Feces sample preservation solution, preparation method thereof and feces sample preservation method

Abstract

The invention provides an excrement sample preservation solution, a preparation method thereof and an excrement sample preservation method, and belongs to the technical field of excrement detection. The invention provides an excrement sample preserving fluid which comprises the following components in concentration: 1 to 4 mol / L of guanidine isothiocyanate, 0.1 to 1 vol% of Tween, 10 to 100 mmol / L of disodium ethylene diamine tetraacetate, 1 to 5 mmol / L of diazolidinyl urea, 1 to 10 mmol / L of dithiothreitol, 1 to 20 mmol / L of sodium chloride, 10 to 50 mmol / L of lauryl sodium sulfate, a tris (hydroxymethyl) aminomethane buffer solution and water, the dosage of the tris (hydroxymethyl) aminomethane buffer solution enables the pH value of the fecal sample preservation solution to be 8. According to the invention, Tween, diazolidinyl urea and dithiothreitol are creatively used, and guanidinium isothiocyanate, disodium ethylene diamine tetraacetate and other components are reasonably proportioned, so that the excrement preservation effect is better, and the method is more suitable for NGS detection.

Description

Technical Field

[0001] The present invention relates to the technical field of stool detection, and in particular to a stool sample preservation solution and a preparation method thereof, and a stool sample preservation method. Background Art

[0002] Gastrointestinal infections are caused by bacteria, viruses and parasites, which can cause inflammation of the gastrointestinal tract, including the stomach and small intestine. Symptoms include diarrhea and dehydration, which are the main hazards of gastrointestinal infections. Although gastroenteritis is not very serious in healthy adults and only manifests as discomfort and inconvenience, it can cause life-threatening dehydration and electrolyte imbalance in seriously ill, weak, young and elderly patients.

[0003] Common diagnostic methods for etiological examination of infectious diarrhea include smear microscopy, culture, immunoassay and molecular biology methods. Although traditional microbial diagnostic methods are commonly used clinical detection methods, they are time-consuming and labor-intensive, with low sensitivity, and may also miss certain cases. Molecular biology techniques are usually more sensitive and time-saving than culture. High-throughput sequencing technology (Next-Generation Sequencing, NGS) does not rely on traditional microbial culture. It directly enriches nucleic acids in clinical samples, then performs high-throughput sequencing, and then compares and analyzes with the database. The types of pathogenic microorganisms contained in the sample are determined based on the compared sequence information. It can quickly and objectively detect more pathogenic microorganisms (including viruses, bacteria and parasites, etc.) in clinical samples, providing a basis for clinical diagnosis.

[0004] However, many hospitals currently do not have the conditions for NGS testing and need to transfer samples to institutions that have the conditions for testing. How to preserve stool samples is a crucial issue. Since stool contains a large amount of enzymes and bacteria, these active substances will quickly degrade the nucleic acids in the samples, resulting in a decrease in sample quality, affecting the accuracy of subsequent test results, and further affecting clinical treatment. Therefore, it is particularly important to develop a preservation solution suitable for NGS that can stably preserve stool samples. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a stool sample preservation solution and a preparation method thereof, and a stool sample preservation method. The stool sample preservation solution provided by the present invention has a better stool preservation effect and is more suitable for NGS detection.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a stool sample preservation solution, comprising components with the following concentrations:

[0008] 1-4 mol / L guanidine thiocyanate, 0.1-1 vol%, Tween, 10-100 mmol / L disodium ethylenediaminetetraacetic acid, 1-5 mmol / L diimidazole urea, 1-10 mmol / L dithiothreitol, 1-20 mmol / L sodium chloride, 10-50 mmol / L sodium dodecyl sulfate, tris(hydroxymethyl)aminomethane buffer and water, wherein the amount of tris(hydroxymethyl)aminomethane buffer is such that the pH value of the stool sample preservation solution is 8.

[0009] Preferably, the stool sample preservation solution comprises components in the following concentrations:

[0010] Guanidine isothiocyanate 2.5mol / L, Tween 0.5vol%, ethylenediaminetetraacetic acid disodium 50mmol / L, diimidazole urea 2-3mmol / L, dithiothreitol 5mmol / L, sodium chloride 10mmol / L, sodium dodecyl sulfate 35mmol / L.

[0011] Preferably, the stool sample preservation solution comprises components in the following concentrations:

[0012] Guanidine isothiocyanate 2.5mol / L, Tween 0.5vol%, EDTA disodium 50mmol / L, diimidazole urea 3mmol / L, dithiothreitol 5mmol / L, sodium chloride 10mmol / L, sodium dodecyl sulfate 35mmol / L.

[0013] Preferably, the stool sample preservation solution comprises components in the following concentrations:

[0014] Guanidine isothiocyanate 2.5mol / L, Tween 0.5vol%, EDTA disodium 50mmol / L, diimidazole urea 2mmol / L, dithiothreitol 5mmol / L, sodium chloride 10mmol / L, sodium dodecyl sulfate 35mmol / L.

[0015] Preferably, the stool sample preservation solution comprises components in the following concentrations:

[0016] Guanidine isothiocyanate 4 mol / L, Tween 0.1 vol%, EDTA disodium 10 mmol / L, diimidazole urea 1 mmol / L, dithiothreitol 10 mmol / L, sodium chloride 1 mmol / L, sodium dodecyl sulfate 10 mmol / L.

[0017] Preferably, the stool sample preservation solution comprises components in the following concentrations:

[0018] Guanidine isothiocyanate 1mol / L, Tween 1vol%, EDTA disodium 100mmol / L, diimidazole urea 5mmol / L, dithiothreitol 1mmol / L, sodium chloride 20mmol / L, sodium dodecyl sulfate 50mmol / L.

[0019] Preferably, the Tween comprises Tween-20.

[0020] The present invention also provides a method for preparing the stool sample preservation solution described in the above technical solution, comprising the following steps:

[0021] Guanidine isothiocyanate, Tween, disodium ethylenediaminetetraacetate, diimidazole lignocerium, dithiothreitol, sodium chloride, sodium dodecyl sulfate, tris(hydroxymethyl)aminomethane) buffer and water are mixed and filtered to obtain the stool sample preservation solution.

[0022] The present invention also provides a stool sample preservation method, which comprises adding the stool sample preservation solution described in the above technical solution into the stool sample.

[0023] Preferably, 5 to 15 mL of the stool sample preservation solution is added to each gram of stool sample.

[0024] The invention provides a stool sample preservation solution, comprising the following components in concentrations: 1-4 mol / L of guanidine isothiocyanate, 0.1-1 vol% of Tween, 10-100 mmol / L of disodium ethylenediaminetetraacetate, 1-5 mmol / L of diimidazole urea, 1-10 mmol / L of dithiothreitol, 1-20 mmol / L of sodium chloride, 10-50 mmol / L of sodium dodecyl sulfate, tris(hydroxymethyl)aminomethane buffer and water, wherein the amount of the tris(hydroxymethyl)aminomethane buffer is such that the pH value of the stool sample preservation solution is 8.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention uses guanidine isothiocyanate (GITC) as a protein denaturant and Tween as an auxiliary agent. The combination of the two can quickly dissolve protein and inhibit the degradation of nucleic acid by nuclease; disodium ethylenediaminetetraacetic acid (EDTA-2Na) is used as a chelating agent to chelate metal ions in the sample and inhibit the reaction of nuclease; diimidazole lignocerium is used as an antibacterial agent, mainly utilizing its broad-spectrum microbial inhibition effect to effectively inhibit the proliferation of bacteria and fungi in feces; dithiothreitol (DTT) can not only reduce the disulfide bond in RNase (ribonuclease) protein and denature RNase enzyme, but also inhibit the breakage of phosphodiester bond caused by phenol oxidation and the cross-linking of RNA and DNA; sodium chloride (NaCl ) can stabilize the ion concentration of the solution; sodium dodecyl sulfate (SDS) changes the conformation of proteins and can also destroy cell membranes and dissociate nucleic acids; tris-hydroxymethylaminomethane buffer (Tris-HCl) can maintain a stable pH value and provide a suitable buffer system for nucleic acids; the present invention creatively uses Tween, diimidazole lignourea and dithiothreitol, combined with a reasonable ratio of guanidine isothiocyanate, disodium ethylenediaminetetraacetic acid and other ingredients, so that the stool preservation effect is better and more suitable for NGS detection. It can effectively protect the nucleic acid in the stool sample for at least 5 days at room temperature to prevent it from degrading during transportation and storage, ensure the stability of the sample, and provide more flexibility and convenience for the transportation and storage of the sample.

[0027] Furthermore, the guanidine isothiocyanate in the present invention can also inactivate viruses, release nucleic acids, and reduce the risk of biological leakage and transmission. DETAILED DESCRIPTION

[0028] The present invention provides a stool sample preservation solution, comprising components with the following concentrations:

[0029] 1-4 mol / L guanidine thiocyanate, 0.1-1 vol%, Tween, 10-100 mmol / L disodium ethylenediaminetetraacetic acid, 1-5 mmol / L diimidazole urea, 1-10 mmol / L dithiothreitol, 1-20 mmol / L sodium chloride, 10-50 mmol / L sodium dodecyl sulfate, tris(hydroxymethyl)aminomethane buffer and water, wherein the amount of tris(hydroxymethyl)aminomethane buffer is such that the pH value of the stool sample preservation solution is 8.

[0030] In the present invention, the vol% refers to volume %.

[0031] In the present invention, unless otherwise specified, the raw materials used are all commercially available products in the art.

[0032] In the present invention, the stool sample preservation solution preferably includes the following components in concentration:

[0033] Guanidine isothiocyanate 2.5mol / L, Tween 0.5vol%, ethylenediaminetetraacetic acid disodium 50mmol / L, diimidazole urea 2-3mmol / L, dithiothreitol 5mmol / L, sodium chloride 10mmol / L, sodium dodecyl sulfate 35mmol / L.

[0034] In a specific embodiment of the present invention, the stool sample preservation solution comprises components with the following concentrations:

[0035] Guanidine isothiocyanate 2.5mol / L, Tween 0.5vol%, EDTA disodium 50mmol / L, diimidazole urea 3mmol / L, dithiothreitol 5mmol / L, sodium chloride 10mmol / L, sodium dodecyl sulfate 35mmol / L.

[0036] In another specific embodiment of the present invention, the stool sample preservation solution comprises components with the following concentrations:

[0037] Guanidine isothiocyanate 2.5mol / L, Tween 0.5vol%, EDTA disodium 50mmol / L, diimidazole urea 2mmol / L, dithiothreitol 5mmol / L, sodium chloride 10mmol / L, sodium dodecyl sulfate 35mmol / L.

[0038] In another specific embodiment of the present invention, the stool sample preservation solution comprises components with the following concentrations:

[0039] Guanidine isothiocyanate 4 mol / L, Tween 0.1 vol%, EDTA disodium 10 mmol / L, diimidazole urea 1 mmol / L, dithiothreitol 10 mmol / L, sodium chloride 1 mmol / L, sodium dodecyl sulfate 10 mmol / L.

[0040] In another specific embodiment of the present invention, the stool sample preservation solution comprises components with the following concentrations:

[0041] Guanidine isothiocyanate 1mol / L, Tween 1vol%, EDTA disodium 100mmol / L, diimidazole urea 5mmol / L, dithiothreitol 1mmol / L, sodium chloride 20mmol / L, sodium dodecyl sulfate 50mmol / L.

[0042] In the present invention, the Tween preferably includes Tween-20.

[0043] In the present invention, guanidine isothiocyanate is used as a protein denaturant, and Tween is used as an auxiliary agent. The combination of the two can quickly dissolve protein and inhibit the degradation of nucleic acid by nuclease; disodium ethylenediaminetetraacetic acid is used as a chelating agent to chelate metal ions in the sample and inhibit the reaction of nuclease; diimidazole lignocerium is used as an antibacterial agent, mainly utilizing its broad-spectrum microbial inhibition effect to effectively inhibit the proliferation of bacteria and fungi in feces; dithiothreitol can not only reduce the disulfide bonds in RNase (ribonuclease) protein and denature RNase enzyme, but also inhibit The oxidation of phenols causes the breakage of phosphodiester bonds and the cross-linking of RNA and DNA; the sodium chloride shown can stabilize the ion concentration of the solution; the sodium dodecyl sulfate shown changes the conformation of proteins and can also destroy cell membranes and dissociate nucleic acids; the tris(hydroxymethyl)aminomethane buffer shown can maintain a stable pH value and provide a suitable buffer system for nucleic acids; the present invention creatively uses Tween, diimidazole lignourea and dithiothreitol, combined with a reasonable ratio of guanidine isothiocyanate, disodium ethylenediaminetetraacetic acid and other ingredients, so that the feces preservation effect is better and more suitable for NGS detection.

[0044] The present invention also provides a method for preparing the stool sample preservation solution described in the above technical solution, comprising the following steps:

[0045] Guanidine isothiocyanate, Tween, disodium ethylenediaminetetraacetate, diimidazole lignocerium, dithiothreitol, sodium chloride, sodium dodecyl sulfate, tris(hydroxymethyl)aminomethane) buffer and water are mixed and filtered to obtain the stool sample preservation solution.

[0046] The present invention has no particular limitation on the specific manner of mixing and filtering, and any manner familiar to those skilled in the art may be used.

[0047] The present invention also provides a stool sample preservation method, which comprises adding the stool sample preservation solution described in the above technical solution into the stool sample.

[0048] In the present invention, preferably 5 to 15 mL of the stool sample preservative solution is added to each gram of the stool sample, and more preferably 10 mL of the stool sample preservative solution is added.

[0049] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] In the embodiments of the present invention, M is the unit of molar concentration, representing mol / L.

[0051] Example 1

[0052] 1. The composition and preparation method of stool sample preservation solution are as follows:

[0053] Guanidine isothiocyanate 2.5M, Tween-20 0.5vol%, ethylenediaminetetraacetic acid disodium 50mM, diimidazole lignocerium 3mM, dithiothreitol 5mM, sodium chloride 10mM, sodium dodecyl sulfate 35mM, each component was mixed in a certain proportion, the pH value was adjusted to 8.0 with tris(hydroxymethyl)aminomethane buffer, the volume was made up to 100mL with deionized water, and filtered.

[0054] 2. Sample preparation:

[0055] The two stool samples were treated differently: control group 1: 0.2 g of stool was taken and stored at room temperature for 0 days and 5 days; control group 2: 0.2 g of stool was taken and stored at -20°C for 0 days and 5 days; experimental group: 0.2 g of stool was mixed with 2 mL of stool sample preservation solution (prepared in Example 1) and stored at room temperature for 0 days and 5 days.

[0056] 3. Nucleic acid extraction, library construction and sequencing:

[0057] (1) Nucleic acid extraction

[0058] DNA / RNA was extracted from stool samples using a nucleic acid extraction and purification kit.

[0059] (2) tNGS library construction and sequencing

[0060] 1) The extracted nucleic acid is synthesized using reverse transcriptase. Primers of the target pathogen are designed, and multiplex PCR amplification is performed using multiplex amplification enzymes to enrich specific nucleic acid fragments of common pathogenic pathogens in the nucleic acid. The PCR program is shown in Table 1.

[0061] Table 1 PCR program

[0062]

[0063] 2) Purify the PCR product using magnetic bead separation.

[0064] 3) After adding the sequencing adapter primer to the purified product, PCR amplification was performed again. The amplification program is shown in Table 2.

[0065] Table 2 Amplification procedures

[0066]

[0067] 4) The above PCR products are purified, the concentration of the purified products is measured, and equal masses are mixed to serve as a sequencing library.

[0068] 5) The library was denatured with 0.1 mol / L sodium hydroxide for 5 min, and then 1 pmol was sequenced using a KMMiniSeqDx-CN sequencer.

[0069] 4. Results

[0070] The stool samples were stored in different ways for 0 days and 5 days, and nucleic acids were extracted. The rpk (normalized reads number) of pathogen detection in different storage methods and storage times was compared by tNGS detection. The results are shown in Table 3: At 0 days, the rpk of the experimental group was slightly higher than that of the control groups 1 and 2. The stool sample preservation solution of the present invention was helpful for nucleic acid extraction and tNGS detection; at 5 days, the rpk of the experimental group was higher than that of the control group 1, and there was no significant difference with the control group 2, and there was no significant difference between the control group 2 and the experimental group at 0 days and 5 days, indicating that the effect of the stool sample preservation solution of the present invention on preserving stool samples at room temperature is equivalent to the effect of -20°C freezing preservation, and can stably preserve the nucleic acids in stool samples for at least 5 days without degradation. In summary, the present invention provides a stool sample preservation solution that can effectively protect stool samples for up to 5 days at room temperature, which is equivalent to -20°C freezing preservation, and provides more flexibility and convenience for the transportation and preservation of samples.

[0071] Table 3 tNGS test results (rpk) of different storage methods and storage times

[0072]

[0073] Example 2

[0074] The composition and preparation method of stool sample preservation solution are as follows:

[0075] Guanidine isothiocyanate 2.5M, Tween-20 0.5vol%, ethylenediaminetetraacetic acid disodium 50mM, diimidazole lignocerium 2mM, dithiothreitol 5mM, sodium chloride 10mM, sodium dodecyl sulfate 35mM, each component was mixed in a certain proportion, the pH value was adjusted to 8.0 with tris(hydroxymethyl)aminomethane buffer, the volume was made up to 100mL with deionized water, and filtered.

[0076] Example 3

[0077] The composition and preparation method of stool sample preservation solution are as follows:

[0078] Guanidine thiocyanate 4M, Tween-20 0.1vol%, ethylenediaminetetraacetic acid disodium 10mM, diimidazole lignocerium 1mM, dithiothreitol 10mM, sodium chloride 1mM, sodium dodecyl sulfate 10mM, each component was mixed in a certain proportion, the pH value was adjusted to 8.0 with tris(hydroxymethyl)aminomethane buffer, the volume was made up to 100mL with deionized water, and filtered.

[0079] Example 4

[0080] The composition and preparation method of stool sample preservation solution are as follows:

[0081] Guanidine thiocyanate 1M, Tween-20 1vol%, ethylenediaminetetraacetic acid disodium 100mM, diimidazole lignocerium 5mM, dithiothreitol 1mM, sodium chloride 20mM, sodium dodecyl sulfate 50mM, each component was mixed in a certain proportion, the pH value was adjusted to 8.0 with tris(hydroxymethyl)aminomethane buffer, the volume was made up to 100mL with deionized water, and filtered.

[0082] Comparative Example

[0083] The stool preservation solution disclosed in Example 1 of CN109371015A.

[0084] The pathogen detection rpk (normalized reads number) of the storage solution of Examples 2 to 4 and the comparative example was tested, and the steps were as follows:

[0085] Sample preparation:

[0086] Two stool samples were selected and evenly divided into 4 portions, each weighing 0.2 g. The samples were mixed with 2 mL of the preservation solution of Examples 2 to 4 and the comparative example, respectively, and stored at room temperature for 0 days and 5 days.

[0087] Nucleic acid extraction, library construction and sequencing:

[0088] DNA / RNA was extracted from stool samples using a nucleic acid extraction and purification kit. The extracted nucleic acid was synthesized into cDNA using reverse transcriptase, and then tNGS library was constructed and sequenced using a KM MiniSeqDx-CN sequencer.

[0089] The results are shown in Table 4. It can be seen that there is no significant difference in the detection effect of Examples 2 to 4 for storage for 0 days and storage for 5 days. The stool sample preservation solution of Example 2 has a slightly better effect when stored at room temperature for 5 days. Compared with the preservation solution of the comparative example, the rpk of fungal detection has a slight upward trend after 5 days of storage. In contrast, the stool sample preservation solution of Example 2 can better maintain the original microbial composition ratio in the sample. In summary, the present invention provides a stool sample preservation solution, which has a better preservation effect on stool samples at room temperature compared to the prior art, can better maintain the original microbial composition ratio in the sample, and is more suitable for tNGS detection.

[0090] Table 4 tNGS test results (rpk) of different preservation solutions, preservation methods and preservation times

[0091]

[0092] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A stool sample preservation solution, characterized in that: Includes components in the following concentrations: 1-4 mol / L guanidine thiocyanate, 0.1-1 vol%, Tween, 10-100 mmol / L disodium ethylenediaminetetraacetic acid, 1-5 mmol / L diimidazole urea, 1-10 mmol / L dithiothreitol, 1-20 mmol / L sodium chloride, 10-50 mmol / L sodium dodecyl sulfate, tris(hydroxymethyl)aminomethane buffer and water, wherein the amount of tris(hydroxymethyl)aminomethane buffer is such that the pH value of the stool sample preservation solution is 8.

2. The stool sample preservation solution according to claim 1, characterized in that: The stool sample preservation solution includes components with the following concentrations: Guanidine isothiocyanate 2.5mol / L, Tween 0.5vol%, ethylenediaminetetraacetic acid disodium 50mmol / L, diimidazole urea 2-3mmol / L, dithiothreitol 5mmol / L, sodium chloride 10mmol / L, sodium dodecyl sulfate 35mmol / L.

3. The stool sample preservation solution according to claim 2, characterized in that: The stool sample preservation solution includes components with the following concentrations: Guanidine isothiocyanate 2.5mol / L, Tween 0.5vol%, EDTA disodium 50mmol / L, diimidazole urea 3mmol / L, dithiothreitol 5mmol / L, sodium chloride 10mmol / L, sodium dodecyl sulfate 35mmol / L.

4. The stool sample preservation solution according to claim 2, characterized in that: The stool sample preservation solution includes components with the following concentrations: Guanidine isothiocyanate 2.5mol / L, Tween 0.5vol%, EDTA disodium 50mmol / L, diimidazole urea 2mmol / L, dithiothreitol 5mmol / L, sodium chloride 10mmol / L, sodium dodecyl sulfate 35mmol / L.

5. The stool sample preservation solution according to claim 1, characterized in that: The stool sample preservation solution includes components with the following concentrations: Guanidine isothiocyanate 4 mol / L, Tween 0.1 vol%, EDTA disodium 10 mmol / L, diimidazole urea 1 mmol / L, dithiothreitol 10 mmol / L, sodium chloride 1 mmol / L, sodium dodecyl sulfate 10 mmol / L.

6. The stool sample preservation solution according to claim 1, characterized in that: The stool sample preservation solution includes components with the following concentrations: Guanidine isothiocyanate 1mol / L, Tween 1vol%, EDTA disodium 100mmol / L, diimidazole urea 5mmol / L, dithiothreitol 1mmol / L, sodium chloride 20mmol / L, sodium dodecyl sulfate 50mmol / L.

7. The stool sample storage solution according to any one of claims 1 to 6, characterized in that: The Tween includes Tween-20.

8. The method for preparing a stool sample preservation solution according to any one of claims 1 to 7, characterized in that: The following steps are involved: Guanidine isothiocyanate, Tween, disodium ethylenediaminetetraacetate, diimidazole lignocerium, dithiothreitol, sodium chloride, sodium dodecyl sulfate, tris(hydroxymethyl)aminomethane) buffer and water are mixed and filtered to obtain the stool sample preservation solution.

9. A method for preserving stool samples, characterized in that: The stool sample storage solution according to any one of claims 1 to 7 is added to the stool sample.

10. The stool sample preservation method according to claim 9, characterized in that: Add 5 to 15 mL of the stool sample preservation solution to each gram of stool sample.

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