Method and reagent for directly detecting urinary tract infection pathogenic bacteria after treatment

Abstract

The invention provides a method and a reagent for directly detecting urinary tract infection pathogenic bacteria after treatment, the reagent can effectively and selectively split and dissolve interferents such as casts, blood cells and white blood cells in a urine sample, and only target pathogenic bacteria in the sample are reserved after treatment. The product can be matched with microorganisms MALDI-TOF MS to directly carry out identification and detection on pathogenic bacteria, and can also be used as a template after simple nucleic acid extraction to carry out amplification on pathogenic bacteria or drug-resistant genes in combination with a PCR / qPCR / dPCR (Polymerase Chain Reaction / Quantitative Polymerase Chain Reaction / dPCR method. According to the method and the reagent, the time for identifying and analyzing clinical urinary tract infection pathogenic bacteria is shortened, the detection efficiency of capsular bacteria and fungi can be improved, and a rapid and accurate guidance basis can be provided for clinical medication and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of measurement and testing, and in particular to a method and a reagent for directly detecting urinary tract infection pathogens after treatment. Background Art

[0002] Urinary tract infection is caused by direct invasion of bacteria, fungi, protozoa and viruses. The incidence of urinary tract infection is quite high, and urinary tract infection is a relatively common disease. In the initial outpatient visit, the main complaint is 1% to 1.8%. At present, the etiological examination of midstream urine specimen culture is the gold standard for diagnosing urinary tract infection in clinical practice, but not all urinary tract infection pathogens can be well cultured. In addition, urine culture usually takes about 48 hours, and clinically it is generally believed that the colony count after culture is >10 5 CFU / mL is clinically significant, but the colony count in urine culture can be affected by many factors. In some patients, even if the number of colonies cultured is small, it has obvious clinical significance. Therefore, there are some shortcomings in using urine culture to identify pathogens for urinary tract infection detection.

[0003] MALDI-TOF MS is a new type of bacterial identification technology developed in recent years. It is currently mainly used for the identification of pure microorganisms. It can quickly and accurately identify common clinical bacteria and fungi, and is considered to be one of the most efficient identification methods. However, for urine, the clinic currently needs to culture the urine on a plate first, and then pick the colonies of pathogens for detection. However, the culture step usually leads to the failure of some bacteria or fungi that are difficult to culture, or the primary and secondary relationship of mixed infection bacteria changes due to culture, which leads to errors in the doctor's original judgment of the patient's condition. At present, there have been reports on the use of urine for direct MALDI-TOF MS detection. The MALDI-TOF MS direct detection method can also overcome the defect that the urine culture results are affected by the previous empirical medication, help clinicians change the treatment plan in time, reduce empirical medication, and prevent the generation of drug-resistant bacteria. However, due to the large number of impurities in urine and the limitations of the detection sensitivity of MALDI-TOF MS, clinical samples of urine cannot be directly subjected to MALDI-TOF MS microbial identification. In addition, because the main target of microbial MALDI-TOF MS detection is ribosomal protein, the sensitivity and accuracy are not high when distinguishing between resistant bacteria and sensitive bacteria. Therefore, other methods are needed to detect resistance genes or virulence genes.

[0004] PCR / qPCR / dPCR and other methods can not only identify or quantify the types of bacteria and fungi, but also detect drug-resistant genes and virulence genes. However, before the pathogens in urine can be directly detected using PCR / qPCR / dPCR and other methods, they need to go through complex nucleic acid purification steps, which is very laborious and time-consuming. Moreover, during the nucleic acid extraction process, a large amount of nucleic acid will be gradually lost in the complex purification steps, and it is impossible to remove the human genome residue alone, which will cause interference with PCR detection and reduce sensitivity.

[0005] Therefore, it is very necessary to develop an efficient, labor-saving and compatible pretreatment method to facilitate direct MALDI-TOF MS or PCR / qPCR / dPCR detection of pathogens in urine.

[0006] Patent application CN201210180123.9 is a MALDI-TOF MS direct detection of urine pathogens pretreatment method, the separation method of urine pathogens is to use differential centrifugation for bacterial enrichment, and use formic acid and acetonitrile to dissolve the bacteria. This method takes 1ml of urine and first centrifuges it at 2000×g for 30 seconds to remove somatic cells, etc., and then centrifuges it at 15500rpm for 5 minutes to obtain a precipitate. This method first uses 2000×g centrifugation for 30 seconds to remove somatic cells. 2000×g is equivalent to about 4000rpm on a common centrifuge adapted for 1.5ml centrifuge tubes. Many molecular biological experiments use this speed to centrifuge bacteria. Therefore, this speed can easily cause a large number of bacteria to be lost, resulting in subsequent detection failure or a significant decrease in detection sensitivity. Secondly, in the clinical urine samples of urinary tract infections, there are often insoluble purulent suspended protein substances or hemoglobin residues. After direct high-speed centrifugation without reagent treatment, a large number of impurities that interfere with the detection remain. Even high-speed centrifugation cannot be well recovered for bacterial precipitation, which significantly affects mass spectrometry detection. In addition, the amount of urine used is relatively small, and the concentration of clinically significant microorganisms is approximately 10 5 CFU / mL or lower, even if the method does not lose bacteria, it does not meet the requirements of mass spectrometry detection object 10 5 ~10 7 The detection requirements of CFU bacteria, the bacteria separated by this method can be directly used for mass spectrometry detection only in high-concentration samples, which is easy to cause missed detection. In addition, this method mentions the use of formic acid and acetonitrile to dissolve bacterial proteins. This method is an optimization of the MALDI-TOF MS processing method. There are corresponding operating requirements for mass spectrometers from different companies and bacteria with different structures (such as Gram-positive bacteria or fungi).

[0007] That is, the existing technology only uses differential centrifugation, which is unable to remove some impurities in the sample that are not easy to remove by centrifugation; the separation and enrichment method using separation gel coagulation tubes is prone to bacterial loss in low-concentration samples, and the separation gel coagulation tubes are expensive, mainly for samples after culture, and there are problems such as poor removal of cell debris impurities and interference with detection; pathogens or direct urine samples separated by the above two methods must be used in conjunction with PCR / qPCR / dPCR before detection, because reagents have not been used to remove impurities, etc., and there are many residual impurities in the samples, and it is impossible to obtain a PCR-amplifiable template through simple nucleic acid extraction, and complex nucleic acid extraction and purification steps are required, especially for Gram-positive bacteria and fungi, which have cell wall structures that are difficult to lyse, which require more time-consuming extraction steps.

[0008] Therefore, an efficient, labor-saving and compatible pretreatment method is needed to facilitate direct MALDI-TOF MS or PCR / qPCR / dPCR detection of pathogens in urine. Summary of the invention

[0009] The present invention is to solve the problem of efficiency and sensitivity of pathogen detection in urine, and provides a method and reagent for direct detection of urinary tract infection pathogens after treatment, which can effectively select and lyse and dissolve the interferences such as casts, blood cells, and white blood cells in the urine sample, and only retain the target pathogens in the sample after treatment, and then use sterile purified water to wash the precipitate to obtain a purer pathogen precipitation product, which can be used with microbial MALDI-TOF MS to directly identify and detect pathogens, or can be used as a template with PCR / qPCR / dPCR method after simple nucleic acid extraction to amplify pathogens or drug resistance genes. This method and reagent not only shortens the time for clinical urinary tract infection pathogen identification and analysis, but also improves the detection efficiency of capsule-containing bacteria and fungi, and can provide a fast and accurate guidance basis for clinical medication, etc.

[0010] The present invention provides a method for directly detecting urinary tract infection pathogens after treatment, comprising the following steps:

[0011] S1, collect 3-10 mL urine sample and mix it by inversion, and choose whether to perform preliminary centrifugation according to the characteristics of the urine sample. If there is blood or granular precipitation or flocculent in the urine sample, centrifuge at 200-800×g for 30 seconds, aspirate the supernatant to obtain the sample after preliminary treatment, and enter step S2, otherwise, directly enter step S2;

[0012] S2. Add 0.5 to 2 times the volume of the first pretreatment reagent to the sample after preliminary treatment or the urine sample, vortex mix for 30 seconds to 2 minutes, lyse and dissolve urine impurities, including red blood cells, white blood cells, phagocytes, epithelial cells and casts, centrifuge at 12000 to 15000 × g for 1 to 3 minutes, discard the supernatant, and retain the precipitate to obtain the first lysed sample;

[0013] S3, add 1-3 mL of the second pretreatment reagent to the first lysed sample, vortex mix for 30 seconds to 2 minutes, lyse and dissolve the cell fragments and protein impurities that were not completely lysed in step S2, centrifuge at 12000-15000×g for 1-3 minutes, discard the supernatant, and retain the precipitate to obtain the second lysed sample;

[0014] S4. Add 1-3 mL of sterile purified water to the second lysed sample, vortex mix for 30 s-2 min, centrifuge at 12000-15000 × g for 1-3 min, discard the supernatant, and retain the precipitate to obtain the first washed sample;

[0015] S5. Add 1-3 mL of sterile purified water to the sample after the first washing, vortex mix for 30 seconds to 2 minutes, centrifuge at 12,000 to 15,000 × g for 1 to 3 minutes, discard the supernatant, and retain the precipitate to obtain the urinary tract infection pathogen isolation product;

[0016] Perform MALDI-TOF MS analysis and proceed to step S6, perform PCR / qPCR / dPCR analysis and proceed to step S7;

[0017] S6. Use a 1 μL inoculation loop to directly scrape a small amount of the urinary tract infection pathogen separation product onto the target plate, or resuspend the target separation product with 5 μL sterile purified water and take 1 to 2 μL onto the target plate, add the matrix solution, dry it, and then perform MALDI-TOFMS detection on the machine to obtain the mass spectrometry detection results of the urinary tract infection pathogen;

[0018] S7. Add 100 μL of TE buffer with a pH of 8.0 and 50-100 mg of 0.1 mm and 1 mm 1:1 mixed acid-washed glass beads to the urinary tract infection pathogen isolation product, oscillate for 5 minutes, heat at 95-100°C for 5 minutes, cool and centrifuge, take the supernatant as a template, add it to the PCR / qPCR / dPCR system at a ratio of 10-20% to perform specific amplification detection of pathogens or drug-resistant genes to obtain the PCR detection results of urinary tract infection pathogens.

[0019] The method for directly detecting urinary tract infection pathogens after treatment according to the present invention is, as a preferred embodiment, in step S2, adding 1 volume of a first pretreatment reagent to the sample after preliminary treatment or the urine sample;

[0020] In steps S2, S3 and S4, the vortex mixing time is 1 min, and the centrifugation time is 2 min.

[0021] The method for directly using the processed urinary tract infection pathogens for detection described in the present invention is, as a preferred embodiment, in step S6, the pathogen sample to be analyzed and the matrix solution are both 1 μL.

[0022] The method for directly detecting urinary tract infection pathogens after treatment described in the present invention is, as a preferred embodiment, in step S1, the urine sample includes any one or more of the following pathogens: Gram-negative bacteria, Gram-positive bacteria and fungi, and both Gram-negative bacteria and Gram-positive bacteria can contain capsules.

[0023] In the method for directly using the treated urinary tract infection pathogens for detection described in the present invention, as a preferred embodiment, the Gram-negative bacteria include Escherichia coli, Escherichia coli and Klebsiella pneumoniae; and the Gram-positive bacteria include Streptococcus pneumoniae.

[0024] The present invention provides a reagent for a method of directly using the treated urinary tract infection pathogens for detection, wherein the first pretreatment reagent is composed of a buffer solution, a surfactant, salts for promoting the dissolution of impurities, a bacteria precipitation promoting reagent, a metal ion chelating agent and sterilized purified water; the pH value of the first pretreatment reagent is 7.0-9.5;

[0025] The second pretreatment reagent is composed of a buffer, salts that promote the dissolution of impurities, a bacterial precipitation promoting reagent, a metal ion chelating agent, and sterilized purified water; the pH value of the second pretreatment reagent is 6.0 to 9.0, and the concentration of the second pretreatment reagent is less than or equal to that of the first pretreatment reagent;

[0026] The buffer substances in the buffer solution are tris(hydroxymethyl)aminomethane hydrochloride) and sodium hydroxide or phosphate buffer and sodium hydroxide, the sodium hydroxide is used to adjust the pH value of the first pretreatment reagent and the second pretreatment reagent, the concentration of the buffer solution in the first pretreatment reagent is 10 to 150 mM, and the concentration of the buffer solution in the second pretreatment reagent is 10 to 100 mM;

[0027] The surfactant is one or more of sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecyl sarcosine, Triton X-100, Tween 20, Tween 80, and ethylphenyl polyethylene glycol, and the concentration is 0.01-2% v / v;

[0028] The salts that promote the dissolution of impurities are one or more of sodium chloride, ammonium chloride, lithium chloride, urea, guanidine hydrochloride, guanidine isothiocyanate, and sodium hypochlorite;

[0029] The bacterial precipitation promoting agent is one or more of PEG6000, PEG8000, ethanol, and isopropanol;

[0030] The metal ion chelating agent is disodium ethylenediaminetetraacetic acid or trisodium citrate. The concentration of the metal ion chelating agent in the first pretreatment reagent is 1.5-30 mM, and the concentration of the metal ion chelating agent in the second pretreatment reagent is 1-10 mM.

[0031] The reagents of the method for directly using the treated urinary tract infection pathogens for detection according to the present invention are preferably such that the pH value of the first pretreatment reagent is 8.0 to 9.0, and the pH value of the second pretreatment reagent is 6.5 to 8.0;

[0032] The concentration of the buffer in the first pretreatment reagent is 20 to 100 mM, and the concentration of the buffer in the second pretreatment reagent is 10 to 50 mM;

[0033] Surfactants are a combination of two types of surfactants;

[0034] The salt that promotes the dissolution of impurities is a combination of sodium chloride and guanidine hydrochloride, the concentration of the salt that promotes the dissolution of impurities in the first pretreatment reagent is 0.1-7M, and the concentration of the salt that promotes the dissolution of impurities in the second pretreatment reagent is 0.1-5M;

[0035] The bacterial precipitation promoting agent is PEG8000, the concentration of the bacterial precipitation promoting agent in the first pretreatment agent is 0.5-20% m / v, and the concentration of the bacterial precipitation promoting agent in the second pretreatment agent is 0.5-10% m / v;

[0036] The metal ion chelating agent is disodium ethylenediaminetetraacetate, the concentration of disodium ethylenediaminetetraacetate in the first pretreatment reagent is 2-10 mM, and the concentration of disodium ethylenediaminetetraacetate in the second pretreatment reagent is 2-5 mM.

[0037] The reagent of the method for directly using the treated urinary tract infection pathogens for detection according to the present invention is preferably used in the first pretreatment reagent, wherein the buffer is 20mM to 150mM tris(hydroxymethyl)aminomethane hydrochloride or 100mM phosphate buffer, and the pH value of the pretreatment reagent is adjusted to 7.5 to 9.5 using NaOH; the surfactant is 0.008 to 0.5% v / v sodium lauryl sarcosine and 0.5 to 2% v / v ethylphenyl polyethylene glycol, or 0.001% v / v sodium lauryl sulfate and 1% v / v Triton X-100, or 0. 5% v / v sodium lauryl sarcosine and 0.5% v / v Tween 20, or 0.005% v / v sodium lauryl sulfate and 0.2% v / v Tween 20, or 0.2% v / v sodium lauryl sarcosine and 0.5% v / v ethylphenyl polyethylene glycol, or 0.01% v / v lithium lauryl sulfate and 1% v / v ethylphenyl polyethylene glycol; the salts that promote the dissolution of impurities are 0.15-1.5M sodium chloride and 2-6 guanidine hydrochloride, or 0.4M NH4Cl and 3M guanidine isothiocyanate, or 0.15M NaCl and 3M guanidine isothiocyanate, or 0.5M NaCl and 5M urea, or 4M lithium chloride; the precipitation aid is PEG8000 with a concentration of 2-10%, or PEG6000 with a concentration of 10%, or ethanol with a concentration of 10-20%, or isopropanol with a concentration of 5%; the metal ion chelating agent is 1.5-15mM disodium ethylenediaminetetraacetate or 5-15mM trisodium citrate.

[0038] The reagent of the method for directly using the urinary tract infection pathogens for detection after treatment described in the present invention is, as a preferred embodiment, in the second pretreatment reagent, the buffer is 10-50mM tris(hydroxymethyl)aminomethane hydrochloride or 10-50mM phosphate buffer, and the pH value of the pretreatment reagent is adjusted to 7.0-8.0 using NaOH; the salts promoting the dissolution of impurities are 0.15-1M sodium chloride and 2-3M guanidine hydrochloride, or 0.15M NaCl and 2-3M guanidine isothiocyanate, or 0.3M NaCl and 5M urea, or 4M lithium chloride; the precipitation aid is 2%-8% PEG8000, or 7.5% PEG6000, or 5% ethanol, or 5% isopropanol; the metal ion chelator is 1-10mM disodium ethylenediaminetetraacetic acid or 2-5mM trisodium citrate.

[0039] The reagent for the method of directly using the urinary tract infection pathogens for detection after treatment described in the present invention is, as a preferred embodiment, the method and reagent for directly using the urinary tract infection pathogens for detection after treatment can also be applied to pus, pleural and abdominal effusions, alveolar lavage fluid, and cultured blood or cerebrospinal fluid.

[0040] The invention provides a method for directly using urinary tract infection pathogens for detection after treatment. First, some sediment impurities are removed at a low speed, and then a specific pre-treatment reagent is used to treat the urine supernatant to lyse and dissolve the cells and insoluble protein impurities that have not been treated cleanly in the previous step, so as to facilitate the removal of impurities after centrifugation. At the same time, high salt, surfactant, precipitation aid reagent and the like are used in combination to enhance the polarity and hydrophilicity of capsule polysaccharides on the surface of bacteria with capsule structures and the like, so that they are easy to be precipitated through subsequent high-speed centrifugation, and also to destroy structures such as cell walls of pathogens to a certain extent, so that bacterial proteins or nucleic acids are more easily released when MALDI-TOF MS and nucleic acid extraction are subsequently performed, so as to facilitate detection.

[0041] The invention provides a pretreatment reagent and method for a urine sample of urinary tract infection, which can be directly used for MALDI-TOFMS or PCR amplification after being treated without urine culture. The method comprises the following steps: taking no less than 3 mL (preferably 10 mL to 20 mL) of urine and first centrifuging at 200 to 800×g for 30 seconds, aspirating the supernatant and discarding a large amount of cell debris, stone particles, insoluble protein precipitation, etc. that may exist in the urine; then adding a first pretreatment reagent (the added amount is 0.5 to 2 times the volume of the supernatant) that can effectively selectively lyse red blood cells, white blood cells, phagocytes, epithelial cells and casts that may exist in the supernatant, and centrifuging at 12000 to 15000×g for 2 minutes to discard the supernatant and retain the precipitation. Add 1-3 mL of the second pretreatment reagent, vortex and mix for 2 minutes, lyse and dissolve the cell fragments and protein impurities that were not completely lysed in the previous step, centrifuge at 12000-15000×g for 2 minutes, discard the supernatant and retain the precipitate; then use sterile purified water (1-3 mL) to resuspend and mix the precipitate, centrifuge at 12000-15000×g for 2 minutes, discard the supernatant and retain the precipitate, repeat this step once, and if MALDI-TOF MS microbial identification is required, resuspend the precipitate with about 5 μL of sterile purified water and take 1 μL of the target plate, then add matrix liquid and other equipment for detection according to the requirements of the mass spectrometer, if PCR-type molecular detection is required, add 100 μL TE buffer (pH 8.0) and acid-washed glass beads, oscillate and break the pathogens, and then heat at high temperature for simple nucleic acid extraction and amplification as a PCR template.

[0042] The urine samples of urinary tract infection that have been quickly processed can be used with MALDI-TOF MS to directly detect pathogens, or can be used with PCR / qPCR / dPCR methods to amplify nucleic acids of pathogens or drug-resistant genes after simple nucleic acid extraction. This method and reagent not only shortens the time for pathogen identification, but also improves the detection efficiency of fungal and mixed infection pathogens, and can provide fast and accurate guidance for clinical medication, etc. This solution can be used not only for urine infections, but also for samples such as blood or cerebrospinal fluid, pleural and peritoneal effusions, alveolar lavage fluid, and pus after culture.

[0043] The preparation and amplification procedures of the probe PCR detection system are as follows:

[0044]

[0045] The primer and probe sequences in the probe PCR system are as follows:

[0046]

[0047]

[0048] The present invention has the following advantages:

[0049] (1) The present invention provides a method and reagent for directly using the treated urinary tract infection pathogens for detection. Clinical urine samples can be directly processed without urine culture. The method is combined with a pretreatment reagent with selective lysis to remove human somatic cells and impurities in the sample. The pathogens can be moderately denatured and will not be broken and dissolved into the supernatant and lost in large quantities. The pathogens can also be easily collected by centrifugation. The obtained pathogens can be directly used for MALDI-TOF MS detection or suitable for PCR / qPCR / dPCR detection after simple nucleic acid crude extraction. There is no need to specially adjust solutions and supporting matrix solutions for promoting bacterial protein release for Gram-negative bacteria, Gram-positive bacteria or fungi. In particular, for bacteria with capsules (negative bacteria such as Kpn or positive bacteria such as Spn), the centrifugal effect caused by the capsule is eliminated after the reagent treatment, and the bacterial collection effect is good and not easy to be lost.

[0050] (2) The samples can be directly processed without culture for MALDI-TOF MS, PCR / qPCR / dPCR and other detections, with good product impurity removal and high detection efficiency. This method and reagent can be applied to samples such as cultured blood or cerebrospinal fluid, pleural effusion, bronchoalveolar lavage fluid and pus. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 A flow chart of a method for directly using urinary tract infection pathogens for detection after treatment;

[0052] Figure 2 This is a mass spectrometry detection result diagram of a method and reagent embodiment 1 for directly using the urinary tract infection pathogens for detection after treatment;

[0053] Figure 3 This is a qPCR test result diagram of a method and reagent embodiment 1 for directly using the urinary tract infection pathogens for detection after treatment;

[0054] Figure 4 This is a qPCR test result diagram of Example 8 of a method and reagent for directly using the urinary tract infection pathogens for detection after treatment;

[0055] Figure 5 This is a graph showing the qPCR detection results of Example 9 of a method and reagent for directly detecting urinary tract infection pathogens after treatment.

[0056] Figure 6 A method and reagent for directly detecting urinary tract infection pathogens after treatment; Example 10, step S1, mass spectrometry detection result of 200×g centrifugation;

[0057] Figure 7 A method and reagent for directly detecting urinary tract infection pathogens after treatment; a mass spectrometry detection result diagram of 800×g centrifugation in step S1 of Example 10;

[0058] Figure 8 It is a mass spectrometry detection result diagram of a method and reagent embodiment 10 for directly using the urinary tract infection pathogens for detection after treatment;

[0059] Fig. 9 A method and reagent for directly detecting urinary tract infection pathogens after treatment; Example 11, step S1, mass spectrometry detection result of 200×g centrifugation;

[0060] Fig.10 A method and reagent for directly detecting urinary tract infection pathogens after treatment; Example 11, step S1, mass spectrometry detection result of 800×g centrifugation;

[0061] Fig.11 This is a mass spectrometry detection result diagram of a method and reagent embodiment 11 for directly detecting urinary tract infection pathogens after treatment. DETAILED DESCRIPTION

[0062] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0063] Examples 1 to 9

[0064] like Figure 1 As shown, a method and reagent for directly detecting urinary tract infection pathogens after treatment, the reagents directly used for detection after treatment of urinary tract infection pathogens include a first pretreatment reagent and a second pretreatment reagent, wherein the first pretreatment reagent effectively selects and lyses red blood cells, white blood cells, macrophages, epithelial cells and casts that may exist therein, and the second pretreatment reagent further lyses and dissolves cell fragments and protein impurities that were not completely lysed in the previous step, thereby further reducing interference from impurities.

[0065] The first pretreatment reagent includes the following components: buffer salt, surfactant, salts that promote the dissolution of impurities, bacteria precipitation promoting reagent, metal ion chelating agent and sterilized purified water. Buffer salt and the like adjust the pH of the pretreatment reagent to 7.0-9.5, the buffer substance is tris-HCl and sodium hydroxide or phosphate buffer (PB), the concentration of the buffer is 10-150mM, preferably 20mM-100mM, the pH value is further preferably 8.0-9.0, and the most preferably pH8.5. The surfactant is one or more of sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LiDS), sodium lauryl sarcosine (SLS), Triton X-100, Tween 20, Tween 80, ethylphenyl polyethylene glycol (NP-40), the concentration is 0.001%-2% (v / v), and it is further preferred that two types of surfactants are compatible, such as 0.001% SLS combined with 0.5% NP-40. The salts that promote the dissolution of impurities are one or more of sodium chloride, ammonium chloride, lithium chloride, urea, guanidine hydrochloride, guanidine isothiocyanate, and sodium hypochlorite, preferably sodium chloride and guanidine hydrochloride, with a concentration of 0.1 to 7 M. The bacterial precipitation promoting agent is one or more of PEG6000, PEG8000, ethanol, and isopropanol, preferably PEG8000, with a concentration of 0.5% to 20% (m / v). The metal ion chelating agent is disodium ethylenediaminetetraacetic acid (EDTA) or trisodium citrate, with a concentration of 1.5 to 30 mM, preferably 2 to 10 mM EDTA.

[0066] The amount of the first pretreatment reagent added is 0.5 to 2 times the sample volume (the urine sample volume is recommended to be at least 3 mL, preferably 10 mL), and the preferred amount of the pretreatment reagent added is 1 times the sample volume.

[0067] The second pretreatment reagent includes the following components: buffer salts, salts that promote the dissolution of impurities, bacteria precipitation promoting reagents, metal ion chelating agents and sterilized purified water. Buffer salts and the like adjust the pH of the pretreatment reagent to 6.0-9.0. The buffer substances are tris(hydroxymethyl)aminomethane hydrochloride and sodium hydroxide or phosphate buffer. The concentration of the buffer is 10-100mM, preferably 10mM-50mM, and the pH value is further preferably 6.5-8.0, and most preferably pH7.0. The salts that promote the dissolution of impurities are one or more of sodium chloride, ammonium chloride, lithium chloride, urea, guanidine hydrochloride, guanidine isothiocyanate, and sodium hypochlorite, preferably sodium chloride and guanidine hydrochloride, with a concentration of 0.1-5M. The bacteria precipitation promoting reagent is one or more of PEG6000, PEG8000, ethanol, and isopropanol, preferably PEG8000, with a concentration of 0.5%-15% (m / v). The metal ion chelating agent is disodium ethylenediaminetetraacetate (EDTA) or trisodium citrate, and the concentration is 1-10 mM, preferably 2-5 mM EDTA. The amount of the second pretreatment reagent added is 1-3 mL.

[0068] The method for directly using the urinary tract infection pathogens for detection after processing includes the following steps:

[0069] S1. Mix 3 mL of collected urine by inversion. Choose whether to perform preliminary centrifugation based on the urine properties. If the urine is visually turbid or has flocculent precipitation, centrifuge it at 300×g for 30 seconds. Aspirate the supernatant into a clean centrifuge tube and wait for the next step. If the sample is visually clear, proceed directly to the next step.

[0070] S2. Add 1.5 mL of the first pretreatment reagent, vortex mix for 1 min, centrifuge at 13,000 × g for 2 min, and discard the supernatant.

[0071] S3. Add 1 mL of the second pretreatment reagent, vortex mix for 1 min, centrifuge at 13,000 × g for 2 min, and discard the supernatant. To prevent the loss of bacteria, about 5 μL of supernatant can be retained.

[0072] S4. Add 2 mL of sterile purified water, vortex mix for 1 min, centrifuge at 13,000 × g for 2 min, and discard the supernatant. To prevent the loss of bacteria, about 5 μL of supernatant can be retained.

[0073] S5. Repeat the sterile purified water washing step once. The remaining precipitate after discarding the supernatant is the target separation product.

[0074] S6. For MALDI-TOF MS detection, directly use 1μL inoculation loop to coat the target plate or use 5μL sterile purified water to resuspend the bacteria and take 1μL to coat the target plate. After the bacteria are dry, add 1μL CHCA matrix solution and test on the machine after drying.

[0075] S7. For PCR / qPCR / dPCR and other tests, add 100 μL TE buffer (pH 8.0) and 100 mg of 0.1 mm and 1 mm 1:1 acid-washed glass beads, shake vigorously for 5 min, heat at 100°C for 5 min, cool and centrifuge at 12,000 × g for 2 min, take the supernatant as a template, take 2 μL and add it to 20 μL PCR system for amplification.

[0076] Note: The preferred vortex mixing time is 1 min and the centrifugation time is 2 min.

[0077] The pretreatment reagent formulas used in Examples 1 to 10 are shown in the following table:

[0078]

[0079]

[0080] Examples 1 to 7 all used simulated E. coli urine with a concentration of 2E5 / mL as the sample, and the amplification primer probe was an E. coli specific primer probe. The results are summarized in the following table:

[0081]

[0082] The results of Examples 1 to 7 are not much different. The mass spectrometry and amplification diagram are taken as an example of the result diagram of Example 1. Figures 2-3 , Figure 3 Eco-1 and Eco-2 are the product amplification results of Example 1, and NTC-1 and NTC-2 are the negative control qPCR results without adding template. The negative control is used to indicate that there is no background amplification contamination in the reagents used.

[0083] Analysis of the results of Examples 1 to 7: The pretreatment reagents and methods of Examples 1 to 7 were used to isolate Escherichia coli at concentrations that could be detected by MALDI-TOF MS, and the probe qPCR amplification results were not much different. That is, the method and reagents of the present invention for directly using the urinary tract infection pathogens for detection after treatment are stable.

[0084] Example 8 is a comparison of the separation effect of adding or not adding a pretreatment reagent on Gram-negative capsule-containing Klebsiella pneumoniae, verifying the advantage of using the pretreatment reagent treatment method in this example in the separation effect on capsule-containing Gram-negative bacteria. 10mL of simulated Klebsiella pneumoniae urine with a concentration of 1.3E6 / mL was used as a sample. The mass spectrometry results are shown in the following table:

[0085] Sample name Microorganism name Confidence value No pretreatment reagents added - Repeat 1 Klebsiella pneumoniae 99.9 No pre-treatment reagents added - Repeat 2 Klebsiella pneumoniae 99.6 Add pretreatment reagent - repeat 1 Klebsiella pneumoniae 96.9 Add pretreatment reagent - repeat 2 Klebsiella pneumoniae 99.9 Without pretreatment reagent - dilute 10 times - repeat 1 / / Without pretreatment reagent - dilute 10 times - repeat 2 times / / Add pretreatment reagent - dilute 10 times - repeat 1 Klebsiella pneumoniae 99.7 Add pretreatment reagent - dilute 10 times - repeat 2 times Klebsiella pneumoniae 91.9

[0086] Analysis of mass spectrometry results: From the perspective of the original treatment products, the mass spectrometry of the separation products with or without pre-treatment reagents can obtain correct and reliable results, and it is impossible to tell the advantages and disadvantages of the two methods. The products obtained by the two separation methods were diluted 10 times and then subjected to mass spectrometry. The detection without pre-treatment reagents failed, while the separation with pre-treatment reagents met the confidence standards and the correct bacteria could be detected.

[0087] Amplification result analysis: Probe method qPCR amplification diagram is as follows Figure 4 As shown, the CT values ​​of the two replicates with the pre-treatment reagent are small, and the CT of the amplification without the pre-treatment reagent is obviously shifted back. At the same time, negative amplification does not work, indicating that the reagent is not contaminated and the amplification result is reliable.

[0088] Comprehensive analysis of mass spectrometry and amplification results: Combining the two test results, it can be clearly seen that the use of pretreatment reagents significantly increased the enrichment rate of Klebsiella pneumoniae containing capsules.

[0089] Example 9 is a comparison of the separation effect of adding or not adding a pretreatment reagent on Gram-positive capsule-containing Streptococcus pneumoniae, verifying the advantages of the separation method containing a pretreatment liquid described in this patent on the separation effect of capsule-containing Gram-positive bacteria. The separation effect of adding or not adding a pretreatment reagent is compared, using 10mL of urine with a simulated concentration of E6 / mL Streptococcus pneumoniae as a sample.

[0090] Sample name Microorganism name Confidence value Without pretreatment reagent-1 / / No pre-treatment reagent-2 / / Add pretreatment reagent-1 Streptococcuspneumoniae 99.9 Add pretreatment reagent-2 / /

[0091] Analysis of mass spectrometry results: Only one of the two replicates in the separation group with pretreatment reagents could detect the correct and reliable bacteria, and the detection without pretreatment reagents failed completely (it is speculated that the Spn loss in the group without pretreatment reagents was too much, and the recovered bacteria were below the minimum detection limit of the mass spectrometer, resulting in the failure of mass spectrometry detection).

[0092] Amplification result analysis: Probe method qPCR amplification diagram is as follows Figure 5 As shown, the two repeated CT values ​​with the pre-treatment reagent are small, and the amplification CT without the pre-treatment reagent is obviously shifted backward. At the same time, negative amplification does not work, indicating that the reagent is not contaminated and the amplification result is reliable. Comprehensive analysis of mass spectrometry and amplification results: Combining the two test results, it can be clearly seen that the use of pre-treatment reagents significantly improves the enrichment rate of capsule-containing Streptococcus pneumoniae.

[0093] Example 10

[0094] like Figure 1 As shown, a method and reagent for directly detecting urinary tract infection pathogens after treatment, the detection method and detection reagent are the same as those in Example 1, the only difference is that: the detection object is a 3mL urine sample of simulated E6CFU / mL Escherichia coli, and centrifugation is performed at 200×g and 800×g in step S1; the result of mass spectrometry detection in step S6 is shown in Figure 6 , 7 As shown, the detection is successful;

[0095] The control example is a MALDI-TOF MS pretreatment method for directly detecting pathogenic bacteria in infected urine according to CN102749233A. The detection object is a 3mL urine sample of simulated E6CFU / mL Escherichia coli. The supernatant is taken after centrifugation at 2000×g for 30 seconds, and then the supernatant is centrifuged at 15500rpm for 5 minutes. The precipitate is washed once with sterile purified water and subjected to MALDI-TOF MS detection. Figure 8 As shown, the mass spectrometry detection of the control example failed.

[0096] The results show that the recovery rate of Escherichia coli by the method of this embodiment is higher than that of the control example.

[0097] Embodiment 11

[0098] like Figure 1As shown, a method and reagent for directly detecting urinary tract infection pathogens after treatment, the detection method and detection reagent are the same as those in Example 1, the only difference is that: the detection object is 3mL of urine sample of Klebsiella pneumoniae simulating E6CFU / mL, and centrifugation is performed at 200×g and 800×g respectively in step S1; the result of mass spectrometry detection in step S6 is as shown Fig. 9 , 10 As shown, the detection is successful;

[0099] The control example is a MALDI-TOF MS pretreatment method for directly detecting pathogenic bacteria in infected urine according to CN102749233A. The detection object is a 3mL urine sample of Klebsiella pneumoniae simulating E6 CFU / mL. The supernatant is taken after centrifugation at 2000×g for 30 seconds, and then the supernatant is centrifuged at 15500rpm for 5 minutes. The precipitate is washed once with sterile purified water and MALDI-TOF MS detection is performed. Fig.11 As shown, mass spectrometry detection failed.

[0100] The results showed that the recovery rate of Klebsiella pneumoniae by the method of this embodiment was higher than that of the control example.

[0101] In the present invention, urinary tract infection (UTI) refers to inflammation caused by pathogens growing and multiplying in the urinary tract and invading the urinary tract mucosa or tissue, and is the most common type of bacterial infection.

[0102] MALDI-TOF MS: English name Matrix-Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry, which means matrix-assisted laser desorption ionization time of flight mass spectrometry.

[0103] PCR: Polymerase chain reaction is a method that uses a segment of DNA as a template and, with the participation of DNA polymerase and nucleotide substrate, amplifies the segment of DNA to a sufficient amount for structural and functional analysis.

[0104] qPCR: Quantitative Real-time PCR is a method that uses fluorescent chemicals to measure the total amount of products after each polymerase chain reaction (PCR) cycle in a DNA amplification reaction.

[0105] dPCR: The abbreviation of digital PCR, which is a method of dividing a sample into tens to tens of thousands of parts and assigning them to different reaction units. Each unit contains at least one copy of the target molecule (DNA template). PCR amplification is performed on the target molecule in each reaction unit. After the amplification, the fluorescent signals of each reaction unit are statistically analyzed.

[0106] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for directly detecting urinary tract infection pathogens after treatment, characterized in that: The following steps are involved: S1, collect 3-10 mL urine sample and mix it by inversion, and choose whether to perform preliminary centrifugation according to the characteristics of the urine sample. When there is blood or granular precipitation or floccules in the urine sample, centrifuge at 200-800×g for 30 seconds, aspirate the supernatant to obtain a sample after preliminary treatment, and enter step S2, otherwise, directly enter step S2; S2, adding 0.5 to 2 times the volume of the first pretreatment reagent to the sample after preliminary treatment or the urine sample, vortex mixing for 30 seconds to 2 minutes, lysing and dissolving urine impurities, wherein the urine impurities include red blood cells, white blood cells, phagocytes, epithelial cells and casts, centrifuging at 12000 to 15000 × g for 1 to 3 minutes, discarding the supernatant, retaining the precipitate to obtain the first lysed sample; S3, adding 1-3 mL of the second pretreatment reagent to the first lysed sample, vortexing and mixing for 30 s-2 min, lysing and dissolving the cell fragments and protein impurities that were not completely lysed in step S2, centrifuging at 12000-15000×g for 1-3 min, discarding the supernatant, retaining the precipitate to obtain the second lysed sample; S4, adding 1-3 mL of sterile purified water to the second lysed sample, vortex mixing for 30 s-2 min, centrifuging at 12000-15000×g for 1-3 min, discarding the supernatant, retaining the precipitate to obtain the first washed sample; S5, adding 1-3 mL of sterile purified water to the sample after the first washing, vortex mixing for 30 s-2 min, centrifuging at 12000-15000×g for 1-3 min, discarding the supernatant, retaining the precipitate to obtain a urinary tract infection pathogen separation product; Perform MALDI-TOF MS analysis and proceed to step S6, perform PCR / qPCR / dPCR analysis and proceed to step S7; S6, directly scrape a small amount of the urinary tract infection pathogen separation product with a 1 μL inoculation loop to coat the target plate, or resuspend the target separation product with 5 μL sterile purified water and take 1-2 μL of the target plate, add matrix solution, dry it, and then perform MALDI-TOF MS detection on the machine to obtain the mass spectrometry detection result of urinary tract infection pathogens; S7. Add 100 μL of TE buffer with a pH of 8.0 and 50-100 mg of 0.1 mm and 1 mm 1:1 mixed acid-washed glass beads to the urinary tract infection pathogen isolation product, oscillate for 5 minutes, heat at 95-100°C for 5 minutes, cool and centrifuge, take the supernatant as a template, and add it to the PCR / qPCR / dPCR system at a ratio of 10-20% to perform specific amplification detection of pathogens or drug-resistant genes to obtain the PCR detection results of urinary tract infection pathogens.

2. The method for directly detecting urinary tract infection pathogens after treatment according to claim 1, characterized in that: In step S2, adding 1 volume of the first pretreatment reagent to the preliminarily treated sample or the urine sample; In steps S2, S3 and S4, the vortex mixing time is 1 min, and the centrifugation time is 2 min.

3. The method for directly detecting urinary tract infection pathogens after treatment according to claim 1, characterized in that: In step S1, the urine sample includes any one or more of the following pathogens: Gram-negative bacteria, Gram-positive bacteria and fungi, and both the Gram-negative bacteria and the Gram-positive bacteria may contain capsules.

4. A method for directly detecting urinary tract infection pathogens after treatment according to claim 3, characterized in that: The Gram-negative bacteria include Escherichia coli, Escherichia coli and Klebsiella pneumoniae; the Gram-positive bacteria include Streptococcus pneumoniae.

5. The reagent for the method of directly using the treated urinary tract infection pathogens for detection according to claim 3, characterized in that: The first pretreatment reagent is composed of a buffer, a surfactant, a salt that promotes the dissolution of impurities, a bacterial precipitation promoting agent, a metal ion chelating agent and sterilized purified water; the pH value of the first pretreatment reagent is 7.0 to 9.5; The second pretreatment reagent is composed of the buffer, the salts promoting the dissolution of impurities, the bacteria precipitation promoting reagent, the metal ion chelating agent and sterilized purified water; the pH value of the second pretreatment reagent is 6.0-9.0, and the concentration of the second pretreatment reagent is less than or equal to that of the first pretreatment reagent; The buffer substance in the buffer is tris(hydroxymethyl)aminomethane hydrochloride and sodium hydroxide or phosphate buffer and sodium hydroxide, the sodium hydroxide is used to adjust the pH value of the first pretreatment reagent and the second pretreatment reagent, the concentration of the buffer in the first pretreatment reagent is 10-150 mM, and the concentration of the buffer in the second pretreatment reagent is 10-100 mM; The surfactant is one or more of sodium dodecyl sulfate, lithium dodecyl sulfate, sodium dodecyl sarcosine, Triton X-100, Tween 20, Tween 80, and ethylphenyl polyethylene glycol, and the concentration is 0.01-2% v / v; The salts promoting the dissolution of impurities are one or more of sodium chloride, ammonium chloride, lithium chloride, urea, guanidine hydrochloride, guanidine isothiocyanate, and sodium hypochlorite; The bacterial precipitation promoting agent is one or more of PEG6000, PEG8000, ethanol, and isopropanol; The metal ion chelating agent is disodium ethylenediaminetetraacetic acid or trisodium citrate. The concentration of the metal ion chelating agent in the first pretreatment reagent is 1.5-30 mM, and the concentration of the metal ion chelating agent in the second pretreatment reagent is 1-10 mM.

6. The reagent for the method of directly using the treated urinary tract infection pathogens for detection according to claim 5, characterized in that: The pH value of the first pretreatment reagent is 8.0 to 9.0, and the pH value of the second pretreatment reagent is 6.5 to 8.0; The concentration of the buffer in the first pretreatment reagent is 20-100 mM, and the concentration of the buffer in the second pretreatment reagent is 10-50 mM; The surfactant is a combination of two types of surfactants; The salt that promotes the dissolution of impurities is a combination of sodium chloride and guanidine hydrochloride, the concentration of the salt that promotes the dissolution of impurities in the first pretreatment reagent is 0.1-7M, and the concentration of the salt that promotes the dissolution of impurities in the second pretreatment reagent is 0.1-5M; The bacterial precipitation promoting reagent is PEG8000, the concentration of the bacterial precipitation promoting reagent in the first pretreatment reagent is 0.5-20% m / v, and the concentration of the bacterial precipitation promoting reagent in the second pretreatment reagent is 0.5-10% m / v; The metal ion chelating agent is disodium ethylenediaminetetraacetate, the concentration of disodium ethylenediaminetetraacetate in the first pretreatment reagent is 2-10 mM, and the concentration of disodium ethylenediaminetetraacetate in the second pretreatment reagent is 2-5 mM.

7. The reagent for the method of directly using the treated urinary tract infection pathogens for detection according to claim 5, characterized in that: In the first pretreatment reagent, the buffer is 20mM to 150mM tris(hydroxymethyl)aminomethane hydrochloride or 100mM phosphate buffer, and the pH value of the pretreatment reagent is adjusted to 7.5 to 9.5 using NaOH; the surfactant is 0.008 to 0.5% v / v sodium lauryl sarcosine and 0.5 to 2% v / v ethylphenyl polyethylene glycol, or 0.001% v / v sodium lauryl sulfate and 1% v / v Triton X-100, Or 0.5% v / v sodium lauryl sarcosinate and 0.5% v / v Tween 20, or 0.005% v / v sodium lauryl sulfate and 0.2% v / v Tween 20, or 0.2% v / v sodium lauryl sarcosinate and 0.5% v / v ethylphenyl polyethylene glycol, or 0.01% v / v lithium lauryl sulfate and 1% v / v ethylphenyl polyethylene glycol; the salt that promotes the dissolution of impurities is 0.15-1.5M sodium chloride and 2-6% guanidine hydrochloride, or 0.4M NH4Cl and 3M guanidine isothiocyanate, or 0.15MNaCl and 3M guanidine isothiocyanate, or 0.5M NaCl and 5M urea, or 4M lithium chloride; the precipitation aid reagent is PEG8000 with a concentration of 2-10%, or PEG6000 with a concentration of 10%, or ethanol with a concentration of 10-20%, or isopropanol with a concentration of 5%; the metal ion chelating agent is 1.5-15mM disodium ethylenediaminetetraacetate or 5-15mM trisodium citrate.

8. The reagent for the method of directly using the treated urinary tract infection pathogens for detection according to claim 7, characterized in that: In the second pretreatment reagent, the buffer is tris(hydroxymethylaminomethane) hydrochloride or 10-50mM phosphate buffer with a concentration of 10-50mM, and the pH value of the pretreatment reagent is adjusted to 7.0-8.0 using NaOH; the salts that promote the dissolution of impurities are 0.15-1M sodium chloride and 2-3M guanidine hydrochloride, or 0.15M NaCl and 2-3M guanidine isothiocyanate, or 0.3M NaCl and 5M urea, or 4M lithium chloride; the precipitation aid is PEG8000 with a concentration of 2-8%, or PEG6000 with a concentration of 7.5%, or ethanol with a concentration of 5%, or isopropanol with a concentration of 5%; the metal ion chelator is 1-10mM disodium ethylenediaminetetraacetic acid or 2-5mM trisodium citrate.

9. A reagent for a method of directly using the treated urinary tract infection pathogens for detection according to any one of claims 5 to 8, characterized in that: The method and reagent for directly detecting the urinary tract infection pathogens after treatment can also be applied to pus, pleural and abdominal effusions, alveolar lavage fluid, and cultured blood or cerebrospinal fluid.

Images