Stomach juice sample preserving fluid as well as preparation method and application thereof

Abstract

The invention provides gastric juice sample preserving fluid as well as a preparation method and application thereof, and belongs to the technical field of biology. The gastric juice sample preserving fluid takes water as a solvent and comprises the following components: 0.5 to 4.5 M of guanidine isothiocyanate, 4.5 to 55 mM of disodium ethylene diamine tetraacetate, 0.5 to 22 mM of tris (2-carboxyethyl) phosphine and a tris (hydroxymethyl) aminomethane buffer solution. The preservation solution can improve the detection effect of the gastric juice sample, can effectively protect the gastric juice sample for 5 days at room temperature, has the effect equivalent to that of-20 DEG C cryopreservation, and provides more flexibility and convenience for transportation and preservation of the sample.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a gastric juice sample preservation solution, a preparation method thereof, and an application thereof. Background Art

[0002] Helicobacter pylori (Hp) infection is one of the most common chronic infections in humans. Almost all Hp-infected individuals will develop chronic active gastritis. Some infected individuals may develop peptic ulcers or indigestion, and a small number of infected individuals may progress to gastric cancer. Common detections of Helicobacter pylori infection include breath test, serological detection, culture, and molecular biology detection, etc. Among them, the culture method and the molecular biology method can be used for bacterial drug susceptibility testing. The drug sensitivity test is the gold standard for drug resistance detection and is a commonly used detection method in clinical practice, but it is time-consuming and laborious, with low sensitivity, and there may be certain missed detections. In recent years, important progress has been made in the detection of Helicobacter pylori drug resistance by molecular biology methods. The molecular biology method has the advantages of being fast, highly sensitive, and highly specific, and is superior to the traditional bacterial culture and drug sensitivity test; using the molecular biology detection method to detect the clarithromycin and levofloxacin drug resistance genotypes has important guiding value for Hp eradication treatment. Based on the Target Next-generation Sequencing (tNGS) technology, it does not rely on traditional microbial culture, directly enriches nucleic acids in clinical samples, then performs high-throughput sequencing, and then compares and analyzes with the database. According to the aligned sequence information, it can judge the bacterial identification, virulence, drug resistance gene mutation sites of Helicobacter pylori contained in the sample, as well as the polymorphism of the patient's drug metabolism genes, etc., and can quickly and objectively provide a basis for clinical diagnosis.

[0003] Gastric juice is one of the common sample types for detecting Helicobacter pylori by molecular biology methods. Taking gastric juice for Helicobacter pylori detection has relatively high accuracy. However, at present, many grass-roots hospitals do not have the detection conditions for Next-generation sequencing (NGS) technology, and the samples to be tested need to be transferred to institutions with detection conditions. How to preserve gastric juice samples is a crucial issue. The pH value of gastric juice is about 0.9 - 1.5, showing strong acidity. Gastric juice contains gastric acid, pepsinogen, mucin, etc., and the components are relatively complex, which has a great impact on the nucleic acid extraction of gastric juice samples, thus affecting the accuracy of subsequent detection results and further affecting clinical treatment. Therefore, it is particularly important to develop a preservation solution suitable for NGS that can stably preserve gastric juice samples and improve the nucleic acid extraction efficiency of gastric juice samples. Summary of the Invention

[0004] In view of this, the object of the present invention is to provide a preservation solution for gastric juice samples, its preparation method and application. This preservation solution can improve the nucleic acid extraction efficiency of gastric juice samples and ensure the stability during sample transportation and storage.

[0005] To solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a preservation solution for gastric juice samples, using water as a solvent, and comprising the following components: 0.5 - 4.5 M guanidine isothiocyanate, 4.5 - 55 mM disodium ethylenediaminetetraacetate, 0.5 - 22 mM tris(2 - carboxyethyl)phosphine, and tris(hydroxymethyl)aminomethane buffer solution.

[0007] Preferably, using water as a solvent, it comprises the following components: 1 - 4 M guanidine isothiocyanate, 5 - 50 mM disodium ethylenediaminetetraacetate, 1 - 20 mM tris(2 - carboxyethyl)phosphine, and tris(hydroxymethyl)aminomethane buffer solution.

[0008] Preferably, the water is deionized water.

[0009] Preferably, the pH of the preservation solution for gastric juice samples is 7 - 9.

[0010] The present invention also provides a preparation method for the preservation solution for gastric juice samples, comprising the following steps: mixing guanidine isothiocyanate, disodium ethylenediaminetetraacetate, tris(2 - carboxyethyl)phosphine with water, adjusting the pH to 7 - 9 with tris(hydroxymethyl)aminomethane buffer solution, making up the volume with water, and filtering to obtain the preservation solution for gastric juice samples.

[0011] Preferably, the mixing is until guanidine isothiocyanate, disodium ethylenediaminetetraacetate, and tris(2 - carboxyethyl)phosphine are dissolved.

[0012] Preferably, the volume is made up to 50 - 150 mL.

[0013] Preferably, the filtering is carried out using a 0.15 - 0.35 μm filter membrane.

[0014] The present invention also provides the application of the preservation solution for gastric juice samples in protecting gastric juice.

[0015] Preferably, the volume ratio of the gastric juice to the preservation solution for gastric juice samples is 1:5 - 15.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] In the present invention, guanidine isothiocyanate (GITC) is used as a protein denaturant, which can dissolve proteins and inhibit the degradation of nucleic acids by nucleases; disodium ethylenediaminetetraacetate (EDTA-2Na) is used as a chelating agent to chelate metal ions in the sample and inhibit the reaction of nucleases; tris(2-carboxyethyl)phosphine (TCEP) is an efficient reducing agent that can selectively reduce disulfide bonds in polypeptides or proteins; tris(hydroxymethyl)aminomethane hydrochloride buffer (Tris-HCl) can maintain pH stability and provide a suitable buffer system for nucleic acids. Through the reasonable proportioning and common action of the above components, the preservation solution of the present invention can effectively release nucleic acids in gastric juice samples and effectively protect the nucleic acids in gastric juice samples for 5 days at room temperature, which can not only improve the extraction efficiency of nucleic acids in gastric juice samples, but also ensure the stability during sample transportation and storage. The preservation solution of the present invention is alkaline, which can increase the pH value of gastric juice, provide a suitable buffer system for the nucleic acids released from the sample, and maintain the stability of the sample. Detailed implementation manners

[0018] The present invention provides a preservation solution for gastric juice samples, which uses water as a solvent and includes the following components: 0.5 - 4.5 M guanidine isothiocyanate, 4.5 - 55 mM disodium ethylenediaminetetraacetate, 0.5 - 22 mM tris(2-carboxyethyl)phosphine, and tris(hydroxymethyl)aminomethane buffer; preferably, the preservation solution for gastric juice samples uses water as a solvent and includes the following components: 1 - 4 M guanidine isothiocyanate, 5 - 50 mM disodium ethylenediaminetetraacetate, 1 - 20 mM tris(2-carboxyethyl)phosphine, and tris(hydroxymethyl)aminomethane buffer; more preferably, the preservation solution for gastric juice samples uses water as a solvent and includes the following components: 2 M guanidine isothiocyanate, 20 mM disodium ethylenediaminetetraacetate, 10 mM tris(2-carboxyethyl)phosphine, and tris(hydroxymethyl)aminomethane buffer. In the present invention, guanidine isothiocyanate is used as a protein denaturant, which can dissolve proteins and inhibit the degradation of nucleic acids by nucleases; disodium ethylenediaminetetraacetate is used as a chelating agent to chelate metal ions in the sample and inhibit the reaction of nucleases; tris(2-carboxyethyl)phosphine is an efficient reducing agent that can selectively reduce disulfide bonds in polypeptides or proteins; the tris(hydroxymethyl)aminomethane buffer can maintain pH stability and provide a suitable buffer system for nucleic acids. The tris(hydroxymethyl)aminomethane buffer in the present invention is tris(hydroxymethyl)aminomethane hydrochloride buffer (Tris-HCl).

[0019] In the present invention, the water is deionized water. Unless otherwise specified, the deionized water described in the present invention can be purchased through commercial channels.

[0020] The present invention also provides a method for preparing the gastric juice sample preservation solution, which includes the following steps: Mix guanidine isothiocyanate, disodium ethylenediaminetetraacetate, tris(2-carboxyethyl)phosphine with water, adjust the pH to 7-9 with tris(hydroxymethyl)aminomethane buffer solution, make up the volume with water, and filter to obtain the gastric juice sample preservation solution. The pH adjustment of the tris(hydroxymethyl)aminomethane buffer solution in the present invention is preferably 7.5-8.5.

[0021] In the present invention, guanidine isothiocyanate, disodium ethylenediaminetetraacetate, tris(2-carboxyethyl)phosphine need to be mixed with water until all components are dissolved. The volume is made up to 50-150 mL in the present invention, preferably 60-120 mL, and more preferably 100 mL. The filtration in the present invention is carried out by filtering with a 0.15-0.35 μm filter membrane, preferably filtering with a 0.22 μm filter membrane.

[0022] The present invention also provides the application of the gastric juice sample preservation solution in protecting gastric juice. In the present invention, the preservation solution is added to gastric juice, and gastric juice is protected at room temperature for 4-8 days, which can increase the pH value of gastric juice, provide a suitable buffer system for the nucleic acid released from the sample, and keep the sample stable. When using the preservation solution to protect gastric juice in the present invention, the volume ratio of gastric juice to the gastric juice sample preservation solution is 1:5-15, preferably 1:6-12, and more preferably 1:10.

[0023] In the present invention, unless otherwise specified, all components or reagents or culture media are commercially available products well-known to those skilled in the art.

[0024] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

[0025] Example 1

[0026] A gastric juice sample preservation solution uses deionized water as a solvent and is made of the following components at final concentrations: 1 M guanidine isothiocyanate, 5 mM disodium ethylenediaminetetraacetate, 1 mM tris(2-carboxyethyl)phosphine, and tris(hydroxymethyl)aminomethane hydrochloride buffer solution.

[0027] The preparation method of the gastric juice sample preservation solution is as follows:

[0028] Mix accurately weighed guanidine isothiocyanate, disodium ethylenediaminetetraacetate, and tris(2-carboxyethyl)phosphine, adjust the pH to 8.0 with tris(hydroxymethyl)aminomethane hydrochloride buffer solution, make up the volume to 100 mL with deionized water, filter through a 0.22 μm filter membrane, take the filtrate to obtain a gastric juice sample preservation solution containing guanidine isothiocyanate with a final concentration of 1 M, disodium ethylenediaminetetraacetate with a concentration of 5 mM, and tris(2-carboxyethyl)phosphine with a concentration of 1 mM.

[0029] Example 2

[0030] A gastric juice sample preservation solution uses deionized water as the solvent and is made from the following components with final concentrations: 2 M guanidine isothiocyanate, 20 mM disodium ethylenediaminetetraacetate, 10 mM tris(2-carboxyethyl)phosphine, and tris(hydroxymethyl)aminomethane hydrochloride buffer solution.

[0031] The preparation method of the gastric juice sample preservation solution is the same as that of Example 1.

[0032] Example 3

[0033] A gastric juice sample preservation solution uses deionized water as the solvent and is made from the following components with final concentrations: 4 M guanidine isothiocyanate, 50 mM disodium ethylenediaminetetraacetate, 20 mM tris(2-carboxyethyl)phosphine, and tris(hydroxymethyl)aminomethane hydrochloride buffer solution.

[0034] The preparation method of the gastric juice sample preservation solution is the same as that of Example 1.

[0035] Comparative Example 1

[0036] The difference from Example 2 is that guanidine isothiocyanate is removed, and the other components and steps are the same as those in Example 2.

[0037] Comparative Example 2

[0038] The difference from Example 2 is that tris(2-carboxyethyl)phosphine is removed, and the other components and steps are the same as those in Example 2.

[0039] Comparative Example 3

[0040] The difference from Example 2 is that guanidine isothiocyanate is replaced with urea, and the other components and steps are the same as those in Example 2.

[0041] Comparative Example 4

[0042] The difference from Example 2 is that tris(2-carboxyethyl)phosphine is replaced with dithiothreitol (DTT), and the other components and steps are the same as those in Example 2.

[0043] Test Example 1

[0044] 1. Sample preparation: Divide 2 gastric juice samples (Sample 1 and Sample 2) from Guangdong Provincial People's Hospital into 6 tubes evenly, 50 μL per tube, two tubes in each group, and perform the following different treatments:

[0045] Control group 1: Add 500 μL of normal saline, mix well, and store at room temperature for 0 days and 5 days.

[0046] Control group 2: Add 500 μL of normal saline, mix well, and store at -20 °C for 0 days and 5 days.

[0047] Experimental group: Add 500 μL of the preservation solution prepared in Example 2, mix well, and store at room temperature for 0 days and 5 days.

[0048] 2. Nucleic acid extraction and pathogen-targeted sequencing (tNGS):

[0049] (1) Nucleic acid extraction

[0050] Use a nucleic acid extraction and purification kit to extract nucleic acids from the samples in each group above.

[0051] (2) tNGS library construction and sequencing

[0052] 1) Design primers for the target pathogen / gene, and use a multiplex amplification enzyme to perform multiplex PCR amplification on the extracted nucleic acids to enrich the specific nucleic acid fragments of the target pathogen / gene in the nucleic acids. The PCR amplification program is shown in Table 1.

[0053] Table 1 PCR amplification program

[0054]

[0055] 2) Purify the above PCR products using the magnetic bead separation method.

[0056] 3) After adding sequencing adapter primers to the purified products, perform PCR amplification again, and the amplification program is shown in Table 2.

[0057] Table 2 Repeated PCR amplification program

[0058]

[0059] 4) Purify the above PCR products, measure the concentration of the purified products, and mix them in equal amounts as the sequencing library.

[0060] 5) After the library is denatured with 0.1 mol / L sodium hydroxide for 5 min, sequence it on the machine according to the amount of 1 pmol, and use a KMMiniSeqDx-CN sequencer for sequencing. Statistically analyze the rpk (normalized reads count) values of each pathogenic microorganism in the preserved gastric juice detected.

[0061] 3. Results

[0062] Two gastric juice samples were stored in different ways for 0 days and 5 days, and then nucleic acids were extracted. Detection was performed by tNGS to compare the pathogen detection rpk (normalized reads) of different storage methods and storage times. The results are shown in Table 3: the rpk of the experimental group at 0 day and 5 days was higher than that of control groups 1 and 2, indicating that the preservation solution of the present invention is helpful for nucleic acid extraction and detection; the rpk of control group 1 decreased significantly when stored for 5 days compared with 0 day, and there was only no significant difference in the rpk of control group 2 and the experimental group when stored for 5 days compared with 0 day, indicating that the preservation solution of the present invention can effectively protect gastric juice samples for 5 days at room temperature, and the effect is equivalent to that of freezing storage at -20°C.

[0063] In summary, the gastric juice sample preservation solution prepared by the present invention can improve the detection effect of gastric juice samples, and can effectively protect gastric juice samples for up to 5 days at room temperature. The effect is equivalent to that of freezing storage at -20°C, providing more flexibility and convenience for sample transportation and storage.

[0064] Table 3 tNGS detection results (rpk) of different storage methods and storage times

[0065]

[0066]

[0067] Experimental Example 2

[0068] 1. Sample preparation

[0069] Two gastric juice samples (Sample 3 and Sample 4) from Guangdong Provincial People's Hospital were evenly divided into 3 groups, with 2 tubes in each group and 50 μL in each tube. 500 μL of the preservation solution prepared in Examples 1-3 was added respectively, mixed well, and stored at room temperature for 0 day and 5 days.

[0070] 2. Nucleic acid extraction and tNGS detection

[0071] A nucleic acid extraction and purification kit was used to extract nucleic acids from the above-mentioned groups of samples. Primers for target pathogens / genes were designed for multiplex PCR amplification to construct a library, and the library was sequenced. The steps were the same as those in Experimental Example 1.

[0072] 3. Results

[0073] Two gastric juice samples were stored in different preservation solutions for 0 days and 5 days, and then nucleic acids were extracted. Detection was performed by tNGS to compare the pathogen detection rpk (normalized reads) of different preservation solutions and storage times. The results are shown in Table 4: there was no obvious difference among the preservation solutions prepared in Examples 1-3 when stored for 0 day and 5 days, and the rpk detected by the preservation solution prepared in Example 2 was slightly higher. In summary, the preservation solutions prepared in Examples 1-3 can all better preserve gastric juice samples, and the detection effects are all good. The preservation solution prepared in Example 2 has relatively better effects.

[0074] Table 4 tNGS detection results (rpk) of preservation solutions with different concentrations

[0075]

[0076]

[0077] Experimental Example 3

[0078] 1. Sample preparation

[0079] Select 2 gastric juice samples (Sample 5 and Sample 6) from Guangdong Provincial People's Hospital, evenly divide each into 5 groups, with 2 tubes in each group and 50 μL in each tube. Add 500 μL of the preservation solution prepared in Example 2, the preservation solution prepared in Comparative Example 1, the preservation solution prepared in Comparative Example 2, the preservation solution prepared in Comparative Example 3, and the preservation solution prepared in Comparative Example 4 respectively, mix well, and store at room temperature for 0 days and 5 days.

[0080] 2. Nucleic acid extraction and tNGS detection

[0081] Use a nucleic acid extraction and purification kit to extract nucleic acids from the samples. Design primers for the target pathogen / genes for multiplex PCR amplification to construct a library, and sequence the library. The steps are the same as those in Experimental Example 1.

[0082] 3. Results

[0083] Extract nucleic acids from 2 gastric juice samples after storing them in different preservation solutions for 0 days and 5 days, and compare the pathogen detection rpk (normalized read counts) of different preservation solutions and storage times through tNGS detection. The results are shown in Table 5: The rpk of the preservation solution in Example 2 is higher than that of the preservation solutions prepared in Comparative Examples 1 - 4 when stored for 0 days and 5 days, and the rpk of the preservation solution in Example 2 after storing for 5 days is higher than that of Comparative Examples 1 - 4 after storing for 5 days, indicating that the preservation solution prepared in Example 2 of the present invention is relatively more conducive to nucleic acid extraction and detection of gastric juice samples and the stable preservation of gastric juice samples. In summary, the formula of the preservation solution prepared in Example 2 has a relatively better effect on the preservation of gastric juice samples.

[0084] Table 5 tNGS detection results (rpk) of different preservation solutions

[0085]

[0086]

[0087] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A gastric juice sample preservation solution, characterized in that Using water as a solvent, it comprises the following components: 0.5 - 4.5 M guanidine isothiocyanate, 4.5 - 55 mM disodium ethylenediaminetetraacetate, 0.5 - 22 mM tris(2 - carboxyethyl)phosphine, and tris(hydroxymethyl)aminomethane buffer solution.

2. The gastric juice sample preservation solution according to claim 1, wherein, Using water as a solvent, it comprises the following components: 1 - 4 M guanidine isothiocyanate, 5 - 50 mM disodium ethylenediaminetetraacetate, 1 - 20 mM tris(2 - carboxyethyl)phosphine, and tris(hydroxymethyl)aminomethane buffer solution.

3. The gastric juice sample preservation solution according to claim 1 or 2, characterized in that, The water is deionized water.

4. The gastric juice sample preservation solution according to claim 1 or 2, characterized in that, The pH of the gastric juice sample preservation solution is 7 - 9.

5. The preparation method of the gastric juice sample preservation solution according to claim 1 or 2, characterized in that, It comprises the following steps: mixing guanidine isothiocyanate, disodium ethylenediaminetetraacetate, tris(2 - carboxyethyl)phosphine with water, adjusting the pH to 7 - 9 with tris(hydroxymethyl)aminomethane buffer solution, making up the volume with water, and filtering to obtain the gastric juice sample preservation solution.

6. The preparation method according to claim 5, wherein, The mixing is until guanidine isothiocyanate, disodium ethylenediaminetetraacetate, and tris(2 - carboxyethyl)phosphine are dissolved.

7. The preparation method according to claim 5, characterized in that, The volume is made up to 50 - 150 mL.

8. The preparation method according to claim 5, characterized in that, The filtering is carried out using a 0.15 - 0.35 μm filter membrane.

9. The application of the gastric juice sample preservation solution according to any one of claims 1 - 4 in protecting gastric juice.

10. The application according to claim 9, wherein The volume ratio of the gastric juice to the gastric juice sample preservation solution is 1:5 - 15.

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