PCR (Polymerase Chain Reaction) method for rapidly screening effective donors for FMT (Fibroblast Modeling

Abstract

The invention provides a PCR (polymerase chain reaction) method for rapidly screening effective donors for FMT (fibronectin) treatment of depression, which comprises the following steps: (1) collecting excrement samples of the donors, and extracting total microbial genome DNA (deoxyribonucleic acid) of the excrement samples; (2) designing six primer pairs according to a specific conserved sequence of a target strain; (3) taking the extracted donor excrement DNA as a PCR (Polymerase Chain Reaction) template, and adding a primer pair for amplification; (4) observing a PCR result through electrophoresis; and if each target band is positive in the PCR electrophoresis result of the donor sample, determining that the donor contains the target strain, and determining that the donor is an effective donor for FMT treatment of depression. The effective donor suitable for FMT treatment of depression is screened out by detecting whether the donor faeces contain three key probiotics closely related to depression symptom relieving or not, a basis is provided for microbial therapy of mental diseases such as depression, and important clinical application value is achieved.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology and medical detection technology, and in particular to a polymerase chain reaction (PCR) method for rapidly screening effective donors for fecal microbiota transplantation (FMT) treatment of depression. Background Art

[0002] Depression is a common chronic mental disorder. Studies in recent years have shown that intestinal flora has an important influence on the occurrence and development of depression through the "brain-gut axis". Fecal microbiota transplantation (FMT) is a therapy that transplants the intestinal flora of healthy individuals into the intestines of patients to restore the balance of the patient's intestinal microecology. Clinical and animal experiments have found that FMT from healthy donors can help alleviate the host's depressive-like symptoms and improve the efficacy of antidepressants. However, there are large individual differences in the efficacy of FMT in chronic diseases such as depression, and not all fecal microbes from donors can effectively improve patient symptoms. Some studies have proposed the concept of "super donors", believing that certain donors can significantly improve the success rate of FMT due to the diversity and specific components of their intestinal flora. Further analysis shows that there may be keystone species in the donor flora that determine the therapeutic effect after transplantation.

[0003] In recent years, research on the efficacy of FMT for depression has increasingly focused on the impact of donor microbiome composition on treatment outcomes. Studies have reported that successful colonization of the intestines of patients with depression treated with FMT by certain probiotics from donors is positively correlated with significant relief of depressive symptoms. These microbiota primarily consist of anaerobic probiotics that produce short-chain fatty acids (such as butyrate), including Eubacterium rectale, Lachnospiraeligens, and Enterococcus faecalis SGB15346. Notably, these strains are typically significantly deficient in the intestines of patients with depression. Their introduction and colonization via FMT are expected to improve the host's inflammatory state and metabolic function, thereby alleviating depressive symptoms. Therefore, rapid screening of donors containing these key probiotics before FMT is performed is a key issue in improving the efficacy of FMT for depression.

[0004] However, current conventional donor screening focuses primarily on pathogens and basic health indicators, without specific detection methods for donor "effectiveness" (i.e., whether the donor contains the key probiotics mentioned above). This present invention addresses this gap by providing a rapid PCR-based detection method for identifying FMT donors with potentially high efficacy. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a PCR method for rapidly screening effective donors for FMT treatment of depression. By detecting whether the donor's feces contains three key probiotics (Eubacterium rectale, Lachnospira eligens, Faecalibacterium SGB15346) that are closely related to the relief of depression symptoms, the method can screen out effective donors suitable for FMT treatment of depression, providing a basis for microbial therapy for mental illnesses such as depression, and has important clinical application value.

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] A PCR method for rapidly screening effective donors for FMT treatment of depression comprises the following steps:

[0008] (1) Sample collection and processing

[0009] Fecal samples from candidate donors were collected and their total microbial genomic DNA was extracted under sterile conditions;

[0010] (2) Specific primer design

[0011] Six primer pairs were designed for the specific conserved sequences of the target strains E. rectale, L. eligens, and Faecalibacterium SGB15346, namely primer pairs 1, 2, 3, 4, 5, and 6 (each primer pair includes a forward primer and a reverse primer).

[0012] Among them, primer pair 1 and primer pair 2 are for strain E. rectale, the forward primer and reverse primer sequences of primer pair 1 are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively, and the forward primer and reverse primer sequences of primer pair 2 are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively;

[0013] Primer pair 3 and primer pair 4 are specific for the strain L. eligens. The forward primer and reverse primer sequences of primer pair 3 are shown as SEQ ID NO. 5 and SEQ ID NO. 6, respectively. The forward primer and reverse primer sequences of primer pair 4 are shown as SEQ ID NO. 7 and SEQ ID NO. 8, respectively.

[0014] Primer pair 5 and primer pair 6 are specific for strain Faecalibacterium SGB15346. The forward primer and reverse primer sequences of primer pair 5 are shown as SEQ ID NO.9 and SEQ ID NO.10, respectively. The forward primer and reverse primer sequences of primer pair 6 are shown as SEQ ID NO.11 and SEQ ID NO.12, respectively.

[0015] (3) PCR amplification reaction

[0016] The donor fecal DNA extracted in step (1) is used as a PCR template, and the primer pair of step (2) is added to perform an amplification reaction;

[0017] (4) Product detection and judgment

[0018] PCR results were observed by agarose gel electrophoresis. If all target bands were positive in the PCR electrophoresis results of the donor sample, the donor was determined to contain the three target strains, E. rectale, L. eligens, and Faecalibacterium SGB15346, and was identified as an effective donor for FMT treatment of depression. Conversely, if any target band was missing or significantly weaker than the detection limit, the donor's bacterial flora was deemed to be potentially unsuitable for antidepressant efficacy and was not considered a preferred donor.

[0019] As one of the preferred embodiments of the present invention, in step (1), a stool DNA extraction kit is used to extract total microbial genomic DNA according to the instructions to ensure that high-quality template DNA is obtained.

[0020] As one of the preferred embodiments of the present invention, in step (2), the primers are 20 bp in length, have high specificity, and have no significant homology with non-target bacteria; and each primer is designed to have a different size for the expected amplification product to facilitate subsequent differentiation by gel electrophoresis.

[0021] As one of the preferred embodiments of the present invention, in step (3), a single amplification or multiplex PCR amplification method is used to perform the amplification reaction; after the amplification is completed, if the target bacteria are present, a target band of a specific size will be generated, otherwise no corresponding band will be generated.

[0022] As one of the preferred embodiments of the present invention, a multiplex PCR amplification method is used, in which three pairs of primers are added to one reaction to simultaneously detect three bacteria.

[0023] As one of the preferred embodiments of the present invention, the PCR reaction system (25 μL) for the multiplex PCR amplification is as follows: 1× PCR buffer containing 2.0 mM Mg 2+; dNTP, final concentration 0.2 mM; forward and reverse primers of six primer pairs, final concentration 0.4 μM each; template DNA, 50 ng; Taq DNA polymerase, 1 U; ultrapure water to 25 μL.

[0024] As one of the preferred embodiments of the present invention, the amplification program of the multiplex PCR amplification is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s, for a total of 35 cycles; and extension at 72°C for 5 min.

[0025] As one of the preferred embodiments of the present invention, in step (4), the PCR products are separated by 1.5% agarose gel electrophoresis, and the corresponding results are observed under a UV gel imaging system after being stained with dye.

[0026] As one of the preferred embodiments of the present invention, in step (4), the target band being positive means that a band corresponding to the target strain appears and the size of the band is consistent with expectations.

[0027] The advantages of the present invention over the prior art are:

[0028] The present invention is the first in the field to propose a specific detection method for the "effectiveness" of donors. It uses specific PCR to rapidly screen the donor's microbial flora (whether it contains three key probiotics closely related to the relief of depression symptoms: Eubacterium rectale, Lachnospira eligens, and Faecalibacterium SGB15346). Compared with 16S sequencing or culture methods, the detection time is greatly shortened (sample processing to result interpretation can be completed within 6 hours), and it is low-cost and easy to operate. At the same time, the method of the present invention does not require complex sequencing analysis and can directly determine the presence or absence of key probiotics in the donor's feces. By pre-screening donors lacking key flora, the method of the present invention helps to improve the success rate of FMT clinical trials and reduce the risk of patients being exposed to inefficient transplants.

[0029] In summary, the present invention is used to screen effective donors suitable for FMT treatment of depression, providing a practical donor selection tool for microbial therapy of psychiatric disorders such as depression, and has important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 These are the agarose gel electrophoresis results after PCR testing of donor samples D24 and D25 in Experimental Example 1 (in the figure, Marker is a DNA molecular weight standard; region D24 is the electrophoresis band of the PCR product of donor sample D24; region D24 is the electrophoresis band of the PCR product of donor sample D25). DETAILED DESCRIPTION

[0031] The following embodiments of the present invention are described in detail. These embodiments are implemented based on the technical solution of the present invention, and detailed implementation methods and specific operating procedures are provided. However, the scope of protection of the present invention is not limited to the following embodiments. Meanwhile, unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0032] Example 1

[0033] The PCR method of this embodiment for rapidly screening effective donors for FMT treatment of depression includes the following steps:

[0034] (1) Sample collection and processing

[0035] Collect fresh stool samples from candidate donors.

[0036] Under sterile conditions, place 5 g of each fresh feces in a sterile centrifuge tube and add sterile physiological saline at a ratio of 1:10 (weight / volume) to make a homogenate. After thorough oscillation and mixing, filter out large particles of impurities through gauze or a fine-pore filter and collect the filtrate. Centrifuge the filtrate (8000 rpm, 5 min) to precipitate the microbial cells and discard the supernatant. Use a fecal genomic DNA extraction kit (QIAamp Fast DNA Stool Mini Kit) or phenol-chloroform extraction method to extract total fecal microbial DNA from the precipitate. The extracted DNA was finally dissolved in nuclease-free water, and its concentration and purity were detected (using a nanocolorimeter to measure A 260 / A 280 The ratio ensures DNA purity is between 1.8 and 2.0) and set aside.

[0037] (2) Specific primer design

[0038] Six primer pairs (each pair includes a forward primer and a reverse primer) were designed for the specific conserved sequences of the target strains E. rectale, L. eligens, and Faecalibacterium SGB15346. The relevant sequences are shown in Table 1.

[0039] Table 1. Target strains detected and their specific primer sequences

[0040]

[0041]

[0042] (3) PCR amplification reaction

[0043] The donor fecal DNA extracted in step (1) was used as a PCR template, and the primer pair of step (2) was added to perform multiplex PCR amplification.

[0044] The PCR reaction system (25 μL) was as follows: 1× PCR buffer (containing 2.0 mM Mg 2+ ); each deoxynucleoside triphosphate (dNTP) at a final concentration of 0.2 mM; forward and reverse primers for each of the six primer pairs at a final concentration of 0.4 μM; template DNA, 50 ng (1-2 μL); Taq DNA polymerase, 1 unit; and ultrapure water to a volume of 25 μL. Mix all components on ice and quickly place in a PCR amplifier for reaction.

[0045] PCR amplification was performed on a gradient PCR instrument using the following protocol: pre-denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s, followed by extension at 72°C for 5 min. After completion of the PCR amplification, the product was temporarily stored at 4°C.

[0046] (4) Product detection and judgment

[0047] Take 5 μL of PCR product, mix with 1 μL of 6× loading dye, and then load. Use 1.5% (w / v) agarose gel (pre-prepared in 1× TAE buffer and added with ethidium bromide dye at a final concentration of 0.5 μg / mL to stain DNA) to separate the amplified products. Use 1× TAE as the electrophoresis buffer and run the electrophoresis at 120 V for approximately 30 minutes. Use a commercial DNA molecular weight standard as a control (TaKaRa DL2000 DNA Marker, containing 100-2000 bp gradient fragments) for simultaneous electrophoresis. After the electrophoresis is completed, observe the gel under a UV transilluminator and take pictures to record the position and intensity of the amplified bands.

[0048] According to the molecular weight gradient provided by the DNA marker, the band size of the PCR product of each sample was compared and analyzed to see whether it was consistent with the expected size of the target fragment (the corresponding PCR product size of the target strain is shown in Table 2), thereby determining the presence of the target bacteria in each donor sample.

[0049] Table 2. Target strains detected and their PCR product sizes

[0050]

[0051] Specifically, if all target bands in the PCR electrophoresis results of the donor sample are positive (the corresponding bands for each target strain appear and the band sizes are consistent with expectations), the donor is judged to contain the three target strains of E. rectale, L. eligens and Faecalibacterium SGB15346, and the donor is identified as an effective donor for FMT treatment of depression; conversely, if any target band is missing or significantly weaker than the detection limit, it is considered that the donor's bacterial flora may not have the ideal antidepressant effect and is not the preferred donor.

[0052] Experimental Example 1

[0053] This experimental example is used to verify the feasibility and effectiveness of the method of the present invention.

[0054] Two volunteer donors (codenamed D24 and D25) who met the standard for routine health screening were selected, and 5 g of fresh fecal samples were collected from each of them. The following procedures were followed according to the method in Example 1:

[0055] (1) Extraction of total fecal microbial DNA

[0056] Under sterile conditions, place 5 g of each fresh feces in a sterile centrifuge tube and add sterile physiological saline at a ratio of 1:10 (weight / volume) to make a homogenate. After thorough oscillation and mixing, filter out large particles of impurities through gauze or a fine-pore filter and collect the filtrate. Centrifuge the filtrate (8000 rpm, 5 min) to precipitate the microbial cells and discard the supernatant. Use a fecal genomic DNA extraction kit (QIAamp Fast DNA Stool Mini Kit) or phenol-chloroform extraction method to extract total fecal microbial DNA from the precipitate. The extracted DNA was finally dissolved in nuclease-free water, and its concentration and purity were detected (using a nanocolorimeter to measure A 260 / A 280 The ratio ensures DNA purity is between 1.8 and 2.0) and set aside.

[0057] (2) Multiplex PCR amplification using the primers listed in Table 1

[0058] Multiplex PCR amplification (simultaneous detection of three target bacteria in a single reaction) was used to screen for key strains in stool samples. Primer sequences were based on the primer pairs listed in Table 1 for Eubacterium rectale, Lachnospira eligens, and Faecalibacterium (SGB15346 group).

[0059] The PCR reaction system (25 μL) was as follows: 1× PCR buffer (containing 2.0 mM Mg 2+ ); each deoxynucleoside triphosphate (dNTP) at a final concentration of 0.2 mM; forward and reverse primers for each of the six primer pairs at a final concentration of 0.4 μM; template DNA, 50 ng (1-2 μL); Taq DNA polymerase, 1 unit; and ultrapure water to a volume of 25 μL. Mix all components on ice and quickly place in a PCR amplifier for reaction.

[0060] PCR amplification was performed on a gradient PCR instrument using the following protocol: pre-denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s, followed by extension at 72°C for 5 min. After completion of the PCR amplification, the product was temporarily stored at 4°C.

[0061] (3) Agarose gel electrophoresis detection

[0062] Take 5 μL of PCR product, mix with 1 μL of 6× loading dye, and then load. Use 1.5% (w / v) agarose gel (pre-prepared in 1× TAE buffer and added with ethidium bromide dye at a final concentration of 0.5 μg / mL to stain DNA) to separate the amplified products. Use 1× TAE as the electrophoresis buffer and run the electrophoresis at 120 V for approximately 30 minutes. Use a commercial DNA molecular weight standard as a control (TaKaRa DL2000 DNA Marker, containing 100-2000 bp gradient fragments) for simultaneous electrophoresis. After the electrophoresis is completed, observe the gel under a UV transilluminator and take pictures to record the position and intensity of the amplified bands.

[0063] According to the molecular weight gradient provided by the DNA marker, the band size of the PCR product of each sample was compared and analyzed to see whether it was consistent with the expected size of the target fragment (the corresponding PCR product size of the target strain is shown in Table 2), thereby determining the presence of the target bacteria in each donor sample.

[0064] (4) PCR screening result determination

[0065] The electrophoresis results are as follows Figure 1 shown.

[0066] Depend on Figure 1 The results, combined with Table 2, show that: six bands clearly appeared on the PCR electrophoresis spectrum of the fecal sample of donor D24, and their positions corresponded to the fragments amplified by six pairs of primers of E.rectale, L.eligens, and Faecalibacterium SGB15346, and the band sizes were consistent with expectations, and the target band detection was determined to be positive; in contrast, only one band appeared in the electrophoresis spectrum of the donor D25 sample, and the size and position were incorrect, indicating that these three key bacteria were not detected in the D25 sample. It can be seen that according to the PCR screening method of the present invention, it is possible to quickly identify whether the donor feces contains the expected key functional flora. The above results indicate that donor D24 contains all three key strains and can be judged as a potential high-efficiency donor; while donor D25 lacks these three key flora, and its effectiveness as a fecal microbiota transplant (FMT) donor is questionable. It is necessary to carefully evaluate or consider a better donor before clinical application.

[0067] This experimental example demonstrates the feasibility and effectiveness of the screening method of the present invention.

[0068] Experimental Example 2

[0069] This experimental example is used to further verify the effectiveness of the method of the present invention.

[0070] For donors D24 (key strain positive) and D25 (key strain negative) obtained by screening in Experimental Example 1, this experimental example verified the effectiveness of the donors through high-throughput sequencing and preliminary clinical trials to evaluate the consistency of the PCR screening results of the present invention with the actual functional effects.

[0071] (1) 16S rRNA gene sequencing analysis of donor bacterial flora

[0072] The fecal microbial communities of the above donors D24 and D25 were subjected to 16S rRNA gene high-throughput sequencing.

[0073] In this experimental example, the same stool DNA samples used for PCR screening were extracted for sequencing analysis (fresh stool DNA samples can also be re-extracted to ensure representative sequencing results). A primer pair targeting the conserved region of the bacterial 16S rRNA gene (from the Takara 16S (V3-V4) Metagenomic Library Construction Kit for NGS, No. R161A) was used to amplify the genomic DNA of each sample using PCR to construct a sequencing library.

[0074] The specific steps include:

[0075] The first PCR amplification produces a 16S amplicon fragment of approximately 400 bp, which is then purified (using magnetic bead reagents to remove primer dimers and impurities). A second round of library construction and amplification is then performed, with sequencing adapters (universal adapters P5 / P7) and sample index tags (Index) added to both ends of the amplicon. The library products with adapters and tags are purified again, quantified, and the sample libraries are mixed in equimolar amounts. After the library passes quality control, it is sequenced using the Illumina high-throughput sequencing platform (2×250 bp paired-end sequencing is performed on the Illumina MiSeq platform to cover the full length of the V3-V4 amplicon).

[0076] The generated raw sequencing data are processed through a bioinformatics analysis pipeline: first, the raw reads are quality controlled (low-quality reads are filtered out and adapter sequences are truncated), and high-quality paired reads are spliced ​​into complete amplicon sequences based on overlapping relationships; then, operational units (OTUs) are clustered at 97% sequence similarity, or amplicon sequence variants (ASVs) are obtained using a denoising method; finally, the taxonomic information of each OTU / ASV is annotated based on authoritative databases (such as Silva or Greengenes), and the relative abundance distribution of each bacterial community in the sample is statistically analyzed.

[0077] (2) Sequencing results and analysis

[0078] High-throughput sequencing results showed:

[0079] There were significant differences in the fecal flora composition of donors D24 and D25. Specifically, the three key flora signals targeted for screening in Experimental Example 1 were indeed detected in the D24 sample: E. rectale, L. eligens, and Faecalibacterium (SGB15346 group affiliation). Quantitative analysis showed that the relative abundance of these three types of beneficial bacteria in the D24 fecal flora exceeded 5% (i.e., the number of sequences of each flora accounted for >5% of the total number of valid sequences in the sample), and they were one of the dominant members of the donor's intestinal flora.

[0080] In the fecal microbiota of donor D25, the sequence abundance of the above three target bacteria was extremely low, and no clear E. rectale, L. eligens and Faecalibacterium SGB15346 OTU / ASV signals were detected in the sequencing data (e.g., the proportion of their sequence numbers was lower than the detection threshold or 0).

[0081] This sequencing analysis result was highly consistent with the PCR screening results in Experimental Example 1: Sample D24 contained the target bacterial groups at significant abundance, while sample D25 lacked these groups. This fully demonstrates that the PCR multiplex detection method of the present invention has excellent accuracy and reliability, can truly reflect the presence of target bacteria in the fecal microbiome, and is consistent with the results of "gold standard" analysis methods such as high-throughput sequencing.

[0082] (3) FMT clinical trial design

[0083] To verify the functional significance of the above donor screening, this experiment selected patients with depression to conduct a preliminary fecal microbiota transplantation (FMT) clinical trial. The subjects were two adults who met the diagnostic criteria for moderate to severe depression (aged 30 to 50 years old, with a course of illness of more than 1 year and a score of 100 on the Hamilton Depression Rating Scale at the time of enrollment). <hamd>All patients maintained their original conventional treatment during the trial, with only FMT added as an auxiliary intervention.

[0084] Fecal samples from donors D24 and D25 were used to prepare bacterial suspensions for transplantation. Within 2 hours of collecting the fecal samples, a 10% (w / v) fecal bacterial suspension was prepared in saline under sterile conditions. After filtering out impurities, the suspension was freeze-dried to produce a lyophilized bacterial powder, which was then packaged into enteric-coated capsules for later use. While receiving conventional treatment, the patients took capsules containing intestinal flora prepared from donor samples D24 and D25 for two weeks.

[0085] (4) Evaluation of the efficacy of depressive symptoms

[0086] After FMT treatment, the patients were followed up for 8 weeks, and the Hamilton Depression Rating Scale (HAMD-17) was used by specialists to quantify the patients' depressive symptoms every week.

[0087] The results showed that patients who received the D24 microbiota transplant experienced significant relief of depressive symptoms: before the transplant, the patient's total HAMD score was 24 (indicating moderate depression), which decreased to 12 points in the eighth week after the transplant, a decrease of approximately 50%, and the symptoms were alleviated to a mild level; the patient subjectively reported significant improvements in sleep, appetite, and mood. However, patients who received the D25 microbiota transplant did not experience significant symptom improvement during the same period: the total HAMD score was 23 points before the transplant and 20 points eight weeks later, with only slight fluctuations (a decrease of approximately 13%), still within the moderate depression range, and the patient's subjective feelings did not change much. Neither patient experienced serious adverse reactions during the treatment process, with only occasional transient symptoms such as mild abdominal distension.

[0088] The above clinical observations suggest that the microbiota transplantation obtained from donor D24 showed a positive effect in alleviating depressive symptoms, while the microbiota transplantation from donor D25 had limited efficacy.

[0089] Experimental conclusion: This experimental example verified the effectiveness and predictive value of the donor screening method through laboratory analysis and preliminary clinical trials. First, the high-throughput sequencing results confirmed the accuracy of the PCR screening results: the detection conclusions of the donor containing the key strain (D24) and the donor lacking the key bacteria (D25) were consistent, indicating that the multiplex PCR detection method of the present invention can reliably reflect the presence and abundance differences of the target beneficial bacteria in the donor feces. Secondly, the comparison of the clinical efficacy of FMT further demonstrated the necessity of screening donors and the predictive value of this method: fecal microbiota transplantation from donor D24 containing key bacteria significantly improved the patient's depressive symptoms, while transplantation from donor D25 without key bacteria failed to produce significant therapeutic effects. This result verified the correlation between the key strains determined by the present invention and the efficacy of depression at the functional level, indicating that the "high-efficiency donors" screened by the method of the present invention do have better clinical application value.

[0090] In summary, the PCR screening method for key strains in donor feces provided by the present invention has good accuracy and practicality, and can be used to predict the effectiveness of FMT donors and improve the success rate of fecal microbiota transplantation treatment for diseases such as depression.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.< / hamd>

Claims

1. A PCR method for rapid screening of effective donors for FMT treatment of depression, characterized by: The steps include: (1) Sample collection and processing Fecal samples from candidate donors were collected and their total microbial genomic DNA was extracted under sterile conditions; (2) Specific primer design A total of six primer pairs were designed targeting the specific conserved sequences of the target strains E. rectale, L. eligens, and Faecalibacterium SGB15346, namely primer pairs 1, 2, 3, 4, 5, and 6; Among them, primer pair 1 and primer pair 2 are for strain E. rectale, the forward primer and reverse primer sequences of primer pair 1 are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively, and the forward primer and reverse primer sequences of primer pair 2 are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively; Primer pair 3 and primer pair 4 are specific for the strain L. eligens. The forward primer and reverse primer sequences of primer pair 3 are shown as SEQ ID NO. 5 and SEQ ID NO. 6, respectively. The forward primer and reverse primer sequences of primer pair 4 are shown as SEQ ID NO. 7 and SEQ ID NO. 8, respectively. Primer pair 5 and primer pair 6 are specific for strain Faecalibacterium SGB15346. The forward primer and reverse primer sequences of primer pair 5 are shown as SEQ ID NO.9 and SEQ ID NO.10, respectively. The forward primer and reverse primer sequences of primer pair 6 are shown as SEQ ID NO.11 and SEQ ID NO.12, respectively. (3) PCR amplification reaction The donor fecal DNA extracted in step (1) is used as a PCR template, and the primer pair of step (2) is added to perform an amplification reaction; (4) Product detection and judgment PCR results were observed by agarose gel electrophoresis. If all target bands were positive in the PCR electrophoresis results of the donor sample, the donor was determined to contain the three target strains of E. rectale, L. eligens, and Faecalibacterium SGB15346, and was identified as an effective donor for FMT treatment of depression.

2. The PCR method for rapid screening of effective donors for FMT treatment of depression according to claim 1, characterized in that: In the step (1), a stool DNA extraction kit is used to extract total microbial genomic DNA.

3. The PCR method for rapid screening of effective donors for FMT treatment of depression according to claim 1, characterized in that: In the step (3), a single amplification or multiplex PCR amplification method is used to perform the amplification reaction.

4. The PCR method for rapid screening of effective donors for FMT treatment of depression according to claim 3, characterized in that: When using the multiplex PCR amplification method, three pairs of primers are added to one reaction to detect three bacteria at the same time.

5. The PCR method for rapid screening of effective donors for FMT treatment of depression according to claim 4, characterized in that: The PCR reaction system for the multiplex PCR amplification was as follows: 1× PCR buffer containing 2.0 mM Mg 2+ ; dNTP, final concentration 0.2 mM; forward and reverse primers of six primer pairs, final concentration 0.4 μM each; template DNA, 50 ng; Taq DNA polymerase, 1 U; ultrapure water to 25 μL.

6. The PCR method for rapid screening of effective donors for FMT treatment of depression according to claim 4, characterized in that: The amplification program of the multiplex PCR amplification was as follows: pre-denaturation at 95° C. for 5 min; denaturation at 95° C. for 30 s, annealing at 58° C. for 30 s, and extension at 72° C. for 30 s, for a total of 35 cycles; and extension at 72° C. for 5 min.

7. The PCR method for rapid screening of effective donors for FMT treatment of depression according to claim 1, characterized in that: In the step (4), the PCR products were separated by 1.5% agarose gel electrophoresis, and the corresponding results were observed under a UV gel imaging system after being stained with dye.

8. The PCR method for rapid screening of effective donors for FMT treatment of depression according to claim 1, characterized in that: In step (4), the target band is positive, which means that the band corresponding to the target strain appears and the size of the band is consistent with the expectation.

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