Method for identifying bacillus in soil based on surface enhanced Raman spectroscopy technology

Abstract

The invention relates to G01N 21 / 65, belongs to the technical field of optical detection of enzymes or microorganisms, and particularly relates to a method for identifying bacillus in soil based on a surface enhanced Raman spectroscopy technology. The method for identifying the bacillus in the soil based on the surface enhanced Raman spectroscopy technology comprises the following steps: S1, preparing a nanogold sol substrate; s2, preparation and determination of a bacillus standard map; s3, preparing and measuring a spectrum of bacillus in the soil sample; and S4, identifying and classifying the bacillus subgroups. According to the method, the possibility and potential of qualitative determination of soil microorganisms are explored by starting from a surface enhanced Raman scattering technology and combining a standard map contrast method, biological culture is not needed, the detection time is short, the sensitivity is high, and a basic research support is provided for developing an efficient comprehensive soil detection technology in the future.

Description

Technical Field

[0001] The present invention relates to the technical field of using optical detection of enzymes or microorganisms, specifically to a method for identifying Bacillus in soil based on surface-enhanced Raman spectroscopy technology. Background Technique

[0002] Surface-enhanced Raman spectroscopy has the characteristics of being fast, highly sensitive, and "real-time". Especially when using SERS substrates for bacterial detection, the detection effect is significantly enhanced. For example, Jiajie Xu et al. used gold colloid substrates to interact with Escherichia coli and could detect concentrations of 10~10 5 cfu / mL. Chinese patent document CN 114216894 B discloses a method for rapidly identifying Trichoderma in soil based on surface-enhanced Raman spectroscopy technology, including the following steps: (1) preparing nano-gold sol, (2) measuring the standard spectrum of Trichoderma, (3) measuring the spectrum of Trichoderma in soil samples, (4) identifying and classifying Trichoderma: comparing the spectrum of Trichoderma in soil samples obtained in step (3) with the standard spectrum of Trichoderma obtained in step (2) to determine the species of Trichoderma. This invention is based on surface-enhanced Raman scattering technology combined with a portable Raman spectrometer for detection. The instruments and equipment used in the method are easy to carry, the detection time of the method is short, it is convenient and fast, does not require biological cultivation, can also avoid using large-scale instruments and equipment, the detection cost is low, and the accuracy is high. This invention provides a new idea for the rapid detection of soil microorganisms.

[0003] The genus Bacillus ( Bacillus ), as a type of biocontrol Bacillus with wide applications, commonly includes Bacillus subtilis ( Bacillus subtilis ), Paenibacillus polymyxa ( Paenibacillus polymyxa ), Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ), Bacillus megaterium ( Bacillus megaterium ), etc. The biocontrol bacteria of the genus Bacillus are mostly applied to inhibit harmful fungi in the soil, improve the structure and quantity of soil microbial communities, activate soil nitrogen, phosphorus, and potassium, promote crop growth, and increase crop yields. Therefore, detecting the presence of Bacillus in the soil is of great significance for tracking the dynamics of beneficial Bacillus in the soil and optimizing the application strategy of microbial fertilizers.

[0004] Common techniques for identifying and differentiating microorganisms in soil mainly include genomic analysis techniques, fatty acid methyl ester (FAME) profiling methods, and Biolog techniques. PCR experiments require a large amount of laboratory equipment and highly skilled personnel. For example, extracting genomic DNA, designing primers for target genes, separating PCR amplification products using gel electrophoresis, and simultaneously carrying out cloning and analysis of target genes. In this process, denaturing gradient gel electrophoresis (DGGE) and restriction fragment preparation are time-consuming, requiring not only complex laboratory operation techniques but also relying on special chemicals. Usually, it takes about 2 - 3 days to complete these operations. Therefore, it is particularly important to develop sensitive and rapid detection techniques that can overcome these drawbacks. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide a method for identifying Bacillus in soil based on surface-enhanced Raman spectroscopy, which can quickly and accurately identify subgroups of three Bacillus species, namely Bacillus megaterium, Bacillus amyloliquefaciens, and Bacillus polymyxa.

[0006] To achieve the above object, the technical solution of the present invention is as follows: A method for identifying Bacillus in soil based on surface-enhanced Raman spectroscopy, comprising the following steps: S1. Preparation of a gold nanoparticle sol substrate; S2. Preparation and measurement of the standard spectra of Bacillus; S3. Preparation and measurement of the spectra of Bacillus in soil samples; S4. Identification and classification of Bacillus subgroups.

[0007] As an improvement, the specific steps of step S1 are as follows: Add 100 mL of 0.01% chloroauric acid solution to a 250 mL beaker, then add 1 mL of 1% sodium citrate solution. Place the beaker in an ultrasonic cleaner and simultaneously turn on the mercury lamp light source. React under irradiation for 30 min.

[0008] As an improvement, the specific steps of step S2 are as follows: Inoculate Bacillus megaterium, Bacillus amyloliquefaciens, and Bacillus polymyxa onto sterilized LB medium and culture them in a 30°C constant temperature incubator for 24 - 48 h, maintaining a saturated humidity state until a large bacterial lawn is formed; after the culture is completed, scrape a fixed-size bacterial lawn and disperse it with shaking; then centrifuge at 2000 rpm for 2 minutes, discard the precipitate containing agar fragments, and retain the upper homogeneous suspension to prepare a suspension of standard Bacillus; use a pipette to mix this suspension with the gold nanoparticle sol in a ratio of 1:20, and then measure it with a portable Raman spectrometer.

[0009] As an improvement, the specific steps of step S3 are as follows: Take 1.0 g of the soil sample added with Bacillus, add 10 mL of sterile pure water, place it in a sterile tube and shake it for 2 - 4 minutes to prepare a Bacillus suspension of the soil sample; Use a pipette to take this suspension and mix it with the nano - gold sol in step S2 according to a ratio of 1:20, and then measure it with a portable Raman spectrometer.

[0010] As an improvement, the specific steps of step S4 are as follows: Perform spectral pre - processing on the original spectra of Paenibacillus polymyxa, Bacillus amyloliquefaciens, Bacillus megaterium and the soil sample to obtain the processed spectra. Combine the cell wall structure compositions of these three bacteria and the characteristic peaks with good repeatability in the processed spectra to obtain the characteristic peak groups of the above - mentioned three Bacillus, and compare them with the characteristic peaks of the soil group to determine whether the corresponding bacteria species exist.

[0011] As a further improvement, it also includes the identification steps of nano - gold sol, specifically: Use an ultraviolet - visible spectrophotometer to characterize the optical properties of the gold nanoparticle dispersion system. Set 200 μL of ultrapure water as the control group to complete baseline calibration. Take an equal volume of nano - gold sol and inject it into a quartz cuvette, set the optical path to 1 cm, and measure its ultraviolet - visible absorption spectrum.

[0012] As a further improvement, the specific judgment basis is as follows: Paenibacillus polymyxa shows common peaks at 820, 1141, 1450, 1514, 1682, 1735 cm - 1; Bacillus amyloliquefaciens shows common characteristic peaks at 961, 1041, 1245, 1362, 1454, 1730 cm - 1; Bacillus megaterium shows stable peaks at 1135, 1370, 1450, 1605, 1682, 1740 cm - 1.

[0013] The beneficial effects of the present invention are as follows: Starting from the surface - enhanced Raman scattering technology, this application explores the possibility and potential of qualitative determination of soil microorganisms by combining with the standard spectrum comparison method. It does not require biological culture, has a short detection time and high sensitivity, and provides basic research support for the future development of efficient comprehensive soil detection technology. Description of the Drawings

[0014] Figure 1 is the ultraviolet - visible absorption spectrum of nano - gold sol; Figure 2 is the comparison chart of the spectra of the pure - species group before and after processing: among them, the pure - poly group is pure Paenibacillus polymyxa, the pure - amy group is pure Bacillus amyloliquefaciens, the pure - meg group is pure Bacillus megaterium, and 1, 2, 3 are the parallel tests conducted; Figure 3Comparison chart of soil group spectra before and after processing: Among them, the soil with more Bacillus polymyxa is the soil added with Bacillus polymyxa, the soil with Bacillus amyloliquefaciens is the soil added with Bacillus amyloliquefaciens, the pure Bacillus megaterium group is the soil added with Bacillus megaterium, and 1, 2, and 3 are the parallel tests carried out; Figure 4 Comparison chart of characteristic peaks after treatment of pure strain group and soil group. Specific implementation mode

[0015] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application. Embodiment

[0016] In this experiment, a portable Raman spectrometer was used. Compared with traditional large-scale analytical equipment, it has characteristics such as a compact volume, convenient operation, non-destructive detection, and rapid response. These technical advantages enable it to show significant advantages in drug ingredient screening and environmental pollutant monitoring, and at the same time can meet the immediate detection needs in complex field scenarios, and has outstanding applicability in the fields of mobile laboratories and emergency monitoring. Figure 2-3 This is the operation process diagram of the spectrometer.

[0017] In the experiment, a portable Raman spectroscopy detection system was used, equipped with BWSpec spectroscopy software, configured with a 532 nm wavelength green laser light source and a BAC102-532 type optical fiber probe, and spectral acquisition was carried out under light-shielded conditions. The spectral acquisition parameters were set as follows: the laser intensity was 100%, the single integration time was 10 seconds, the time expansion coefficient was 1, and the signal averaging times was 1. After completing the spectral measurement, the dark current was deducted and then smoothed, baseline corrected, and normalized to obtain the processed spectrum. The characteristic peaks of the processed spectrum were attributed.

[0018] As Figure 1 shown, the method for identifying Bacillus in soil based on surface-enhanced Raman spectroscopy technology described in this scheme specifically includes the following steps: S1. Preparation of nano-gold sol substrate Add 100 mL of 0.01% chloroauric acid solution to a 250 mL beaker, then add 1 mL of 1% sodium citrate solution. Place the beaker in an ultrasonic cleaner, and at the same time turn on the mercury lamp light source. React for 30 min under irradiation. After the reaction is completed, turn off the ultrasonic cleaner and the mercury lamp. Continuously mix the system using magnetic stirring and naturally cool to room temperature. Finally, transfer the synthesized product to a 250 mL blue-capped bottle and store it at low temperature in a 4°C constant temperature refrigeration device.

[0019] Subsequently, an ultraviolet-visible spectrophotometer was used to characterize the optical properties of the gold nanoparticle dispersion system. 200 μL of ultrapure water was set as the control group to complete the baseline calibration. An equal volume of gold nanosol was taken and injected into a quartz cuvette with an optical path of 1 cm, and its ultraviolet-visible absorption spectrum was measured. The measurement results are as Figure 1 shown. The absorption peak is around 522 nm. The typical plasmon resonance absorption peak of AuNPs attributed to the plasmon resonance effect is near 522 nm. Therefore, the formation of AuNPs was proved.

[0020] Preparation and determination of the standard spectra of S2 and Bacillus Different Bacillus strains were inoculated onto sterilized LB medium and cultured in a constant temperature incubator at 30 °C for 24 - 48 h, maintaining a saturated humidity state until a large bacterial lawn was formed. After the culture, a fixed-sized bacterial lawn was scraped off and shaken for about 3 minutes to assist dispersion. Subsequently, it was centrifuged at 2000 rpm for 2 minutes, and the precipitate containing agar fragments was discarded, and the upper homogeneous suspension was retained to prepare a suspension of standard Bacillus. The suspension was taken with a pipette and mixed with the gold nanosol in a ratio of 1:20, and then immediately measured with a portable Raman spectrometer. The original spectra of Paenibacillus polymyxa, Bacillus amyloliquefaciens, and Paenibacillus polymyxa were subjected to dark current subtraction, smoothing, baseline correction, and normalization spectral processing operations to obtain the processed spectral diagrams, and the results are as Figure 2 shown.

[0021] These three bacteria are all Gram-positive bacteria. The peptidoglycan layer of the cell wall of Gram-positive bacteria is thick, with NAW-NAG as the backbone, the tetrapeptide chain contains meso-DAP and has wall teichoic acid, lipoteichoic acid, and surface polysaccharides.

[0022] Paenibacillus polymyxa belongs to the genus Paenibacillus. The degree of peptidoglycan crosslinking is at a medium level among the three. The peptide bridge is connected by meso-DAP. The wall teichoic acid contains ribitol phosphate and secretes levan or β-glucan. Obvious and relatively repeatable characteristic peaks appeared near 820 cm-1, 941 cm-1, 1141 cm-1, 1237 cm-1, 1447 cm-1, 1514 cm-1, 1685 cm-1, 1738 cm-1, etc. for pure Paenibacillus polymyxa. The possible reasons for the generation of these characteristic peaks are protein bands, polysaccharides, amylose, C-H group bending vibration, amide III and CH2 wagging vibration of glycine backbone and proline side chain, CH2 bending mode of protein and lipid, cytochrome, amide I, and lipid respectively. The difference between pure Paenibacillus polymyxa 1 and pure Paenibacillus polymyxa 2 and pure Paenibacillus polymyxa 3 is relatively large, probably because it was not shaken well enough before detection or the probe was aimed at the analyte for too long during the experiment, resulting in the sedimentation of the bacteria.

[0023] Bacillus amyloliquefaciens belongs to the genus Bacillus, with a relatively high degree of peptidoglycan cross-linking. The wall teichoic acid is modified with D-glutamic acid and mainly consists of glycerol phosphate, and some strains are modified with glucose, secreting β-1,3-glucan or fructan. In the pure Bacillus amyloliquefaciens group, relatively obvious and reproducible characteristic peaks appeared near 825 cm-1, 960 cm-1, 1058 cm-1, 1237 cm-1, 1360 cm-1, 1450 cm-1, 1622 cm-1, 1729 cm-1, etc. The possible reasons for the generation of these characteristic peaks are phosphodiester, a large amount of cholesterol, symmetric and asymmetric stretching vibrations of O=S=O, amide III and CH2 wagging vibrations from the glycine backbone and proline side chain, cholesterol, C-H deformation band, tryptophan, and esters, respectively. Among them, the reproducibility of the pure Bacillus amyloliquefaciens group is better than that of the pure multiple group, probably because as the experiment progresses, the operator relatively proficiently masters the experimental operation, reducing some errors.

[0024] Bacillus megaterium belongs to the genus Bacillus, and its cross-linking degree is the highest among these three bacteria. The peptide bridge structure is dense, the wall teichoic acid is modified with a high proportion of glucose, and the surface polysaccharide is thicker. In the pure Bacillus megaterium group, relatively obvious and reproducible characteristic peaks appeared near 1135 cm-1, 1235 cm-1, 1370 cm-1, 1450 cm-1, 1506 cm-1, 1605 cm-1, 1682 cm-1, 1740 cm-1, etc.

[0025] S3. Preparation and determination of the Bacillus map in soil samples Dig several grams of soil 5 cm and below the surface in the back greenhouse of the School of Life Sciences of Huzhou Normal University. After removing larger stones and naturally drying for several days, take 1.0 g of the soil sample added with Bacillus, add 10 mL of sterile pure water, place it in a sterile tube and shake for 2 - 4 minutes to prepare a Bacillus suspension of the soil sample; use a pipette to take this suspension and mix it with the nano-gold sol in step S1 according to a ratio of 1:20, and immediately measure it with a portable Raman spectrometer. The measurement results are as Figure 3 shown.

[0026] The spectral pretreatment results of the pure strain group and the spectral pretreatment results of the soil group were compared. Both spectra of Bacillus polymyxa are flat at both ends, most of which are symmetrical peaks, and a few have multiple peaks and platform peaks near 1237 cm-1 and 1738 cm-1, and three more obvious prominent peaks appear near 820 cm-1, 1141 cm-1, and 1450 cm-1. That is, the overall appearance is flat at both ends and there are about three prominent peaks in the middle. The spectrum of Bacillus amyloliquefaciens is closer to that of Bacillus megaterium and Bacillus polymyxa, and it is also a spectrum with flat ends. Most of them are symmetrical peaks, and a few have tailing peaks near 1620 cm-1 and 1737 cm-1. There are two obvious peaks around 825 cm-1 and 1450 cm-1 in the middle, but there are more dense small peaks between the first and second peaks, such as around 1360 cm-1 and 1040 cm-1. That is, the overall spectrum is flat at both ends, with about 2 protruding peaks in the middle, and there are relatively dense small protruding peaks between the two protruding peaks. The spectrum of Bacillus megaterium is quite different from the previous two. The flat distance will be longer at the right end starting from 600 cm-1. Most of them are symmetrical peaks, and a few have tailing peaks at 1677 cm-1. There are about 2 protruding peaks, near 1450 cm-1 and 1150 cm-1. The overall spectrum shows multiple dense small protruding peaks and a long and gentle spectrum with 2 protruding peaks.

[0027] S4. Identification and classification of Bacillus subgroups After spectral preprocessing of the detected original spectra of Paenibacillus polymyxa, Bacillus amyloliquefaciens, Bacillus megaterium and soil samples, the specific operations are smoothing, baseline correction and normalization, and then the processed spectra are obtained. Combining the cell wall structure composition of these three bacteria and the characteristic peaks with good repeatability in the processed spectra, the characteristic peak groups of the above three Bacillus are obtained and compared with the characteristic peaks of the soil group. It is used to identify whether the corresponding bacteria exist.

[0028] According to the characteristic peak attribution results of the purebred group and the characteristic peak information of the spectrum of the soil group after spectral preprocessing, Figure 4As shown. There are overlapping characteristic peaks in the two types of spectra near 820 cm-1, 1141 cm-1, 1450 cm-1, 1514 cm-1, 1682 cm-1, and 1735 cm-1 for Paenibacillus polymyxa. There are overlapping characteristic peaks in the two types of spectra near 961 cm-1, 1041 cm-1, 1245 cm-1, 1362 cm-1, 1454 cm-1, and 1730 cm-1 for Bacillus amyloliquefaciens. There are overlapping characteristic peaks in the two types of spectra at around 1135 cm-1, 1370 cm-1, 1450 cm-1, 1605 cm-1, 1682 cm-1, and 1740 cm-1 for Bacillus megaterium. However, due to the complex soil composition, the experimental method in this study cannot completely exclude the interference of its complex soil matrix, such as the fusion of peaks, etc. Therefore, the comparison results of the two spectra can only indicate the presence of the corresponding bacteria, but cannot exclude the absence of a certain type of bacteria.

Claims

1. A method for identifying Bacillus in soil based on surface-enhanced Raman spectroscopy technology, characterized in that, It includes the following steps: S1. Preparation of the gold nanoparticle sol substrate; S2. Preparation and determination of the standard spectrum of Bacillus; S3. Preparation and determination of the spectrum of Bacillus in soil samples; S4. Identification and classification of Bacillus subgroups.

2. The method for identifying Bacillus in soil based on surface-enhanced Raman spectroscopy according to claim 1, wherein The specific steps of step S1 are as follows: Add 100 mL of 0.01% chloroauric acid solution to a 250 mL beaker, then add 1 mL of 1% sodium citrate solution. Place the beaker in an ultrasonic cleaner and turn on the mercury lamp source at the same time. React for 30 min under irradiation.

3. The method for identifying Bacillus in soil based on surface-enhanced Raman spectroscopy according to claim 1, wherein The specific steps of step S2 are as follows: Inoculate Bacillus megaterium, Bacillus amyloliquefaciens, and Bacillus polymyxa onto the sterilized LB medium, and culture them in a constant temperature incubator at 30 °C for 24 - 48 h, maintaining a saturated humidity state until a large bacterial lawn is formed; after the culture is completed, scrape the bacterial lawn of a fixed size and disperse it with shaking; then centrifuge at 2000 rpm for 2 minutes, discard the precipitate containing agar fragments, and retain the upper homogeneous suspension to prepare a suspension of standard Bacillus; use a pipette to take this suspension and mix it with the gold nanoparticle sol in a ratio of 1:20, and then measure it with a portable Raman spectrometer.

4. The method for identifying Bacillus in soil based on surface enhanced Raman spectroscopy according to claim 1, characterized in that, The specific steps of step S3 are as follows: Take 1.0 g of the soil sample added with Bacillus, add 10 mL of sterile pure water, place it in a sterile tube and shake it for 2 - 4 minutes to prepare a suspension of Bacillus in the soil sample; use a pipette to take this suspension and mix it with the gold nanoparticle sol in a ratio of 1:20, and then measure it with a portable Raman spectrometer.

5. The method for identifying Bacillus in soil based on surface-enhanced Raman spectroscopy according to claim 1, wherein The specific steps of step S4 are as follows: Perform spectral preprocessing on the detected Paenibacillus polymyxa, Bacillus amyloliquefaciens, Bacillus megaterium, and the original spectrum of the soil sample to obtain the processed spectrum. Combine the cell wall structure compositions of these three bacteria and the characteristic peaks with good repeatability in the processed spectrum to obtain the characteristic peak groups of the above three Bacillus, and compare them with the characteristic peaks of the soil group to determine whether the corresponding bacterial species exist.

6. The method for identifying Bacillus in soil based on surface enhanced Raman spectroscopy according to claim 2, characterized in that, It also includes the identification step of the gold nanoparticle sol, specifically: Use a UV-visible spectrophotometer to characterize the optical properties of the gold nanoparticle dispersion system. Set 200 μL of ultrapure water as the control group to complete baseline calibration. Take an equal volume of the gold nanoparticle sol and inject it into a quartz cuvette with an optical path set to 1 cm, and measure its UV-visible absorption spectrum.

7. The method for identifying Bacillus in soil based on surface-enhanced Raman spectroscopy according to claim 5, wherein The specific judgment basis is as follows: Those showing common peaks at 820, 1141, 1450, 1514, 1682, 1735 cm -1 are Paenibacillus polymyxa; those showing common characteristic peaks at 961, 1041, 1245, 1362, 1454, 1730 cm -1 are Bacillus amyloliquefaciens; those retaining stable peaks at 1135, 1370, 1450, 1605, 1682, 1740 cm -1 are Bacillus megaterium.

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