Micro-plastic exposure health risk detection method with desulfovibrio as biomarker

Abstract

The invention discloses a micro-plastic exposure health risk detection method with desulfovibrio as a biomarker. The method comprises the steps of fecal sample collection, DNA extraction, desulfurization vibrio abundance detection and risk judgment. Based on the experimental conclusion that microplastic exposure can significantly enrich intestinal desulfurization vibrio, and the abundance of the bacterium is positively correlated with neuropathy and cancer related metabolites, 16S rRNA sequencing is adopted to detect the abundance of the desulfurization vibrio, and health risk assessment is achieved through comparison with a control group. The method has the advantages of noninvasive property, specificity and convenience, can be used for human microplastic exposure screening or experimental animal model research, and provides an effective tool for microplastic health risk early warning.

Description

Technical Field

[0001] This invention belongs to the field of environmental exposure health risk detection technology, specifically involving a detection method for rapidly assessing the health risks associated with exposure to microplastics (especially polystyrene microplastics [PS-MPs]) using Desulfovibrio spp. in the gut microbiota as a biomarker. Background Technology

[0002] Microplastics (MPs, <5 mm in diameter) are emerging global pollutants that have permeated water, soil, air, and the food chain, and are ingested by humans through respiration and diet. Long-term exposure to low doses of microplastics is closely associated with health risks such as gut microbiota dysbiosis, metabolic disorders (lipid / amino acid metabolism abnormalities), neuroinflammation, and cancer (colorectal cancer).

[0003] Currently, the detection of microplastic exposure largely relies on instrumental analysis (such as mass spectrometry and spectroscopy) to directly measure microplastic particles in environmental or biological samples. This method is complex, costly, and time-consuming. Furthermore, existing health risk assessments often depend on high-throughput technologies such as metabolomics and genomics, requiring complex data analysis and hindering rapid screening. Therefore, there is an urgent need to develop a specific, convenient, and low-cost biomarker detection method for early warning of health risks from microplastic exposure. Summary of the Invention

[0004] To address the shortcomings of existing microplastic exposure health risk detection methods, such as "complex operation, high cost, and lack of specific biomarkers," this invention provides a method using *Vibrio desulfurans* (*Vibrio* spp.) Desulfovibrio spp. This method uses microplastics as the core biomarker for detection, enabling rapid, non-invasive, and accurate assessment of health risks associated with microplastic exposure.

[0005] The method for detecting the health risks of microplastic exposure using *Desulfovibrio* spp. as a biomarker is as follows: Step 1: Sample Collection Fecal samples were collected from the subjects to be tested (non-invasive sampling to reduce the invasiveness of the test); the subjects to be tested included people who were exposed to high-risk scenarios of microplastics for a long time (such as those who frequently use plastic tableware or drink bottled water) or laboratory animals (such as mice).

[0006] Step 2: Sample pretreatment and nucleic acid extraction Fecal samples were anaerobically washed (PBS buffer, centrifuged at 2000 rpm for 2 min, and the precipitate was collected). Total DNA was extracted from the samples using a DNA extraction kit (such as the Mag Pure Soil DNA LQ Kit) to remove proteins, impurities and other interfering substances, and to obtain high-quality DNA templates.

[0007] Step 3: Quantitative detection of Desulfovibrio spp. The abundance of Desulfovibrio spp. in the DNA template was detected by 16S rRNA gene sequencing: the V3-V4 region primer 338F_806R of 16S rRNA gene was used for high-throughput sequencing by the Illumina HiSeq 2500 platform, the Kraken2 / Bracken software was used for species annotation, and the relative abundance of Desulfovibrio spp. in the intestinal flora was calculated.

[0008] Step 4: Health risk judgment The detected abundance of Desulfovibrio spp. was compared with the reference value of the control group to determine the health risk level of microplastic exposure: (i) If the relative abundance of Desulfovibrio spp. is significantly higher than that of the control group (such as an increase of more than 2 times compared with the control group, p<0.05), it is determined as "high health risk of microplastic exposure", which indicates the risk of intestinal inflammation, metabolic disorder or neuropathy; (ii) If the abundance of Desulfovibrio spp. has no significant difference with the control group (p≥0.05), it is determined as "low health risk of microplastic exposure".

[0009] Among them, the reference value of the control group is the average abundance of Desulfovibrio spp. of healthy people / experimental animals not exposed to microplastics, which is obtained by the same detection method.

[0010] Verification method: detect the content of L-glutamyl gamma-semialdehyde, 8-amino-7-oxononanoic acid or Cer(d18:0 / 16:0) in the serum of the object to be detected, if the content is positively correlated with the abundance of Desulfovibrio spp., p<0.05, then it is further confirmed that the health risk is high.

[0011] The present application has the following beneficial effects: (1) High specificity: Figure 1 , Figure 2 Data shows that only the abundance of Desulfovibrio spp. in the microplastic exposure group (especially the low-dose group) is significantly increased, and it is co-enriched with colorectal cancer and intestinal inflammation-related bacteria (such as Lepagella spp.), which can specifically indicate microplastic exposure; (2) Strong convenience: non-invasive collection of fecal samples, detection relying on mature nucleic acid extraction and sequencing technology, simple operation process, no need for complex instruments (such as electron microscope and mass spectrometer required for direct detection of microplastics); (3) High risk correlation: as Figure 3 shows that the abundance of Desulfovibrio spp. is significantly positively correlated with neuropathy and cancer-related metabolites (L-glutamyl gamma-semialdehyde, 8-amino-7-oxononanoic acid, etc.), which can simultaneously evaluate the linked risk of "exposure-bacteria-metabolism-disease", rather than only detecting microplastics themselves; (4) Wide application range: can be used for microplastic exposure research of human population screening (such as infants, long-term exposure to plastic packaging) or experimental animal models (such as mice, rats), and provides a tool for environmental health risk assessment. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 : Difference in Desulfovibrio abundance in mice under different doses of PS-MPs exposure. *: 0.01 p < 0.05 indicates significant difference; **: 0.001 p < 0.01 indicates extremely significant difference; ***: p < 0.001 indicates highly significant difference. It is proved that microplastic exposure can significantly increase the abundance of Desulfovibrio, which provides data support for "abundance threshold determination".

[0013] Figure 2 : LEfSe analysis results of Desulfovibrio related taxonomic units. It is shown that Desulfovibrio is the characteristic difference bacteria of the microplastic exposure group, which supports its specificity as a marker.

[0014] Figure 3 : Correlation analysis of Desulfovibrio and risk metabolites. It is proved that the abundance of Desulfovibrio is positively correlated with the metabolites related to neuropathy and cancer, which provides the basis for "health risk association". DETAILED DESCRIPTION

[0015] Example 1 Experimental materials:

[0016] (1) Experimental animals: 3-week-old male BALB / c mice (14-18g), divided into control group (n=9, drinking regular water), low-dose group (n=8, drinking 10 μg / ml PS-MPs water), high-dose group (n=8, drinking 100 μg / ml PS-MPs water), exposure period 6 weeks; (2) Reagents: PS-MPs (diameter 4 μm, Shanghai Funtai Suohua), Mag Pure Soil DNA LQ Kit (Magen, D6356-02), Illumina HiSeq sequencing reagents; (3) Instruments: Illumina HiSeq 2500 sequencer, high-speed centrifuge. Experimental steps:

[0017] (1) Sample collection: collect fresh fecal samples of mice in each group every week, about 0.1g per sample, put in sterile centrifuge tube, store at -80℃; (2) DNA extraction: Take the fecal sample, wash with PBS anaerobically (2000 rpm, 2 min), take the precipitate, extract total DNA according to the Mag Pure Soil DNA LQ Kit instructions, and determine the DNA concentration (Nanodrop) and purity (A260 / A280 = 1.8-2.0); (3) 16S rRNA sequencing detection: For 16S rRNA V3-V4 region, forward primer 5'-ACTCCTACGGGAGGCAGCAG-3', reverse primer 5'-GGACTACHVGGGTWTCTAAT-3', PCR amplification of V4-V5 region, product purification to construct Illumina library, sequencing to obtain double-end 150bp reads; use Readfq to remove low-quality reads, Kraken2 to annotate species, and Bracken to quantify the relative abundance of Desulfovibrio. Result determination:

[0018] (i) The average relative abundance of Desulfovibrio in the control group mice was 0.21%±0.08%; (ii) The average relative abundance of Desulfovibrio in the low-dose group (10 μg / ml PS-MPs) was 0.68%±0.15%, which was 3.2 times higher than that of the control group (p<0.01), and was determined as "high health risk"; (iii) The average relative abundance of Desulfovibrio in the high-dose group (100 μg / ml PS-MPs) was 0.52%±0.12%, which was 2.5 times higher than that of the control group (p<0.05), and was determined as "high health risk".

[0019] Verification experiment: The L-glutamyl gamma-semialdehyde content in the serum of the low-dose group mice was detected, and it was found that it was positively correlated with the abundance of Desulfovibrio (R=0.3529, p =0.001), further confirming the correlation of "increase of Desulfovibrio→ increase of risk metabolites→ increase of health risk".

[0020] In this example, the abundance of Desulfovibrio in the microplastic exposure group (low and high dose) mice was significantly higher than that in the control group, which proved that this method could effectively determine the health risk of microplastic exposure through the abundance of Desulfovibrio.

Claims

1. A method for detecting health risks from microplastic exposure using *Desulfovibrio* spp. as a biomarker, characterized in that, The detection method includes the following steps: (i) Collect fecal samples from the subjects to be tested; (ii) The fecal sample from pretreatment step (i) is used to extract total DNA from the sample; (iii) The abundance of *Desulfovibrio* in the total DNA from step (ii) was detected by 16S rRNA gene sequencing. (iv) Compare the abundance of the *Desulfovibrio* genus with the baseline value of the control group. If the abundance is significantly higher than the baseline value of the control group, i.e. p If the value is less than 0.05, the object to be tested is determined to be at high health risk due to microplastic exposure.

2. The detection method according to claim 1, characterized in that, In step (ii), the pretreatment is as follows: the fecal sample is anaerobically washed with PBS buffer, centrifuged at 2000 rpm for 2 min, and the precipitate is collected; the total DNA is extracted using the Mag Pure Soil DNA LQ Kit.

3. The detection method according to claim 1, characterized in that, In step (iii), primers for the V3-V4 region of the 16S rRNA gene are designed for sequencing using the 16S rRNA gene. Sequencing is performed using the Illumina HiSeq platform, and species annotation is performed using Kraken2 / Bracken software to calculate the relative abundance of *Desulfovibrio* in the gut microbiota.

4. The detection method according to claim 1, characterized in that, In step (iv), the significant increase is defined as a relative abundance of *Desulfovibrio* that is ≥2 times higher than the baseline value of the control group.

5. The detection method according to claim 1, characterized in that, Step (iv) also includes detecting the content of L-glutamyl-γ-semialdehyde, 8-amino-7-oxonanoic acid, or Cer(d18:0 / 16:0) in the serum of the subject to be tested. If the content is positively correlated with the abundance of Desulfovibrio spp., p < 0.05, then a high health risk is further confirmed.

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