A new specific molecular target for detecting Bacillus subtilis and its rapid detection method

Abstract

The present invention discloses a new specific molecular target for identifying Bacillus subtilis and a rapid detection method thereof. The nucleotide sequence of the specific molecular target of Bacillus subtilis is shown in SEQ ID NO.1, and the nucleotide sequences of primers for specifically detecting Bacillus subtilis are shown in SEQ ID NO.2 and SEQ ID NO.3. The present invention utilizes a pan-genomic analysis method to obtain a specific molecular target gene for Bacillus subtilis, designs primer sequences capable of specifically detecting Bacillus subtilis, and establishes a new method for rapid identification of Bacillus subtilis. The present invention has the advantages of being simple and quick to operate, having high specificity, and low detection costs.

Description

Technical Field

[0001] The present invention relates to the fields of microbial technology and medical technology, and in particular to a specific new molecular target for detecting Bacillus subtilis and a rapid detection method thereof. Background Art

[0002] Bacillus subtilis, also known as Bacillus subtilis, is Gram-positive. Its cells lack a capsule structure, are motile, and possess peritrichous flagella. They can grow aerobically or facultatively anaerobically. During the late stages of stable growth, Bacillus subtilis typically produces dormant spores, which are round or oval in shape. When grown on solid culture media, Bacillus subtilis colonies are typically white to pale yellow, round or irregular in shape, with a wrinkled, opaque surface. When grown in liquid culture media, a dark, wrinkled biofilm often forms. Bacillus subtilis secretes enzymes such as amylase, lipase, and protease, which can utilize starch, lipids, and proteins. It also produces pectinase and saccharifying enzymes, which break down pectin and polysaccharides in plant tissues. It also secretes various growth hormones and metabolites that promote healthy growth in plants and animals. It can also solubilize phosphorus and potassium, promoting phosphorus and potassium absorption in crops. Therefore, Bacillus subtilis is widely used in microbial fertilizers and feeds.

[0003] Bacillus subtilis is a core microbial fertilizer and feed strain, and its relative abundance is crucial for its vital functions. Therefore, accurate identification of Bacillus subtilis is crucial for product testing and quality evaluation. In recent years, with the rapid advancement of microbial taxonomy and genomics, many species closely related to Bacillus subtilis have been isolated and identified. Phenotypic characterization using physiological and biochemical methods, colony morphology, and fatty acid analysis is cumbersome, costly, and time-consuming. Furthermore, these traditional phenotypic methods struggle to rapidly and accurately distinguish Bacillus subtilis from its related species. Specific PCR technology is currently a key method for rapid and accurate microbial identification. For Bacillus subtilis identification, methods have been established based on genes such as gyrB, rpoB, gyrA, and the β-galactosidase-encoding gene. However, these genes lack species demarcation thresholds, and the low coverage of the original test strains limits their detection range, often leading to false-positive or false-negative results. Therefore, it is urgent to obtain new specific molecular targets for Bacillus subtilis and establish rapid and accurate detection methods based on new specific molecular targets.

[0004] In 2005, Tettelin et al. first proposed the concept of pan-genome when studying different pathogenic strains of Streptococcus agalactiae. Pan-genome refers to the sum of all genomic information within a species, including core genes and accessory genes, and accessory genes include non-essential genes and specific genes. Among them, the genes shared by all samples of the species are called core genes, the genes that only exist in some samples are called non-essential genes, and the genes unique to a certain sample are called specific genes. Therefore, based on the pan-genome analysis of Bacillus subtilis and its closely related species, the specific genes of Bacillus subtilis accessory genes can be obtained, that is, these genes only exist in Bacillus subtilis, but not in closely related species of Bacillus subtilis. Using these specific genes, specific detection primers for Bacillus subtilis are designed, and a rapid and accurate detection method for Bacillus subtilis is established. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a new specific molecular target for identifying and detecting Bacillus subtilis and a rapid and accurate detection method thereof.

[0006] The present invention uses Roary, a prokaryotic microbial pan-genome analysis software, to obtain a set of specific genes for Bacillus subtilis. Taking into account differences in sequence length, similarity, and resolution, each specific gene is evaluated and the optimal specific gene is selected as a specific molecular target for Bacillus subtilis detection. This target, including the nucleotide sequence SEQ ID NO. 1, is used to identify whether the test strain is Bacillus subtilis and whether the test sample contains Bacillus subtilis.

[0007] The present invention provides a specific molecular target for detecting Bacillus subtilis. The nucleotide sequence of the Bacillus subtilis specific molecular target is shown in SEQ ID NO.1.

[0008] The present invention provides a set of specific primers for detecting Bacillus subtilis. The nucleotide sequences of the primers for specifically detecting Bacillus subtilis are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0009] The present invention provides a method for rapidly and accurately detecting Bacillus subtilis. The method comprises extracting genomic DNA from a sample and performing PCR amplification using the primers for detecting Bacillus subtilis. If the band size of the amplified product is 183 bp, the strain to be detected is Bacillus subtilis and the sample to be detected contains Bacillus subtilis.

[0010] Preferably, the PCR reaction system is: PhantaMax Super-Fidelity DNA Polymerase 0.5 μL, 2×Phanta Max Buffer 12.5 μL, dNTP Mix (10 mM) 0.5 μL, upstream primer (10 μM) 0.5 μL, downstream primer (10 μM) 0.5 μL, template DNA (20 ng / μL) 0.5 μL, and ddH2O is added to a total volume of 25 μL.

[0011] Preferably, the PCR reaction procedure is: pre-denaturation, 94°C, 5 min; denaturation, 94°C, 5 min; annealing, 61°C for 30 s; extension, 72°C for 12 s; denaturation-annealing-extension, 30 cycles; re-extension, 72°C for 10 min; termination, 16°C for 10 min.

[0012] The present invention has the following advantages and effects compared to the prior art:

[0013] The present invention adopts a pan-genomic analysis method to obtain specific molecular targets for detecting Bacillus subtilis, and establishes a rapid and accurate PCR detection method for Bacillus subtilis based on specific molecular targets. Compared with the prior art, the molecular targets for detecting Bacillus subtilis determined by the present invention have high specificity and strong resolution. At the same time, the detection method established by the present invention based on specific molecular targets has the advantages of simple operation, high specificity, rapidity, accuracy, and low cost. Therefore, the present invention has a good application prospect in the quality inspection and quality assessment of related products such as microbial fertilizers, microbial feeds, and microbial agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the phylogenomic tree of Bacillus subtilis and its closely related species;

[0015] Figure 2 Heat map of gene distribution from pan-genome analysis of Bacillus subtilis and its related species;

[0016] Figure 3 This figure shows the results of PCR detection of Bacillus subtilis and its related strains. Lane M is Marker 2000; lanes 1-18 correspond to the strain tested, Bacillus subtilis, and electrophoresis analysis of their PCR products reveals a single, bright band of interest. Lanes 19-37 correspond to strains tested, related species of Bacillus subtilis, and electrophoresis analysis of their PCR products reveals no bands. DETAILED DESCRIPTION

[0017] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0018] Example 1: Pan-genome analysis of Bacillus subtilis and its related species

[0019] The genome sequences of Bacillus subtilis and its closely related species were downloaded from the NCBI database, yielding a total of 158 genomes of Bacillus subtilis and its closely related species (Table 1). Of these, 35 genome sequences were from Bacillus subtilis, and 123 genomes were from 20 closely related species. This analysis uses a large number of genomes and comprehensive species coverage.

[0020] Table 1 Species and genome numbers used for analysis in the present invention

[0021]

[0022]

[0023] To ensure the accuracy of pan-genome analysis, the present invention conducted a quality assessment of the genomic sequences used in the analysis. Based on analysis using the CheckM software, the contamination levels of all 158 genomic sequences were less than 2%, and the integrity levels were all above 97%, indicating that the genomic sequences used in the present invention were of high quality.

[0024] To ensure consistency in pan-genome analysis, we re-predicted and annotated all genomes used in the analysis, obtaining their protein sequence files for analysis. Using Roary analysis software, we identified 687 core genes and 33,264 accessory genes across the 158 genomes. A phylogenomic tree based on the core genes was constructed to verify the taxonomic affiliation of the 158 genomes.

[0025] like Figure 1 As shown, the present invention reconstructed a phylogenetic tree of 158 strains of Bacillus subtilis and its closely related species based on a data set of 687 core genes. Based on the clustering of phylogenetic branches, the present invention determined that the phylogenetic relationships of these strains were reliable and the species divisions were correct.

[0026] Example 2: Screening of Bacillus subtilis-specific molecular targets

[0027] Accessory genes include genes specific to each species. Therefore, the present invention screened 33,264 accessory genes for species-specific genes of Bacillus subtilis as potential specific molecular targets. Based on this analysis, the present invention identified a Bacillus subtilis species-specific marker gene, named BSSM (Bacillus subtilis species-specific marker). The Bacillus subtilis species-specific gene BSSM is 291 bp in length (SEQ ID NO. 1) and is annotated as a bacteriophage protein.

[0028] Based on pan-genome analysis, the present invention extracts gene BSSM sequences from 35 genomes of Bacillus subtilis. The obtained gene BSSM sequences are subjected to multiple sequence alignment using the software DNAMAN, the repeated sequences are eliminated, and differential genes are retained for primer design. Based on multiple sequence alignment analysis and combined with the primer design principle, the present invention designs a specific primer pair for amplifying gene BSSM sequences, namely the upstream primer BSSMF (5′-GTTTTTTCTGTACTGGCTCAACT-3′) and the downstream primer BSSMR (5′-ACCAGTTCCAAGTAGACCTATTA-3′). According to the position information of the upstream and downstream primers, the size of the target product amplified by the gene BSSM sequence is 183bp, and its extension time is preset to 12 seconds. Based on T m =4(G+C)+2(A+T) calculation formula to determine the upstream and downstream primers T m The values ​​are all 64°C. Therefore, the present invention preliminarily sets the annealing temperature of the target region for amplification of the gene BSSM sequence to be 60°C.

[0029] Example 3: Determination of Bacillus subtilis-specific molecular target validation strains

[0030] To verify the specificity of the Bacillus subtilis molecular target gene BSSM, the present invention obtained 37 bacterial strains from the Guangdong Provincial Microbial Culture Collection (GDMCC) as reference (Table 2). These 37 reference strains include one model strain of Bacillus subtilis, 17 non-model strains of Bacillus subtilis, and 18 model strains of closely related species of Bacillus subtilis. Because Bacillus subtilis and its closely related species are highly related, in order to ensure the accuracy of subsequent verification experiments, the present invention sequenced the genomes of the 17 Bacillus subtilis strains from the GDMCC to determine their correct taxonomic status.

[0031] The software SPAdes was used to splice the sequencing sequences of 17 strains of Bacillus subtilis after quality inspection, and the spliced ​​sequences were cleaned with reference to the sequence fragment length and coverage, and the obtained genome sequences were used for downstream analysis. The genome sequences cleaned by the reference strain were quality inspected using CheckM. The results showed that the genome sequence integrity of the 17 strains of Bacillus subtilis was higher than 98%, and the contamination was lower than 2.5%, indicating that these genome sequences were of high quality. In order to determine the species classification of the 17 strains of Bacillus subtilis, the present invention used the online tool Genome-to-Genome Distance Calculator 3.0 to calculate the DNA-DNA hybridization value between the genome sequence of each reference strain and the genome sequence of the Bacillus subtilis model strain. As shown in Table 3, the dDDH values ​​between the 17 strains of Bacillus subtilis and the model strain were all higher than the 70% species classification "gold standard", confirming that these strains all belong to Bacillus subtilis and can be used to verify the specificity of the Bacillus subtilis molecular target gene BSSM.

[0032] Table 2 Model strains of Bacillus subtilis and related species

[0033]

[0034] Table 3 Reference strains of Bacillus subtilis

[0035]

[0036] Example 4: Validation of Bacillus subtilis-specific molecular targets

[0037] The validation of the specific molecular target of Bacillus subtilis includes two aspects: (1) confirming the presence of specific molecular targets in 17 reference strains of Bacillus subtilis at the genomic level; and (2) performing PCR validation on all reference strains using the amplification primers of the specific molecular target gene designed by the present invention.

[0038] Local alignment was performed using the specific target gene BSSM as the target and the reference strain genome sequences as the database for comparison. As shown in Table 4, the genome sequences of 17 reference strains of Bacillus subtilis contain homologous fragments to the specific target gene BSSM, with similarities exceeding 90% and coverage exceeding 99%. This indicates that the genome sequences of these reference strains contain the specific target gene BSSM, verifying the specificity of this target gene in Bacillus subtilis.

[0039] Table 4 Specific target gene detection of reference strain genome sequences

[0040]

[0041] PCR verification was performed on 18 strains of Bacillus subtilis and 19 strains of non-Bacillus subtilis bacteria using the target gene BSSM-specific amplification primers designed in this study, according to the determined annealing temperature and extension time; the PCR reaction system was: Phanta Max Super-Fidelity DNA Polymerase 0.5 μL, 2×Phanta Max Buffer 12.5 μL, dNTP Mix (10 mM) 0.5 μL, upstream primer (10 μM) 0.5 μL, downstream primer (10 μM) 0.5 μL, template DNA (20 ng / μL) 0.5 μL, and ddH2O was added to a total volume of 25 μL. The PCR reaction program is as follows: pre-denaturation, 94°C, 5 min; denaturation, 94°C, 5 min; annealing, 61°C for 30 s; extension, 72°C for 12 s; denaturation-annealing-extension, 30 cycles; re-extension, 72°C for 10 min; termination, 16°C for 10 min. Figure 3 As shown, electrophoresis of the PCR products revealed bright, clear, and single target bands in all 18 Bacillus subtilis strains, whereas no target bands were detected in any of the 19 non-Bacillus subtilis strains. These results demonstrate that the specific primers designed for the target gene BSSM and the established PCR conditions can be used for the rapid and accurate identification of Bacillus subtilis bacteria.

[0042] The above results show that the present invention uses a pan-genomic analysis method to obtain a specific molecular target for detecting Bacillus subtilis and establishes a Bacillus subtilis PCR detection method based on a specific molecular target. Compared with the prior art, the Bacillus subtilis detection molecular target determined by the present invention has good specificity and strong resolution. Moreover, the detection method established by the present invention based on the specific molecular target has the advantages of simple operation, high specificity, rapidity and accuracy, and low cost. Therefore, the present invention has great application potential in the quality inspection and quality assessment of related products such as microbial fertilizers, microbial feeds, and microbial agents.

[0043] SEQ ID NO.1

[0044]

[0045] SEQ ID NO.2

[0046] 5′-GTTTTTCTGTACTGGCTCAACT-3′

[0047] SEQ ID NO.3

[0048] 5′-ACCAGTTCCAAGTAGACCTATTA-3′

[0049] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A detection reagent for detecting a specific molecular target of Bacillus subtilis in detecting Bacillus subtilis, characterized in that: The Bacillus subtilis specific molecular target nucleotide sequence is shown in SEQ ID NO.

1.

2. Use of a set of specific primers for detecting Bacillus subtilis in detecting Bacillus subtilis, characterized in that: The nucleotide sequences of the specific primers for detecting Bacillus subtilis are shown in SEQ ID NO.2 and SEQ ID NO.

3.

3. A rapid and accurate detection method for Bacillus subtilis, characterized in that: Extract the genomic DNA of the test strain or the test sample, and then use the specific primers for detecting Bacillus subtilis according to claim 2 to perform PCR amplification. If the band size of the amplified product is 183 bp, it indicates that the test strain is Bacillus subtilis and the test sample contains Bacillus subtilis.

4. The method according to claim 3, characterized in that The PCR reaction system is 0.5 μL of Phanta Max Super-Fidelity DNA Polymerase, 12.5 μL of 2 × Phanta Max Buffer, 0.5 μL of 10 mM dNTPMix, 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, and 0.5 μL of 20 ng / μL template DNA, supplemented with ddH2O to a total volume of 25 μL.

5. The method according to claim 3, characterized in that The PCR reaction procedure is as follows: pre-denaturation, 94°C, 5 min; denaturation, 94°C, 5 min; annealing, 61°C, 30 s; extension, 72°C, 12 s; denaturation-annealing-extension, 30 cycles; re-extension, 72°C, 10 min; termination, 16°C, 10 min.

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