CN116042603A - Method and reagent for separating, enriching and extracting nucleic acid from pathogenic microorganism in blood - Google Patents
Method and reagent for separating, enriching and extracting nucleic acid from pathogenic microorganism in blood Download PDFInfo
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Abstract
The invention provides a method for separating, enriching and extracting nucleic acid of pathogenic microorganism in blood and a reagent thereof. The method can rapidly and accurately extract the target infection pathogen nucleic acid which can meet the molecular biological detection requirement from a blood sample of not more than 10mL, does not need blood culture, directly uses the blood sample to carry out pathogen enrichment and nucleic acid extraction, is also suitable for a post-blood culture sample, has simple nucleic acid extraction steps, does not need complex processing equipment, and can be used for downstream detection of various molecular biology.
Description
The application is a divisional application of patent application of the invention with the application date of 2021, 3 and 31, the application number of 202110346173.9 and the name of 'a method for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood'.
Technical Field
The invention relates to the technical field of detection or inspection methods containing enzymes, nucleic acids or microorganisms, in particular to a method and a reagent for separating, enriching and extracting pathogenic microorganisms in blood.
Background
Blood flow infections refer to systemic inflammatory response syndrome caused by invasion of the blood flow by pathogenic microorganisms such as bacteria, fungi, etc. Pathogenic microorganisms exist in circulating blood in a transient, intermittent or continuous manner, damage is caused to all organs of the organism, such as heart valves, joints and the like, and shock, multiple organ failure, disseminated intravascular coagulation and even death can be caused by serious diseases. Sepsis and bacteremia are currently collectively referred to as blood flow infections. Sepsis is a blood infection caused by a variety of pathogenic microorganisms (bacteria or fungi) and toxins invading the blood stream of humans. Bacteria are classified as primary and secondary if they are only transient human blood and have no clinically significant toxic blood symptoms, called bacteremia. Primary bacteremia is associated with intravenous procedures, and secondary bacteremia is mostly caused by postoperative wound, abdominal, urinary and pulmonary infections. In recent years, due to the wide application of invasive devices and treatments such as intravenous catheter indwelling, mechanical ventilation, parenteral administration, and the like, and the abuse of immunosuppressants and a large amount of antibacterial drugs, the incidence of blood flow infection has increased year by year, and blood flow infection has been paid attention to by more and more doctors and researchers because of not only high death rate but also prolonged hospitalization time, increased hospitalization costs, and the like.
The most common causes of blood flow infections are bacteria such as E.coli, staphylococcus aureus and pneumococcus. In addition to bacterial infections, fungal infections have recently become a significant cause of the disease. The development of diagnostic and therapeutic methods for blood flow infections has been a thirty year history, but only little progress has been made. Culturing microorganisms from a blood sample to increase the microorganism content from 1 to 10CFU/mL to 10 6 ～10 8 CFU/mL remains the gold standard for detecting blood flow infections. However, this method has a number of disadvantages, including a relatively long diagnostic time, typically 1 to 5 days between sampling from the patient and providing the result of the infection. During this time, the usual therapy is to treat the patient with a broad spectrum of non-targeted antibiotics. This may have an effect in the treatment of the disease, but has the serious consequence that many microorganisms develop multi-drug resistance. Second, the culture method requires a large amount of blood samples for diagnosis and has low detection sensitivity. Furthermore, there is a relatively high risk of contamination during blood collection and blood sample use. So, although blood culture is still the main reference method for diagnosing blood flow infection at present, the method is not an ideal gold standard because of the obvious defects of low sensitivity, long time consumption and the like.
With the development of molecular biology techniques, molecular biology diagnostic techniques, such as Polymerase Chain Reaction (PCR) and second generation sequencing techniques, are beginning to be applied to the detection of microorganisms infected with blood flow. The method based on molecular biological diagnosis has the remarkable advantages of high speed, high sensitivity, small sample size, short time (generally not more than 12 hours) for providing relevant information of microbial diagnosis, and subsequent introduction of proper antibiotic treatment (detection of drug resistance genes). However, for a long time, due to the extremely low number of circulating microorganisms (1-10 CFU/mL) in blood, the large amount of human nucleic acid in blood itself may interfere with the detection of PCR and sequencing, and at the same time, components of blood itself, such as iron, hemoglobin, etc., and blood anticoagulants, may severely inhibit PCR and library construction, so the diagnosis of microorganisms directly from whole blood has been limited.
Therefore, the nucleic acid which is rapidly separated from the whole blood, enriched to the pathogenic microorganism and extracted for molecular biology detection can effectively improve the detection efficiency of the pathogenic microorganism infected by blood flow, provide guidance for doctors reasonably and pertinently taking medicine and treating in time, and meanwhile, strive for gold treatment time for patients, thereby increasing the survival rate of the patients.
Disclosure of Invention
The invention aims to provide a method and a reagent for directly separating and enriching infectious pathogenic microorganisms from blood (the blood consumption of a pediatric patient comprises 1-2 mL of neonate and 5-10 mL of adult) of a suspected blood-infected patient and extracting nucleic acid, wherein blood samples are directly used for pathogen enrichment and nucleic acid extraction without blood culture, meanwhile, the method is also applicable to blood post-culture samples, the nucleic acid extraction step is simple, the method can be indifferently applied to gram-negative bacteria, positive bacteria and fungi, the extracted nucleic acid can be matched with a plurality of molecular biological detection methods such as single-weight, multiple PCR/RT-PCR, second-generation sequencing and the like, the requirement for operating an adaptive instrument is not high, and the operation requirement can be met only by a common high-speed centrifuge in a laboratory.
The invention provides a method for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood, which comprises the following steps:
s1, primary cracking of a blood sample: adding a blood selective lysate A and a pathogenic microorganism sedimentation assisting agent into a blood sample, fully and uniformly mixing to obtain a lysate, dissolving macromolecular organic matters in the lysate, and removing part of supernatant after high-speed centrifugation to obtain a blood primary lysis sample; lysates include lysed blood cells, including erythrocytes and leukocytes, and non-lysed but surface-modified pathogenic microorganisms, and high-speed centrifugal sedimentation for the enrichment and precipitation of pathogenic microorganisms;
S2, re-splitting the blood sample: adding blood to select lysate B to further dissolve residual macromolecular organic matters, and removing partial supernatant on the interface of the microbial sedimentation aid and the aqueous solution after high-speed centrifugation to obtain a blood re-lysis sample;
s3, washing: adding a washing liquid, uniformly mixing, centrifuging, removing part of supernatant to obtain a blood lysis sample containing pathogenic microorganisms, wherein the washing liquid is used for cleaning impurities;
s4, extracting nucleic acid: adding a nucleic acid extraction reagent into a blood cracking sample, removing part of supernatant after high-speed centrifugation, performing electric cracking on the residual solution or adding grinding beads with different particle size combinations into the residual solution, oscillating, heating at high temperature, centrifuging to crack pathogenic microorganisms and promote release and dissolution of nucleic acid in the pathogenic microorganisms, thus obtaining a pathogenic microorganism nucleic acid sample.
In the method for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood according to the present invention, in a preferred mode, in step S1, the blood selective lysate a includes: buffer solution with the concentration of 10-100 mM, auxiliary cracking salt with the mass and volume percentage of 0-6.5%, main cracking salt with the mass and volume percentage of 5-65%, surfactant with the mass and volume percentage of 0-5%, chelating agent with the concentration of 0-10 mM and defoaming agent with the volume percentage of 0-0.5%, wherein the pH value of the blood selective cracking solution A is 4-8;
The pathogenic microorganism sedimentation aid is a water-insoluble biological inert organic solvent;
the blood sample is 1-10 ml, after adding the blood selective lysate A and the pathogenic microorganism sedimentation assisting agent, the mixture is uniformly mixed by normal temperature oscillation or uniformly mixed for 1-5 minutes by normal temperature inversion, the dosage of the blood selective lysate A is 0.5-5 times of the volume of the blood sample, and the dosage of the pathogenic microorganism sedimentation assisting agent is 0.5-5% of the volume of the blood sample.
In the method for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood according to the present invention, in a preferred mode, in step S2, the blood selective lysate B includes: buffer solution with the concentration of 10-100 mM, auxiliary cracking salt with the mass and volume percentage of 0-3%, main cracking salt with the mass and volume percentage of 0-40%, surfactant with the mass and volume percentage of 0-2%, chelating agent with the concentration of 0-10 mM and defoaming agent with the volume percentage of 0-0.5%, wherein the pH value of the blood selective cracking solution B is 5-8;
the dosage of the blood selective lysate B is 2-10 times of the volume of the blood primary lysate sample;
in step S3, the washing liquid includes: buffer solution with the concentration of 10-100 mM, impurity dissolution promoting salt with the mass and volume percentage of 0-5% and chelating agent with the concentration of 0-10 mM, and the pH value of the washing solution is 5-9;
The consumption of the washing liquid is 5-10 times of the volume of the blood re-splitting sample, and the washing liquid is used by shaking and mixing at normal temperature or reversing and mixing at normal temperature;
steps S2, S3 may be repeated a number of times.
In a preferred embodiment of the method for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood according to the present invention, in step S4, the nucleic acid extracting solution includes: 0.5-20 mM buffer solution, 0-5% nucleic acid dissolution promoting salt, 0.01-5% surfactant, 0-3 mM chelating agent and 0-0.5% defoaming agent, wherein the pH of the nucleic acid extracting solution is 7-9;
the surfactant is an anionic surfactant and/or a nonionic surfactant;
the dosage of the nucleic acid extracting solution is 5-10 times of the volume of a blood cracking sample, an electric cracking device for MEMS processing is used for electric cracking, the grinding beads are one or more of acid-washing glass beads, zirconia beads and quartz sand with the particle size of 0.1-3 mm, the dosage of the grinding beads is 20-150 mg, the oscillation time is 5-15 minutes, the high-temperature heating temperature is 85-100 ℃, and the heating time is 3-15 minutes.
The invention relates to a method for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood, which is characterized in that a buffer solution is used as a preferable mode, and the buffer solution is any one of the following: phosphate buffer, sodium acetate buffer and tris hydrochloride buffer;
Auxiliary lysis salt, nucleic acid dissolution promoting salt and impurity dissolution promoting salt are one or more of the following: sodium chloride, potassium chloride and ammonium sulfate;
the main cracking salt is 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: polydimethyl siloxane, polypropylene glycol, polyether modified polysiloxane and polyoxypropylene glycerol ether;
the chelating agent is trisodium citrate;
the anionic surfactant is any one of the following: sodium dodecyl sulfate, sodium dodecyl 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 polyoxyethylene ether Brij C20;
in steps S1, S2, S3 and S4, the centrifugal speed is 10000-16000 Xg, and the centrifugal time is 1-5 minutes.
The invention provides a reagent for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood, which comprises a blood selective lysate A with the volume of 0.5-10 times of a blood sample, a pathogenic microorganism sedimentation aid with the volume of 0.5-10% of the blood sample, a blood selective lysate B with the volume of 1-10 times of the blood sample, a washing liquid with the volume of 1-10 times of the blood sample and a nucleic acid extracting liquid with the volume of 0.5-2 times of the blood sample;
The blood selective lysate A and the blood selective lysate B are used for only lysing blood cells to retain pathogenic microorganisms and dissolving macromolecular organic matters released in a blood sample and the lysed blood cells; the pathogenic microorganism sedimentation aid is used for enriching and precipitating pathogenic microorganisms; the washing liquid is used for cleaning impurities, and the nucleic acid extracting liquid is used for enriching and lysing pathogenic microorganisms and promoting the release and dissolution of nucleic acid in the pathogenic microorganisms;
blood selection lysate a includes: buffer solution with the concentration of 10-100 mM, auxiliary cracking salt with the mass and volume percentage of 0-6.5%, main cracking salt with the mass and volume percentage of 5-65%, surfactant with the mass and volume percentage of 0-5%, chelating agent with the concentration of 0-10 mM and defoaming agent with the volume percentage of 0-0.5%, wherein the pH value of the blood selective cracking solution A is 4-8;
the pathogenic microorganism sedimentation aid is a water-insoluble biological inert organic solvent;
blood selection lysate B includes: buffer solution with the concentration of 10-100 mM, auxiliary cracking salt with the mass and volume percentage of 0-3%, main cracking salt with the mass and volume percentage of 0-40%, surfactant with the mass and volume percentage of 0-2%, chelating agent with the concentration of 0-10 mM and defoaming agent with the volume percentage of 0-0.5%, wherein the pH value of the blood selective cracking solution B is 5-8.
The invention relates to a reagent for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood, which is characterized in that a washing solution comprises: buffer solution with the concentration of 10-100 mM, impurity dissolution promoting salt with the mass and volume percentage of 0-5% and chelating agent with the concentration of 0-10 mM, and the pH value of the washing solution is 5-9;
the nucleic acid extraction solution comprises: 0.5-20 mM buffer solution, 0-5% nucleic acid dissolution promoting salt, 0.01-5% surfactant, 0-3 mM chelating agent and 0-0.5% defoaming agent, wherein the pH of the nucleic acid extracting solution is 7-9;
the biologically inert organic solvent is fluorine oil or HFE-7500 or Novec7500 or FC-40.
The invention relates to a reagent for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood, which is characterized in that a buffer solution is used as a preferable mode, and the buffer solution is any one of the following components: phosphate buffer, sodium acetate buffer, tris hydrochloride buffer;
auxiliary lysis salt, nucleic acid dissolution promoting salt and impurity dissolution promoting salt are one or more of the following: sodium chloride, potassium chloride and ammonium sulfate;
the main cracking salt is 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: polydimethyl siloxane, polypropylene glycol, polyether modified polysiloxane and polyoxypropylene glycerol ether;
the chelating agent is trisodium citrate;
the surfactant is an anionic surfactant and/or a nonionic surfactant.
The invention relates to a reagent for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood, which is characterized in that an anionic surfactant is any one of the following materials: sodium dodecyl sulfate, sodium dodecyl 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 polyoxyethylene ether Brij C20.
The invention relates to a reagent for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood, which is characterized in that a preferred mode is that a blood selective lysate A comprises 20mM of tris (hydroxymethyl) aminomethane hydrochloride buffer solution, 0.5% of potassium chloride by mass and volume percent, 65% of guanidine isothiocyanate by mass and volume percent, 0.2% of sarcosyl by mass and volume percent, 10mM of trisodium citrate and 0.02% of polyoxypropylene glycerol ether by volume percent, and the pH value of the blood selective lysate A is 7.0;
The blood selective lysate B comprises 20mM of tris (hydroxymethyl) aminomethane hydrochloride buffer solution, 0.5% of potassium chloride, 30% of guanidine isothiocyanate, 0.1% of sarcosyl, 10mM of trisodium citrate and 0.02% of polyoxypropylene glycerol ether, wherein the pH value of the blood selective lysate B is 7.0;
the microbial sedimentation aid is Novec7500;
the washing liquid comprises 10mM of tris hydrochloride buffer solution, 0.9% of sodium chloride in mass and volume percentage, 10% of ammonium sulfate in mass and volume percentage and 3mM of trisodium citrate, and the pH value of the washing liquid is 7.0;
the nucleic acid extracting solution comprises 10mM of tris hydrochloride buffer solution, 0.45% of sodium chloride by mass and volume percentage, 0.1% of sodium dodecyl sulfate by mass and volume percentage, 2% of Tween 20 by mass and volume percentage, 1mM of trisodium citrate and 0.02% of polyoxypropylene glycerol ether by volume percentage, and the pH value of the nucleic acid extracting solution is 8.8.
The specific scheme is as follows: adding blood selective lysate A and pathogenic microorganism sedimentation aid into blood sample for sample lysis treatment, selectively lysing blood cells (including red blood cells, white blood cells and the like), dissolving protein, sugar, lipid and other impurities in blood, and then settling pathogenic microorganisms by high-speed centrifugation, and carefully discarding part of the supernatant; adding the blood selective lysate B, performing primary or secondary blood selective lysis, removing part of supernatant on the interface of the microbial sedimentation aid and the aqueous solution by high-speed centrifugation, adding a washing solution into the residual solution, uniformly mixing, centrifuging, continuously removing part of supernatant, and repeatedly washing for one or more times; then adding a nucleic acid extraction reagent to treat the residual solution again, and removing part of supernatant after high-speed centrifugation, wherein pathogenic microorganisms are in the residual nucleic acid extraction solution; and (3) performing electric pyrolysis or adding grinding beads with different particle size combinations into the mixture, and performing high-temperature heating and centrifugal extraction on nucleic acid of pathogenic microorganisms.
In a preferred technical scheme of the invention, the dosage of the blood selective lysate A is 0.5-5 times of the volume of a blood sample, the dosage of the microbial sedimentation agent is 0.5-5% of the volume of the blood sample, the condition of the lysis is that the mixture is uniformly mixed at normal temperature in an oscillating way or reversely mixed at normal temperature for 1-5 minutes, the centrifugal speed is 10000-16000 Xg, the centrifugal time is 1-5 minutes, the supernatant is discarded by a pipette or a syringe and the like, the sediment at the junction of the microbial sedimentation agent and the water solution is not touched, and the solution discarding amount is 80-95% of the total solution volume.
In a preferred technical scheme of the invention, the dosage of the blood selective lysate B is 2-10 times of the volume of the residual supernatant, the condition of the lysis is that the mixture is uniformly mixed at normal temperature or reversely mixed at normal temperature for 1-5 minutes, the centrifugal speed is 10000-16000 Xg, the centrifugal time is 1-5 minutes, the supernatant is discarded, a pipettor or a syringe or the like is used, the precipitate at the junction of the microbial sedimentation aid and the aqueous solution is not accessible, the solution discarding amount is equal to the adding amount of the blood selective lysate B, and the lysis step is repeated one or more times according to the sedimentation property.
In a preferred technical scheme of the invention, the consumption of the washing liquid is 5-10 times of the volume of the residual supernatant, the washing condition is that the washing liquid is uniformly mixed at normal temperature in an oscillating way or reversely mixed at normal temperature for 1-5 minutes, the centrifugal speed is 10000-16000 Xg, the centrifugal time is 1-5 minutes, the supernatant is discarded by a liquid transfer device or a syringe and the like, the sediment at the junction of the microbial sedimentation aid and the aqueous solution is inaccessible, the solution discarding amount is equal to the adding amount of the washing liquid, and the cracking step is repeated one or more times according to the sedimentation property.
In a preferred technical scheme of the invention, the dosage of the nucleic acid extracting solution is 5-10 times of the volume of the residual supernatant, the treatment condition is that the solution is uniformly mixed at normal temperature in an oscillating way or is reversely mixed at normal temperature for 1-5 minutes, the centrifugal speed is 10000-16000 Xg, the centrifugal time is 1-5 minutes, the supernatant is discarded by using a liquid-transferring device or a syringe and the like, the precipitate at the junction of the microbial sedimentation aid and the aqueous solution is not accessible, the solution discarding amount is equal to the adding amount of the nucleic acid extracting solution, and pathogenic microorganisms enriched from a sample are contained in the residual supernatant and the precipitate.
In a preferred technical scheme of the invention, the grinding beads are acid-washed glass beads or zirconia beads with different particle sizes of 0.1-3 mm, the dosage is 20-150 mg, the oscillation time is 5-15 minutes, the high-temperature heating temperature is 85-100 ℃, the heating time is 3-15 minutes, the centrifugal speed is 10000-16000 Xg, the centrifugal time is 1-5 minutes, and the supernatant is sucked by a liquid-transfering device or a syringe after centrifugation, thus obtaining the target nucleic acid.
In a preferred embodiment of the present invention, the size of the electric lysis device for nucleic acid extraction may be (0.1 to 1) × (5 to 1000) × (10 to 1000) mm 3 The size can be larger or smaller than the design size according to the requirements. The cracking voltage is 10-2000V.
In a preferred technical scheme of the invention, the blood sample is blood of a patient suspected of blood flow infection, the blood dosage of the pediatric patient comprises 1-2 mL of blood of a neonate and 5-10 mL of adult; the sample can also be a sample after blood culture, and the dosage is 0.2-1 mL.
According to the invention, blood cells (including red blood cells, white blood cells and the like) are ruptured through the osmotic pressure difference between the blood selective lysate A and blood and the addition of the surfactant, macromolecular organic matters and the like released by blood cell rupture are dissolved in the blood as far as possible under proper concentration, and the pathogenic microorganism cell structure is not seriously damaged. And the salts and the surfactant can also promote the enrichment and precipitation of pathogenic microorganisms which are difficult to centrifugally precipitate due to the special extracellular structure (such as bacterial capsule) together with the microbial sedimentation aid, so as to reduce the differential enrichment of the microorganisms. The microbial sedimentation aid can also reduce adhesion between microorganisms and the front of the centrifuge tube, and avoid loss caused by incomplete centrifugal sedimentation of the microorganisms. The addition of the blood selective lysate B compensates for the incomplete lysis treatment due to sample differences and sufficiently dissolves residual impurities. The wash solution removes lysate components and blood insoluble materials present in the lysate that may inhibit downstream detection. The large amount of nucleic acid extracting solution dilutes the salts in the washing solution and provides conditions suitable for the lysis and nucleic acid dissolution of pathogenic microorganisms, and the pathogenic microorganisms are fully lysed to promote the release and dissolution of the nucleic acid under the action of instantaneous electric lysis or physical grinding and high-temperature heating denaturation of grinding beads with different particle diameters.
Macromolecular organic substances include proteins, carbohydrates, lipids, nucleic acids, and the like.
The invention has the following advantages:
(1) Can rapidly and accurately extract target infection pathogen nucleic acid meeting the molecular biological detection requirement from blood samples of not more than 10 mL.
(2) The blood sample is directly used for pathogen enrichment and nucleic acid extraction without blood culture, and is also suitable for samples after blood culture.
(3) The nucleic acid extraction steps are simple, and can be applied to gram-negative bacteria, gram-positive bacteria and fungi indiscriminately.
(4) The extracted nucleic acid can be matched with a plurality of molecular biological detection methods such as single-weight PCR, multiple PCR/RT-PCR, second generation sequencing and the like.
(5) The requirement on operation of the adapting instrument is not high, and the operation requirement can be met only by a high-speed centrifuge common in a laboratory.
Drawings
FIG. 1 is a flow chart of a method for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood;
FIG. 2 is a schematic diagram of a method for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood;
FIG. 3 is an amplification curve of a fluorescent quantitative PCR for a method for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood and a reagent example 5 probe method;
FIG. 4 is an amplification curve of a fluorescent quantitative PCR method for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood and a reagent example 6 probe method;
FIG. 5 shows the amplification curves of a method for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood and a fluorescent quantitative PCR method using the probe of reagent example 7.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments.
Example 1
As shown in fig. 1, a method for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood is characterized in that: the method comprises the following steps:
s1, primary cracking of a blood sample: adding a blood selective lysate A and a pathogenic microorganism sedimentation assisting agent into a blood sample, fully and uniformly mixing to obtain a lysate, dissolving macromolecular organic matters in the lysate, and removing part of supernatant after high-speed centrifugation to obtain a blood primary lysis sample; lysates include lysed blood cells, including erythrocytes and leukocytes, and non-lysed but surface-modified pathogenic microorganisms, and high-speed centrifugal sedimentation for the enrichment and precipitation of pathogenic microorganisms;
s2, re-splitting the blood sample: adding blood to select lysate B to further dissolve residual macromolecular organic matters, and removing partial supernatant on the interface of the microbial sedimentation aid and the aqueous solution after high-speed centrifugation to obtain a blood re-lysis sample;
S3, washing: adding a washing liquid, uniformly mixing, centrifuging, removing part of supernatant to obtain a blood lysis sample containing pathogenic microorganisms, wherein the washing liquid is used for cleaning impurities;
s4, extracting nucleic acid: adding a nucleic acid extraction reagent into a blood cracking sample, removing part of supernatant after high-speed centrifugation, performing electric cracking on the residual solution or adding grinding beads with different particle size combinations into the residual solution, oscillating, heating at high temperature, centrifuging to crack pathogenic microorganisms and promote release and dissolution of nucleic acid in the pathogenic microorganisms, thus obtaining a pathogenic microorganism nucleic acid sample.
Example 2
As shown in fig. 1, a method for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood is characterized in that: the method comprises the following steps:
s1, primary cracking of a blood sample: adding a blood selective lysate A and a pathogenic microorganism sedimentation assisting agent into a blood sample, fully and uniformly mixing to obtain a lysate, dissolving macromolecular organic matters in the lysate, and removing part of supernatant after high-speed centrifugation to obtain a blood primary lysis sample; lysates include lysed blood cells, including erythrocytes and leukocytes, and non-lysed but surface-modified pathogenic microorganisms, and high-speed centrifugal sedimentation for the enrichment and precipitation of pathogenic microorganisms;
Blood selection lysate a includes: buffer solution with the concentration of 10-100 mM, auxiliary cracking salt with the mass and volume percentage of 0-6.5%, main cracking salt with the mass and volume percentage of 5-65%, surfactant with the mass and volume percentage of 0-5%, chelating agent with the concentration of 0-10 mM and defoaming agent with the volume percentage of 0-0.5%, wherein the pH value of the blood selective cracking solution A is 4-8;
the pathogenic microorganism sedimentation aid is a water-insoluble biological inert organic solvent;
adding 1-10 ml of blood sample, adding blood selective lysate A and pathogenic microorganism sedimentation assisting agent, shaking at normal temperature, mixing uniformly or reversing at normal temperature, mixing uniformly for 1-5 minutes, wherein the dosage of the blood selective lysate A is 0.5-5 times of the volume of the blood sample, and the dosage of the pathogenic microorganism sedimentation assisting agent is 0.5-5% of the volume of the blood sample;
s2, re-splitting the blood sample: adding blood to select lysate B to further dissolve residual macromolecular organic matters, and removing partial supernatant on the interface of the microbial sedimentation aid and the aqueous solution after high-speed centrifugation to obtain a blood re-lysis sample;
blood selection lysate B includes: buffer solution with the concentration of 10-100 mM, auxiliary cracking salt with the mass and volume percentage of 0-3%, main cracking salt with the mass and volume percentage of 0-40%, surfactant with the mass and volume percentage of 0-2%, chelating agent with the concentration of 0-10 mM and defoaming agent with the volume percentage of 0-0.5%, wherein the pH value of the blood selective cracking solution B is 5-8;
The dosage of the blood selective lysate B is 2-10 times of the volume of the blood primary lysate sample;
step S2 may be repeated a plurality of times;
s3, washing: adding a washing liquid, uniformly mixing, centrifuging, removing part of supernatant to obtain a blood lysis sample containing pathogenic microorganisms, wherein the washing liquid is used for cleaning impurities;
in step S3, the washing liquid includes: buffer solution with the concentration of 10-100 mM, impurity dissolution promoting salt with the mass and volume percentage of 0-5% and chelating agent with the concentration of 0-10 mM, and the pH value of the washing solution is 5-9;
the consumption of the washing liquid is 5-10 times of the volume of the blood re-splitting sample, and the washing liquid is used by shaking and mixing at normal temperature or reversing and mixing at normal temperature;
step S3 may be repeated a plurality of times;
s4, extracting nucleic acid: adding a nucleic acid extraction reagent into a blood cracking sample, removing part of supernatant after high-speed centrifugation, performing electric cracking on the residual solution or adding grinding beads with different particle size combinations into the residual solution, oscillating, heating at high temperature, centrifuging to crack pathogenic microorganisms and promote release and dissolution of nucleic acid in the pathogenic microorganisms to obtain a pathogenic microorganism nucleic acid sample;
the nucleic acid extraction solution comprises: 0.5-20 mM buffer solution, 0-5% nucleic acid dissolution promoting salt, 0.01-5% surfactant, 0-3 mM chelating agent and 0-0.5% defoaming agent, wherein the pH of the nucleic acid extracting solution is 7-9;
The surfactant is an anionic surfactant and/or a nonionic surfactant;
the dosage of the nucleic acid extracting solution is 5-10 times of the volume of a blood cracking sample, an electric cracking device for MEMS processing is used for electric cracking, grinding beads are one or more of acid-washing glass beads, zirconia beads and quartz sand with the particle size of 0.1-3 mm, the dosage of the grinding beads is 20-150 mg, the oscillation time is 5-15 minutes, the high-temperature heating temperature is 85-100 ℃, and the heating time is 3-15 minutes;
wherein the buffer solution is any one of the following: phosphate buffer, sodium acetate buffer and tris hydrochloride buffer;
auxiliary lysis salt, nucleic acid dissolution promoting salt and impurity dissolution promoting salt are one or more of the following: sodium chloride, potassium chloride and ammonium sulfate;
the main cracking salt is 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: polydimethyl siloxane, polypropylene glycol, polyether modified polysiloxane and polyoxypropylene glycerol ether;
the chelating agent is trisodium citrate;
the anionic surfactant is any one of the following: sodium dodecyl sulfate, sodium dodecyl 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 polyoxyethylene ether Brij C20;
In steps S1, S2, S3 and S4, the centrifugal speed is 10000-16000 Xg, and the centrifugal time is 1-5 minutes.
Example 3
A reagent for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood comprises a blood selective lysate A with the volume of 0.5-10 times of that of a blood sample, a pathogenic microorganism sedimentation aid with the volume of 0.5-10% of that of the blood sample, a blood selective lysate B with the volume of 1-10 times of that of the blood sample, a washing liquid with the volume of 1-10 times of that of the blood sample and a nucleic acid extracting liquid with the volume of 0.5-2 times of that of the blood sample;
the blood selective lysate A and the blood selective lysate B are used for only lysing blood cells to retain pathogenic microorganisms and dissolving macromolecular organic matters released in a blood sample and the lysed blood cells; the pathogenic microorganism sedimentation aid is used for enriching and precipitating pathogenic microorganisms; the washing liquid is used for cleaning impurities, and the nucleic acid extracting liquid is used for enriching and lysing pathogenic microorganisms and promoting the release and dissolution of nucleic acid in the pathogenic microorganisms;
blood selection lysate a includes: buffer solution with the concentration of 10-100 mM, auxiliary cracking salt with the mass and volume percentage of 0-6.5%, main cracking salt with the mass and volume percentage of 5-65%, surfactant with the mass and volume percentage of 0-5%, chelating agent with the concentration of 0-10 mM and defoaming agent with the volume percentage of 0-0.5%, wherein the pH value of the blood selective cracking solution A is 4-8;
The pathogenic microorganism sedimentation aid is a water-insoluble biological inert organic solvent;
blood selection lysate B includes: buffer solution with the concentration of 10-100 mM, auxiliary cracking salt with the mass and volume percentage of 0-3%, main cracking salt with the mass and volume percentage of 0-40%, surfactant with the mass and volume percentage of 0-2%, chelating agent with the concentration of 0-10 mM and defoaming agent with the volume percentage of 0-0.5%, wherein the pH value of the blood selective cracking solution B is 5-8.
Example 4
A reagent for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood comprises a blood selective lysate A with the volume 10 times that of a blood sample, a pathogenic microorganism sedimentation aid with the volume 10% of the blood sample, a blood selective lysate B with the volume 10 times that of the blood sample, a washing liquid with the volume 10 times that of the blood sample and a nucleic acid extracting liquid with the volume 2 times that of the blood sample;
the blood selective lysate A and the blood selective lysate B are used for only lysing blood cells to retain pathogenic microorganisms and dissolving macromolecular organic matters released in a blood sample and the lysed blood cells; the pathogenic microorganism sedimentation aid is used for enriching and precipitating pathogenic microorganisms; the washing liquid is used for cleaning impurities, and the nucleic acid extracting liquid is used for enriching and lysing pathogenic microorganisms and promoting the release and dissolution of nucleic acid in the pathogenic microorganisms;
Blood selection lysate a includes: buffer solution with the concentration of 10-100 mM, auxiliary cracking salt with the mass and volume percentage of 0-6.5%, main cracking salt with the mass and volume percentage of 5-65%, surfactant with the mass and volume percentage of 0-5%, chelating agent with the concentration of 0-10 mM and defoaming agent with the volume percentage of 0-0.5%, wherein the pH value of the blood selective cracking solution A is 4-8;
the pathogenic microorganism sedimentation aid is a water-insoluble biological inert organic solvent;
the biological inert organic solvent is fluorine oil or HFE-7500 or Novec7500 or FC-40;
blood selection lysate B includes: buffer solution with the concentration of 10-100 mM, auxiliary cracking salt with the mass and volume percentage of 0-3%, main cracking salt with the mass and volume percentage of 0-40%, surfactant with the mass and volume percentage of 0-2%, chelating agent with the concentration of 0-10 mM and defoaming agent with the volume percentage of 0-0.5%, wherein the pH value of the blood selective cracking solution B is 5-8; the washing liquid comprises: buffer solution with the concentration of 10-100 mM, impurity dissolution promoting salt with the mass and volume percentage of 0-5% and chelating agent with the concentration of 0-10 mM, and the pH value of the washing solution is 5-9;
the nucleic acid extraction solution comprises: 0.5-20 mM buffer solution, 0-5% nucleic acid dissolution promoting salt, 0.01-5% surfactant, 0-3 mM chelating agent and 0-0.5% defoaming agent, wherein the pH of the nucleic acid extracting solution is 7-9;
The buffer solution is any one of the following: phosphate buffer, sodium acetate buffer, tris hydrochloride buffer;
auxiliary lysis salt, nucleic acid dissolution promoting salt and impurity dissolution promoting salt are one or more of the following: sodium chloride, potassium chloride and ammonium sulfate;
the main cracking salt is 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: polydimethyl siloxane, polypropylene glycol, polyether modified polysiloxane and polyoxypropylene glycerol 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 dodecyl sulfate, sodium dodecyl 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 polyoxyethylene ether Brij C20.
Example 5
In order to verify the effectiveness of the method and the reagent, the reagent and the method are used for separating and extracting nucleic acid of pathogenic microorganisms simulating blood flow infection, meanwhile, the nucleic acid extracted by adding the same quantity of pathogenic microorganisms into a simulated sample is used as a reference, and the extracted DNA is detected by a probe method fluorescence quantitative PCR, so that the feasibility of the method is verified.
In the following examples, the PCR detection target is DNA of a pathogenic microorganism in the nucleic acid, the isolated nucleic acid of the present invention also includes RNA of a pathogenic microorganism, and qRT-PCR or the like can be performed depending on the design sites of the detection primer and the probe, and the detection method is not limited to other downstream application modes of the present invention.
Reagents for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood are as follows: blood selection lysate A was 20mM tris hydrochloride buffer (pH 7.0), 0.5% potassium chloride, 65% guanidine isothiocyanate, 0.2% sarcosyl, 10mM trisodium citrate and 0.02% polyoxypropylene glycerol ether. The microbial sedimentation aid is Novec7500. Blood selection lysate B was 20mM tris hydrochloride buffer (pH 7.0), 0.5% potassium chloride, 30% guanidine isothiocyanate, 0.1% sodium lauroyl sarcosinate, 10mM trisodium citrate and 0.02% polyoxypropylene glycerol ether. The wash solution was 10mM tris hydrochloride buffer (pH 7.0), 0.9% sodium chloride, 10% ammonium sulfate and 3mM trisodium citrate. The nucleic acid extract was 10mM tris hydrochloride buffer (pH 8.8), 0.45% sodium chloride, 0.1% sodium dodecyl sulfate, 2% Tween 20, 1mM trisodium citrate and 0.02% polyoxypropylene glycerol ether.
The method for separating, enriching and extracting the nucleic acid from the escherichia coli in the blood comprises the following steps:
preparing a simulation sample containing escherichia coli, respectively adding 10CFU/100CFU/1000CFU escherichia coli into 3 pieces of 1mL negative blood, and uniformly mixing for later use; meanwhile, preparing a control sample, respectively adding 10CFU/100CFU/1000CFU of escherichia coli into 3 100 mu L of nucleic acid extracting solution, and uniformly mixing for later use. Reagents are added to the prepared simulated sample, and operations such as microorganism separation, enrichment, nucleic acid extraction and the like are performed. Specifically, 1mL of blood selection lysate A and 20. Mu.L of a pathogenic microorganism auxiliary reagent were added to 1mL of the simulated blood sample, the mixture was shaken for 1 minute, and the mixture was centrifuged at 12000 Xg for 2 minutes, and 1.8mL of the supernatant (the remaining 200. Mu.L) was discarded. Then 1mL of blood selection lysate B was added, shaking was performed for 1 minute, 12000 Xg was centrifuged for 2 minutes, and 1mL of the supernatant was discarded. This step is repeated one or more times. 1mL of the washing solution was added to the sample, the mixture was shaken for 1 minute, centrifuged at 12000 Xg for 2 minutes, and 1mL of the supernatant was discarded. This step is repeated one or more times. Finally, 1mL of the nucleic acid extract was added, the mixture was shaken for 1 minute, centrifuged at 12000 Xg for 2 minutes, and 1mL of the supernatant was discarded. 30mg of 0.5mm zirconia beads were added, vortexed for 5 minutes, heated at 95℃for 5 minutes, and centrifuged at 12000 Xg for 2 minutes, and the supernatant was collected to obtain a mock sample nucleic acid. Meanwhile, 30mg of 0.5mm zirconia beads were added to the control sample, vortexed and oscillated for 5 minutes, heated at 95℃for 5 minutes, centrifuged at 12000 Xg for 2 minutes, and the supernatant was collected to obtain nucleic acid of the control sample. In parallel, a group of blank blood separation and extraction controls without E.coli was made. The nucleic acid is prepared by a probe method PCR detection system according to the table 1 and is subjected to on-machine amplification program setting, and the specific primer probe sequences are shown in the table 2. As shown in FIG. 3, on three detection gradients, the amplification overlapping performance of the control group and the experimental group is good at the levels of 100CFU and 1000CFU per milliliter, the amplification of the two groups is slightly different at the level of 10CFU, but the amplification of the two groups can be stably detected, and the amplification results are reliable because the negative blood extraction control and the template-free control are not amplified. Thus, the reagent and the method can be used for separating and extracting the E.coli DNA on the order of 10CFU in blood, and meet the detection requirement of blood samples infected by blood flow.
And (3) comparing the fluorescence quantitative PCR amplification curve of the probe method for separating 10/100/1000CFU of escherichia coli from the blood sample and directly extracting 10/100/1000CFU of escherichia coli. Wherein C-E1, C-E2, C-E3 in the left legend of FIG. 3 correspond to 10, 100, 1000CFU, respectively, of the control group without isolation, E1, E2, E3 correspond to 10, 100, 1000CFU, respectively, of the pathogen experimental group isolated from the blood sample; BNTC is negative blood separation and extraction control, and NTC is template-free amplification control.
Example 6
In order to verify the effectiveness of the method and the reagent, the reagent and the method are used for separating and extracting nucleic acid of pathogenic microorganisms of the type of streptococcus pneumoniae for simulating blood infection, meanwhile, the nucleic acid extracted by adding the same quantity of pathogenic microorganisms into a simulated sample is used as a reference, and the extracted DNA is detected by a probe method fluorescence quantitative PCR, so that the feasibility of the method is verified.
Reagents for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood are as follows: blood selection lysate A was 5mM phosphate buffer (pH 7.5), 1.5% ammonium sulfate, 12% sodium iodide, 10mM trisodium citrate, 0.5% triton X-100, and 0.03% polyoxypropylene glycerol ether. The microbial sedimentation aid is FC-40. Blood selection lysate B was 5mM phosphate buffer (pH 7.5), 0.9% sodium chloride, 4% sodium iodide, 5mM trisodium citrate, and 0.03% polyoxypropylene glycerol ether. The wash solution was 2mM phosphate buffer (pH 8.5), 0.9% sodium chloride, 3% ammonium sulfate, 5mM trisodium citrate. The nucleic acid extracts were 5mM phosphate buffer (pH 8.0), 0.45% sodium chloride, 0.1% sarcosyl, 5% Tween 20, 0.5mM trisodium citrate, and 0.1% polypropylene glycol.
The separation, enrichment and nucleic acid extraction method of streptococcus pneumoniae in blood comprises the following steps:
preparing a simulation sample containing streptococcus pneumoniae, respectively adding 10CFU/100CFU/1000CFU streptococcus pneumoniae into 3 pieces of 1mL negative blood, and uniformly mixing for later use; meanwhile, preparing a control sample, respectively adding 10CFU/100CFU/1000CFU streptococcus pneumoniae into 3 100 mu L nucleic acid extracting solutions, and uniformly mixing for later use. Reagents are added to the prepared simulated sample, and operations such as microorganism separation, enrichment, nucleic acid extraction and the like are performed. Specifically, 1mL of the selective lysate A and 20. Mu.L of the microbial sedimentation aid were added to 1mL of the simulated blood sample, the mixture was shaken for 1 minute, and the mixture was centrifuged at 12000 Xg for 2 minutes, and 1.8mL of the supernatant (the remaining 200. Mu.L) was discarded. Then 1mL of blood selection lysate B was added, shaking was performed for 1 minute, 12000 Xg was centrifuged for 2 minutes, and 1mL of the supernatant was discarded. This step is repeated one or more times. 1mL of the washing solution was added to the sample, the mixture was shaken for 1 minute, centrifuged at 12000 Xg for 2 minutes, and 1mL of the supernatant was discarded. This step is repeated one or more times. Finally, 1mL of the nucleic acid extract was added, the mixture was shaken for 1 minute, centrifuged at 12000 Xg for 2 minutes, and 1mL of the supernatant was discarded. 30mg of 0.5mm zirconia beads were added, vortexed for 5 minutes, heated at 95℃for 5 minutes, and centrifuged at 12000 Xg for 2 minutes, and the supernatant was collected to obtain a mock sample nucleic acid. Meanwhile, 30mg of 0.5mm zirconia beads were added to the control sample, vortexed and oscillated for 5 minutes, heated at 95℃for 5 minutes, centrifuged at 12000 Xg for 2 minutes, and the supernatant was collected to obtain nucleic acid of the control sample. In parallel, a set of blank blood separation and extraction controls without Streptococcus pneumoniae was made. The nucleic acid is prepared by a probe method PCR detection system according to the table 1 and is subjected to on-machine amplification program setting, and the specific primer probe sequences are shown in the table 2. The results are shown in FIG. 4, in which the control group is delayed in comparison with the CT value of the experimental group compared with the amplification of the experimental group on three different detection gradients, but the control group can be stably detected, and the negative blood extraction control and the template-free control are not amplified, so that the amplification results are reliable and repeatable. Thus, it has been shown that 10CFU per milliliter of streptococcus pneumoniae DNA in blood can be isolated and extracted using the reagents and methods of the invention, meeting the blood sample detection requirements of gram-negative pathogens in blood stream infections.
And (3) comparing the nucleic acid extracted after 10/100/1000CFU streptococcus pneumoniae in the blood sample with a probe method needle fluorescence quantitative PCR amplification curve of directly extracting the nucleic acid of 10/100/1000CFU streptococcus pneumoniae. Wherein C-E1, C-E2, C-E3 in the left legend of FIG. 4 correspond to 10, 100, 1000CFU, respectively, of the control group without isolation, E1, E2, E3 correspond to 10, 100, 1000CFU, respectively, of the pathogen experimental group isolated from the blood sample; BNTC is negative blood separation and extraction control, and NTC is template-free amplification control.
Example 7
In order to verify the effectiveness of the method and the reagent, the reagent and the method are used for separating and extracting nucleic acid of the candida albicans pathogenic microorganism simulating blood flow infection, meanwhile, the nucleic acid extracted by adding the same quantity of pathogenic microorganisms into a simulated sample is used as a reference, and the extracted DNA is detected by a probe method fluorescence quantitative PCR, so that the feasibility of the method is verified.
Reagents for separating, enriching and extracting nucleic acid from pathogenic microorganisms in blood are as follows: blood selection lysate A was 5mM phosphate buffer (pH 8), 1% sodium chloride, 20% guanidine hydrochloride, 0.5% Tween 20, 5mM trisodium citrate, and 0.05% polypropylene glycol. The microbial sedimentation aid is Novec7500. Blood selection lysate B was 5mM phosphate buffer (pH 8), 0.5% potassium chloride, 5mM trisodium citrate. The washing solution was 0.1% phosphate buffer (pH 7.5), 0.9% sodium chloride, 3% ammonium sulfate, and 0.1% trisodium citrate. The nucleic acid extract was 0.2% tris hydrochloride buffer (pH 9), 0.15% potassium chloride, 3% triton X-100 and 0.05% polypropylene glycol.
The separation, enrichment and nucleic acid extraction method of candida albicans in blood comprises the following steps:
preparing a simulation sample containing candida albicans, respectively adding 10CFU/100CFU/1000CFU candida albicans into 3 pieces of 1mL negative blood, and uniformly mixing for later use; meanwhile, preparing a control sample, respectively adding 10CFU/100CFU/1000CFU candida albicans into 3 100 mu L of nucleic acid extracting solution, and uniformly mixing for later use. Reagents are added to the prepared simulated sample, and operations such as microorganism separation, enrichment, nucleic acid extraction and the like are performed. Specifically, 1mL of the selective lysate A and 20. Mu.L of the microbial sedimentation aid were added to 1mL of the simulated blood sample, the mixture was shaken for 1 minute, and the mixture was centrifuged at 12000 Xg for 2 minutes, and 1.8mL of the supernatant (the remaining 200. Mu.L) was discarded. Then 1mL of blood selection lysate B was added, shaking was performed for 1 minute, 12000 Xg was centrifuged for 2 minutes, and 1mL of the supernatant was discarded. This step is repeated one or more times. 1mL of the washing solution was added to the sample, the mixture was shaken for 1 minute, centrifuged at 12000 Xg for 2 minutes, and 1mL of the supernatant was discarded. This step is repeated one or more times. Finally, 1mL of the nucleic acid extract was added, the mixture was shaken for 1 minute, centrifuged at 12000 Xg for 2 minutes, and 1mL of the supernatant was discarded. 30mg of 0.5mm zirconia beads were added, vortexed for 5 minutes, heated at 95℃for 5 minutes, and centrifuged at 12000 Xg for 2 minutes, and the supernatant was collected to obtain a mock sample nucleic acid. Meanwhile, 30mg of 0.5mm zirconia beads were added to the control sample, vortexed and oscillated for 5 minutes, heated at 95℃for 5 minutes, centrifuged at 12000 Xg for 2 minutes, and the supernatant was collected to obtain nucleic acid of the control sample. In parallel, a set of blank blood separation and extraction controls without Streptococcus pneumoniae was made.
The nucleic acid is prepared by a probe method PCR detection system according to the table 1 and is subjected to on-machine amplification program setting, and the specific primer probe sequences are shown in the table 2. As shown in FIG. 5, the amplification differences between the control group and the experimental group are not obvious in the simulated samples of 100CFU and 1000CFU per milliliter on the three detection gradients, and the two groups of experimental amplifications have good overlapping property and can be stably detected in the simulated samples of 10CFU per milliliter. The negative blood extraction control group and the template-free control group are not amplified, and the amplification result is reliable. Thus, it has been shown that 10CFU per milliliter of candida albicans DNA can be isolated and extracted into blood using the reagents and methods of the invention, which meet the blood sample testing requirements of fungal pathogens in blood stream infections.
Probe-based fluorescence quantitative PCR amplification curve comparison diagram of 10/100/1000CFU candida albicans extracted nucleic acid and 10/100/1000CFU candida albicans directly extracted nucleic acid in blood sample. Wherein C-E1, C-E2, C-E3 in the left legend of FIG. 5 correspond to 10, 100, 1000CFU, respectively, of the control group without isolation, E1, E2, E3 correspond to 10, 100, 1000CFU, respectively, of the pathogen experimental group isolated from the blood sample; BNTC is negative blood separation and extraction control, and NTC is template-free amplification control.
TABLE 1 preparation of probe method PCR detection System and on-machine amplification procedure
TABLE 2 primers and probe sequences in a Probe PCR System
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (10)
1. A method for extracting a pathogenic microorganism nucleic acid sample in blood is characterized by comprising the following steps: the method comprises the following steps:
s1) primary lysis of blood samples: adding a blood selective lysate A and a pathogenic microorganism sedimentation aid into a blood sample, and fully and uniformly mixing to obtain a lysate; dissolving macromolecular organic matters in the lysate, and removing part of supernatant after high-speed centrifugation to obtain a primary blood lysis sample;
wherein the lysate comprises lysed blood cells, including erythrocytes and leukocytes, and non-lysed but surface-modified pathogenic microorganisms, and high-speed centrifugal sedimentation is used to enrich and sediment the pathogenic microorganisms;
the blood selection lysate a comprises: buffer solution with the concentration of 10-100 mM, auxiliary cracking salt with the mass and volume percentage of 0-6.5%, main cracking salt with the mass and volume percentage of 5-65%, surfactant with the mass and volume percentage of 0-5%, chelating agent with the concentration of 0-10 mM and defoaming agent with the volume percentage of 0-0.5%, wherein the pH value of the blood selective cracking solution A is 4-8;
S2) re-lysis of blood samples: adding a blood selective lysate B into the blood primary lysis sample in the step S1 to further dissolve residual macromolecular organic matters, and removing part of supernatant on the interface of the microbial sedimentation agent and the aqueous solution after high-speed centrifugation to obtain a blood secondary lysis sample;
the blood selective lysate B comprises: buffer solution with the concentration of 10-100 mM, auxiliary cracking salt with the mass and volume percentage of 0-3%, main cracking salt with the mass and volume percentage of 0-40%, surfactant with the mass and volume percentage of 0-2%, chelating agent with the concentration of 0-10 mM and defoaming agent with the volume percentage of 0-0.5%, wherein the pH value of the blood selective cracking solution B is 5-8;
s3) washing: adding a washing solution into the blood re-lysis sample in the step S2, uniformly mixing, centrifuging, and removing part of supernatant to obtain a blood lysis sample containing the pathogen microorganisms which are not lysed but have changed surface characteristics, wherein the washing solution is used for cleaning impurities;
s4) nucleic acid extraction: adding a nucleic acid extraction reagent into the blood re-lysis sample washed in the step S3, removing part of supernatant after high-speed centrifugation, performing electric lysis on the residual solution or adding grinding beads with different particle size combinations for shaking, heating at high temperature, centrifuging to crack the uncleaved pathogenic microorganisms with changed surface characteristics, and promoting release and dissolution of nucleic acid in the uncleaved pathogenic microorganisms with changed surface characteristics to obtain a pathogenic microorganism nucleic acid sample;
The primary cleavage salt is selected from one or more of the following: lithium chloride, sodium iodide, potassium iodide, guanidine hydrochloride, and guanidine isothiocyanate;
the auxiliary cracking salt is selected from one or more of the following: sodium chloride, potassium chloride and ammonium sulfate;
preferably, the blood sample of the extraction method is a sample that is not subjected to blood culture or a sample after blood culture;
more preferably, the blood cells are ruptured by the osmotic pressure difference between the blood selective lysate A and blood and the addition of a surfactant; preferably, the salts, surfactants and microbial sedimentation aids in the blood selective lysate A promote the enrichment and sedimentation of pathogenic microorganisms which are difficult to centrifugally sediment due to special extracellular structures (such as bacterial capsules), so as to reduce the differential enrichment of microorganisms;
more preferably, the addition of the blood-selective lysate B compensates for incomplete lysis treatment due to sample differences and sufficiently dissolves residual impurities.
2. The extraction method according to claim 1, characterized in that: in the step S1, the blood selective lysate A consists of buffer solution with the concentration of 10-100 mM, auxiliary lysate with the mass and volume percentage of 0-6.5%, main lysate with the mass and volume percentage of 5-65%, surfactant with the mass and volume percentage of 0-5%, chelating agent with the concentration of 0-10 mM and defoamer with the volume percentage of 0-0.5%;
The pathogenic microorganism sedimentation aid is a water-insoluble biological inert organic solvent;
the blood sample is 1-10 ml, the blood selective lysate A and the pathogenic microorganism sedimentation assisting agent are added and mixed uniformly by normal temperature oscillation or mixed uniformly by normal temperature inversion for 1-5 minutes, the dosage of the blood selective lysate A is 0.5-5 times of the volume of the blood sample, and the dosage of the pathogenic microorganism sedimentation assisting agent is 0.5-5% of the volume of the blood sample;
preferably, the macromolecular organic matter includes proteins, carbohydrates, lipids, nucleic acids, and the like.
3. The extraction method according to claim 1, characterized in that:
in the step S2, the blood selective lysate B consists of buffer solution with the concentration of 10-100 mM, auxiliary lysate with the mass and volume percentage of 0-3%, main lysate with the mass and volume percentage of 0-40%, surfactant with the mass and volume percentage of 0-2%, chelating agent with the concentration of 0-10 mM and defoamer with the volume percentage of 0-0.5%;
the dosage of the blood selective lysate B is 2-10 times of the volume of the blood primary lysis sample;
in step S3, the washing solution includes: the concentration of the buffer solution is 10-100 mM, the mass and volume percentage of the impurity dissolution promoting salt is 0-5%, the concentration of the chelating agent is 0-10 mM, and the pH value of the washing solution is 5-9;
The consumption of the washing liquid is 5-10 times of the volume of the blood re-splitting sample, and the use method of the washing liquid is that the washing liquid is uniformly mixed by normal-temperature vibration or uniformly mixed by normal-temperature inversion;
steps S2, S3 may be repeated a number of times.
4. The extraction method according to claim 1, characterized in that:
in step S4, the nucleic acid extraction solution includes: 0.5-20 mM buffer solution, 0-5% nucleic acid dissolution promoting salt, 0.01-5% surfactant, 0-3 mM chelating agent and 0-0.5% defoaming agent, wherein the pH of the nucleic acid extracting solution is 7-9;
the surfactant is an anionic surfactant and/or a nonionic surfactant;
the dosage of the nucleic acid extracting solution is 5-10 times of the volume of the blood cracking sample, the electric cracking is performed by using an electric cracking device processed by MEMS, the grinding beads are one or more of acid-washing glass beads, zirconia beads and quartz sand with the particle size of 0.1-3 mm, the dosage of the grinding beads is 20-150 mg, the oscillation time is 5-15 minutes, the high-temperature heating temperature is 85-100 ℃, and the heating time is 3-15 minutes.
5. The extraction method according to any one of claims 1 to 4, characterized in that:
The buffer solution is any one of the following: phosphate buffer, sodium acetate buffer and tris hydrochloride buffer;
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;
the defoamer is any one of the following: polydimethyl siloxane, polypropylene glycol, polyether modified polysiloxane and polyoxypropylene glycerol 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, wherein the nonionic surfactant is one or more of the following: tween 20, tween 80, triton X-100, ethylphenyl polyethylene glycol and cetyl polyoxyethylene ether Brij C20;
in steps S1, S2, S3 and S4, the centrifugal speed is 10000-16000 Xg, and the centrifugal time is 1-5 minutes.
6. An extraction reagent for a pathogenic microorganism nucleic acid sample in blood, which is characterized in that:
the extraction reagent comprises a blood selective lysate A with the volume of 0.5-10 times of that of a blood sample, a pathogenic microorganism sedimentation aid with the volume of 0.5-10% of that of the blood sample, a blood selective lysate B with the volume of 1-10 times of that of the blood sample, a washing liquid with the volume of 1-10 times of that of the blood sample and a nucleic acid extraction liquid with the volume of 0.5-2 times of that of the blood sample;
The blood selective lysate A and the blood selective lysate B are used for only lysing blood cells to retain pathogenic microorganisms and dissolving macromolecular organic matters released in the blood sample and the lysed blood cells; the pathogenic microorganism sedimentation aid is used for enriching and precipitating pathogenic microorganisms; the washing liquid is used for washing impurities, and the nucleic acid extracting liquid is used for enriching, lysing and promoting the release and dissolution of nucleic acid in the pathogenic microorganisms;
the blood selection lysate a comprises: buffer solution with the concentration of 10-100 mM, auxiliary cracking salt with the mass and volume percentage of 0-6.5%, main cracking salt with the mass and volume percentage of 5-65%, surfactant with the mass and volume percentage of 0-5%, chelating agent with the concentration of 0-10 mM and defoaming agent with the volume percentage of 0-0.5%, wherein the pH value of the blood selective cracking solution A is 4-8;
the blood selective lysate B comprises: the pH value of the blood selective lysate B is 5-8, wherein the concentration of the buffer solution is 10-100 mM, the mass and volume percentage of the auxiliary lysate is 0-3%, the mass and volume percentage of the main lysate is 0-40%, the mass and volume percentage of the surfactant is 0-2%, the concentration of the chelating agent is 0-10 mM, and the volume percentage of the antifoaming agent is 0-0.5%.
7. The extraction reagent according to claim 6, wherein:
the washing liquid comprises: the concentration of the buffer solution is 10-100 mM, the mass and volume percentage of the impurity dissolution promoting salt is 0-5%, the concentration of the chelating agent is 0-10 mM, and the pH value of the washing solution is 5-9;
preferably, the nucleic acid extraction solution comprises: the concentration of the buffer solution is 0.5-20 mM, the mass and volume percentage of the nucleic acid dissolution promoting salt is 0-5%, the mass and volume percentage of the surfactant is 0.01-5%, the concentration of the chelating agent is 0-3 mM, the volume percentage of the antifoaming agent is 0-0.5%, and the pH value of the nucleic acid extracting solution is 7-9;
preferably, the biologically inert organic solvent is a fluorooil or HFE-7500 or Novec7500 or FC-40.
8. The extraction reagent according to claim 6 or 7, wherein:
the buffer solution is any one of the following: phosphate buffer, sodium acetate buffer, tris hydrochloride buffer;
the surfactant is an anionic surfactant and/or a nonionic surfactant;
preferably, the anionic surfactant is selected from any one of the following: sodium lauryl sulfate, sodium lauryl sarcosinate and sodium deoxycholate, the nonionic surfactant being selected from one or more of the following: tween 20, tween 80, triton X-100, ethylphenyl polyethylene glycol and cetyl polyoxyethylene ether Brij C20.
9. The extraction reagent of claim 8, wherein: the blood selective lysate A comprises 20mM of tris (hydroxymethyl) aminomethane hydrochloride buffer solution, 0.5% of potassium chloride, 65% of guanidine isothiocyanate, 0.2% of sarcosyl, 10mM of trisodium citrate and 0.02% of polyoxypropylene glycerol ether, and the pH value of the blood selective lysate A is 7.0;
the blood selective lysate B comprises 20mM of tris (hydroxymethyl) aminomethane hydrochloride buffer solution, 0.5% of potassium chloride, 30% of guanidine isothiocyanate, 0.1% of sarcosyl, 10mM of trisodium citrate and 0.02% of polyoxypropylene glycerol ether, wherein the pH value of the blood selective lysate B is 7.0;
the microbial sedimentation aid is Novec7500;
the washing liquid comprises 10mM of tris hydrochloride buffer solution, 0.9% of sodium chloride in mass and volume percentage, 10% of ammonium sulfate in mass and volume percentage and 3mM of trisodium citrate, and the pH value of the washing liquid is 7.0;
the nucleic acid extracting solution comprises 10mM of tris hydrochloride buffer solution, 0.45% of sodium chloride in mass and volume percentage, 0.1% of sodium dodecyl sulfate in mass and volume percentage, 2% of Tween 20 in mass and volume percentage, 1mM of trisodium citrate and 0.02% of polyoxypropylene glycerol ether in volume percentage, and the pH value of the nucleic acid extracting solution is 8.8.
10. Use of the extraction reagent of any one of claims 6-9 for the extraction of a pathogenic microbial nucleic acid sample in blood;
preferably, the blood is a sample that is not cultured with blood or a sample that is cultured with blood.
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| CN113502317A (en) * | 2021-07-08 | 2021-10-15 | 华东医院 | Blood sample processing method for blood flow infected patient |
| CN113699144A (en) * | 2021-08-27 | 2021-11-26 | 厦门佶银生物科技有限公司 | New application of PT-1 or its effective component in pathogenic microorganism detection and method for processing pathogenic microorganism detection sample |
| CN113980956A (en) * | 2021-11-25 | 2022-01-28 | 中科欧蒙未一(北京)医学技术有限公司 | Extraction method for removing host nucleic acid of respiratory tract pathogenic microorganism metagenome |
| CN115960885B (en) * | 2022-10-09 | 2023-12-12 | 南京诺唯赞生物科技股份有限公司 | Method and composition for extracting nucleic acid from heparin sodium sample |
| CN115735902A (en) * | 2022-11-16 | 2023-03-07 | 苏州颐坤生物科技有限公司 | Cell preservation solution and preparation method and application thereof |
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| AU4586096A (en) * | 1995-03-10 | 1996-09-19 | Becton Dickinson & Company | Sample processing method for whole blood |
| JP3602071B2 (en) * | 2001-06-05 | 2004-12-15 | 株式会社日立製作所 | Purification and separation method of nucleic acid |
| WO2013091102A1 (en) * | 2011-12-21 | 2013-06-27 | Geneohm Sciences Canada Inc. | Enrichment & isolation of microbial cells & microbial nucleic acids from a biological sample |
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| CN113073096B (en) * | 2021-03-31 | 2023-02-03 | 北京源微生物科技有限公司 | Method and reagent for separating and enriching pathogenic microorganisms in blood and extracting nucleic acid |
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