WO2022206726A1 - 一种血液中病原微生物分离、富集和核酸提取方法及试剂 - Google Patents

一种血液中病原微生物分离、富集和核酸提取方法及试剂 Download PDF

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WO2022206726A1
WO2022206726A1 PCT/CN2022/083559 CN2022083559W WO2022206726A1 WO 2022206726 A1 WO2022206726 A1 WO 2022206726A1 CN 2022083559 W CN2022083559 W CN 2022083559W WO 2022206726 A1 WO2022206726 A1 WO 2022206726A1
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blood
nucleic acid
solution
volume
pathogenic microorganisms
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French (fr)
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许雪娇
贺翠婷
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北京源微生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the present invention relates to the technical field of assay or testing methods comprising enzymes, nucleic acids or microorganisms, in particular to a method and reagent for the separation, enrichment and nucleic acid extraction of pathogenic microorganisms in blood.
  • Bloodstream infection is a systemic inflammatory response syndrome caused by bacterial, fungal and other pathogenic microorganisms invading the bloodstream.
  • Pathogenic microorganisms exist in the circulating blood temporarily, intermittently or continuously, causing damage to all organs of the body, such as heart valves, joints, etc. In severe cases, it can lead to shock, multiple organ failure, disseminated intravascular coagulation, and even death. .
  • Sepsis and bacteremia are currently collectively referred to as bloodstream infections.
  • Sepsis is a blood infection caused by various pathogenic microorganisms (bacteria or fungi) and toxins invading the bloodstream.
  • bacteremia which can be divided into two types: primary and secondary.
  • Primary bacteremia is related to intravenous procedures, and secondary bacteremia is mostly caused by postoperative wounds, abdominal cavity, urinary tract and lung infections.
  • secondary bacteremia is mostly caused by postoperative wounds, abdominal cavity, urinary tract and lung infections.
  • invasive devices and treatments such as venous catheter indwelling, mechanical ventilation, and parenteral administration, as well as the abuse of immunosuppressants and a large number of antibiotics
  • the incidence of bloodstream infections has increased year by year.
  • the high mortality rate, prolonged hospital stay, and increased hospitalization costs have attracted more and more attention from doctors, patients and researchers.
  • the usual therapy is to treat patients with broad-spectrum, non-targeted antibiotics. This may be effective in treating the disease, but with the serious consequence that many microorganisms develop multidrug resistance.
  • the culture method requires a large number of blood samples required for diagnosis and the detection sensitivity is low. In addition, there is a relatively high risk of contamination during blood collection and blood sample use. Therefore, although blood culture is still the main reference method for the diagnosis of bloodstream infection, it is not an ideal gold standard due to its significant shortcomings such as low sensitivity and long time.
  • molecular biology diagnostic technology represented by polymerase chain reaction (PCR) and next-generation sequencing technology has been applied to the microbial detection of bloodstream infection.
  • the method based on molecular biology diagnosis has the significant advantages of high speed, high sensitivity, small sample size, providing relevant information on microbial diagnosis in a short time (generally no more than 12 hours), and then introducing appropriate antibiotic treatment (detecting drug resistance genes) and other significant advantages.
  • PCR polymerase chain reaction
  • next-generation sequencing technology has been applied to the microbial detection of bloodstream infection.
  • the method based on molecular biology diagnosis has the significant advantages of high speed, high sensitivity, small sample size, providing relevant information on microbial diagnosis in a short time (generally no more than 12 hours), and then introducing appropriate antibiotic treatment (detecting drug resistance genes) and other significant advantages.
  • the large amount of human nucleic acid in the blood itself may interfere with the detection of PCR and sequencing.
  • the components of the blood itself such as iron, hemoglobin, etc. Coagulants can severely inhibit
  • the present invention is to propose a method and reagent for directly separating and enriching infectious pathogenic microorganisms and extracting nucleic acid from the blood of patients suspected of bloodstream infection (the blood volume of pediatric patients including neonates is 1-2 mL, and the amount of adult blood is 5-10 mL).
  • the blood volume of pediatric patients including neonates is 1-2 mL, and the amount of adult blood is 5-10 mL.
  • No blood culture is required, and blood samples are directly used for pathogen enrichment and nucleic acid extraction. It is also suitable for samples after blood culture.
  • the nucleic acid extraction steps are simple, and can be applied to Gram-negative bacteria, positive bacteria and fungi indiscriminately.
  • the extracted nucleic acid can be With a variety of molecular biology detection methods such as single-plex, multiplex PCR/RT-PCR and next-generation sequencing, the requirements for operating and adapting instruments and equipment are not high, and only a high-speed centrifuge common in laboratories can meet the operating needs.
  • the present invention provides a method for separating, enriching and extracting nucleic acid of pathogenic microorganisms in blood, comprising the following steps:
  • Blood sample re-lysing adding blood selection lysing solution B to further dissolve the residual macromolecular organic matter, and discarding part of the supernatant on the interface between the microbial precipitation aid and the aqueous solution after high-speed centrifugation to obtain a blood re-lysing sample;
  • washing adding washing solution and mixing, centrifuging and discarding part of the supernatant to obtain a blood lysate sample containing pathogenic microorganisms, and the washing solution is used to clean impurities;
  • nucleic acid extraction Add nucleic acid extraction reagents to the blood lysed samples, discard part of the supernatant after high-speed centrifugation, electrolyze the remaining solution or add grinding beads of different particle size combinations, oscillate at high temperature, and centrifuge to lyse pathogenic microorganisms And promote the release and dissolution of nucleic acid in pathogenic microorganisms to obtain nucleic acid samples of pathogenic microorganisms.
  • the blood selection lysate A includes: a buffer with a concentration of 10-100 mM, a mass-volume percentage of 0% ⁇ 6.5% of auxiliary cleavage salt, 5% ⁇ 65% of main cleavage salt by mass volume, 0% ⁇ 5% by mass of surfactant, chelating agent at concentration of 0 ⁇ 10mM and 0% ⁇ 0.5% defoamer, the pH of blood selection lysate A is 4-8;
  • the pathogenic microorganism precipitation aid is a biologically inert organic solvent insoluble in water
  • the blood sample is 1-10ml. After adding blood-selective lysate A and pathogenic microorganism precipitation aid, shake and mix at room temperature or invert at room temperature for 1-5 minutes.
  • the dosage of blood-selective lysate A is 0.5-5 times the volume of the blood sample.
  • the dosage of pathogenic microorganism precipitation aid is 0.5% to 5% of the volume of the blood sample.
  • the blood selection lysate B comprises: a buffer with a concentration of 10-100 mM, a mass-volume percentage of 0% ⁇ 3% of auxiliary cleavage salt, 0% ⁇ 40% of main cleavage salt by mass volume, 0% ⁇ 2% by mass of surfactant, chelating agent at concentration of 0 ⁇ 10mM, and 0% ⁇ 0.5% defoamer, the pH of blood selection lysate B is 5-8;
  • the dosage of blood selection lysis solution B is 2 to 10 times the volume of the initial blood lysis sample
  • the washing liquid includes: a buffer with a concentration of 10-100 mM, a salt promoting impurity dissolution with a mass volume percentage of 0-5% and a chelating agent with a concentration of 0-10 mM, and the pH of the washing liquid is 5-9;
  • the amount of the washing solution is 5-10 times the volume of the blood re-lysed sample, 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 may be repeated multiple times.
  • the nucleic acid extraction solution comprises: a buffer solution with a concentration of 0.5-20 mM, a mass-volume percentage of 0-5% Nucleic acid-promoting salts, surfactants with a mass volume percentage of 0.01% to 5%, chelating agents with a concentration of 0 to 3 mM, and antifoaming agents with a volume percentage of 0% to 0.5%, the pH of the nucleic acid extraction solution is 7 to 9 ;
  • the surfactant is an anionic surfactant and/or a nonionic surfactant
  • the amount of nucleic acid extraction solution is 5 to 10 times the volume of the blood lysis sample.
  • Electrolysis uses a MEMS electrolysis device.
  • the grinding beads are one of acid-washed glass beads, zirconia beads, and quartz sand with a particle size of 0.1 to 3 mm. Or several kinds, the amount of grinding beads is 20-150 mg, the shaking time is 5-15 minutes, the high-temperature heating temperature is 85-100°C, and the heating time is 3-15 minutes.
  • the buffer is any one of the following: phosphate buffer, sodium acetate acetic acid buffer and tris-hydroxymethylaminomethane hydrochloric acid buffer liquid;
  • the auxiliary cleavage salt, the nucleic acid-promoting salt and the impurity-promoting salt are one or more of the following: sodium chloride, potassium chloride and ammonium sulfate;
  • the main cracking salts are one or more of the following: lithium chloride, sodium iodide, potassium iodide, guanidine hydrochloride and guanidine isothiocyanate;
  • the defoamer is any one of the following: polydimethylsiloxane, polypropylene glycol, polyether-modified polysiloxane and polyoxypropylene glyceryl ether;
  • the chelating agent is trisodium citrate
  • the anionic surfactant is any one of the following: sodium lauryl sulfate, sodium lauryl sarcosinate and sodium deoxycholate, and the nonionic surfactant is one or more of the following: Tween 20, Tween 80. Triton X-100, ethyl phenyl polyethylene glycol and cetyl alcohol polyoxyethylene ether Brij C20;
  • the centrifugation speed is 10000-16000 ⁇ g, and the centrifugation time is 1-5 minutes.
  • the present invention provides a reagent for the separation, enrichment and nucleic acid extraction of pathogenic microorganisms in blood, comprising a blood selection lysate A with a volume of 0.5-10 times the blood sample, a pathogenic microorganism sedimentation aid with a volume of 0.5-10% of the blood sample, Blood selection lysis solution B with a volume of 1 to 10 times the blood sample, washing solution with a volume of 1 to 10 times of the blood sample, and nucleic acid extraction solution of 0.5 to 2 times the volume of the blood sample;
  • Blood selection lysis solution A and blood selection lysis solution B are used to lyse only blood cells to retain pathogenic microorganisms and dissolve macromolecular organic matter released in blood samples and lysed blood cells; pathogenic microorganism precipitation aids are used to enrich and precipitate pathogenic microorganisms;
  • the washing solution is used to clean impurities, and the nucleic acid extraction solution is used to enrich and lyse pathogenic microorganisms and promote the release and dissolution of nucleic acids in pathogenic microorganisms;
  • Blood selection lysis solution A includes: buffer with a concentration of 10-100 mM, auxiliary lysis salt with a mass volume percentage of 0% to 6.5%, a main lysis salt with a mass volume percentage of 5% to 65%, and a mass volume percentage of 0% ⁇ 5% surfactant, chelating agent with a concentration of 0 ⁇ 10mM, and antifoaming agent with a volume percentage of 0% ⁇ 0.5%, the pH of blood selection lysate A is 4 ⁇ 8;
  • the pathogenic microorganism precipitation aid is a biologically inert organic solvent insoluble in water
  • the blood selection lysis solution B includes: buffer with a concentration of 10-100 mM, auxiliary lysis salt with a mass volume percentage of 0% to 3%, a main lysis salt with a mass volume percentage of 0% to 40%, and a mass volume percentage of 0% ⁇ 2% surfactant, chelating agent with concentration of 0 ⁇ 10mM and antifoaming agent with volume percentage of 0% ⁇ 0.5%, pH of blood selection lysate B is 5 ⁇ 8.
  • the washing solution includes: a buffer solution with a concentration of 10-100 mM, and a mass-volume percent of 0%-5% to promote the dissolution of impurities Salt and chelating agent with a concentration of 0 to 10 mM, and the pH of the washing solution is 5 to 9;
  • the nucleic acid extraction solution includes: a buffer with a concentration of 0.5-20 mM, a salt promoting nucleic acid dissolution with a concentration of 0-5% by volume, a surfactant with a concentration of 0.01-5% by volume, and a chelating agent with a concentration of 0-3 mM and 0% to 0.5% of the defoaming agent by volume, the pH of the nucleic acid extraction solution is 7 to 9;
  • the biologically inert organic solvent is fluorine oil or HFE-7500 or Novec7500 or FC-40.
  • the buffer is any one of the following: phosphate buffer, sodium acetate acetic acid buffer, tris-hydroxymethylaminomethane hydrochloric acid buffer liquid;
  • the auxiliary cleavage salt, the nucleic acid-promoting salt and the impurity-promoting salt are one or more of the following: sodium chloride, potassium chloride and ammonium sulfate;
  • the main cracking salts are one or more of the following: lithium chloride, sodium iodide, potassium iodide, guanidine hydrochloride and guanidine isothiocyanate;
  • the defoamer is any one of the following: polydimethylsiloxane, polypropylene glycol, polyether-modified polysiloxane and polyoxypropylene glyceryl ether;
  • the chelating agent is trisodium citrate
  • the surfactants are anionic surfactants and/or nonionic surfactants.
  • the anionic surfactant is any one of the following: sodium lauryl sulfate, sodium lauryl sarcosinate and Sodium deoxycholate
  • nonionic surfactant is one or more of the following: Tween 20, Tween 80, Triton X-100, ethylphenyl polyethylene glycol and cetyl ethoxylate Brij C20 .
  • the blood selection lysate A includes 20mM tris hydrochloric acid buffer, 0.5% potassium chloride by mass and volume , 65% by volume of guanidine isothiocyanate, 0.2% by volume of sodium lauryl sarcosinate, 10 mM trisodium citrate and 0.02% by volume of polyoxypropylene glycerol, blood selection lysate A
  • the pH is 7.0;
  • Blood selection lysate B includes 20 mM Tris hydrochloride buffer, 0.5 wt% potassium chloride, 30 wt% guanidine isothiocyanate, 0.1 wt% dodecyl muscle Sodium citrate, 10 mM trisodium citrate and 0.02% volume percent polyoxypropylene glyceryl ether, the pH of blood selection lysate B is 7.0;
  • the microbial precipitation aid is Novec7500
  • the washing solution includes 10 mM tris hydrochloride buffer, 0.9% sodium chloride by mass and volume, 10% ammonium sulfate by mass and 3 mM trisodium citrate, and the pH of the washing solution is 7.0;
  • the nucleic acid extraction solution includes 10 mM tris hydrochloride buffer, 0.45% wt sodium chloride, 0.1 wt% sodium dodecyl sulfate, 2 wt% Tween 20, 1mM trisodium citrate and 0.02% by volume polyoxypropylene glycerol ether, the pH of the nucleic acid extraction solution is 8.8.
  • the specific plan is: adding blood selection lysis solution A and pathogenic microorganism precipitation aid to the blood sample for sample lysis treatment, selectively lysing blood cells (including red blood cells and white blood cells, etc.), and dissolving impurities such as proteins, carbohydrates and lipids in the blood. After that, the pathogenic microorganisms were sedimented by high-speed centrifugation, and part of the supernatant was carefully discarded; blood selective lysis solution B was added to perform one or two blood selective lysis and high-speed centrifugation to discard part of the supernatant on the interface between the microbial sedimentation aid and the aqueous solution.
  • nucleic acid extraction reagent to process the residual solution again, discard part of the supernatant after high-speed centrifugation, pathogenic microorganisms In the remaining nucleic acid extraction solution; electrolysis or adding grinding beads with different particle size combinations, after high temperature heating and centrifugation, the nucleic acid of pathogenic microorganisms is extracted.
  • the dosage of the blood selection lysate A is 0.5-5 times the volume of the blood sample
  • the dosage of the microbial precipitation aid is 0.5%-5% of the volume of the blood sample
  • the lysis condition is normal temperature oscillation Mix well or invert at room temperature for 1 to 5 minutes, centrifuge at 10,000 to 16,000 ⁇ g, and centrifuge for 1 to 5 minutes. Discard the supernatant and use a pipette or syringe. Do not touch the two phases of the microbial precipitation aid and the aqueous solution. For the precipitate at the junction, the amount of solution discarded is 80% to 95% of the total solution volume. .
  • the amount of blood selection lysis solution B is 2 to 10 times the volume of the remaining supernatant, and the conditions of lysis are vibration mixing at room temperature or inversion mixing at room temperature for 1 to 5 minutes, and the centrifugal speed is 10,000 ⁇ 16000 ⁇ g, centrifugation time is 1-5 minutes, discard the supernatant and use a pipette or a syringe, etc., do not touch the sediment at the junction of the microbial precipitation aid and the aqueous solution, the amount of solution discarded is equivalent to the blood selection lysate
  • the amount of B added, this cracking step is repeated one or more times according to the precipitation properties.
  • the dosage of the washing solution is 5-10 times the volume of the remaining supernatant
  • the washing conditions are shaking and mixing at room temperature or inverting and mixing at room temperature for 1-5 minutes
  • the centrifugal speed is 10,000-16,000 ⁇ g.
  • the centrifugation time is 1 to 5 minutes, discard the supernatant and use a pipette or a syringe, etc., do not touch the sediment located at the junction of the microbial precipitation aid and the aqueous solution, and the amount of solution discarded is equal to the amount of washing solution added.
  • the lysis step is repeated one or more times, depending on the precipitation profile.
  • the amount of the nucleic acid extraction solution is 5-10 times the volume of the remaining supernatant
  • the processing conditions are shaking and mixing at room temperature or inverting and mixing at room temperature for 1-5 minutes
  • the centrifugal speed is 10,000-16,000 ⁇ g.
  • the centrifugation time is 1 to 5 minutes. Discard the supernatant and use a pipette or syringe. Do not touch the sediment located at the junction of the microbial precipitation aid and the aqueous solution.
  • the amount of solution discarded is equal to the amount of nucleic acid extraction solution added.
  • the remaining supernatant and pellet contain pathogenic microorganisms enriched from the sample.
  • the grinding beads are acid-washed glass beads or zirconia beads with a combination of different particle sizes of 0.1-3 mm, the dosage is 20-150 mg, the oscillation time is 5-15 minutes, and the high-temperature heating temperature is 85 ⁇ 100°C, heating time is 3 ⁇ 15 minutes, centrifugal speed is 10000 ⁇ 16000 ⁇ g, centrifugation time is 1 ⁇ 5 minutes, after centrifugation, use a pipette or a syringe to suck the supernatant to be the target nucleic acid.
  • a microfabricated electrolysis device is used for nucleic acid extraction, and the size can be (0.1 ⁇ 1) ⁇ (5 ⁇ 1000) ⁇ (10 ⁇ 1000) mm 3 , and the size can be larger or smaller according to requirements Size of this design.
  • the cracking voltage is 10-2000V.
  • the blood sample is the blood of a patient suspected of bloodstream infection, the amount of blood for pediatric patients including neonates is 1-2 mL, and the amount of blood for adults is 5-10 mL; the sample can also be a sample after blood culture, and the dosage is 0.2-2 mL 1mL.
  • the present invention disrupts blood cells (including red blood cells and white blood cells, etc.) by selecting the osmotic pressure difference between the lysate A and blood and the addition of surfactants, and at the same time dissolves the blood and the large amount released by the rupture of blood cells as much as possible at a suitable concentration.
  • Molecular organic matter, etc. and will not cause serious damage to the cellular structure of pathogenic microorganisms.
  • the salts and surfactants can also promote the enrichment and sedimentation of pathogenic microorganisms that are difficult to be centrifuged due to their special extracellular structure (such as bacterial capsule) together with microbial precipitation aids, reducing the differential enrichment of microorganisms.
  • the microbial sedimentation aid can also reduce the adhesion of microorganisms to the centrifuge tube, and avoid the loss of microorganisms due to incomplete centrifugal sedimentation.
  • the addition of blood selection lysis solution B makes up for incomplete lysis treatment caused by sample differences and fully dissolves residual impurities.
  • the wash solution removes lysate components and blood insoluble substances present in the lysate that may inhibit downstream assays.
  • a large amount of nucleic acid extraction solution dilutes the salts in the washing solution and provides conditions suitable for the lysis of pathogenic microorganisms and the dissolution and preservation of nucleic acids. Under the action of instantaneous electrolysis or physical grinding of grinding beads of different sizes and high temperature heating denaturation, pathogenic microorganisms are fully lysed Promote the release and solubilization of nucleic acids therein.
  • Macromolecular organic matter includes proteins, carbohydrates, lipids and nucleic acids.
  • the nucleic acid of the target infectious pathogen that can meet the requirements of molecular biology detection can be quickly and accurately extracted from a blood sample of no more than 10 mL.
  • Pathogen enrichment and nucleic acid extraction can be carried out directly using blood samples without blood culture, and it is also suitable for samples after blood culture.
  • the nucleic acid extraction procedure is simple, and can be applied to Gram-negative bacteria, positive bacteria and fungi indiscriminately.
  • the extracted nucleic acid can be matched with a variety of molecular biology detection methods such as single-plex, multiplex PCR/RT-PCR and next-generation sequencing.
  • Fig. 1 is a flow chart of a method for separating, enriching and extracting nucleic acid of pathogenic microorganisms in blood;
  • FIG. 2 is a schematic diagram of a method for separating, enriching and extracting nucleic acid of pathogenic microorganisms in blood;
  • Fig. 3 is the amplification curve of a kind of pathogenic microorganism separation, enrichment and nucleic acid extraction method and reagent embodiment 5 in blood by probe method fluorescence quantitative PCR;
  • Fig. 4 is the amplification curve of a kind of blood pathogenic microorganism separation, enrichment and nucleic acid extraction method and reagent Embodiment 6 probe method fluorescence quantitative PCR;
  • FIG. 5 is an amplification curve of a method for isolating, enriching, and nucleic acid extraction of pathogenic microorganisms in blood and reagents Example 7 of probe-based fluorescence quantitative PCR.
  • a method for separating, enriching and extracting nucleic acid of pathogenic microorganisms in blood comprises the following steps:
  • Blood sample re-lysing adding blood selection lysing solution B to further dissolve the residual macromolecular organic matter, and discarding part of the supernatant on the interface between the microbial precipitation aid and the aqueous solution after high-speed centrifugation to obtain a blood re-lysing sample;
  • washing adding washing solution and mixing, centrifuging and discarding part of the supernatant to obtain a blood lysate sample containing pathogenic microorganisms, and the washing solution is used to clean impurities;
  • nucleic acid extraction Add nucleic acid extraction reagents to the blood lysed samples, discard part of the supernatant after high-speed centrifugation, electrolyze the remaining solution or add grinding beads of different particle size combinations, oscillate at high temperature, and centrifuge to lyse pathogenic microorganisms And promote the release and dissolution of nucleic acid in pathogenic microorganisms to obtain nucleic acid samples of pathogenic microorganisms.
  • a method for separating, enriching and extracting nucleic acid of pathogenic microorganisms in blood comprises the following steps:
  • Blood selection lysis solution A includes: buffer with a concentration of 10-100 mM, auxiliary lysis salt with a mass volume percentage of 0% to 6.5%, a main lysis salt with a mass volume percentage of 5% to 65%, and a mass volume percentage of 0% ⁇ 5% surfactant, chelating agent with a concentration of 0 ⁇ 10mM, and antifoaming agent with a volume percentage of 0% ⁇ 0.5%, the pH of blood selection lysate A is 4 ⁇ 8;
  • the pathogenic microorganism precipitation aid is a biologically inert organic solvent insoluble in water
  • the blood sample is 1-10ml. After adding blood-selective lysate A and pathogenic microorganism precipitation aid, shake and mix at room temperature or invert at room temperature for 1-5 minutes.
  • the dosage of blood-selective lysate A is 0.5-5 times the volume of the blood sample.
  • the dosage of pathogenic microorganism precipitation aid is 0.5% to 5% of the blood sample volume;
  • Blood sample re-lysing adding blood selection lysing solution B to further dissolve the residual macromolecular organic matter, and discarding part of the supernatant on the interface between the microbial precipitation aid and the aqueous solution after high-speed centrifugation to obtain a blood re-lysing sample;
  • the blood selection lysis solution B includes: buffer with a concentration of 10-100 mM, auxiliary lysis salt with a mass volume percentage of 0% to 3%, a main lysis salt with a mass volume percentage of 0% to 40%, and a mass volume percentage of 0% ⁇ 2% surfactant, chelating agent with concentration of 0 ⁇ 10mM and antifoaming agent with volume percentage of 0% ⁇ 0.5%, pH of blood selection lysate B is 5 ⁇ 8;
  • the dosage of blood selection lysis solution B is 2 to 10 times the volume of the initial blood lysis sample
  • Step S2 can be repeated many times
  • washing adding washing solution and mixing, centrifuging and discarding part of the supernatant to obtain a blood lysate sample containing pathogenic microorganisms, and the washing solution is used to clean impurities;
  • the washing liquid includes: a buffer with a concentration of 10-100 mM, a salt promoting impurity dissolution with a mass volume percentage of 0-5% and a chelating agent with a concentration of 0-10 mM, and the pH of the washing liquid is 5-9;
  • the amount of the washing solution is 5-10 times the volume of the blood re-lysed sample, and the washing solution is used by shaking and mixing at room temperature or by inverting and mixing at room temperature;
  • Step S3 can be repeated many times
  • nucleic acid extraction Add nucleic acid extraction reagents to the blood lysed samples, discard part of the supernatant after high-speed centrifugation, electrolyze the remaining solution or add grinding beads of different particle size combinations, oscillate at high temperature, and centrifuge to lyse pathogenic microorganisms And promote the release and dissolution of nucleic acid in pathogenic microorganisms to obtain nucleic acid samples of pathogenic microorganisms;
  • the nucleic acid extraction solution includes: a buffer with a concentration of 0.5-20 mM, a salt promoting nucleic acid dissolution with a concentration of 0-5% by volume, a surfactant with a concentration of 0.01-5% by volume, and a chelating agent with a concentration of 0-3 mM and 0% to 0.5% of the defoaming agent by volume, the pH of the nucleic acid extraction solution is 7 to 9;
  • the surfactant is an anionic surfactant and/or a nonionic surfactant
  • the amount of nucleic acid extraction solution is 5 to 10 times the volume of the blood lysis sample.
  • Electrolysis uses a MEMS electrolysis device.
  • the grinding beads are one of acid-washed glass beads, zirconia beads, and quartz sand with a particle size of 0.1 to 3 mm. or several kinds, the amount of grinding beads is 20-150 mg, the oscillation time is 5-15 minutes, the high-temperature heating temperature is 85-100°C, and the heating time is 3-15 minutes;
  • the buffer is any one of the following: phosphate buffer, sodium acetate acetic acid buffer and tris hydrochloric acid buffer;
  • the auxiliary cleavage salt, the nucleic acid-promoting salt and the impurity-promoting salt are one or more of the following: sodium chloride, potassium chloride and ammonium sulfate;
  • the main cracking salts are one or more of the following: lithium chloride, sodium iodide, potassium iodide, guanidine hydrochloride and guanidine isothiocyanate;
  • the defoamer is any one of the following: polydimethylsiloxane, polypropylene glycol, polyether-modified polysiloxane and polyoxypropylene glyceryl ether;
  • the chelating agent is trisodium citrate
  • the anionic surfactant is any one of the following: sodium lauryl sulfate, sodium lauryl sarcosinate and sodium deoxycholate, and the nonionic surfactant is one or more of the following: Tween 20, Tween 80. Triton X-100, ethyl phenyl polyethylene glycol and cetyl alcohol polyoxyethylene ether Brij C20;
  • the centrifugation speed is 10000-16000 ⁇ g, and the centrifugation time is 1-5 minutes.
  • a reagent for separation, enrichment and nucleic acid extraction of pathogenic microorganisms in blood comprising a blood selection lysate A with a volume of 0.5 to 10 times that of a blood sample, a pathogenic microorganism sedimentation aid with a volume of 0.5 to 10 percent of the blood sample, and a volume of blood Blood selection lysate B with a volume of 1 to 10 times the sample, washing solution with a volume of 1 to 10 times of the blood sample, and nucleic acid extraction solution of 0.5 to 2 times the volume of the blood sample;
  • Blood selection lysis solution A and blood selection lysis solution B are used to lyse only blood cells to retain pathogenic microorganisms and dissolve macromolecular organic matter released in blood samples and lysed blood cells; pathogenic microorganism precipitation aids are used to enrich and precipitate pathogenic microorganisms;
  • the washing solution is used to clean impurities, and the nucleic acid extraction solution is used to enrich and lyse pathogenic microorganisms and promote the release and dissolution of nucleic acids in pathogenic microorganisms;
  • Blood selection lysis solution A includes: buffer with a concentration of 10-100 mM, auxiliary lysis salt with a mass volume percentage of 0% to 6.5%, a main lysis salt with a mass volume percentage of 5% to 65%, and a mass volume percentage of 0% ⁇ 5% surfactant, chelating agent with a concentration of 0 ⁇ 10mM, and antifoaming agent with a volume percentage of 0% ⁇ 0.5%, the pH of blood selection lysate A is 4 ⁇ 8;
  • the pathogenic microorganism precipitation aid is a biologically inert organic solvent insoluble in water
  • the blood selection lysis solution B includes: buffer with a concentration of 10-100 mM, auxiliary lysis salt with a mass volume percentage of 0% to 3%, a main lysis salt with a mass volume percentage of 0% to 40%, and a mass volume percentage of 0% ⁇ 2% surfactant, chelating agent with concentration of 0 ⁇ 10mM and antifoaming agent with volume percentage of 0% ⁇ 0.5%, pH of blood selection lysate B is 5 ⁇ 8.
  • a reagent for separation, enrichment and nucleic acid extraction of pathogenic microorganisms in blood comprising a blood selection lysate A with a volume of 10 times the blood sample, a pathogenic microorganism sedimentation aid with a volume of 10% of the blood sample, and blood with a volume of 10 times the blood sample Select lysis solution B, washing solution with a volume of 10 times that of the blood sample, and nucleic acid extraction solution with a volume of 2 times that of the blood sample;
  • Blood selection lysis solution A and blood selection lysis solution B are used to lyse only blood cells to retain pathogenic microorganisms and dissolve macromolecular organic matter released in blood samples and lysed blood cells; pathogenic microorganism precipitation aids are used to enrich and precipitate pathogenic microorganisms;
  • the washing solution is used to clean impurities, and the nucleic acid extraction solution is used to enrich and lyse pathogenic microorganisms and promote the release and dissolution of nucleic acids in pathogenic microorganisms;
  • Blood selection lysis solution A includes: buffer with a concentration of 10-100 mM, auxiliary lysis salt with a mass volume percentage of 0% to 6.5%, a main lysis salt with a mass volume percentage of 5% to 65%, and a mass volume percentage of 0% ⁇ 5% surfactant, chelating agent with a concentration of 0 ⁇ 10mM, and antifoaming agent with a volume percentage of 0% ⁇ 0.5%, the pH of blood selection lysate A is 4 ⁇ 8;
  • the pathogenic microorganism precipitation aid is a biologically inert organic solvent insoluble in water
  • Biologically inert organic solvent is fluorine oil or HFE-7500 or Novec7500 or FC-40;
  • the blood selection lysis solution B includes: buffer with a concentration of 10-100 mM, auxiliary lysis salt with a mass volume percentage of 0% to 3%, a main lysis salt with a mass volume percentage of 0% to 40%, and a mass volume percentage of 0% ⁇ 2% surfactant, chelating agent with concentration of 0 ⁇ 10mM and antifoaming agent with volume percentage of 0% ⁇ 0.5%, pH of blood selection lysate B is 5 ⁇ 8; washing solution includes: concentration of 10 ⁇ 100mM Buffer solution, 0% to 5% mass volume percentage of salts that promote the dissolution of impurities and chelating agents with a concentration of 0 to 10mM, and the pH of the washing solution is 5 to 9;
  • the nucleic acid extraction solution includes: a buffer with a concentration of 0.5-20 mM, a salt promoting nucleic acid dissolution with a concentration of 0-5% by volume, a surfactant with a concentration of 0.01-5% by volume, and a chelating agent with a concentration of 0-3 mM and 0% to 0.5% of the defoaming agent by volume, the pH of the nucleic acid extraction solution is 7 to 9;
  • the buffer is any one of the following: phosphate buffer, sodium acetate acetic acid buffer, tris hydrochloric acid buffer;
  • the auxiliary cleavage salt, the nucleic acid-promoting salt and the impurity-promoting salt are one or more of the following: sodium chloride, potassium chloride and ammonium sulfate;
  • the main cracking salts are one or more of the following: lithium chloride, sodium iodide, potassium iodide, guanidine hydrochloride and guanidine isothiocyanate;
  • the defoamer is any one of the following: polydimethylsiloxane, polypropylene glycol, polyether-modified polysiloxane and polyoxypropylene glyceryl ether;
  • the chelating agent is trisodium citrate
  • the surfactant is an anionic surfactant and/or a nonionic surfactant
  • the anionic surfactant is any one of the following: sodium lauryl sulfate, sodium lauryl sarcosinate and sodium deoxycholate, and the nonionic surfactant is one or more of the following: Tween 20, Tween 80. Triton X-100, ethylphenyl polyethylene glycol and cetyl alcohol polyoxyethylene ether Brij C20.
  • the reagents and methods described in the present invention were used to isolate and extract the nucleic acid of Escherichia coli-type pathogenic microorganisms simulating bloodstream infection, and at the same time, the nucleic acid extracted by adding the same amount of pathogenic microorganisms to the simulated samples was used as the sample.
  • the extracted DNA is detected by the probe method fluorescent quantitative PCR, so as to verify the feasibility of the present invention.
  • the PCR detection target is the DNA of the pathogenic microorganism in the extracted nucleic acid.
  • the isolated nucleic acid of the present invention also includes the RNA of the pathogenic microorganism. According to the different detection primers and probe design sites, detection such as qRT-PCR can also be performed.
  • detection method here does not limit other downstream applications of the present invention.
  • a reagent for separation, enrichment and nucleic acid extraction of pathogenic microorganisms in blood the reagents used are as follows: blood selection lysate A is 20mM tris hydrochloric acid buffer solution (pH 7.0), 0.5% potassium chloride, 65% isobaric acid Guanidine thiocyanate, 0.2% sodium lauryl sarcosinate, 10 mM trisodium citrate, and 0.02% polyoxypropylene glyceryl ether.
  • the microbial precipitation aid is Novec7500.
  • Blood selection lysate B is 20 mM Tris-HCl 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 solution was 10 mM Tris hydrochloride buffer (pH 7.0), 0.9% sodium chloride, 10% ammonium sulfate and 3 mM trisodium citrate.
  • Nucleic acid extraction solution was 10 mM Tris 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.
  • 1 mL of blood selection lysate A and 20 ⁇ L of pathogenic microorganism sedimentation aid were added to 1 mL of simulated blood sample, shaken for 1 minute, centrifuged at 12,000 ⁇ g for 2 minutes, and 1.8 mL of supernatant (remaining 200 ⁇ L) was discarded. Then add 1 mL of blood selection lysate B, shake for 1 minute, centrifuge at 12,000 ⁇ g for 2 minutes, and discard 1 mL of supernatant. Repeat this step one or more times. Add 1 mL of washing solution to the sample, shake for 1 minute, centrifuge at 12,000 ⁇ g for 2 minutes, and discard 1 mL of supernatant.
  • nucleic acid extraction solution was added, shaken for 1 minute, centrifuged at 12,000 ⁇ g for 2 minutes, and 1 mL of supernatant was discarded.
  • the negative blood extraction control and the control without template were not amplified, and the amplification results were reliable. Therefore, it is shown that the reagent and method of the present invention can be used to separate and extract E. coli DNA of the order of 10 CFU in blood, which meets the requirements for blood sample detection of bloodstream infection.
  • C ⁇ E1, C ⁇ E2, C ⁇ E3 in the legend on the left side of Figure 3 correspond to 10, 100, 1000 CFU of the control group without separation, respectively, and E1, E2, and E3 correspond to the 10, 100, 100, 1000CFU; BNTC is the control for negative blood separation and extraction, and NTC is the control without template amplification.
  • the reagents and methods of the present invention were used to isolate and extract nucleic acid from pathogenic microorganisms of Streptococcus pneumoniae type simulating bloodstream infection.
  • the extracted DNA was detected by the probe method fluorescence quantitative PCR to verify the feasibility of the present invention.
  • a reagent for separation, enrichment and nucleic acid extraction of pathogenic microorganisms in blood the reagents used are as follows: blood selection lysate A is 5mM phosphate buffer solution (pH7.5), 1.5% ammonium sulfate, 12% sodium iodide, 10mM citric acid trisodium Sodium, 0.5% Triton X-100 and 0.03% Polyoxypropylene Glyceryl Ether. The microbial precipitation aid is FC-40.
  • Blood Selection Lysate B was 5 mM phosphate buffer (pH 7.5), 0.9% sodium chloride, 4% sodium iodide, 5 mM trisodium citrate and 0.03% polyoxypropylene glyceryl ether.
  • the washing solution was 2 mM phosphate buffer (pH 8.5), 0.9% sodium chloride, 3% ammonium sulfate, 5 mM trisodium citrate.
  • the nucleic acid extraction solution was 5 mM phosphate buffer (pH 8.0), 0.45% sodium chloride, 0.1% sodium lauryl sarcosinate, 5% Tween 20, 0.5 mM trisodium citrate and 0.1% polypropylene glycol.
  • the isolation, enrichment and nucleic acid extraction methods of Streptococcus pneumoniae in blood are as follows:
  • 1 mL of selected lysate A and 20 ⁇ L of microbial sedimentation aid were added to 1 mL of simulated blood sample, shaken for 1 minute, centrifuged at 12,000 ⁇ g for 2 minutes, and 1.8 mL of supernatant (the remaining 200 ⁇ L) was discarded. Then add 1 mL of blood selection lysate B, shake for 1 minute, centrifuge at 12,000 ⁇ g for 2 minutes, and discard 1 mL of supernatant. Repeat this step one or more times. Add 1 mL of washing solution to the sample, shake for 1 minute, centrifuge at 12,000 ⁇ g for 2 minutes, and discard 1 mL of supernatant. Repeat this step one or more times.
  • nucleic acid extraction solution was added, shaken for 1 minute, centrifuged at 12,000 ⁇ g for 2 minutes, and 1 mL of supernatant was discarded.
  • 30 mg of 0.5 mm zirconia beads were added to the control sample, vortexed for 5 minutes, heated at 95°C for 5 minutes, centrifuged at 12,000 ⁇ g for 2 minutes, and the supernatant was collected to obtain the nucleic acid of the control sample.
  • a blank blood separation extraction control without S. pneumoniae was performed.
  • the proposed nucleic acid was prepared according to Table 1 for the preparation of the probe-based PCR detection system and the setting of the amplification program on the computer.
  • the specific primer and probe sequences are shown in Table 2.
  • the results are shown in Figure 4.
  • the CT value of the control group compared with the experimental group was delayed compared with the control group, although the CT value of the experimental group was delayed, but they could be stably detected, and the negative blood extraction control and No template control was added without amplification, and the amplification results were reliable and reproducible.
  • the reagent and method of the present invention can be used to separate and extract Streptococcus pneumoniae DNA with 10 CFU per milliliter of blood in blood, which can meet the requirement of blood sample detection of Gram-negative pathogens in bloodstream infection.
  • C-E1, C-E2, and C-E3 in the legend on the left side of Figure 4 correspond to 10, 100, and 1000 CFU of the control group without separation, respectively, and E1, E2, and E3 correspond to the 10, 100, 100, 1000CFU; BNTC is the control for negative blood separation and extraction, and NTC is the control without template amplification.
  • the reagent and method of the present invention were used to isolate and extract the nucleic acid of the pathogenic microorganisms of Candida albicans in the simulated bloodstream infection.
  • the extracted DNA was detected by the probe method fluorescence quantitative PCR to verify the feasibility of the present invention.
  • a reagent for separation, enrichment and nucleic acid extraction of pathogenic microorganisms in blood the reagents used are as follows: blood selection lysate A is 5mM phosphate buffer solution (pH8), 1% sodium chloride, 20% guanidine hydrochloride, 0.5% Tween 20, 5mM trisodium citrate and 0.05% polypropylene glycol. The microbial precipitation aid is Novec7500. Blood selection lysate B was 5 mM phosphate buffer (pH 8), 0.5% potassium chloride, 5 mM trisodium citrate.
  • the washing solution was 0.1% phosphate buffer (pH 7.5), 0.9% sodium chloride, 3% ammonium sulfate, 0.1% trisodium citrate.
  • the nucleic acid extraction solution was 0.2% tris buffer (pH 9), 0.15% potassium chloride, 3% triton X-100 and 0.05% polypropylene glycol.
  • the isolation, enrichment and nucleic acid extraction methods of Candida albicans in blood are as follows:
  • a mock sample containing Candida albicans prepare a mock sample containing Candida albicans, add 10CFU/100CFU/1000CFU of Candida albicans to three 1mL negative blood, respectively, mix well for later use; at the same time prepare a control sample and add it to three 100 ⁇ L nucleic acid extracts respectively 10CFU/100CFU/1000CFU of Candida albicans, mix well for use. Add reagents to the prepared mock samples and perform operations such as microbial isolation, enrichment, and nucleic acid extraction.
  • 1 mL of selected lysate A and 20 ⁇ L of microbial sedimentation aid were added to 1 mL of simulated blood sample, shaken for 1 minute, centrifuged at 12,000 ⁇ g for 2 minutes, and 1.8 mL of supernatant (the remaining 200 ⁇ L) was discarded. Then add 1 mL of blood selection lysate B, shake for 1 minute, centrifuge at 12,000 ⁇ g for 2 minutes, and discard 1 mL of supernatant. Repeat this step one or more times. Add 1 mL of washing solution to the sample, shake for 1 minute, centrifuge at 12,000 ⁇ g for 2 minutes, and discard 1 mL of supernatant. Repeat this step one or more times.
  • nucleic acid extraction solution was added, shaken for 1 minute, centrifuged at 12,000 ⁇ g for 2 minutes, and 1 mL of supernatant was discarded.
  • 30 mg of 0.5 mm zirconia beads were added to the control sample, vortexed for 5 minutes, heated at 95°C for 5 minutes, centrifuged at 12,000 ⁇ g for 2 minutes, and the supernatant was collected to obtain the nucleic acid of the control sample.
  • a blank blood separation extraction control without the addition of Streptococcus pneumoniae was performed.
  • the proposed nucleic acid was prepared according to Table 1 for the preparation of the probe-based PCR detection system and the setting of the amplification program on the computer.
  • the specific primer and probe sequences are shown in Table 2.
  • the results are shown in Figure 5.
  • the amplification difference between the control group and the experimental group was not obvious. Good and stable detection.
  • the negative blood extraction control and the control group without template were not amplified, and the amplification results were reliable. This shows that the reagent and method of the present invention can be used to separate and extract Candida albicans DNA with 10 CFU per milliliter of blood in blood, and the reagent and method meet the blood sample detection requirements for fungal pathogens in bloodstream infections.
  • C-E1, C-E2, and C-E3 in the legend on the left side of Figure 5 correspond to 10, 100, and 1000 CFU of the control group without separation, respectively, and E1, E2, and E3 correspond to the 10, 100, 1000CFU; BNTC is the control for negative blood separation and extraction, and NTC is the control without template amplification.

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Abstract

提供一种血液中病原微生物分离、富集和核酸提取方法及试剂。该方法首先将血液样本中加入血液选择裂解液A和病原微生物助沉剂选择性裂解血细胞并通过高速离心沉降病原微生物，然后加入血液选择裂解液B进行再次血液样本选择裂解，再加入洗涤液混匀后洗涤，最后加入核酸提取试剂，离心富集病原微生物后配合物理方法裂解，得到病原微生物核酸样本。该核酸提取方法可从不大于10mL的血液样品中，快速准确的提取到能满足分子生物学检测要求的目标感染病原体核酸，核酸提取步骤简单，无需复杂处理设备，所得核酸可用于多种分子生物学下游检测。

Description

一种血液中病原微生物分离、富集和核酸提取方法及试剂 技术领域
本发明涉及包含酶、核酸或微生物的测定或检验方法技术领域，具体涉及一种血液中病原微生物分离、富集和核酸提取方法及试剂。
背景技术
血流感染是指由细菌、真菌等病原微生物入侵血流所致的全身性炎性反应综合征。病原微生物在循环血液中呈一过性、间歇性或持续性存在，对机体所有器官，如心脏瓣膜、关节等造成损害，严重者可导致休克、多器官衰竭，弥散性血管内凝血，甚至死亡。目前将败血症和菌血症统称为血流感染。败血症是由各种病原微生物(细菌或真菌)和毒素侵人血流所引起的血液感染。若细菌仅短暂人血而无临床明显的毒血症状则称为菌血症，可分为原发性和继发性两种。原发性菌血症与静脉内操作有关，继发性菌血症大多数由术后伤口、腹腔、尿道及肺部感染引起。近年来由于静脉导管留置、机械通气、肠外给药等侵入性设备及治疗的广泛应用，以及免疫抑制剂和大量抗菌药物的滥用，血流感染的发病率逐年上升，且由于血流感染不仅病死率高而且延长住院时间、增加住院费用等，因而受到了越来越多医患及研究者的重视。
引起血流感染最常见原因是细菌，例如大肠杆菌，金黄色葡萄球菌和肺炎球菌。除细菌感染外，真菌感染在最近已成为该疾病的重要原因。血流感染诊断和治疗方法的发展已有三十年历史，却仅有很少的进展。从血液样本中培养微生物，使微生物含量从1～10CFU/mL增加到10 6～10 8CFU/mL仍然是检测血流感染的金标准。但是这种方法有很多缺点，首先是诊断时间较长，从病人取样到提供样品的感染结果之间通常需要1～5天的诊断时间。在这段时间内，通常疗法是给病人使用广谱的非靶向性的抗生素进行治疗。这可能会在治疗该疾病方 面取得效果，但会有很严重的后果，就是很多微生物会产生多重抗药性。其次，培养方法需要大量的诊断所需的血液样品且检测灵敏度很低。此外，在采血和血样使用过程中有相对较高的污染风险。所以虽然目前血培养仍然为血流感染诊断的主要参考方法，但因其灵敏度低且耗时长等显著缺点的存在，因此并不是一个理想的金标准。
随着分子生物学技术的发展，以聚合酶链式反应(PCR)及二代测序技术为代表的分子生物学诊断技术开始被应用于血流感染的微生物检测。基于分子生物学诊断的方法具有速度快，高灵敏度，小样本量，短时间(一般不超过12小时)内提供微生物诊断相关信息及随后可引入适当的抗生素治疗(检测耐药基因)等显著优势。但是长期以来，由于血液中极低的循环微生物数量(1～10CFU/mL)，血液本身大量人类核酸可能会干扰PCR及测序的检测，同时，血液本身的成分,如铁、血红蛋白等以及血液抗凝剂会对PCR及文库构建产生严重的抑制，因此，直接从全血对微生物进行微生物诊断一直受到限制。
因此快速的从全血中分离、富集到病原微生物并提取到可以用于分子生物学检测的核酸将有效的提高血流感染病原微生物的检测效率，及时为医生合理、针对性用药及治疗提供指导，同时给病人争取到黄金治疗时间，从而增加病人的生存率。
发明内容
本发明是为了提出一种从疑似血流感染病人的血液(小儿患者包括新生儿的血液用量1～2mL,成人5～10mL)中直接分离、富集感染病原微生物并提取核酸的方法及试剂，无需血液培养，直接使用血液样本进行病原体富集和核酸提取，同时也适用于血培后样本，核酸提取步骤简单，且可以无差别适用于革兰阴性菌、阳性菌及真菌，提取的核酸可搭配单重、多重PCR/RT～PCR及二代测序等多种分子生物学检测方法，操作适配仪器设备要求不高，仅需实验室常见的高速离心机即可满足操作需求。
本发明提供一种血液中病原微生物分离、富集和核酸提取方法， 包括如下步骤：
S1、血液样本初次裂解：将血液样本中加入血液选择裂解液A和病原微生物助沉剂充分混匀后得到裂解物并使裂解物中的大分子有机物溶解，高速离心后弃除部分上清液，得到血液初次裂解样本；裂解物包括裂解的血细胞和未裂解但表面特性改变的病原微生物，血细胞包括红细胞和白细胞，高速离心沉降用于使病原微生物富集沉淀；
S2、血液样本再裂解：加入血液选择裂解液B使残留的大分子有机物进一步溶解，高速离心后弃除微生物助沉剂与水性溶液界面上的部分上清液，得到血液再次裂解样本；
S3、洗涤：加入洗涤液混匀后离心弃除部分上清液得到包含病原微生物的血液裂解样本，洗涤液用于清洗杂质；
S4、核酸提取：在血液裂解样本中加入核酸提取试剂，高速离心后弃除部分上清，将剩余溶液进行电裂解或加入不同粒径组合的研磨珠振荡后高温加热、离心，使病原微生物裂解并促使病原微生物中的核酸释放、溶解，得到病原微生物核酸样本。
本发明所述的一种血液中病原微生物分离、富集和核酸提取方法，作为优选方式，步骤S1中，血液选择裂解液A包括：浓度为10～100mM的缓冲液、质量体积百分比为0％～6.5％的辅助裂解盐、质量体积百分比为5％～65％的主要裂解盐、质量体积百分比为0％～5％的表面活性剂、浓度为0～10mM的螯合剂和体积百分比0％～0.5％的消泡剂，血液选择裂解液A的pH为4～8；
病原微生物助沉剂为不溶于水的生物惰性有机溶剂；
血液样本为1～10ml，加入血液选择裂解液A和病原微生物助沉剂后常温振荡混匀或常温颠倒混匀1～5分钟，血液选择裂解液A的用量为血液样本体积的0.5～5倍，病原微生物助沉剂的用量为血液样本体积的0.5％～5％。
本发明所述的一种血液中病原微生物分离、富集和核酸提取方法，作为优选方式，步骤S2中，血液选择裂解液B包括：浓度为10～100mM的缓冲液、质量体积百分比为0％～3％的辅助裂解盐、质量体积百分比为0％～40％的主要裂解盐、质量体积百分比为0％～2％的表面活性剂、 浓度为0～10mM的螯合剂和体积百分比0％～0.5％的消泡剂，血液选择裂解液B的pH为5～8；
血液选择裂解液B的用量为血液初次裂解样本体积的2～10倍；
步骤S3中，洗涤液包括：浓度为10～100mM的缓冲液、质量体积百分比为0％～5％的促进杂质溶解盐和浓度为0～10mM的螯合剂，洗涤液的pH为5～9；
洗涤液的用量为血液再次裂解样本体积的5～10倍，洗涤液的使用方法为常温震荡混匀或常温颠倒混匀；
步骤S2、S3可重复多次。
本发明所述的一种血液中病原微生物分离、富集和核酸提取方法，作为优选方式，步骤S4中，核酸提取液包括：浓度为0.5～20mM的缓冲液、质量体积百分比为0～5％的促进核酸溶解盐、质量体积百分比为0.01％～5％的表面活性剂、浓度为0～3mM的螯合剂和体积百分比0％～0.5％的消泡剂，核酸提取液的pH为7～9；
表面活性剂为阴离子表面活性剂和/或非离子表面活性剂；
核酸提取液的用量为血液裂解样本体积的5～10倍，电裂解使用MEMS加工的电裂解装置，研磨珠为粒径0.1～3mm的酸洗玻璃珠、氧化锆珠、石英砂中的一种或几种，研磨珠的用量为20～150mg、振荡时间为5～15分钟，高温加热的温度为85～100℃、加热时间为3～15分钟。
本发明所述的一种血液中病原微生物分离、富集和核酸提取方法，作为优选方式，缓冲液为以下任意一种：磷酸缓冲液、乙酸钠乙酸缓冲液和三羟甲基氨基甲烷盐酸缓冲液；
辅助裂解盐、促进核酸溶解盐和促进杂质溶解盐为以下一种或多种：氯化钠、氯化钾和硫酸铵；
主要裂解盐为以下一种或多种：氯化锂、碘化钠、碘化钾、盐酸胍和异硫氰酸胍；
消泡剂为以下任意一种：聚二甲基硅氧烷、聚丙二醇、聚醚改性聚硅氧烷和聚氧丙烯甘油醚；
螯合剂为柠檬酸三钠；
阴离子表面活性剂为以下任意一种：十二烷基硫酸钠、十二烷基肌氨酸钠和脱氧胆酸钠，非离子表面活性剂为以下一种或多种：吐温20、吐温80、曲拉通X-100、乙基苯基聚乙二醇和十六醇聚氧乙烯醚Brij C20；
步骤S1、S2、S3和S4中，离心转速为10000～16000×g，离心时间为1～5分钟。
本发明提供一种血液中病原微生物分离、富集和核酸提取试剂，包括体积为血液样本0.5～10倍的血液选择裂解液A、体积为血液样本0.5％～10％的病原微生物助沉剂、体积为血液样本1～10倍的血液选择裂解液B、体积为血液样本1～10倍的洗涤液和体积为血液样本0.5～2倍的核酸提取液；
血液选择裂解液A和血液选择裂解液B用于仅裂解血细胞保留病原微生物并将血液样本中以及裂解血细胞中释放的大分子有机物进行溶解；病原微生物助沉剂用于使病原微生物富集沉淀；洗涤液用于清洗杂质，核酸提取液用于使病原微生物富集、裂解并促使病原微生物中的核酸释放和溶解；
血液选择裂解液A包括：浓度为10～100mM的缓冲液、质量体积百分比为0％～6.5％的辅助裂解盐、质量体积百分比为5％～65％的主要裂解盐、质量体积百分比为0％～5％的表面活性剂、浓度为0～10mM的螯合剂和体积百分比0％～0.5％的消泡剂，血液选择裂解液A的pH为4～8；
病原微生物助沉剂为不溶于水的生物惰性有机溶剂；
血液选择裂解液B包括：浓度为10～100mM的缓冲液、质量体积百分比为0％～3％的辅助裂解盐、质量体积百分比为0％～40％的主要裂解盐、质量体积百分比为0％～2％的表面活性剂、浓度为0～10mM的螯合剂和体积百分比0％～0.5％的消泡剂，血液选择裂解液B的pH为5～8。
本发明所述的一种血液中病原微生物分离、富集和核酸提取试剂，作为优选方式，洗涤液包括：浓度为10～100mM的缓冲液、质量体积百分比为0％～5％的促进杂质溶解盐和浓度为0～10mM的螯合剂，洗 涤液的pH为5～9；
核酸提取液包括：浓度为0.5～20mM的缓冲液、质量体积百分比为0～5％的促进核酸溶解盐、质量体积百分比为0.01％～5％的表面活性剂、浓度为0～3mM的螯合剂和体积百分比0％～0.5％的消泡剂，核酸提取液的pH为7～9；
生物惰性有机溶剂为氟油或HFE-7500或Novec7500或FC-40。
本发明所述的一种血液中病原微生物分离、富集和核酸提取试剂，作为优选方式，缓冲液为以下任意一种：磷酸缓冲液、乙酸钠乙酸缓冲液、三羟甲基氨基甲烷盐酸缓冲液；
辅助裂解盐、促进核酸溶解盐和促进杂质溶解盐为以下一种或多种：氯化钠、氯化钾和硫酸铵；
主要裂解盐为以下一种或多种：氯化锂、碘化钠、碘化钾、盐酸胍和异硫氰酸胍；
消泡剂为以下任意一种：聚二甲基硅氧烷、聚丙二醇、聚醚改性聚硅氧烷和聚氧丙烯甘油醚；
螯合剂为柠檬酸三钠；
表面活性剂为阴离子表面活性剂和/或非离子表面活性剂。
本发明所述的一种血液中病原微生物分离、富集和核酸提取试剂，作为优选方式，阴离子表面活性剂为以下任意一种：十二烷基硫酸钠、十二烷基肌氨酸钠和脱氧胆酸钠，非离子表面活性剂为以下一种或多种：吐温20、吐温80、曲拉通X-100、乙基苯基聚乙二醇和十六醇聚氧乙烯醚Brij C20。
本发明所述的一种血液中病原微生物分离、富集和核酸提取试剂，作为优选方式，血液选择裂解液A包括20mM三羟甲基氨基甲烷盐酸缓冲液、质量体积百分比0.5％的氯化钾、质量体积百分比65％的异硫氰酸胍、质量体积百分比0.2％的十二烷基肌氨酸钠、10mM柠檬酸三钠和体积百分比0.02％的聚氧丙烯甘油醚，血液选择裂解液A的pH为7.0；
血液选择裂解液B包括20mM三羟甲基氨基甲烷盐酸缓冲液，质量体积百分比0.5％的氯化钾、质量体积百分比30％的异硫氰酸胍、质 量体积百分比的0.1％十二烷基肌氨酸钠、10mM柠檬酸三钠和体积百分比0.02％的聚氧丙烯甘油醚，血液选择裂解液B的pH为7.0；
微生物助沉剂为Novec7500；
洗涤液包括10mM三羟甲基氨基甲烷盐酸盐缓冲液、质量体积百分比的0.9％氯化钠、质量体积百分比的10％硫酸铵和3mM柠檬酸三钠，洗涤液的pH为7.0；
核酸提取液包括10mM三羟甲基氨基甲烷盐酸盐缓冲液、质量体积百分比0.45％的氯化钠、质量体积百分比0.1％的十二烷基硫酸钠、质量体积百分比2％的吐温20、1mM柠檬酸三钠和体积百分比0.02％的聚氧丙烯甘油醚，核酸提取液的pH为8.8。
具体方案为：将血液样本中加入血液选择裂解液A和病原微生物助沉剂进行样本裂解处理，选择性裂解血细胞(包括红细胞和白细胞等)，溶解血液中的蛋白质、糖类和脂类等杂质，之后通过高速离心沉降病原微生物，小心弃除部分上清；加入血液选择裂解液B再进行一次或二次血液选择裂解、高速离心弃除微生物助沉剂与水性溶液界面上的部分上清液，之后在剩余溶液中加入洗涤液混匀后离心并继续弃除部分上清液，重复洗涤一次或多次；之后加入核酸提取试剂再次处理残留溶液，高速离心后弃除部分上清，病原微生物在剩余核酸提取液；电裂解或加入不同粒径组合的研磨珠振荡后高温加热、离心提取病原微生物的核酸。
在本发明的一个优选技术方案中，血液选择裂解液A的用量为血液样本体积的0.5～5倍，微生物助沉剂用量为血液样本的体积的0.5％～5％，裂解的条件为常温振荡混匀或常温颠倒混匀1～5分钟，离心转速10000～16000×g，离心时间为1～5分钟，弃上清使用移液器或注射器等，不可触及位于微生物助沉剂与水溶液两相交界处的沉淀物，溶液弃除量为总溶液体积的80％～95％。。
在本发明的一个优选技术方案中，血液选择裂解液B的用量为剩余上清体积的2～10倍，裂解的条件为常温振荡混匀或常温颠倒混匀1～5分钟，离心转速10000～16000×g，离心时间为1～5分钟，弃上清使用移液器或注射器等，不可触及位于微生物助沉剂与水溶液两相 交界处的沉淀物，溶液弃除量等同于血液选择裂解液B的加入量，本裂解步骤根据沉淀性状，重复一次或多次。
在本发明的一个优选技术方案中，洗涤液的用量为剩余上清体积的5～10倍，洗涤的条件为常温振荡混匀或常温颠倒混匀1～5分钟，离心转速10000～16000×g，离心时间为1～5分钟，弃上清使用移液器或注射器等，不可触及位于微生物助沉剂与水溶液两相交界处的沉淀物，溶液弃除量等同于洗涤液的加入量，本裂解步骤根据沉淀性状，重复一次或多次。
在本发明的一个优选技术方案中，核酸提取液的用量为剩余上清体积的5～10倍，处理的条件为常温振荡混匀或常温颠倒混匀1～5分钟，离心转速10000～16000×g，离心时间为1～5分钟，弃上清使用移液器或注射器等，不可触及位于微生物助沉剂与水溶液两相交界处的沉淀物，溶液弃除量等同于核酸提取液的加入量，剩余上清和沉淀中包含从样本中富集的病原微生物。
在本发明的一个优选技术方案中，研磨珠为0.1～3mm不同粒径组合的酸洗玻璃珠或氧化锆珠，用量为20～150mg，振荡时间为5～15分钟，高温加热的温度为85～100℃，加热时间为3～15分钟，离心转速10000～16000×g，离心时间为1～5分钟，离心后用移液器或注射器吸取上清液即为目的核酸。
在本发明的一个优选技术方案中，核酸提取使用微加工的电裂解装置，尺寸可为(0.1～1)x(5～1000)x(10～1000)mm 3,根据需求尺寸可大于或小于本设计尺寸。裂解电压为10～2000V。
在本发明的一个优选技术方案中，血液样本为疑似血流感染病人的血液，小儿患者包括新生儿的血液用量1～2mL,成人5～10mL；样本也可为血液培养后样本，用量0.2～1mL。
本发明通过血液选择裂解液A与血液的渗透压差和表面活性剂的添加破裂血细胞(包括红细胞和白细胞等)，同时在适宜的浓度下尽可能的溶解血液中及血细胞破裂所释放出的大分子有机物等，且不会对病原微生物细胞结构造成严重损伤。并且其中的盐类、表面活性剂还可以与微生物助沉剂一起促进那些因具有特殊细胞外结构(如细菌 荚膜)而难以离心沉淀的病原微生物富集沉淀，减少微生物的差异化富集。微生物助沉剂还可以减少微生物与离心管之前的黏附，避免微生物离心沉降不完全而产生丢失。血液选择裂解液B的添加弥补因样本差异造成的裂解处理不完全并充分溶解残留杂质。洗涤液去除裂解液中存在的可能对下游检测造成抑制的裂解液成分和血液难溶物质。大量的核酸提取液稀释洗涤液中的盐类并提供适合病原微生物裂解和核酸溶解、保存的条件，在瞬时电裂解或者不同粒径研磨珠的物理研磨和高温加热变性作用下，充分裂解病原微生物促进其中核酸的释放和溶解。
大分子有机物包括蛋白质、糖类、脂类和核酸等。
本发明具有以下优点：
(1)可从不大于10mL的血液样品中，快速、准确的提取到能满足分子生物学检测要求的目标感染病原体核酸。
(2)无需血液培养，直接使用血液样本进行病原体富集和核酸提取，同时也适用于血培后样本。
(3)核酸提取步骤简单，且可以无差别适用于革兰阴性菌、阳性菌及真菌。
(4)提取的核酸可搭配单重、多重PCR/RT～PCR及二代测序等多种分子生物学检测方法。
(5)操作适配仪器设备要求不高，仅需实验室常见的高速离心机即可满足操作需求。
附图说明
图1为一种血液中病原微生物分离、富集和核酸提取方法流程图；
图2为一种血液中病原微生物分离、富集和核酸提取方法示意图；
图3为一种血液中病原微生物分离、富集和核酸提取方法及试剂实施例5探针法荧光定量PCR的扩增曲线；
图4为一种血液中病原微生物分离、富集和核酸提取方法及试剂 实施例6探针法荧光定量PCR的扩增曲线；
图5为一种血液中病原微生物分离、富集和核酸提取方法及试剂实施例7探针法荧光定量PCR的扩增曲线。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。
实施例1
如图1所示，一种血液中病原微生物分离、富集和核酸提取方法，其特征在于：包括如下步骤：
S1、血液样本初次裂解：将血液样本中加入血液选择裂解液A和病原微生物助沉剂充分混匀后得到裂解物并使裂解物中的大分子有机物溶解，高速离心后弃除部分上清液，得到血液初次裂解样本；裂解物包括裂解的血细胞和未裂解但表面特性改变的病原微生物，血细胞包括红细胞和白细胞，高速离心沉降用于使病原微生物富集沉淀；
S2、血液样本再裂解：加入血液选择裂解液B使残留的大分子有机物进一步溶解，高速离心后弃除微生物助沉剂与水性溶液界面上的部分上清液，得到血液再次裂解样本；
S3、洗涤：加入洗涤液混匀后离心弃除部分上清液得到包含病原微生物的血液裂解样本，洗涤液用于清洗杂质；
S4、核酸提取：在血液裂解样本中加入核酸提取试剂，高速离心后弃除部分上清，将剩余溶液进行电裂解或加入不同粒径组合的研磨珠振荡后高温加热、离心，使病原微生物裂解并促使病原微生物中的核酸释放、溶解，得到病原微生物核酸样本。
实施例2
如图1所示，一种血液中病原微生物分离、富集和核酸提取方法，其特征在于：包括如下步骤：
S1、血液样本初次裂解：将血液样本中加入血液选择裂解液A和病原微生物助沉剂充分混匀后得到裂解物并使裂解物中的大分子有机物溶解，高速离心后弃除部分上清液，得到血液初次裂解样本；裂解物包括裂解的血细胞和未裂解但表面特性改变的病原微生物，血细胞包括红细胞和白细胞，高速离心沉降用于使病原微生物富集沉淀；
血液选择裂解液A包括：浓度为10～100mM的缓冲液、质量体积百分比为0％～6.5％的辅助裂解盐、质量体积百分比为5％～65％的主要裂解盐、质量体积百分比为0％～5％的表面活性剂、浓度为0～10mM的螯合剂和体积百分比0％～0.5％的消泡剂，血液选择裂解液A的pH为4～8；
病原微生物助沉剂为不溶于水的生物惰性有机溶剂；
血液样本为1～10ml，加入血液选择裂解液A和病原微生物助沉剂后常温振荡混匀或常温颠倒混匀1～5分钟，血液选择裂解液A的用量为血液样本体积的0.5～5倍，病原微生物助沉剂的用量为血液样本体积的0.5％～5％；
S2、血液样本再裂解：加入血液选择裂解液B使残留的大分子有机物进一步溶解，高速离心后弃除微生物助沉剂与水性溶液界面上的部分上清液，得到血液再次裂解样本；
血液选择裂解液B包括：浓度为10～100mM的缓冲液、质量体积百分比为0％～3％的辅助裂解盐、质量体积百分比为0％～40％的主要裂解盐、质量体积百分比为0％～2％的表面活性剂、浓度为0～10mM的螯合剂和体积百分比0％～0.5％的消泡剂，血液选择裂解液B的pH为5～8；
血液选择裂解液B的用量为血液初次裂解样本体积的2～10倍；
步骤S2可重复多次；
S3、洗涤：加入洗涤液混匀后离心弃除部分上清液得到包含病原微生物的血液裂解样本，洗涤液用于清洗杂质；
步骤S3中，洗涤液包括：浓度为10～100mM的缓冲液、质量体积百分比为0％～5％的促进杂质溶解盐和浓度为0～10mM的螯合剂，洗涤液的pH为5～9；
洗涤液的用量为血液再次裂解样本体积的5～10倍，洗涤液的使用方法为常温震荡混匀或常温颠倒混匀；
步骤S3可重复多次；
S4、核酸提取：在血液裂解样本中加入核酸提取试剂，高速离心后弃除部分上清，将剩余溶液进行电裂解或加入不同粒径组合的研磨珠振荡后高温加热、离心，使病原微生物裂解并促使病原微生物中的核酸释放、溶解，得到病原微生物核酸样本；
核酸提取液包括：浓度为0.5～20mM的缓冲液、质量体积百分比为0～5％的促进核酸溶解盐、质量体积百分比为0.01％～5％的表面活性剂、浓度为0～3mM的螯合剂和体积百分比0％～0.5％的消泡剂，核酸提取液的pH为7～9；
表面活性剂为阴离子表面活性剂和/或非离子表面活性剂；
核酸提取液的用量为血液裂解样本体积的5～10倍，电裂解使用MEMS加工的电裂解装置，研磨珠为粒径0.1～3mm的酸洗玻璃珠、氧化锆珠、石英砂中的一种或几种，研磨珠的用量为20～150mg、振荡时间为5～15分钟，高温加热的温度为85～100℃、加热时间为3～15分钟；
其中，缓冲液为以下任意一种：磷酸缓冲液、乙酸钠乙酸缓冲液和三羟甲基氨基甲烷盐酸缓冲液；
辅助裂解盐、促进核酸溶解盐和促进杂质溶解盐为以下一种或多种：氯化钠、氯化钾和硫酸铵；
主要裂解盐为以下一种或多种：氯化锂、碘化钠、碘化钾、盐酸胍和异硫氰酸胍；
消泡剂为以下任意一种：聚二甲基硅氧烷、聚丙二醇、聚醚改性聚硅氧烷和聚氧丙烯甘油醚；
螯合剂为柠檬酸三钠；
阴离子表面活性剂为以下任意一种：十二烷基硫酸钠、十二烷基肌氨酸钠和脱氧胆酸钠，非离子表面活性剂为以下一种或多种：吐温20、吐温80、曲拉通X-100、乙基苯基聚乙二醇和十六醇聚氧乙烯醚Brij C20；
步骤S1、S2、S3和S4中，离心转速为10000～16000×g，离心 时间为1～5分钟。
实施例3
一种血液中病原微生物分离、富集和核酸提取试剂，包括体积为血液样本0.5～10倍的血液选择裂解液A、体积为血液样本0.5％～10％的病原微生物助沉剂、体积为血液样本1～10倍的血液选择裂解液B、体积为血液样本1～10倍的洗涤液和体积为血液样本0.5～2倍的核酸提取液；
血液选择裂解液A和血液选择裂解液B用于仅裂解血细胞保留病原微生物并将血液样本中以及裂解血细胞中释放的大分子有机物进行溶解；病原微生物助沉剂用于使病原微生物富集沉淀；洗涤液用于清洗杂质，核酸提取液用于使病原微生物富集、裂解并促使病原微生物中的核酸释放和溶解；
血液选择裂解液A包括：浓度为10～100mM的缓冲液、质量体积百分比为0％～6.5％的辅助裂解盐、质量体积百分比为5％～65％的主要裂解盐、质量体积百分比为0％～5％的表面活性剂、浓度为0～10mM的螯合剂和体积百分比0％～0.5％的消泡剂，血液选择裂解液A的pH为4～8；
病原微生物助沉剂为不溶于水的生物惰性有机溶剂；
血液选择裂解液B包括：浓度为10～100mM的缓冲液、质量体积百分比为0％～3％的辅助裂解盐、质量体积百分比为0％～40％的主要裂解盐、质量体积百分比为0％～2％的表面活性剂、浓度为0～10mM的螯合剂和体积百分比0％～0.5％的消泡剂，血液选择裂解液B的pH为5～8。
实施例4
一种血液中病原微生物分离、富集和核酸提取试剂，包括体积为血液样本10倍的血液选择裂解液A、体积为血液样本10％的病原微生物助沉剂、体积为血液样本10倍的血液选择裂解液B、体积为血液样本10倍的洗涤液和体积为血液样本2倍的核酸提取液；
血液选择裂解液A和血液选择裂解液B用于仅裂解血细胞保留病原微生物并将血液样本中以及裂解血细胞中释放的大分子有机物进行溶解；病原微生物助沉剂用于使病原微生物富集沉淀；洗涤液用于清洗杂质，核酸提取液用于使病原微生物富集、裂解并促使病原微生物中的核酸释放和溶解；
血液选择裂解液A包括：浓度为10～100mM的缓冲液、质量体积百分比为0％～6.5％的辅助裂解盐、质量体积百分比为5％～65％的主要裂解盐、质量体积百分比为0％～5％的表面活性剂、浓度为0～10mM的螯合剂和体积百分比0％～0.5％的消泡剂，血液选择裂解液A的pH为4～8；
病原微生物助沉剂为不溶于水的生物惰性有机溶剂；
生物惰性有机溶剂为氟油或HFE-7500或Novec7500或FC-40；
血液选择裂解液B包括：浓度为10～100mM的缓冲液、质量体积百分比为0％～3％的辅助裂解盐、质量体积百分比为0％～40％的主要裂解盐、质量体积百分比为0％～2％的表面活性剂、浓度为0～10mM的螯合剂和体积百分比0％～0.5％的消泡剂，血液选择裂解液B的pH为5～8；洗涤液包括：浓度为10～100mM的缓冲液、质量体积百分比为0％～5％的促进杂质溶解盐和浓度为0～10mM的螯合剂，洗涤液的pH为5～9；
核酸提取液包括：浓度为0.5～20mM的缓冲液、质量体积百分比为0～5％的促进核酸溶解盐、质量体积百分比为0.01％～5％的表面活性剂、浓度为0～3mM的螯合剂和体积百分比0％～0.5％的消泡剂，核酸提取液的pH为7～9；
缓冲液为以下任意一种：磷酸缓冲液、乙酸钠乙酸缓冲液、三羟甲基氨基甲烷盐酸缓冲液；
辅助裂解盐、促进核酸溶解盐和促进杂质溶解盐为以下一种或多种：氯化钠、氯化钾和硫酸铵；
主要裂解盐为以下一种或多种：氯化锂、碘化钠、碘化钾、盐酸胍和异硫氰酸胍；
消泡剂为以下任意一种：聚二甲基硅氧烷、聚丙二醇、聚醚改性 聚硅氧烷和聚氧丙烯甘油醚；
螯合剂为柠檬酸三钠；
表面活性剂为阴离子表面活性剂和/或非离子表面活性剂；
阴离子表面活性剂为以下任意一种：十二烷基硫酸钠、十二烷基肌氨酸钠和脱氧胆酸钠，非离子表面活性剂为以下一种或多种：吐温20、吐温80、曲拉通X-100、乙基苯基聚乙二醇和十六醇聚氧乙烯醚Brij C20。
实施例5
为验证本方法和试剂的有效性，使用本发明所述试剂和方法进行模拟血流感染大肠杆菌型病原微生物的分离和核酸提取，同时将模拟样本中添加相同数量的病原微生物所提取核酸作为为对照，再通过探针法荧光定量PCR对所提DNA进行检测，从而验证本发明的可行性。
以下实施例中PCR检测目标为所提核酸中的病原微生物的DNA，本发明所分离核酸中也包括病原微生物的RNA，根据检测引物及探针设计位点不同也可进行qRT～PCR等检测，此处检测方法不对本发明的其它下游应用方式产生限制。
一种血液中病原微生物分离、富集和核酸提取试剂，所用试剂如下：血液选择裂解液A为20mM三羟甲基氨基甲烷盐酸缓冲液(pH7.0)、0.5％氯化钾、65％异硫氰酸胍、0.2％十二烷基肌氨酸钠、10mM柠檬酸三钠和0.02％聚氧丙烯甘油醚。微生物助沉剂为Novec7500。血液选择裂解液B为20mM三羟甲基氨基甲烷盐酸缓冲液(pH7.0)，0.5％氯化钾、30％异硫氰酸胍、0.1％月桂酰肌氨酸钠、10mM柠檬酸三钠和0.02％聚氧丙烯甘油醚。洗涤液为10mM三羟甲基氨基甲烷盐酸盐缓冲液(pH7.0)，0.9％氯化钠、10％硫酸铵和3mM柠檬酸三钠。核酸提取液为10mM三羟甲基氨基甲烷盐酸盐缓冲液(pH8.8)、0.45％氯化钠、0.1％十二烷基硫酸钠、2％吐温20、1mM柠檬酸三钠和0.02％聚氧丙烯甘油醚。
血液中大肠杆菌的分离、富集及核酸提取方法如下：
制备含有大肠杆菌的模拟样本，在3支1mL阴性血液中分别添 加10CFU/100CFU/1000CFU的大肠杆菌，混匀备用；同时制备对照样本，在3支100μL核酸提取液中分别添加10CFU/100CFU/1000CFU的大肠杆菌，混匀备用。在准备好的模拟样本中添加试剂并进行微生物分离、富集和核酸提取等操作。具体为在1mL模拟血液样本中添加1mL血液选择裂解液A和20μL病原微生物助沉剂，振荡1分钟，12000×g离心2分钟，弃去1.8mL上清(剩余200μL)。然后加入1mL血液选择裂解液B，振荡1分钟，12000×g离心2分钟，弃去1mL上清。重复本步骤一次或多次。样品中加入1mL洗涤液，振荡1分钟，12000×g离心2分钟，弃去1mL上清。重复本步骤一次或多次。最后加入1mL核酸提取液，振荡1分钟，12000×g离心2分钟，弃去1mL上清。加入30mg 0.5mm氧化锆珠，涡旋振荡5分钟，95℃加热5分钟，12000×g离心2分钟，收集上清得到模拟样本核酸。同时对照样本中加入30mg 0.5mm氧化锆珠，涡旋振荡5分钟，95℃加热5分钟，12000×g离心2分钟，收集上清得到对照样本的核酸。并行的，做一组不添加大肠杆菌的空白血液分离提取对照。所提核酸按表1进行探针法PCR检测体系配制和上机扩增程序设置，具体引物探针序列见表2。结果如图3显示，在三个检测梯度上，100CFU和1000CFU每毫升级别上对照组与实验组扩增重叠性很好，10CFU级别上两组扩增略有不同但均可稳定检出，且阴性血液提取对照和不加模板对照均未扩增，扩增结果可靠。由此表明使用本发明的试剂和方法可分离并提取到血液中10CFU数量级的大肠杆菌DNA，符合血流感染的血液样本检测需求。
分离血液样本中10/100/1000CFU大肠杆菌后提取的核酸与直接提取10/100/1000CFU大肠杆菌的核酸的探针法荧光定量PCR扩增曲线对比图。其中图3左侧图例中C～E1、C～E2、C～E3分别对应无需分离对照组的10、100、1000CFU，E1、E2、E3分别对应从血样中分离病原实验组的10、100、1000CFU；BNTC是阴性血液分离、提取对照，NTC为不加模板扩增对照。
实施例6
为验证本方法和试剂的有效性，使用本发明所述试剂和方法进行模拟血流感染肺炎链球菌类型病原微生物的分离和核酸提取，同时将模拟样本中添加相同数量的病原微生物所提取核酸作为为对照，再通过探针法荧光定量PCR对所提DNA进行检测，从而验证本发明的可行性。
一种血液中病原微生物分离、富集和核酸提取试剂，所用试剂如下：血液选择裂解液A为5mM磷酸缓冲液(pH7.5)、1.5％硫酸铵、12％碘化钠、10mM柠檬酸三钠、0.5％曲拉通X-100和0.03％聚氧丙烯甘油醚。微生物助沉剂为FC-40。血液选择裂解液B为5mM磷酸缓冲液(pH7.5)、0.9％氯化钠、4％碘化钠、5mM柠檬酸三钠和0.03％聚氧丙烯甘油醚。洗涤液为2mM磷酸缓冲液(pH8.5)，0.9％氯化钠、3％硫酸铵、5mM柠檬酸三钠。核酸提取液为5mM磷酸缓冲液(pH8.0)、0.45％氯化钠、0.1％十二烷基肌氨酸钠、5％吐温20、0.5mM柠檬酸三钠和0.1％聚丙二醇。
血液中肺炎链球菌的分离、富集及核酸提取方法如下：
制备含有肺炎链球菌的模拟样本，在3支1mL阴性血液中分别添加10CFU/100CFU/1000CFU的肺炎链球菌，混匀备用；同时制备对照样本，在3支100μL核酸提取液中分别添加10CFU/100CFU/1000CFU的肺炎链球菌，混匀备用。在准备好的模拟样本中添加试剂并进行微生物分离、富集和核酸提取等操作。具体为在1mL模拟血液样本中添加1mL选择裂解液A和20μL微生物助沉剂，振荡1分钟，12000×g离心2分钟，弃去1.8mL上清(剩余200μL)。然后加入1mL血液选择裂解液B，振荡1分钟，12000×g离心2分钟，弃去1mL上清。重复本步骤一次或多次。样品中加入1mL洗涤液，振荡1分钟，12000×g离心2分钟，弃去1mL上清。重复本步骤一次或多次。最后加入1mL核酸提取液，振荡1分钟，12000×g离心2分钟，弃去1mL上清。加入30mg 0.5mm氧化锆珠，涡旋振荡5分钟，95℃加热5分钟，12000×g离心2分钟，收集上清得到模拟样本核酸。同时对照样本中加入30mg 0.5mm氧化锆珠，涡旋振荡5分钟，95℃加热5分钟，12000×g离心2分钟，收集上清得到对照样本的核酸。并 行的，做一组不添加肺炎链球菌的空白血液分离提取对照。所提核酸按表1进行探针法PCR检测体系配制和上机扩增程序设置，具体引物探针序列见表2。结果如图4显示，在三个不同的检测梯度上，对照组与实验组扩增相比实验组CT值相比对照组虽有延后，但均可稳定检出，且阴性血液提取对照和不加模板对照均未扩增，扩增结果可靠且可重复。因此表明使用本发明的试剂和方法可分离并提取到血液中10CFU每毫升血液的肺炎链球菌DNA，达到血流感染中革兰氏阴性病原体的血液样本检测需求。
分离血液样本中10/100/1000CFU肺炎链球菌后提取的核酸与直接提取10/100/1000CFU肺炎链球菌的核酸的探针法针法荧光定量PCR扩增曲线对比图。其中图4左侧图例中C-E1、C-E2、C-E3分别对应无需分离对照组的10、100、1000CFU，E1、E2、E3分别对应从血样中分离病原实验组的10、100、1000CFU；BNTC是阴性血液分离、提取对照，NTC为不加模板扩增对照。
实施例7
为验证本方法和试剂的有效性，使用本发明所述试剂和方法进行模拟血流感染白色念珠菌类型病原微生物的分离和核酸提取，同时将模拟样本中添加相同数量的病原微生物所提取核酸作为为对照，再通过探针法荧光定量PCR对所提DNA进行检测，从而验证本发明的可行性。
一种血液中病原微生物分离、富集和核酸提取试剂，所用试剂如下：血液选择裂解液A为5mM磷酸缓冲液(pH8)，1％氯化钠、20％盐酸胍、0.5％吐温20、5mM柠檬酸三钠和0.05％聚丙二醇。微生物助沉剂为Novec7500。血液选择裂解液B为5mM磷酸缓冲液(pH8)、0.5％氯化钾、5mM柠檬酸三钠。洗涤液为0.1％磷酸缓冲液(pH7.5)，0.9％氯化钠、3％硫酸铵、0.1％柠檬酸三钠。核酸提取液为0.2％三羟甲基氨基甲烷盐酸缓冲液(pH9)、0.15％氯化钾、3％曲拉通X-100和0.05％聚丙二醇。
血液中白色念珠菌的分离、富集及核酸提取方法如下：
同前，制备含有白色念珠菌的模拟样本，在3支1mL阴性血液中分别添加10CFU/100CFU/1000CFU的白色念珠菌，混匀备用；同时制备对照样本，在3支100μL核酸提取液中分别添加10CFU/100CFU/1000CFU的白色念珠菌，混匀备用。在准备好的模拟样本中添加试剂并进行微生物分离、富集和核酸提取等操作。具体为在1mL模拟血液样本中添加1mL选择裂解液A和20μL微生物助沉剂，振荡1分钟，12000×g离心2分钟，弃去1.8mL上清(剩余200μL)。然后加入1mL血液选择裂解液B，振荡1分钟，12000×g离心2分钟，弃去1mL上清。重复本步骤一次或多次。样品中加入1mL洗涤液，振荡1分钟，12000×g离心2分钟，弃去1mL上清。重复本步骤一次或多次。最后加入1mL核酸提取液，振荡1分钟，12000×g离心2分钟，弃去1mL上清。加入30mg 0.5mm氧化锆珠，涡旋振荡5分钟，95℃加热5分钟，12000×g离心2分钟，收集上清得到模拟样本核酸。同时对照样本中加入30mg 0.5mm氧化锆珠，涡旋振荡5分钟，95℃加热5分钟，12000×g离心2分钟，收集上清得到对照样本的核酸。并行的，做一组不添加肺炎链球菌的空白血液分离提取对照。
所提核酸按表1进行探针法PCR检测体系配制和上机扩增程序设置，具体引物探针序列见表2。结果如图5显示，在三个检测梯度上，100CFU和1000CFU每毫升的模拟样本中，对照组与实验组扩增差异不明显，10CFU每毫升的模拟样本中，两组实验扩增重叠性较好且均可稳定检出。阴性血液提取对照和不加模板对照组均未扩增，扩增结果可靠。由此表明使用本发明的试剂和方法可分离并提取到血液中10CFU每毫升血液的白色念珠菌DNA，本试剂和方法达到血流感染中真菌病原体的血液样本检测需求。
分离血液样本中10/100/1000CFU白色念珠菌后提取的核酸与直接提取10/100/1000CFU白色念珠菌的核酸的探针法针法荧光定量PCR扩增曲线对比图。其中图5左侧图例中C-E1、C-E2、C-E3分别对应无需分离对照组的10、100、1000CFU，E1、E2、E3分别对应从血样中分离病原实验组的10、100、1000CFU；BNTC是阴性 血液分离、提取对照，NTC为不加模板扩增对照。
表1探针法PCR检测体系配制和上机扩增程序
Figure PCTCN2022083559-appb-000001
表2探针法PCR体系中引物、探针序列
Figure PCTCN2022083559-appb-000002
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。

Claims (10)

  1. 一种血液中病原微生物分离、富集和核酸提取方法，其特征在于：包括如下步骤：
    S1、血液样本初次裂解：将血液样本中加入血液选择裂解液A和病原微生物助沉剂充分混匀后得到裂解物并使所述裂解物中的大分子有机物溶解，高速离心后弃除部分上清液，得到血液初次裂解样本；所述裂解物包括裂解的血细胞和未裂解但表面特性改变的病原微生物，所述血细胞包括红细胞和白细胞，高速离心沉降用于使所述病原微生物富集沉淀；
    S2、血液样本再裂解：加入血液选择裂解液B使残留的大分子有机物进一步溶解，高速离心后弃除所述微生物助沉剂与水性溶液界面上的部分上清液，得到血液再次裂解样本；
    S3、洗涤：加入洗涤液混匀后离心弃除部分上清液得到包含所述病原微生物的血液裂解样本，所述洗涤液用于清洗杂质；
    S4、核酸提取：在所述血液裂解样本中加入核酸提取试剂，高速离心后弃除部分上清，将剩余溶液进行电裂解或加入不同粒径组合的研磨珠振荡后高温加热、离心，使所述病原微生物裂解并促使所述病原微生物中的核酸释放、溶解，得到病原微生物核酸样本。
  2. 根据权利要求1所述的一种血液中病原微生物分离、富集和核酸提取方法，其特征在于：步骤S1中，所述血液选择裂解液A包括：浓度为10～100mM的缓冲液、质量体积百分比为0％～6.5％的辅助裂解盐、质量体积百分比为5％～65％的主要裂解盐、质量体积百分比为0％～5％的表面活性剂、浓度为0～10mM的螯合剂和体积百分比0％～0.5％的消泡剂，所述血液选择裂解液A的pH为4～8；
    所述病原微生物助沉剂为不溶于水的生物惰性有机溶剂；
    所述血液样本为1～10ml，加入所述血液选择裂解液A和所述病原微生物助沉剂后常温振荡混匀或常温颠倒混匀1～5分钟，所述血液选择裂解液A的用量为所述血液样本体积的0.5～5倍，所述病原微生物助沉剂的用量为所述血液样本体积的0.5％～5％。
  3. 根据权利要求1所述的一种血液中病原微生物分离、富集和核酸提取方法，其特征在于：步骤S2中，所述血液选择裂解液B包括： 浓度为10～100mM的缓冲液、质量体积百分比为0％～3％的辅助裂解盐、质量体积百分比为0％～40％的主要裂解盐、质量体积百分比为0％～2％的表面活性剂、浓度为0～10mM的螯合剂和体积百分比0％～0.5％的消泡剂，所述血液选择裂解液B的pH为5～8；
    所述血液选择裂解液B的用量为所述血液初次裂解样本体积的2～10倍；
    步骤S3中，所述洗涤液包括：浓度为10～100mM的所述缓冲液、质量体积百分比为0％～5％的促进杂质溶解盐和浓度为0～10mM的所述螯合剂，所述洗涤液的pH为5～9；
    所述洗涤液的用量为所述血液再次裂解样本体积的5～10倍，所述洗涤液的使用方法为常温震荡混匀或常温颠倒混匀；
    步骤S2、S3可重复多次。
  4. 根据权利要求1所述的一种血液中病原微生物分离、富集和核酸提取方法，其特征在于：步骤S4中，所述核酸提取液包括：浓度为0.5～20mM的缓冲液、质量体积百分比为0～5％的促进核酸溶解盐、质量体积百分比为0.01％～5％的表面活性剂、浓度为0～3mM的螯合剂和体积百分比0％～0.5％的消泡剂，所述核酸提取液的pH为7～9；
    所述表面活性剂为阴离子表面活性剂和/或非离子表面活性剂；
    所述核酸提取液的用量为所述血液裂解样本体积的5～10倍，所述电裂解使用MEMS加工的电裂解装置，所述研磨珠为粒径0.1～3mm的酸洗玻璃珠、氧化锆珠、石英砂中的一种或几种，所述研磨珠的用量为20～150mg、振荡时间为5～15分钟，所述高温加热的温度为85～100℃、加热时间为3～15分钟。
  5. 根据权利要求1～4任意一项所述的一种血液中病原微生物分离、富集和核酸提取方法，其特征在于：
    所述缓冲液为以下任意一种：磷酸缓冲液、乙酸钠乙酸缓冲液和三羟甲基氨基甲烷盐酸缓冲液；
    所述辅助裂解盐、所述促进核酸溶解盐和所述促进杂质溶解盐为以下一种或多种：氯化钠、氯化钾和硫酸铵；
    所述主要裂解盐为以下一种或多种：氯化锂、碘化钠、碘化钾、 盐酸胍和异硫氰酸胍；
    所述消泡剂为以下任意一种：聚二甲基硅氧烷、聚丙二醇、聚醚改性聚硅氧烷和聚氧丙烯甘油醚；
    所述螯合剂为柠檬酸三钠；
    所述阴离子表面活性剂为以下任意一种：十二烷基硫酸钠、十二烷基肌氨酸钠和脱氧胆酸钠，所述非离子表面活性剂为以下一种或多种：吐温20、吐温80、曲拉通X-100、乙基苯基聚乙二醇和十六醇聚氧乙烯醚Brij C20；
    步骤S1、S2、S3和S4中，离心转速为10000～16000×g，离心时间为1～5分钟。
  6. 一种血液中病原微生物分离、富集和核酸提取试剂，其特征在于：包括体积为血液样本0.5～10倍的血液选择裂解液A、体积为所述血液样本0.5％～10％的病原微生物助沉剂、体积为所述血液样本1～10倍的血液选择裂解液B、体积为所述血液样本1～10倍的洗涤液和体积为所述血液样本0.5～2倍的核酸提取液；
    所述血液选择裂解液A和所述血液选择裂解液B用于仅裂解血细胞保留病原微生物并将所述血液样本中以及所述裂解血细胞中释放的大分子有机物进行溶解；所述病原微生物助沉剂用于使病原微生物富集沉淀；所述洗涤液用于清洗杂质，所述核酸提取液用于使所述病原微生物富集、裂解并促使所述病原微生物中的核酸释放和溶解；
    所述血液选择裂解液A包括：浓度为10～100mM的缓冲液、质量体积百分比为0％～6.5％的辅助裂解盐、质量体积百分比为5％～65％的主要裂解盐、质量体积百分比为0％～5％的表面活性剂、浓度为0～10mM的螯合剂和体积百分比0％～0.5％的消泡剂，所述血液选择裂解液A的pH为4～8；
    所述病原微生物助沉剂为不溶于水的生物惰性有机溶剂；
    所述血液选择裂解液B包括：浓度为10～100mM的所述缓冲液、质量体积百分比为0％～3％的所述辅助裂解盐、质量体积百分比为0％～40％的所述主要裂解盐、质量体积百分比为0％～2％的所述表面活性剂、浓度为0～10mM的所述螯合剂和体积百分比0％～0.5％的所述消 泡剂，所述血液选择裂解液B的pH为5～8。
  7. 根据权利要求6所述的一种血液中病原微生物分离、富集和核酸提取试剂，其特征在于：所述洗涤液包括：浓度为10～100mM的所述缓冲液、质量体积百分比为0％～5％的促进杂质溶解盐和浓度为0～10mM的所述螯合剂，所述洗涤液的pH为5～9；
    所述核酸提取液包括：浓度为0.5～20mM的所述缓冲液、质量体积百分比为0～5％的促进核酸溶解盐、质量体积百分比为0.01％～5％的表面活性剂、浓度为0～3mM的所述螯合剂和体积百分比0％～0.5％的所述消泡剂，所述核酸提取液的pH为7～9；
    所述生物惰性有机溶剂为氟油或HFE-7500或Novec7500或FC-40。
  8. 根据权利要求6或7任意一项所述的一种血液中病原微生物分离、富集和核酸提取试剂，其特征在于：
    所述缓冲液为以下任意一种：磷酸缓冲液、乙酸钠乙酸缓冲液、三羟甲基氨基甲烷盐酸缓冲液；
    所述辅助裂解盐、所述促进核酸溶解盐和所述促进杂质溶解盐为以下一种或多种：氯化钠、氯化钾和硫酸铵；
    所述主要裂解盐为以下一种或多种：氯化锂、碘化钠、碘化钾、盐酸胍和异硫氰酸胍；
    所述消泡剂为以下任意一种：聚二甲基硅氧烷、聚丙二醇、聚醚改性聚硅氧烷和聚氧丙烯甘油醚；
    所述螯合剂为柠檬酸三钠；
    所述表面活性剂为阴离子表面活性剂和/或非离子表面活性剂。
  9. 根据权利要求8所述的一种血液中病原微生物分离、富集和核酸提取试剂，其特征在于：所述阴离子表面活性剂为以下任意一种：十二烷基硫酸钠、十二烷基肌氨酸钠和脱氧胆酸钠，所述非离子表面活性剂为以下一种或多种：吐温20、吐温80、曲拉通X-100、乙基苯基聚乙二醇和十六醇聚氧乙烯醚Brij C20。
  10. 根据权利要求9所述的一种血液中病原微生物分离、富集和核酸提取试剂，其特征在于：所述血液选择裂解液A包括20mM三羟 甲基氨基甲烷盐酸缓冲液、质量体积百分比0.5％的氯化钾、质量体积百分比65％的异硫氰酸胍、质量体积百分比0.2％的十二烷基肌氨酸钠、10mM柠檬酸三钠和体积百分比0.02％的聚氧丙烯甘油醚，所述血液选择裂解液A的pH为7.0；
    所述血液选择裂解液B包括20mM三羟甲基氨基甲烷盐酸缓冲液，质量体积百分比0.5％的氯化钾、质量体积百分比30％的异硫氰酸胍、质量体积百分比的0.1％十二烷基肌氨酸钠、10mM柠檬酸三钠和体积百分比0.02％的聚氧丙烯甘油醚，所述血液选择裂解液B的pH为7.0；
    所述微生物助沉剂为Novec7500；
    所述洗涤液包括10mM三羟甲基氨基甲烷盐酸盐缓冲液、质量体积百分比的0.9％氯化钠、质量体积百分比的10％硫酸铵和3mM柠檬酸三钠，所述洗涤液的pH为7.0；
    所述核酸提取液包括10mM三羟甲基氨基甲烷盐酸盐缓冲液、质量体积百分比0.45％的氯化钠、质量体积百分比0.1％的十二烷基硫酸钠、质量体积百分比2％的吐温20、1mM柠檬酸三钠和体积百分比0.02％的聚氧丙烯甘油醚，所述核酸提取液的pH为8.8。
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