A method and reagent for separating, enriching and extracting nucleic acid of pathogenic microorganism in blood

Abstract

The application provides a method and reagent for separating, enriching and extracting nucleic acid of pathogenic microorganism in blood. First, blood sample is added with blood selective lysing solution A and pathogenic microorganism settling agent to selectively lyse blood cells and centrifuge pathogenic microorganism by high speed, then blood selective lysing solution B is added to perform secondary selective lysis of blood sample, washing liquid is added and mixed to wash, finally nucleic acid extraction reagent is added, pathogenic microorganism is enriched by centrifugation, then is lysed by physical method, and nucleic acid sample of pathogenic microorganism is obtained. The application can quickly and accurately extract nucleic acid of target infectious pathogenic microorganism which can meet the requirement of molecular biology detection from blood sample of not more than 10 mL, without blood culture, directly using blood sample to enrich pathogenic microorganism and extract nucleic acid, and is also suitable for blood culture sample. The nucleic acid extraction step is simple, without complex processing equipment, and the obtained nucleic acid can be used for various downstream detection of molecular biology.

Description

[0001] This application is a divisional application of the invention patent application filed on March 31, 2021, with application number 202110346173.9 and invention title "A method and reagent for isolating, enriching and extracting nucleic acids from pathogenic microorganisms in blood". Technical Field

[0002] This invention relates to the technical field of methods for determining or testing enzymes, nucleic acids, or microorganisms, and specifically to a method and reagent for isolating, enriching, and extracting nucleic acids from pathogenic microorganisms in blood. Background Technology

[0003] Bloodstream infection refers to a systemic inflammatory response syndrome caused by the invasion of the bloodstream by pathogenic microorganisms such as bacteria and fungi. These pathogens can exist transiently, intermittently, or persistently in the circulating blood, damaging all organs of the body, such as heart valves and joints. In severe cases, it can lead to shock, multiple organ failure, disseminated intravascular coagulation, and even death. Currently, sepsis and bacteremia are collectively referred to as bloodstream infection. Sepsis is a blood infection caused by various pathogenic microorganisms (bacteria or fungi) and toxins entering the bloodstream. If bacteria only briefly enter the bloodstream without clinically obvious toxemia symptoms, it is called bacteremia, which can be divided into primary and secondary types. Primary bacteremia is related to intravenous procedures, while secondary bacteremia is mostly caused by postoperative wound, abdominal cavity, urinary tract, and lung infections. In recent years, due to the widespread use of invasive devices and treatments such as indwelling intravenous catheters, mechanical ventilation, and parenteral administration, as well as the overuse of immunosuppressants and large amounts of antibiotics, the incidence of bloodstream infections has been rising year by year. Moreover, because bloodstream infections not only have a high mortality rate but also prolong hospital stays and increase hospitalization costs, they have received increasing attention from doctors, patients, and researchers.

[0004] The most common causes of bloodstream infections are bacteria, such as Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae. Besides bacterial infections, fungal infections have recently become an important cause of this disease. Despite thirty years of development in the diagnosis and treatment of bloodstream infections, there has been very little progress. Microorganisms are cultured from blood samples, increasing the microbial concentration from 1–10 CFU / mL to 10… 6 ~10 8CFU / mL remains the gold standard for detecting bloodstream infections. However, this method has several drawbacks. First, the diagnostic time is long, typically 1–5 days from patient sampling to receiving infection results. During this period, treatment usually involves broad-spectrum, non-targeted antibiotics. While this may be effective in treating the disease, it has a serious consequence: many microorganisms develop multidrug resistance. Second, culture methods require large quantities of blood samples for diagnosis and have low sensitivity. Furthermore, there is a relatively high risk of contamination during blood collection and use. Therefore, although blood culture remains the primary reference method for diagnosing bloodstream infections, it is not an ideal gold standard due to its significant drawbacks, including low sensitivity and long processing time.

[0005] With the development of molecular biology techniques, molecular biological diagnostic technologies, represented by polymerase chain reaction (PCR) and next-generation sequencing, have begun to be applied to the detection of microorganisms in bloodstream infections. Molecular biological diagnostic methods offer significant advantages such as speed, high sensitivity, small sample size, and the ability to provide microbial diagnostic information within a short time (generally no more than 12 hours), followed by the introduction of appropriate antibiotic treatment (detection of drug resistance genes). However, for a long time, the extremely low number of circulating microorganisms in blood (1–10 CFU / mL), the large amount of human nucleic acid in blood itself, which may interfere with PCR and sequencing detection, and the fact that blood components such as iron, hemoglobin, and blood anticoagulants can severely inhibit PCR and library construction, have limited the direct microbial diagnosis from whole blood.

[0006] Therefore, rapidly isolating and enriching pathogenic microorganisms from whole blood and extracting nucleic acids that can be used for molecular biological detection will effectively improve the detection efficiency of bloodstream infection pathogens, provide timely guidance for doctors to prescribe drugs and treat patients rationally and in a targeted manner, and at the same time buy patients the golden treatment time, thereby increasing the patient's survival rate. Summary of the Invention

[0007] This invention provides a method and reagents for directly isolating and enriching infectious pathogens and extracting nucleic acids from the blood of suspected bloodstream infection patients (1-2 mL for pediatric patients, including newborns, and 5-10 mL for adults). It eliminates the need for blood culture, allowing direct use of blood samples for pathogen enrichment and nucleic acid extraction. It is also applicable to samples after blood culture. The nucleic acid extraction procedure is simple and can be applied indiscriminately to Gram-negative bacteria, Gram-positive bacteria, and fungi. The extracted nucleic acids can be used with various molecular biology detection methods, including singlet and multiplex PCR / RT-PCR and next-generation sequencing. The operation requires minimal equipment; a common laboratory high-speed centrifuge suffices.

[0008] This invention provides a method for isolating, enriching, and extracting nucleic acids from pathogenic microorganisms in blood, comprising the following steps:

[0009] S1. Initial lysis of blood samples: Blood selective lysis buffer A and pathogenic microorganism sedimentation agent are added to the blood sample and mixed thoroughly to obtain lysate. The large molecular organic matter in the lysate is dissolved. After high-speed centrifugation, part of the supernatant is discarded to obtain the initial lysate of blood sample. The lysate includes lysed blood cells and unlysed pathogenic microorganisms with altered surface properties. Blood cells include red blood cells and white blood cells. High-speed centrifugation is used to enrich and precipitate pathogenic microorganisms.

[0010] S2, Blood sample re-lysis: Add blood selective lysis buffer B to further dissolve the residual macromolecular organic matter. After high-speed centrifugation, discard part of the supernatant at the interface between the microbial flocculant and the aqueous solution to obtain a blood sample that has been re-lysed.

[0011] S3. Washing: After adding washing solution and mixing well, centrifuge and discard part of the supernatant to obtain a blood lysate sample containing pathogenic microorganisms. The washing solution is used to clean impurities.

[0012] S4. Nucleic acid extraction: Add nucleic acid extraction reagent to the blood lysate sample, centrifuge at high speed and discard part of the supernatant. Electrolyze the remaining solution or add grinding beads of different particle sizes, shake, heat at high temperature and centrifuge to lyse the pathogenic microorganism and promote the release and dissolution of nucleic acid in the pathogenic microorganism to obtain the pathogenic microorganism nucleic acid sample.

[0013] The present invention discloses a method for isolating, enriching, and extracting nucleic acids from pathogenic microorganisms in blood. In a preferred embodiment, step S1 involves the blood selective lysis buffer A comprising: a buffer solution with a concentration of 10–100 mM, an auxiliary lysis salt with a mass-volume percentage of 0%–6.5%, a major lysis salt with a mass-volume percentage of 5%–65%, a surfactant with a mass-volume percentage of 0%–5%, a chelating agent with a concentration of 0–10 mM, and an antifoaming agent with a volume percentage of 0%–0.5%. The pH of the blood selective lysis buffer A is 4–8.

[0014] The pathogenic microorganism flocculant is a water-insoluble, biologically inert organic solvent;

[0015] The blood sample is 1-10 ml. After adding blood selective lysis buffer A and pathogenic microorganism sedimentation agent, mix by shaking at room temperature or by inverting at room temperature for 1-5 minutes. The amount of blood selective lysis buffer A is 0.5-5 times the volume of the blood sample, and the amount of pathogenic microorganism sedimentation agent is 0.5%-5% of the volume of the blood sample.

[0016] The present invention provides a method for isolating, enriching, and extracting nucleic acids from pathogenic microorganisms in blood. In a preferred embodiment, step S2 involves the blood selective lysis buffer B comprising: a buffer solution with a concentration of 10–100 mM, an auxiliary lysis salt with a mass-volume percentage of 0%–3%, a major lysis salt with a mass-volume percentage of 0%–40%, a surfactant with a mass-volume percentage of 0%–2%, a chelating agent with a concentration of 0–10 mM, and an antifoaming agent with a volume percentage of 0%–0.5%. The pH of the blood selective lysis buffer B is 5–8.

[0017] The volume of blood selective lysis buffer B is 2 to 10 times the volume of the initial blood lysis sample;

[0018] In step S3, the washing solution includes: a buffer solution with a concentration of 10-100 mM, a salt that promotes the dissolution of impurities with a mass-volume percentage of 0%-5%, and a chelating agent with a concentration of 0-10 mM. The pH of the washing solution is 5-9.

[0019] The amount of washing solution used is 5 to 10 times the volume of the blood sample after lysis. The washing solution is used by shaking and mixing at room temperature or by inverting and mixing at room temperature.

[0020] Steps S2 and S3 can be repeated multiple times.

[0021] The present invention provides a method for isolating, enriching, and extracting nucleic acids from pathogenic microorganisms in blood. In a preferred embodiment, step S4 comprises the following nucleic acid extraction solution: a buffer solution with a concentration of 0.5–20 mM, a nucleic acid dissolution-promoting salt with a mass-volume percentage of 0–5%, a surfactant with a mass-volume percentage of 0.01%–5%, a chelating agent with a concentration of 0–3 mM, and an antifoaming agent with a volume percentage of 0%–0.5%. The pH of the nucleic acid extraction solution is 7–9.

[0022] The surfactant is anionic surfactant and / or nonionic surfactant;

[0023] The amount of nucleic acid extraction solution used is 5 to 10 times the volume of the blood lysed sample. Electrolysis is performed using an electrolysis device fabricated by MEMS. The grinding beads are one or more of the following: acid-washed glass beads, zirconia beads, and quartz sand with a particle size of 0.1 to 3 mm. The amount of grinding beads used is 20 to 150 mg, the shaking time is 5 to 15 minutes, and the high-temperature heating temperature is 85 to 100°C for 3 to 15 minutes.

[0024] The method for isolating, enriching and extracting nucleic acids from pathogenic microorganisms in blood according to the present invention, wherein the buffer solution is any one of the following: phosphate buffer, sodium acetate buffer, and tris(hydroxymethyl)aminomethane hydrochloride buffer;

[0025] The auxiliary lysis salt, the nucleic acid dissolution promoting salt, and the impurity dissolution promoting salt are one or more of the following: sodium chloride, potassium chloride, and ammonium sulfate;

[0026] The main cleavage salts are one or more of the following: lithium chloride, sodium iodide, potassium iodide, guanidine hydrochloride, and guanidine isothiocyanate;

[0027] The defoamer is any one of the following: polydimethylsiloxane, polypropylene glycol, polyether-modified polysiloxane, and polyoxypropylene glycerol ether;

[0028] The chelating agent is trisodium citrate;

[0029] The anionic surfactant is any one of the following: sodium dodecyl sulfate, sodium dodecyl sarcosinate and sodium deoxycholate; the nonionic surfactant is one or more of the following: Tween 20, Tween 80, Triton X-100, ethyl phenyl polyethylene glycol and hexadecyl polyoxyethylene ether Brij C20.

[0030] In steps S1, S2, S3 and S4, the centrifugation speed is 10000 to 16000 × g, and the centrifugation time is 1 to 5 minutes.

[0031] This invention provides a reagent for isolating, enriching, and extracting nucleic acids from pathogenic microorganisms in blood, comprising: blood selective lysis buffer A (0.5-10 times the volume of the blood sample); pathogenic microorganism flocculant (0.5%-10% the volume of the blood sample); blood selective lysis buffer B (1-10 times the volume of the blood sample); washing solution (1-10 times the volume of the blood sample); and nucleic acid extraction solution (0.5-2 times the volume of the blood sample).

[0032] Blood selective lysis buffer A and blood selective lysis buffer B are used to lyse only blood cells, retain pathogenic microorganisms, and dissolve large organic molecules released from the blood sample and lysed blood cells; pathogenic microorganism flocculant is used to enrich and precipitate pathogenic microorganisms; washing solution is used to remove impurities; and nucleic acid extraction solution is used to enrich and lyse pathogenic microorganisms and promote the release and dissolution of nucleic acids in pathogenic microorganisms.

[0033] Blood selective lysis buffer A comprises: a buffer solution with a concentration of 10–100 mM, an auxiliary lysis salt with a mass-volume percentage of 0%–6.5%, a major lysis salt with a mass-volume percentage of 5%–65%, a surfactant with a mass-volume percentage of 0%–5%, a chelating agent with a concentration of 0–10 mM, and an antifoaming agent with a volume percentage of 0%–0.5%. The pH of blood selective lysis buffer A is 4–8.

[0034] The pathogenic microorganism flocculant is a water-insoluble, biologically inert organic solvent;

[0035] Selective blood lysis buffer B comprises: a buffer solution with a concentration of 10–100 mM, an auxiliary lysis salt with a volume percentage of 0%–3%, a major lysis salt with a volume percentage of 0%–40%, a surfactant with a volume percentage of 0%–2%, a chelating agent with a concentration of 0–10 mM, and an antifoaming agent with a volume percentage of 0%–0.5%. The pH of selective blood lysis buffer B is 5–8.

[0036] The present invention provides a reagent for isolating, enriching, and extracting nucleic acids from pathogenic microorganisms in blood. In a preferred embodiment, the washing solution comprises: a buffer solution with a concentration of 10–100 mM, a salt for promoting the dissolution of impurities with a mass-volume percentage of 0%–5%, and a chelating agent with a concentration of 0–10 mM. The pH of the washing solution is 5–9.

[0037] The nucleic acid extraction solution includes: a buffer solution with a concentration of 0.5–20 mM, a nucleic acid dissolution promoting salt with a mass-volume percentage of 0–5%, a surfactant with a mass-volume percentage of 0.01%–5%, a chelating agent with a concentration of 0–3 mM, and an antifoaming agent with a volume percentage of 0%–0.5%. The pH of the nucleic acid extraction solution is 7–9.

[0038] The biologically inert organic solvent is fluorinated oil, HFE-7500, Novec 7500, or FC-40.

[0039] The reagent for isolating, enriching and extracting nucleic acids from pathogenic microorganisms in blood according to the present invention, wherein the buffer solution is any one of the following: phosphate buffer, sodium acetate buffer, or tris(hydroxymethyl)aminomethane hydrochloride buffer;

[0040] The auxiliary lysis salt, the nucleic acid dissolution promoting salt, and the impurity dissolution promoting salt are one or more of the following: sodium chloride, potassium chloride, and ammonium sulfate;

[0041] The main cleavage salts are one or more of the following: lithium chloride, sodium iodide, potassium iodide, guanidine hydrochloride, and guanidine isothiocyanate;

[0042] The defoamer is any one of the following: polydimethylsiloxane, polypropylene glycol, polyether-modified polysiloxane, and polyoxypropylene glycerol ether;

[0043] The chelating agent is trisodium citrate;

[0044] The surfactant is anionic surfactant and / or nonionic surfactant.

[0045] The reagent for isolating, enriching and extracting nucleic acids from pathogenic microorganisms in blood according to the present invention, as a preferred embodiment, uses any one of the following anionic surfactants: sodium dodecyl sulfate, sodium dodecyl sarcosinate and sodium deoxycholate, and one or more of the following nonionic surfactants: Tween 20, Tween 80, Triton X-100, ethyl phenyl polyethylene glycol and hexadecyl polyoxyethylene ether Brij C20.

[0046] The present invention provides a reagent for isolating, enriching, and extracting nucleic acids from pathogenic microorganisms in blood. In a preferred embodiment, blood selective lysis buffer A comprises 20 mM tris(hydroxymethyl)aminomethane hydrochloride buffer, 0.5% potassium chloride (w / v), 65% guanidine isothiocyanate (w / v), 0.2% sodium dodecyl sarcosinate (w / v), 10 mM trisodium citrate, and 0.02% polyoxypropylene glycerol ether (w / v). The pH of blood selective lysis buffer A is 7.0.

[0047] Blood selective lysis buffer B consists of 20 mM tris(hydroxymethyl)aminomethane hydrochloride buffer, 0.5% potassium chloride, 30% guanidine isothiocyanate, 0.1% sodium dodecyl sarcosinate, 10 mM trisodium citrate, and 0.02% polyoxypropylene glycerol ether. The pH of blood selective lysis buffer B is 7.0.

[0048] The microbial settling agent is Novec 7500;

[0049] The washing solution consists of 10 mM tris(hydroxymethyl)aminomethane hydrochloride buffer, 0.9% sodium chloride (by weight / volume), 10% ammonium sulfate (by weight / volume), and 3 mM trisodium citrate. The pH of the washing solution is 7.0.

[0050] The nucleic acid extraction buffer consisted of 10 mM tris(hydroxymethyl)aminomethane hydrochloride buffer, 0.45% sodium chloride, 0.1% sodium dodecyl sulfate, 2% Tween 20, 1 mM trisodium citrate, and 0.02% polyoxypropylene glycerol ether. The pH of the nucleic acid extraction buffer was 8.8.

[0051] The specific procedure is as follows: Blood selective lysis buffer A and pathogenic microorganism sedimentation agent are added to the blood sample for sample lysis treatment, selectively lysing blood cells (including red blood cells and white blood cells, etc.), dissolving impurities such as proteins, sugars, and lipids in the blood. Then, the pathogenic microorganisms are settled by high-speed centrifugation, and part of the supernatant is carefully discarded. Blood selective lysis buffer B is added for one or two more blood selective lysis treatments, and high-speed centrifugation is performed to discard part of the supernatant at the interface between the microorganism sedimentation agent and the aqueous solution. Then, washing buffer is added to the remaining solution, mixed, centrifuged, and part of the supernatant is discarded again. This washing process is repeated once or multiple times. Then, nucleic acid extraction reagent is added to treat the remaining solution again, and after high-speed centrifugation, part of the supernatant is discarded. The pathogenic microorganisms are in the remaining nucleic acid extraction solution. The nucleic acid of the pathogenic microorganisms is extracted by electrolysis or by adding grinding beads with different particle size combinations, shaking, heating at high temperature, and centrifugation.

[0052] In a preferred embodiment of the present invention, the amount of blood selective lysis buffer A is 0.5 to 5 times the volume of the blood sample, the amount of microbial flocculant is 0.5% to 5% of the volume of the blood sample, the lysis conditions are: room temperature shaking or inverting mixing for 1 to 5 minutes, centrifugation speed of 10000 to 16000 × g, centrifugation time of 1 to 5 minutes, discarding the supernatant using a pipette or syringe, etc., and avoiding touching the precipitate located at the interface between the microbial flocculant and the aqueous solution, and discarding 80% to 95% of the total solution volume.

[0053] In a preferred embodiment of the present invention, the amount of blood selective lysis buffer B is 2 to 10 times the volume of the remaining supernatant. The lysis conditions are: room temperature shaking or inverting mixing for 1 to 5 minutes, centrifugation speed of 10,000 to 16,000 × g, centrifugation time of 1 to 5 minutes, discarding the supernatant using a pipette or syringe, etc., and avoiding touching the precipitate located at the interface between the microbial precipitant and the aqueous solution. The amount of solution discarded is equal to the amount of blood selective lysis buffer B added. This lysis step is repeated once or multiple times depending on the precipitate characteristics.

[0054] In a preferred embodiment of the present invention, the amount of washing liquid is 5 to 10 times the volume of the remaining supernatant. The washing conditions are: room temperature shaking and mixing or room temperature inverting and mixing for 1 to 5 minutes, centrifugation speed of 10,000 to 16,000 × g, centrifugation time of 1 to 5 minutes, discarding the supernatant using a pipette or syringe, etc., and avoiding touching the precipitate located at the interface between the microbial precipitant and the aqueous solution. The amount of solution discarded is equal to the amount of washing liquid added. This lysis step is repeated once or multiple times depending on the precipitate characteristics.

[0055] In a preferred embodiment of the present invention, the amount of nucleic acid extraction solution used is 5 to 10 times the volume of the remaining supernatant. The processing conditions are: room temperature shaking and mixing or room temperature inverting and mixing for 1 to 5 minutes, centrifugation speed of 10,000 to 16,000 × g, centrifugation time of 1 to 5 minutes, discarding the supernatant using a pipette or syringe, etc., and avoiding touching the precipitate located at the interface between the microbial precipitant and the aqueous solution. The amount of solution discarded is equal to the amount of nucleic acid extraction solution added. The remaining supernatant and precipitate contain pathogenic microorganisms enriched from the sample.

[0056] In a preferred embodiment of the present invention, the grinding beads are acid-washed glass beads or zirconia beads with different particle sizes of 0.1-3 mm, the amount used is 20-150 mg, the shaking time is 5-15 minutes, the high temperature heating is 85-100℃, the heating time is 3-15 minutes, the centrifugation speed is 10000-16000×g, the centrifugation time is 1-5 minutes, and the supernatant is aspirated by a pipette or syringe after centrifugation, which is the target nucleic acid.

[0057] In a preferred embodiment of the present invention, nucleic acid extraction uses a microfabricated electrolysis device with dimensions of (0.1~1)×(5~1000)×(10~1000)mm. 3 The dimensions may be larger or smaller than the dimensions specified in this design, depending on the required size. The decomposition voltage is 10–2000V.

[0058] In a preferred embodiment of the present invention, the blood sample is the blood of a patient suspected of having a bloodstream infection. For pediatric patients, including newborns, the blood volume is 1-2 mL, and for adults, it is 5-10 mL. The sample can also be a blood culture sample, with a volume of 0.2-1 mL.

[0059] This invention utilizes the osmotic pressure difference between blood selective lysis buffer A and blood, along with the addition of surfactants, to rupture blood cells (including red blood cells and white blood cells). Simultaneously, at an appropriate concentration, it dissolves as much of the large organic molecules in the blood and released during cell rupture as possible without severely damaging the cellular structure of pathogenic microorganisms. Furthermore, the salts and surfactants in the buffer, together with the microbial sedimentation aid, promote the enrichment and precipitation of pathogenic microorganisms that are difficult to centrifuge due to their special extracellular structures (such as bacterial capsules), reducing differential microbial enrichment. The microbial sedimentation aid also reduces adhesion between microorganisms and centrifuge tubes, preventing loss due to incomplete centrifugation sedimentation. The addition of blood selective lysis buffer B compensates for incomplete lysis caused by sample differences and fully dissolves residual impurities. The washing buffer removes lysis buffer components and poorly soluble substances in the blood that may inhibit downstream detection. A large amount of nucleic acid extract dilutes the salts in the washing solution and provides suitable conditions for the lysis of pathogenic microorganisms and the dissolution and preservation of nucleic acids. Under the action of instantaneous electrolysis or physical grinding with grinding beads of different particle sizes and high-temperature heating denaturation, pathogenic microorganisms are fully lysed, promoting the release and dissolution of nucleic acids.

[0060] Macromolecular organic compounds include proteins, carbohydrates, lipids, and nucleic acids.

[0061] The present invention has the following advantages:

[0062] (1) It can quickly and accurately extract the target infectious pathogen nucleic acid that meets the requirements of molecular biology detection from blood samples of no more than 10 mL.

[0063] (2) No blood culture is required. Blood samples can be used directly for pathogen enrichment and nucleic acid extraction. It is also applicable to samples after blood culture.

[0064] (3) The nucleic acid extraction steps are simple and can be applied indiscriminately to Gram-negative bacteria, Gram-positive bacteria and fungi.

[0065] (4) The extracted nucleic acid can be combined with a variety of molecular biological detection methods such as single-particle and multiplex PCR / RT-PCR and next-generation sequencing.

[0066] (5) The requirements for operating and adapting instruments and equipment are not high; only a common high-speed centrifuge in the laboratory is needed to meet the operating requirements. Attached Figure Description

[0067] Figure 1 This is a flowchart of a method for isolating, enriching, and extracting nucleic acids from pathogenic microorganisms in blood.

[0068] Figure 2 This is a schematic diagram of a method for isolating, enriching, and extracting nucleic acids from pathogenic microorganisms in blood.

[0069] Figure 3 Example 5 shows the amplification curve of probe-based real-time PCR for a method and reagents for isolating, enriching and extracting nucleic acids from pathogenic microorganisms in blood.

[0070] Figure 4 Example 6: Amplification curve of probe-based real-time PCR for a method and reagents for isolating, enriching and extracting nucleic acids from pathogenic microorganisms in blood;

[0071] Figure 5 Example 7 shows the amplification curve of probe-based real-time PCR, which is a method and reagent for isolating, enriching and extracting nucleic acids from pathogenic microorganisms in blood. Detailed Implementation

[0072] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0073] Example 1

[0074] like Figure 1 As shown, a method for isolating, enriching, and extracting nucleic acids from pathogenic microorganisms in blood is characterized by the following steps:

[0075] S1. Initial lysis of blood samples: Blood selective lysis buffer A and pathogenic microorganism sedimentation agent are added to the blood sample and mixed thoroughly to obtain lysate. The large molecular organic matter in the lysate is dissolved. After high-speed centrifugation, part of the supernatant is discarded to obtain the initial lysate of blood sample. The lysate includes lysed blood cells and unlysed pathogenic microorganisms with altered surface properties. Blood cells include red blood cells and white blood cells. High-speed centrifugation is used to enrich and precipitate pathogenic microorganisms.

[0076] S2, Blood sample re-lysis: Add blood selective lysis buffer B to further dissolve the residual macromolecular organic matter. After high-speed centrifugation, discard part of the supernatant at the interface between the microbial flocculant and the aqueous solution to obtain a blood sample that has been re-lysed.

[0077] S3. Washing: After adding washing solution and mixing well, centrifuge and discard part of the supernatant to obtain a blood lysate sample containing pathogenic microorganisms. The washing solution is used to clean impurities.

[0078] S4. Nucleic acid extraction: Add nucleic acid extraction reagent to the blood lysate sample, centrifuge at high speed and discard part of the supernatant. Electrolyze the remaining solution or add grinding beads of different particle sizes, shake, heat at high temperature and centrifuge to lyse the pathogenic microorganism and promote the release and dissolution of nucleic acid in the pathogenic microorganism to obtain the pathogenic microorganism nucleic acid sample.

[0079] Example 2

[0080] like Figure 1 As shown, a method for isolating, enriching, and extracting nucleic acids from pathogenic microorganisms in blood is characterized by the following steps:

[0081] S1. Initial lysis of blood samples: Blood selective lysis buffer A and pathogenic microorganism sedimentation agent are added to the blood sample and mixed thoroughly to obtain lysate. The large molecular organic matter in the lysate is dissolved. After high-speed centrifugation, part of the supernatant is discarded to obtain the initial lysate of blood sample. The lysate includes lysed blood cells and unlysed pathogenic microorganisms with altered surface properties. Blood cells include red blood cells and white blood cells. High-speed centrifugation is used to enrich and precipitate pathogenic microorganisms.

[0082] Blood selective lysis buffer A comprises: a buffer solution with a concentration of 10–100 mM, an auxiliary lysis salt with a mass-volume percentage of 0%–6.5%, a major lysis salt with a mass-volume percentage of 5%–65%, a surfactant with a mass-volume percentage of 0%–5%, a chelating agent with a concentration of 0–10 mM, and an antifoaming agent with a volume percentage of 0%–0.5%. The pH of blood selective lysis buffer A is 4–8.

[0083] The pathogenic microorganism flocculant is a water-insoluble, biologically inert organic solvent;

[0084] The blood sample is 1-10 ml. After adding blood selective lysis buffer A and pathogenic microorganism sedimentation agent, mix by shaking at room temperature or by inverting at room temperature for 1-5 minutes. The amount of blood selective lysis buffer A is 0.5-5 times the volume of the blood sample, and the amount of pathogenic microorganism sedimentation agent is 0.5%-5% of the volume of the blood sample.

[0085] S2, Blood sample re-lysis: Add blood selective lysis buffer B to further dissolve the residual macromolecular organic matter. After high-speed centrifugation, discard part of the supernatant at the interface between the microbial flocculant and the aqueous solution to obtain a blood sample that has been re-lysed.

[0086] Blood selective lysis buffer B comprises: a buffer solution with a concentration of 10–100 mM, an auxiliary lysis salt with a volume percentage of 0%–3%, a major lysis salt with a volume percentage of 0%–40%, a surfactant with a volume percentage of 0%–2%, a chelating agent with a concentration of 0–10 mM, and an antifoaming agent with a volume percentage of 0%–0.5%. The pH of blood selective lysis buffer B is 5–8.

[0087] The volume of blood selective lysis buffer B is 2 to 10 times the volume of the initial blood lysis sample;

[0088] Step S2 can be repeated multiple times;

[0089] S3. Washing: After adding washing solution and mixing well, centrifuge and discard part of the supernatant to obtain a blood lysate sample containing pathogenic microorganisms. The washing solution is used to clean impurities.

[0090] In step S3, the washing solution includes: a buffer solution with a concentration of 10-100 mM, a salt that promotes the dissolution of impurities with a mass-volume percentage of 0%-5%, and a chelating agent with a concentration of 0-10 mM. The pH of the washing solution is 5-9.

[0091] The amount of washing solution used is 5 to 10 times the volume of the blood sample after lysis. The washing solution is used by shaking or inverting at room temperature to mix.

[0092] Step S3 can be repeated multiple times;

[0093] S4. Nucleic acid extraction: Add nucleic acid extraction reagent to the blood lysate sample, centrifuge at high speed and discard part of the supernatant. Electrolyze the remaining solution or add grinding beads of different particle sizes, shake, heat at high temperature and centrifuge to lyse the pathogenic microorganism and promote the release and dissolution of nucleic acid in the pathogenic microorganism to obtain the pathogenic microorganism nucleic acid sample.

[0094] The nucleic acid extraction solution includes: a buffer solution with a concentration of 0.5–20 mM, a nucleic acid dissolution promoting salt with a mass-volume percentage of 0–5%, a surfactant with a mass-volume percentage of 0.01%–5%, a chelating agent with a concentration of 0–3 mM, and an antifoaming agent with a volume percentage of 0%–0.5%. The pH of the nucleic acid extraction solution is 7–9.

[0095] The surfactant is anionic surfactant and / or nonionic surfactant;

[0096] The amount of nucleic acid extraction solution used is 5 to 10 times the volume of the blood lysed sample. Electrolysis is performed using an electrolysis device fabricated by MEMS. The grinding beads are one or more of the following: acid-washed glass beads, zirconium oxide beads, and quartz sand with a particle size of 0.1 to 3 mm. The amount of grinding beads used is 20 to 150 mg, the shaking time is 5 to 15 minutes, and the high-temperature heating temperature is 85 to 100°C for 3 to 15 minutes.

[0097] The buffer solution is any one of the following: phosphate buffer, sodium acetate buffer, and tris(hydroxymethyl)aminomethane hydrochloride buffer;

[0098] The auxiliary lysis salt, the nucleic acid dissolution promoting salt, and the impurity dissolution promoting salt are one or more of the following: sodium chloride, potassium chloride, and ammonium sulfate;

[0099] The main cleavage salts are one or more of the following: lithium chloride, sodium iodide, potassium iodide, guanidine hydrochloride, and guanidine isothiocyanate;

[0100] The defoamer is any one of the following: polydimethylsiloxane, polypropylene glycol, polyether-modified polysiloxane, and polyoxypropylene glycerol ether;

[0101] The chelating agent is trisodium citrate;

[0102] The anionic surfactant is any one of the following: sodium dodecyl sulfate, sodium dodecyl sarcosinate and sodium deoxycholate; the nonionic surfactant is one or more of the following: Tween 20, Tween 80, Triton X-100, ethyl phenyl polyethylene glycol and hexadecyl polyoxyethylene ether Brij C20.

[0103] In steps S1, S2, S3 and S4, the centrifugation speed is 10000 to 16000 × g, and the centrifugation time is 1 to 5 minutes.

[0104] Example 3

[0105] A reagent for isolating, enriching, and extracting nucleic acids from pathogenic microorganisms in blood includes a blood selective lysis buffer A (0.5–10 times the volume of the blood sample), a pathogenic microorganism flocculant (0.5%–10% the volume of the blood sample), a blood selective lysis buffer B (1–10 times the volume of the blood sample), a washing solution (1–10 times the volume of the blood sample), and a nucleic acid extraction solution (0.5–2 times the volume of the blood sample).

[0106] Blood selective lysis buffer A and blood selective lysis buffer B are used to lyse only blood cells, retain pathogenic microorganisms, and dissolve large organic molecules released from the blood sample and lysed blood cells; pathogenic microorganism flocculant is used to enrich and precipitate pathogenic microorganisms; washing solution is used to remove impurities; and nucleic acid extraction solution is used to enrich and lyse pathogenic microorganisms and promote the release and dissolution of nucleic acids in pathogenic microorganisms.

[0107] Blood selective lysis buffer A comprises: a buffer solution with a concentration of 10–100 mM, an auxiliary lysis salt with a mass-volume percentage of 0%–6.5%, a major lysis salt with a mass-volume percentage of 5%–65%, a surfactant with a mass-volume percentage of 0%–5%, a chelating agent with a concentration of 0–10 mM, and an antifoaming agent with a volume percentage of 0%–0.5%. The pH of blood selective lysis buffer A is 4–8.

[0108] The pathogenic microorganism flocculant is a water-insoluble, biologically inert organic solvent;

[0109] Selective blood lysis buffer B comprises: a buffer solution with a concentration of 10–100 mM, an auxiliary lysis salt with a volume percentage of 0%–3%, a major lysis salt with a volume percentage of 0%–40%, a surfactant with a volume percentage of 0%–2%, a chelating agent with a concentration of 0–10 mM, and an antifoaming agent with a volume percentage of 0%–0.5%. The pH of selective blood lysis buffer B is 5–8.

[0110] Example 4

[0111] A reagent for isolating, enriching, and extracting nucleic acids from pathogenic microorganisms in blood includes a blood selective lysis buffer A (10 times the volume of the blood sample), a pathogenic microorganism flocculant (10% of the volume of the blood sample), a blood selective lysis buffer B (10 times the volume of the blood sample), a washing solution (10 times the volume of the blood sample), and a nucleic acid extraction solution (2 times the volume of the blood sample).

[0112] Blood selective lysis buffer A and blood selective lysis buffer B are used to lyse only blood cells, retain pathogenic microorganisms, and dissolve large organic molecules released from the blood sample and lysed blood cells; pathogenic microorganism flocculant is used to enrich and precipitate pathogenic microorganisms; washing solution is used to remove impurities; and nucleic acid extraction solution is used to enrich and lyse pathogenic microorganisms and promote the release and dissolution of nucleic acids in pathogenic microorganisms.

[0113] Blood selective lysis buffer A comprises: a buffer solution with a concentration of 10–100 mM, an auxiliary lysis salt with a mass-volume percentage of 0%–6.5%, a major lysis salt with a mass-volume percentage of 5%–65%, a surfactant with a mass-volume percentage of 0%–5%, a chelating agent with a concentration of 0–10 mM, and an antifoaming agent with a volume percentage of 0%–0.5%. The pH of blood selective lysis buffer A is 4–8.

[0114] The pathogenic microorganism flocculant is a water-insoluble, biologically inert organic solvent;

[0115] The biologically inert organic solvent is fluorinated oil, HFE-7500, Novec 7500, or FC-40;

[0116] Selective blood lysis buffer B comprises: a buffer solution with a concentration of 10–100 mM, an auxiliary lysis salt with a volume percentage of 0%–3%, a major lysis salt with a volume percentage of 0%–40%, a surfactant with a volume percentage of 0%–2%, a chelating agent with a concentration of 0–10 mM, and an antifoaming agent with a volume percentage of 0%–0.5%. The pH of selective blood lysis buffer B is 5–8. Washing buffer comprises: a buffer solution with a concentration of 10–100 mM, a salt that promotes the dissolution of impurities with a volume percentage of 0%–5%, and a chelating agent with a concentration of 0–10 mM. The pH of washing buffer is 5–9.

[0117] The nucleic acid extraction solution includes: a buffer solution with a concentration of 0.5–20 mM, a nucleic acid dissolution promoting salt with a mass-volume percentage of 0–5%, a surfactant with a mass-volume percentage of 0.01%–5%, a chelating agent with a concentration of 0–3 mM, and an antifoaming agent with a volume percentage of 0%–0.5%. The pH of the nucleic acid extraction solution is 7–9.

[0118] The buffer solution is any one of the following: phosphate buffer, sodium acetate buffer, or tris(hydroxymethyl)aminomethane hydrochloride buffer;

[0119] The auxiliary lysis salt, the nucleic acid dissolution promoting salt, and the impurity dissolution promoting salt are one or more of the following: sodium chloride, potassium chloride, and ammonium sulfate;

[0120] The main cleavage salts are one or more of the following: lithium chloride, sodium iodide, potassium iodide, guanidine hydrochloride, and guanidine isothiocyanate;

[0121] The defoamer is any one of the following: polydimethylsiloxane, polypropylene glycol, polyether-modified polysiloxane, and polyoxypropylene glycerol ether;

[0122] The chelating agent is trisodium citrate;

[0123] The surfactant is anionic surfactant and / or nonionic surfactant;

[0124] The anionic surfactant is any one of the following: sodium dodecyl sulfate, sodium dodecyl sarcosinate, and sodium deoxycholate; the nonionic surfactant is one or more of the following: Tween 20, Tween 80, Triton X-100, ethyl phenyl polyethylene glycol, and hexadecyl polyoxyethylene ether Brij C20.

[0125] Example 5

[0126] To verify the effectiveness of the method and reagents, the reagents and methods described in this invention were used to isolate and extract nucleic acids from simulated bloodstream-infected Escherichia coli pathogens. Simultaneously, the nucleic acids extracted from the simulated samples were added with the same amount of pathogens as a control. The extracted DNA was then detected by probe-based real-time PCR to verify the feasibility of this invention.

[0127] In the following examples, the PCR detection target is the DNA of pathogenic microorganisms in the extracted nucleic acid. The nucleic acid isolated by this invention also includes the RNA of pathogenic microorganisms. Depending on the design sites of the detection primers and probes, qRT-PCR and other detection methods can also be performed. The detection method here does not limit other downstream applications of this invention.

[0128] A reagent for isolating, enriching, and extracting nucleic acids from pathogenic microorganisms in blood, comprising the following reagents: Blood selective lysis buffer A consists of 20 mM tris(hydroxymethyl)aminomethane hydrochloride buffer (pH 7.0), 0.5% potassium chloride, 65% guanidine isothiocyanate, 0.2% sodium lauryl sarcosinate, 10 mM trisodium citrate, and 0.02% polyoxypropylene glycerol ether. The microbial sedimentation aid is Novec 7500. Blood selective lysis buffer B consists of 20 mM tris(hydroxymethyl)aminomethane hydrochloride buffer (pH 7.0), 0.5% potassium chloride, 30% guanidine isothiocyanate, 0.1% sodium lauroyl sarcosinate, 10 mM trisodium citrate, and 0.02% polyoxypropylene glycerol ether. The washing buffer consists of 10 mM tris(hydroxymethyl)aminomethane hydrochloride buffer (pH 7.0), 0.9% sodium chloride, 10% ammonium sulfate, and 3 mM trisodium citrate. The nucleic acid extraction buffer consisted of 10 mM tris(hydroxymethyl)aminomethane hydrochloride buffer (pH 8.8), 0.45% sodium chloride, 0.1% sodium dodecyl sulfate, 2% Tween 20, 1 mM trisodium citrate, and 0.02% polyoxypropylene glycerol ether.

[0129] The methods for isolating, enriching, and extracting nucleic acids from Escherichia coli in blood are as follows:

[0130] Prepare simulated samples containing *E. coli* by adding 10 CFU / 100 CFU / 1000 CFU of *E. coli* to three 1 mL aspirin-contaminated blood samples, respectively, and mixing thoroughly. Simultaneously prepare control samples by adding 10 CFU / 100 CFU / 1000 CFU of *E. coli* to three 100 μL aspirin-contaminated nucleic acid extraction solutions, respectively, and mixing thoroughly. Add reagents to the prepared simulated samples and perform microbial isolation, enrichment, and nucleic acid extraction. Specifically, add 1 mL of blood selective lysis buffer A and 20 μL of pathogenic microorganism sedimentation agent to 1 mL of simulated blood sample, vortex for 1 minute, centrifuge at 12000 × g for 2 minutes, and discard 1.8 mL of supernatant (remaining 200 μL). Then add 1 mL of blood selective lysis buffer B, vortex for 1 minute, centrifuge at 12000 × g for 2 minutes, and discard 1 mL of supernatant. Repeat this step once or multiple times. Add 1 mL of washing buffer to the sample, vortex for 1 minute, centrifuge at 12000 × g for 2 minutes, and discard 1 mL of supernatant. Repeat this step once or multiple times. Finally, add 1 mL of nucleic acid extraction buffer, vortex for 1 minute, centrifuge at 12000×g for 2 minutes, and discard 1 mL of supernatant. Add 30 mg of 0.5 mm zirconia beads, vortex for 5 minutes, heat at 95℃ for 5 minutes, centrifuge at 12000×g for 2 minutes, and collect the supernatant to obtain the simulated sample nucleic acid. Simultaneously, add 30 mg of 0.5 mm zirconia beads to the control sample, vortex for 5 minutes, heat at 95℃ for 5 minutes, centrifuge at 12000×g for 2 minutes, and collect the supernatant to obtain the control sample nucleic acid. In parallel, perform a blank blood separation and extraction control without the addition of E. coli. The extracted nucleic acid was used for probe-based PCR detection system preparation and amplification program settings according to Table 1. Specific primer and probe sequences are shown in Table 2. Results are as follows: Figure 3 The results showed that at the three detection gradients, the control and experimental groups exhibited excellent amplification overlap at the 100 CFU and 1000 CFU / mL levels. At the 10 CFU level, the two groups showed slight differences in amplification, but both were stably detected. Furthermore, neither the negative blood extraction control nor the template-free control showed amplification, indicating reliable amplification results. This demonstrates that the reagents and methods of this invention can isolate and extract E. coli DNA at the 10 CFU level from blood, meeting the requirements for blood sample detection of bloodstream infections.

[0131] A comparison of probe-based quantitative PCR amplification curves of nucleic acid extracted from 10 / 100 / 1000 CFU of E. coli isolated from blood samples and nucleic acid directly extracted from 10 / 100 / 1000 CFU of E. coli. Figure 3 In the left-hand legend, C~E1, C~E2, and C~E3 correspond to 10, 100, and 1000 CFU of the control group that does not require separation, respectively; E1, E2, and E3 correspond to 10, 100, and 1000 CFU of the experimental group that isolates the pathogen from the blood sample, respectively; BNTC is the negative blood separation and extraction control, and NTC is the amplification control without template.

[0132] Example 6

[0133] To verify the effectiveness of the method and reagents, the reagents and methods described in this invention were used to isolate and extract nucleic acids from pathogenic microorganisms of the type Streptococcus pneumoniae that cause bloodstream infection. At the same time, the nucleic acids extracted from the simulated samples were added with the same amount of pathogenic microorganisms as controls. The extracted DNA was then detected by probe-based real-time PCR to verify the feasibility of this invention.

[0134] A reagent for isolating, enriching, and extracting nucleic acids from pathogenic microorganisms in blood, comprising the following reagents: Blood selective lysis buffer A consists of 5 mM phosphate buffer (pH 7.5), 1.5% ammonium sulfate, 12% sodium iodide, 10 mM trisodium citrate, 0.5% Triton X-100, and 0.03% polyoxypropylene glycerol ether. The microbial flocculant is FC-40. Blood selective lysis buffer B consists of 5 mM phosphate buffer (pH 7.5), 0.9% sodium chloride, 4% sodium iodide, 5 mM trisodium citrate, and 0.03% polyoxypropylene glycerol ether. The washing buffer consists of 2 mM phosphate buffer (pH 8.5), 0.9% sodium chloride, 3% ammonium sulfate, and 5 mM trisodium citrate. The nucleic acid extraction buffer consists of 5 mM phosphate buffer (pH 8.0), 0.45% sodium chloride, 0.1% sodium dodecyl sarcosinate, 5% Tween 20, 0.5 mM trisodium citrate, and 0.1% polypropylene glycol.

[0135] The methods for isolating, enriching, and extracting nucleic acids from Streptococcus pneumoniae in blood are as follows:

[0136] Prepare simulated samples containing Streptococcus pneumoniae by adding 10 CFU / 100 CFU / 1000 CFU of Streptococcus pneumoniae to three 1 mL aspirin-negative blood samples, respectively, and mixing well. Simultaneously prepare control samples by adding 10 CFU / 100 CFU / 1000 CFU of Streptococcus pneumoniae to three 100 μL aspirin-containing nucleic acid extraction buffers, respectively, and mixing well. Add reagents to the prepared simulated samples and perform microbial isolation, enrichment, and nucleic acid extraction. Specifically, add 1 mL of selective lysis buffer A and 20 μL of microbial flocculant to 1 mL of simulated blood sample, vortex for 1 minute, centrifuge at 12000×g for 2 minutes, and discard 1.8 mL of supernatant (remaining 200 μL). Then add 1 mL of blood selective lysis buffer B, vortex for 1 minute, centrifuge at 12000×g for 2 minutes, and discard 1 mL of supernatant. Repeat this step once or multiple times. Add 1 mL of washing buffer to the sample, vortex for 1 minute, centrifuge at 12000×g for 2 minutes, and discard 1 mL of supernatant. Repeat this step once or multiple times. Finally, add 1 mL of nucleic acid extraction buffer, vortex for 1 minute, centrifuge at 12000×g for 2 minutes, and discard 1 mL of supernatant. Add 30 mg of 0.5 mm zirconia beads, vortex for 5 minutes, heat at 95℃ for 5 minutes, centrifuge at 12000×g for 2 minutes, and collect the supernatant to obtain the simulated sample nucleic acid. Simultaneously, add 30 mg of 0.5 mm zirconia beads to the control sample, vortex for 5 minutes, heat at 95℃ for 5 minutes, centrifuge at 12000×g for 2 minutes, and collect the supernatant to obtain the control sample nucleic acid. In parallel, perform a blank blood isolation and extraction control without adding Streptococcus pneumoniae. The extracted nucleic acid was prepared according to Table 1 for probe-based PCR detection system preparation and amplification program settings; specific primer and probe sequences are shown in Table 2. Results are as follows: Figure 4 The results showed that, at three different detection gradients, although the CT values ​​of the experimental group were delayed compared to the control group, they were still consistently detectable. Furthermore, neither the negative blood extraction control nor the template-free control showed amplification, indicating that the amplification results were reliable and reproducible. Therefore, the reagents and methods of this invention can isolate and extract 10 CFU / mL of Streptococcus pneumoniae DNA from blood, meeting the blood sample detection requirements for Gram-negative pathogens in bloodstream infections.

[0137] A comparison of probe-based and needle-based quantitative PCR amplification curves of nucleic acid extracted from 10 / 100 / 1000 CFU of Streptococcus pneumoniae isolated from blood samples and nucleic acid directly extracted from 10 / 100 / 1000 CFU of Streptococcus pneumoniae. Figure 4 In the left-hand legend, C-E1, C-E2, and C-E3 correspond to 10, 100, and 1000 CFU of the control group that does not require separation, respectively; E1, E2, and E3 correspond to 10, 100, and 1000 CFU of the experimental group that isolates the pathogen from the blood sample, respectively; BNTC is the negative blood separation and extraction control, and NTC is the amplification control without template.

[0138] Example 7

[0139] To verify the effectiveness of the method and reagents, the reagents and methods described in this invention were used to isolate and extract nucleic acids from simulated bloodstream infection of Candida albicans type pathogens. At the same time, the same number of pathogens were added to the simulated sample as a control. The extracted DNA was then detected by probe-based real-time PCR to verify the feasibility of the invention.

[0140] A reagent for isolating, enriching, and extracting nucleic acids from pathogenic microorganisms in blood, comprising the following reagents: Blood selective lysis buffer A consists of 5 mM phosphate buffer (pH 8), 1% sodium chloride, 20% guanidine hydrochloride, 0.5% Tween 20, 5 mM trisodium citrate, and 0.05% polypropylene glycol. The microbial flocculant is Novec 7500. Blood selective lysis buffer B consists of 5 mM phosphate buffer (pH 8), 0.5% potassium chloride, and 5 mM trisodium citrate. The washing buffer consists of 0.1% phosphate buffer (pH 7.5), 0.9% sodium chloride, 3% ammonium sulfate, and 0.1% trisodium citrate. The nucleic acid extraction buffer consists of 0.2% tris(hydroxymethyl)aminomethane hydrochloride buffer (pH 9), 0.15% potassium chloride, 3% Triton X-100, and 0.05% polypropylene glycol.

[0141] The methods for isolating, enriching, and extracting nucleic acids from Candida albicans in blood are as follows:

[0142] As before, simulated samples containing Candida albicans were prepared by adding 10 CFU / 100 CFU / 1000 CFU of Candida albicans to three 1 mL aspirin-contaminated blood samples, respectively, and mixing thoroughly. Simultaneously, control samples were prepared by adding 10 CFU / 100 CFU / 1000 CFU of Candida albicans to three 100 μL aspirin-contaminated nucleic acid extraction solutions, respectively, and mixing thoroughly. Reagents were added to the prepared simulated samples, and microbial isolation, enrichment, and nucleic acid extraction were performed. Specifically, 1 mL of selective lysis buffer A and 20 μL of microbial sedimentation agent were added to 1 mL of simulated blood sample, vortexed for 1 minute, centrifuged at 12000×g for 2 minutes, and 1.8 mL of supernatant was discarded (200 μL remaining). Then, 1 mL of blood selective lysis buffer B was added, vortexed for 1 minute, centrifuged at 12000×g for 2 minutes, and 1 mL of supernatant was discarded. This step was repeated once or multiple times. 1 mL of washing buffer was added to the sample, vortexed for 1 minute, centrifuged at 12000×g for 2 minutes, and 1 mL of supernatant was discarded. Repeat this step once or multiple times. Finally, add 1 mL of nucleic acid extraction buffer, vortex for 1 minute, centrifuge at 12000×g for 2 minutes, and discard 1 mL of supernatant. Add 30 mg of 0.5 mm zirconia beads, vortex for 5 minutes, heat at 95℃ for 5 minutes, centrifuge at 12000×g for 2 minutes, and collect the supernatant to obtain the nucleic acid of the simulated sample. Simultaneously, add 30 mg of 0.5 mm zirconia beads to the control sample, vortex for 5 minutes, heat at 95℃ for 5 minutes, centrifuge at 12000×g for 2 minutes, and collect the supernatant to obtain the nucleic acid of the control sample. In parallel, perform a blank blood separation and extraction control without the addition of Streptococcus pneumoniae.

[0143] The extracted nucleic acids were prepared according to Table 1 for probe-based PCR detection system preparation and amplification program settings. Specific primer and probe sequences are shown in Table 2. Results are as follows: Figure 5 The results showed that at the three detection gradients, there was no significant difference in amplification between the control and experimental groups in simulated samples of 100 CFU / mL and 1000 CFU / mL. In simulated samples of 10 CFU / mL, the amplification overlap between the two experimental groups was good, and both groups could be stably detected. Negative blood extraction controls and the control group without template showed no amplification, indicating reliable amplification results. This demonstrates that the reagents and methods of this invention can isolate and extract Candida albicans DNA at a concentration of 10 CFU / mL from blood, meeting the requirements for detecting fungal pathogens in bloodstream infections.

[0144] A comparison of probe-based and needle-based quantitative PCR amplification curves of nucleic acid extracted from 10 / 100 / 1000 CFU of Candida albicans isolated from blood samples and nucleic acid directly extracted from 10 / 100 / 1000 CFU of Candida albicans. Figure 5In the left-hand legend, C-E1, C-E2, and C-E3 correspond to 10, 100, and 1000 CFU of the control group that does not require separation, respectively; E1, E2, and E3 correspond to 10, 100, and 1000 CFU of the experimental group that isolates the pathogen from the blood sample, respectively; BNTC is the negative blood separation and extraction control, and NTC is the amplification control without template.

[0145] Table 1. Preparation and amplification procedures for probe-based PCR detection system

[0146]

[0147] Table 2 Primer and probe sequences in the probe-based PCR system

[0148]

[0149]

[0150] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for extracting nucleic acid samples of pathogenic microorganisms from blood, characterized in that: Includes the following steps: S1) Initial lysis of blood samples: Blood selective lysis buffer A and pathogenic microorganism sedimentation agent are added to the blood sample and mixed thoroughly to obtain lysate; the macromolecular organic matter in the lysate is dissolved, and after high-speed centrifugation, part of the supernatant is discarded to obtain the initial lysed blood sample. The lysate includes lysed blood cells and unlysed pathogenic microorganisms with altered surface properties. The blood cells include red blood cells and white blood cells. High-speed centrifugation is used to enrich and precipitate the pathogenic microorganisms. The pathogenic microorganisms mentioned are selected from Escherichia coli type pathogenic microorganisms; The blood selective lysis buffer A consists of 20 mM, pH 7.0 tris(hydroxymethyl)aminomethane hydrochloride buffer, 0.5% by volume of potassium chloride, 65% by volume of guanidine isothiocyanate, 0.2% by volume of sodium dodecyl sarcosinate, 10 mM trisodium citrate, and 0.02% by volume of polyoxypropylene glycerol ether. The pathogenic microorganism sedimentation aid is Novec7500; S2) Blood sample re-lysis: Add blood selective lysis buffer B to the blood sample initially lysed in step S1 to further dissolve the residual macromolecular organic matter. After high-speed centrifugation, discard part of the supernatant at the interface between the microbial flocculant and the aqueous solution to obtain the blood sample re-lysed. The blood selective lysis buffer B consists of 20 mM, pH 7.0 tris(hydroxymethyl)aminomethane hydrochloride buffer, 0.5% potassium chloride, 30% guanidine isothiocyanate, 0.1% sodium lauroyl sarcosinate, 10 mM trisodium citrate, and 0.02% polyoxypropylene glycerol ether. S3) Washing: Add washing solution to the blood lysate sample from step S2, mix well, centrifuge, discard part of the supernatant, and obtain a blood lysate sample containing the unlysaturated pathogenic microorganisms with altered surface characteristics. The washing solution is used to clean impurities. The washing solution consists of 10 mM, pH 7.0 tris(hydroxymethyl)aminomethane hydrochloride buffer, 0.9% sodium chloride, 10% ammonium sulfate, and 3 mM trisodium citrate. S4) Nucleic acid extraction: In the blood lysate sample washed in step S3, add nucleic acid extraction reagent, centrifuge at high speed and discard part of the supernatant. Electrolyze the remaining solution or add grinding beads of different particle sizes, oscillate, heat at high temperature and centrifuge to lyse the pathogenic microorganisms that have not been lysed but whose surface characteristics have been altered, and promote the release and dissolution of nucleic acids in the pathogenic microorganisms that have not been lysed but whose surface characteristics have been altered, to obtain a pathogenic microorganism nucleic acid sample. The nucleic acid extraction reagent consists of 10mM, pH 8.8 tris(hydroxymethyl)aminomethane hydrochloride buffer, 0.45% sodium chloride, 0.1% sodium dodecyl sulfate, 2% Tween 20, 1mM trisodium citrate and 0.02% polyoxypropylene glycerol ether.

2. The extraction method according to claim 1, characterized in that: In step S1, the blood sample is 1-10 ml. After adding the blood selective lysis buffer A and the pathogenic microorganism sedimentation agent, it is shaken and mixed at room temperature or inverted and mixed at room temperature for 1-5 minutes. The amount of blood selective lysis buffer A is 0.5-5 times the volume of the blood sample, and the amount of pathogenic microorganism sedimentation agent is 0.5%-5% of the volume of the blood sample.

3. The extraction method according to claim 1, characterized in that: In step S2, The amount of selective lysis buffer B used in the blood is 2 to 10 times the volume of the initial lysed blood sample. In step S3, The amount of washing solution used is 5 to 10 times the volume of the blood sample after lysis, and the washing solution is used by shaking and mixing at room temperature or by inverting and mixing at room temperature. Steps S2 and S3 can be repeated multiple times.

4. The extraction method according to claim 1, characterized in that: In step S4, The amount of nucleic acid extraction reagent used is 5 to 10 times the volume of the blood lysed sample. The electrolysis is performed using an electrolysis device fabricated by MEMS. The grinding beads are one or more of acid-washed glass beads, zirconia beads, and quartz sand with a particle size of 0.1 to 3 mm. The amount of grinding beads used is 20 to 150 mg, and the shaking time is 5 to 15 minutes. The high-temperature heating temperature is 85 to 100°C, and the heating time is 3 to 15 minutes.

5. The extraction method according to any one of claims 1 to 4, characterized in that: In steps S1, S2, S3 and S4, the centrifugation speed is 10000~16000×g and the centrifugation time is 1~5 minutes.

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