Pretreatment method of resistance gene detection sample in breeding waste

Abstract

The pretreatment method of the resistance gene detection sample in the breeding waste comprises the following steps: suspending the breeding waste in sterile water, and treating the breeding waste for 1-2 hours by adopting a pretreatment agent I; the pretreating agent I is prepared from aluminum sulfate and ferrous sulfate according to the weight ratio of 5: (1-3); after centrifuging, adding a pretreating agent II into a solid phase for treating; the pretreatment agent II is a sterile aqueous solution composed of 8-12% w / w of PVP30 and 0.5-1.5% w / w of sodium acetate; after centrifugation, adding a pretreatment agent III into a solid phase for treatment; the pretreatment agent III is a sterile aqueous solution composed of potassium acetate with the concentration of 3-5 M and sodium chloride with the concentration of 0.5-0.8 M. According to the pretreatment method provided by the invention, DNA in the breeding waste can be effectively reserved, the content of DNA obtained by subsequent DNA extraction work is effectively increased, and detection of resistance genes in the breeding waste is facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of resistance gene detection sample processing, and in particular relates to a pretreatment method for resistance gene detection samples in aquaculture waste. Background Art

[0002] Antibiotic resistance genes are one of the emerging biological contaminants, often enriched in livestock and poultry farming waste. While the technology for detecting resistance genes in water is relatively mature, there are still some challenges in detecting resistance genes in solid-phase pollutants with more complex compositions, such as livestock and poultry farming waste. For example, impurities in solid-phase pollutants are difficult to remove, which can easily lead to DNA degradation during the removal process. Furthermore, when the DNA concentration in solid-phase pollutants is low, it is difficult to concentrate and enrich them for subsequent DNA extraction, as is the case with water pollutants.

[0003] Therefore, how to provide a pretreatment method for detecting resistance genes in aquaculture waste is urgently needed in this field. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a pretreatment method for resistance gene detection samples in aquaculture waste. The method is simple and can be widely used in the sample pretreatment process of different DNA extraction kits before testing samples, and can improve the extraction concentration of DNA.

[0005] The present invention provides the following technical solutions to achieve the aforementioned objectives.

[0006] A method for pretreating a sample for detecting resistance genes in aquaculture waste, wherein the pretreatment is a process of treating the sample before using a DNA extraction kit, and the pretreatment method comprises the following steps:

[0007] (1) suspending the aquaculture waste in sterile water and treating it with a pretreatment agent I for 1 to 2 hours; the pretreatment agent I is composed of aluminum sulfate and ferrous sulfate in a weight ratio of 5:1 to 3;

[0008] (2) After centrifugation, adding pretreatment agent II to the solid phase for treatment; the pretreatment agent II is a sterile aqueous solution composed of 8-12% w / w PVP30 and 0.5-1.5% w / w sodium acetate;

[0009] (3) After centrifugation, pretreatment agent III is added to the solid phase for treatment; the pretreatment agent III is a sterile aqueous solution consisting of potassium acetate with a concentration of 3 to 5 M and sodium chloride with a concentration of 0.5 to 0.8 M.

[0010] Specifically, the breeding waste includes livestock manure.

[0011] Preferably, the pretreatment agent I is composed of aluminum sulfate and ferrous sulfate in a weight ratio of 5:2.

[0012] Preferably, the concentration of the aquaculture waste in sterile water is 20% w / w, and the weight ratio of the aquaculture waste to the pretreatment agent I is 30:1.

[0013] Preferably, the pretreatment agent II is a sterile aqueous solution consisting of 10% w / w PVP30 and 1% w / w sodium acetate.

[0014] Preferably, the pretreatment agent II is a sterile aqueous solution consisting of 10% w / w PVP30 and 1% w / w sodium acetate.

[0015] Preferably, in step (2), the weight ratio of the solid phase to the pretreatment agent II is 2:8.

[0016] Preferably, in step (2), the treatment time is 0.5 hours.

[0017] Preferably, the pretreatment agent III is a sterile aqueous solution consisting of 4M potassium acetate and 0.6M sodium chloride.

[0018] Preferably, in step (3), the weight ratio of the solid phase to the pretreatment agent III is 2:8.

[0019] Preferably, in step (3), the treatment time is 0.5 hours.

[0020] Beneficial effects of the present invention:

[0021] The pretreatment method for detecting resistance genes in aquaculture waste provided by the present invention can effectively retain the DNA in the aquaculture waste, effectively increase the DNA content obtained in subsequent DNA extraction work, and facilitate the detection of resistance genes in aquaculture waste; at the same time, the pretreatment method provided by the present invention can be applied to different existing DNA extraction kits. DETAILED DESCRIPTION

[0022] The following descriptions of specific embodiments are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0023] Example 1

[0024] The breeding waste in this embodiment is taken from chicken manure from a local chicken farm;

[0025] Pretreatment agent I: composed of aluminum sulfate and ferrous sulfate in a weight ratio of 5:2;

[0026] Pretreatment agent II: a sterile aqueous solution consisting of 10% w / w PVP30 and 1% w / w sodium acetate;

[0027] Pretreatment agent III: a sterile aqueous solution consisting of 4 M potassium acetate and 0.6 M sodium chloride;

[0028] The above chemicals were all analytically pure.

[0029] Chicken manure was used as the test sample and pretreated as follows:

[0030] (1) The aquaculture waste was suspended in sterile water at a concentration of 20% w / w, and pretreatment agent I was added at a weight ratio of 30:1 to the aquaculture waste and treated for 2 hours;

[0031] (2) After centrifugation, pretreatment agent II was added to the solid phase at a weight ratio of 2:8 for 0.5 hours;

[0032] (3) After centrifugation, pretreatment agent III was added to the solid phase at a weight ratio of 2:8 for 0.5 hours.

[0033] Example 2

[0034] The breeding waste in this embodiment was taken from chicken manure from a local chicken farm, which is consistent with Example 1;

[0035] Pretreatment agent I: composed of aluminum sulfate and ferrous sulfate in a weight ratio of 5:1;

[0036] Pretreatment agent II: a sterile aqueous solution consisting of 12% w / w PVP30 and 0.5% w / w sodium acetate;

[0037] Pretreatment agent III: a sterile aqueous solution consisting of 5 M potassium acetate and 0.5 M sodium chloride;

[0038] The above chemicals were all analytically pure.

[0039] Chicken manure was used as the test sample and pretreated as follows:

[0040] (1) The aquaculture waste was suspended in sterile water at a concentration of 20% w / w, and pretreatment agent I was added at a weight ratio of 30:1 to the aquaculture waste and treated for 2 hours;

[0041] (2) After centrifugation, pretreatment agent II was added to the solid phase at a weight ratio of 2:8 for 0.5 hours;

[0042] (3) After centrifugation, pretreatment agent III was added to the solid phase at a weight ratio of 2:8 for 0.5 hours.

[0043] Example 3

[0044] The breeding waste in this embodiment was taken from chicken manure from a local chicken farm, which is consistent with Example 1;

[0045] Pretreatment agent I: composed of aluminum sulfate and ferrous sulfate in a weight ratio of 5:3;

[0046] Pretreatment agent II: a sterile aqueous solution consisting of 8% w / w PVP30 and 1.5% w / w sodium acetate;

[0047] Pretreatment agent III: a sterile aqueous solution consisting of 3 M potassium acetate and 0.8 M sodium chloride;

[0048] The above chemicals were all analytically pure.

[0049] Chicken manure was used as the test sample and pretreated as follows:

[0050] (1) The aquaculture waste was suspended in sterile water at a concentration of 20% w / w, and pretreatment agent I was added at a weight ratio of 30:1 to the aquaculture waste and treated for 2 hours;

[0051] (2) After centrifugation, pretreatment agent II was added to the solid phase at a weight ratio of 2:8 for 0.5 hours;

[0052] (3) After centrifugation, pretreatment agent III was added to the solid phase at a weight ratio of 2:8 for 0.5 hours.

[0053] Comparative Example 1

[0054] Compared with Example 1, except that the pretreatment agent I is only aluminum sulfate, the rest is the same as Example 1.

[0055] Comparative Example 2

[0056] Compared with Example 1, except for the lack of step (2), the rest is consistent with Example 1. The specific scheme is:

[0057] The breeding waste in this embodiment is taken from chicken manure from a local chicken farm;

[0058] Pretreatment agent I: composed of aluminum sulfate and ferrous sulfate in a weight ratio of 5:2;

[0059] Pretreatment agent III: a sterile aqueous solution consisting of 4 M potassium acetate and 0.6 M sodium chloride;

[0060] The above chemicals were all analytically pure.

[0061] Chicken manure was used as the test sample and pretreated as follows:

[0062] (1) The aquaculture waste was suspended in sterile water at a concentration of 20% w / w, and pretreatment agent I was added at a weight ratio of 30:1 to the aquaculture waste and treated for 2 hours;

[0063] (2) After centrifugation, pretreatment agent III was added to the solid phase at a weight ratio of 2:8 for 0.5 hours.

[0064] Comparative Example 3

[0065] Compared with Example 1, except for the lack of step (3), the rest is consistent with Example 1. The specific scheme is:

[0066] The breeding waste in this embodiment is taken from chicken manure from a local chicken farm;

[0067] Pretreatment agent I: composed of aluminum sulfate and ferrous sulfate in a weight ratio of 5:2;

[0068] Pretreatment agent II: a sterile aqueous solution consisting of 10% w / w PVP30 and 1% w / w sodium acetate;

[0069] The above chemicals were all analytically pure.

[0070] Chicken manure was used as the test sample and pretreated as follows:

[0071] (1) The aquaculture waste was suspended in sterile water at a concentration of 20% w / w, and pretreatment agent I was added at a weight ratio of 30:1 to the aquaculture waste and treated for 2 hours;

[0072] (2) After centrifugation, pretreatment agent II was added to the solid phase at a weight ratio of 2:8 for 0.5 hours.

[0073] Experimental Example 1

[0074] This experiment uses FastDNA TM The DNA concentrations obtained after pretreatment with the methods described in Examples 1 to 3 and Comparative Examples 1 to 3 were investigated using the SPIN Kit for Soil kit. TMAccording to the SPINKit for Soil kit instructions, three extractions were performed for each Example or Comparative Example, and the average value was calculated. A control group, without the pretreatment methods of each Example or Comparative Example, followed the QIAamp Fast DNAStool Mini Kit protocol throughout the entire extraction process, also performing three extractions, with the average value calculated. The experimental results are shown in Table 1.

[0075] Table 1

[0076] DNA concentration (ng / μl) Example 1 63.2 Example 2 62.7 Example 3 62.3 Comparative Example 1 41.8 Comparative Example 2 31.0 Comparative Example 3 28.4 control group 26.9

[0077] Experimental Example 2

[0078] This experimental example used the Magnetic Soil And Stool DNA Kit for extraction, examining the DNA concentrations obtained after pretreatment using the methods described in Examples 1-3 and Comparative Examples 1-3. The extraction steps were followed as described in the Magnetic Soil And Stool DNA Kit instructions. Three extractions were performed for each Example or Comparative Example, and the average value was calculated. A control group, excluding the pretreatment methods of each Example or Comparative Example, followed the QIAampFast DNA Stool Mini Kit procedures throughout the entire process, with the average value calculated. The experimental results are shown in Table 2.

[0079] Table 2

[0080] DNA concentration (ng / μl) Example 1 58.6 Example 2 56.9 Example 3 57.4 Comparative Example 1 35.9 Comparative Example 2 27.1 Comparative Example 3 26.4 control group 26.7

[0081] As shown in Experimental Examples 1 and 2, the pretreatment method for detecting resistance genes in aquaculture waste provided by the present invention is not only applicable to different existing commercial test kits, but also significantly improves the extraction concentration of DNA, overcoming the defect that the DNA concentration is often insufficient when detecting resistance genes in aquaculture waste.

Claims

1. A method for pre-treating a sample for detecting resistance genes in aquaculture waste, wherein the pre-treatment is a process of treating the sample before using a DNA extraction kit, characterized in that: The pretreatment method comprises the following steps: (1) suspending the aquaculture waste in sterile water and treating it with a pretreatment agent I for 1 to 2 hours; the pretreatment agent I is composed of aluminum sulfate and ferrous sulfate in a weight ratio of 5:1 to 3; (2) After centrifugation, pretreatment agent II is added to the solid phase for treatment; The pretreatment agent II is a sterile aqueous solution composed of 8-12% w / w PVP30 and 0.5-1.5% w / w sodium acetate; (3) After centrifugation, pretreatment agent III is added to the solid phase for treatment; the pretreatment agent III is a sterile aqueous solution consisting of potassium acetate with a concentration of 3 to 5 M and sodium chloride with a concentration of 0.5 to 0.8 M.

2. The pretreatment method according to claim 1, characterized in that The breeding waste includes livestock excrement.

3. The pretreatment method according to claim 2, characterized in that The pretreatment agent I is composed of aluminum sulfate and ferrous sulfate in a weight ratio of 5:

2.

4. The pretreatment method according to claim 3, characterized in that The concentration of the aquaculture waste in sterile water is 20% w / w, and the weight ratio of the aquaculture waste to the pretreatment agent I is 30:

1.

5. The pretreatment method according to claim 1 or 4, characterized in that The pretreatment agent II is a sterile aqueous solution consisting of 10% w / w PVP30 and 1% w / w sodium acetate.

6. The pretreatment method according to claim 5, characterized in that In step (2), the weight ratio of the solid phase to the pretreatment agent II is 2:

8.

7. The pretreatment method according to claim 6, characterized in that In step (2), the treatment time is 0.5 hours.

8. The pretreatment method according to claim 1 or 6, characterized in that The pretreatment agent III is a sterile aqueous solution consisting of 4M potassium acetate and 0.6M sodium chloride.

9. The pretreatment method according to claim 8, characterized in that In step (3), the weight ratio of the solid phase to the pretreatment agent III is 2:

8.

10. The pretreatment method according to claim 9, characterized in that: In step (3), the treatment time is 0.5 hours.