Method, primer and kit for detecting versicolor aspergillus producing bacteria polluted on surfaces of traditional Chinese medicinal materials and decoction pieces

Abstract

The invention discloses a method, a primer and a kit for detecting sterigmatocystin producing bacteria polluted on the surfaces of traditional Chinese medicinal materials and decoction pieces. Based on an LAMP detection method, the kit comprises a primer pair capable of performing LAMP reaction in a reaction tube. The invention establishes an LAMP rapid detection system for the toxic fungi polluted on the surfaces of the traditional Chinese medicinal materials and the decoction pieces, which can be amplified within 1 hour only by using simple constant-temperature instruments (such as a water bath kettle and a metal bath) without thermal cycle equipment, and the LAMP rapid detection system can be used for detecting the toxic fungi polluted on the surfaces of the traditional Chinese medicinal materials and the decoction pieces; the primer specificity is high, the detection limit of the method is equivalent to 102-104 CFU / g traditional Chinese medicine, and the detection sensitivity is high. Meanwhile, after the reaction is finished, the color change can be directly observed by naked eyes to judge the result, agarose gel electrophoresis and gel imaging system photographing observation are not needed, and the method is simple, rapid and particularly suitable for rapid detection of base-layer or field samples.

Description

Technical Field

[0001] This invention relates to methods, primers, and kits for detecting Aspergillus versicolor producing bacteria, belonging to the field of biotechnology detection, and particularly to methods, LAMP primers, and kits for detecting Aspergillus versicolor producing bacteria contaminated with Chinese medicinal materials and decoction pieces. Background Technology

[0002] Many traditional Chinese medicine (TCM) varieties, due to their high content of starch, polysaccharides, fats, and other nutrients, are easily contaminated by fungi and prone to mold growth during production, processing, transportation, and storage. This not only affects their medicinal value but can also cause poisoning or even death after consumption. To address the problem of fungal contamination in TCM materials and processed medicinal slices, my country has formulated several management regulations. For example, the 2020 edition of the Chinese Pharmacopoeia specifies the testing for aflatoxin in 24 kinds of TCM processed medicinal slices, including cypress seed kernel; the 2025 edition of the Chinese Pharmacopoeia adds the testing for ochratoxin A in herbs such as astragalus and areca nut, and adds testing for aflatoxin and zearalenone under the item for stir-fried coix seed slices. Sterigmatocystin (ST) is another type of fungal toxin commonly found in nature, and ST contamination in TCM materials and processed medicinal slices is serious. ST has a basic structure very similar to aflatoxin, mainly composed of… Aspergillus versicolor , A. nidulans When produced by fungal metabolism, it is also A. flavus and A. parasiticus ST is an intermediate in the metabolism of aflatoxin. ST is second only to aflatoxin in toxicity and is classified as a Group 2B carcinogen by the IARC. Although the pharmacopoeia currently does not specify a limit for its content, it has already attracted national attention regarding food safety (GB 5009.25-2016 National Food Safety Standard: Determination of Aflatoxin in Food). Therefore, establishing rapid and accurate detection methods for ST contamination in Chinese medicinal materials and processed medicinal slices, as well as its producing bacteria, is essential and will help standardize the supervision of ST.

[0003] Traditional fungal identification primarily relies on morphological characteristics and physiological and biochemical properties. However, due to the complexity of fungal morphology and the instability of their physiological and biochemical characteristics, accurate identification requires specialized mycologists. With the development of molecular biology, methods based on polymerase chain reaction (PCR) are widely used for the detection of mycotoxins. By designing primers or probes, specific DNA or RNA sequences of the target fungus are amplified to achieve rapid detection. Examples include traditional PCR, multiplex PCR, quantitative real-time PCR, and droplet digital PCR. However, these PCR methods still require expensive temperature control equipment, resulting in relatively long reaction times. To reduce equipment requirements and shorten detection time, isothermal amplification technology has gradually developed. This technology avoids the repeated thermal cycling process of denaturation, annealing, and extension in PCR, allowing the reaction to occur at a constant temperature. This eliminates the reliance on precise temperature cycling equipment and provides a new direction for the miniaturization and portability of nucleic acid amplification detection technology. Common isothermal amplification techniques include sequence-dependent amplification (NASBA), helicase-dependent amplification (HDA), rolling circle amplification (RCA), strand displacement amplification (SDA), recombinase polymerase amplification (RPA), and loop-mediated isothermal amplification (LAMP). Among these, LAMP technology is widely used for the identification of aflatoxin, ochratoxin, and fumonisin-producing bacteria due to its advantages such as high specificity, high sensitivity, simple instrumentation (constant temperature water bath or thermos), high detection efficiency, and visualized results. Douksouna et al., based on the aflatoxin biosynthesis pathway... nor -1 Gene-designed primers were used to successfully identify aflatoxin-producing and non-aflatoxin-producing Aspergillus strains isolated from rice using LAMP. Liu et al. based their work on the internal transcriptional spacer (ITS) region of conserved fungal regions and genes involved in aflatoxin biosynthesis. afl P designed primers and successfully detected Aspergillus flavus and aflatoxin-producing strains in food for the first time using LAMP. ITS and afl The LAMP detection limits for P were 10 fg and 1 pg, respectively. Storari et al. designed specific primers targeting the PKS gene in the ochratoxin biosynthesis pathway and established two LAMP detection systems, successfully identifying ochratoxin-producing strains of *Aspergillus anthocyanin* and *Aspergillus niger* isolated from grapes. The amplified products used hydroxynaphthol blue as an endpoint indicator, and the detection limits were comparable to those of conventional PCR. Massimo et al., based on the fumonisin biosynthesis pathway... fum 10 gene primers were designed to establish a LAMP detection technique for detecting fumonisin-producing Aspergillus niger and Aspergillus cerevisiae, and this technique was applied to the detection of fumonisin-producing Aspergillus in maize.

[0004] In summary, the discovery of potential toxin-producing fungal contamination is of great significance for ensuring the quality and safety of Chinese medicinal materials and processed medicinal slices, reducing potential risks, and avoiding economic losses. However, existing detection technologies, such as PCR nucleic acid amplification, suffer from time-consuming, costly, or equipment-dependent issues. Rapid, accurate, and convenient detection methods for fungal contamination of Chinese medicinal materials and processed medicinal slices still need development. LAMP technology has been applied to the identification of aflatoxin, ochratoxin, and fumonisin-producing bacteria in nuts, fruits, spices, and feed, demonstrating promising application prospects. However, it has not yet been used to identify toxin-producing fungi contaminating Chinese medicinal materials and processed medicinal slices, especially aspergillus versicolor-producing bacteria. Therefore, the main research objective of this invention is to apply LAMP technology to achieve rapid and accurate detection of aspergillus versicolor-producing bacteria contaminating the surface of Chinese medicinal materials and processed medicinal slices (hereinafter collectively referred to as Chinese medicine). Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the purpose of this invention is primarily to provide LAMP primers, methods, and kits for detecting aflatoxin-producing bacteria contaminating traditional Chinese medicine (TCM) with aflatoxin as the main toxin. This invention establishes a rapid LAMP detection system for toxin-producing fungi contaminating the surface of TCM, requiring no thermal cycling equipment but only a simple constant temperature instrument, and capable of amplifying the toxin-producing fungi within one hour. After the reaction, the result can be determined by visually observing the color change.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides LAMP primers for detecting Aspergillus versicolor-producing bacteria contaminating traditional Chinese medicine, used to amplify genes related to Aspergillus versicolor synthesis. Flu G, including inner primers F1 and B1, and outer primers F2 and B2; inner primer F1 is 5'-TACACGGGCAGCCCTCGATTGAGCGCCACCGTAATGAC-3', inner primer B1 is 5'-TTGGTATCCAAGCCACTTGCGGTGGACGGGTCTGTGATCG-3', outer primer F2 is 5'-GCCCAAATTCCGGACTTGA-3', and outer primer F3 is 5'-CGTTACGCTAGGAACGAAGT-3'; the amplification products turn yellow-green after staining with SYBR Green I fluorescent dye, and the gel electrophoresis results show continuous diffuse ladder-shaped bands in the range of 100bp-1500bp.

[0008] This invention provides the application of the above-mentioned LAMP primers in the preparation of reagents or instruments for detecting Aspergillus violaceus-producing bacteria contaminating traditional Chinese medicine.

[0009] This invention provides a LAMP method for detecting aflatoxin-producing bacteria contaminating traditional Chinese medicine. The LAMP method includes the following steps: 1) Extract and purify the DNA of the toxin-producing bacteria using a modified CTAB method as template DNA; the modified CTAB method is a CTAB method combined with a nucleic acid purification column; 2) LAMP amplification of template DNA was performed using two sets of primer pairs and the LAMP system; The primer pair used to amplify the synthetic gene FLuG of Aspergillus versicolor includes inner primers F1 and B1, and outer primers F2 and B2; inner primer F1 is 5'-TACACGGGCAGCCCTCGATTGAGCGCCACCGTAATGAC-3', inner primer B1 is 5'-TTGGTATCCAAGCCACTTGCGGTGGACGGGTCTGTGATCG-3', outer primer F2 is 5'-GCCCAAATTCCGGACTTGA-3', and outer primer B2 is 5'-CGTTACGCTAGGAACGAAGT-3'; the amplification product is a mixture of DNA with different numbers of stem-loop structures and polycyclic cauliflower-like structures.

[0010] 3) Detect the amplification products; after staining with SYBR Green I fluorescent dye, they turn yellow-green, and the gel electrophoresis results show diffuse ladder-like bands at 100bp-1500bp.

[0011] This allows for the analysis of the presence or absence of aflatoxin-producing bacteria in the aforementioned traditional Chinese medicine.

[0012] Further, in step 1), the modified CTAB method includes the following steps: collect the traditional Chinese medicine in an EP tube, add an appropriate amount of 0.1% Tween 20, shake vigorously, filter the suspension, collect the filtrate, centrifuge, discard the supernatant, add the bacterial cells to the lysis buffer, shake, place in a constant temperature water bath, let stand, add an equal amount of chloroform:isoamyl alcohol (24:1), shake, centrifuge, collect the supernatant, add 2 / 3 V / V isopropanol and mix well, transfer to a nucleic acid DNA purification column, let stand, centrifuge at high speed, wash with 70% ethanol, elute with TE solution, collect the eluent, and store at -20℃.

[0013] Further, in step 2), the PCR system is as follows: 1 μL BstII DNA polymerase; 2-5 μL 5×LAMP reaction buffer; 2-4 μL each of inner primers F1 and B1 (10 µM); 0.5-1 μL each of outer primers F2 and B2 (10 µM); 1-2 μL template DNA; and sterile water to a total reaction volume of 25 μL.

[0014] The reaction conditions for LAMP amplification were: 65℃, reaction time 1h.

[0015] Further, in step 3), the detection of the amplification product is as follows: ① SYBR Green Ⅰ fluorescent dye color development; the specific steps are as follows: after the reaction is completed, add 2μL of 1000×SYBR Green Ⅰ, and observe with the naked eye that the amplified product shows a clear yellow-green color; ② Separation by agarose gel electrophoresis and observation by gel imaging system; the specific steps are as follows: prepare 1.5% agarose gel, add 5 µL of LAMP amplification product to the gel sample well, and perform electrophoretic separation at 220 V voltage and 110 mA current; image the electrophoretic products in the gel imaging system, use DNA DL2000 Marker to determine whether there are diffuse ladder-like bands at 100bp-1500bp, and record and save the data.

[0016] This invention provides a LAMP kit for detecting Aspergillus violaceus-producing bacteria contaminating traditional Chinese medicine. Based on the LAMP detection method, it includes two primer pairs, a reaction buffer (containing four deoxynucleoside triphosphates), DNA polymerase, and a 1000×SYBR Green I fluorescent dye solution. Furthermore, it also includes negative control and positive control DNA; the negative control is ultrapure water; the positive control DNA is derived from Aspergillus versicolor. A. versicolor As3.4413.

[0017] Furthermore, it also includes buffer solutions for the reaction and necessary operating tools.

[0018] This invention provides an application method for detecting Aspergillus violaceus-producing bacteria contaminating the surface of traditional Chinese medicine. The method described above is used to detect the bacteria using primers or kits, and the results can be determined by visual color change or gel imaging.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1) Currently, it has been found that traditional Chinese medicine is susceptible to contamination by various fungal toxin-producing bacteria, among which the contamination by Aspergillus versicolor (a type II carcinogen)-producing bacteria is the most common, seriously threatening the life safety of users; this invention establishes a simple, rapid, and on-site detection method for Aspergillus versicolor-producing bacteria contaminating traditional Chinese medicine based on LAMP technology.

[0020] 2) This invention is based on the LAMP detection method and includes primer pairs capable of performing LAMP reactions in reaction tubes. This invention establishes a rapid LAMP detection system for toxin-producing fungi contaminating the surface of traditional Chinese medicine, requiring no thermal cycling equipment but only simple temperature control instruments (such as water baths or metal baths), and can complete amplification within 1 hour. The primers have high specificity, and the method's detection limit is equivalent to 10. 2 ~10 4The method offers high sensitivity for detecting CFU / g of traditional Chinese medicine. Furthermore, after the reaction, the results can be determined directly by visually observing the color change, eliminating the need for agarose gel electrophoresis and gel imaging system photography. The method is simple, fast, and particularly suitable for rapid detection of samples from grassroots or on-site locations.

[0021] 3) This invention first establishes a LAMP detection system for aspergillus versicolor-producing bacteria based on pure cultures, with a detection limit of 10. 2 CFU / mL; based on this, the effect of 17 kinds of Chinese herbal medicine matrices on the sensitivity of the LAMP detection method was investigated. The detection sensitivity of the method for the 17 kinds of herbs was within 10. 2 ~10 4 Between CFU / g. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the LAMP reaction system of the present invention; Figure 2 This is a comparison chart of color changes in the LAMP reaction detection results of this invention; Figure 3 These are typical images of LAMP reaction gel electrophoresis detection according to the present invention; Figure 4-1 , 4-2 4-3 are the strains of Aspergillus versicolor that were established for LAMP detection in Example 1; Figure 4-1 In the middle, Lanes 1~8: 55℃; 58℃; 60℃; 63℃; 65℃; 68℃; 70℃; sterile water (negative control); Figure 4-2 In the middle, the following time steps were taken: Lanes 1-7: 20 min; 30 min; 40 min; 50 min; 60 min; 70 min; sterile water (negative control); Figure 4-3 In the study, the ratios of Lanes 1-10 were 1:1; 2:1; 3:1; 4:1; 5:1; 6:1; 7:1; 8:1; 9:1; 10:1; sterile water (negative control).

[0023] Figure 5 This study investigates the specificity of the two primer pairs F1 / B1 and F2 / B2 in Example 1. In the figure, Lanes 1-9: As3.4413; ST bacteria 1; ST bacteria 2; ST bacteria 3; As3.4410; As3.6156; As3.4520; OTA bacteria 1; sterile water (negative control); Figure 6 To assess the sensitivity of LAMP detection of aflatoxin-producing bacteria in Example 1, In the image, Lanes 1-8:107 CFU / mL; 10 6 CFU / mL; 10 5 CFU / mL; 10 4 CFU / mL; 10 3 CFU / mL; 10 2 CFU / mL; 10 1 CFU / mL; sterile water (negative control); Figure 7 This is an example of investigating the specificity of LAMP detection based on malt matrix in Example 2. In the figure, Lines 1-35: DNA from a single strain or a mixture of DNA from different strains; Lines 36: sterile water (negative control). Figure 8 This illustrates the effect of the silkworm matrix on the LAMP detection sensitivity in Example 2. In the image, Lanes 1-7:10 6 CFU / mL; 10 5 CFU / mL; 10 4 CFU / mL; 10 3 CFU / mL; 10 2 CFU / mL; 10 1 CFU / mL; sterile silkworm pupae (negative control); Figure 9 The effect of 17 medicinal material matrices on LAMP detection sensitivity in Example 2; Figure 10 The results of LAMP assays for the 17 artificially infected medicinal materials in Example 3 are shown. Figure 11-1 , 11-2 11-3, 11-4, 11-5, and 11-6 are the LAMP detection results of 102 batches of 17 kinds of commercially available Chinese medicine samples in Example 4; Figure 11-1 The results are from samples tested at Hospital 01. Lines 1-34 are samples, Line 35 is a positive strain, and Line 36 is sterile water (negative control). Figure 11-2 The results are for samples from Hospital 02. Lines 1-34 are samples, Line 35 is a positive strain, and Line 36 is sterile water (negative control). Figure 11-3 The results are from samples tested at Hospital 03. Lines 1-34 are samples, Line 35 is a positive strain, and Line 36 is sterile water (negative control). Figure 11-4The results are for samples from pharmacy 01. Lines 1-34 are samples, Line 35 is a positive strain, and Line 36 is sterile water (negative control). Figure 11-5 The results are for samples from pharmacy 02. Lines 1-34 are samples, Line 35 is a positive strain, and Line 36 is sterile water (negative control). Figure 11-6 The results are for samples from pharmacy 03. Lines 1-34 are samples, Line 35 is a positive strain, and Line 36 is sterile water (negative control). Figure 12 The contamination rate of 102 batches of 17 kinds of commercially available Chinese medicine samples was determined by ST-producing bacteria. Detailed Implementation

[0024] Currently, LAMP technology has not been used to detect ST-producing bacteria. This invention targets genes related to ST toxin synthesis. Flu G. Two sets of LAMP primer pairs were developed, and an optimized LAMP system (reaction temperature, reaction time, ratio of inner and outer primer concentrations, etc.) was used to identify this type of toxin-producing fungus.

[0025] This invention discloses a LAMP detection method for detecting aflatoxin-producing bacteria contaminating the surface of traditional Chinese medicine. The LAMP detection method includes the following steps: 1) Extract and purify the DNA of the toxin-producing bacteria using a modified CTAB method as template DNA; the modified CTAB method is a CTAB method combined with a nucleic acid purification column; 2) LAMP amplification of template DNA was performed using two pairs of primers and a LAMP reaction system; The two pairs of primers include primers for the LAMP reaction carried out in a reaction tube, wherein, Primers were used to amplify the FluG gene, which is responsible for the synthesis of Aspergillus versicolor. The primers included a pair of inner primers, F1 and B1, and a pair of outer primers, F2 and B2. Inner primer F1 was 5'-TACACGGGCAGCCCTCGATTGAGCGCCACCGTAATGAC-3', inner primer B1 was 5'-TTGGTATCCAAGCCACTTGCGGTGGACGGGTCTGTGATCG-3', outer primer F2 was 5'-GCCCAAATTCCGGACTTGA-3', and outer primer B2 was 5'-CGTTACGCTAGGAACGAAGT-3'. 3) Detect the amplification products; analyze the presence or absence of toxin-producing fungi in the tested traditional Chinese medicine.

[0026] Furthermore, in step 1), the improved CTAB method includes the following steps: Collect the medicinal materials in an EP tube, add 3 / 5 of the tube volume of 0.1% Tween 20, shake vigorously, filter the suspension, collect the filtrate, centrifuge, discard the supernatant, add the bacterial cells to the lysis buffer, shake, incubate in a constant temperature water bath, let stand, add an equal volume of chloroform:isoamyl alcohol (24:1), shake, centrifuge, collect the supernatant, add 2 / 3 V / V isopropanol and mix well, transfer to a nucleic acid DNA purification column, let stand, centrifuge at high speed, wash with 70% ethanol, elute with TE solution, collect the eluent, and store at -20℃.

[0027] Furthermore, in step 2), the LAMP system is: Bst II. DNA Polymerase: 1 µL; 5×LAMP Reaction Mix: 2~5uL; The inner primer pair F1 / B1 at a concentration of 10 µM: 2~4 µL Primers at a concentration of 10 µM for external primers F2 / B2: 0.5~1 µL Template DNA: 2 µL Sterile water: Add to 25 µL; The reaction conditions for LAMP amplification were: 65℃, reaction time 1h.

[0028] Furthermore, in step 3), the detection of the amplification product involves adding 1000×SYBR Green I fluorescent dye for color development and observation; separating by agarose gel electrophoresis; and taking pictures using a gel imaging system.

[0029] The specific steps are as follows: After the reaction is complete, add 2 μL of 1000×SYBR Green Ⅰ fluorescent dye and observe with the naked eye whether the color of the bottom of the tube is yellow-green; prepare a 1.5% agarose gel, add the LAMP amplification product to the gel sample well, and perform electrophoresis separation; image the electrophoresis product in the gel imaging system to confirm whether there are continuous diffuse ladder-like bands.

[0030] In the parallel LAMP reaction assay, negative and positive control DNA were further included; the negative control was ultrapure water, and the positive control DNA was derived from Aspergillus versicolor. Aspergillus versicolor As3.4413.

[0031] The schematic diagram of the LAMP reaction system of this invention is shown below. Figure 1 As shown in the image, the color change comparison chart of the detection results is as follows: Figure 2 As shown, a typical image of gel electrophoresis detection is as follows: Figure 3 As shown.

[0032] Currently, LAMP detection technology is mainly used for the detection of food microorganisms, with very few reports on its application in traditional Chinese medicine. Based on the LAMP principle, specific ST synthesis-related genes are amplified, and the results are visualized using fluorescent dyes or gel electrophoresis. This allows for accurate identification of ST-producing bacteria on the surfaces of various traditional Chinese medicines contaminated by common susceptible fungi. Traditional PCR technology requires expensive temperature control equipment, has a long amplification time, and the amplified products need to be observed using agarose gel electrophoresis and gel imaging systems, making the operation quite complex. In contrast, the method of this invention allows for direct detection with only a simple temperature control device, and the amplification results can be directly visualized, significantly improving detection efficiency. It is particularly suitable for rapid detection at the grassroots level and in the field. The invention will now be further described with reference to the accompanying drawings and specific embodiments.

[0033] The experimental materials used in this invention are ST-producing bacteria and negative control strains, including *Aspergillus versicolor*. A. versicolor As3.4413, ST bacteria 1, ST bacteria 2 (isolated from Buddha's Hand), ST bacteria 3 (isolated from Panax notoginseng), Aspergillus flavus A. flavus As3.4410, A. flavus ZD, Aspergillus parasiticus A. parasiticus As3.6156, Ochratus A. ochraceus As3.4520, OTA strain 1 (isolated from *Ilex chinensis*), and other strains. These strains were purchased from the Genetic Culture Collection Center (GCMCC) of the Institute of Microbiology, Chinese Academy of Sciences, or isolated from the surface of traditional Chinese medicine; among them, *Aspergillus flavus* (… A. flavus As3.4410, Aspergillus parasiticus ( A. parasiticus As3.6156, Ochratus ( A. ochraceus As3.4520 and OTA bacteria 1 (isolated from Ilex chinensis) were used as negative controls.

[0034] Example 1: Establishment of LAMP detection method and methodological investigation 1. Extraction and purification methods for toxin-producing bacterial DNA (modified CTAB method).

[0035] Take a concentration of 2×10 7 1 mL of CFU / mL ST-generating bacterial culture was centrifuged at 12000 rpm for 5 min, and the supernatant was discarded. The fungal genomic DNA was extracted and purified using a modified CTAB method. The specific steps are as follows: The cultured ST-producing bacteria were eluted from PDA plates and collected by centrifugation. 0.3 g of glass beads and fungal lysis buffer were added to the bacterial cells, followed by shaking, incubation in a constant temperature water bath, and standing. An equal volume of chloroform:isoamyl alcohol (24:1) was added, followed by shaking and centrifugation. The supernatant was collected, mixed with 2 / 3 V / V isopropanol, transferred to a nucleic acid / DNA purification column, allowed to stand, and centrifuged at high speed for 1 min. The cells were washed with 70% ethanol, eluted with TE buffer, and the eluent was collected and stored at -20°C for later use.

[0036] 2. Primers The primers used in this invention are shown in Table 1.

[0037] Table 1 Primer Sequences

[0038] 3. Establish a LAMP system for detecting Aspergillus versicolor toxin-producing strains.

[0039] ① Seven reaction temperature gradients were set: 55℃, 58℃, 60℃, 63℃, 65℃, 68℃, and 70℃. The optimal reaction temperature was investigated, and 65℃ was determined to be the optimal reaction temperature. Results are shown below. Figure 4-1 .

[0040] ② Six reaction time gradients were set: 20 min, 30 min, 40 min, 50 min, 60 min, and 70 min, to investigate the optimal reaction time. The optimal reaction time was determined to be 60 min. Results are shown below. Figure 4-2 .

[0041] ③ Ten primer-inner-primer concentration ratios were set (with the outer primer concentration remaining constant): 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, and 10:1. The optimal primer-inner-primer concentration ratio was investigated, and it was determined to be 8:1. Results are shown below. Figure 4-3 .

[0042] 4. Methodological Examination 4.1 Specificity Four ST-producing strains were collected, including *Aspergillus versicolor* As3.4413, ST strain 1, ST strain 2 (isolated from *Buddha's Hand*), and ST strain 3 (isolated from *Panax notoginseng*), as well as four negative control strains, including *Aspergillus flavus*. A. flavus As3.4410, Aspergillus parasiticus ( A. parasiticus As3.6156, Ochratus ( A. ochraceusAs3.4520 and OTA strain 1 (isolated from *Ilex chinensis*). Each strain was plated on a PDA plate and cultured. DNA was then extracted using a modified CTAB method and used as a template for LAMP amplification. The LAMP reaction conditions and system were as described in section 3 above. The obtained LAMP products were detected by SYBR Green I staining and agarose gel electrophoresis.

[0043] See results Figure 5 The results showed that all ST strains amplified continuous, diffuse ladder-shaped bands, and stained yellow-green with SYBR Green I, while the negative control strains showed no bands and stained orange. This indicates... Flu G can serve as a specific sequence for identifying various ST-producing bacteria.

[0044] 4.2 Sensitivity The ST toxin-producing strain (As3.4413) was inoculated onto a PDA plate, cultured, and the strain was collected. An appropriate amount of 0.1% Tween 20 was added to dilute the strain to 2 × 10⁻⁶. 7 CFU / mL bacterial culture, then stepwise dilution using the 10-fold method (2×10⁻⁶). 1 ~10 7 DNA (CFU / mL) was extracted using a modified CTAB method and used as a template for LAMP sensitivity testing. The LAMP reaction conditions and system are as described in section 3 above.

[0045] The obtained LAMP products were observed by color development with SYBR Green I dye and separated by agarose gel electrophoresis, and then photographed using a gel imaging system.

[0046] See results Figure 6 The results showed that LAMP had a sensitivity of 10 for detecting ST-producing bacteria. 2 CFU / mL.

[0047] 5. Summary Using ST-producing bacteria as the research object, a modified CTAB method was used to extract fungal genomic DNA as a template, and a LAMP method for detecting ST-producing bacteria was established. Methodological evaluation results showed that LAMP detection of ST bacteria is highly specific, does not interfere with by other non-target strains, and has high sensitivity, reaching 10. 2 CFU / mL.

[0048] Example 2: Establishment of a LAMP detection system for Aspergillus flavus-producing bacteria on the surface of traditional Chinese medicine. 1. Chinese medicinal herbs The following herbs were used: Coix seed, Quisqualis indica, dried tangerine peel, peach kernel, arborvitae seed, jujube, areca nut, sour jujube seed, cassia seed, polygala root, lotus seed, malt, sterculia lychnophora, bee honeycomb, silkworm pupae, ground beetle, and earthworm. Using malt and silkworm pupae as representative Chinese medicinal herbs, and employing the LAMP detection method for ST-generating bacteria established based on pure bacteria as described in "Example 1," a LAMP detection system for ST-generating bacteria contaminating the surface of Chinese medicinal herbs was established.

[0049] 2. Specificity assessment of LAMP detection based on traditional Chinese medicine Weigh 2.0 g of blank malt medicinal material that is not contaminated by fungi, and extract the medicinal material matrix using the modified CTAB method (according to the method under item 1 in Example 1); take 2 ST-producing strains and 5 interfering strains (including 3 AFs-producing strains and 2 OTA-producing strains), spread each strain on PDA plates, culture them, and then extract genomic DNA. Using single strain DNA or mixed DNA of different strains as templates, mix the templates with the malt medicinal material matrix at a ratio of 1:1 (V / V) and perform LAMP amplification (according to the method under item 3 in Example 1). The obtained LAMP products are detected by SYBR Green I staining and agarose gel electrophoresis.

[0050] See results Figure 7 The results show that in the detection system containing malt medicinal material matrix, LAMP detection has good specificity, and coexisting interfering bacterial DNA does not affect the detection.

[0051] 3. Sensitivity assessment of LAMP detection based on traditional Chinese medicine Weigh 2.0 g of silkworm pupae and extract the medicinal matrix using the modified CTAB method (according to the method under item 1 in Example 1), denoted as A; extract ST-producing bacteria (As3.4413, bacterial count 2×10~10). 6 Genomic DNA (CFU / mL) was denoted as B; equal amounts of A and B (unit: ng / g) were mixed and used as a template for LAMP amplification (according to the method in item 3 of Example 1). The resulting LAMP product was then stained with SYBR Green I and detected by agarose gel electrophoresis.

[0052] See results Figure 8 The results show that in detection systems containing silkworm pupae medicinal material matrix, the LAMP detection sensitivity can reach 10. 2 Compared with the results of pure bacteria detection, the sensitivity of CFU / mL did not show a significant increase or decrease.

[0053] Similarly, experiments were conducted to investigate the effects of the matrix of 16 other medicinal materials, including Coix seed, on the sensitivity of LAMP detection. Figure 9 The effects of 17 medicinal herb matrices on the LAMP detection sensitivity in Example 2 were summarized, showing that the LAMP detection sensitivity was between 10 and 10. 2 ~104 CFU / mL, equivalent to 10 2 ~10 4 CFU / g medicinal materials.

[0054] 4. Summary A LAMP detection system was established for 17 Chinese medicinal herbs, including *Bombyx mori*, which are susceptible to contamination by toxin-producing fungi. The methodological results showed that the primers had high specificity, and the LAMP detection sensitivity reached 10. 2 Compared with the results of pure strains, the medicinal material matrix has a lower impact on sensitivity (CFU / mL); the method has a wide range of applications.

[0055] Example 3: Detection of artificially infected medicinal materials using the established LAMP detection system. 1. Preparation of artificially infected traditional Chinese medicine samples Take blank Chinese herbal medicine that is not contaminated with fungi and soak it in a solution with a concentration of 2×10 4 ST-producing bacteria at CPU / mL ( A. versicolor Immerse in As3.4413 solution for 30 s, remove, blot dry, place in sterile petri dish, air dry, and set aside for later use. Two replicates for each herb.

[0056] 2. Detection of Traditional Chinese Medicine in Artificial Infection Take 2.0 g of artificially infected traditional Chinese medicine and place it in a 50 mL EP tube. Add 0.1% Tween 20, shake vigorously, filter the suspension, collect the filtrate, centrifuge, discard the supernatant, and add lysis buffer to the bacterial cells. Extract the genomic DNA of the bacteria contaminating the surface of the medicinal material using the modified CTAB method (according to the method in section 1 of Example 1). Use this genomic DNA as a template for LAMP amplification (according to the method in section 3 of Example 1). The obtained LAMP product is detected by SYBR Green I staining and agarose gel electrophoresis.

[0057] See results Figure 10 The results showed that all 17 artificially infected Chinese herbal medicine samples with ST toxin-producing bacteria had positive LAMP test results, showing a yellow-green color, while the negative control was orange.

[0058] 3. Summary: All 17 artificially infected traditional Chinese medicine samples were successfully detected, indicating that the method is reliable, highly specific, and sensitive.

[0059] Example 4: The established LAMP detection system was applied to analyze multiple batches of traditional Chinese medicine samples from different sources and types. Detection 1. DNA was extracted from 102 samples of 17 different traditional Chinese medicines collected from 3 pharmacies and 3 hospitals using a modified CTAB method (following the method described in section 1 of Example 1), followed by LAMP amplification (following the method described in section 3 of Example 1). The resulting LAMP products were then stained with SYBR Green I and detected by agarose gel electrophoresis. Each sample was measured twice in parallel.

[0060] See results Figures 11-1 to 11-6 The results showed that 102 batches of 17 types of traditional Chinese medicine samples were severely contaminated by *ST*-producing bacteria. The contamination rate statistics are shown below. Figure 12 The data showed that the pollution rate reached 78.4%, with the pollution rates of cypress seed kernel, polygala root, and honeycomb reaching 100%.

[0061] 2. Summary: The LAMP assay results of 102 commercially available Chinese herbal medicine samples from 17 different types showed that the method had good stability and the limit of detection reached 10. 2 The concentration of CFU / g meets the screening requirements for common contamination levels in traditional Chinese medicine. It shows no reaction to uncontaminated samples and other non-Aspergillus chromis strains, exhibiting high specificity and significantly outperforming traditional PCR detection (which requires longer reaction times and higher equipment costs).

[0062] This invention significantly improves the detection speed and ease of operation of Aspergillus versicolor toxin-producing bacteria in traditional Chinese medicine decoction pieces, achieving the following technical advantages: Fast: Results are available within 1 hour, greatly shortening the testing cycle.

[0063] Applicable to the field: No complicated instruments are required; a constant temperature chamber and visual observation are sufficient for diagnosis.

[0064] Sensitivity: The detection limit reaches 10^2 CFU / g, which is close to the clinical and industry safety requirements.

[0065] Specificity: High specificity, effectively distinguishing different strains and reducing false positives.

[0066] Low cost: The reagent kit is simple in design and easy to promote and apply to grassroots testing sites.

[0067] In summary, this invention provides an efficient, simple, and on-site feasible detection technology for Aspergillus versicolor contamination of Chinese medicinal materials, offering a new approach for the safety evaluation of Chinese medicinal materials and possessing broad application prospects and promotional value.

[0068] This invention is not limited to the above-described embodiments. If any modifications or variations to this invention do not depart from the spirit and scope of this invention, and if such modifications and variations fall within the scope of the claims and equivalent technologies of this invention, then this invention also intends to include such modifications and variations.

Claims

1. A method for detecting aspergillus-producing bacteria contaminating the surface of Chinese medicinal materials and processed medicinal slices, characterized in that, Includes the following steps: 1) Extraction and purification of toxin-producing bacterial DNA using a modified CTAB method, wherein the modified CTAB method includes: Collect samples of Chinese medicinal materials or decoction pieces, add 0.1% Tween 20, shake vigorously, filter to obtain a suspension, centrifuge to remove the supernatant, take the bacterial cells, add lysis buffer, shake, react in a constant temperature water bath, add an equal volume of chloroform:isoamyl alcohol (24:1), shake and centrifuge, take the supernatant, add 2 / 3 volume of isopropanol and mix well, then transfer to a nucleic acid purification column, wash by high efficiency centrifugation, wash with 70% ethanol, and elute with TE buffer to obtain purified DNA samples; 2) The FluG gene, which is related to the synthesis of aflatoxin, was amplified using two sets of LAMP primer pairs, including inner primer F1: 5'-TACACGGGCAGCCCTCGATTGAGCGCCACCGTAATGAC-3' as shown in SEQ ID No. 1, inner primer B1: 5'-TTGGTATCCAAGCCACTTGCGGTGGACGGGTCTGTGATCG-3' as shown in SEQ ID No. 2, and outer primers F2: 5'-GCCCAAATTCCGGACTTGA-3' as shown in SEQ ID No. 3 and B2: 5'-CGTTACGCTAGGAACGAAGT-3' as shown in SEQ ID No.

4. The reaction was carried out at 65°C for 1 hour using a specific LAMP reaction system. 3) A positive result is indicated by visual observation of the reaction system after the addition of SYBR Green I fluorescent dye; or by observing the diffuse ladder-like bands in the range of 100bp-1500bp using agarose gel electrophoresis.

2. The method according to claim 1, characterized in that, In step 2), the LAMP amplification reaction system includes: Bst II DNA polymerase 1 μL; 2-5 μL of 5×LAMP reaction buffer; 2-4 μL each of inner primers F1 and B1 (10 µM); 0.5-1 μL each of outer primers F2 and B2 (10 µM); Template DNA 1-2 μL; Add sterile water to a total reaction volume of 25 μL.

3. The method according to claim 1, characterized in that, In step 3), the SYBR Green I fluorescent dye is at a concentration of 1000×, and 2 μL is added.

4. The method according to claim 1, characterized in that, In step 3), the agarose gel electrophoresis involves adding 5 μL of the amplification product to the gel and electrophoresis under the conditions of 220V voltage and 110mA current. Then, a gel imaging system is used to observe the diffuse ladder-like bands in the range of 100bp-1500bp.

5. The method according to claim 1, characterized in that, It also includes negative control and positive control DNA; the negative control is ultrapure water; the positive control DNA is derived from Aspergillus versicolor. Aspergillus versicolor As3.4413.

6. A set of primers for detecting aspergillus violaceus-producing bacteria contaminating the surface of Chinese medicinal materials and processed medicinal slices, characterized in that, It includes a pair of inner primers and a pair of outer primers: Inner primer F1: 5'-TACACGGGCAGCCCTCGATTGAGCGCCACCGTAATGAC-3', as shown in SEQ ID No. 1; Inner primer B1: 5'-TTGGTATCCAAGCCACTTGCGGTGGACGGGTCTGTGATCG-3', as shown in SEQ ID No. 2; Outer primer F2: 5'-GCCCAAATTCCGGACTTGA-3', as shown in SEQ ID No. 3; Outer primer B2: 5'-CGTTACGCTAGGAACGAAGT-3', as shown in SEQ ID No.

4.

7. A detection kit for detecting aspergillus violaceus-producing bacteria contaminating the surface of Chinese medicinal materials and processed medicinal slices, characterized in that, include: A pair of inner primers and a pair of outer primers are used to specifically amplify the FluG gene, which is related to the synthesis of aflatoxin varisome. Reaction buffer containing four deoxynucleoside triphosphates (dNTPs); DNA polymerase; SYBR Green I fluorescent dye at a concentration of 1000×; Negative control DNA; Positive control DNA, derived from strains known to produce aflatoxin.

8. The detection kit according to claim 7, characterized in that: It also includes buffer solutions for the reaction and the necessary tools for handling the reaction.

9. A method for detecting aspergillus violaceus-producing bacteria contaminating the surface of Chinese medicinal materials and processed medicinal slices, characterized in that: The detection is performed using the method described in any one of claims 1 to 8, primers or kits, and the results can be determined by visual color change or gel imaging.

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