Marker for quantifying excrement pollution and detection method

Abstract

The invention belongs to the field of biological environment quantitative detection, and particularly relates to a marker for quantifying excrement pollution and a detection method. The invention discloses an abundance-stable excrement pollution marker and a detection method, and is characterized in that the marker is obtained by metagenome analysis, a specific primer is designed, a real-time fluorescent quantitative PCR (qPCR) detection method is established, field sampling finds that the marker is detected in large quantities in excrement of different individuals, and the detection process only needs 3-4 hours, so that the detection efficiency is greatly improved, and the detection cost is reduced. Compared with the most common escherichia coli culture method at present, the method has high timeliness. In addition, according to the invention, the biomarker is highly related to the attenuation rate of human viruses and can reflect the real trend of virus changes. The biomarker detection method is convenient to operate, pollution evaluation can be rapidly completed, and the application potential in excrement pollution monitoring is shown.

Description

Technical Field

[0001] The invention belongs to the field of quantitative detection of biological environment, and specifically relates to a marker for quantitative fecal pollution and a detection method. Background Art

[0002] Inadequate sanitation and poor fecal management can lead to fecal contamination and the spread of related pathogens in the environment. It contains a large number of enteric pathogens, protozoa and viruses, which can cause diseases such as diarrhea, cholera, typhoid and polio, resulting in thousands of deaths each year. Therefore, tracking and quantifying fecal contamination in water environments is crucial to reducing the potential risks that enteric pathogens may pose to human, animal and environmental health. However, due to the large number of pathogens in feces, direct detection is time-consuming and labor-intensive and impractical. People have proposed fecal markers, hoping that the concentration of such indicators can reflect fecal pathogens and thus reflect the risk of disease. Currently commonly used fecal markers include fecal indicator bacteria (FIB) and human-related viruses. FIB (such as E. coli) will proliferate in the natural environment, while human-related virus markers vary greatly in the feces of different individuals, resulting in inconsistencies between the marker measurement results and the amount of fecal contamination, making it impossible to achieve quantitative / semi-quantitative fecal contamination. Therefore, it is necessary to develop a biomarker that is stable in different individuals to assess fecal contamination. Summary of the invention

[0003] In order to solve the above problems, the present invention aims to provide a detection method using a novel biomarker that can quantify the contamination level of feces in natural water bodies and reveal the changing trends of other human viruses.

[0004] The purpose of the first aspect of the present invention is to provide a marker for use in assessing environmental fecal pollution.

[0005] The second aspect of the present invention aims to provide a reagent for detecting a marker for use in preparing a product for evaluating environmental fecal pollution.

[0006] The third aspect of the present invention aims to provide a reagent for detecting a marker.

[0007] The fourth aspect of the present invention aims to provide a method for detecting environmental fecal contamination.

[0008] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0009] The first aspect of the present invention provides an application of a marker in detecting environmental fecal pollution.

[0010] In some embodiments of the present invention, the sequence of the marker is shown as SEQ ID NO:1.

[0011] The second aspect of the present invention provides the use of a reagent for detecting a fecal marker in the preparation of a product for detecting fecal environmental pollution, wherein the sequence of the marker is shown in SEQ ID NO:1.

[0012] In some embodiments of the present invention, the product includes at least one of a kit, a test paper, and a chip.

[0013] In some embodiments of the present invention, the reagent is selected from the following group of reagents for one or more detection methods: high-throughput sequencing, in situ hybridization, digital PCR, and fluorescent quantitative PCR.

[0014] The third aspect of the present invention provides a kit, which includes the reagent for detecting stool markers according to the second aspect of the present invention.

[0015] In some embodiments of the invention, the reagents include PCR primers.

[0016] In some embodiments of the present invention, the sequences of the primers are shown in SEQ ID NOs: 2 and 3.

[0017] In some embodiments of the invention, the kit comprises a buffer.

[0018] A fourth aspect of the present invention provides a method for detecting environmental fecal contamination, comprising the following steps:

[0019] 1) Extract sample DNA;

[0020] 2) Detection using the kit described in the third aspect of the present invention

[0021] In some embodiments of the present invention, the detection method is PCR detection.

[0022] In some embodiments of the present invention, the primer sequences for PCR detection are shown in SEQ ID NOs: 1 and 2.

[0023] In some embodiments of the present invention, the sample is a water sample.

[0024] In some embodiments of the present invention, the water sample includes at least one of a river water sample, a lake water sample, a sea water sample, and a sewage sample.

[0025] In some embodiments of the present invention, the qPCR reaction temperature is 95°C / 30s, 1 cycle; denaturation 95°C / 10s, annealing 60°C / 30s, 39 cycles, melting curve: 60°C-95°C, heating 0.5°C every 5s.

[0026] The beneficial effects of the present invention are:

[0027] 1. The markers of the present invention can be widely used in different regions: The biomarkers used in the present invention have stable abundance in the feces of different individuals, the coefficient of variation of relative abundance (standard deviation / mean value) is only 0.73, and the coefficient of variation of qPCR absolute abundance is only 1.50, while the most commonly used crAssphage is 3.91 and 4.18, respectively, which are 5 times and 3 times that of the markers of the present invention.

[0028] 2. The markers of the present invention are highly abundant in samples: in 2058 metagenomic samples, the transcript per kilobase per million mapped reads (RPKM) was 49.06, while crAssphage was only 24.65. In 200 real fecal samples, the average log gene copies (GC) per gram of feces was 8.3, while the most commonly used crAssphage was only 5.7.

[0029] 3. The markers of the present invention are highly correlated with the virus decay rate: The biomarkers of the present invention are significantly correlated with the decay rate of human polyomaviruses (HPyVs) and human adenoviruses (HAdVs) within 168 hours (Pearson R with HPyVs and HAdVs are 0.935 and 0.948, respectively), while the Pearson R with crAssphage and HPyVs and HAdVs are 0.723 and 0.631, respectively. It can be seen that the markers of the present invention can be used to predict major public health events. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0031] Figure 1 It is the relative abundance result of the marker of the present invention in the stool sample.

[0032] Figure 2 This is the melting curve result of the primers used in the present invention.

[0033] Figure 3 It is the absolute quantitative standard curve result of the detection method of the present invention.

[0034] Figure 4This is the abundance detection result of the markers of the present invention and crAssphage in real samples.

[0035] Figure 5 This is the relationship result of the attenuation rate between the marker of the present invention and human enteroviruses HAdVs and HpyVs. DETAILED DESCRIPTION

[0036] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0037] Example 1 Identification of markers

[0038]

[0039] Common fecal marker sequences were downloaded from NCBI GenBank, including crAssphage (NC_024711.1), NC_003197.2, HE608841.1_Bacteroides_phage, Lachno12, NC_001526.4_HPyV, DQ282121.1_nifH1, Lachno3, NC_015440.3_AiV-1, MK241690.1_HAdV, HAdV, Salivirus, A B480173.1_HBoV, BKPyV, KP710212.1_Cosavirus, KT027852.1_nifH, LC636334.1_HPyV, M81413.1_PMMoV, NC_034385.1_HCoSV, NC_039477.1_Norovirus_GII, Norovirus_GII, PMMoV, their abundance (RPKM) in 2058 human feces was calculated and compared with the markers of the present invention.

[0040] Figure 1 The fecal markers of the present invention and other existing fecal markers are shown in the figure. The fecal markers used in the present invention have stable abundance in the feces of different individuals, and the coefficient of variation (standard deviation / mean) of relative abundance is only 0.73, while the most commonly used crAssphage is 3.91.

[0041] Example 2 Primer Design

[0042] Based on the marker sequences obtained from the fecal metagenome, a pair of DNA primers (amplification length 118 bp) that can specifically identify fecal contamination were designed. The designed forward primers are shown below:

[0043] Forward primer: 5'-GCTGCACCCGTAGTCAAGCA-3' (SEQ ID NO: 2).

[0044] Reverse primer: 5'-TGCGGTATTCCAGCGTTCCC-3' (SEQ ID NO: 3).

[0045] Influent water from Guangzhou Lijiao Wastewater Treatment Plant and Guangzhou Zengcheng Wastewater Treatment Plant was collected and DNA was extracted. qPCR experiments were performed as complex environmental samples to detect primer specificity.

[0046] The reaction system was as follows: 10 μl ChamQ Universal SYBR qPCR Master Mix (Vazyme), 5 μl ddwater, 0.5 μl forward primer, 0.5 μl reverse primer, 4 μl sample DNA with a final concentration of 20 ng / μl. The qPCR reaction temperature was 95°C / 30s, 1 cycle; denaturation 95°C / 10s, annealing 60°C / 30s, 39 cycles, melting curve: 60°C-95°C, 0.5°C rise every 5s.

[0047] Figure 2 The melting curve diagram shows that each reaction shows a single peak, indicating that the product of each reaction is single, which verifies the effectiveness and specificity of primers SEQ ID NO: 2 and 3.

[0048] The synthetic marker plasmid (completed by Beijing Qingke Biotechnology Co., Ltd.) was diluted in a 10-fold gradient, with a total of 9 gradient dilutions as the standard. The reaction system was as follows: 10μl ChamQ Universal SYBR qPCR MasterMix (Vazyme), 5μl ddwater, 0.5μl forward primer, 0.5μl reverse primer, 4μl standard. The reaction temperature cycle was the same as above. Figure 3 The figure shows the absolute quantitative standard curve. After calculation, the minimum detection limit (LOQs) of SEQ ID NO: 2, 3 is 195.90 copies / μL, indicating that the primers have high sensitivity. The standard curve function is: y=-3.0535x+11.934.

[0049] Example 3 Stability Verification

[0050] 186 human fecal samples were collected from 186 volunteers by Shenzhen Longhua District People's Hospital, including 61 aged 0-18, 100 aged 19-65, and 25 aged 65 and above, with a male to female ratio of 106:80. The present invention was approved by the Medical Ethics Committee of the School of Public Health (Shenzhen) of Sun Yat-sen University (Zhongda Public Health (Shenzhen) Medical Ethics {2024} No. 107), and the informed consent of the subjects was obtained.

[0051] The collected stool samples were placed in sterile stool collection tubes and transported to the laboratory at -20°C for DNA extraction. 0.25 g of frozen stool samples were taken from each portion, and genomic DNA was extracted using the FastDNA SPIN Kit for Soil (MP Biomedicals) DNA kit. Fluorescence quantitative PCR was used for detection (qTOWER 3 Analytik Jena).

[0052] The reaction system was as follows: 10 μl ChamQ Universal SYBR qPCR Master Mix (Vazyme), 5 μl ddwater, 0.5 μl forward primer, 0.5 μl reverse primer, and 4 μl sample DNA with a final concentration of 20 ng / μl.

[0053] The reaction conditions were as follows: 95°C / 30s, 1 cycle; denaturation 95°C / 10s, annealing 60°C / 30s, 39 cycles, melting curve: 60°C-95°C, heating 0.5°C every 5s.

[0054] The crAssphage CPQ_056 fragment in the patent "A quantitative detection method for human fecal indicators in aquatic environments" (application publication number: CN110819699A) was selected as a comparison, and its qPCR method and primers were referred to, and the abundance of CPQ_056 in the above-mentioned different fecal samples was detected at the same time.

[0055] like Figure 4 The figure shows the abundance of the markers in feces. In 186 samples, the average log gene copy number (GC) of the biomarker of the present invention per gram of feces is 8.3, while the commonly used crAssphage is 5.68; the abundance variation coefficient is only 1.50, while that of crAssphage is 4.18.

[0056] Example 4 Attenuation Rate Relationship

[0057] The five feces were mixed evenly and placed in a dialysis bag (Dialysis Tube MD34 Mw: 7,000) (the dialysis tape only allows salt and small molecules to pass through, and biological macromolecules are trapped in the bag) for a decay rate experiment with three parallel groups.

[0058] Placed in a natural river with a water temperature of 19.6°C. Nine identical sampling time points were taken for each group, namely 0, 12, 24, 48, 96h, 120h, 144h, and 168h.

[0059] Two common human enteroviruses, HAdVs and HpyVs, were monitored simultaneously to study the relationship between the markers and the attenuation rate of human viruses. The primers and amplification procedures were based on McQuaig SM, Scott TM, Harwood VJ, Farrah SR, Lukasik JO. Detection of human-derived fecal pollution in environmental waters by use of a PCR-based human polyomavirus assay. Appl Environ Microbiol. 2006 Dec; 72(12): 7567-74. doi: 10.1128 / AEM.01317-06. Epub 2006 Sep 22. PMID: 16997988; PMCID: PMC1694258. and Staggemeier R, Bortoluzzi M, Heck TM, Spilki FR, Almeida SE. QUANTITATIVE VS. CONVENTIONAL PCR FOR DETECTION OF HUMAN ADENOVIRUSES INWATER AND SEDIMENT SAMPLES. Rev Inst Med Trop Sao Paulo. 2015Jul-Aug; 57(4):299-303.doi:10.1590 / S0036-46652015000400005.PMID:26422153; PMCID: PMC4616914.

[0060] crAssphage was also detected as a marker control.

[0061] like Figure 5 As shown, the biomarkers of the present invention were significantly correlated with the decay rate of human polyomaviruses (HPyVs) and human adenoviruses (HAdVs) within 168 hours (Pearson R with HPyVs and HAdVs were 0.935 and 0.948, respectively), while the Pearson R with crAssphage and HPyVs and HAdVs were 0.723 and 0.631, respectively.

[0062] It can be seen that the biomarkers of the present invention have a significant correlation with HPyVs and HAdVs, and the dynamic changes and transmission risks of the virus can be quickly and indirectly evaluated through the detection of the markers, providing efficient and economical technical support for public health management.

Claims

1. Application of a marker in detecting environmental fecal pollution, characterized in that: The sequence of the marker is shown in SEQ ID NO:

1.

2. Use of a reagent for detecting fecal markers in the preparation of a product for detecting fecal environmental pollution, characterized in that: The sequence of the marker is shown in SEQ ID NO:

1.

3. The use according to claim 2, characterized in that: The product includes at least one of a test kit, a test paper, and a chip.

4. The use according to claim 2, characterized in that: The reagents are selected from the following group of reagents for one or more detection methods: high-throughput sequencing, in situ hybridization, digital PCR, and fluorescent quantitative PCR.

5. A kit, characterized in that: The kit comprises the reagent for detecting stool markers as described in any one of claims 2 to 4.

6. The use according to claim 5, characterized in that: The reagents include PCR primers; Preferably, the sequences of the primers are shown in SEQ ID NOs: 2 and 3.

7. A method for detecting environmental fecal contamination, comprising the following steps: 1) Extract sample DNA; 2) Using the kit according to any one of claims 5 to 6 for detection.

8. The use according to claim 7, characterized in that: The detection method is PCR detection.

9. The use according to claim 8, characterized in that: The primer sequences for PCR detection are shown in SEQ ID NOs: 2 and 3.

10. The use according to claim 7, characterized in that: The sample comprises a water sample.

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