Kit for extracting and purifying fungal nucleic acid in sputum sample and use method thereof

Abstract

The invention relates to the technical field of in-vitro diagnosis, in particular to a kit for extracting and purifying fungal nucleic acid in a sputum sample and a use method. The nucleic acid extraction and purification kit provided by the invention comprises a sputum treatment solution, a pre-lysis solution, a nucleic acid releasing agent, magnetic beads, a lysis solution, a rinsing solution I, a rinsing solution II and an eluent. The invention provides a method capable of quickly liquefying sputum and efficiently, simply and conveniently extracting fungal nucleic acid from the sputum, nucleic acid in fungi is released by providing a buffer environment and using a nucleic acid releasing agent to quickly break walls, and the nucleic acid is treated and purified step by step, so that amplification inhibitor residues can be removed, and the yield of the fungal nucleic acid is increased. The yield and the purity of nucleic acid can be greatly improved. The nucleic acid extraction and purification kit can be matched with an automatic nucleic acid extractor for nucleic acid extraction and purification, pollution is reduced, and the kit has important significance in nucleic acid extraction of respiratory tract infection samples.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of in vitro diagnosis, in particular to a fungal nucleic acid extraction and purification kit for sputum samples and a use method thereof. BACKGROUND

[0002] Fungi are microorganisms widely distributed in the natural environment, and are also commonly found on the skin and mucosal surfaces of humans, and can coexist with the host by avoiding the immune system. Fungal infection has become the most threatening infectious disease to public health, and pulmonary fungal infection is also increasing day by day, but it is often misdiagnosed as bacterial infection in clinic, or identified through sputum culture, but it is time-consuming and difficult, with a low positive rate, and it is difficult to determine whether it is colonization, contamination or infection, making diagnosis extremely difficult.

[0003] Due to the continuous change of the environment, the pathogenic spectrum of the main pathogenic fungi is also changing, and it is difficult to completely separate and identify using ordinary means. Nucleic acid detection can become a reliable and convenient means for accurate diagnosis of fungal infection, and efficient and complete extraction of DNA is a necessary prerequisite for nucleic acid detection. Due to its unique cell structure, fungal DNA is not easily fully released during nucleic acid extraction.

[0004] In the diagnosis of respiratory system diseases, sputum samples are mostly used, which have the advantage of convenient sampling. However, sputum contains a large amount of mucin, inflammatory cells, and shed necrotic mucosal epithelial cells, and the composition is complex, which also causes certain interference to molecular diagnosis. SUMMARY

[0005] In view of this, the present application provides a fungal nucleic acid extraction and purification kit for sputum samples and a use method thereof, which solves the problems of difficulty and low efficiency in extracting fungal genomes from sputum samples, and can conveniently and efficiently extract infected fungal genomes from sputum.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0007] The present application provides a composition comprising a DNA protective agent and Chelex-100.

[0008] The DNA protective agent comprises protamine and / or calf thymus DNA.

[0009] The present application also provides the use of the above-mentioned composition in the preparation of a pre-lysis solution for extracting and / or purifying fungal nucleic acid.

[0010] The present application also provides a pre-lysis solution for extracting and / or purifying fungal nucleic acid, comprising the above-mentioned composition and acceptable adjuvants or aids.

[0011] In some embodiments of the present application, the pre-lysis solution comprises EDTA, Tris-HCl, a DNA protectant, and Chelex-100;

[0012] The DNA protectant comprises protamine and / or calf thymus DNA;

[0013] The pre-lysis solution has a pH of 7.2-8.5.

[0014] In some embodiments of the present application, the pre-lysis solution comprises:

[0015] The EDTA has a concentration of 5-50 mM; and / or

[0016] The Tris-HCl has a concentration of 5-50 mM; and / or

[0017] The DNA protectant has a concentration of 50-500 μg / mL; and / or

[0018] The Chelex-100 has a weight ratio of 1%-10%.

[0019] The present application also provides a kit for extracting and / or purifying fungal nucleic acid from a sputum sample, comprising the pre-lysis solution.

[0020] In some embodiments of the present application, the kit comprises a sample pre-treatment reagent and a nucleic acid extraction and purification reagent.

[0021] The sample pre-treatment reagent comprises a sputum treatment solution, the pre-lysis solution, and a nucleic acid release reagent.

[0022] The nucleic acid extraction and purification reagent comprises magnetic beads, a lysis solution, a rinse solution I, a rinse solution II, and an elution solution.

[0023] The sputum treatment solution has a pH of 7.5-13.0.

[0024] The sputum treatment solution comprises one or two or more of NaOH, sodium citrate dihydrate, or L-cysteine.

[0025] The nucleic acid release reagent comprises glass beads.

[0026] The lysis solution comprises one or two or more of Tris-HCl, EDTA, NaCl, a surfactant, guanidine salt, or isopropanol.

[0027] The rinse solution I comprises one or two or more of Tris-HCl, guanidine salt, a surfactant, or anhydrous ethanol.

[0028] The rinse solution II comprises one or two or more of Tris-HCl, a surfactant, or anhydrous ethanol.

[0029] The eluent comprises Tris-HCl and / or EDTA.

[0030] In some embodiments of the present application, the above-mentioned kit comprises:

[0031] The surfactant is one or two or more of Tween 20, sodium lauroyl sarcosinate, SDS, Triton X-100, fatty alcohol polyoxyethylene ether or NP40; and / or

[0032] The guanidinium salt comprises one or two or more of guanidinium isothiocyanate, guanidinium hydrochloride or guanidinium thiocyanate.

[0033] In some embodiments of the present application, the above-mentioned kit comprises:

[0034] The concentration of NaOH in the sputum treatment solution is 50-500 mM; and / or

[0035] The concentration of sodium citrate dihydrate in the sputum treatment solution is 15-150 mM; and / or

[0036] The weight percentage of L-cysteine in the sputum treatment solution is 0.1%-5%; and / or

[0037] The concentration of Tris-HCl in the lysis solution is 1-10 mM; and / or

[0038] The concentration of EDTA in the lysis solution is 0.01-2 mM; and / or

[0039] The concentration of NaCl in the lysis solution is 1-5 mM; and / or

[0040] The volume percentage of surfactant in the lysis solution is 5%-10%; and / or

[0041] The concentration of guanidinium salt in the lysis solution is 2-5 mM; and / or

[0042] The volume percentage of isopropanol in the lysis solution is 20%-50%; and / or

[0043] The concentration of Tris-HCl in the rinse solution I is 1-10 mM; and / or

[0044] The concentration of guanidinium salt in the rinse solution I is 1-2 mM; and / or

[0045] The volume percentage of surfactant in the rinse solution I is 0.1%-1%; and / or

[0046] The volume percentage of anhydrous ethanol in the rinsing solution I is 40-60%; and / or

[0047] The concentration of Tris-HCl in the rinsing solution II is 0.1-1 mM; and / or

[0048] The volume percentage of surfactant in the rinsing solution II is 0.05%-1%; and / or

[0049] The volume percentage of anhydrous ethanol in the rinsing solution II is 60-80%; and / or

[0050] The concentration of Tris-HCl in the elution solution is 2-100 mM; and / or

[0051] The concentration of EDTA in the elution solution is 2-100 mM.

[0052] The application also provides a method for extracting and / or purifying fungal nucleic acid in a sputum sample, which extracts fungal nucleic acid in a sputum sample based on the above composition, the above pre-lysis solution or the above kit.

[0053] In some specific embodiments of the application, the above method comprises the following steps:

[0054] Step (1): mix the sputum sample to be extracted with the sputum treatment solution uniformly for 10-60 min, centrifuge at 13000 rpm for 3-5 min, discard the supernatant and obtain the precipitate;

[0055] Step (2): mix the precipitate in step (1) with the pre-lysis solution, incubate at 50-60℃ for 10-30 min, and obtain a mixed solution;

[0056] Step (3): mix the mixed solution in step (2) with the nucleic acid release agent, shake at high speed for 5 min, incubate at 95-100℃ for 5-15 min, centrifuge at 13000 rpm for 2-5 min, and obtain the supernatant;

[0057] Step (4): divide the lysis solution, the magnetic beads, the rinsing solution I, the rinsing solution II and the elution solution into extraction grooves, the volume of the lysis solution is 1.5-3 mL, the volume of the magnetic beads is 25-45 μL, the volume of the rinsing solution I and the rinsing solution II is 500-750 μL respectively, the volume of the elution solution is 60-120 μL, and mix the supernatant in step (3) with the lysis solution;

[0058] Use a full-automatic nucleic acid extractor, set the program according to the conditions shown in Table 1 below, and extract nucleic acid.

[0059] The sputum sample fungal nucleic acid extraction and purification kit and the use method of the application have the following effects:

[0060] The kit does not need to participate in extraction by proteinase K, is easy to store and transport, saves time and labor; after fully liquefying the sputum sample, the fungus is fully settled at the bottom by centrifugation, only the precipitate is used for nucleic acid extraction, the purity of the nucleic acid is improved, the specificity of molecular diagnosis is stronger; the sputum sample fungus nucleic acid extraction can be carried out with the nucleic acid automatic extraction instrument, large batches of samples can be automatically extracted, the throughput is improved, and the extraction efficiency is higher; the extraction efficiency of the fungus in the sputum sample is higher. BRIEF DESCRIPTION OF DRAWINGS

[0061] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below.

[0062] Figure 1 Digital PCR results of target gene 1 in Example 1;

[0063] Figure 2 Digital PCR results of target gene 2 in Example 1;

[0064] Figure 3 Digital PCR results of target gene 1 in Comparative Example 1;

[0065] Figure 4 Digital PCR results of target gene 2 in Comparative Example 1;

[0066] Figure 5 Digital PCR results of target gene 1 in Example 2;

[0067] Figure 6 Digital PCR results of target gene 2 in Example 2;

[0068] Figure 7 Digital PCR results of target gene 1 in Comparative Example 2;

[0069] Figure 8 Digital PCR results of target gene 2 in Comparative Example 2. DETAILED DESCRIPTION

[0070] The present application discloses a kind of fungus nucleic acid extraction and purification kit in sputum sample and use method, and those skilled in the art can learn from the content of this paper, and appropriately improve process parameters to realize.It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are regarded as including in the present application.The method and application of the present application have been described by preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described in this paper without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0071] In order to solve the problems of low efficiency and difficulty in extracting fungal genome from sputum samples, the present application provides a fungal nucleic acid extraction and purification kit for sputum samples, which can conveniently and efficiently extract the infected fungal genome from sputum.

[0072] The present application also provides a method for using the sputum fungal extraction and purification kit, which is simple to operate and has a high degree of automation.

[0073] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application comprises:

[0074] In the first aspect, the present application provides a fungal nucleic acid extraction and purification kit for sputum samples, which comprises the following components: sample pretreatment reagent and nucleic acid extraction and purification reagent.

[0075] The sample pretreatment reagent comprises sputum treatment solution, pre-lysis solution and nucleic acid release agent.

[0076] The nucleic acid extraction and purification reagent comprises magnetic beads, lysis solution, rinse solution I, rinse solution II and elution solution.

[0077] The pre-lysis solution comprises 0.5-50 mM EDTA, 1-100 mM Tris-HCl, 50-500 μg / mL DNA protective agent and 1-10% (by weight) Chelex-100; and the pH value of the pre-lysis solution is 7.2-8.5.

[0078] The fungal nucleic acid extraction and purification kit can fully liquefy the sputum, fully break the fungal cell wall, and optimize some components, so as to remove the metal ions that catalyze the degradation of DNA and protect the degradation of the DNA template. In the extraction process, the components and the sample are not viscous, the sputum sample is fully liquefied and the cell wall is broken at the beginning of the extraction process, which can avoid the loss of pathogenic bacteria in the sample during the extraction process, and achieve the purpose of efficient extraction of nucleic acid.

[0079] Preferably, in the pre-lysis solution, the concentration of EDTA is 5-50 mM, for example, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM or 50 mM.

[0080] Preferably, in the pre-lysis solution, the concentration of Tris-HCl is 5-50 mM, for example, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM or 50 mM.

[0081] Preferably, the concentration of the DNA protection agent in the pre-lysis solution is 50-500 μg / mL, such as 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, 500 μg / mL, etc.

[0082] Preferably, the concentration of Chelex-100 in the pre-lysis solution is 1-10%, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc.

[0083] Preferably, the sputum processing solution comprises the following components at the working concentrations: 50-500 mM NaOH, 15-150 mM sodium citrate dihydrate, 0.1%-5% L-cysteine, and pH 7.5-13.0.

[0084] Preferably, the lysis solution comprises the following components at the working concentrations: 1-10 mM Tris-HCl, 0.01-2 mM EDTA, 1-5 M NaCl, 5-10% (volume percentage) surfactant, 2-5 mM guanidinium salt, and 20%-50% (volume percentage) isopropanol.

[0085] Specifically, the surfactant is composed of Tween 20, sodium lauroyl sarcosinate, SDS, Triton X-100, and fatty alcohol polyoxyethylene ether.

[0086] Preferably, the rinse solution I comprises the following components at the working concentrations: 1-10 mM Tris-HCl, 1-2 mM guanidinium salt, 0.1-1% (volume percentage) surfactant, and 40%-60% (volume percentage) anhydrous ethanol.

[0087] Specifically, the surfactant is Tween 20, and the guanidinium salt is guanidinium isothiocyanate.

[0088] Preferably, the rinse solution II comprises the following components at the working concentrations: 0.1-1 mM Tris-HCl, 0.05-1% (volume percentage) surfactant, and 60%-80% (volume percentage) anhydrous ethanol.

[0089] Specifically, the surfactant is Tween 20.

[0090] Preferably, the elution solution mainly comprises 2-100 mM Tris-HCl and 2-100 mM EDTA.

[0091] Specifically, the DNA protective agent is one or both of protamine and / or calf thymus, and the raw material supplier is sigma.

[0092] The fungus extraction and purification kit for sputum samples of the present application, the main role of Chelex-100 in the pre-lysis solution is to chelate some metal ions that catalyze the degradation of DNA under high temperature and low ionic strength, avoiding the degradation of DNA in the process of high-temperature wall breaking. The purpose of adding a nucleic acid release agent in the pretreatment process is to hit and break the fungus, so that the nucleic acid inside the sample is exposed. The appropriate concentration of guanidine hydrochloride, Tween 20, sodium lauroyl sarcosinate, SDS, Triton X-100, fatty alcohol polyoxyethylene ether and other components in the lysis solution are used together, which can make the sample quickly and fully lyse, and cooperate with the subsequent rinse solution to obtain high-purity nucleic acid and improve the sensitivity of molecular diagnosis.

[0093] In the second aspect, the present application provides a method for using a fungus nucleic acid extraction and purification kit for sputum samples, and the specific operation is as follows:

[0094] S1: Add the sputum sample to be extracted into a 1.5 mL centrifuge tube, add sputum treatment solution, cover the tube cap, shake and mix for 10-60 min, and then centrifuge at 13000 rpm for 3-5 min to discard the supernatant;

[0095] S2: Add pre-lysis solution to the precipitate after S1 treatment, cover the tube cap, and incubate in a water bath at 50-60℃ for 10-30 min, and then centrifuge instantly;

[0096] S3: Add a nucleic acid release agent to the sample in S2, shake at high speed for 5 min to hit and break the fungal cell wall to release the nucleic acid, centrifuge instantly, incubate in a water bath at 95-100℃ for 5-15 min, and then centrifuge at 13000 rpm for 2-5 min;

[0097] S4: Divide each component in the fungus nucleic acid extraction and purification kit into an extraction tank, the volume of the lysis solution is 1.5-3 mL, the volume of the magnetic bead solution is 25-45 μL, the volume of the rinse solution I and the rinse solution II is 500-750 μL, the volume of the elution solution is 60-120 μL, and the supernatant in S3 is mixed with the lysis solution;

[0098] Use a full-automatic nucleic acid extractor, and set the program according to the conditions shown in Table 1.

[0099] Table 1: Automatic extraction program

[0100]

[0101] After the extraction is completed, the nucleic acid sample eluted can be transferred to a nuclease-free centrifuge tube and stored at -20±5℃.

[0102] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present application are ordinary commercially available products, which can be purchased from the market.

[0103] The present application is further described below in conjunction with examples:

[0104] Example 1

[0105] The sputum fungus nucleic acid extraction and purification kit of the present embodiment comprises a sputum treatment solution, a pre-lysis solution, a nucleic acid release agent, magnetic beads, a lysis solution, a rinse solution I, a rinse solution II, and an elution solution. The pre-lysis solution is composed of 50 mM EDTA, 50 mM Tris-HCl, 250 μg / mL of a DNA protection agent, and 5% Chelex-100 by weight, and the solvents thereof are all sterile ultrapure water, wherein the DNA protection agent is calf thymus DNA. The sputum treatment solution is composed of 200 mM NaOH, 50 mM sodium citrate dihydrate, and 0.5% L-cysteine; the lysis solution is composed of 1 mM Tris-HCl, 2 mM EDTA, 1 mM NaCl, 5% sodium lauroyl sarcosinate by total volume percentage, and Triton X-100, 5 mM guanidine isothiocyanate, 35% isopropanol by volume percentage; the rinse solution I is composed of 1 mM Tris-HCl, 1.5 mM guanidine isothiocyanate, 0.5% Tween 20 by volume percentage, and 60% anhydrous ethanol by volume percentage; the rinse solution II is composed of 1 mM Tris-HCl, 0.05% Tween 20 by volume percentage, and 60% anhydrous ethanol by volume percentage; and the elution solution is composed of 20 mM EDTA and 20 mM Tris-HCl.

[0106] Comparative Example 1

[0107] In order to explore the influence of the Chelex-100 component in the kit of the present application on the extraction results of fungal nucleic acid in sputum samples, the pre-lysis solution component of the present comparative example is different from that of Example 1, and the Chelex-100 component is removed from the component, and the remaining components are the same as those of Example 1.

[0108] Effect Example 1

[0109] The pre-lysis solution was prepared according to the components of Example 1 and Comparative Example 1, and the remaining components such as the sputum treatment solution, the nucleic acid release agent, the magnetic beads, the lysis solution, the rinse solution I, the rinse solution II, and the elution solution were consistent.

[0110] The lysis solution, magnetic beads, rinse solution I, rinse solution II, and elution solution were pre-packaged into extraction well plates for use. The volume of the pre-packaged lysis solution was 2 mL, the volume of the pre-packaged rinse solution I and rinse solution II was 800 μL, and the volume of the pre-packaged elution solution was 60 μL.

[0111] Using a pipette to add 500 μL of positive sputum sample A into a 1.5 mL centrifuge tube, add 1 mL of sputum treatment solution, shake well and stand for 20 min, then centrifuge at 13000 rpm for 3-5 min and discard the supernatant;

[0112] Add 300 μL of pre-lysis solution in Example 1 and Comparative Example 1 respectively, cover the tube cap, and incubate in a water bath at 50-60°C for 20 min, then centrifuge instantaneously;

[0113] To the above 1.5 mL nucleic acid release agent, beat and break the fungal cell wall to release nucleic acid by high-speed shaking for 5-15 min, centrifuge instantaneously, incubate in a water bath at 95-100°C for 5 min, and centrifuge at 13000 rpm for 2-5 min;

[0114] Transfer the supernatant after centrifugation to the pre-packaged lysis solution tank of the extraction well plate, and run the automatic extraction program shown in Table 1.

[0115] After the extraction is completed, the eluted nucleic acid sample can be transferred to a nuclease-free centrifuge tube and stored at -20±5°C.

[0116] The extracted sample is subjected to digital PCR detection using the lung invasion detection kit produced by Navigation Genomics (Hangzhou) Co., Ltd., and the results shown in Table 2 are obtained. Figures 1-4

[0117] Table 2: Data analysis table for amplification of nucleic acid extracted and purified by the kits in Example 1 and Comparative Example 1

[0118]

[0119] As shown in Table 2, the concentration and purity of the nucleic acid extracted from sputum in Example 1 are higher, and the fifth column in the table indicates the purity of the extracted nucleic acid, and the A260 / A280 value should be between 1.8 and 2.0, indicating that the nucleic acid sample has high quality, and the nucleic acid sample in Comparative Example 1 is contaminated with certain organic matter such as protein. From the results in Table 2, it can be concluded that the consistency of the sputum sample fungal extraction kit of the present application is good, and the CV is less than 5%, and the extraction efficiency of Example 1 containing Chelex-100 is higher.

[0120] Example 2

[0121] ​The sputum fungus nucleic acid extraction and purification kit of the embodiment comprises a sputum treatment solution, a pre-lysis solution, a nucleic acid release agent, magnetic beads, a lysis solution, a rinsing solution I, a rinsing solution II, and an elution solution. The pre-lysis solution is composed of 50 mM EDTA, 50 mM Tris-HCl, 100 μg / mL of a DNA protection agent, and 10% Chelex-100 by weight, and the solvents thereof are all sterile ultrapure water, wherein the DNA protection agent is calf thymus DNA. The sputum treatment solution is composed of 200 mM NaOH, 50 mM sodium citrate dihydrate, and 0.5% L-cysteine; the lysis solution is composed of 1 mM Tris-HCl, 2 mM EDTA, 1M NaCl, 5% sodium lauroyl sarcosinate by total volume percentage, and Triton X-100, 5 mM guanidine isothiocyanate, and 35% isopropanol by volume percentage; the rinsing solution I is composed of 1 mM Tris-HCl, 1.5 mM guanidine isothiocyanate, 0.5% Tween 20 by volume percentage, and 60% anhydrous ethanol by volume percentage; the rinsing solution II is composed of 1 mM Tris-HCl, 0.05% Tween 20 by volume percentage, and 60% anhydrous ethanol by volume percentage; and the elution solution is composed of 20 mM EDTA and 20 mM Tris-HCl.

[0122] Comparative Example 2

[0123] In order to explore the influence of the DNA protection agent component in the kit of the present application on the extraction results of fungal nucleic acid in sputum samples, the following comparative example is obtained.

[0124] The pre-lysis solution of this comparative example is different from that of Example 2 in that the DNA protection agent component is removed from the composition, and the remaining components are the same as those of Example 2.

[0125] Effect Example 2

[0126] The pre-lysis solution is prepared according to the components of Example 2 and Comparative Example 2, and the remaining components such as the sputum treatment solution, the nucleic acid release agent, the magnetic beads, the lysis solution, the rinsing solution I, the rinsing solution II, and the elution solution are consistent.

[0127] The lysis solution, the magnetic beads, the rinsing solution I, the rinsing solution II, and the elution solution are divided into extraction well plates in advance for use. The volume of the divided lysis solution is 2 mL, the volume of the divided rinsing solution I and rinsing solution II is 800 μL, and the volume of the divided elution solution is 60 μL.

[0128] 500 μL of positive sputum sample B is added to a 1.5 mL centrifuge tube using a pipette, 1 mL of sputum treatment solution is added, and after shaking and mixing, it is left to stand for 20 min. After centrifugation at 13000 rpm for 3-5 min, the supernatant is discarded;

[0129] Add 300 μL of pre-lysis solution in Example 2 and Comparative Example 2 respectively, cover the tube cap, and incubate in a water bath at 50-60℃ for 20 min, then centrifuge instantaneously;

[0130] To the above 1.5 mL nucleic acid release agent, shake and beat the fungal cell wall at high speed for 5-15 min to release the nucleic acid, centrifuge instantaneously, incubate in a water bath at 95-100℃ for 5 min, and centrifuge at 13000 rpm for 2-5 min;

[0131] Transfer the supernatant after centrifugation to the lysis solution tank of the pre-packaged extraction well plate, and run the automatic extraction program shown in Table 1.

[0132] After the extraction is completed, the eluted nucleic acid sample can be transferred to a nuclease-free centrifuge tube and stored at -20±5℃.

[0133] Using the extracted sample, add sample according to the instructions of the lung invasion detection kit, and perform digital PCR detection to obtain the results shown in Table 3. Figures 5-8

[0134] Table 3: Data analysis table for amplification of nucleic acid extracted and purified by the kits in Example 2 and Comparative Example 2 for the same sample

[0135]

[0136] As can be seen from Table 3, the concentration of the nucleic acid extracted from the sputum in Example 2 is higher, and it can be concluded that the consistency of the sputum sample fungal extraction kit of the present application is good, and the CV is less than 5%, and the extraction efficiency of Example 2 containing the DNA protective agent is significantly higher than that of Comparative Example 2.

[0137] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.​

Claims

1. A composition, characterized in that, Including DNA protectants and Chelex-100; The DNA protectant includes protamine and / or calf thymus DNA.

2. The use of the composition of claim 1 in the preparation of a pre-lysate for the extraction and / or purification of fungal nucleic acids.

3. A pre-lysis buffer for extracting and / or purifying fungal nucleic acids, characterized in that, Includes the composition as described in claim 1 and acceptable excipients or additives.

4. The pre-lysis solution as described in claim 3, characterized in that, Including EDTA, Tris-HCl, DNA protectants, and Chelex-100; The DNA protectant includes protamine and / or calf thymus DNA; The pH value of the pre-lysis solution is 7.2 to 8.

5.

5. The pre-lysis solution as described in claim 4, characterized in that, include: The concentration of EDTA is 5–50 mM; and / or The concentration of Tris-HCl is 5–50 mM; and / or The concentration of the DNA protectant is 50–500 μg / mL; and / or The weight percentage of Chelex-100 is 1% to 10%.

6. A kit for extracting and / or purifying fungal nucleic acids from sputum samples, characterized in that, Includes the pre-lysis solution as described in any one of claims 3 to 5.

7. The kit according to claim 6, characterized in that, Includes sample pretreatment reagents and nucleic acid extraction and purification reagents; The sample pretreatment reagent includes sputum treatment solution, prelysis buffer as described in any one of claims 3 to 5, and nucleic acid release agent; The nucleic acid extraction and purification reagents include magnetic beads, lysis buffer, washing buffer I, washing buffer II, and elution buffer; The pH of the sputum treatment solution is 7.5–13.0; The sputum treatment solution includes one or more of NaOH, sodium citrate dihydrate, or L-cysteine. The nucleic acid releasing agent includes glass beads; The lysis solution includes one or more of Tris-HCl, EDTA, NaCl, surfactant, guanidine salt, or isopropanol; The rinsing solution I includes one or more of Tris-HCl, guanidine salt, surfactant, or anhydrous ethanol; The rinsing solution II includes one or more of Tris-HCl, surfactants, or anhydrous ethanol; The eluent includes Tris-HCl and / or EDTA.

8. The kit according to claim 7, characterized in that, include: The surfactant is one or more of Tween 20, sodium lauroyl sarcosinate, SDS, Triton X-100, fatty alcohol polyoxyethylene ether, or NP40; and / or The guanidine salt includes one or more of guanidine isothiocyanate, guanidine hydrochloride, or guanidine thiocyanate.

9. The kit as described in claim 7 or 8, characterized in that, include: The concentration of NaOH in the sputum treatment solution is 50–500 mM; and / or The concentration of sodium citrate dihydrate in the sputum treatment solution is 15–150 mM; and / or The weight percentage of L-cysteine ​​in the sputum treatment solution is 0.1% to 5%; and / or The concentration of Tris-HCl in the lysis buffer is 1–10 mM; and / or The concentration of EDTA in the lysis buffer is 0.01–2 mM; and / or The concentration of NaCl in the lysis buffer is 1–5 mM; and / or The surfactant in the lysis buffer has a volume percentage of 5% to 10%; and / or The concentration of guanidine salt in the lysis buffer is 2–5 mM; and / or The volume percentage of isopropanol in the lysis buffer is 20%–50%; and / or The concentration of Tris-HCl in the rinsing solution I is 1–10 mM; and / or The concentration of guanidine salt in the rinsing solution I is 1–2 mM; and / or The volume percentage of surfactant in the rinsing solution I is 0.1% to 1%; and / or The volume percentage of anhydrous ethanol in the rinsing solution I is 40%–60%; and / or The concentration of Tris-HCl in the rinsing solution II is 0.1–1 mM; and / or The volume percentage of surfactant in the rinsing solution II is 0.05% to 1%; and / or The volume percentage of anhydrous ethanol in the rinsing solution II is 60%–80%; and / or The concentration of Tris-HCl in the eluent is 2–100 mM; and / or The concentration of EDTA in the eluent is 2–100 mM.

10. A method for extracting and / or purifying fungal nucleic acids from sputum samples, characterized in that, Fungal nucleic acids are extracted from sputum samples using the composition as described in claim 1, the pre-lysis buffer as described in any one of claims 3 to 5, or the kit as described in any one of claims 6 to 9.

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