Universal nucleic acid extraction reagent for pathogenic fungi and bacteria and application of universal nucleic acid extraction reagent

Abstract

The invention discloses a universal nucleic acid extraction reagent for pathogenic fungi and bacteria and application of the universal nucleic acid extraction reagent, and belongs to the technical field of nucleic acid extraction. The invention develops an efficient nucleic acid extraction method for pathogenic fungi and bacteria from the perspective of innovative use of products and reasonable optimization of the method by deeply researching the structural characteristics of the pathogenic fungi and bacteria. Wherein the extraction reagent comprises a lysis solution I (containing 2% of a defoaming agent), a lysis solution II, a washing solution I, a washing solution II, an eluent, a grinding tube (containing 50g of 200 [mu] m and 150g of 500 [mu] m pickling glass beads) and a nucleic acid adsorption material, and the extraction method comprises the steps of grinding (the optimal time is 15 minutes), splitting, washing and eluting. Evaluation of various technical parameters shows that compared with an existing nucleic acid extraction method, the yield (concentration), purity and integrity of nucleic acid are all remarkably improved; compared with a commercialized kit, the sensitivity and the specificity are greatly improved, and performance parameters are excellent.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid extraction, and particularly relates to a general nucleic acid extraction reagent for pathogenic fungi and bacteria and its application. Background Art

[0002] Early diagnosis is a prerequisite for the precise treatment of pathogenic fungi and bacteria. Commonly used clinical diagnostic techniques for pathogenic fungi and bacteria include culture observation, microscopy, electron microscopy examination, serological detection, and histopathological examination, etc. These techniques often lead to missed diagnoses and misdiagnoses respectively due to low sensitivity, poor specificity, complex operation, long diagnostic cycle, high requirements for technical personnel, etc., especially the missed diagnoses of critically ill patients, missing the golden period of treatment, which is an important reason for the increased mortality rate of pathogenic fungal and bacterial infections. Compared with these methods, nucleic acid detection has obvious technical advantages: (1) Strong specificity: It can accurately identify specific nucleic acid sequences and other molecular markers of pathogens, accurately distinguish different pathogens, and reduce misdiagnosis. (2) High sensitivity: It can detect extremely small amounts of pathogen nucleic acid components, which is conducive to early diagnosis. (3) Rapid detection: Compared with traditional culture and other methods, it can produce results in a short time, facilitating timely diagnosis and treatment. (4) Quantitative analysis: It can accurately quantify the pathogen load, which is conducive to monitoring the condition and evaluating the treatment effect.

[0003] The quality prerequisite for the detection of pathogenic fungi and bacteria is to obtain complete nucleic acids of pathogens with high purity and high concentration. Since the cell walls of pathogenic fungi and Gram-positive bacteria are thick and the structures are complex, the nucleic acid extraction protocols for pathogenic fungi and Gram-positive bacteria in the laboratory are basically to break the cell walls first and then extract and purify the nucleic acids. Commonly used cell wall breaking methods include: (1) Liquid nitrogen grinding is a traditional method for breaking the cell walls of fungi. The cell wall breaking effect is relatively good and the technology is reliable. However, liquid nitrogen is not easy to obtain, the operation is dangerous, and it is extremely easy to cause frostbite to the operator. Moreover, the intense mechanical force is likely to cause nucleic acid breakage and reduce the integrity of the nucleic acid; (2) Ultrasonic cell wall breaking is a commonly used method in fermentation engineering and can also be used for nucleic acid extraction of fungi. However, it requires a relatively high ultrasonic power, and the ordinary ultrasonic crushing power cannot meet the requirements for breaking the cell walls of fungi, resulting in a low yield (concentration) of extracted nucleic acids, reducing the sensitivity of subsequent nucleic acid detection techniques and making it difficult to be applied in diagnostic laboratories; if the ultrasonic power is increased, it will increase the temperature and vibration intensity during the breaking process, easily destroying the nucleic acid and affecting the integrity; (3) Lysozyme, lysing enzyme, and snail enzyme can hydrolyze the cell wall structure through biochemical methods to lyse the cell wall. However, the enzymes are expensive, increasing the reagent cost; the stability of the enzymes themselves is low, not suitable for storage at room temperature, affecting reagent storage and transportation; the incubation treatment time of the enzymes is long, which will inevitably lengthen the test time and cause a delay in the diagnostic report; if the incubation time is not well controlled, it will also cause nucleic acid degradation and reduce the integrity of the nucleic acid, etc.

[0004] Meanwhile, the above method also includes the following components: phenol-chloroform, high-concentration guanidine salts, and nucleic acid adsorption materials. Among them, phenol-chloroform is highly toxic and volatile, and needs to be operated in a well-ventilated environment such as a fume hood or a biosafety cabinet. The combined use of high-concentration guanidine salts and nucleic acid adsorption materials is a commonly used component in current nucleic acid extraction. It is still necessary to add biological enzymes such as proteinase K for extraction treatment, which has a long extraction time and high raw material costs.

[0005] Nucleic acid detection technology has become the gold standard for the diagnosis of pathogenic fungi and bacteria. However, due to the special cell wall structures of pathogenic fungi and Gram-positive bacteria, the above existing technologies have defects such as complex operation, long time consumption, high cost, use of harmful substances, and low nucleic acid yield (concentration). Therefore, how to quickly, stably, and efficiently extract complete nucleic acids of pathogenic fungi and Gram-positive bacteria from samples, improve the sensitivity of nucleic acid detection technology for pathogenic fungi, and thus promote the wide application of nucleic acid detection technology in the field of pathogenic fungi diagnosis is an urgent problem to be solved. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a universal nucleic acid extraction reagent for pathogenic fungi and bacteria and its application. Through in-depth research on the structural characteristics of pathogenic fungi and bacteria, the present invention has designed and developed an efficient method for extracting nucleic acids of pathogenic fungi and bacteria. Through the evaluation of various technical parameters, compared with the existing nucleic acid extraction methods, the nucleic acid yield (concentration), purity, and integrity have all been significantly improved; compared with commercial nucleic acid detection kits for pathogenic fungi, the sensitivity and specificity have both been greatly improved, and the performance parameters are excellent.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] One of the objectives of the present invention is to provide a general nucleic acid extraction reagent for pathogenic fungi and bacteria, which comprises lysis solution I, lysis solution II, washing solution I, washing solution II, elution solution, grinding tube and nucleic acid adsorption material; the lysis solution I contains: 0.4-0.6% SDS, 0.8-1.2% TritonX-100, 18-22 mM Tris-HCl and 1-3% defoaming agent; the lysis solution II contains: 4-6 M guanidine isothiocyanate, 180-220 mM sodium chloride, 18-22 mM sodium citrate and 18-22 mM Tris-HCl; the washing solution I contains: 1.8-2.2 M guanidine hydrochloride, 0.4-0.6% TritonX-100, 18-22 mM Tris-HCl and 30%-50% ethanol; the washing solution II contains: 18-22 mM sodium chloride, 18-22 mM Tris-HCl and 60%-80% ethanol; the elution solution contains: 0.8-1.2 mM EDTA and 8-12 mM Tris-HCl; the grinding tube contains: 40-60 g of 180-220 μm acid-washed glass beads, 140-160 g of 480-520 μm acid-washed glass beads and a centrifuge tube with a conical bottom and a lid; the nucleic acid adsorption material contains: a nucleic acid adsorption column or carboxyl magnetic beads.

[0009] Further, the pH value of the Tris-HCl is 7.5-8.5.

[0010] Furthermore, the defoaming agent is any one or more of non-ionic T-F composite defoaming agent for fermentation and silicone defoaming agent.

[0011] Still further, the defoaming agent is non-ionic T-F composite defoaming agent for fermentation, and the concentration is 2%.

[0012] Even further, the weight ratio of the 180-220 μm acid-washed glass beads to the 480-520 μm acid-washed glass beads used in the grinding tube is 1:3.

[0013] Another objective of the present invention is to provide a general nucleic acid extraction kit for pathogenic fungi and bacteria, which contains the above-mentioned general nucleic acid extraction reagent for pathogenic fungi and bacteria.

[0014] A third object of the present invention is to provide a nucleic acid extraction method, which is carried out by using the general nucleic acid extraction reagent for pathogenic fungi and bacteria or the general nucleic acid extraction kit for pathogenic fungi and bacteria, and includes the following steps: (1) Grinding: Add 180 - 220 μL of the sample into a grinding tube, and mix it with 180 - 220 μL of lysis solution I, and grind for 10 - 20 minutes; (2) Lysis: Add 180 - 220 μL of lysis solution II to the ground sample, and then add 380 - 420 μL of ethanol or isopropanol to make the final concentration of ethanol or isopropanol in the lysed sample reach 35 - 45%; (3) Washing: Use 480 - 520 μL of washing solution I and washing solution II to remove the residual proteins and impurities during the nucleic acid adsorption process; (4) Elution: Use 80 - 120 μL of elution solution to elute the nucleic acid to obtain pure biological nucleic acid.

[0015] Further, the grinding instrument used in the step (1) is a vortex shaker or a tissue grinder, and the grinding speeds of the two are 3200 RPM and 30 Hz respectively, and the grinding time is 15 minutes.

[0016] A fourth object of the present invention is to provide the application of the general nucleic acid extraction reagent for pathogenic fungi and bacteria, the general nucleic acid extraction kit for pathogenic fungi and bacteria or the nucleic acid extraction method in extracting nucleic acids of pathogenic fungi and bacteria.

[0017] Further, the pathogenic fungi and bacteria are any one or more of the following: (1) Candida albicans; (2) Candida parapsilosis; (3) Candida tropicalis; (4) Candida glabrata; (5) Aspergillus fumigatus; (6) Cryptococcus neoformans; (7) Mucor; (8) Klebsiella pneumoniae; (9) Pseudomonas aeruginosa; (10) Escherichia coli; (11) Staphylococcus aureus; (12) Group A Streptococcus; (13) Streptococcus agalactiae; (14) Enterococcus faecium.

[0018] Compared with the prior art, the present invention has the following technical effects:

[0019] 1. Advantages of the present invention: (1) The extraction reagent has a simple composition. The product and method are highly efficient, easy to operate, and low in cost. Nucleic acid extraction can be completed without excessive special instruments, and it is not prone to cross-contamination caused by frequent opening of the lid. The operation steps are few and the time is short. (2) Different extraction methods are compatible with different nucleic acid adsorption materials, and it can be applied to different downstream nucleic acid purification and detection methods, showing good compatibility. (3) Versatile: Different grinding methods and extraction methods can be combined according to different usage scenarios; it is applicable to the nucleic acid extraction of various pathogenic fungi and bacteria; it is applicable to various clinical specimens, including blood, sputum, bronchoalveolar lavage fluid, cerebrospinal fluid, and vaginal swabs. (3) Good safety, using common salt reagents without substances that are harmful to operators such as liquid nitrogen, chloroform, formamide, potassium perchlorate, and mercaptoethanol. (4) Good economy, low cost, no need to use tool enzymes for treatment, and no need to use expensive auxiliary equipment. (5) High nucleic acid yield (concentration), high extraction purity and concentration, and good integrity, which is beneficial to improving the sensitivity of downstream molecular tests after nucleic acid extraction.

[0020] 2. Aiming at the performance defects of the existing technical methods, the present invention first optimizes the operation procedure by reducing cumbersome steps such as tube transfer and incubation, combines the structural characteristics of microorganisms, uses glass beads of different particle sizes, and achieves the crushing and lysis of cells from the inside out through mechanical force. Secondly, a defoaming agent with a certain concentration is added to the grinding environment to inhibit the generation of bubbles and reduce the influence of bubbles on the grinding of glass beads. Furthermore, the use of unstable enzyme substances is reduced to reduce the incubation time and also lower the reagent cost. Finally, a general-purpose extraction and purification reagent is optimized according to different nucleic acid adsorption materials, improving the nucleic acid yield (concentration) and integrity, greatly enhancing the sensitivity of detection, and laying a foundation for achieving a higher degree of automation. That is, the present invention effectively solves the problems of complex operation, high cost, long time consumption, low nucleic acid yield (concentration), and poor integrity existing in the current nucleic acid extraction reagents for fungi. At the same time, it is also applicable to the nucleic acid extraction of three pathogens, namely pathogenic fungi and bacteria; it is applicable to a variety of different types of samples, etc. Description of the Drawings

[0021] Figure 1 Electrophoresis diagrams of different grinding times in Example 3, M: DL2000 Maker; 1 - 5: Candida albicans; 6 - 10: Aspergillus fumigatus; 11 - 15: Cryptococcus neoformans; 16 - 20: Escherichia coli; 21 - 25: Staphylococcus aureus; among which the grinding times are 30, 15, 10, 8, and 5 minutes in sequence.

[0022] Figure 2Electrophoresis diagram for comparing the grinding effects of two grinders in Example 4. M: DL2000 Maker; 1-9: vortex oscillation grinding; 10-18: tissue grinder grinding; among which 1-9 and 10-18 are Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Aspergillus fumigatus, Mucor, Cryptococcus neoformans, Escherichia coli, and Staphylococcus aureus in sequence.

[0023] Figure 3 Another electrophoresis diagram for comparing the grinding effects of two grinders in Example 4. M: DL2000 Maker; 1-5: vortex oscillation grinding; 6-10: tissue grinder grinding; among which 1-5 and 6-10 are Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus group A, Streptococcus agalactiae, and Enterococcus faecium in sequence.

[0024] Figure 4 Electrophoresis diagram for comparing the extraction effects of magnetic beads and nucleic acid adsorption columns in Example 5. M: DL2000 Maker; 1-7: nucleic acid adsorption column extraction; 8-14: magnetic bead extraction; among which 1-7 and 8-14 are Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Aspergillus fumigatus, Mucor, Cryptococcus neoformans, Escherichia coli, and Staphylococcus aureus in sequence.

[0025] Figure 5 Another electrophoresis diagram for comparing the extraction effects of magnetic beads and nucleic acid adsorption columns in Example 5. M: DL2000 Maker; 1-5: nucleic acid adsorption column extraction; 6-10: magnetic bead extraction; among which 1-5 and 8-10 are Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus group A, Streptococcus agalactiae, and Enterococcus faecium in sequence.

[0026] Figure 6 Results of nucleic acid integrity detection in Example 6. M: DL2000 Maker; 1-3: Candida albicans; 4-6: Candida parapsilosis; 7-9: Candida glabrata; 10-12: Candida tropicalis; 13-15: Aspergillus fumigatus; 16-18: Mucor; 19-21: Cryptococcus neoformans; 22-24: Escherichia coli; 25-27: Staphylococcus aureus; 28-30: Klebsiella pneumoniae; 31-33: Pseudomonas aeruginosa; 34-36: Enterococcus faecium; 37-39: Streptococcus group A; 40-42: Streptococcus agalactiae.

[0027] Figure 7 Electrophoresis diagram for comparing the extraction effects of the present invention and a comparative reagent in Comparative Example 1. M: DL2000 Maker; 1-7: comparative reagent; 8-14: reagent of the present invention; among which 1-7 and 8-14 are Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Aspergillus fumigatus, Mucor, Cryptococcus neoformans, Escherichia coli, and Staphylococcus aureus in sequence.

[0028] Figure 8 Another comparative electrophoresis diagram of the extraction effects of the present invention and the comparative reagent in Comparative Example 1. M: DL2000 Maker; 1-5: Comparative reagent; 6-10: Reagent of the present invention. Among them, 1-5 and 6-10 are Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus group A, Streptococcus agalactiae, and Enterococcus faecium in sequence. Detailed implementation manners

[0029] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement made to the methods, steps or conditions of the present invention shall fall within the scope of the present invention. The reagents, reagent kits and instruments used in the following examples can all be obtained commercially. Unless otherwise specified, the methods used in the examples are the same as those commonly used.

[0030] The reagents used in the following examples are: (1) SDS (Sangon Biotech (Shanghai) Co., Ltd., A600485-0500); (2) Triton X-100 (Sangon Biotech (Shanghai) Co., Ltd., A600198-0500); (3) Tris-HCl (pH = 8.0) (Sangon Biotech (Shanghai) Co., Ltd., B548127-0500); (4) guanidine isothiocyanate (Sangon Biotech (Shanghai) Co., Ltd., A610244-0500); (5) sodium chloride (Sangon Biotech (Shanghai) Co., Ltd., A610476-0001); (6) sodium citrate (Sangon Biotech (Shanghai) Co., Ltd., A610035-0500); (7) guanidine hydrochloride (Sangon Biotech (Shanghai) Co., Ltd., A610242-0500); (8) EDTA (Sangon Biotech (Shanghai) Co., Ltd., A610185-0500); (9) non-ionic T-F composite antifoaming agent for fermentation (Sangon Biotech (Shanghai) Co., Ltd., A504007-0500); (10) nucleic acid adsorption column (Sangon Biotech (Shanghai) Co., Ltd., B615005-0100); (11) carboxyl magnetic microspheres (Suzhou Weidu Biotechnology Co., Ltd., MS05HC-3); (12) 200 μm acid-washed glass beads (Sigma-Aldrich, G1277); (13) 500 μm acid-washed glass beads (Sigma-Aldrich, G8772); (14) Qiagen QIAamp UCP Pathogen Mini Kit (Qiagen, 50214) and the supporting reagent Qiagen Pathogen Lysis Tubes L (Qiagen, 19092); (15) TAKARA Probe qPCR Mix, with UNG (TAKARA, RR392); (16) agarose (BIOWEST AGAROSE, BY-R0100); (17) TAE buffer (Sangon Biotech (Shanghai) Co., Ltd., B548101-0500); (18) PBS buffer (Beijing Solarbio Science & Technology Co., Ltd., P1020).

[0031] The instruments used in the following examples are: (1) Refrigerated high-speed centrifuge (Eppendorf 5424R); (2) Vortex shaker (Vortex-Genie 2); (3) Tissue grinder (Qiagen TissueLyser II); (4) Magnetic stand (Invitrogen DynaMag-2); (5) Micro-spectrophotometer (Thermo Scientific NanoDrop One); (6) Nucleic acid quantifier (Invitrogen Qubit 4); (7) Electrophoresis power supply (Bio-Red PowerPac basical); (8) Horizontal agarose electrophoresis apparatus (Beijing Liuyi DYCP-31DN); (8) Gel imaging system (Bio-Red ChemiDoc XRS+); (9) Real-time fluorescence quantitative PCR instrument (Roche LightCycler 480II); (10) Dry bath (Labnet Dry Bath, D1302-230V).

[0032] The strains used in the following examples are: (1) Candida albicans; (2) Candida parapsilosis; (3) Candida tropicalis; (4) Candida glabrata / Nakaseomyces glabrate; (5) Aspergillus fumigatus; (6) Cryptococcus neoformans; (7) Mucorales; (8) Klebsiella pneumoniae; (9) Pseudomonas aeruginosa; (10) Escherichia coli; (11) Staphylococcus aureus; (12) Streptococcus pyogenes group A; (13) Streptococcus agalactiae; (14) Enterococcus faecium. Standardization of culture concentration: The absorbance A600 value of various bacterial and fungal cultures is 2 when detected at a wavelength of 600 nm in visible light.

[0033] (W / V) used in the following examples is the mass / volume (1 g / 1 mL * 100% = 100%) percentage, and (V / V) is the volume percentage.

[0034] The technical solution of the present invention will be further elaborated in detail below in conjunction with embodiments.

[0035] Example 1: Explore the optimal concentration of defoamer to improve nucleic acid extraction efficiency

[0036] During the grinding and shaking process, the generated bubbles affect the grinding effect. To eliminate and inhibit the generation of bubbles, an appropriate amount of defoamer is added to improve nucleic acid extraction efficiency.

[0037] 1. Samples used: Candida albicans suspension.

[0038] 2. Reagent preparation: (1) Lysis buffer I: 0.5% (W / V) SDS, 1% (V / V) Triton X-100, 20 mM Tris-HCl (pH = 8.0). (2) Lysis buffer II: 5 M guanidine isothiocyanate, 20 mM sodium chloride, 20 mM sodium citrate, 20 mM Tris-HCl (pH = 8.0). (3) Wash buffer I: 2 M guanidine hydrochloride, 0.5% (V / V) Triton X-100, 20 mM Tris-HCl (pH = 8.0), 40% (V / V) ethanol. (4) Wash buffer II: 20 mM sodium chloride, 20 mM Tris-HCl (pH = 8.0), 70% (V / V) ethanol. (5) Elution buffer: 1 mM EDTA, 10 mM Tris-HCl (pH = 8.0). (6) Grinding tube: 2.0 mL EP tube containing 200 μg acid-washed glass beads with a ratio of 200 μm:500 μm of 1:1. (7) Nucleic acid adsorption material: Nucleic acid adsorption column. (8) Defoamer: Non-ionic T-F composite fermentation defoamer, silicone defoamer.

[0039] 3. Extraction steps:

[0040] (1) Add defoamer to lysis solution I and PBS buffer respectively to prepare treatment solutions with final defoamer concentrations of 0%, 1%, and 2% (V / V). (2) Add 200 μL of the suspension of Candida albicans culture to the grinding tube, and then add 200 μL of lysis solution I and PBS buffer with defoamer concentrations of 0%, 1%, and 2% (V / V). (3) Place the grinding tube in a vortex shaker (Vortex - Genie 2) and vortex - grind at the highest speed (10th gear, 3200 RPM) for 15 minutes. (4) Centrifuge instantaneously, add 200 μL of lysis solution II and 400 μL of isopropanol, mix well, and centrifuge instantaneously. (5) Carefully aspirate 700 μL of the supernatant and place it in a new nucleic acid adsorption column, centrifuge at 12000g for 1 minute, and discard the waste liquid. (6) Add 500 μL of washing solution I to the nucleic acid adsorption column, centrifuge at 12000g for 1 minute, and discard the waste liquid. (7) Add 500 μL of washing solution II to the nucleic acid adsorption column, centrifuge at 12000g for 1 minute, and discard the waste liquid. (8) Add 500 μL of washing solution II to the nucleic acid adsorption column, centrifuge at the highest speed for 3 minutes, and discard the waste liquid. (9) Transfer the nucleic acid adsorption column to a clean 1.5 mL EP tube, add 100 μL of elution solution to the nucleic acid adsorption column, let it stand at room temperature for 3 minutes, centrifuge at 6000g for 1 minute, and use the eluted sample as the sample to be tested for standby.

[0041] 4. Nucleic acid analysis:

[0042] (1) Purity analysis: Use Nanodrop One to detect the eluted sample to be tested and record the OD260 / OD280 ratio. (2) Concentration analysis: Use Qubit HS DNA Assay Kit to measure the concentration of the eluted sample to be tested and record the sample concentration.

[0043] 5. Evaluation criteria:

[0044] (1) Purity: When the OD260 / OD280 ratio is between 1.6 and 2.2, it proves that the nucleic acid purity meets the requirements. (2) Concentration: The higher the concentration, the higher the nucleic acid yield, indicating that the method is better.

[0045] 6. The results are shown in Table 1.

[0046] Table 1 Nucleic acid extraction results with different concentrations of defoamer

[0047]

[0048] 7. Conclusion

[0049] When comparing the addition of different defoamers, for defoamers with the same addition amount, the nucleic acid extraction concentration of the non-ionic T-F composite fermentation defoamer is higher than that of the silicone defoamer. Therefore, the non-ionic T-F composite fermentation defoamer is used as the defoamer in the present invention.

[0050] In the case of not using a defoamer, due to a large number of bubbles generated by violent shaking, it affects the grinding of the glass beads on the surface of the bacteria, and the extraction effect of the experimental group without adding a defoamer is poor compared with the experimental group adding a defoamer.

[0051] When comparing the treatment solutions with the same defoamer concentration of 1% (V / V), the extraction effect of PBS as the treatment solution is higher than that of Lysis Buffer I. Exactly because during the grinding process, the surfactant in Lysis Buffer I will generate a large number of bubbles, affecting the grinding and cell wall breaking effect of fungi. Therefore, adding a defoamer can improve the grinding effect.

[0052] When comparing the treatment solutions with the same defoamer concentration of 2% (V / V), whether it is PBS or Lysis Buffer I, the nucleic acid concentration obtained by extraction has been significantly improved. However, when comparing the two treatment solutions, the nucleic acid concentration obtained from the same concentration of samples by Lysis Buffer I is higher.

[0053] In summary, under the action of the high-frequency mechanical force of oscillatory grinding, the bubbles generated by PBS are sufficient to affect the effect of glass bead grinding and cell wall breaking. Moreover, in the environment of Lysis Buffer I containing a surfactant, but facing complex sample situations, the addition of a surfactant is beneficial for sample treatment. At this time, adding a certain amount of defoamer can not only retain the addition of the surfactant, solve the sample treatment problem, but also inhibit the bubbles generated by the viscosity of the liquid itself, avoiding affecting the grinding effect; combined with the results explored in Comparative Example 1, the defoamer concentration of Lysis Buffer I is 2% (V / V) as the optimal defoamer concentration.

[0054] Example 2: Explore the best glass bead ratio to improve the grinding efficiency

[0055] During oscillatory grinding, the particle size affects the grinding efficiency, and matching glass beads with different particle sizes is beneficial for treating a wider spectrum of bacteria and fungi.

[0056] 1. Samples used: Suspensions of Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, Escherichia coli, and Staphylococcus aureus cultures.

[0057] 2. Reagent preparation:

[0058] Lysis Buffer I: 0.5% (W / V) SDS, 1% (V / V) Triton X-100, 20 mM Tris-HCl (pH = 8.0), 2% (V / V) defoamer. Lysis Buffer II, Wash Buffer I, Wash Buffer II, Elution Buffer, and nucleic acid adsorption material are the same as in Example 1.

[0059] 3. Extraction steps:

[0060] (1) The method for preparing grinding tubes (200 mg / tube) is shown in Table 2.

[0061] Table 2 Configuration methods of pickled glass beads with different particle sizes in different grinding tubes

[0062]

[0063]

[0064] (2) Add 200 μL of suspension of Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, Escherichia coli, and Staphylococcus aureus cultures to the grinding tubes respectively. Other steps are the same as in Example 1.

[0065] 4. Nucleic acid analysis is the same as in Example 1.

[0066] 5. The evaluation criteria are the same as in Example 1.

[0067] 6. The purity results are shown in Table 3, and the concentration results are shown in Table 4, unit: ng / μL.

[0068] Table 3 Purity results of different configuration methods of pickled glass beads

[0069]

[0070] Table 4 Concentration results of different configuration methods of pickled glass beads

[0071]

[0072] 7. Conclusion

[0073] As the proportion of large - sized glass beads (500 μm) increases continuously, the nucleic acid extraction concentration of fungal samples also increases. When it reaches 100% of 500 - μm glass beads, the nucleic acid concentration of fungal samples is the highest in this example, which proves that large - sized glass beads have a good effect on grinding fungi. On the contrary, for bacteria, as the proportion of large - sized glass beads (500 μm) increases continuously, the nucleic acid concentration of bacterial samples is continuously decreasing. When it reaches 100% of 500 - μm glass beads, the nucleic acid concentration of bacterial samples is the lowest in this example, which proves that small - sized glass beads have a better grinding effect on bacteria.

[0074] Analyzing the increasing and decreasing trends of nucleic acid concentration, the increasing trend of nucleic acid concentration in fungi is relatively large, but when it reaches a ratio of 1:3, the increasing trend slows down. While for bacteria, due to the relatively simple cell wall structure, the decreasing trend is relatively small and tends to be stable. Considering the nucleic acid extraction concentrations of pathogenic fungi and bacteria comprehensively, the mixed glass beads with a particle size ratio of 200 μm:500 μm of 1:3 are used as the general glass bead matching formula of the present invention.

[0075] Example 3: Explore the optimal grinding time for maintaining nucleic acid integrity

[0076] The grinding time has a great impact on the degree of cell wall disruption by glass beads. The longer the time, the better the cell wall disruption. However, when the highest grinding degree is reached, continued grinding will damage the integrity of nucleic acids. Therefore, an optimal grinding time needs to be found.

[0077] 1. Samples used: Suspensions of Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, Escherichia coli, and Staphylococcus aureus cultures.

[0078] 2. Reagent preparation:

[0079] Lysis buffer I: 0.5% (W / V) SDS, 1% (V / V) Triton X-100, 20 mM Tris-HCl (pH = 8.0), 2% (V / V) antifoaming agent. Grinding tube: A 2.0 mL Eppendorf tube containing 200 μg of acid-washed glass beads with a ratio of 200 μm:500 μm of 1:3. Lysis buffer II, Wash buffer I, Wash buffer II, Elution buffer, and nucleic acid adsorption material are the same as in Example 1.

[0080] 3. Extraction steps:

[0081] (1) Add 200 μL of the suspensions of Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, Escherichia coli, and Staphylococcus aureus cultures to the grinding tubes respectively. (2) Place the grinding tubes in a vortex shaker (Vortex-Genie 2) and vortex-grind at the highest speed (10th gear, 3200 RPM) for 5 minutes, 8 minutes, 10 minutes, 15 minutes, and 30 minutes. Other steps are the same as in Example 1.

[0082] 4. Nucleic acid analysis:

[0083] (1) The purity analysis method is the same as in Example 1. (2) The concentration analysis method is the same as in Example 1. (3) Integrity analysis: Use 1% agarose gel to perform electrophoresis detection and analysis on the elution samples to be tested, and observe the electrophoresis band situation of nucleic acids.

[0084] 5. Evaluation criteria:

[0085] (1) The purity evaluation criteria are the same as in Example 1. (2) The concentration evaluation criteria are the same as in Example 1. (3) Integrity: The electrophoresis nucleic acid bands are clearly visible, without obvious diffused bands, and those with clear genomic bands and ribosomal RNA bands are excellent.

[0086] 6. The purity results are shown in Table 5, the concentration results are shown in Table 6, unit: ng / μL, and the integrity results are as Figure 1 shown.

[0087] Table 5 Purity Results at Different Grinding Times

[0088] Strain 5 minutes 8 minutes 10 minutes 15 minutes 30 minutes Candida albicans 1.90 1.79 2.10 2.05 2.10 Aspergillus fumigatus 1.86 1.75 2.11 1.96 2.07 Cryptococcus neoformans 1.95 1.75 2.08 2.01 1.84 Escherichia coli 1.93 1.69 1.98 1.96 1.86 Staphylococcus aureus 1.86 1.69 1.92 1.79 1.71

[0089] Table 6 Concentration Results at Different Grinding Times

[0090]

[0091]

[0092] 7. Conclusion

[0093] From the perspective of extraction purity, there is no difference in extraction purity with time, which proves that the nucleic acid extraction reagent of the present invention has high stability in purification.

[0094] From the perspective of the yield (concentration) of extracted nucleic acid, with the increase of time, the yield (concentration) of nucleic acid gradually increases, and the yield (concentration) of nucleic acid is the highest at 30 minutes. However, by analyzing the 15-minute and 30-minute periods, it can be seen that at 30 minutes of grinding time, the yield (concentration) of nucleic acid does not increase multiplicatively, which proves that in the grinding time range from 15 minutes to 30 minutes, it has almost reached the plateau of the grinding degree; from the integrity analysis, it can be seen that there is no obvious difference in the brightness of the nucleic acid bands ground for 30 minutes and those ground for 15 minutes, and the integrity is very good, with obvious and single genomic and ribosomal RNA bands.

[0095] Based on the above results, the present invention aims to save the cumbersome and long enzyme treatment time through sufficient grinding, thereby simplifying the operation and shortening the extraction time. Exploring the grinding time with the least time and the best effect through experiments is one of the key steps of this goal. Considering the concentration, purity and efficiency of nucleic acid extraction from pathogenic fungi and bacteria, 15 minutes of grinding time is taken as the optimal grinding time of the present invention.

[0096] Example 4: Comparison of the Effects of Nucleic Acid Extraction by a Tissue Grinder and a Vortex Shaker

[0097] The vortex shaker is an essential homogenization auxiliary instrument in today's clinical departments. The present invention mainly realizes the lysis of the fungal cell wall by the mechanical force provided by the vortex shaker. The tissue grinder is a new generation of homogenization grinding auxiliary instrument that can achieve stable and high-throughput homogenization treatment effects. Example 4 will compare the differences in the effects of nucleic acid extraction between grinding with a tissue grinder and grinding with a vortex shaker.

[0098] 1. Samples Used: Suspensions of cultures of Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Aspergillus fumigatus, Cryptococcus neoformans, Mucor, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Group A Streptococcus, Streptococcus agalactiae, Enterococcus faecium

[0099] 2. Reagent preparation:

[0100] Lysis buffer I: 0.5% (W / V) SDS, 1% (V / V) Triton X-100, 20 mM Tris-HCl (pH = 8.0), 2% (V / V) antifoaming agent. Grinding tube: A 2.0 mL Eppendorf tube containing 200 μg of acid-washed glass beads with a ratio of 200 μm:500 μm of 1:3. Lysis buffer II, Wash buffer I, Wash buffer II, Elution buffer, and nucleic acid adsorption material are the same as in Example 1.

[0101] 3. Extraction steps:

[0102] (1) Add 200 μL of suspension of Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Aspergillus fumigatus, Cryptococcus neoformans, Mucor, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus group A, Streptococcus agalactiae, and Enterococcus faecalis cultures into the grinding tubes respectively, with 2 tubes for each sample. (2) Place one grinding tube in a Vortex-Genie 2 and vortex-grind at the highest rotation speed (10th gear, 3200 RPM) for 15 minutes, and place the other grinding tube in a Qiagen Tissuelyser II and homogenize at the highest rotation speed of 30 Hz for 15 minutes. Other steps are the same as in Example 1.

[0103] 4. Nucleic acid analysis:

[0104] (1) The purity analysis method is the same as in Example 1. (2) The concentration analysis method is the same as in Example 1. (3) Integrity analysis: Use a 1% agarose gel to perform electrophoresis detection and analysis on the elution sample to be tested, and observe the electrophoresis band situation of the nucleic acid.

[0105] 5. Evaluation criteria:

[0106] (1) The purity evaluation criteria are the same as in Example 1. (2) The concentration evaluation criteria are the same as in Example 1. (3) Integrity evaluation: The electrophoresis nucleic acid bands are clearly visible, without obvious diffused bands, and those with clear genomic bands and ribosomal RNA bands are excellent. (4) Time: When the nucleic acid integrity meets the requirements and the nucleic acid concentrations differ by less than 10%, the shorter the time, the higher the efficiency.

[0107] 6. The purity results are shown in Table 7, the concentration results are shown in Table 8, unit: ng / μL, and the integrity results are as Figures 2 to 3 shown.

[0108] Table 7 Purity results of different grinding instruments

[0109] Strain Vortex shaker Tissue grinder Candida albicans 2.01 2.09 Candida parapsilosis 2.03 2.08 Candida glabrata 2.19 2.11 Candida tropicalis 2.14 2.11 Aspergillus fumigatus 1.91 2.07 Cryptococcus neoformans 1.95 2.15 Mucor 1.99 2.15 Escherichia coli 1.90 1.84 Klebsiella pneumoniae 1.84 1.83 Pseudomonas aeruginosa 2.04 2.06 Staphylococcus aureus 1.84 1.74 Group A Streptococcus 2.16 2.20 Streptococcus agalactiae 2.11 2.14 Enterococcus faecium 1.99 1.92

[0110] Table 8 Concentration results of different grinding instruments

[0111]

[0112]

[0113] 7. Conclusions

[0114] By analyzing the purity, concentration, and integrity results of the extracts obtained using a vortex shaker and a tissue grinder, it was found that the OD260 / OD280 ratios of the nucleic acids of all pathogenic fungi and bacteria were between 1.6 and 2.2, and the purity met the requirements. In terms of concentration and integrity, there was no significant difference in the grinding effects between the two instruments. There were slight differences in height among different samples, but the results were not significantly different, indicating that both different grinding methods could achieve the grinding and cell wall breaking work of the present invention well.

[0115] Qiagen Tissuelyser II is an instrument that oscillates horizontally back and forth, while the vortex shaker can achieve vertical or horizontal vortex oscillation according to different modules. In this example, vertical and horizontal oscillation modules were used, and under sufficient oscillation intensity, cell wall breaking of microorganisms could be achieved. Moreover, on the premise that the vortex shaker selects a module with a bayonet or fixing device, both instruments can achieve automation, thereby increasing the extraction efficiency and reducing errors caused by operations among different technicians.

[0116] Example 5: Comparison of the Effects of Magnetic Beads and Nucleic Acid Adsorption Columns in Nucleic Acid Extraction

[0117] Example 5 mainly compared the differences in the effects of nucleic acid adsorption columns and carboxyl magnetic microspheres in nucleic acid extraction.

[0118] 1. Samples used: Suspensions of cultures of Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Aspergillus fumigatus, Cryptococcus neoformans, Mucor, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Group A Streptococcus, Streptococcus agalactiae, and Enterococcus faecium.

[0119] 2. Reagent preparation:

[0120] Lysis buffer I: 0.5% (W / V) SDS, 1% (V / V) Triton X-100, 20 mM Tris-HCl (pH = 8.0), 2% (V / V) antifoaming agent. Grinding tube: A 2.0 mL Eppendorf tube containing 200 μg of acid-washed glass beads with a ratio of 200 μm:500 μm of 1:3. Nucleic acid adsorption materials: Nucleic acid adsorption column, hydroxyl magnetic beads. Lysis buffer II, Wash buffer I, Wash buffer II, and Elution buffer were the same as in Example 1.

[0121] 3. Extraction steps:

[0122] (1) Add 200 μL of the culture suspension of Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Aspergillus fumigatus, Cryptococcus neoformans, Mucor, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus group A, Streptococcus agalactiae, and Enterococcus faecium into the grinding tube, with 2 tubes for each sample. The other steps are the same as in Example 1.

[0123] 3.1. The operation steps of the nucleic acid adsorption column are the same as in Example 1.

[0124] 3.2. The operation steps of the magnetic beads:

[0125] (1) Add 20 μL of magnetic beads into the EP tube containing the sample, mix well, let stand for 2 minutes, then place it in the magnetic rack and aspirate all the liquid. (2) Add 500 μL of Wash Buffer I into the EP tube, gently pipette to mix evenly, place it in the magnetic rack and aspirate all the liquid. (3) Add 500 μL of Wash Buffer II into the EP tube, gently pipette to mix evenly, place it in the magnetic rack and aspirate all the liquid. (4) Add 500 μL of Wash Buffer II into the EP tube, gently pipette to mix evenly, place it in the magnetic rack and aspirate all the liquid. (5) Keep the EP tube in the magnetic rack, open the tube cap and dry at room temperature for 5 minutes. (6) Remove the EP tube, use a pipette to aspirate 100 μL of elution buffer, wash down the magnetic beads adhering to the tube wall and pipette to mix evenly, let stand at room temperature for 3 minutes, then place it in the magnetic rack. After the magnetic beads are completely adhered to the wall, transfer all the supernatant to a new 1.5 mL EP tube, and the eluted sample is used as the test sample for standby.

[0126] 4. Nucleic acid analysis:

[0127] (1) The purity analysis method is the same as in Example 1. (2) The concentration analysis method is the same as in Example 1. (3) Integrity analysis: Use 1% agarose gel to perform electrophoresis detection and analysis on the test eluted sample, and observe the electrophoresis band situation of the nucleic acid.

[0128] 5. Evaluation criteria:

[0129] (1) The purity evaluation criteria are the same as in Example 1. (2) The concentration evaluation criteria are the same as in Example 1. (3) Integrity evaluation: The electrophoresis nucleic acid bands are clearly visible, without obvious diffused bands, and those with clear genomic bands and ribosomal RNA bands are excellent.

[0130] 6. The purity results are shown in Table 9, the concentration results are shown in Table 10, unit: ng / μL, and the integrity results are as Figures 4 to 5 shown.

[0131] Table 9 Purity Results of Different Adsorption Materials

[0132]

[0133]

[0134] Table 10 Concentration Results of Different Adsorption Materials

[0135] Strain Nucleic acid adsorption column Magnetic beads Candida albicans 33.94 37.06 Candida parapsilosis 41.93 45.23 Candida glabrata 63.72 69.76 Candida tropicalis 54.58 58.30 Aspergillus fumigatus 36.20 39.39 Cryptococcus neoformans 46.57 50.05 Mucor 39.57 42.86 Escherichia coli 27.58 30.27 Klebsiella pneumoniae 27.28 27.49 Pseudomonas aeruginosa 30.44 29.25 Staphylococcus aureus 38.46 40.56 Group A Streptococcus 29.73 30.39 Streptococcus agalactiae 48.85 51.35 Enterococcus faecium 26.96 24.58

[0136] 7. Conclusions

[0137] From the purity results, for both the adsorption column and magnetic bead methods, the OD260 / OD280 ratios of all pathogenic fungal and bacterial nucleic acids are between 1.6 and 2.2, and there is no obvious difference between the two nucleic acid adsorption materials; in terms of concentration analysis, the nucleic acids purified by magnetic beads are all higher than those by the nucleic acid adsorption column, but it is not a doubling relationship, with an increase of 5% - 10%; from the integrity results, both the nucleic acid adsorption column and magnetic beads can obtain single genomes and ribosomal RNAs, meeting the requirements.

[0138] The concentration of nucleic acid extracted by magnetic beads is higher than that by the nucleic acid adsorption column because the magnetic beads added in this example are 20 μL, a relatively high amount, so the limit of nucleic acid adsorption capacity will increase. The disadvantage is that there is impurity residue and multiple washes are required to ensure high purity. However, there is no qualitative difference between the two. In the case of a laboratory lacking an automatic extractor or magnetic stand, the nucleic acid extraction operation of the present invention can be completed simply using a high-speed centrifuge with the nucleic acid adsorption column; while using magnetic beads as the nucleic acid adsorption material, high-throughput automatic nucleic acid extraction can be achieved, greatly increasing the efficiency.

[0139] This example compares two commonly used clinical nucleic acid adsorption materials, and the results show that both materials can well achieve the nucleic acid extraction of the present invention, providing more method choices for high-throughput and automation in clinical laboratories.

[0140] Example 6: Evaluation of the Differences in Nucleic Acid Extraction from Common Clinical Pathogenic Fungi and Bacteria

[0141] Example 6 studies the purity, concentration, and integrity of nucleic acids extracted from common clinical pathogenic fungi and bacteria. The implementation steps are as follows:

[0142] 1. Samples used: Bacterial and fungal culture suspensions of Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Aspergillus fumigatus, Cryptococcus neoformans, Mucor, Staphylococcus aureus, Group A Streptococcus, Streptococcus agalactiae, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa.

[0143] 2. Reagent preparation:

[0144] Lysis solution Ⅰ: 0.5% (W / V) SDS, 1% (V / V) Triton X-100, 20 mM Tris-HCl (pH = 8.0), 2% (W / V) antifoaming agent. Grinding tube: A 2.0 mL EPP tube containing 200 μg acid-washed glass beads with a ratio of 200 μm:500 μm of 1:3. Lysis solution Ⅱ, washing solution Ⅰ, washing solution Ⅱ, elution solution and nucleic acid adsorption material are the same as in Example 1.

[0145] 3. Extraction steps: (1) Add 200 μL of suspension of pathogenic fungal and bacterial cultures of Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Aspergillus fumigatus, Cryptococcus neoformans, Mucor, Staphylococcus aureus, Streptococcus group A, Streptococcus agalactiae, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa into the grinding tube, and then add 200 μL of lysis solution Ⅰ thereto and mix well. Other steps are the same as in Example 1.

[0146] 4. Nucleic acid analysis

[0147] (1) The purity analysis method is the same as in Example 1. (2) The concentration analysis method is the same as in Example 1. (3) Integrity analysis: Use 1% agarose gel to perform electrophoresis detection and analysis on the elution sample to be tested, and observe the electrophoresis band situation of the nucleic acid.

[0148] 5. Evaluation criteria

[0149] (1) The purity evaluation criteria are the same as in Example 1. (2) The concentration evaluation criteria are the same as in Example 1. (3) Integrity evaluation: The electrophoresis nucleic acid bands are clearly visible, without obvious diffused bands, and those with clear genomic bands and ribosomal RNA bands are excellent.

[0150] 6. The purity results are shown in Table 11, the concentration results are shown in Table 12, unit: ng / μL, and the integrity results are as Figure 6 shown.

[0151] Table 11 Purity results

[0152]

[0153]

[0154] Table 12 Concentration results

[0155] Strain Replicate 1 Replicate 2 Replicate 3 Candida albicans 47.32 47.22 47.69 Candida parapsilosis 52.51 52.43 52.84 Candida glabrata 44.36 44.00 44.58 Candida tropicalis 50.52 50.17 50.84 Aspergillus fumigatus 46.26 46.05 46.88 Cryptococcus neoformans 51.94 51.55 51.39 Mucor 55.93 55.36 55.19 Staphylococcus aureus 58.31 58.05 58.38 Group A Streptococcus 27.19 28.50 28.49 Streptococcus agalactiae 27.58 29.03 29.29 Enterococcus faecium 50.11 50.96 50.73 Escherichia coli 49.59 49.34 49.55 Klebsiella pneumoniae 29.06 28.52 28.85 Pseudomonas aeruginosa 28.91 29.02 28.46

[0156] 7. Conclusion:

[0157] In the purity detection, the OD260 / OD280 ratios of all pathogenic fungal and bacterial nucleic acids are between 1.6 and 2.2.

[0158] As can be seen from the concentration detection, the present invention has no preference for the extraction of the above-mentioned pathogenic fungi and bacteria. The extraction concentrations of various pathogenic fungi are relatively consistent, and it also has a good extraction effect on Gram-positive bacteria with relatively thick cell walls.

[0159] In terms of integrity, clear genomic bands and some ribosomal RNA bands can be seen in 1% gel electrophoresis. The reagent of the present invention can better maintain the integrity of nucleic acids during the nucleic acid extraction process, which lays a good foundation for downstream molecular experiments.

[0160] In summary, the reagent of the present invention can efficiently and completely extract and purify common clinical pathogenic fungi and bacteria (including Gram-positive bacteria with relatively difficult nucleic acid extraction), and has no preference.

[0161] Comparative Example 1: Comparison of nucleic acid extraction effects between the present invention and a commercialized reagent

[0162] Comparative Example 1 studied the comparison of the purity, concentration and integrity of nucleic acid extraction between the present invention and the commercialized reagent Qiagen QIAamp UCP Pathogen Mini Kit.

[0163] 1. Samples used: Bacterial and fungal suspensions of Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Aspergillus fumigatus, Cryptococcus neoformans, Mucor, Staphylococcus aureus, Group A Streptococcus, Streptococcus agalactiae, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa.

[0164] 2. Preparation of the reagent of the present invention:

[0165] Lysis buffer I: 0.5% (W / V) SDS, 1% (V / V) Triton X-100, 20 mM Tris-HCl (pH = 8.0), 2% (V / V) antifoaming agent. Grinding tube: A 2.0 mL Eppendorf tube containing 200 μg of acid-washed glass beads with a ratio of 200 μm:500 μm of 1:3. Lysis buffer II, Wash buffer I, Wash buffer II, Elution buffer and nucleic acid adsorption material are the same as in Example 1.

[0166] 3. Extraction steps of the present invention:

[0167] (1) Respectively add 200 μL of the bacterial and fungal suspensions of Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Aspergillus fumigatus, Cryptococcus neoformans, Mucor, Staphylococcus aureus, Group A Streptococcus, Streptococcus agalactiae, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa into the grinding tube, and then add 200 μL of Lysis buffer I thereto and mix well. Other steps are the same as in Example 1.

[0168] 4. Extraction steps of Qiagen QIAamp UCP Pathogen Mini Kit:

[0169] (1) Add 100 μL of Reagent DX to 15 mL of Buffer ATL and mix well for later use. (2) Add 200 μL of the bacterial suspension to the Pathogen Lysis tube, then add 1.3 mL of PBS buffer, mix well, centrifuge at 14000 g for 5 minutes, and carefully remove the supernatant with a pipette. (3) Add the pre-prepared Buffer ATL to the Pathogen Lysis tube, mix well, place the Pathogen Lysis tube in a vortex shaker (Vortex-Genie 2), and vortex and grind at the highest speed (10th gear, 3200 RPM) for 10 minutes. (4) Centrifuge briefly, carefully pipette 400 μL of the supernatant, and place it in a clean 2.0 mL EP tube. (5) Add 40 μL of proteinase K to the EP tube, mix well, and incubate at 56 °C for 10 minutes. (6) Add 200 μL of Buffer APL2 to the EP tube, mix well, and incubate at 70 °C for 10 minutes. (7) Centrifuge briefly and add 300 μL of absolute ethanol, mix well. (8) Carefully pipette 600 μL of the lysed sample and place it in a new QIAamp UCP Mini spin column, centrifuge at 6000 g for 1 minute, and transfer the upper centrifugal column to a new 2 mL collection tube. (9) Repeat step (8) to adsorb all the lysed samples onto the centrifugal column. (10) Add 600 μL of Buffer APW1 to the centrifugal column, centrifuge at 6000 g for 1 minute, and transfer the upper centrifugal column to a new 2 mL collection tube. (11) Add 750 μL of Buffer APW2 to the centrifugal column, centrifuge at 20000 g for 3 minutes, and transfer the upper centrifugal column to a new 2 mL collection tube. (12) Open the lid of the centrifugal column and place the entire centrifugal column kit in a 56 °C dry bath for 3 minutes to dry the centrifugal column. (13) Transfer the centrifugal column to a clean 1.5 mL EP tube, add 100 μL of Buffer AEV to the silicon-based membrane of the centrifugal column, let it stand at room temperature for 1 minute, centrifuge at 20000 g for 1 minute, and elute the sample for use as the sample to be tested.

[0170] 5. Comparative determination

[0171] (1) The purity determination method is the same as that in Example 1. (2) The concentration determination method is the same as that in Example 1. (3) Integrity analysis: Use 1% agarose gel electrophoresis to perform electrophoresis analysis on the eluted sample to be tested and observe the band situation.

[0172] 6. Judgment criteria

[0173] (1) The purity determination standard is the same as that in Example 1. (2) The concentration determination standard is the same as that in Example 1. (3) Integrity determination: The nucleic acid bands in electrophoresis are clearly visible, without obvious diffused bands. It is better if clear genomic bands and ribosomal RNA bands can be seen.

[0174] 7. The purity results are shown in Table 13, the concentration results are shown in Table 14, unit: ng / μL, and the integrity results are as Figures 7 to 8 shown.

[0175] Table 13 Nucleic acid extraction concentration results of the present invention and commercialized reagents

[0176] Strain Comparison reagent The present invention Candida albicans 2.11 2.02 Candida parapsilosis 2.09 2.02 Candida glabrata 2.10 2.07 Candida tropicalis 2.10 2.04 Aspergillus fumigatus 2.11 2.01 Cryptococcus neoformans 2.11 2.04 Mucor 2.11 2.04 Staphylococcus aureus 2.04 1.97 Group A Streptococcus 2.20 2.13 Streptococcus agalactiae 2.13 2.10 Enterococcus faecium 1.93 1.93 Escherichia coli 2.08 1.98 Klebsiella pneumoniae 1.75 1.82 Pseudomonas aeruginosa 2.04 2.02

[0177] Table 14 Nucleic acid extraction purity results of the present invention and commercialized reagents

[0178]

[0179]

[0180] 8. Conclusion

[0181] Analyzing the results, the OD260 / OD280 ratios of the nucleic acids extracted by the comparative reagent and the reagent of the present invention are both between 1.6 and 2.2, and the purities all meet the requirements; in terms of concentration (yield), the reagent of the present invention is better than the comparative reagent, with a higher extraction concentration and an average yield increase of up to 20%; in terms of integrity, there is no obvious difference between the comparative reagent and the reagent of the present invention, and both have good integrity, which can be seen from the band depths in the electrophoresis diagram.

[0182] The comparative reagent is a benchmark in the nucleic acid extraction industry and can show excellent levels in terms of nucleic acid extraction purity, concentration, and integrity. However, its extraction operation is relatively traditional, with cumbersome procedures, long time consumption, and it only comes with nucleic acid adsorption column purification materials, and can only use a centrifuge to complete the nucleic acid extraction operation, with poor compatibility with nucleic acid extraction methods and difficulty in achieving automated extraction. The above defects make it not conducive to clinical diagnosis. In contrast, the reagent of the present invention is easy to operate, time-consuming, and low-cost, and is applicable to a variety of grinding and extraction purification methods. To solve the defects of the comparative reagent, we have invented an extraction reagent suitable for clinical diagnosis.

[0183] Comparative Example 2: Evaluation of sample applicability of the present invention and commercialized reagents

[0184] Comparative Example 2 studied the applicability of the reagent of the present invention and the commercialized reagent Qiagen QIAamp UCP Pathogen Mini Kit for different clinical samples. The commonly detected clinical samples include: blood, sputum, nasopharyngeal swab, bronchoalveolar lavage fluid, cerebrospinal fluid, vaginal swab.

[0185] 1. Sample preparation

[0186] (1) Blood: 4 cases of anticoagulated blood were taken, and Candida albicans, Candida parapsilosis, Candida glabrata, and Candida tropicalis were added respectively to make their final concentration 1×10 6 CFU / mL. After mixing evenly, red blood cell lysate was added. After lysing red blood cells, centrifugation was carried out, and then physiological saline was added for resuspension for standby. (2) Sputum: 5 cases, the pathogenic microorganisms were Aspergillus fumigatus, Candida albicans, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Group A Streptococcus respectively. Equal volume of sputum digestive juice was added for homogenization for standby. (3) Bronchoalveolar lavage fluid: 2 cases, the pathogenic microorganisms were Aspergillus fumigatus and Candida albicans respectively. (4) Cerebrospinal fluid: 1 case, the pathogenic microorganism was positive for Cryptococcus neoformans. (5) Vaginal swab: 3 cases, the pathogenic microorganisms were Candida albicans, Candida glabrata, and Streptococcus agalactiae respectively. The swab was soaked in 1 mL of physiological saline and shaken evenly for standby.

[0187] 2. Preparation of the reagent of the present invention:

[0188] Lysis buffer I: 0.5% (W / V) SDS, 1% (V / V) Triton X-100, 20 mM Tris-HCl (pH = 8.0), 2% (V / V) antifoaming agent. Grinding tube: A 2.0 mL Eppendorf tube containing 200 μg of acid-washed glass beads with a ratio of 200 μm:500 μm of 1:3. Lysis buffer II, washing buffer I, washing buffer II, elution buffer, and nucleic acid adsorption material were the same as in Example 1.

[0189] 3. Extraction steps of the present invention:

[0190] (1) 200 μL of the sample was added to the grinding tube respectively, and then 200 μL of lysis buffer I was added and mixed evenly. Other steps were the same as in Example 1.

[0191] 4. The extraction steps of Qiagen QIAamp UCP Pathogen Mini Kit were the same as in Comparative Example 1.

[0192] 5. The nucleic acid was measured by fluorescence quantitative PCR, and the specific primer sequences are shown in Table 15.

[0193] Table 15 Primer details

[0194]

[0195]

[0196] Prepare the reaction system using TAKARA Probe qPCR Mix with UNG reagent. The PCR reaction system is shown in Table 16 below.

[0197] Table 16 PCR Reaction System Formulation

[0198] Reagent Dosage Final concentration ProbeqPCRMix, with UNG (2×) 10 μL 1× Forward primer (10 μM) 0.5 μL 0.25 μM Reverse primer (10 μM) 0.5 μL 0.25 μM Probe (10 μM) 0.2 μL 0.1 μM Water 3.8 μL / Template 5 μL / Total volume 20 μL /

[0199] The PCR program is shown in Table 17 below.

[0200] Table 17 Specific PCR Program

[0201]

[0202] Perform PCR detection using Roche LightCycler 480.

[0203] 6. Judgment Criteria

[0204] If Ct ≤ 37.00, the target gene is determined to be positive. Moreover, the smaller the Ct value, the higher the copy number of the corresponding sample, indicating a higher nucleic acid yield (concentration) of the extracted nucleic acid.

[0205] 7. The results are shown in Table 18.

[0206] Table 18 Judgment Results of the Invention and Commercialized Reagents

[0207]

[0208]

[0209] 8. Conclusion

[0210] Nucleic acid extraction from clinical samples is a key upstream step in clinical nucleic acid detection tests, directly affecting the quality of downstream molecular experiments. In this comparative example, through fluorescence quantitative PCR experiments, simulating real usage scenarios, it can be seen from the PCR results that under the premise of the same primers, the reagent of the present invention has lower Ct values. Although it is not very obvious in strongly positive samples (Ct value < 30.00), in relatively clean samples such as cerebrospinal fluid, there are obvious differences in pathogens with lower copy numbers. It can be seen that the PCR result of Cryptococcus neoformans of the comparative reagent is 36.62, close to the detection limit, with a relatively high risk of false negatives. The PCR result of Cryptococcus neoformans of the reagent of the present invention is 33.78, showing better performance in nucleic acid extraction from clinical samples than the comparative reagent.

[0211] Comparative Example 3: "Comparison of Parameters of the Prior Art (Patent)"

[0212] Comparative Example 3 will compare the advantages and highlights of the reagent of the present invention in terms of cost, operation time consumption, and presence or absence of toxic and harmful substances by comparing the patent and existing marketed products. The detailed comparison is shown in Table 19 below.

[0213] Table 19 Comparison Results of the Present Invention with Existing Patents and Products

[0214]

[0215]

[0216] The comparative reagent is the extraction reagent of the industry benchmark brand Qiagen, but due to its high price, it is generally not used in general clinical diagnostic laboratories.

[0217] The comparative patents are all patents related to fungal nucleic acid extraction. According to the formula and extraction method described in the claims, they respectively have the defects of high cost and long time consumption; moreover, the reagent formula of one patent uses formamide, which is classified as a substance with reproductive toxicity by the European Chemicals Agency and is a toxic and harmful substance; one patent formula uses potassium perchlorate, which is easy to make explosives and is a controlled product, making it difficult to purchase; one patent culture uses 12-3-12 type Gemini quaternary ammonium salt, which is a cationic surfactant and a special raw material with scarce supply and difficult procurement; one patent formula uses mercaptoethanol, which is volatile, has an irritating odor, and is easy to cause mucosal damage and is a toxic and harmful substance.

[0218] In summary, aiming at the defects of the existing technology such as complex operation, long time consumption, use of harmful substances, and high cost, the reagent of the present invention optimizes the components, removes toxic and harmful reagents, and uses raw materials with stable physical and chemical properties and extremely easy to purchase on the market; optimizes the extraction steps, and according to the key role of the extraction steps, combines with the reagent characteristics to shorten the incubation and lysis time. Finally, a general fungal nucleic acid extraction reagent with the advantages of low cost, short time consumption, and no toxic and harmful substances is developed.

[0219] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solution of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A general nucleic acid extraction reagent for pathogenic fungi and bacteria, characterized in that, It includes Lysis Solution I, Lysis Solution II, Wash Solution I, Wash Solution II, Elution Solution, grinding tube and nucleic acid adsorption material; The Lysis Solution I contains: 0.4 - 0.6% SDS, 0.8 - 1.2% TritonX-100, 18 - 22 mM Tris-HCl and 1 - 3% defoamer; The Lysis Solution II contains: 4 - 6 M guanidine isothiocyanate, 180 - 220 mM sodium chloride, 18 - 22 mM sodium citrate and 18 - 22 mM Tris-HCl; The Wash Solution I contains: 1.8 - 2.2 M guanidine hydrochloride, 0.4 - 0.6% TritonX-100, 18 - 22 mM Tris-HCl and 30% - 50% ethanol; The Wash Solution II contains: 18 - 22 mM sodium chloride, 18 - 22 mM Tris-HCl and 60% - 80% ethanol; The Elution Solution contains: 0.8 - 1.2 mM EDTA and 8 - 12 mM Tris-HCl; The grinding tube contains: 40 - 60 g of acid-washed glass beads with a diameter of 180 - 220 μm, 140 - 160 g of acid-washed glass beads with a diameter of 480 - 520 μm and a conical bottom centrifuge tube with a lid; The nucleic acid adsorption material contains: nucleic acid adsorption column or carboxyl magnetic beads.

2. The general nucleic acid extraction reagent for pathogenic fungi and bacteria according to claim 1, wherein The pH value of the Tris-HCl is 7.5 - 8.

5.

3. The universal nucleic acid extraction reagent for pathogenic fungi and bacteria according to claim 2, wherein The defoamer is any one or more of non-ionic T-F composite fermentation defoamer and silicone defoamer.

4. The general nucleic acid extraction reagent for pathogenic fungi and bacteria according to claim 3, characterized in that, The defoamer is non-ionic T-F composite fermentation defoamer with a concentration of 2%.

5. The general nucleic acid extraction reagent for pathogenic fungi and bacteria according to claim 4, wherein, The weight ratio of the acid-washed glass beads with a diameter of 180 - 220 μm to the acid-washed glass beads with a diameter of 480 - 520 μm used in the grinding tube is 1:

3.

6. A universal nucleic acid extraction kit for pathogenic fungi and bacteria, characterized in that, It contains the universal nucleic acid extraction reagent for pathogenic fungi and bacteria according to any one of claims 1 - 5.

7. A nucleic acid extraction method, characterized in that, It is carried out by using the universal nucleic acid extraction reagent for pathogenic fungi and bacteria according to any one of claims 1 - 5 or the universal nucleic acid extraction kit for pathogenic fungi and bacteria according to claim 6, and includes the following steps: (1) Grinding: Add 180 - 220 μL of the sample into the grinding tube, and mix with 180 - 220 μL of Lysis Solution I, and grind for 10 - 20 minutes; (2) Lysis: Add 180 - 220 μL of Lysis Solution II to the ground sample, and then add 380 - 420 μL of ethanol or isopropanol to make the final concentration of ethanol or isopropanol in the lysed sample reach 35% - 45%; (3) Washing: Use 480 - 520 μL of Wash Solution I and Wash Solution II to remove the residual proteins and impurities during nucleic acid adsorption; (4) Elution: Use 80 - 120 μL of Elution Solution to elute nucleic acid to obtain pure biological nucleic acid.

8. The nucleic acid extraction method according to claim 7, wherein The grinding instrument used in step (1) is a vortex shaker or a tissue grinder, and their grinding speeds are 3200 RPM and 30 Hz respectively, and the grinding time is 15 minutes.

9. Use of the general nucleic acid extraction reagent for pathogenic fungi and bacteria according to any one of claims 1 to 5, the general nucleic acid extraction kit for pathogenic fungi and bacteria according to claim 6, or the nucleic acid extraction method according to any one of claims 7 to 8 in extracting nucleic acids of pathogenic fungi and bacteria.

10. The application according to claim 9, wherein The pathogenic fungi and bacteria are any one or more of the following: (1) Candida albicans; (2) Candida parapsilosis; (3) Candida tropicalis; (4) Candida glabrata / Nakaseomyces glabrate; (5) Aspergillus fumigatus; (6) Cryptococcus neoformans; (7) Mucorales; (8) Klebsiella pneumoniae; (9) Pseudomonas aeruginosa; (10) Escherichia coli; (11) Staphylococcus aureus; (12) Streptococcus pyogenes; (13) Streptococcus agalactiae; (14) Enterococcus faecium.

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