Reagent and method for pretreatment of tissue sample and wall-breaking-difficult microorganisms and application of reagent and method

Abstract

The invention relates to a pretreatment reagent and method for a tissue sample and microorganisms difficult to break walls and application. The pretreatment reagent comprises a first enzyme mixed solution and a second enzyme mixed solution, the first enzyme mixed solution contains protease K and collagenase I, and the second enzyme mixed solution contains lywallzyme and beta-glucanase. A specific pretreatment reagent and a treatment method are designed, a tissue sample is efficiently dissociated into a dispersed cell state by utilizing a first enzyme mixed solution, subsequent grinding and second enzyme mixed solution treatment are facilitated, and the second enzyme mixed solution can greatly improve the release effect of nucleic acid in the sample and has a relatively great promotion effect on subsequent nucleic acid extraction; in addition, specific grinding condition parameters are designed, nucleic acid release can be further promoted, the sample detection performance can be improved, and the probability of false negative in detection is reduced.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and relates to a tissue sample and a pretreatment reagent, method and application for difficult-to-break microorganisms, particularly to a tissue sample and a pretreatment reagent, method and application for targeted pathogen next-generation sequencing. Background Technology

[0002] Infectious diseases are diseases caused by pathogens (such as bacteria, viruses, fungi, and parasites) entering the human body through various transmission routes, growing and multiplying within the body, and causing a series of physiological and pathological reactions. For infectious diseases, timely and accurate identification of the causative microorganism is of great significance for clinical medication, prognosis and treatment, and the epidemiological monitoring and management of infectious diseases, as well as transmission control. Therefore, rapidly and accurately identifying the pathogen causing the infection has become a primary challenge in the treatment of infectious diseases, playing a crucial role in assisting precise clinical medication, reducing hospitalization costs, and lowering the risk of serious complications and mortality.

[0003] Traditional methods for detecting infectious pathogens include microscopic examination, culture, serological tests, and PCR. However, these methods have limitations. With the clinical translation and application of molecular detection technologies, targeted next-generation sequencing (tNGS) has developed rapidly. tNGS is a technique based on multiplex PCR specific amplification to enrich specific target nucleic acids in samples, enabling the simultaneous detection of dozens to thousands or even tens of thousands of specific pathogens, their virulence genes, and drug resistance genes. Compared to metagenomic next-generation sequencing (mNGS), tNGS offers advantages such as high specificity, high sensitivity, and cost-effectiveness, enabling rapid and accurate differentiation and identification of infectious pathogens, thereby effectively guiding clinical medication and prognostic assessment.

[0004] Currently, in the field of tNGS research, the detection of tissue samples and difficult-to-break cell-wall microorganisms within them, and whether the tissue samples and these microorganisms can fully release pathogenic nucleic acids during pretreatment, have a significant impact on subsequent tNGS library construction experiments and the detection performance of pathogenic microorganisms. The standard pretreatment procedure for tissue samples and difficult-to-break cell-wall microorganisms involves adding sodium dodecyl sulfate (SDS) to the sample, followed by high-speed grinding. SDS disrupts the cell membrane and cell wall structure during high-speed grinding, leading to cell lysis and the release of pathogenic microorganism nucleic acids. However, conventional pretreatment procedures for tissue samples suffer from low lysis efficiency and insufficient release of nucleic acids from pathogenic microorganisms, especially those with difficult-to-break cell walls, such as Clostridium, Cryptococcus, Aspergillus, and Mycobacterium. The cell walls of these pathogenic microorganisms are typically composed of chitin, glucan, and mannan, resulting in complex cell wall structures. Some also possess unique cell wall structures, such as the cell walls of Mycobacterium, which contain large amounts of mycobacterial acids and waxy substances. These unique cell wall structures make Mycobacterium resistant to conventional treatment methods, often requiring special lysis methods or combinations thereof to improve lysis efficiency.

[0005] In summary, current routine pretreatment procedures for tissue samples and recalcitrant microorganisms cannot meet the performance requirements of subsequent tNGS detection methods, significantly impacting the precision treatment of critically ill patients. Therefore, there is an urgent need to develop a more efficient and convenient pretreatment procedure for tissue samples and recalcitrant microorganisms to meet the requirements of subsequent tNGS detection methods. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides a reagent, method, and application for pretreatment of tissue samples and difficult-to-break microorganisms in the samples, aiming to significantly improve the release effect of nucleic acids in tissue samples and difficult-to-break microorganisms in the samples.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a pretreatment reagent for tissue samples and difficult-to-break microorganisms, the pretreatment reagent comprising a first enzyme mixture and a second enzyme mixture, wherein the first enzyme mixture contains proteinase K and collagenase I, and the second enzyme mixture contains lysing enzyme and β-glucanase.

[0009] In this invention, specific tissue samples and pretreatment reagents for difficult-to-break microorganisms are designed. The first enzyme mixture enables the tissue sample to be efficiently dissociated into a dispersed cellular state, which is beneficial for subsequent grinding and second enzyme mixture treatment. The second enzyme mixture can greatly enhance the release effect of nucleic acids (especially nucleic acids of difficult-to-break microorganisms) in the sample, which has a significant promoting effect on subsequent nucleic acid extraction, helps to improve the sample detection performance (such as targeted pathogen detection), and reduces the probability of false negatives in the detection.

[0010] Preferably, the concentration of proteinase K in the first enzyme mixture is 100-200 U / mL, for example, it can be 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 195, 196, 197, 198 or 199 U / mL, and the concentration of collagenase I is 10-30 U / mL, for example, it can be 11, 12, 13, 14, 15, 20, 25, 26, 27, 28 or 29 U / mL.

[0011] Preferably, the concentration of the lysozyme in the second enzyme mixture is 10-30 U / mL, for example, it can be 11, 12, 13, 14, 15, 20, 25, 26, 27, 28 or 29 U / mL, and the concentration of β-glucanase is 150-250 U / mL, for example, it can be 151, 152, 153, 154, 155, 160, 170, 180, 190, 200, 210, 220, 230, 240, 245, 246, 247, 248 or 249 U / mL.

[0012] Preferably, the solvents for the first enzyme mixture and the second enzyme mixture are each independently a PBS solution or a DPBS solution.

[0013] Preferably, the pretreatment reagent further includes SDS lysis buffer.

[0014] Preferably, the SDS lysis solution contains sodium dodecyl sulfate.

[0015] Preferably, the sodium dodecyl sulfate content (concentration) in the SDS lysis solution is 15~25% (m / v).

[0016] In a second aspect, the present invention provides a method for pretreatment of tissue samples and difficult-to-break microorganisms, wherein the method uses the tissue sample and difficult-to-break microorganism pretreatment reagent described in the first aspect to treat the sample, and includes the following steps:

[0017] (1) Mix the sample to be treated with the first enzyme mixture for the first treatment;

[0018] (2) Take the sample after step (1) and mix it with SDS lysis buffer for grinding. Take the sample after grinding and mix it with the second enzyme mixture for the second treatment.

[0019] This invention features a specific processing flow, which sequentially performs first enzyme mixture treatment, grinding treatment, and second enzyme mixture treatment. It requires no additional equipment, is easy to operate, and is inexpensive, making it highly valuable and suitable for widespread application in clinical infection laboratories.

[0020] Preferably, the first processing time is 10 to 30 minutes, for example, 11, 12, 13, 14, 15, 20, 25, 26, 27, 28 or 29 minutes, and the temperature is 15 to 35°C, for example, 16, 17, 18, 19, 20, 25, 30, 31, 32, 33 or 34°C.

[0021] Preferably, the grinding conditions are as follows: grinding speed of 4700~4900 rpm, for example, 4750, 4800 or 4850 rpm, grinding 2~3 times, and grinding time of 45~60 s each time, for example, 46, 47, 48, 49, 50, 55, 56, 57, 58 or 59 s.

[0022] This invention designs specific grinding conditions that can further promote the release of nucleic acids from pathogenic microorganisms.

[0023] Preferably, the second treatment time is 10 to 20 minutes, for example, 11, 12, 13, 14, 15, 16, 17, 18 or 19 minutes, and the temperature is 15 to 35°C, for example, 16, 17, 18, 19, 20, 25, 30, 31, 32, 33 or 34°C.

[0024] Thirdly, the present invention provides the application of the tissue sample and difficult-to-break microbial pretreatment reagent described in the first aspect or the tissue sample and difficult-to-break microbial pretreatment method described in the second aspect in the construction of nucleic acid libraries and / or sequencing.

[0025] This invention designs specific tissue sample and difficult-to-break cell wall microorganism pretreatment reagents and methods, which can greatly improve the release effect of pathogenic microorganism nucleic acid in the sample, and have a significant promoting effect on subsequent pathogenic microorganism nucleic acid extraction. This helps to improve the detection performance of tissue samples and difficult-to-break cell wall microorganisms in them in targeted pathogen detection, and reduce the probability of false negatives in subsequent targeted pathogen detection.

[0026] Fourthly, the present invention provides a method for constructing a nucleic acid library, the method comprising:

[0027] The tissue sample and the pretreatment reagent for difficult-to-break microorganisms described in the first aspect or the pretreatment method for tissue sample and difficult-to-break microorganisms described in the second aspect are used to process the sample. The processed sample is then subjected to nucleic acid extraction and PCR amplification to obtain a nucleic acid library.

[0028] Fifthly, the present invention provides a sequencing method, the sequencing method comprising:

[0029] A nucleic acid library is constructed using the method for constructing a nucleic acid library described in the fourth aspect, and the nucleic acid library is then sequenced.

[0030] The sequencing method of this invention can also be used for mechanism research and source control of infection for purposes other than disease diagnosis.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] This invention designs specific tissue sample and difficult-to-break microbial cell pretreatment reagents and methods. It utilizes a first enzyme mixture to efficiently dissociate the tissue sample into a dispersed cellular state, which is beneficial for subsequent grinding and second enzyme mixture treatment. The second enzyme mixture can greatly enhance the release of nucleic acids (especially nucleic acids of difficult-to-break pathogenic microorganisms) in the sample, and has a significant promoting effect on subsequent nucleic acid extraction. In addition, the design of specific grinding conditions can further promote nucleic acid release, which helps to improve the sample detection performance (such as targeted pathogen detection) and reduce the probability of false negatives in the detection. Detailed Implementation

[0033] The technical solution of the present invention will be further illustrated below through specific embodiments. However, the examples below are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0034] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0035] This invention addresses the problem of low nucleic acid release efficiency of pathogenic microorganisms in current tNGS research pretreatment schemes for tissue samples and difficult-to-break microorganisms. It designs a novel comprehensive treatment scheme, including specific pretreatment solutions and methods. The pretreatment solutions include a first enzyme mixture and a second enzyme mixture. The first enzyme mixture contains proteinase K and collagenase I, which can effectively separate cells from tissues. The second enzyme mixture contains cell-lysing enzymes and β-glucanase, which can effectively promote the release of nucleic acids from pathogenic microorganisms (especially difficult-to-break microorganisms). A specific processing flow is designed, sequentially performing treatment with the first enzyme mixture, grinding, and second enzyme mixture. Specific grinding conditions are designed for synergistic effect, further promoting the release of pathogenic microorganism nucleic acids. The grinding conditions are: grinding speed 4700~4900 rpm, grinding times 2~3 times.

[0036] In one embodiment of the present invention, a tissue sample pretreatment reagent can be provided, the reagent comprising a first enzyme mixture and a second enzyme mixture, wherein the first enzyme mixture contains collagenase I (100~200 U / mL) and proteinase K (10~30 U / mL), and PBS can be used as the solvent; the second enzyme mixture contains lysozyme (10~30 U / mL) and β-glucanase (150~250 U / mL), and PBS can be used as the solvent; the treatment reagent may further include SDS lysis buffer, wherein the SDS lysis buffer contains sodium dodecyl sulfate.

[0037] Specifically, the tissue sample may be lung tissue.

[0038] In one embodiment of the present invention, a tissue sample pretreatment method is provided, the method comprising:

[0039] (1) Mix the tissue sample with the first enzyme mixture for the first treatment; (2) Take the sample after treatment in step (1) and mix it with SDS lysis buffer for grinding, and take the sample after grinding and mix it with the second enzyme mixture for the second treatment. The time for the first treatment is 10~30 min, and the temperature is 15~35℃; the grinding conditions are: grinding speed 4700~4900 rpm, grinding times 2~3 times; the time for the second treatment is 10~20 min, and the temperature is 15~35℃.

[0040] In one embodiment of the present invention, a targeted pathogen detection method is provided to improve the detection performance of pathogenic microorganisms in tissue samples. The method involves processing the tissue sample using the above-mentioned tissue sample pretreatment method to fully release the nucleic acid of pathogenic microorganisms in the sample to be tested. Finally, the sample undergoes nucleic acid extraction and targeted pathogen sequencing, and the detection results of pathogenic microorganisms in the sample are analyzed using high-throughput sequencing technology.

[0041] In a specific embodiment of the present invention, the SDS lysis buffer was purchased from MedChemExpress (MCE), catalog number HY-Y0316, and was an aqueous solution with a concentration of 15~25% (m / v).

[0042] In the specific embodiments of the present invention, proteinase K was purchased from NEW ENGLAND Biolabs (catalog number P8107S), collagenase I was purchased from Thermo Fisher Scientific (catalog number 17100017), lysozyme was purchased from Merck KGaA (catalog number L2524), and β-glucanase was purchased from Solarbio LIFE SCIENCES (catalog number G9220).

[0043] Example 1

[0044] In this embodiment, the sample to be tested is a simulated sample (Jurkat cell line + pathogenic microorganism culture), wherein the concentration of the Jurkat cell line is 10^6 cells / mL, and the pathogenic microorganism cultures are Haemophilus influenzae (10^3 CFU / mL), Legionella pneumophila (10^3 CFU / mL), Streptococcus pneumoniae (10^3 CFU / mL), Pseudomonas aeruginosa (10^3 CFU / mL), Acinetobacter baumannii (10^3 CFU / mL), Cryptococcus neoformans (500 CFU / mL), Aspergillus fumigatus (500 CFU / mL), Aspergillus niger (500 CFU / mL), Aspergillus flavus complex (10^4 CFU / mL), and Mycobacterium tuberculosis complex, etc., to prepare the sample to be tested. Among them, Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus complex, and Mycobacterium tuberculosis complex are all difficult-to-break cell walls bacteria.

[0045] 1. Enzyme treatment for dissociation:

[0046] (1) For control group sample 1: the sample to be tested was processed according to the routine procedure. 0.5 mL of the sample was added to a 1.5 mL centrifuge tube without adding enzyme mixture I. The mixture was vortexed and treated at room temperature.

[0047] (2) For control group sample 2: the test sample was processed according to the conventional procedure. 0.5 mL of the sample was added to a 1.5 mL centrifuge tube without adding enzyme mixture I. The mixture was vortexed and treated at room temperature.

[0048] (3) For the control group 3 samples: The test samples were processed according to the enzyme mixture I of the present invention (collagenase I (150 U / mL) + proteinase K (20 U / mL) + PBS). 0.5 mL of the sample was added to a 1.5 mL centrifuge tube, 0.5 mL of enzyme mixture I was added, vortexed and mixed, and treated at room temperature.

[0049] (4) For the experimental group samples: The samples to be tested were processed according to the enzyme mixture I of the present invention (collagenase I (150 U / mL) + proteinase K (20 U / mL) + PBS). 0.5 mL of the sample was added to a 1.5 mL centrifuge tube, 0.5 mL of enzyme mixture I was added, vortexed and mixed, and treated at room temperature.

[0050] 2. Grinding and enzyme treatment to break cell walls:

[0051] (1) For control group sample 1: 0.5 mL of the sample to be tested was transferred to a grinding tube, 70 μL of SDS lysis buffer was added, and the cell wall was broken by grinding according to the conventional protocol. The grinding was performed twice, for a grinding time of 45 s and a rotation speed of 4700 rpm. After grinding, the sample was centrifuged at 13000 rpm for 3 min, and the supernatant was taken to extract nucleic acid.

[0052] (2) For control group sample 2: 0.5 mL of the sample to be tested was transferred to a grinding tube, 70 μL of SDS lysis buffer was added, the grinding was performed 3 times, the grinding time was 45 s, and the rotation speed was 4900 rpm. After grinding, 100 μL of enzyme mixture II (20 U / mL of wall lysin + β-glucanase (100 U / mL) + PBS) was added for treatment. After treatment, the mixture was centrifuged at 13000 rpm for 3 min, and the supernatant was taken to extract nucleic acid.

[0053] (3) For the control group of 3 samples: 0.5 mL of the sample to be tested was transferred to a grinding tube, 70 μL of SDS lysis buffer was added, and the cell wall was broken by grinding according to the conventional protocol. The grinding was performed twice, for a grinding time of 45 s and a rotation speed of 4700 rpm. After grinding, the sample was centrifuged at 13000 rpm for 3 min, and the supernatant was taken to extract nucleic acid.

[0054] (4) For the experimental group samples: Transfer 0.5 mL of the sample to be tested to a grinding tube, add 70 μL of SDS lysis buffer, grind 3 times, grind for 45 s, and rotate at 4900 rpm. After grinding, add 100 μL of enzyme mixture II (20 U / mL of lysozyme) + β-glucanase (100 U / mL) + PBS for treatment. After treatment, centrifuge at 13000 rpm for 3 min and take the supernatant to extract nucleic acid.

[0055] 3. Nucleic acid extraction and tNGS library construction:

[0056] (1) Nucleic acid extraction and purification kit (Guangzhou Jinqirui Biotechnology Co., Ltd., KS132-TNGSTQA96) was used to extract and purify nucleic acid from the pretreated sample.

[0057] (2) Library construction was performed using a targeted pathogen detection kit (Guangzhou Jinqirui Biotechnology Co., Ltd., KS608-100HXD96), and pathogens were enriched by multiplex PCR. The PCR program is shown in Table 1.

[0058] Table 1

[0059]

[0060] (3) The above PCR products were purified using magnetic bead separation method.

[0061] (4) After adding the sequencing adapter sequence to the purified nucleic acid, PCR amplification was performed again. The amplification program is shown in Table 2.

[0062] Table 2

[0063]

[0064] (5) The PCR products were purified, and the concentration of the purified nucleic acid was determined by Qubit 3.0 / 4.0. The nucleic acid of each sample was mixed in a quantity of 60 ng to form a sequencing library.

[0065] 4. Sequencing

[0066] After denaturing the library with 0.1 mol / L sodium hydroxide for 5 min, it was sequenced at a rate of 1 pmol using a KM MiniSeqDx-CN sequencer.

[0067] 5. Results

[0068] Bioinformatics analysis was performed on the targeted pathogen detection results of different pretreatment schemes to compare the pathogen detection RPK (reads per thousand sequence reads) of different enzyme dissociation treatment, grinding and enzyme cell disruption schemes. The results are shown in Tables 3 and 4.

[0069] Table 3

[0070]

[0071] Table 4

[0072]

[0073] As shown in Tables 3 and 4, for simulated samples, the conventional scheme of control group 1 (enzyme-free treatment + conventional grinding and cell disruption), the scheme of control group 2 (enzyme-free treatment + grinding and enzyme treatment cell disruption of the present invention), the scheme of control group 3 (enzyme treatment dissociation + conventional grinding and cell disruption), and the scheme designed in this invention (enzyme treatment dissociation + grinding and enzyme treatment cell disruption of the present invention) showed that the enzyme mixture I of the present invention, after dissociation, grinding, and cell disruption treatment with enzyme mixture II, exhibited the best performance in subsequent tNGS detection of target pathogens, and showed a significant improvement over the conventional scheme of control group 1.

[0074] This indicates that the processing scheme designed in this invention effectively improves the release effect of nucleic acids of pathogenic microorganisms (including difficult-to-break cell wall microorganisms) in simulated samples, thereby improving the detection effect of pathogenic microorganisms (including difficult-to-break cell wall microorganisms) in simulated samples, and thus reducing the probability of false negatives in subsequent targeted pathogen sequencing detection of pathogenic microorganisms (including difficult-to-break cell wall microorganisms).

[0075] Example 2

[0076] In this embodiment, the samples were 5 clinical tissue samples (numbered S1-S5, the samples came from China-Japan Friendship Hospital, and the infection status was known).

[0077] 1. Enzyme treatment for dissociation:

[0078] (1) For control group sample 1: the test sample was processed according to the routine procedure. 0.5 mL of the sample was added to a 1.5 mL centrifuge tube without adding enzyme mixture I. The mixture was vortexed and treated at room temperature.

[0079] (2) For control group 2 samples: the test samples were treated with enzyme mixture (collagenase I (150 U / mL), collagenase IV (150 U / mL), PBS). 0.5 mL of the sample was added to a 1.5 mL centrifuge tube, 0.5 mL of enzyme mixture was added, vortexed and mixed, and treated at room temperature.

[0080] (3) For the control group 3 samples: the test samples were treated with enzyme mixture (collagenase I (150 U / mL), collagenase II (150 U / mL), collagenase IV (150 U / mL), PBS). 0.5 mL of the sample was added to a 1.5 mL centrifuge tube, 0.5 mL of enzyme mixture was added, vortexed and mixed, and treated at room temperature.

[0081] (4) For the experimental group samples: The test samples were processed according to the enzyme mixture I of the present invention (collagenase I (150U / mL) + proteinase K (20U / mL) + PBS). 0.5 mL of the sample was added to a 1.5 mL centrifuge tube, 0.5 mL of enzyme mixture I was added, vortexed and mixed, and treated at room temperature.

[0082] 2. Grinding and enzyme treatment to break cell walls:

[0083] (1) For control group sample 1: 0.5 mL of test sample was transferred to a grinding tube, 70 μL of SDS lysis buffer was added, and the cell wall was broken by grinding according to the conventional protocol. The grinding was performed twice, for 45 seconds, and at a speed of 4700 rpm. After grinding, the sample was centrifuged at 13000 rpm for 3 minutes, and the supernatant was taken to extract nucleic acid.

[0084] (2) For control group 2 samples: 0.5 mL of test sample was transferred to a grinding tube, 70 μL of SDS lysis buffer was added, and the cell wall was broken by grinding according to the conventional protocol. The grinding was performed twice, for a grinding time of 45 s and a rotation speed of 4700 rpm. After grinding, the sample was centrifuged at 13000 rpm for 3 min, and the supernatant was taken to extract nucleic acid.

[0085] (3) For the control group of 3 samples: 0.5 mL of test sample was transferred to a grinding tube, 70 μL of SDS lysis buffer was added, and the cell wall was broken by grinding according to the conventional protocol. The grinding was performed twice, for 45 seconds, and at a speed of 4700 rpm. After grinding, the sample was centrifuged at 13000 rpm for 3 minutes, and the supernatant was taken to extract nucleic acid.

[0086] (4) For the experimental group samples: Transfer 0.5 mL of test sample to a grinding tube, add 70 μL of LDS lysis buffer, grind 3 times, grind for 45 s, and rotate at 4900 rpm. After grinding, add 100 μL of enzyme mixture II (20 U / mL of wall lysin + β-glucanase (100 U / mL) + PBS) for treatment. After treatment, centrifuge at 13000 rpm for 3 min and take the supernatant to extract nucleic acid.

[0087] 3. Nucleic acid extraction and tNGS library construction:

[0088] (1) Nucleic acid extraction and purification kit (Guangzhou Jinqirui Biotechnology Co., Ltd., KS132-TNGSTQA96) was used to extract and purify nucleic acid from the pretreated sample.

[0089] (2) Library construction was performed using a targeted pathogen detection kit (Guangzhou Jinqirui Biotechnology Co., Ltd., KS608-100HXD96), and pathogens were enriched by multiplex PCR. The PCR program is shown in Table 5.

[0090] Table 5

[0091]

[0092] (3) The above PCR products were purified using magnetic bead separation method.

[0093] (4) After adding the sequencing adapter sequence to the purified nucleic acid, PCR amplification was performed again. The amplification program is shown in Table 6.

[0094] Table 6

[0095]

[0096] (5) The PCR products were purified, and the concentration of the purified nucleic acid was determined by Qubit 3.0 / 4.0. The nucleic acid of each sample was mixed in a quantity of 60 ng to form a sequencing library.

[0097] 4. Sequencing

[0098] After denaturing the library with 0.1 mol / L sodium hydroxide for 5 min, it was sequenced at a rate of 1 pmol using a KM MiniSeqDx-CN sequencer.

[0099] 5. Results

[0100] Bioinformatics analysis was performed on the targeted pathogen detection results of different pretreatment schemes to compare the RPK counts of pathogens detected by different enzyme dissociation, grinding and enzyme cell disruption schemes. The results are shown in Table 7.

[0101] Table 7

[0102]

[0103] As shown in Table 7, for clinical tissue samples, compared with the conventional protocol of control group 1, control group 2, control group 3 and the protocol of the present invention, the enzyme mixture I of the present invention, after dissociation, grinding and cell wall disruption treatment of clinical tissue samples, showed the best performance in subsequent tNGS target pathogen detection, and showed a more significant improvement than the conventional protocol of control group 1.

[0104] In summary, this invention designs specific pretreatment reagents and methods for tissue samples and difficult-to-break microorganisms. A first enzyme mixture efficiently dissociates the tissue sample into dispersed cells, facilitating subsequent grinding and second enzyme mixture processing. The second enzyme mixture significantly enhances the release of nucleic acids from the sample and the difficult-to-break microorganisms, greatly promoting subsequent nucleic acid extraction. Furthermore, specific grinding conditions further promote nucleic acid release, improving sample detection performance and reducing the probability of false negatives.

[0105] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A tissue sample and recalcitrant microorganism pretreatment reagent, characterized by, The pretreatment reagent comprises a first enzyme mixture and a second enzyme mixture, the first enzyme mixture contains protease K and collagenase I, and the second enzyme mixture contains lyticase and beta-glucanase.

2. The tissue sample and resistant microorganism pre-treatment reagent according to claim 1, wherein, The concentration of protease K in the first enzyme mixture is 100-200 U / mL, and the concentration of collagenase I is 10-30 U / mL.

3. The tissue sample and resistant microorganism pre-treatment reagent according to claim 1 or 2, characterized by, The concentration of lyticase in the second enzyme mixture is 10-30 U / mL, and the concentration of beta-glucanase is 150-250 U / mL.

4. The tissue sample and resistant microorganism pretreatment reagent according to any one of claims 1 to 3, characterized in that, The solvent of the first enzyme mixture and the second enzyme mixture is independently PBS solution or DPBS solution.

5. The tissue sample and resistant microorganism pre-treatment reagent according to any one of claims 1 to 4, characterized in that, The pretreatment reagent further comprises an SDS lysis solution. Preferably, the SDS lysis solution contains sodium dodecyl sulfate.

6. A method for pretreating a tissue sample and a hard-to-crack microbial organism, characterized in that, The tissue sample and difficult-to-break microbial pretreatment method uses the tissue sample and difficult-to-break microbial pretreatment reagent of any one of claims 1-4 to treat the sample, comprising the following steps: (1) mixing the sample to be treated with the first enzyme mixture for first treatment; (2) grinding the sample after step (1) with the SDS lysis solution, and mixing the ground sample with the second enzyme mixture for second treatment.

7. The tissue sample and resistant microorganism pre-treatment method of claim 6, wherein, The first treatment time is 10-30 min, and the temperature is 15-35℃. Preferably, the grinding treatment condition parameters are: grinding speed 4700-4900 rpm, grinding times 2-3 times, and each grinding time 45-60 s. Preferably, the second treatment time is 10-20 min, and the temperature is 15-35℃.

8. Use of the tissue sample and difficult-to-break microbial pretreatment reagent of any one of claims 1-5 or the tissue sample and difficult-to-break microbial pretreatment method of claim 6 or 7 in constructing a nucleic acid library and / or sequencing.

9. A method of constructing a library of nucleic acids, characterized by, The method comprises: treating the sample using the tissue sample and difficult-to-break microbial pretreatment reagent of any one of claims 1-5 or the tissue sample and difficult-to-break microbial pretreatment method of claim 6 or 7, extracting nucleic acid from the treated sample, and performing PCR amplification to obtain a nucleic acid library.

10. A sequencing method, comprising: The sequencing method comprises: constructing a nucleic acid library using the method of claim 9, and sequencing the nucleic acid library.

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