A lysis buffer, kit, and gene detection method for Candida albicans.

Abstract

This invention belongs to the field of biomedical detection technology, specifically a lysis buffer, kit, and gene detection method for Candida albicans. This invention balances lysis efficiency and background interference by regulating the concentration of saponins; too much saponin micelles will encapsulate nucleic acids, while too little will result in incomplete lysis. Although citrate buffering to regulate the acidic environment may promote RNA degradation, the negative impact is offset by rapid lysis with high salt and proteinase K to clear nucleases. Pre-activated proteinase K synchronizes with membrane rupture, ensuring that the peak enzyme activity matches the exposure time of the target protein, thus improving degradation efficiency. This invention develops a lysis technology that can simultaneously achieve high yield, high-functionality nucleic acid production, and is adapted to rapid detection procedures.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically a lysis buffer, kit, and gene detection method for Candida albicans. Background Technology

[0002] In the field of nucleic acid extraction from pathogenic microorganisms, existing technologies generally face a yield-quality paradox: traditional mechanical disruption methods (such as bead milling and ultrasonication) can achieve high nucleic acid yields, but are accompanied by severe DNA fragmentation, leading to a decrease in qPCR amplification efficiency and a decrease in mass spectrometry detection signal intensity; while enzymatic methods (such as lysozyme and lysozyme) can maintain nucleic acid integrity, but are inefficient at lysing pathogens encapsulated in thick-walled spores or biofilms, and have drawbacks such as being time-consuming and having complex operation steps (requiring multiple pretreatment steps).

[0003] While current mainstream chemical lysis methods (such as the CTAB method) can partially balance yield and quality, key bottlenecks still exist. For example, conventional NaCl concentrations (0.5-1M) cannot simultaneously achieve effective cell membrane expansion and optimized DNA binding efficiency on silica gel columns, leading to fluctuations in functional nucleic acid recovery rates. Although high concentrations of surfactants improve lysis efficiency, they severely interfere with downstream PCR reactions and mass spectrometry ionization processes. Unactivated proteinase K has insufficient disulfide bond reduction, resulting in nucleosome dissociation efficiency of less than 60%, causing false negatives for key drug resistance genes.

[0004] Therefore, developing a lysis technology that can simultaneously achieve high yield and high-functionality nucleic acid production and is compatible with rapid detection procedures has become a core issue that urgently needs to be addressed in this field. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a lysis buffer, kit, and gene detection method for Candida albicans.

[0006] This invention provides a lysis buffer for Candida albicans, used for Candida albicans gene detection.

[0007] The lysis buffer consists of the following components: saponins, sodium citrate, citric acid, sodium chloride, preactivated proteinase K, and deionized water.

[0008] Preferably, the lysis buffer comprises, by mass percentage, the following components:

[0009] Saponin 0.8 wt%;

[0010] Sodium citrate 0.62 wt%;

[0011] Citric acid 0.52 wt%;

[0012] Sodium chloride 7.01 wt%;

[0013] Pre-activated proteinase K 0.05 wt%;

[0014] The remainder is deionized water.

[0015] The method for preparing the preactivated proteinase K includes the following steps: adding proteinase K to 5 mol / L dithiothreitol, wherein the concentration of proteinase K is 0.5 mg / mL, and incubating at 37°C for 10 min to obtain the preactivated proteinase K.

[0016] The present invention also provides a kit for the detection of Candida albicans genes, comprising the lysis buffer as described above.

[0017] This invention also provides a method for detecting Candida albicans genes, using the lysis buffer and the kit for Candida albicans gene detection as described above, comprising the following steps:

[0018] (1) Sample processing: Take 10 5 CFU of Candida albicans colonies were suspended in 200 μL of sterile physiological saline, centrifuged at 12000 r / min for 2 min and the supernatant was discarded; then 100 μL of lysis buffer was added and vortexed for 30 s to obtain Candida albicans colony samples.

[0019] (2) Lysis reaction: The Candida albicans colony sample obtained in step (1) was placed in a 65°C metal bath and lysed by shaking at 1000 r / min for 15 min; then the lysate was immediately placed at 0°C to cool for 5 min to inhibit the activity of residual protease and obtain the lysate.

[0020] (3) Nucleic acid purification: Transfer the lysis buffer 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 buffer at 65℃, let stand at room temperature for 2 min, centrifuge at 10000 r / min for 1 min, and collect the centrifuged liquid to obtain nucleic acid;

[0021] (4) Target gene amplification: The Candida Resistance RT-PCR Kit was used for one-step reverse transcription and PCR reaction. The reaction system for reverse transcription-multiplex PCR was prepared as follows: 50℃ for 30 min; 94℃ for 3 min; then 45 cycles were performed, each cycle including 94℃ for 30 sec, 58℃ for 30 sec, 72℃ for 30 sec, and 72℃ for 5 min after the cycle to obtain PCR products;

[0022] Each sample was prepared using the PCR reaction solution as follows:

[0023]

[0024] (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℃ for 30 min, and then inactivate at 85℃ for 15 min to obtain the purified product; then take 10 μL of the above purified product and mix it with 10 μL of 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 positive ion mode.

[0025] The binding buffer in step (3) is a 70% ethanol aqueous solution, and the pH is adjusted to 5.2 by acetate-sodium acetate buffer; the washing buffer is 80% ethanol, 20 mmol / L Tris-HCl, and the pH is adjusted to 7.5 by acetate-sodium acetate buffer; the nucleic acid elution buffer is 10 mmol / L Tris-HCl, and the pH is adjusted to 8.5 by acetate-sodium acetate buffer.

[0026] 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%.

[0027] Step (5) Using a MALDI-TOF mass spectrometer, the parameters for data acquisition in positive ion mode are set as follows: mass range: 4,000~10,000 Da, laser intensity: 60%, number of accumulations: 200.

[0028] The beneficial effects of this invention are:

[0029] A concentration of 0.8 wt% saponin in the lysis buffer exceeds its critical micelle concentration, forming stable micelles. These micelles insert their hydrophobic ends to disrupt the fungal cell membrane (phospholipid bilayer), forming nanoscale channels and releasing intracellular substances. While ensuring sufficient saponin monomer concentration for efficient lysis, the micelles encapsulate hydrophobic fragments, reducing non-specific binding of cell debris to subsequent detection reagents (such as fluorescent dyes) and lowering background interference.

[0030] Using a citrate-sodium citrate buffer system to maintain the acidity of the lysis buffer helps inhibit exogenous nucleases. At the same time, it chelates free calcium and magnesium ions, inhibiting the destruction of nucleic acids by endogenous nucleases (such as fungal nucleases) that depend on divalent metal ions (such as fungal nucleases) and host defense enzymes (such as lysozyme), thereby protecting the integrity of nucleic acids.

[0031] Sodium chloride increases the osmotic pressure of the lysis buffer, triggering rapid cell dehydration and accelerating membrane collapse through saponins in the pores, releasing intracellular substances. At the same time, high salt concentration can also inhibit the binding of negatively charged nucleases and substrates through charge shielding, thus protecting nucleic acids.

[0032] Preactivated proteinase K is active at the initial stage of lysis and can act synchronously with saponins-dominated membrane rupture, preferentially degrading fungal intracellular proteins (such as histones and nucleic acid-binding proteins) to release nucleic acids, and rapidly decomposing potentially released endogenous interfering enzymes (such as chitinases and nucleases). At the same time, after completing the degradation of the target protein, excess proteinase K can be inactivated through autohydrolysis and conformational degradation under acidic conditions, avoiding residual enzyme activity from interfering with downstream applications such as PCR or sequencing.

[0033] In summary, this invention balances cleavage efficiency and background interference by regulating the concentration of saponins. Too many saponin micelles will encapsulate nucleic acids, while too few will result in incomplete cleavage. Although the acidic environment regulated by citrate buffer may promote RNA degradation, the negative effects are offset by rapid cleavage with high salt and the removal of nucleases by proteinase K. Pre-activation of proteinase K is synchronized with membrane rupture to ensure that the peak enzyme activity matches the exposure time of the target protein, thereby improving degradation efficiency.

[0034] The lysis buffer of this invention employs a multi-mechanism linkage of saponins (physical lysis), high salt (chemical regulation), buffer solution (ion / pH control), and proteinase K (enzymatic hydrolysis), replacing the complex steps of traditional enzymatic hydrolysis of cell walls, simplifying the process and reducing costs. Detailed Implementation

[0035] Saponins: CAS No.: 20310-89-8.

[0036] Proteinase K, CAS: 39450-01-6, Product No.: GPK003001, Jiangsu Jinpu Nuoan Biotechnology Co., Ltd.

[0037] Dithiothreitol, CAS: 3483-12-3.

[0038] Candida albicans, CGMCC 2.4159, was purchased from the China General Microbiological Culture Collection Center.

[0039] Nucleic acid purification column, catalog number: N1010, Beijing Solarbio Science & Technology Co., Ltd.

[0040] Shrimp alkaline phosphatase, product number: KMC125113, Wenzhou Kemiao Biotechnology Co., Ltd.

[0041] Exonuclease I, catalog number: XG-P3062, Shanghai Siger Biotechnology Co., Ltd.

[0042] 3-Hydroxy-2-pyridinecarboxylic acid, CAS: 874-24-8.

[0043] Lysozyme, catalog number: E0077, Nanjing Dulai Biotechnology Co., Ltd.

[0044] Snail enzyme, product number: 161790, Shanghai Koraman Reagent Co., Ltd. Example 1

[0045] A lysis buffer comprising the following components: saponins, sodium citrate, citric acid, sodium chloride, preactivated proteinase K, and deionized water.

[0046] The pyrolysis solution, by mass percentage, consists of the following components:

[0047] Saponin 0.8 wt%;

[0048] Sodium citrate 0.62 wt%;

[0049] Citric acid 0.52 wt%;

[0050] Sodium chloride 7.01 wt%;

[0051] Pre-activated proteinase K 0.05 wt%;

[0052] The remainder is deionized water.

[0053] The method for preparing the preactivated proteinase K includes the following steps: adding proteinase K to 5 mol / L dithiothreitol, wherein the concentration of proteinase K is 0.5 mg / mL, and incubating at 37°C for 10 min to obtain the preactivated proteinase K.

[0054] A kit for the detection of Candida albicans genes, comprising the lysis buffer as described above.

[0055] A method for detecting Candida albicans genes, using the lysis buffer and the kit for Candida albicans gene detection as described above, includes the following steps:

[0056] (1) Sample processing: Take 10 5 CFU of Candida albicans colonies were suspended in 200 μL of sterile physiological saline, centrifuged at 12000 r / min for 2 min and the supernatant was discarded; then 100 μL of lysis buffer was added and vortexed for 30 s to obtain Candida albicans colony samples.

[0057] (2) Lysis reaction: The Candida albicans colony sample obtained in step (1) was placed in a 65°C metal bath and lysed by shaking at 1000 r / min for 15 min; then the lysate was immediately placed at 0°C to cool for 5 min to inhibit the activity of residual protease and obtain the lysate.

[0058] (3) Nucleic acid purification: Transfer the lysis buffer 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 buffer at 65℃, let stand at room temperature for 2 min, centrifuge at 10000 r / min for 1 min, and collect the centrifuged liquid to obtain nucleic acid;

[0059] (4) Target gene amplification: The Candida Resistance RT-PCR Kit was used for one-step reverse transcription and PCR reaction. The reaction system for reverse transcription-multiplex PCR was prepared as follows: 50℃ for 30 min; 94℃ for 3 min; then 45 cycles were performed, each cycle including 94℃ for 30 sec, 58℃ for 30 sec, 72℃ for 30 sec, and 72℃ for 5 min after the cycle to obtain PCR products;

[0060] Each sample was prepared using the PCR reaction solution as follows:

[0061]

[0062] (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℃ for 30 min, and then inactivate at 85℃ for 15 min to obtain the purified product; then take 10 μL of the above purified product and mix it with 10 μL of 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 positive ion mode.

[0063] The binding buffer in step (3) is a 70% ethanol aqueous solution, and the pH is adjusted to 5.2 by acetate-sodium acetate buffer; the washing buffer is 80% ethanol, 20 mmol / L Tris-HCl, and the pH is adjusted to 7.5 by acetate-sodium acetate buffer; the nucleic acid elution buffer is 10 mmol / L Tris-HCl, and the pH is adjusted to 8.5 by acetate-sodium acetate buffer.

[0064] 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%.

[0065] Step (5) Using a MALDI-TOF mass spectrometer, the parameters for data acquisition in positive ion mode are set as follows: mass range: 4,000~10,000 Da, laser intensity: 60%, number of accumulations: 200. Example 2

[0066] The difference from Example 1 is that the citrate buffer in the lysis buffer formulation is replaced with phosphate buffer;

[0067] A lysis buffer comprising the following components: saponins, sodium hydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, preactivated proteinase K, and deionized water.

[0068] The pyrolysis solution, by mass percentage, consists of the following components:

[0069] Saponin 0.8 wt%;

[0070] Sodium hydrogen phosphate 0.62 wt%;

[0071] Potassium dihydrogen phosphate 0.52 wt%;

[0072] Sodium chloride 7.01 wt%;

[0073] Pre-activated proteinase K 0.05 wt%;

[0074] The remainder is deionized water.

[0075] The method for preparing the preactivated proteinase K includes the following steps: adding proteinase K to 5 mol / L dithiothreitol, wherein the concentration of proteinase K is 0.5 mg / mL, and incubating at 37°C for 10 min to obtain the preactivated proteinase K. Example 3

[0076] The difference from Example 1 is that the pyrolysis temperature was adjusted to 60°C;

[0077] A lysis buffer comprising the following components: saponins, sodium citrate, citric acid, sodium chloride, preactivated proteinase K, and deionized water.

[0078] The pyrolysis solution, by mass percentage, consists of the following components:

[0079] Saponin 0.8 wt%;

[0080] Sodium citrate 0.62 wt%;

[0081] Citric acid 0.52 wt%;

[0082] Sodium chloride 7.01 wt%;

[0083] Pre-activated proteinase K 0.05 wt%;

[0084] The remainder is deionized water.

[0085] The method for preparing the preactivated proteinase K includes the following steps: adding proteinase K to 5 mol / L dithiothreitol, wherein the concentration of proteinase K is 0.5 mg / mL, and incubating at 37°C for 10 min to obtain the preactivated proteinase K.

[0086] A kit for the detection of Candida albicans genes, comprising the lysis buffer as described above.

[0087] A method for detecting Candida albicans genes, using the lysis buffer and the kit for Candida albicans gene detection as described above, includes the following steps:

[0088] (1) Sample processing: Take 10 5 CFU of Candida albicans colonies were suspended in 200 μL of sterile physiological saline, centrifuged at 12000 r / min for 2 min and the supernatant was discarded; then 100 μL of lysis buffer was added and vortexed for 30 s to obtain Candida albicans colony samples.

[0089] (2) Lysis reaction: The Candida albicans colony sample obtained in step (1) was placed in a 60℃ metal bath and lysed by shaking at 1000r / min for 15min; then the lysate was immediately placed at 0℃ and cooled for 5min to inhibit the activity of residual protease, and the lysate was obtained.

[0090] (3) Nucleic acid purification: Transfer the lysis buffer 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 buffer at 65℃, let stand at room temperature for 2 min, centrifuge at 10000 r / min for 1 min, and collect the centrifuged liquid to obtain nucleic acid;

[0091] (4) Target gene amplification: The Candida Resistance RT-PCR Kit was used for one-step reverse transcription and PCR reaction. The reaction system for reverse transcription-multiplex PCR was prepared as follows: 50℃ for 30 min; 94℃ for 3 min; then 45 cycles were performed, each cycle including 94℃ for 30 sec, 58℃ for 30 sec, 72℃ for 30 sec, and 72℃ for 5 min after the cycle to obtain PCR products;

[0092] Each sample was prepared using the PCR reaction solution as follows:

[0093]

[0094] (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℃ for 30 min, and then inactivate at 85℃ for 15 min to obtain the purified product; then take 10 μL of the above purified product and mix it with 10 μL of 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 positive ion mode.

[0095] The binding buffer in step (3) is a 70% ethanol aqueous solution, and the pH is adjusted to 5.2 by acetate-sodium acetate buffer; the washing buffer is 80% ethanol, 20 mmol / L Tris-HCl, and the pH is adjusted to 7.5 by acetate-sodium acetate buffer; the nucleic acid elution buffer is 10 mmol / L Tris-HCl, and the pH is adjusted to 8.5 by acetate-sodium acetate buffer.

[0096] 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%.

[0097] Step (5) Using a MALDI-TOF mass spectrometer, the parameters for data acquisition in positive ion mode are set as follows: mass range: 4,000~10,000 Da, laser intensity: 60%, number of accumulations: 200. Example 4

[0098] The difference from Example 1 is that the concentration of saponins is 0.5 wt%.

[0099] A lysis buffer comprising the following components: saponins, sodium citrate, citric acid, sodium chloride, preactivated proteinase K, and deionized water.

[0100] The pyrolysis solution, by mass percentage, consists of the following components:

[0101] Saponin 0.5 wt%;

[0102] Sodium citrate 0.62 wt%;

[0103] Citric acid 0.52 wt%;

[0104] Sodium chloride 7.01 wt%;

[0105] Pre-activated proteinase K 0.05 wt%;

[0106] The remainder is deionized water.

[0107] The method for preparing the preactivated proteinase K includes the following steps: adding proteinase K to 5 mol / L dithiothreitol, wherein the concentration of proteinase K is 0.5 mg / mL, and incubating at 37°C for 10 min to obtain the preactivated proteinase K. Example 5

[0108] The difference from Example 1 is that the concentration of saponins is 1.2 wt%.

[0109] A lysis buffer comprising the following components: saponins, sodium citrate, citric acid, sodium chloride, preactivated proteinase K, and deionized water.

[0110] The pyrolysis solution, by mass percentage, consists of the following components:

[0111] Saponins 1.2 wt%;

[0112] Sodium citrate 0.62 wt%;

[0113] Citric acid 0.52 wt%;

[0114] Sodium chloride 7.01 wt%;

[0115] Pre-activated proteinase K 0.05 wt%;

[0116] The remainder is deionized water.

[0117] The method for preparing the preactivated proteinase K includes the following steps: adding proteinase K to 5 mol / L dithiothreitol, wherein the concentration of proteinase K is 0.5 mg / mL, and incubating at 37°C for 10 min to obtain the preactivated proteinase K. Example 6

[0118] The difference from Example 1 is that the proteinase K incubation time is 5 minutes;

[0119] A lysis buffer comprising the following components: saponins, sodium citrate, citric acid, sodium chloride, preactivated proteinase K, and deionized water.

[0120] The pyrolysis solution, by mass percentage, consists of the following components:

[0121] Saponin 0.8 wt%;

[0122] Sodium citrate 0.62 wt%;

[0123] Citric acid 0.52 wt%;

[0124] Sodium chloride 7.01 wt%;

[0125] Pre-activated proteinase K 0.05 wt%;

[0126] The remainder is deionized water.

[0127] The method for preparing the preactivated proteinase K includes the following steps: adding proteinase K to 5 mol / L dithiothreitol, wherein the concentration of proteinase K is 0.5 mg / mL, and incubating at 37°C for 5 min to obtain the preactivated proteinase K. Example 7

[0128] The difference from Example 1 is that the proteinase K incubation time is 15 min;

[0129] A lysis buffer comprising the following components: saponins, sodium citrate, citric acid, sodium chloride, preactivated proteinase K, and deionized water.

[0130] The pyrolysis solution, by mass percentage, consists of the following components:

[0131] Saponin 0.8 wt%;

[0132] Sodium citrate 0.62 wt%;

[0133] Citric acid 0.52 wt%;

[0134] Sodium chloride 7.01 wt%;

[0135] Pre-activated proteinase K 0.05 wt%;

[0136] The remainder is deionized water.

[0137] The method for preparing the preactivated proteinase K includes the following steps: adding proteinase K to 5 mol / L dithiothreitol, wherein the concentration of proteinase K is 0.5 mg / mL, and incubating at 37°C for 15 min to obtain the preactivated proteinase K. Comparative Example 1

[0138] The difference from Example 1 is that: mechanical glass bead crushing method + CTAB lysis solution;

[0139] A method for detecting Candida albicans genes includes the following steps:

[0140] (1) Sample processing: Take 10 5 CFU Candida albicans colonies were suspended in 200 μL of TE buffer (pH=8.0) containing 2 wt% β-mercaptoethanol. 0.1 g of 0.5 mm acid-washed glass beads were added, and the mixture was vortexed for 5 min. After centrifugation at 12000 r / min for 2 min, the supernatant was discarded to obtain the Candida albicans colony sample.

[0141] (2) Lysis reaction: Add 100 μL of CTAB lysis buffer at 65℃ to the Candida albicans colony sample obtained in step (1), and place it in a 65℃ water bath for 30 min, vortexing for 30 s every 10 min during the process. After the process, add an equal volume of isopropanol chloroform solution and shake for 2 min. Finally, centrifuge at 12000 r / min for 10 min and take the supernatant to obtain the lysis buffer.

[0142] (3) Nucleic acid purification: Transfer the lysis buffer 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 buffer at 65℃, let stand at room temperature for 2 min, centrifuge at 10000 r / min for 1 min, and collect the centrifuged liquid to obtain nucleic acid;

[0143] (4) Target gene amplification: The Candida Resistance RT-PCR Kit was used for one-step reverse transcription and PCR reaction. The reaction system for reverse transcription-multiplex PCR was prepared as follows: 50℃ for 30 min; 94℃ for 3 min; then 45 cycles were performed, each cycle including 94℃ for 30 sec, 58℃ for 30 sec, 72℃ for 30 sec, and 72℃ for 5 min after the cycle to obtain PCR products;

[0144] Each sample was prepared using the PCR reaction solution as follows:

[0145]

[0146] (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℃ for 30 min, and then inactivate at 85℃ for 15 min to obtain the purified product; then take 10 μL of the above purified product and mix it with 10 μL of 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 positive ion mode.

[0147] In step (2), the concentration of CTAB in the CTAB lysis buffer 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 the pH is 8.0; the volume ratio of chloroform to isoamyl alcohol in the isopropanol chloroform solution is 24:1.

[0148] The binding buffer in step (3) is a 70% ethanol aqueous solution, and the pH is adjusted to 5.2 by acetate-sodium acetate buffer; the washing buffer is 80% ethanol, 20 mmol / L Tris-HCl, and the pH is adjusted to 7.5 by acetate-sodium acetate buffer; the nucleic acid elution buffer is 10 mmol / L Tris-HCl, and the pH is adjusted to 8.5 by acetate-sodium acetate buffer.

[0149] 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%.

[0150] Step (5) Using a MALDI-TOF mass spectrometer, the parameters for data acquisition in positive ion mode are set as follows: mass range: 4,000~10,000 Da, laser intensity: 60%, number of accumulations: 200. Comparative Example 2

[0151] The difference from Example 1 is that it uses a combined enzymatic hydrolysis method of lysing enzyme and snail enzyme;

[0152] A method for detecting Candida albicans genes includes the following steps:

[0153] (1) Sample processing: Take 10 5 CFU of Candida albicans colonies were suspended in 200 μL of PBS solution (pH 7.4) containing 20 mmol / L DTT and pretreated at 37°C for 30 min. After centrifugation at 12000 rpm for 2 min, the supernatant was discarded. Then, 100 μL of enzymatic digestion buffer was added, and the mixture was vortexed at 37°C for 2 h. The reaction was terminated by adding 1 wt% SDS and heating at 70°C for 10 min to denature the protein. After centrifugation at 12000 rpm for 2 min, the supernatant was collected to obtain the lysis buffer.

[0154] (2) Nucleic acid purification: Transfer the lysis buffer 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℃, let stand at room temperature for 2 min, centrifuge at 10000 r / min for 1 min, and collect the centrifuged liquid to obtain nucleic acid;

[0155] (3) Target gene amplification: The Candida Resistance RT-PCR Kit was used for one-step reverse transcription and PCR reaction. The reaction system for reverse transcription-multiplex PCR was prepared. The reaction program for the one-step RT-PCR reaction was as follows: 50℃ for 30 min; 94℃ for 3 min; then 45 cycles were performed, each cycle including 94℃ for 30 sec, 58℃ for 30 sec, 72℃ for 30 sec, and 72℃ for 5 min after the cycle to obtain the PCR product.

[0156] Each sample was prepared using the PCR reaction solution as follows:

[0157]

[0158] (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℃ for 30 min, and then inactivate at 85℃ for 15 min to obtain the purified product; then take 10 μL of the above purified product and mix it with 10 μL of 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 positive ion mode.

[0159] The enzymatic hydrolysate in step (1) contains 10 mg / mL lysozyme, 5 mg / mL snailase, 0.6 mol / L KCl, 10 mmol / L MgCl2, and pH=6.0.

[0160] The binding buffer in step (2) is a 70% ethanol aqueous solution, and the pH is adjusted to 5.2 by acetate-sodium acetate buffer; the washing buffer is 80% ethanol, 20 mmol / L Tris-HCl, and the pH is adjusted to 7.5 by acetate-sodium acetate buffer; the nucleic acid elution buffer is 10 mmol / L Tris-HCl, and the pH is adjusted to 8.5 by acetate-sodium acetate buffer.

[0161] The matrix solution in step (4) consists 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%.

[0162] Step (4) Using a MALDI-TOF mass spectrometer, the parameters for data acquisition in positive ion mode are set as follows: mass range: 4,000~10,000 Da, laser intensity: 60%, number of accumulations: 200. Comparative Example 3

[0163] The difference from Example 1 is that the lysis buffer does not contain saponins; Comparative Example 4

[0164] The difference from Example 1 is that proteinase K was not pre-activated;

[0165] A lysis buffer comprising the following components: saponins, sodium citrate, citric acid, sodium chloride, proteinase K, and deionized water.

[0166] The pyrolysis solution, by mass percentage, consists of the following components:

[0167] Saponin 0.8 wt%;

[0168] Sodium citrate 0.62 wt%;

[0169] Citric acid 0.52 wt%;

[0170] Sodium chloride 7.01 wt%;

[0171] Proteinase K 0.05 wt%;

[0172] The remainder is deionized water. Comparative Example 5

[0173] The difference from Example 1 is that the lysis solution does not contain sodium chloride; Comparative Example 6

[0174] Nucleic acid yield detection:

[0175] By generating a signal through the specific binding of fluorescent dye to double-stranded DNA / RNA, and comparing it with a standard of known concentration, the nucleic acid concentration can be calculated, thus avoiding the drawback of Nanodrop being affected by contaminants.

[0176] Preparation of standard curve: Using λDNA standards (0, 1, 10, 50, 100 ng / μL gradient), perform 3 replicates for each concentration point to establish a standard curve with R²>0.99;

[0177] Sample processing: Mix 2 μL of purified nucleic acid with 198 μL of Qubit working solution, incubate in the dark for 2 min, and then perform the analysis.

[0178] Total yield (ng) = detection concentration (ng / μL) × elution volume (50μL) × dilution factor;

[0179] Quality control requirements:

[0180] The CV value of the standard product is <5%;

[0181] The difference between repeated tests of samples is <10%.

[0182] The test results are shown in Table 1.

[0183] Table 1: Nucleic Acid Yield Detection Results

[0184]

[0185] As can be seen from the test results in Table 1, Example 1 employed a multi-mechanism linkage approach to lyse cells using saponins (physical lysis), high salt (chemical regulation), buffer (ion / pH control), and proteinase K (enzymatic digestion). This resulted in the nucleic acid yield closest to 100%, and the lowest Ct value for qPCR. A lower Ct value in qPCR indicates a higher nucleic acid content and the strongest mass spectrometry signal. Example 1 exhibited the strongest signal due to the integrity and high functionality of the DNA.

[0186] While Comparative Example 1 achieved a nucleic acid yield of 145.3%, the mechanical fragmentation resulted in severe DNA fragmentation. Therefore, although the yield was high, the amount of functional DNA was likely low, leading to a higher Ct value in qPCR and a weaker mass spectrometry signal, as large fragments were likely destroyed. Example 5 achieved a nucleic acid yield of 118.5%, but the excessively high concentration of saponins may have introduced inhibitors, increasing the PCR inhibition rate and causing a higher Ct value. The mass spectrometry signal was also affected, but it was still better than mechanical fragmentation.

[0187] The low nucleic acid yields in Examples 2 to 4 were due to factors such as insufficient buffer system, lysis temperature, and saponin concentration, leading to low lysis efficiency, low DNA extraction, high Ct values, and weak mass spectrometry signals. In Example 6, insufficient proteinase K pre-activation time resulted in inadequate proteinase K activity, leading to insufficient nucleoprotein dissociation, low DNA release, and a high Ct value. Although Example 7 had a longer pre-activation time and increased activity, there was a risk of autolysis, with some DNA being degraded. Therefore, the Ct value was slightly lower than in Example 6, but the mass spectrometry signal might be weaker due to degradation. Comparative Example 4 lacked pre-activated proteinase K, resulting in low activity and a likely high Ct value, but also a weak mass spectrometry signal. Comparative Example 5 lacked NaCl, affecting lysozyme activity and DNA binding, resulting in low yield, high Ct value, and a weak signal.

[0188] In summary, this invention balances cleavage efficiency and background interference by regulating the concentration of saponins. Too many saponin micelles will encapsulate nucleic acids, while too few will result in incomplete cleavage. Although the acidic environment regulated by citrate buffer may promote RNA degradation, the negative effects are offset by rapid cleavage with high salt and the removal of nucleases by proteinase K. Pre-activation of proteinase K is synchronized with membrane rupture to ensure that the peak enzyme activity matches the exposure time of the target protein, thereby improving degradation efficiency.

Claims

1. A lysis buffer for Candida albicans gene detection, characterized in that, It consists of the following components: saponins, sodium citrate, citric acid, sodium chloride, preactivated proteinase K, and deionized water; The lysis buffer for Candida albicans gene detection is characterized in that the preparation method of the preactivated proteinase K includes the following steps: adding proteinase K to 5 mol / L dithiothreitol, wherein the concentration of proteinase K is 0.5 mg / mL, and incubating at 37°C for 10 min to obtain the preactivated proteinase K.

2. The lysis buffer for Candida albicans gene detection as described in claim 1, characterized in that, It 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 remainder is deionized water.

3. A kit for detecting Candida albicans genes, characterized in that, It contains the lysis buffer for Candida albicans gene detection as described in any one of claims 1-2.

4. A method for gene detection of Candida albicans samples for non-diagnostic purposes, characterized in that, Candida albicans was lysed using the lysis buffer for Candida albicans gene detection as described in any one of claims 1-2.

5. The gene detection method for Candida albicans samples for non-diagnostic purposes as described in claim 4, characterized in that, Includes the following steps: (1) Sample processing: Take 10 5 CFU of Candida albicans colonies were suspended in 200 μL of sterile physiological saline, centrifuged at 12000 r / min for 2 min and the supernatant was discarded; then 100 μL of lysis buffer was added and vortexed for 30 s to obtain Candida albicans colony samples. (2) Lysis reaction: The Candida albicans colony sample obtained in step (1) was placed in a 65°C metal bath and lysed by shaking at 1000 r / min for 15 min; then the lysate for Candida albicans gene detection as described in any one of claims 1-2 was immediately placed at 0°C and cooled for 5 min to inhibit residual protease activity, and the lysate was obtained. (3) Nucleic acid purification: Transfer the lysis buffer 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 65℃ nucleic acid elution buffer, let stand at room temperature for 2 min, centrifuge at 10000 r / min for 1 min, collect the centrifuged liquid, and obtain the nucleic acid; (4) Target gene amplification: The Candida Resistance RT-PCR Kit was used for one-step reverse transcription and PCR reaction. The reaction system for reverse transcription-multiplex PCR was prepared. The reaction program for the one-step RT-PCR reaction was as follows: 50℃ for 30 min; 94℃ for 3 min; then 45 cycles were performed, each cycle including 94℃ for 30 sec, 58℃ for 30 sec, 72℃ for 30 sec, and 72℃ for 5 min after the cycle to obtain the PCR product. Each sample was prepared using the PCR reaction solution as follows: (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℃ for 30 min, and then inactivate at 85℃ for 15 min to obtain the purified product; then take 10 μL of the above purified product and mix it with 10 μL of 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 positive ion mode.

6. The gene detection method for Candida albicans samples for non-diagnostic purposes as described in claim 5, characterized in that, The binding buffer in step (3) is a 70% ethanol aqueous solution, and the pH is adjusted to 5.2 by acetate-sodium acetate buffer; the washing buffer is 80% ethanol, 20 mmol / L Tris-HCl, and the pH is adjusted to 7.5 by acetate-sodium acetate buffer; the nucleic acid elution buffer is 10 mmol / L Tris-HCl, and the pH is adjusted to 8.5 by acetate-sodium acetate buffer.

7. The gene detection method for Candida albicans samples for non-diagnostic purposes as described in claim 5, 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%.

8. The gene detection method for Candida albicans samples for non-diagnostic purposes as described in claim 5, characterized in that, Step (5) Using a MALDI-TOF mass spectrometer, the parameters for data acquisition in positive ion mode are set as follows: mass range: 4,000~10,000 Da, laser intensity: 60%, number of accumulations: 200.

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