Sample pretreatment solutions and methods

Abstract

The present invention discloses a solution and method for sample pretreatment, wherein the solution comprises the following components: 2wt%-5wt% guanidine hydrochloride, 2 mM-10mM EDTA, 1%-5% sodium citrate, 0.5wt%-4wt% sodium hydroxide, 0.5wt%-3wt% N-acetyl-L-cysteine, and 2wt%-10wt% trehalose. The sample pretreatment solution of the present invention can realize pretreatment of various complex samples, and after pretreatment, sample nucleic acid extraction is not affected and has high quality; the sample pretreatment method of the present invention is time-consuming, and the treated sample can be effectively preserved without tube transfer.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical detection, and more specifically, to a solution and method for sample pretreatment. Background Art

[0002] There are many types of pathogenic microorganism samples in clinical practice, such as sputum, thick alveolar lavage fluid, feces, pus secretions, blood, dry throat swabs, dry genital tract samples, anal swabs, etc. These sample types are highly viscous or contain particulate matter, and generally require pre-treatment before proceeding to the next step of conventional nucleic acid extraction or microfluidic device extraction and other experimental operations.

[0003] The existing method of pre-treatment of samples is usually to use a specific treatment method for each specific microbial pathogen. For example, for sputum, it is generally treated with a liquefier such as 4% sodium hydroxide, which includes a heating step. This method takes about 30 minutes to process, which is time-consuming, and there is still the problem that some samples are difficult to liquefy. Other studies have shown that samples treated with 4% sodium hydroxide will also affect subsequent experiments such as nucleic acid extraction, making subsequent experimental operations troublesome. For fecal samples, due to the presence of a large amount of particulate matter, the commonly used treatment method is to perform low-speed centrifugation or stand for a period of time and take the supernatant. This method requires strict control of the centrifugation speed or standing time, because the pathogens will settle, and taking the supernatant will affect the inaccuracy of the detection results. For blood samples, because they are prone to coagulation, if the blood samples are not treated, it is likely to cause blockage of the microfluidic channel, making the instrument unable to operate normally. Red blood cell lysis solution is usually used for lysis. Other samples such as thick alveolar lavage fluid, pus secretions, and reproductive tract samples contain sticky substances, which are difficult to absorb during the experiment and are prone to laboratory contamination due to stringing. They are generally liquefied using relevant reagents, or sampled directly after continuous oscillation, which is not effective and is prone to sampling errors.

[0004] The existing method for pre-treatment of samples can only prepare a specific solution for each specific sample and perform specific treatment. The operation is complicated, the sample treatment effect is not good, and the treated samples cannot be effectively preserved. Therefore, it is very meaningful to provide a universal method that can process multiple samples. Summary of the invention

[0005] Based on this, an object of the present invention is to provide a sample pretreatment solution and a preparation method thereof and a sample pretreatment method, wherein the sample pretreatment solution can realize pretreatment of a variety of samples.

[0006] The technical solutions for achieving the above-mentioned invention objectives include the following.

[0007] The first aspect of the present invention provides a sample pretreatment solution, which includes the following components: 2wt%-5wt% guanidine hydrochloride, 2 mM~10mM EDTA, 1%~5% sodium citrate, 0.5wt%~4wt% sodium hydroxide, 0.5wt%~3wt% N-acetyl-L-cysteine, and 2wt%~10wt% trehalose.

[0008] The second aspect of the present invention provides a method for preparing the sample pretreatment solution, comprising the following steps: mixing the components, and adjusting the pH to 8-11.

[0009] The third aspect of the present invention provides a method for sample pretreatment, comprising the following steps: soaking the sample in the sample pretreatment solution and letting it stand.

[0010] The sample pretreatment solution of the present invention can realize pretreatment of various complex samples (inactivating samples, preventing blood coagulation, liquefying viscous samples such as sputum, etc.), and after pretreatment, the nucleic acid extraction of the samples is not affected and the quality is high; the sample pretreatment method of the present invention is time-saving, and the treated samples can be effectively preserved without the need for tube transfer. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 It is a filtering device used in the sample pretreatment method of the present invention, wherein a is a filter with a filter membrane; b is a collection tube with a tube cover; and c is a filtering device composed of a and b. DETAILED DESCRIPTION

[0012] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0013] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0014] The experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions, such as those described in Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0015] In some embodiments of the present invention, a sample pretreatment solution is disclosed, comprising the following components: 2wt%-5wt% guanidine hydrochloride, 2 mM~10mM EDTA, 1%~5% sodium citrate, 0.5wt%~4wt% sodium hydroxide, 0.5wt%~3wt% N-acetyl-L-cysteine, and 2wt%~10wt% trehalose.

[0016] Although sodium hydroxide alone is added to the sample pretreatment solution, it can liquefy viscous samples such as sputum, but it will affect the quality of the sample nucleic acid. The inventors of the present invention have found that by adding sodium hydroxide and N-acetyl-L-cysteine ​​in a specific ratio at the same time, the quality of the sample nucleic acid after pretreatment can be guaranteed while effectively liquefying viscous samples such as sputum. This may be because the acidic N-acetyl-L-cysteine ​​neutralizes part of the alkalinity of sodium hydroxide, so that the quality of the sample nucleic acid after pretreatment will not be damaged. The simultaneous addition of sodium hydroxide and N-acetyl-L-cysteine ​​also enhances the stability of the pretreatment solution. On the basis of sodium hydroxide and N-acetyl-L-cysteine, it is combined with specific concentrations of guanidine hydrochloride (protein denaturant, which can be used to dissolve proteins, causing damage to cell structure and secondary structure of nucleoprotein, dissociating from nucleic acid, and inactivating samples), EDTA (inhibiting nucleases, preventing DNA or RNA from being degraded, and protecting nucleic acids in samples), sodium citrate (preventing blood contained in samples from coagulation), and trehalose (further protecting nucleic acids). Under the joint action of various components, the solution can achieve pre-treatment of various samples, and the quality of the treated nucleic acids is high.

[0017] In some embodiments, the solution includes the following components: guanidine hydrochloride 3wt%-4wt%, EDTA 4 mM~8mM, sodium citrate 2%~4%, sodium hydroxide 1wt~3wt%, N-acetyl-L-cysteine ​​1wt%~2wt%, trehalose 4wt%~8wt%.

[0018] In some embodiments, the solution includes the following components: guanidine hydrochloride 3.4wt%-3.6wt%, EDTA 5mM~6mM, sodium citrate 2.5%~3.5%, sodium hydroxide 1.5wt%~2.5wt%, N-acetyl-L-cysteine ​​1.4wt%~1.6wt%, trehalose 5wt%~6wt%.

[0019] In other embodiments of the present invention, a method for preparing a sample pretreatment solution is disclosed, comprising the following steps: mixing the components, and adjusting the pH to 8-11.

[0020] In other embodiments of the present invention, a sample pretreatment method is disclosed, comprising the following steps: soaking the sample in the sample pretreatment solution and letting it stand.

[0021] In some embodiments, the sample includes a liquid sample or a dry sample, the liquid sample includes sputum, thick alveolar lavage fluid, pus secretion, blood or feces; the dry sample includes a throat swab, a genital tract sample or an anal swab.

[0022] In some embodiments, the method for processing the liquid sample includes the following steps: mixing the liquid sample with a sample pre-treatment solution in a volume ratio of 1:1 to 4, letting it stand for 15 min to 30 min, and then placing the sample in a filtration device for filtering and collecting the filtrate.

[0023] In some embodiments, the dry sample processing method includes the following steps: adding the sample pre-treatment solution to the dry sample to completely immerse the dry sample, standing for 8 min to 12 min, and then placing the sample in a filtration device for filtering and collecting the filtrate.

[0024] In some embodiments, the filtering device includes a collection tube and a filter placed in the collection tube, the collection tube has a tube cover, and the filter includes a filter membrane with a pore size of 5 to 25 μm. The sample after standing is filtered through the filter to further remove sticky substances and particulate matter without causing loss of the sample. After a brief centrifugation using a palm-type centrifuge, the sample is collected in the collection tube. Since the collection tube has a tube cover, the cover can be buckled for storage. Compared with conventional filter tubes without covers, it is more convenient to transfer the tube for storage.

[0025] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0026] Example 1 A sample pretreatment solution

[0027] A sample pretreatment solution of this embodiment is composed of the following components:

[0028] Guanidine hydrochloride 3.5wt%

[0029] EDTA 5.5 mM

[0030] Sodium Citrate 3%

[0031] Sodium hydroxide 2wt%

[0032] N-acetyl-L-cysteine ​​1.5wt%

[0033] Trehalose 5.5wt%.

[0034] The method for preparing the sample pretreatment solution of this embodiment includes the following steps: using an analytical balance to weigh 3.5 g of guanidine hydrochloride, 1.848 g of EDTA, 3 g of sodium citrate, 2 g of sodium hydroxide, 1.5 g of N-acetyl-L-cysteine, and 5.5 g of trehalose in a beaker, adding deionized water to melt, and finally quantifying to 100 mL with a volumetric flask.

[0035] Example 2 A sample pretreatment solution

[0036] A sample pretreatment solution of this embodiment is composed of the following components:

[0037] Guanidine hydrochloride 3wt%

[0038] EDTA 8 mM

[0039] Sodium citrate 4%

[0040] Sodium hydroxide 1wt%

[0041] N-acetyl-L-cysteine ​​2wt%

[0042] Trehalose 4 wt%.

[0043] The preparation method of the sample pretreatment solution in this embodiment is the same as that in Example 1.

[0044] Example 3 A sample pretreatment solution

[0045] A sample pretreatment solution of this embodiment is composed of the following components:

[0046] Guanidine hydrochloride 4wt%

[0047] EDTA 4 mM

[0048] Sodium Citrate 2%

[0049] Sodium hydroxide 3wt%

[0050] N-acetyl-L-cysteine ​​1wt%

[0051] Trehalose 8 wt%.

[0052] The preparation method of the sample pretreatment solution in this embodiment is the same as that in Example 1.

[0053] Example 4 A sample pretreatment solution

[0054] A sample pretreatment solution of this embodiment is composed of the following components:

[0055] Guanidine hydrochloride 5wt%

[0056] EDTA 2mM

[0057] Sodium citrate 1%

[0058] Sodium hydroxide 4wt%

[0059] N-acetyl-L-cysteine ​​0.5wt%

[0060] Trehalose 10wt%.

[0061] The preparation method of the sample pretreatment solution in this embodiment is the same as that in Example 1.

[0062] Example 5 A sample pretreatment solution

[0063] A sample pretreatment solution of this embodiment is composed of the following components:

[0064] Guanidine hydrochloride 2wt%

[0065] EDTA 10 mM

[0066] Sodium Citrate 5%

[0067] Sodium hydroxide 0.5wt%

[0068] N-acetyl-L-cysteine ​​3wt%

[0069] Trehalose 2wt%.

[0070] The preparation method of the sample pretreatment solution in this embodiment is the same as that in Example 1.

[0071] Comparative Example 1 A sample pretreatment solution

[0072] A sample pretreatment solution of this comparative example is composed of the following components:

[0073] Guanidine hydrochloride 3.5wt%

[0074] EDTA 5.5 mM

[0075] Sodium Citrate 3%

[0076] Trehalose 5.5wt%.

[0077] The preparation method of the sample pretreatment solution of this comparative example is the same as that of Example 1.

[0078] Comparative Example 2 A sample pretreatment solution

[0079] A sample pretreatment solution of this comparative example is composed of the following components:

[0080] Guanidine hydrochloride 3.5wt%

[0081] EDTA 5.5 mM

[0082] Sodium Citrate 3%

[0083] Sodium hydroxide 2wt%

[0084] Trehalose 5.5wt%.

[0085] The preparation method of the sample pretreatment solution of this comparative example is the same as that of Example 1.

[0086] Comparative Example 3 A sample pretreatment solution

[0087] A sample pretreatment solution of this comparative example is composed of the following components:

[0088] Guanidine hydrochloride 3.5wt%

[0089] EDTA 5.5 mM

[0090] Sodium Citrate 3%

[0091] N-acetyl-L-cysteine ​​1.5wt%

[0092] Trehalose 5.5wt%.

[0093] The preparation method of the sample pretreatment solution of this comparative example is the same as that of Example 1.

[0094] Comparative Example 4 A sample pretreatment solution

[0095] A sample pretreatment solution of this comparative example is composed of the following components:

[0096] Guanidine hydrochloride 3.5wt%

[0097] EDTA 5.5 mM

[0098] Sodium Citrate 3%

[0099] Sodium hydroxide 2wt%

[0100] N-acetyl-L-cysteine ​​1.5wt%.

[0101] The preparation method of the sample pretreatment solution of this comparative example is the same as that of Example 1.

[0102] Experimental Example 1 Effect of the sample pretreatment solution of the present invention on the physical properties of various samples after treatment

[0103] Various samples were treated using the sample pretreatment solution of Example 1 to examine the physical and chemical properties before and after the treatment.

[0104] 1. For sputum, thick alveolar lavage fluid, pus secretions, blood, and stool samples, mix the sample with the sample pretreatment solution in a volume ratio of 1:2. After mixing, let it stand for 20 minutes. Then take the treated sample, put it into the filter device for filtration, use a palm centrifuge for brief centrifugation, collect it in the collection tube, and close the lid to complete the sample pretreatment.

[0105] 2. For dry throat swabs, dry genital tract samples, and anal swab samples, add the sample pretreatment solution to the above dry swabs until the swab head is completely soaked, let it stand for 10 minutes, then take the treated sample, place it in a filter device for filtration, use a palm-type centrifuge for brief centrifugation, collect it in a collection tube, close the lid, and complete the sample pretreatment.

[0106] The physical properties of each sample before and after treatment are shown in Table 1.

[0107] Table 1

[0108]

[0109] Experimental Example 2 Effect of the sample pretreatment solution of the present invention on the quality of nucleic acid after various sample treatments

[0110] The sample pretreatment solution of Example 1 was used to treat various samples, and then nucleic acids were extracted to examine the quality of the nucleic acids before and after the treatment.

[0111] Six clinical samples of each of the eight sample types were selected and processed using the method of Experimental Example 1. The nucleic acid was then extracted using the same nucleic acid extraction kit (Tian Gen-Bacterial Genomic DNA Extraction Kit DP302). The Ct values ​​of the nucleic acid before and after treatment are shown in Tables 2 to 9.

[0112] Table 2 Nucleic acid Ct values ​​before and after sputum treatment

[0113]

[0114] Table 3 Nucleic acid Ct values ​​before and after treatment with thick bronchoalveolar lavage fluid

[0115]

[0116] Table 4 Nucleic acid Ct values ​​before and after pus secretion treatment

[0117]

[0118] Note: NoCt, weak positive sample, the extraction effect before treatment was very poor, so no nucleic acid was detected.

[0119] Table 5 Nucleic acid Ct values ​​before and after blood treatment

[0120]

[0121] Table 6 Nucleic acid Ct values ​​before and after feces treatment

[0122]

[0123] Table 7 Nucleic acid Ct values ​​before and after dry throat swab treatment

[0124]

[0125] Table 8 Nucleic acid Ct values ​​of dry genital tract samples before and after treatment

[0126]

[0127] Table 9 Nucleic acid Ct values ​​before and after anal swab treatment

[0128]

[0129] From the results in Tables 2 to 9, it can be seen that the sample pretreatment solution of Example 1 of the present invention is used to pre-treat 8 types of samples, which can perform good lysis and liquefaction treatment on the samples. After treatment, the nucleic acid extraction effect is enhanced, and the nucleic acid Ct values ​​after treatment are lower than the nucleic acid Ct values ​​before treatment.

[0130] Experimental Example 3 Effects of various sample pretreatment solutions on the physicochemical quality and nucleic acid quality of the sample after treatment

[0131] Five clinical sputum samples were selected and treated with the sample pretreatment solutions of Example 1 and Comparative Examples 1 to 3, respectively, and the physicochemical properties and nucleic acid Ct values ​​of the treated samples were investigated. The results are shown in Table 10.

[0132] Table 10

[0133]

[0134] Note: NoCt, weak positive sample, the extraction effect before treatment was very poor, so no nucleic acid was detected.

[0135] The results in Table 10 show that the sample pretreatment solution of Example 1 of the present invention can effectively liquefy the sample, and the nucleic acid Ct value of the extracted sample is the smallest. When sodium hydroxide and N-acetyl-L-cysteine ​​are not added to the sample pretreatment solution, the sample cannot be effectively liquefied (Comparative Example 1); when only sodium hydroxide is added to the sample pretreatment solution (other components are the same as in Example 1), although the sample can be liquefied, it has an impact on the nucleic acid extraction effect, and the detected Ct value is large (Comparative Example 2); when only N-acetyl-L-cysteine ​​is added to the sample pretreatment solution (other components are the same as in Example 1), the sample can also be liquefied, and the nucleic acid Ct value is relatively large (Comparative Example 3). Since N-acetyl-L-cysteine ​​itself is not very stable and has a short storage time, the use of N-acetyl-L-cysteine ​​alone can easily cause the sample pretreatment solution to fail. Therefore, the combined use of sodium hydroxide and N-acetyl-L-cysteine ​​can effectively liquefy the sample while making the quality of the sample nucleic acid after pretreatment high.

[0136] Experimental Example 4 Protective Effect of Trehalose on Nucleic Acids in Samples

[0137] Five clinical sputum samples were selected for nucleic acid extraction, and diluted with the sample pretreatment solutions of Example 1 and Comparative Example 4, respectively, and the concentrations at 0 days, 5 days, 10 days, 15 days, 25 days, and 35 days were investigated. The results are shown in Table 11.

[0138] Table 11

[0139]

[0140] The results in Table 11 show that compared with Comparative Example 4, the concentration of the nucleic acid in Example 1 decreased significantly after being placed for 35 days, indicating that adding trehalose to the sample pretreatment solution has a protective effect on the sample nucleic acid and has higher stability.

[0141] Experimental Example 5: Effect of Filtration of Samples in Pre-treatment on Sample Quality

[0142] This test example verifies whether the sample nucleic acid will be lost during sample pretreatment by leaving the sample to stand in a pretreatment solution and then filtering it (using a filter with a pore size of 5 to 25 μm).

[0143] Five sputum samples were collected after pretreatment (the sample and the sample pretreatment solution were mixed in a volume ratio of 1:2, mixed evenly, and allowed to stand for 20 minutes), and filtered and unfiltered samples were respectively processed. The nucleic acid of the samples was then extracted and the Ct value was detected. The results are shown in Table 12.

[0144] Table 12

[0145]

[0146] From the results in Table 12, it can be seen that the Ct values ​​of nucleic acid in the sputum samples after pre-treatment are not much different when filtered or not, indicating that filtering will not cause loss to the sample. However, filtering can remove impurities (such as food residues, granular solids, etc.), which is beneficial to the extraction of the microfluidic device and will not cause channel blockage.

[0147] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0148] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A sample pretreatment solution, characterized in that: The solution consists of the following components: 3wt%-4wt% of guanidine hydrochloride, 4 mM-8 mM of EDTA, 2wt%-4wt% of sodium citrate, 1wt%-3wt% of sodium hydroxide, 1wt%-2wt% of N-acetyl-L-cysteine ​​and 4wt%-8wt% of trehalose.

2. The sample pretreatment solution according to claim 1, characterized in that: The solution consists of the following components: 3.4wt%-3.6wt% of guanidine hydrochloride, 5mM-6mM of EDTA, 2.5wt%-3.5wt% of sodium citrate, 1.5wt%-2.5wt% of sodium hydroxide, 1.4wt%-1.6wt% of N-acetyl-L-cysteine ​​and 5wt%-6wt% of trehalose.

3. The method for preparing a sample pretreatment solution according to claim 1 or 2, characterized in that: The following steps are involved: After mixing all the components, adjust the pH to 8-11.

4. A method for sample pretreatment, characterized in that: The method comprises the following steps: soaking the sample in the sample pretreatment solution according to claim 1 or 2, and then letting it stand.

5. The sample pre-treatment method according to claim 4, characterized in that: The sample includes a liquid sample or a dry sample.

6. The sample pre-processing method according to claim 5, characterized in that: The liquid sample includes sputum, thick alveolar lavage fluid, purulent secretions, blood or feces; the dry sample includes throat swab, genital tract sample or anal swab.

7. The sample pre-treatment method according to claim 5, characterized in that: The liquid sample processing method comprises the following steps: mixing the liquid sample with a sample pre-treatment solution in a volume ratio of 1:1-4, standing for 15 min-30 min, placing the sample in a filtering device for filtering, and collecting the filtrate to obtain the sample.

8. The sample pre-treatment method according to claim 5, characterized in that: The dry sample processing method comprises the following steps: adding the sample pre-treatment solution into the dry sample to completely immerse the dry sample, standing for 8 min to 12 min, and then placing the sample in a filtering device for filtering and collecting the filtrate to obtain the dry sample.

9. The sample pretreatment method according to claim 7 or 8, characterized in that: The filtering device comprises a collecting tube and a filter placed in the collecting tube, wherein the collecting tube has a tube cover, and the filter comprises a filter membrane with a pore size of 5-25 μm.

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