Lysate for candida albicans, kit and gene detection method

Abstract

The invention belongs to the technical field of biomedical detection, and particularly relates to a lysis solution for candida albicans, a kit and a gene detection method. The cleavage efficiency and background interference are balanced by regulating and controlling the concentration of saponin, nucleic acid can be wrapped by too many saponin micelles, and cleavage is incomplete if too few saponin micelles exist; citric acid buffers to adjust the acidic environment, although RNA degradation may be promoted, nuclease is cleared through high-salt rapid cracking and protease K, and negative effects are counteracted; the pre-activated protease K is synchronous with membrane rupture, so that the enzyme activity peak is matched with the exposure time of the target protein, the degradation efficiency is improved, and a cracking technology which can simultaneously realize high-yield and high-function nucleic acid output and is adaptive to a rapid detection process is developed.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection, and specifically relates to a lysis solution, a kit and a gene detection method for Candida albicans. Background Art

[0002] In the field of nucleic acid extraction of pathogenic microorganisms, the existing technologies generally face the yield-quality paradox: Although traditional mechanical fragmentation methods (such as bead milling, ultrasound) can obtain a relatively high nucleic acid yield, they are accompanied by serious DNA fragmentation phenomena, resulting in a decrease in qPCR amplification efficiency and attenuation of mass spectrometry detection signal intensity; while enzymatic digestion methods (such as lywallzyme, lysozyme) can maintain the integrity of nucleic acids, but have low lysis efficiency for pathogens wrapped by chlamydospores or biofilms, and have defects such as long time consumption and complex operation steps (requiring multiple pretreatment steps).

[0003] At present, although the mainstream chemical lysis method (such as CTAB method) can partially balance the yield and quality, there are still key bottlenecks. For example, conventional NaCl concentrations (0.5-1M) cannot simultaneously achieve effective cell membrane swelling and optimization of the DNA binding efficiency of silica columns, resulting in fluctuations in the recovery rate of functional nucleic acids; although high-concentration surfactants improve the lysis efficiency, they seriously interfere with downstream PCR reactions and the mass spectrometry ionization process; the protease K that has not been pre-activated has insufficient reduction of disulfide bonds, and the nucleosome dissociation efficiency is less than 60%, resulting in false negatives of key drug resistance genes.

[0004] Therefore, developing a lysis technology that can simultaneously achieve high yield, high output of functional nucleic acids and adapt to rapid detection processes has become a core problem that urgently needs to be broken through in this field. Summary of the Invention

[0005] In view of the deficiencies in the existing technology, the present invention provides a lysis solution, a kit and a gene detection method for Candida albicans.

[0006] The present invention provides a lysis solution for Candida albicans, which is used for gene detection of Candida albicans.

[0007] The lysis solution is composed of the following components: saponin, sodium citrate, citric acid, sodium chloride, pre-activated protease K and deionized water.

[0008] Preferably, the lysis solution is composed of the following components by mass percentage: Saponin 0.8 wt%; Sodium citrate 0.62 wt%; Citric acid 0.52 wt%; Sodium chloride 7.01 wt%; Pre-activated protease K 0.05 wt%; The balance is deionized water.

[0009] The preparation method of the pre-activated proteinase K comprises the following steps: adding proteinase K into 5 mol / L dithiothreitol, wherein the concentration of the proteinase K is 0.5 mg / mL, incubating at 37°C for 10 min to obtain the pre-activated proteinase K.

[0010] The present invention also provides a kit for Candida albicans gene detection, which comprises the lysis solution as described above.

[0011] The present invention also provides a method for Candida albicans gene detection. Using the lysis solution and the kit for Candida albicans gene detection as described above, the method comprises the following steps: (1) Sample treatment: Take 10 5 CFU Candida albicans colonies, suspend them in 200 μL of sterile physiological saline, centrifuge at 12000 r / min for 2 min, and discard the supernatant; then add 100 μL of lysis solution, vortex and mix well for 30 s to obtain a Candida albicans colony sample; (2) Lysis reaction: Place the Candida albicans colony sample obtained in step (1) in a 65°C metal bath, shake and lyse at 1000 r / min for 15 min; then immediately cool the lysis solution at 0°C for 5 min to inhibit the activity of residual protease to obtain a lysis solution; (3) Nucleic acid purification: Transfer the lysis solution obtained in step (2) to a nucleic acid purification column, add 200 μL of binding buffer, centrifuge at 10000 r / min for 1 min, and discard the filtrate; then add 500 μL of washing buffer, centrifuge at 10000 r / min for 1 min, remove the washing buffer, and repeat the washing twice; finally, centrifuge the nucleic acid purification column at 12000 r / min for 2 min to remove residual ethanol, add 50 μL of nucleic acid elution solution at 65°C, let it stand at room temperature for 2 min, and then centrifuge at 10000 r / min for 1 min to take the centrifugate to obtain nucleic acid; (4) Target gene amplification: Use the Candida Resistance RT-PCR Kit for one-step reverse transcription and PCR reaction. Configure the reaction system for reverse transcription-multiplex PCR. The reaction procedure for one-step RT-PCR reaction is as follows: 50°C for 30 min; 94°C for 3 min; then perform 45 cycles, each cycle including 94°C for 30 sec, 58°C for 30 sec, 72°C for 30 sec, and after the cycle ends, 72°C for 5 min to obtain a PCR product; Each sample is prepared with the PCR reaction solution as follows:

[0012] (5) Nucleic acid mass spectrometry detection: Add 1 μL of shrimp alkaline phosphatase and 1 U of exonuclease I to the PCR product obtained in step (4), incubate at 37 °C for 30 min, and then inactivate at 85 °C for 15 min to obtain a purified product; then take 10 μL of the above purified product and mix it with 10 μL of the matrix solution, spot it onto the MALDI target plate, and crystallize at room temperature; finally, use a MALDI-TOF mass spectrometer to collect data in the positive ion mode.

[0013] The binding buffer described in step (3) is a 70% ethanol aqueous solution, and the pH is adjusted to 5.2 with acetic acid-sodium acetate buffer; the washing buffer is 80% ethanol containing 20 mmol / L Tris-HCl, and the pH is adjusted to 7.5 with acetic acid-sodium acetate buffer; the nucleic acid elution buffer is 10 mmol / L Tris-HCl, and the pH is adjusted to 8.5 with acetic acid-sodium acetate buffer.

[0014] The matrix solution described in step (5) is composed of 3-hydroxy-2-pyridinecarboxylic acid, acetonitrile, citric acid, and deionized water, where the concentration of 3-hydroxy-2-pyridinecarboxylic acid is 0.05 g / mL, the acetonitrile concentration is 50 wt%, and the citric acid concentration is 0.1 wt%.

[0015] The parameter settings for using a MALDI-TOF mass spectrometer to collect data in the positive ion mode in step (5): mass range: 4,000~10,000 Da, laser intensity: 60%, number of accumulations: 200 times.

[0016] The beneficial effects of the present invention: The concentration of 0.8 wt% saponarin in the lysis solution exceeds its critical micelle concentration, forming stable micelles. The hydrophobic end inserts to disrupt the fungal cell membrane (phospholipid bilayer), forming nanoscale pores and releasing intracellular substances; while ensuring a sufficient monomer concentration of saponarin for efficient lysis, the micelles are used to encapsulate hydrophobic fragments, reducing the non-specific binding of cell debris to subsequent detection reagents (such as fluorescent dyes) and reducing background interference.

[0017] A buffer system is formed with citric acid-sodium citrate to keep the lysis solution slightly acidic, which is beneficial for inhibiting exogenous nucleases and chelating free calcium and magnesium ions to inhibit the destruction of nucleic acids by endogenous nucleases (such as fungal nucleases) and host defense enzymes (such as lysozyme) that depend on divalent metal ions, thereby protecting the integrity of nucleic acids.

[0018] Sodium chloride increases the osmotic pressure of the lysis solution, causing rapid cell dehydration, and synergistically with the saponarin pores to accelerate the collapse of the membrane structure and release intracellular substances; at the same time, high salt can also inhibit the binding of negatively charged nucleases and substrates through the charge shielding effect, playing a role in protecting nucleic acids.

[0019] Pre-activated proteinase K is active at the initial stage of cleavage and can act synchronously with saponin-led membrane rupture, preferentially degrading fungal intracellular proteins (such as histones and nucleic acid-binding proteins) to release nucleic acids, and quickly decomposing endogenous interfering enzymes (such as chitinase and nuclease) that may be released. At the same time, after completing the degradation of the target protein, excess proteinase K can be inactivated by self-hydrolysis and conformational inactivation in an acidic environment, avoiding residual enzyme activity interfering with downstream PCR or sequencing applications.

[0020] In summary, the present invention balances the cleavage efficiency and background interference by regulating the saponin concentration. Too many saponin micelles will wrap the nucleic acid, while too few will result in incomplete cleavage. Although the acidic environment regulated by citric acid buffer may promote RNA degradation, the negative effects are offset by high-salt rapid cleavage and proteinase K to remove nucleases. Pre-activation of proteinase K is synchronized with membrane rupture to ensure that the peak of enzyme activity matches the exposure time of the target protein, thereby improving the degradation efficiency.

[0021] The lysis solution of the present invention adopts saponin (physical lysis), high salt (chemical regulation), buffer (ion / pH control), proteinase K (enzymatic hydrolysis) multi-mechanism linkage to replace the complex steps of traditional enzymatic hydrolysis of cell walls, simplify the process and reduce costs. DETAILED DESCRIPTION

[0022] Saponin: CAS number: 20310-89-8.

[0023] Proteinase K, CAS: 39450-01-6, Catalog No.: GPK003001, Jiangsu Jinpu Noan Biotechnology Co., Ltd.

[0024] Dithiothreitol, CAS: 3483-12-3.

[0025] Candida albicans , CGMCC 2.4159, was purchased from China General Microbiological Culture Collection Center.

[0026] Nucleic acid purification column, product number: N1010, Beijing Solebow Technology Co., Ltd.

[0027] Shrimp alkaline phosphatase, product number: KMC125113, Wenzhou Kemiao Biotechnology Co., Ltd.

[0028] Exonuclease I, product number: XG-P3062, Shanghai Xige Biotechnology Co., Ltd.

[0029] 3-Hydroxy-2-pyridinecarboxylic acid, CAS: 874-24-8.

[0030] Lytic enzyme, product number: E0077, Nanjing Dulai Biotechnology Co., Ltd.

[0031] Snail enzyme, product number: 161790, Shanghai Kelaman Reagent Co., Ltd. Example 1

[0032] A lysis solution consists of the following components: saponin, sodium citrate, citric acid, sodium chloride, pre-activated proteinase K and deionized water.

[0033] The lysis solution consists of the following components by mass percentage: Saponin 0.8 wt%; Sodium citrate 0.62 wt%; Citric acid 0.52 wt%; Sodium chloride 7.01 wt%; Pre-activated proteinase K 0.05 wt%; The balance is deionized water.

[0034] The preparation method of the pre-activated proteinase K includes the following steps: adding proteinase K into 5 mol / L dithiothreitol, the concentration of the proteinase K is 0.5 mg / mL, incubating at 37 °C for 10 min to obtain the pre-activated proteinase K.

[0035] A kit for Candida albicans gene detection includes the lysis solution as described above.

[0036] A method for Candida albicans gene detection, using the lysis solution as described above and the kit for Candida albicans gene detection as described above, includes the following steps: (1) Sample treatment: Take 10 5 CFU Candida albicans colonies, suspend them in 200 μL of sterile physiological saline, centrifuge at 12000 r / min for 2 min and discard the supernatant; then add 100 μL of lysis solution, vortex and mix well for 30 s to obtain a Candida albicans colony sample; (2) Lysis reaction: Place the Candida albicans colony sample obtained in step (1) in a 65 °C metal bath, shake and lyse at 1000 r / min for 15 min; then immediately cool the lysis solution at 0 °C for 5 min to inhibit the activity of residual protease to obtain the lysis solution; (3) Nucleic acid purification: Transfer the lysis solution obtained in step (2) to a nucleic acid purification column, add 200 μL of binding buffer, centrifuge at 10000 r / min for 1 min and discard the filtrate; then add 500 μL of washing buffer, centrifuge at 10000 r / min for 1 min to remove the washing buffer, repeat the washing twice; finally centrifuge the nucleic acid purification column at 12000 r / min for 2 min to remove the residual ethanol, add 50 μL of nucleic acid elution buffer at 65 °C, let it stand at room temperature for 2 min and then centrifuge at 10000 r / min for 1 min, take the centrifugate to obtain the nucleic acid; (4)Target gene amplification: Use the Candida Resistance RT-PCR Kit to perform one-step reverse transcription and PCR reactions. Configure the reaction system for reverse transcription-multiplex PCR. The reaction procedure for the one-step RT-PCR reaction is as follows: 50 °C for 30 min; 94 °C for 3 min; then perform 45 cycles, each cycle including 94 °C for 30 sec, 58 °C for 30 sec, 72 °C for 30 sec. After the cycle ends, incubate at 72 °C for 5 min to obtain the PCR product; Each sample is prepared with the PCR reaction solution as follows:

[0037] (5)Nucleic acid mass spectrometry detection: Add 1 μL of shrimp alkaline phosphatase and 1 U of exonuclease I to the PCR product obtained in step (4), incubate at 37 °C for 30 min, and then inactivate at 85 °C for 15 min to obtain the purified product; then take 10 μL of the above purified product and mix it with 10 μL of the matrix solution, spot it onto the MALDI target plate, and crystallize at room temperature; finally, use a MALDI-TOF mass spectrometer to collect data in the positive ion mode.

[0038] The binding buffer described in step (3) is a 70% ethanol aqueous solution, and the pH is adjusted to 5.2 with acetic acid-sodium acetate buffer; the washing buffer is a solution containing 80% ethanol, 20 mmol / L Tris-HCl, and the pH is adjusted to 7.5 with acetic acid-sodium acetate buffer; the nucleic acid elution buffer is 10 mmol / L Tris-HCl, and the pH is adjusted to 8.5 with acetic acid-sodium acetate buffer.

[0039] The matrix solution described in step (5) is composed of 3-hydroxy-2-pyridinecarboxylic acid, acetonitrile, citric acid, and deionized water, where the concentration of 3-hydroxy-2-pyridinecarboxylic acid is 0.05 g / mL, the acetonitrile concentration is 50 wt%, and the citric acid concentration is 0.1 wt%.

[0040] The parameter settings for using the MALDI-TOF mass spectrometer to collect data in the positive ion mode in step (5): mass range: 4,000~10,000 Da, laser intensity: 60%, number of accumulations: 200 times. Example 2

[0041] The difference from Example 1 is that the citrate buffer in the lysis solution formula is replaced with a phosphate buffer; A lysis solution, which consists of the following components: saponin, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, pre-activated proteinase K, and deionized water.

[0042] The lysis solution, by mass percentage, consists of the following components: Saponin 0.8 wt%; Sodium hydrogen phosphate 0.62 wt%; Potassium dihydrogen phosphate 0.52 wt%; Sodium chloride 7.01 wt%; Pre-activated Proteinase K 0.05 wt%; The balance is deionized water.

[0043] The preparation method of the pre-activated Proteinase K includes the following steps: Add Proteinase K to 5 mol / L dithiothreitol, the concentration of the Proteinase K is 0.5 mg / mL, incubate at 37 °C for 10 min to obtain the pre-activated Proteinase K. Example 3

[0044] The difference from Example 1 is that the lysis temperature is adjusted to 60 °C; A lysis solution is composed of the following components: saponin, sodium citrate, citric acid, sodium chloride, pre-activated Proteinase K and deionized water.

[0045] The lysis solution is composed of the following components by mass percentage: Saponin 0.8 wt%; Sodium citrate 0.62 wt%; Citric acid 0.52 wt%; Sodium chloride 7.01 wt%; Pre-activated Proteinase K 0.05 wt%; The balance is deionized water.

[0046] The preparation method of the pre-activated Proteinase K includes the following steps: Add Proteinase K to 5 mol / L dithiothreitol, the concentration of the Proteinase K is 0.5 mg / mL, incubate at 37 °C for 10 min to obtain the pre-activated Proteinase K.

[0047] A kit for Candida albicans gene detection includes the lysis solution as described above.

[0048] A method for Candida albicans gene detection using the lysis solution as described above and the kit for Candida albicans gene detection as described above includes the following steps: (1) Sample treatment: Take 10 5 CFU Candida albicans colonies, suspend them in 200 μL of sterile physiological saline, centrifuge at 12000 r / min for 2 min and then discard the supernatant; then add 100 μL of the lysis solution, vortex and mix well for 30 s to obtain a Candida albicans colony sample; (2) Lysis reaction: Place the Candida albicans colony sample obtained in step (1) in a 60°C metal bath and shake it at 1000 r / min for 15 min; then immediately cool the lysate at 0°C for 5 min to inhibit the activity of residual protease, obtaining the lysate; (3) Nucleic acid purification: Transfer the lysate obtained in step (2) to a nucleic acid purification column, add 200 μL of binding buffer, centrifuge at 10000 r / min for 1 min, and discard the filtrate; then add 500 μL of washing buffer, centrifuge at 10000 r / min for 1 min, remove the washing buffer, and repeat the washing twice; finally, centrifuge the nucleic acid purification column at 12000 r / min for 2 min to remove the residual ethanol, add 50 μL of nucleic acid elution buffer at 65°C, let it stand at room temperature for 2 min, then centrifuge at 10000 r / min for 1 min, and take the centrifugate to obtain the nucleic acid; (4) Target gene amplification: Use the Candida Resistance RT-PCR Kit for one-step reverse transcription and PCR reaction, configure the reaction system for reverse transcription-multiplex PCR, and the reaction program for one-step RT-PCR is as follows: 50°C for 30 min; 94°C for 3 min; then perform 45 cycles, each cycle including 94°C for 30 sec, 58°C for 30 sec, 72°C for 30 sec, and after the cycle ends, 72°C for 5 min to obtain the PCR product; Each sample is prepared with the PCR reaction solution as follows:

[0049] (5) Nucleic acid mass spectrometry detection: Add 1 μL of shrimp alkaline phosphatase and 1 U of exonuclease I to the PCR product obtained in step (4), incubate at 37°C for 30 min, then inactivate at 85°C for 15 min to obtain the purified product; then take 10 μL of the above purified product and mix it with 10 μL of the matrix solution, spot it onto the MALDI target plate, and crystallize at room temperature; finally, use a MALDI-TOF mass spectrometer to collect data in the positive ion mode.

[0050] The binding buffer described in step (3) is a 70% ethanol aqueous solution, adjusted to pH = 5.2 with acetic acid-sodium acetate buffer; the washing buffer is a solution containing 80% ethanol, 20 mmol / L Tris-HCl, adjusted to pH 7.5 with acetic acid-sodium acetate buffer; the nucleic acid elution buffer is 10 mmol / L Tris-HCl, adjusted to pH 8.5 with acetic acid-sodium acetate buffer.

[0051] The matrix solution described in step (5) is composed of 3-hydroxy-2-pyridinecarboxylic acid, acetonitrile, citric acid, and deionized water. Among them, the concentration of 3-hydroxy-2-pyridinecarboxylic acid is 0.05 g / mL, the concentration of acetonitrile is 50 wt%, and the concentration of citric acid is 0.1 wt%.

[0052] In step (5), a MALDI-TOF mass spectrometer is used, and the parameter settings for data acquisition in the positive ion mode are as follows: mass range: 4,000~10,000 Da, laser intensity: 60%, number of accumulations: 200 times. Example 4

[0053] The difference from Example 1 is that the concentration of saponarin is 0.5 wt%. A lysis solution is composed of the following components: saponarin, sodium citrate, citric acid, sodium chloride, pre-activated proteinase K, and deionized water.

[0054] The lysis solution is composed of the following components by mass percentage: Saponarin 0.5 wt%; Sodium citrate 0.62 wt%; Citric acid 0.52 wt%; Sodium chloride 7.01 wt%; Pre-activated proteinase K 0.05 wt%; The balance is deionized water.

[0055] The preparation method of the pre-activated proteinase K includes the following steps: Add proteinase K to 5 mol / L dithiothreitol. The concentration of the proteinase K is 0.5 mg / mL, and it is incubated at 37°C for 10 min to obtain the pre-activated proteinase K. Example 5

[0056] The difference from Example 1 is that the concentration of saponarin is 1.2 wt%. A lysis solution is composed of the following components: saponarin, sodium citrate, citric acid, sodium chloride, pre-activated proteinase K, and deionized water.

[0057] The lysis solution is composed of the following components by mass percentage: Saponarin 1.2 wt%; Sodium citrate 0.62 wt%; Citric acid 0.52 wt%; Sodium chloride 7.01 wt%; Pre-activated proteinase K 0.05 wt%; The balance is deionized water.

[0058] The preparation method of the pre-activated proteinase K comprises the following steps: adding proteinase K into 5 mol / L dithiothreitol, wherein the concentration of the proteinase K is 0.5 mg / mL, and incubating at 37°C for 10 min to obtain the pre-activated proteinase K. Example 6

[0059] It is different from Example 1 in that: the incubation time of proteinase K is 5 min; A lysis solution is composed of the following components: saponin, sodium citrate, citric acid, sodium chloride, pre-activated proteinase K and deionized water.

[0060] The lysis solution is composed of the following components by mass percentage: Saponin 0.8 wt%; Sodium citrate 0.62 wt%; Citric acid 0.52 wt%; Sodium chloride 7.01 wt%; Pre-activated proteinase K 0.05 wt%; The balance is deionized water.

[0061] The preparation method of the pre-activated proteinase K comprises the following steps: adding proteinase K into 5 mol / L dithiothreitol, wherein the concentration of the proteinase K is 0.5 mg / mL, and incubating at 37°C for 5 min to obtain the pre-activated proteinase K. Example 7

[0062] It is different from Example 1 in that: the incubation time of proteinase K is 15 min; A lysis solution is composed of the following components: saponin, sodium citrate, citric acid, sodium chloride, pre-activated proteinase K and deionized water.

[0063] The lysis solution is composed of the following components by mass percentage: Saponin 0.8 wt%; Sodium citrate 0.62 wt%; Citric acid 0.52 wt%; Sodium chloride 7.01 wt%; Pre-activated proteinase K 0.05 wt%; The balance is deionized water.

[0064] The preparation method of the pre-activated proteinase K comprises the following steps: adding proteinase K into 5 mol / L dithiothreitol, wherein the concentration of the proteinase K is 0.5 mg / mL, and incubating at 37°C for 15 min to obtain the pre-activated proteinase K. Comparative Example 1

[0065] The difference from Example 1 lies in: glass bead mechanical fragmentation method + CTAB lysis buffer; A method for detecting Candida albicans genes, comprising the following steps: (1) Sample treatment: Take 10 5 CFU Candida albicans colonies, suspend them in 200 μL of TE buffer with a pH of 8.0 containing 2 wt% β-mercaptoethanol, add 0.1 g of acid-washed glass beads with a diameter of 0.5 mm, vortex for 5 min, centrifuge at 12,000 r / min for 2 min, and then discard the supernatant to obtain a Candida albicans colony sample; (2) Lysis reaction: Add 100 μL of CTAB lysis buffer at 65 °C to the Candida albicans colony sample obtained in step (1), and place it in a 65 °C water bath for 30 min. During this period, vortex for 30 s every 10 min. After completion, add an equal volume of isopropanol-chloroform solution and shake for 2 min. Finally, centrifuge at 12,000 r / min for 10 min and take the supernatant to obtain the lysis solution; (3) Nucleic acid purification: Transfer the lysis solution obtained in step (2) to a nucleic acid purification column, add 200 μL of binding buffer, centrifuge at 10,000 r / min for 1 min, and discard the filtrate; then add 500 μL of washing buffer, centrifuge at 10,000 r / min for 1 min, remove the washing buffer, and repeat the washing twice; finally, centrifuge the nucleic acid purification column at 12,000 r / min for 2 min to remove residual ethanol, add 50 μL of nucleic acid elution buffer at 65 °C, let it stand at room temperature for 2 min, and then centrifuge at 10,000 r / min for 1 min to take the centrifugate to obtain nucleic acid; (4) Target gene amplification: Use the Candida Resistance RT-PCR Kit for one-step reverse transcription and PCR reactions. Configure the reaction system for reverse transcription-multiplex PCR. The reaction procedure for one-step RT-PCR is as follows: 50 °C for 30 min; 94 °C for 3 min; then perform 45 cycles, each cycle including 94 °C for 30 sec, 58 °C for 30 sec, 72 °C for 30 sec. After the cycle ends, incubate at 72 °C for 5 min to obtain the PCR product; Each sample is prepared with the PCR reaction solution as follows:

[0066] (5) Nucleic acid mass spectrometry detection: Add 1 μL of shrimp alkaline phosphatase and 1 U of exonuclease I to the PCR product obtained in step (4), incubate at 37 °C for 30 min, and then inactivate at 85 °C for 15 min to obtain the purified product; then take 10 μL of the above purified product and mix it with 10 μL of the matrix solution, spot it onto the MALDI target plate, and crystallize at room temperature; finally, use a MALDI-TOF mass spectrometer to collect data in the positive ion mode.

[0067] The concentration of CTAB in the CTAB lysis solution described in step (2) is 2 wt%, the concentration of sodium chloride is 1.4 mol / L, the concentration of EDTA is 20 mmol / L, the concentration of Tris-HCl is 100 mmol / L, and pH = 8.0; the volume ratio of chloroform to isoamyl alcohol in the isopropanol-chloroform solution is 24:1.

[0068] The binding buffer described in step (3) is a 70% ethanol aqueous solution, and the pH is adjusted to 5.2 with an acetic acid-sodium acetate buffer; the washing buffer is 80% ethanol containing 20 mmol / L Tris-HCl, and the pH is adjusted to 7.5 with an acetic acid-sodium acetate buffer; the nucleic acid elution solution is 10 mmol / L Tris-HCl, and the pH is adjusted to 8.5 with an acetic acid-sodium acetate buffer.

[0069] The matrix solution described in step (5) is composed of 3-hydroxy-2-pyridinecarboxylic acid, acetonitrile, citric acid, and deionized water, wherein the concentration of 3-hydroxy-2-pyridinecarboxylic acid is 0.05 g / mL, the concentration of acetonitrile is 50 wt%, and the concentration of citric acid is 0.1 wt%.

[0070] In step (5), a MALDI-TOF mass spectrometer is used, and the parameter settings for data acquisition in the positive ion mode are as follows: mass range: 4,000~10,000 Da, laser intensity: 60%, number of accumulations: 200 times. Comparative Example 2

[0071] The difference from Example 1 is: the combined enzymolysis method of lywallzyme + snailase; A Candida albicans gene detection method includes the following steps: (1) Sample treatment: Take 10 5 CFU Candida albicans colonies, suspend them in 200 μL of PBS solution with a pH of 7.4 containing 20 mmol / L DTT, pre-treat at 37°C for 30 min, centrifuge at 12,000 r / min for 2 min, and discard the supernatant; then add 100 μL of the enzymolysis solution, vortex at 37°C for 2 h, add SDS with a final concentration of 1 wt% to terminate the reaction, and heat at 70°C for 10 min to denature the protein, centrifuge at 12,000 r / min for 2 min, and take the supernatant to obtain the lysis solution; (2)Nucleic acid purification: Transfer the lysate obtained in step (1) to a nucleic acid purification column, add 200 μL of binding buffer, centrifuge at 10000 r / min for 1 min, and discard the filtrate; then add 500 μL of washing buffer, centrifuge at 10000 r / min for 1 min, remove the washing buffer, and repeat the washing twice; finally, centrifuge the nucleic acid purification column at 12000 r / min for 2 min to remove residual ethanol, add 50 μL of nucleic acid elution buffer at 65 °C, let it stand at room temperature for 2 min, then centrifuge at 10000 r / min for 1 min, and take the centrifugate to obtain nucleic acid; (3)Target gene amplification: Use the Candida Resistance RT-PCR Kit for one-step reverse transcription and PCR reactions. Configure the reaction system for reverse transcription-multiplex PCR. The reaction program for one-step RT-PCR is as follows: 50 °C for 30 min; 94 °C for 3 min; then perform 45 cycles, each cycle including 94 °C for 30 sec, 58 °C for 30 sec, 72 °C for 30 sec. After the cycle ends, incubate at 72 °C for 5 min to obtain the PCR product; Each sample is prepared with the PCR reaction solution as follows:

[0072] (4)Nucleic acid mass spectrometry detection: Add 1 μL of shrimp alkaline phosphatase and 1 U of exonuclease I to the PCR product obtained in step (3), incubate at 37 °C for 30 min, then inactivate at 85 °C for 15 min to obtain the purified product; then take 10 μL of the above purified product and mix it with 10 μL of the matrix solution, spot it onto the MALDI target plate, and crystallize at room temperature; finally, use a MALDI-TOF mass spectrometer to collect data in the positive ion mode.

[0073] The enzyme digestion solution described in step (1) contains 10 mg / mL of lysing enzyme, 5 mg / mL of snail enzyme, 0.6 mol / L of KCl, 10 mmol / L of MgCl 2 , pH = 6.0.

[0074] The binding buffer described in step (2) is a 70% ethanol aqueous solution, adjusted to pH = 5.2 with acetic acid-sodium acetate buffer; the washing buffer is a solution containing 80% ethanol, 20 mmol / L of Tris-HCl, adjusted to pH 7.5 with acetic acid-sodium acetate buffer; the nucleic acid elution buffer is 10 mmol / L of Tris-HCl, adjusted to pH 8.5 with acetic acid-sodium acetate buffer.

[0075] The matrix solution described in step (4) consists of 3-hydroxy-2-pyridinecarboxylic acid, acetonitrile, citric acid, and deionized water. The concentration of 3-hydroxy-2-pyridinecarboxylic acid is 0.05 g / mL, the concentration of acetonitrile is 50 wt%, and the concentration of citric acid is 0.1 wt%.

[0076] In step (4), a MALDI-TOF mass spectrometer was used, and the parameter settings for data acquisition in the positive ion mode were as follows: mass range: 4,000 - 10,000 Da, laser intensity: 60%, number of accumulations: 200 times. Comparative Example 3

[0077] The difference from Example 1 is that the lysis solution does not contain saponarin; Comparative Example 4

[0078] The difference from Example 1 is that Proteinase K was not pre-activated; A lysis solution consists of the following components: saponarin, sodium citrate, citric acid, sodium chloride, Proteinase K, and deionized water.

[0079] The lysis solution consists of the following components by mass percentage: Saponarin 0.8 wt%; Sodium citrate 0.62 wt%; Citric acid 0.52 wt%; Sodium chloride 7.01 wt%; Proteinase K 0.05 wt%; The balance is deionized water. Comparative Example 5

[0080] The difference from Example 1 is that the lysis solution does not contain sodium chloride; Comparative Example 6

[0081] Nucleic acid yield detection: Specific binding of a fluorescent dye to double-stranded DNA / RNA generates a signal, and the nucleic acid concentration is calculated by comparing with a standard product of known concentration, avoiding the defect of Nanodrop being interfered by contaminants.

[0082] Standard curve preparation: Use λDNA standard products (0, 1, 10, 50, 100 ng / μL gradients), and make 3 replicates for each concentration point to establish a standard curve with R² > 0.99; Sample processing: Take 2 μL of purified nucleic acid and mix it with 198 μL of Qubit working solution, incubate in the dark for 2 min and then detect on the machine; Total yield (ng) = detection concentration (ng / μL) × elution volume (50 μL) × dilution factor; Quality control requirements: CV value of the standard product < 5%; The difference between repeated sample tests was <10%.

[0083] The test results are shown in Table 1.

[0084] Table 1: Nucleic acid yield test results

[0085] From the test results in Table 1, it can be seen that Example 1 uses saponin (physical lysis), high salt (chemical regulation), buffer (ion / pH control), and proteinase K (enzymatic hydrolysis) to lyse cells, and its nucleic acid yield is closest to 100%, and the Ct value of qPCR is the lowest. The lower the Ct value of qPCR, the higher the nucleic acid content, and the strongest mass spectrometry signal is displayed. Example 1 has the strongest signal due to its integrity and high functional DNA.

[0086] Although the nucleic acid yield of Comparative Example 1 is as high as 145.3%, the mechanical disruption causes serious DNA fragmentation. Therefore, although the yield is high, the functional DNA may be less, the Ct value of qPCR is higher, and the mass spectrometry signal will be weak because the large fragments may be destroyed. The nucleic acid yield of Example 5 is 118.5%, but the high saponin concentration may introduce inhibitors, which will increase the inhibition rate of PCR, resulting in an increase in the Ct value and affecting the mass spectrometry signal, but it is better than mechanical disruption.

[0087] The nucleic acid yields of Examples 2 to 4 are low because the buffer system, lysis temperature, insufficient saponin concentration and other factors lead to low lysis efficiency, so the amount of DNA extracted is small, the Ct value is high, and the mass spectrometry signal is weak. The pre-activation time of Proteinase K in Example 6 is insufficient, and the activity of Proteinase K is insufficient, resulting in insufficient nucleoprotein dissociation, less DNA release, and a higher Ct value. Although the pre-activation time of Example 7 is long and the activity is improved, there is a risk of self-hydrolysis, and part of the DNA is degraded, so the Ct value is slightly lower than that of Example 6, but the mass spectrometry signal may be weakened due to degradation. Comparative Example 4 does not pre-activate Proteinase K, has low activity, the Ct value should be very high, and the mass spectrometry signal is weak. Comparative Example 5 does not have NaCl, which affects the activity of lysozyme and DNA binding, and has a low yield, a high Ct value, and a weak signal.

[0088] In summary, the present invention balances the cleavage efficiency and background interference by regulating the saponin concentration. Too many saponin micelles will wrap the nucleic acid, while too few will result in incomplete cleavage. Although the acidic environment regulated by citric acid buffer may promote RNA degradation, the negative effects are offset by high-salt rapid cleavage and proteinase K to remove nucleases. Pre-activation of proteinase K is synchronized with membrane rupture to ensure that the peak of enzyme activity matches the exposure time of the target protein, thereby improving the degradation efficiency.

Claims

1. A lysate for gene detection of Candida albicans, characterized in that: It is composed of the following components: saponin, sodium citrate, citric acid, sodium chloride, pre-activated proteinase K and deionized water.

2. The lysate for gene detection of Candida albicans according to claim 1, characterized in that: By mass percentage, it is composed of the following components: Saponin 0.8wt%; Sodium citrate 0.62wt%; Citric acid 0.52wt%; Sodium chloride 7.01wt%; Pre-activated proteinase K 0.05wt%; The balance was deionized water.

3. The lysate for gene detection of Candida albicans according to any one of claims 1 or 2, characterized in that: The method for preparing the preactivated proteinase K comprises the following steps: adding proteinase K to 5 mol / L dithiothreitol, wherein the concentration of the proteinase K is 0.5 mg / mL, and incubating at 37°C for 10 minutes to obtain the preactivated proteinase K.

4. A kit for detecting Candida albicans genes, characterized in that: A lysis solution containing the lysis solution for Candida albicans gene detection as claimed in any one of claims 1 to 3.

5. A method for detecting Candida albicans genes, characterized in that: The kit for detecting the gene of Candida albicans as claimed in claim 4 is used.

6. The method for detecting Candida albicans genes according to claim 5, characterized in that: The following steps are involved: (1) Sample processing: Take 10 5 CFU of Candida albicans colonies were suspended in 200 μL of sterile saline, centrifuged at 12000 r / min for 2 min, and the supernatant was discarded; then 100 μL of lysis buffer was added, and the mixture was vortexed and mixed for 30 s to obtain a Candida albicans colony sample; (2) Lysis reaction: the Candida albicans colony sample obtained in step (1) is placed in a 65°C metal bath and oscillated at 1000 r / min for 15 min; the lysate is then immediately cooled at 0°C for 5 min to inhibit the activity of residual protease, thereby obtaining a lysate; (3) Nucleic acid purification: transfer the lysate obtained in step (2) to a nucleic acid purification column, add 200 μL of binding buffer, centrifuge at 10,000 rpm for 1 min, and discard the filtrate; Then add 500 μL of washing buffer, centrifuge at 10,000 r / min for 1 min, remove the washing buffer, and repeat the washing twice; finally, centrifuge the nucleic acid purification column at 12,000 r / min for 2 min to remove the residual ethanol, add 50 μL of 65°C nucleic acid eluent, let stand at room temperature for 2 min, centrifuge at 10,000 r / min for 1 min, take the centrifuge liquid, and obtain the nucleic acid; (4) Target gene amplification: Use Candida Resistance RT-PCR Kit for one-step reverse transcription and PCR reaction, configure the reverse transcription-multiplex PCR reaction system, and the reaction procedure of the one-step RT-PCR reaction is as follows: 50°C for 30 min; 94°C for 3 min; followed by 45 cycles, each cycle including 94°C for 30 sec, 58°C for 30 sec, and 72°C for 30 sec, and 72°C for 5 min after the cycle to obtain the PCR product; The PCR reaction solution for each sample was prepared as follows:

7. (5) Nucleic acid mass spectrometry detection: Add 1 μL shrimp alkaline phosphatase and 1 U exonuclease I to the PCR product obtained in step (4), incubate at 37°C for 30 min, and then inactivate at 85°C for 15 min to obtain a purified product. Then, take 10 μL of the purified product and mix it with 10 μL of matrix solution, spot it on a MALDI target plate, and crystallize it at room temperature. Finally, use a MALDI-TOF mass spectrometer to collect data in positive ion mode.

8. The method for detecting Candida albicans genes according to claim 6, characterized in that: The binding buffer in step (3) is 70% ethanol in water, and the pH value is adjusted to 5.2 by acetic acid-sodium acetate buffer; the washing buffer is 80% ethanol, 20 mmol / L Tris-HCl, and the pH value is adjusted to 7.5 by acetic acid-sodium acetate buffer; the nucleic acid elution solution is 10 mmol / L Tris-HCl, and the pH value is adjusted to 8.5 by acetic acid-sodium acetate buffer.

9. The method for detecting Candida albicans genes according to claim 6, characterized in that: The matrix solution in step (5) is composed of 3-hydroxy-2-pyridinecarboxylic acid, acetonitrile, citric acid, and deionized water, wherein the concentration of 3-hydroxy-2-pyridinecarboxylic acid is 0.05 g / mL, the concentration of acetonitrile is 50 wt%, and the concentration of citric acid is 0.1 wt%.

10. The method for detecting Candida albicans genes according to claim 6, characterized in that: Step (5) Using a MALDI-TOF mass spectrometer, the parameters for collecting data in positive ion mode were as follows: mass range: 4,000-10,000 Da, laser intensity: 60%, accumulation times: 200 times.

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