The whole genome sequence used for the screening of the specific molecular targets of Bacteroides multocida is derived from the genome sequence of the strain in a public database (https:// www.ncbi.nlm.nih.gov /). Total genome data of 10 bacteroides dorsalis (Bacteroides dorei) and total genome data of 20 other bacteroides were selected and subjected to flood genome analysis using Roary v 3.11.2. The core genome of each strain adopts a 99% threshold value, the BLASTP homology threshold value is set to be 95%, and 100% genes existing in all bacteroides dorsalis and bacteroides paradenticola are screened out, namely the genes belong to the core genome, but can be separated at the threshold value of 95% of homology. And further comparing the screened core genes through the local Blast to obtain conserved genes containing specific molecular targets, and verifying through NCBI database Blast. And finally obtaining the specific molecular target of the bacteroides dorsalis strain with the nucleotide sequence shown as SEQ ID NO.1 after PCR amplification verification.
The nucleotide sequence of the specific molecular target of the bacteroides dorsalis strain is shown as SEQ ID NO.1, and the specific sequence is as follows:CGGCTGCGCGAAATGGGATTCATCCTGACTCTCAATACGAACGGCACACTCATCGACAACGAAATGGTACGCA TCTTGCAGACCCACAAGCCGCGGCGGATAAACGTCACCCTGTATGGAGACAGCAGAGAAACTTACGGACGCTTGTG CCACAATCCGCAAGGATACACCCTGTGCATGGAAGCCCTGAAAC。
example 2 establishment of a method for Rapid detection of specific molecular targets of Bacteroides Persiana species
(1) The embodiment provides a method for detecting bacteroides dorsalis, which comprises the following specific operations:
a pair of specific amplification primers is designed according to the sequence SEQ ID NO.1, and the sequences of the primers are as follows:
forward primer F30:5'-CGGCTGCGCGAAATGGGATTCATCCTGACT-3' (SEQ ID NO. 2);
forward primer R30:5'-GTTTCAGGGCTTCCATGCACAGGGTGTATC-3' (SEQ ID NO. 3).
PCR was performed using genomic DNA of Bacteroides multocida isolated from human fecal samples in the laboratory as a template (the Bacteroides multocida strain had completed whole genome sequencing and was subjected to average nucleotide homology analysis with the Bacteroides dorei standard strain UHGG_MGYG-HGUT-02478, with an ANI value > 99%). And (3) extracting the genome DNA of the microorganism to be detected by using a bacterial DNA extraction kit (Michao, china), and adding the extracted genome DNA into a PCR detection reaction system.
The PCR reaction system was 25. Mu.L and was configured as follows: dream Taq Green PCR Master Mix (2X) 12.5. Mu.L, 10. Mu. Mol/L forward and reverse primers each 0.5. Mu.L, 1. Mu.L DNA solution, 10.5. Mu.L sterilized double distilled water.
The PCR reaction procedure was: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 68℃for 30s, extension at 72℃for 30s, and a total of 34 cycles; extending at 72℃for 5min.
After the PCR is finished, taking 6 mu L of PCR reaction liquid, detecting a PCR product by agarose gel electrophoresis (agarose concentration is 2%), judging whether the gel electrophoresis result shows that an amplification band appears at 193bp of an amplification product, and if so, indicating that the bacteroides dorsalis is detected in a sample; if the corresponding amplified band does not appear, the fact that the bacteroides dorsalis is not detected in the sample is indicated. In the invention, the target band size of the bacteroides dorsalis is 193bp, and the nucleotide sequence shown as the underlined sequence of SEQ ID NO.1 can be obtained by Sanger sequencing.
(2) Specificity evaluation of Paecilomyces dorsalis strain specificity molecular target rapid detection method
24 common non-bacteroides dorsalis strains are selected, and the specific coverage is as follows: the probiotic strains such as bacteroides and paramecilomyces strains, bifidobacteria and lactobacillus which are common in human intestinal tracts or strains which are reported to be beneficial to human health, normal colonising bacteria in animal intestinal tracts such as Escherichia coli (Escherichia coli) and enterococcus faecalis (Enterococcus faecalis), common intestinal conditional pathogenic bacteria, gram negative bacteria and gram positive bacteria are used for comparison among the same genus species and non-same species in the detection of bacteroides dorsalis. The above steps are adopted to detect the Paecilomyces dorsalis isolates and 24 common Paecilomyces dorsalis, the electrophoresis result is shown in figure 1, and the result interpretation is shown in table 1.
Table 1: detection result of specific molecular target of bacteroides dorsalis species
(3) Application of Paecilomyces dorsalis strain specific molecular target rapid detection method in food system
A detection model using a solution, a turbid liquid and a complex food system having a plurality of dispersions as a matrix was constructed using 1 strain of Bacteroides multocida and 11 strains of other species of microorganisms shown in Table 2, and combinations of microorganisms in the detection model are shown in Table 3. The strains of Table 2 were cultured under conditions suitable for use in the culture, and centrifuged at 12000gCollecting thallus for 1min, re-suspending with physiological saline, and diluting to about 10 7 Bacterial suspension with CFU/mL concentration is ready for use. Then mixing the above bacterial suspensions according to the same ratio according to the combination of Table 3 (combination No. A-F), centrifuging to obtain bacterial cells, adding into three food systems (mineral water, milk, 10% bean dreg suspension) and re-suspending to obtain final concentration of each bacterial in food system of 10 6 CFU/mL, the detection model taking the food system as the matrix is obtained.
Table 2: strain information used for constructing detection model
Note that: in the table, class I is Bacteroides multocida, class II is Bacteroides or Paramycolatopsis strains other than Bacteroides multocida, and class III is a strain other than Bacteroides or Paramycolatopsis.
Table 3: detection of combinations of microorganisms in a model
Extracting the microbial genome DNA of a sample to be detected by adopting a water boiling method, and then adding the microbial genome DNA into a PCR detection reaction system to detect the bacteroides dorsalis.
The method for extracting the genome DNA of the microorganism by the water boiling method comprises the following steps: centrifuging the sample to be detected at 12000g for 3min, washing twice with an equal volume of physiological saline, centrifuging at 12000g for 3min, then re-suspending the precipitate in an equal volume of sterile water, placing in a boiling water bath for 15min, cooling to room temperature, placing in a-20 ℃ for refrigeration for 5min, and finally centrifuging at 12000g for 3min, wherein the supernatant is the DNA solution.
The PCR reaction system and the PCR reaction program are as in the step (1), 6 mu L of PCR reaction liquid is taken after the PCR is finished, the PCR product detection is carried out by agarose gel electrophoresis (the agarose concentration is 2%), whether the gel electrophoresis result shows that the amplified product has amplified bands at 193bp or not is judged, if the positive group has the bands, and meanwhile, the negative group has no bands, the food system does not interfere with the detection of the bacteroides dorsalis. The detection results of the PCR electrophoresis products of the detection model are shown in FIG. 2, and the results are shown in Table 4.
Table 4: detection results of bacteroides dorsalis in different detection models
Example 3 quantitative detection method of Bacteroides multocida species-specific molecular target
(1) The embodiment provides a method for quantitatively detecting bacteroides dorsalis in a sample, wherein the adopted qPCR amplification primer is F30:5'-CGGCTGCGCGAAATGGGATTCATCCTGACT-3' and R30:5'-GTTTCAGGGCTTCCATGCACAGGGTGTATC-3', the specific operation is as follows:
(1) template DNA preparation: and extracting microbial DNA in the sample to be detected by adopting a bacterial DNA extraction kit so as to obtain a DNA solution sample to be detected.
(2) qPCR detection system and amplification procedure:
the qPCR amplification system was 20. Mu.L, which included: TB Green Premix EX Taq II (2X) 10. Mu.L, 10. Mu. Mol/L forward and reverse primers each 0.2. Mu.L, sterilized double distilled water 7.6. Mu.L, and a DNA solution to be measured 2. Mu.L.
By RocheThe 96 real-time fluorescent quantitative PCR instrument carries out amplification detection on the system, and the qPCR reaction program is a two-step PCR reaction program: preheating at 95 ℃ for 30s; denaturation at 95℃for 5s and annealing at 68℃for 30s were performed for 45 cycles in total.
(3) qPCR result analysis:
using96Software 1.1 readAnalyzing the amplification detection result, when generating fluorescent signal, if the melting curve is single peak and T m The value is between 86 and 87 ℃, which indicates that the bacteroides dorsalis is detected in the sample; if no fluorescent signal is generated, or T m If the value is not within the above range, bacteroides thetaiotaomicron was not detected in the sample.
(2) Quantitative detection method evaluates detection sensitivity of bacteroides dorsalis
The concentration is 1X 10 by using physiological saline 8 Diluting the CFU/mL Bacteroides multocida according to a gradient of 10 times to obtain the concentration of 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1 After bacterial DNA extraction kit is adopted to extract bacterial strain DNA in CFU/mL bacterial solution, quantitative detection of bacteroides dorsalis is carried out according to the qPCR scheme, and three parallel experiments are respectively carried out on each concentration sample.
Drawing a standard curve: and taking the logarithmic value of the concentration of the bacteroides dorsalis liquid in the sample as an abscissa, and taking the corresponding qPCR average Ct value as an ordinate, wherein the fitting curve is the standard curve for the quantification detection of the bacteroides dorsalis. As shown in FIG. 3, the detection limit of the primer pair in the sample is 1×10 1 CFU/mL, the fitting standard curve of the Bacteroides multocida detection is y= -3.0671x+35.027, and the correlation coefficient R 2 0.9817.
(3) qPCR quantitative detection of bacteroides dorsalis in actual sample
(1) Adding fresh bacterial liquid of Bacteroides multocida, bifidobacterium and Lactobacillus plantarum in the same proportion into milk in an exogenous manner, and fully suspending to ensure that the final concentration of each bacterium in the milk is about 10 6 CFU/mL (wherein the concentration of Bacteroides multocida was 9.1X10 by the method of reference GB 4789.2-2022) 5 CFU/mL). Centrifuging the milk sample in 12000g for 3min, washing twice with equal volume of physiological saline, centrifuging in 12000g for 3min, extracting microorganism DNA with bacterial DNA extraction kit, qPCR detecting according to example 3 (1), wherein Ct value of the detected sample is 16.70+ -0.07, and calculating according to standard curve to obtain milk sample containing Bacteroides dorsalis bacteria 1×10 5.98 CFU/mL (i.e., 9.5X10) 5 CFU/mL milk), seeThe quantitative detection of the bacteroides dorsalis by adopting the method is close to the result of the classical plate counting method;
(2) collecting human feces sample 7g, suspending in 5 times of physiological saline, centrifuging for 5min at 450g, collecting supernatant 1mL, extracting microorganism DNA in feces with bacterial DNA extraction kit, qPCR detecting according to example 3 (1), and calculating according to standard curve to obtain sample with Ct value of 19.38+ -0.22 and Bacteroides multocida bacterial load of 1×10 5.10 CFU/mL (i.e., 6.29×10 5 CFU/g faeces).
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.
Claims (10)
1. A specific detection molecular target for detecting bacteroides dorsalis is characterized in that the bacteroides dorsalis molecular target has a nucleotide sequence shown as SEQ ID NO. 1.
2. A set of primers for detecting a molecular target according to claim 1, wherein the primers comprise a forward primer with a nucleotide sequence shown as SEQ ID NO.2 and a reverse primer with a nucleotide sequence shown as SEQ ID NO. 3.
3. A kit for detecting bacteroides dorsalis, comprising the primer of claim 2.
4. Use of the molecular target of claim 1, the primer of claim 2 or the kit of claim 3 for detecting bacteroides dorsalis.
5. A method for detecting bacteroides dorsalis for non-disease diagnosis and treatment purposes, comprising the steps of:
extracting DNA of a microorganism in a sample to be detected, carrying out PCR amplification by using the primer of claim 2, carrying out gel electrophoresis on an amplified product, judging that the bacteroides dorsalis is detected in the sample if a band with the size of 193bp is obtained, and judging that the bacteroides dorsalis is not detected in the sample if the band with the size of 193bp is not obtained.
6. The method of claim 5, wherein the PCR amplification system is 25 μl, comprising: 2X Dream Taq Green PCR Master Mix 12.5.5. Mu.L, 10. Mu. Mol/L forward primer and reverse primer each 0.5. Mu.L, 1. Mu.L DNA template, 10.5. Mu.L sterilized double distilled water.
7. The method of claim 5, wherein the PCR reaction procedure is: pre-denaturation at 95℃for 3min;
denaturation at 95℃for 30s, annealing at 68℃for 30s, extension at 72℃for 30s, and a total of 34 cycles; extending at 72℃for 5min.
8. A qPCR detection method for quantifying bacteroides dorsalis for non-disease diagnosis and treatment purposes, comprising the steps of:
(1) Drawing a standard curve using the primer of claim 2;
(2) qPCR amplification is carried out on a sample by using the primer of claim 2, a Ct value of the sample is obtained, and the content of the bacteroides dorsalis in the sample is calculated according to a standard curve.
9. The qPCR detection method according to claim 8, wherein the qPCR amplification system is 20 μl, comprising: 2X TB Green Premix EX Taq II 10. Mu.L, 10. Mu. Mol/L forward and reverse primers each 0.2. Mu.L, sterilized double distilled water 7.6. Mu.L, and DNA solution to be measured 2. Mu.L.
10. The qPCR detection method according to claim 8, wherein the qPCR reaction procedure is a two-step PCR reaction procedure: preheating at 95 ℃ for 30s; denaturation at 95℃for 5s and annealing at 68℃for 30s were performed for 45 cycles in total.
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