Kit for quantitatively detecting human feces in polluted water and detection method thereof

Abstract

The invention provides a qPCR (quantitative polymerase chain reaction) kit for quantitatively detecting human feces in polluted water and a detection method thereof, and relates to the field of biotechnology detection. The kit contains three groups of qPCR primers with the sequences shown as SEQ ID NO: 1-6, can quantitatively detect the human excrement pollution intensity in the water body, can detect trace human excrement pollution in the water body, and has very high application value in the aspects of water body excrement pollution detection, pollution prevention and control early warning and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology detection, and particularly relates to a kit for quantitatively detecting human feces in polluted water and a detection method thereof. Background Art

[0002] Human and animal feces are one of the main factors causing water - borne pathogen pollution. Quickly and correctly identifying the source of water pollution is particularly important for ensuring the safety of drinking water and environmental water bodies. In recent years, using microorganisms for the source - tracing analysis of water - borne pathogen pollution has become a research hotspot internationally. In foreign countries, the indicator microorganisms used in microbial source tracking (MST) are mainly bacteria. Due to different living environments and dietary structures, there are certain genetic differences in the indicator microorganisms in the feces of different hosts. Microbial source tracking uses these differences to determine the source of the polluting host. Biomarkers have developed rapidly in foreign countries in recent years, such as Escherichia coli - specific genes, Bacteroidales biomarkers, and mitochondrial - encoded genes. Microbial source tracking is currently in the stage of continuous exploration, discovery, and verification. Some relevant work has been carried out in the United States, Australia, and some European Union countries. However, in China, microbial source tracking mainly focuses on Bacteroidales markers and mainly on qualitative detection, that is, it can only analyze the source of fecal pollution and cannot analyze the degree of pollution. The high technical difficulty and cost also affect the popularization and application of this method. At present, the human fecal biomarkers discovered in China are limited to Bacteroidales. Bacteroidales biomarkers can only prove whether a polluted sample has the biomarkers of a certain animal fecal pollution and cannot indicate whether there is a connection between different polluted samples. Other types of (viruses, phages) host - specific biomarkers remain to be studied and developed. Summary of the Invention

[0003] To solve the above problems, the present invention provides a qPCR kit for quantitatively detecting human feces in polluted water and a detection method thereof. On the one hand, the present invention provides a primer set for quantitatively detecting human feces in polluted water.

[0004] Specifically, the primer set includes three groups of primers: the Bacteroidales primer set with sequences shown in SEQ ID NO: 1 - 2; the phage primer set with sequences shown in SEQ ID NO: 3 - 4; the human polyomavirus primer set with sequences shown in SEQ ID NO: 5 - 6.

[0005] On the other hand, the present invention provides the application of the above - mentioned primer set in preparing a kit for quantitatively detecting human feces in polluted water.

[0006] On the other hand, the present invention provides a kit for quantitatively detecting human feces in polluted water bodies.

[0007] Specifically, the kit includes the above primer set.

[0008] More specifically, the kit further includes PCR buffer, dNTP, and Mg 2+ .

[0009] On another aspect, the present invention provides a detection method for quantitatively detecting human feces in polluted water bodies using the kit according to claim 3.

[0010] Specifically, the steps of the detection method are as follows: (1) Sample DNA extraction; (2) PCR amplification; (3) Result and data analysis.

[0011] More specifically, the sample in step (1) is sewage.

[0012] More specifically, in step (2), the PCR is qPCR.

[0013] More specifically, in step (2), the qPCR includes: SYBR Green I dye method, TaqMan probe method, and digital PCR.

[0014] More specifically, in step (2), the PCR system is: 2.5 μL of 10×PCR buffer; 2.5 μL dNTP; 2 μL Mg 2+ ; 0.5 μL of upstream and downstream primers with a concentration of 10 μmol / L, 1 μL of template DNA; sterilized ddH 2 O is supplemented to 20 μL.

[0015] More specifically, in step (2), the PCR amplification conditions are: 95°C, pre-denaturation for 10 min; 95°C for 15 s, 62°C for 15 s, 72°C for 45 s, 40 cycles; 72°C for 7 min.

[0016] Specifically, the result and data analysis in step (3) are obtained by detecting the PCR amplification product.

[0017] More specifically, the PCR product is electrophoresed on a 1% agarose gel, 1×TAE is used as the electrophoresis buffer, stained with EB, observed under ultraviolet light. If bands of 127, 235, and 426 bp are present respectively, it indicates that the water body is polluted by human feces. Description of the Drawings

[0018] Figure 1Standard curve of the biomarker for the Bacteroides primers in Example 1.

[0019] Figure 2 Standard curve of the biomarker for the phage primers in Example 1.

[0020] Figure 3 Standard curve of the biomarker for the human polyomavirus primers in Example 1.

[0021] Figure 4 PCR results of microbial source tracking using Bacteroides vulgatus in pig feces in Comparative Example 1.

[0022] Figure 5 PCR results of microbial source tracking using Bacteroides vulgatus in bovine feces in Comparative Example 1.

[0023] Figure 6 PCR results of microbial source tracking using Bacteroides vulgatus in chicken feces in Comparative Example 1. Detailed implementation manners

[0024] The present invention will be further described in detail below in conjunction with specific embodiments. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments are generally carried out under conventional conditions unless otherwise specified. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0025] Example 1 Detection steps of human feces in polluted water: (1) Extract DNA from the water sample to be tested as a template; (2) Perform qPCR amplification. The primer sequences are shown in Table 1: The PCR system is as follows: 2.5 μL of 10×PCR buffer; 2.5 μL of dNTP; 2 μL of Mg 2+ ; 0.5 μL each of three sets of upstream and downstream primers with a concentration of 10 μmol / L, 1 μL of template DNA; sterilized ddH 2 O is added to make up to 20 μL, and amplification is carried out separately.

[0026] The amplification conditions are: pre-denaturation at 95°C for 10 min; 95°C for 15 s, 62°C for 15 s, 72°C for 45 s, 40 cycles; 72°C for 7 min.

[0027] (3) Examine the PCR amplification product: 5 μL of the PCR product is electrophoresed on a 1% agarose gel, 1×TAE is used as the electrophoresis buffer, stained with EB, and observed under an ultraviolet lamp. If bands of 127, 235, and 426 bp are present respectively, it indicates that the water body is contaminated with human feces.

[0028] (4) Construct the PUC57 plasmid with the amplified product, perform gradient dilution with the above system for qPCR detection, construct a gene standard curve, and the standard curve is shown in Figures 1 - 3 .

[0029] (5) Substitute the qPCR results of the test samples into the standard curve for quantitative analysis of the human fecal pollution degree, and the test results are shown in Table 2.

[0030] Table 1

[0031] Table 2 Water body detection concentration (Copy Number / L)

[0032] We can see that in the actual water body, compared with the traditional Bacteroides indicator, the new biomarker has similar sensitivity and avoids the problem of false positives.

[0033] Comparative Example 1 Use the human Bacteroides primers with sequences shown in SEQ ID NO.1-2 alone for microbial source tracing in pig feces, cattle feces and chicken feces. The detection method is the same as that in Example 1, and the PCR results are shown in Table 3 and Figures 4 - 6 .

[0034] According to the data in Table 3 and Figures 4 - 6 , it can be seen that when using the human Bacteroides primers alone, the specificity is poor, but when using three groups of primers simultaneously, the specificity is high.

[0035] Table 3

[0036] Experimental Example 1 Accuracy (1) Collect human fecal samples from three regions, extract human fecal DNA, take 1 μL of the human fecal DNA solution as a template, and perform PCR amplification with the primers of SEQ ID NO.1-6. The PCR system is: 2.5 μL of 10×PCR buffer; 2.5 μL of dNTP; 2 μL of Mg 2+ ; 0.5 μL of upstream and downstream primers with a concentration of 10 μmol / L, 1 μL of template DNA; sterilized ddH 2 O is added to make up to 20 μL.

[0037] The amplification conditions are: 95°C, pre-denaturation for 10 min; 95°C for 15 s, 62°C for 15 s, 72°C for 45 s, 40 cycles; 72°C for 7 min.

[0038] (2) Substitute the qPCR results into the standard curve, and the test results are shown in Table 4.

[0039] Table 4 Human feces detection concentration (Copy Number / L)

[0040] Experimental Example 2 Specificity 1. Collect rabbit feces, pig feces, poultry feces and sheep feces.

[0041] 2. Detect the collected samples. The detection method is the same as that in Example 1. The results of the phage marker concentration are shown in Table 5.

[0042] Table 5 Fecal phage marker concentration (Copy Number / L)

[0043] According to the detection results, it can be seen that the detection rate of the method of the present invention in human samples is as high as 95%, while the detection rates in sheep feces, cattle feces, pig feces and rabbit feces are relatively low, only 10%.

[0044] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than limiting the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A primer set for quantitative detection of human feces in polluted water, characterized in that: The primer set includes three sets of primers: The Bacteroides primer set has the sequence shown in SEQ ID NO: 1-2; A phage primer set having a sequence as shown in SEQ ID NO:3-4; The human polyomavirus primer set has the sequence shown in SEQ ID NO:5-6.

2. Use of the primer set according to claim 1 in preparing a kit for quantitatively detecting human feces in polluted water.

3. A kit for quantitatively detecting human feces in polluted water, characterized in that: The kit comprises the primer set according to claim 1.

4. The kit according to claim 3, characterized in that The kit also includes PCR buffer, dNTP and Mg 2+ .

5. A method for quantitatively detecting human feces in polluted water using the kit according to claim 3.

6. The detection method according to claim 5, characterized in that: The detection method steps are: (1) Sample DNA extraction; (2) PCR amplification; (3) Results and data analysis 7. The detection method according to claim 6, characterized in that: The sample in step (1) is sewage.

8. The detection method according to claim 6, characterized in that: In step (2), the PCR is qPCR.

9. The detection method according to claim 8, characterized in that: In step (2), the qPCR includes: SYBR Green I dye method, TaqMan probe method and digital PCR.

10. The detection method according to claim 6, characterized in that: In step (2), the 2.5 μL 10× PCR buffer; 2.5 μL dNTP; 2 μL Mg 2+ ; 0.5 μL of upstream and downstream primers with a concentration of 10 μmol / L; 1 μL of template DNA; Make up to 20 μL with sterile ddH2O.

11. The detection method according to claim 10, characterized in that: In step (2), the PCR amplification conditions are: 95°C, pre-denaturation for 10 min; 95°C for 15 s, 62°C for 15 s, 72°C for 45 s, 40 cycles; 72°C for 7 min.

12. The detection method according to claim 6, characterized in that: In step (3), the results and data analysis are performed by electrophoresis to detect the amplified product of step (2). If bands of 127, 235, and 426 bp are present, respectively, it indicates that the water body is contaminated by human feces.

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