A method and kit for detecting a nucleic acid of a pathogenic microorganism
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-27
- Publication Date
- 2026-06-30
AI Technical Summary
Existing technologies have low efficiency in extracting nucleic acids from pathogenic microorganisms when processing respiratory samples, resulting in easy loss and insufficient detection sensitivity and specificity. Furthermore, traditional methods are costly and time-consuming, making it difficult to meet the needs of rapid detection.
A combination of pretreatment reagents containing buffers, organic polycarboxylic acid compounds, and surfactants, along with thiol-based reducing agents, was used to treat samples through gentle incubation, followed by nucleic acid extraction and amplification. Specific primers and probes were then used to detect pathogenic microorganisms.
It achieves efficient extraction and rapid detection of nucleic acids from pathogenic microorganisms, improves detection sensitivity and specificity, reduces reagent costs, and has good compatibility with existing nucleic acid extraction methods, thus reducing nucleic acid loss.
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Figure CN122303452A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial nucleic acid detection, and more specifically to a method and kit for detecting pathogenic microorganism nucleic acid. Background Technology
[0002] With the continuous advancement of urbanization, the incidence of respiratory diseases such as tuberculosis, chronic obstructive pulmonary disease, and pulmonary fungal infections is gradually increasing, seriously threatening human health. Common respiratory pathogens currently include Streptococcus pneumoniae, Mycoplasma pneumoniae, Klebsiella pneumoniae, Mycobacterium tuberculosis, and Aspergillus.
[0003] Currently, nucleic acid detection for many pathogenic microorganisms still relies on traditional methods such as microscopic examination, isolation and culture, serological testing, and immunological detection. These methods suffer from drawbacks such as low sensitivity, poor specificity, and high subjectivity. With the increasing variety of pathogenic microorganisms and the widespread use of antibiotics, traditional detection methods are no longer adequate for current clinical needs. In recent years, nucleic acid amplification has become a more mature method for disease detection, enabling the objective detection of pathogenic microorganisms in clinical samples within a short timeframe. It has gradually become an important tool for the precise diagnosis of infectious diseases.
[0004] However, clinical samples commonly used in the detection of respiratory infectious diseases are characterized by their complex composition and diverse morphology. Furthermore, they contain mucin and various other proteins, enzymes, microorganisms, inflammatory cells, and necrotic cells, resulting in high viscosity. Because of these characteristics, clinical samples such as sputum or bronchoalveolar lavage fluid require pretreatment before nucleic acid extraction to ensure the full release of pathogenic nucleic acids during the subsequent extraction process.
[0005] The current processing methods are mainly as follows: (1) Sodium hydroxide treatment: Under certain concentrations of sodium hydroxide, the proteins and other components in clinical samples such as sputum or bronchoalveolar lavage fluid can undergo irreversible denaturation, greatly reducing the viscosity of clinical samples. However, when using this method for pretreatment of clinical samples, the loss of pathogenic microbial nucleic acids may lead to missed detection of diseases due to the following two factors. Firstly, some pathogenic microorganisms will lyse under strong alkaline conditions, resulting in premature release of nucleic acids and degradation of nucleic acids; secondly, after sodium hydroxide treatment, the supernatant generally needs to be discarded by centrifugation to avoid the influence of excess alkaline substances on subsequent nucleic acid extraction. However, the loss of pathogenic microorganisms and their nucleic acids is inevitable during centrifugation. (2) Dithiothreitol method: This method mainly uses dithiothreitol, which has reducing properties, to reduce viscous mucoproteins. Since this method is relatively mild and will not damage the structural integrity of cells, it is widely used for clinical sputum samples. However, since dithiothreitol is difficult to reduce the disulfide bonds inside proteins, and its reduction requires certain pH conditions, the clinical sample processing status often becomes poor. (3) Protease method: This method uses proteases to fully hydrolyze mucin in clinical samples. However, due to the limited rate of enzyme incubation, the time required for protease treatment is relatively long, which is not conducive to the rapid detection of pathogenic microorganisms. In addition, this method usually depends on high concentrations of protease, which makes its cost higher than other methods, further limiting its use.
[0006] Therefore, there is still a need to provide an effective method for processing clinical samples and for the efficient extraction and detection of nucleic acids from pathogenic microorganisms.
[0007] The information in the background section is merely intended to illustrate the general background of the invention and should not be construed as an admission or implication in any way that such information constitutes prior art known to those skilled in the art. Summary of the Invention
[0008] To address at least some of the technical problems in the prior art, the present invention provides a method and kit for detecting nucleic acid in pathogenic microorganisms. Specifically, the present invention includes the following:
[0009] A first aspect of the present invention provides a method for detecting nucleic acid of pathogenic microorganisms, comprising:
[0010] (1) The pretreatment reagent is mixed with the sample and incubated at room temperature. The pretreatment reagent includes a first reagent and a second reagent. The first reagent includes a buffer, an organic polycarboxylic acid compound containing multiple carboxyl groups and nitrogen atoms, and a surfactant. The second reagent is a thiol-based reducing agent.
[0011] (2) Nucleic acid extraction was performed on the pretreated sample using nucleic acid extraction reagents;
[0012] (3) Amplification is performed using amplification primers and probes for the pathogenic microorganism to determine the presence of the pathogenic microorganism or to quantify it.
[0013] In some embodiments, according to the pathogenic microorganism nucleic acid detection method of the present invention, the pathogenic microorganism includes at least one of Streptococcus pneumoniae, Mycoplasma pneumoniae, Klebsiella pneumoniae, Mycobacterium tuberculosis, and Aspergillus.
[0014] In some embodiments, according to the method for detecting pathogenic microorganism nucleic acid according to the present invention, the primer sequences for detecting Streptococcus pneumoniae are shown in SEQ ID No. 1-2, and the probe sequences are shown in SEQ ID No. 3; the primer sequences for detecting Mycoplasma pneumoniae are shown in SEQ ID No. 4-5, and the probe sequences are shown in SEQ ID No. 6; the primer sequences for detecting Klebsiella pneumoniae are shown in SEQ ID No. 7-8, and the probe sequences are shown in SEQ ID No. 9; the primer sequences for detecting Mycobacterium tuberculosis are shown in SEQ ID No. 10-11, and the probe sequences are shown in SEQ ID No. 12; and the primer sequences for detecting Aspergillus are shown in SEQ ID No. 13-14, and the probe sequences are shown in SEQ ID No. 15.
[0015] In some embodiments, according to the pathogenic microorganism nucleic acid detection method of the present invention, in step (3), the nucleic acid is subjected to fluorescence quantitative PCR to obtain the Ct value of the microorganism.
[0016] In some embodiments, according to the pathogenic microorganism nucleic acid detection method of the present invention, the second reagent includes at least one of sulfur-containing amino acids, acetyl-containing amino acid derivatives, thiol-containing organic compounds, and organophosphorus compounds.
[0017] In some embodiments, according to the pathogenic microorganism nucleic acid detection method of the present invention, the first reagent comprises: 50-600 mM of buffer, 1-50 mM of organic polycarboxylic acid compound containing multiple carboxyl groups and nitrogen atoms, and 0.5-5% of surfactant.
[0018] In some embodiments, according to the pathogenic microorganism nucleic acid detection method of the present invention, the pH of the first reagent is 6-10.
[0019] In some embodiments, according to the pathogenic microorganism nucleic acid detection method of the present invention, the buffer includes at least one selected from glycine, acetate, phosphate, tris(hydroxymethyl)aminomethane, 3-morpholine propanesulfonic acid, and 4-hydroxyethylpiperazine ethanesulfonic acid.
[0020] In some embodiments, according to the pathogenic microorganism nucleic acid detection method of the present invention, the surfactant includes at least one of saponins, surfactants, ethyl phenyl polyethylene glycol, polyoxyethylene sorbitan monolaurate, polyethylene glycol octylphenyl ether, polyoxyethylene ether, and sodium dodecyl sulfate.
[0021] In some embodiments, according to the pathogenic microorganism nucleic acid detection method of the present invention, in step (1), the incubation temperature is 20-42℃ and the incubation time is 3-30min.
[0022] In some embodiments, according to the pathogenic microorganism nucleic acid detection method of the present invention, the incubation is performed by shaking at 80-1200 rpm.
[0023] In some embodiments, according to the pathogenic microorganism nucleic acid detection method of the present invention, the volume ratio of the first reagent to the sample is 0.5:1 to 3:1, preferably 0.5:1 to 1.5:1.
[0024] In some embodiments, according to the pathogenic microorganism nucleic acid detection method of the present invention, the volume ratio of the second reagent to the sample is 0.1:1 to 0.5:1, preferably 0.1:1 to 0.45:1.
[0025] In some embodiments, according to the pathogenic microorganism nucleic acid detection method of the present invention, the final concentration of the second reagent is 5-300 mM, preferably 5-150 mM.
[0026] A second aspect of the present invention provides a pathogenic microorganism nucleic acid detection kit, comprising:
[0027] (1) A pretreatment reagent, comprising a first reagent and a second reagent, wherein the first reagent comprises: a buffer, an organic polycarboxylic acid compound containing multiple carboxyl groups and nitrogen atoms, and a surfactant; and the second reagent is a thiol-based reducing agent; and
[0028] (2) Amplification primers and / or probes for the pathogenic microorganism and optional nucleic acid extraction reagents.
[0029] This invention has the following advantages:
[0030] (1) The processing method provided by this invention achieves pretreatment of clinical samples in a relatively mild manner. It can not only rapidly process clinical samples such as sputum or bronchoalveolar lavage fluid, but also directly perform subsequent nucleic acid extraction. It can be used in conjunction with high-throughput nucleic acid extraction. Furthermore, the mild processing reagents will not damage the structural integrity of pathogenic microorganisms, reduce the degree of nucleic acid degradation, and improve the nucleic acid yield (Table 1 and...). Figure 1 Furthermore, the processing method provided by this invention has good compatibility with column-based and magnetic bead-based nucleic acid extraction reagents, eliminating the need for centrifugation and further avoiding the loss of pathogenic microorganism nucleic acids, thereby significantly improving the positive detection rate of pathogenic microorganisms.
[0031] (2) The method for processing clinical samples such as sputum or bronchoalveolar lavage fluid provided by the present invention requires only a short time. It can fully process viscous sputum or bronchoalveolar lavage fluid in no more than 15 minutes, which greatly shortens the processing time required for clinical samples such as sputum or bronchoalveolar lavage fluid.
[0032] (3) The reagents required for the processing method provided by the present invention are significantly reduced compared with processing methods such as protease hydrolysis, thus reducing the reagent cost required for the processing steps. Attached Figure Description
[0033] Figure 1 The image shows the electrophoresis results of the performance comparison of different treatment methods in Example 7. Lane 1 represents the treatment method provided by the present invention (sample O), Lane 2 represents the dithiothreitol treatment method (sample M), and Lane 3 represents the sodium hydroxide treatment method (sample N).
[0034] Figure 2 This is the nucleic acid detection amplification curve of sample A in Example 1.
[0035] Figure 3 This is the nucleic acid detection amplification curve of sample B in Example 1.
[0036] Figure 4 This is a comparison of the nucleic acid detection amplification curves of sample A and sample B in Example 1, where the bold curve is the nucleic acid amplification curve of sample B.
[0037] Figure 5 This is the nucleic acid detection amplification curve of sample C in Example 2.
[0038] Figure 6 This is the nucleic acid detection amplification curve of sample D in Example 2.
[0039] Figure 7 This is a comparison of the nucleic acid detection amplification curves of sample C and sample D in Example 2, where the bold curve is the nucleic acid amplification curve of sample D.
[0040] Figure 8 This is the nucleic acid detection amplification curve of sample E in Example 3.
[0041] Figure 9 This is the nucleic acid detection amplification curve of sample F in Example 3.
[0042] Figure 10 This is a comparison of the nucleic acid detection amplification curves of sample E and sample F in Example 3. The bolded curve is the nucleic acid amplification curve of sample F.
[0043] Figure 11 This is the nucleic acid detection amplification curve of sample G in Example 4.
[0044] Figure 12 This is the nucleic acid detection amplification curve of sample H in Example 4.
[0045] Figure 13 This is a comparison of the nucleic acid detection amplification curves of sample G and sample H in Example 4, where the bold curve is the nucleic acid amplification curve of sample H.
[0046] Figure 14 This is the nucleic acid detection amplification curve of sample I in Example 5.
[0047] Figure 15 This is the nucleic acid detection amplification curve of sample J in Example 5.
[0048] Figure 16 This is a comparison of the nucleic acid detection amplification curves of sample I and sample J in Example 5, where the bold curve is the nucleic acid amplification curve of sample J.
[0049] Figure 17 This is the nucleic acid detection amplification curve of sample K in Example 6.
[0050] Figure 18 This is the nucleic acid detection amplification curve of sample L in Example 6.
[0051] Figure 19 This is a comparison of the nucleic acid detection amplification curves of sample K and sample L in Example 6, where the bold curve is the nucleic acid amplification curve of sample L.
[0052] Figure 20 This is the nucleic acid detection amplification curve of sample M in Example 7.
[0053] Figure 21 This is the nucleic acid detection amplification curve of sample N in Example 7.
[0054] Figure 22 This is the nucleic acid detection amplification curve of sample O in Example 7.
[0055] Figure 23This is a comparison of the nucleic acid detection amplification curves of samples M, N, and O in Example 7, where the bold curve is the nucleic acid amplification curve of sample O. Detailed Implementation
[0056] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0057] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0058] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0059] Nucleic acid testing methods
[0060] One aspect of the present invention provides a method for detecting nucleic acid of pathogenic microorganisms, which includes steps (1)-(3), and will be described in detail below.
[0061] Step (1) of the present invention is to mix the pretreatment reagent with the sample and incubate it at room temperature. The pretreatment reagent includes a first reagent and a second reagent. The first reagent includes a buffer, an organic polycarboxylic acid compound containing multiple carboxyl groups and nitrogen atoms, and a surfactant. The second reagent is a thiol-based reducing agent.
[0062] In this invention, the samples include clinical samples, and examples of clinical samples include, but are not limited to, sputum, bronchoalveolar lavage fluid, and bronchial aspirate.
[0063] To better extract the nucleic acid of pathogenic microorganisms from the sample to be tested, the pretreatment reagent and the sample need to be incubated under suitable conditions. In a preferred embodiment, the incubation temperature is 20-42℃, preferably 25-40℃, such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40℃, and the incubation time is 3-30 min, preferably 5-28 min, even more preferably 7-26 min, even more preferably 9-24 min, further preferably 11-22 min, and more preferably 13-20 min, such as 13, 14, 15, 16, 17, 18, 19, 20 min. Understandably, in order to better extract the nucleic acid of pathogenic microorganisms from the sample to be tested, the sample can be shaken and mixed during the incubation of the pretreatment reagent. The shaking frequency is 80-1200 rpm, preferably 100-1100 rpm, and even more preferably 120-1000 rpm, such as 160, 180, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, and 1000 rpm.
[0064] To better extract nucleic acids from pathogenic microorganisms in the sample to be tested, the volume ratio of the pretreatment reagent to the sample should not be too high or too low. For example, if the volume ratio of the pretreatment reagent is too low, incomplete nucleic acid extraction will occur. In a preferred embodiment, the volume ratio of the first reagent to the sample in the pretreatment reagent is 0.5:1 to 3:1, preferably 0.5:1 to 2.8:1, even more preferably 0.5:1 to 2.6:1, even more preferably 0.5:1 to 2.4:1, further preferably 0.5:1 to 2.2:1, more preferably 0.5:1 to 2:1, and even more preferably 0.5:1 to 1.5:1; for example, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, and 1.5:1. In a preferred embodiment, the volume ratio of the second reagent to the sample in the pretreatment reagent is 0.1:1 to 0.5:1, preferably 0.1:1 to 0.45:1; for example, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.45:1.
[0065] To better extract nucleic acids from pathogenic microorganisms in the test sample, the final concentration of the second reagent in the reaction system after mixing with the sample should not be too high or too low. If the final concentration of the second reagent is too low, incomplete nucleic acid extraction will occur; if the final concentration is too high, the nucleic acid extraction rate will be reduced. In a preferred embodiment, the final concentration of the second reagent is 5-300 mM, preferably 5-150 mM, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, and 150 mM.
[0066] In a preferred embodiment, the first reagent comprises a buffer of 50-600 mM, preferably 60-590 mM, even more preferably 70-580 mM, further preferably 80-570 mM, more preferably 90-560 mM, and even more preferably 100-550 mM, such as 100, 150, 200, 250, 300, 350, 400, 450, 500, and 550 mM; and 1-50 mM, preferably 2-48 mM, even more preferably 4-46 mM, further preferably 6-44 mM, more preferably 8-42 mM, and even more preferably... Select an organic polycarboxylic acid compound containing multiple carboxyl groups and nitrogen atoms at a concentration of 10-40 mM, such as 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40 mM, and 0.5-5%, preferably 0.6-4.9%, even more preferably 0.7-4.8%, further preferably 0.8-4.7%, more preferably 0.9-4.6%, and even more preferably 1-4.5%, such as 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, or 4.5%, of a surfactant. In a preferred embodiment, the pH of the first reagent is 6-10, preferably 6-9, such as 6, 6.5, 7, 7.5, 8, 8.5, or 9.
[0067] In this invention, the buffer includes at least one of glycine, acetate, phosphate, tris(hydroxymethyl)aminomethane, 3-morpholinopropanesulfonic acid, and 4-hydroxyethylpiperazine ethanesulfonic acid.
[0068] In this invention, the organic polycarboxylic acid compound containing multiple carboxyl groups and nitrogen atoms includes at least one of disodium ethylenediaminetetraacetate and ethylene glycol-bis-(2-aminoethyl)tetraacetic acid.
[0069] In this invention, the surfactant includes at least one selected from saponins, surfactants, ethylphenyl polyethylene glycol, polyoxyethylene sorbitan monolaurate, polyethylene glycol octylphenyl ether, polyoxyethylene ether, and sodium dodecyl sulfate. In one specific embodiment, the surfactant is polyethylene glycol octylphenyl ether.
[0070] In a preferred embodiment, the second reagent comprises at least one of the following: 0.001-1.5M, preferably 0.1-1.5M, even more preferably 0.5-1.5M, and more preferably 0.7-1.5M, such as 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5M sulfur-containing amino acids (e.g., but not limited to cysteine), acetyl-containing amino acid derivatives (e.g., but not limited to N-acetylcysteine), thiol-containing organic compounds (e.g., but not limited to dithioerythritol, dithiothreitol, 6-mercapto-1-hexanol), and organophosphorus compounds (e.g., but not limited to tris(2-carboxyethyl)phosphine). It is understood that the second reagent may comprise pure water or a buffer solution containing acetate. In a preferred embodiment, the second reagent is an aqueous solution containing 0.7-1.5M dithiothreitol.
[0071] Step (2) of the present invention is to extract nucleic acid from the pretreated sample using a nucleic acid extraction reagent.
[0072] In this invention, the method of nucleic acid extraction is not particularly limited. Methods known in the art or conventional nucleic acid extraction kits can be used to extract nucleic acid according to the manufacturer's instructions. Examples of nucleic acid extraction reagents include, but are not limited to, sodium dodecyl sulfate, hexadecyltrimethylammonium bromide, disodium ethylenediaminetetraacetate dihydrate, chloroform, etc.
[0073] Step (3) of the present invention is to amplify the pathogenic microorganism by using amplification primers and probes for the pathogenic microorganism in order to determine the presence of the pathogenic microorganism or to quantify it.
[0074] In this invention, the amplification method is not particularly limited, and methods known in the art can be used for amplification, including but not limited to polymerase chain reaction, loop-mediated isothermal amplification, helicase-dependent isothermal amplification, and strand displacement amplification. In a preferred embodiment, the nucleic acid is subjected to real-time quantitative PCR to obtain the Ct value of the microorganism, and the content of pathogenic microorganism nucleic acid in the sample is further determined based on the Ct value.
[0075] In a preferred embodiment, the pathogenic microorganism includes at least one of Streptococcus pneumoniae, Mycoplasma pneumoniae, Klebsiella pneumoniae, Mycobacterium tuberculosis, and Aspergillus.
[0076] In a preferred embodiment, the primer sequences for detecting Streptococcus pneumoniae are shown in SEQ ID No. 1-2, and the probe sequence is shown in SEQ ID No. 3; the primer sequences for detecting Mycoplasma pneumoniae are shown in SEQ ID No. 4-5, and the probe sequence is shown in SEQ ID No. 6; the primer sequences for detecting Klebsiella pneumoniae are shown in SEQ ID No. 7-8, and the probe sequence is shown in SEQ ID No. 9; the primer sequences for detecting Mycobacterium tuberculosis are shown in SEQ ID No. 10-11, and the probe sequence is shown in SEQ ID No. 12; and the primer sequences for detecting Aspergillus are shown in SEQ ID No. 13-14, and the probe sequence is shown in SEQ ID No. 15.
[0077] Reagent test kit
[0078] One aspect of the present invention provides a pathogenic microorganism nucleic acid detection kit, comprising:
[0079] (1) Pretreatment reagent, wherein the pretreatment reagent includes a first reagent and a second reagent, the first reagent includes: a buffer, an organic polycarboxylic acid compound containing multiple carboxyl groups and nitrogen atoms and a surfactant, and the second reagent is a thiol-based reducing agent;
[0080] (2) Amplification primers and / or probes for the pathogenic microorganism, and optional nucleic acid extraction reagents.
[0081] In addition to the components described above, the kit of the present invention may also include precautions related to the regulation of manufacturing, use, or sale of the diagnostic kit. Furthermore, the kit of the present invention may also provide detailed instructions for storage and troubleshooting. The kit may optionally be housed in a suitable device, preferably for high-throughput robotic operation.
[0082] In some embodiments, the components of the kit of the present invention may be disposed in a container. The container typically includes at least one vial, test tube, flask, bottle, syringe, and / or other container means, wherein the solvent may optionally be placed in equal portions. The kit may also include means for containing a second container of sterile, pharmaceutically acceptable buffers and / or other solvents.
[0083] In some embodiments, the components of the kit of the present invention may be provided in solution form, such as an aqueous solution. When present in an aqueous solution, the concentration or content of these components can be readily determined by those skilled in the art according to different needs.
[0084] Example 1
[0085] The following shows a comparison of the nucleic acid extraction effects of different processing methods for sputum samples.
[0086] 1. Experimental Methods
[0087] (1) Prepare sputum samples that are positive for pathogenic microorganisms by microscopic examination from patients with pulmonary fungal disease (collected from Changsha First Hospital), and divide them into sample A and sample B in 200 μL each, and place them in 1.5 mL centrifuge tubes respectively;
[0088] (2) Add twice the volume of commercial sodium hydroxide treatment reagent to sample A for treatment. After treatment, centrifuge carefully to discard 500 μL of supernatant, and add 100 μL of PBS to neutralize the sodium hydroxide. Then, use the Qiagen 51104 kit for subsequent nucleic acid extraction, and perform nucleic acid detection according to the Aspergillus detection kit provided by Shanghai Geneobio Biotechnology Co., Ltd.
[0089] (3) Add 100 μL of the first reagent and 30 μL of the second reagent to sample B, mix well, and incubate at 37°C for 10 min. After processing, use the Qiagen 51104 kit to extract relevant nucleic acids, and perform nucleic acid detection according to the Aspergillus detection kit provided by Shanghai Geneobio Biotechnology Co., Ltd. The first reagent includes 350 mM glycine, 25 mM disodium ethylenediaminetetraacetate, 2.5% polyethylene glycol octylphenyl ether, and 0.5% surfactant, with a pH of 9.0; the second reagent includes 100 mM tris(2-carboxyethyl)phosphine.
[0090] 2. Experimental Results
[0091] The test results for sample A are shown below. Figure 2 The test results for sample B are shown below. Figure 3 The comparison results between sample A and sample B are shown in [link to comparison results]. Figure 4 .
[0092] The results showed that the detection results obtained by the commercial sodium hydroxide treatment method were consistent with those obtained by the treatment method provided in this embodiment, and were also consistent with the clinical detection results. Furthermore, the Ct value obtained by the treatment method provided in this embodiment was lower than that obtained by the commercial reagent, indicating that the treatment method proposed in this embodiment can effectively reduce nucleic acid loss and improve the detection rate of trace pathogenic microorganisms.
[0093] Example 2
[0094] The following shows a comparison of the nucleic acid extraction effects of different processing methods for bronchoalveolar lavage fluid samples.
[0095] 1. Experimental Methods
[0096] (1) Prepare bronchoalveolar lavage fluid samples that are positive for pathogenic microorganisms by microscopic examination from patients with pulmonary fungal disease (collected from Changsha First Hospital), and divide them into sample C and sample D in 200 μL each, and place them in 1.5 mL centrifuge tubes respectively;
[0097] (2) Add two volumes of 4% sodium hydroxide treatment reagent to sample C and treat for 10 min. After treatment, centrifuge carefully and discard 500 μL of supernatant. Add 0.5 times the sample volume of PBS to neutralize the sodium hydroxide. Then, use the Qiagen 51104 kit for subsequent nucleic acid extraction and refer to the Aspergillus detection kit provided by Shanghai Geneobio Biotechnology Co., Ltd. for pathogen nucleic acid detection.
[0098] (3) Add 0.5 times the sample volume of the first reagent to sample D, and add 20 μL of the second reagent. Mix well and incubate at room temperature for 10 min. After processing, use the Qiagen 51104 kit to extract relevant nucleic acids, and perform nucleic acid detection according to the Aspergillus detection kit provided by Shanghai Geneobio Biotechnology Co., Ltd. The first reagent includes 200 mM tris(hydroxymethyl)aminomethane, 40 mM ethylene glycol-bis-(2-aminoethyl)tetraacetic acid, and 1.5% surfactant, with a pH of 8.0; the second reagent includes 1.2 M dithiothreitol.
[0099] 2. Experimental Results
[0100] The test results for sample C are shown below. Figure 5 The detection results for sample D are shown below. Figure 6 The comparison results between sample C and sample D are shown in [the original text]. Figure 7 .
[0101] The results showed that the detection results using the processing method provided in this embodiment were consistent with the clinical detection results, while the detection results using the sodium hydroxide treatment method were negative. The experimental results indicate that the processing solution provided in this embodiment has a significant promoting effect on improving the detection rate of pathogenic microorganisms in bronchoalveolar lavage fluid.
[0102] Example 3
[0103] The following shows a comparison of the nucleic acid extraction effects of different processing methods for bronchoalveolar lavage fluid samples.
[0104] 1. Experimental Methods
[0105] (1) Prepare bronchoalveolar lavage fluid samples that are positive for pathogenic microorganisms from patients with pulmonary fungal disease (collected from Changsha First Hospital), and divide them into two equal parts, E and F, at 200 μL each, and place them in 1.5 mL centrifuge tubes respectively.
[0106] (2) Sample E was treated with 400 μL of commercial sodium hydroxide treatment reagent. After treatment, 500 μL of supernatant was carefully discarded by centrifugation, and 0.5 times the sample volume of PBS was added to neutralize the sodium hydroxide. Subsequently, nucleic acid extraction was performed using the Qiagen 51104 kit, and pathogenic microorganism nucleic acid detection was performed according to the Aspergillus detection kit provided by Shanghai Geneobio Biotechnology Co., Ltd.
[0107] (3) Add 0.5 times the sample volume of the first reagent to sample F, and add 20 μL of the second reagent. After mixing evenly, incubate at 37°C for 10 min at 1000 rpm. After processing, use the Qiagen 51104 kit to extract relevant nucleic acids, and perform nucleic acid detection according to the Aspergillus detection kit provided by Shanghai Geneobio Biotechnology Co., Ltd. The first reagent includes 300 mM tris(hydroxymethyl)aminomethane, 30 mM disodium ethylenediaminetetraacetate, 1% ethylphenyl polyethylene glycol, and 1% sodium dodecyl sulfate, with a pH of 8.5; the second reagent includes 0.8 M N-acetylcysteine and 50 mM tris(2-carboxyethyl)phosphine.
[0108] 2. Experimental Results
[0109] The detection results for sample E are shown below. Figure 8 The detection results for sample F are shown in Figure 9 The comparison results between sample E and sample F are shown in [the original text]. Figure 10 .
[0110] The results showed that the final test result using the sodium hydroxide treatment method was negative, while the test result using the treatment method of this embodiment indicated that the patient in this sample had an Aspergillus infection, consistent with the clinical diagnosis. These experimental results further demonstrate that this embodiment not only improves the treatment process but also significantly increases the detection rate of pathogenic microorganisms.
[0111] Example 4
[0112] The following shows a comparison of the nucleic acid extraction effects of different processing methods for sputum samples.
[0113] 1. Experimental Methods
[0114] (1) Prepare sputum samples that are positive for pathogenic microorganisms by microscopic examination from patients with pulmonary fungal disease (collected from Changsha First People's Hospital), and divide them into 200 μL samples G and H, which are placed in 1.5 mL centrifuge tubes respectively;
[0115] (2) Sample G was treated with 30 μL of proteinase K for sputum processing. After processing, Qiagen 51104 kit was used for subsequent nucleic acid extraction, and the Aspergillus detection kit provided by Shanghai Geneobio Biotechnology Co., Ltd. was used for pathogen nucleic acid detection.
[0116] (3) Add 0.5 times the sample volume of the first reagent to sample H, and add 30 μL of the second reagent. After mixing well, incubate at 37°C for 10 min at 1000 rpm. After processing, use the Qiagen 51104 kit to extract relevant nucleic acids, and perform nucleic acid detection according to the Aspergillus detection kit provided by Shanghai Geneobio Biotechnology Co., Ltd. The first reagent includes 150 mM 3-morpholinopropanesulfonic acid, 20 mM disodium ethylenediaminetetraacetate, and 4% polyoxyethylene sorbitan monolaurate, with a pH of 7.0; the second reagent includes 0.5 M dithiothreitol.
[0117] 2. Experimental Results
[0118] The detection results for sample G are shown below. Figure 11 The detection results for sample H are shown in Figure 12 The comparison results between sample G and sample H are shown in [the original text]. Figure 13 .
[0119] The results showed that the detection results using the processing method provided in this embodiment were consistent with the clinical detection results, while the experimental group treated with proteinase K had negative results. These experimental results further demonstrate that this embodiment can effectively avoid missed detection of pathogenic microorganisms compared to the proteinase treatment method.
[0120] Example 5
[0121] The following shows a comparison of the nucleic acid extraction effects of different processing methods for sputum samples.
[0122] 1. Experimental Methods
[0123] (1) Prepare sputum samples that are negative for pathogenic microorganisms (collected from Changsha First People's Hospital, negative samples), and divide them into two equal parts, 200 μL each, into sample I and sample J, and place them in 1.5 mL centrifuge tubes respectively;
[0124] (2) Sample I was treated with 30 μL of commercial dithiothreitol solution (1M) for sputum processing. After processing, Qiagen 51104 kit was used for subsequent nucleic acid extraction, and the Aspergillus detection kit provided by Shanghai Geneobio Biotechnology Co., Ltd. was used for pathogen nucleic acid detection.
[0125] (3) Add 0.5 times the sample volume of the first reagent to sample J, and add 30 μL of the second reagent. Mix well and incubate at room temperature for 10 min. After processing, use the Qiagen 51104 kit to extract relevant nucleic acids, and perform nucleic acid detection according to the Aspergillus detection kit provided by Shanghai Geneobio Biotechnology Co., Ltd. The first reagent includes 350 mM glycine, 20 mM disodium ethylenediaminetetraacetate, and 3% polyoxyethylene sorbitan monolaurate, with a pH of 8.0; the second reagent includes 200 mM tris(2-carboxyethyl)phosphine.
[0126] 2. Experimental Results
[0127] The test results for sample I are shown below. Figure 14 The test results for sample J are shown in [link to test results]. Figure 15 The comparison results between sample I and sample J are shown in [the original text]. Figure 16 .
[0128] The results showed that the detection results using the treatment method provided in this embodiment and the dithiothreitol treatment method were consistent with the clinical detection results. However, the Ct value of the internal reference gene using the first reagent in this embodiment was significantly lower than that using dithiothreitol alone. These experimental results further demonstrate that this embodiment can effectively improve the nucleic acid extraction efficiency compared to using dithiothreitol alone.
[0129] Example 6
[0130] The following shows a comparison of the nucleic acid extraction effects of different sputum sample processing methods adapted to the magnetic bead method.
[0131] 1. Experimental Methods
[0132] (1) Prepare sputum samples that are positive for pathogenic microorganisms by microscopic examination (collected from Changsha First People's Hospital), and divide them into sample K and sample L in 200 μL and place them in 1.5 mL centrifuge tubes respectively;
[0133] (2) Sample K was treated with 400 μL of commercial sodium hydroxide treatment reagent for 20 min. After treatment, it was centrifuged at 12000 rpm for 5 min and 500 μL of supernatant was carefully discarded. 0.5 times the sample volume of PBS was added to neutralize the sodium hydroxide. After treatment, the remaining sample was resuspended and nucleic acid extraction was performed according to the instructions of the magnetic bead nucleic acid extraction reagent (Shanghai Jienuo GF1). The pathogen nucleic acid was detected by referring to the Aspergillus detection kit provided by Shanghai Jienuo Biotechnology Co., Ltd.
[0134] (3) Add 0.5 times the sample volume of the first reagent to sample L, and add 30 μL of the second reagent. Mix well and incubate at 37°C for 5 min at 1000 rpm. After processing, perform nucleic acid extraction directly according to the instructions of the magnetic bead nucleic acid extraction reagent (Shanghai Jienuo GF1), and perform nucleic acid detection according to the Aspergillus detection kit provided by Shanghai Jienuo Biotechnology Co., Ltd. The first reagent includes 200 mM tris(hydroxymethyl)aminomethane, 30 mM disodium ethylenediaminetetraacetate, and 2% ethylphenyl polyethylene glycol, with a pH of 8.5; the second reagent includes 1.2 M dithiothreitol.
[0135] 2. Experimental Results
[0136] The detection results for sample K are shown below. Figure 17 The detection results for sample L are shown in Figure 18 The comparison results between sample K and sample L are shown in [the original text]. Figure 19 .
[0137] The results show that the detection results using the processing method provided in this embodiment are consistent with the clinical detection results, while the detection results using the sodium hydroxide treatment method are negative. The above results indicate that the processing method provided in this embodiment is more suitable for the sample pretreatment process of magnetic bead nucleic acid extraction reagent than the sodium hydroxide treatment method. At the same time, since the processing method provided in this embodiment does not require centrifugation, it can significantly increase the throughput of automated detection of sputum and bronchoalveolar lavage fluid samples.
[0138] Example 7
[0139] The following shows a comparison of the nucleic acid extraction effects of different processing methods for bronchoalveolar lavage fluid samples adapted to the magnetic bead method.
[0140] 1. Experimental Methods
[0141] (1) Prepare bronchoalveolar lavage fluid samples that are positive for pathogenic microorganisms by microscopic examination (collected from Changsha First People's Hospital), and divide them into samples M, N and O in 200 μL each, and place them in 1.5 mL centrifuge tubes respectively;
[0142] (2) Sample M was treated with 30 μL of commercial dithiothreitol solution (1M) and treated at room temperature for 30 min. Then, nucleic acid extraction was performed according to the instructions of the magnetic bead nucleic acid extraction reagent (Shanghai Jienuo GF1). The pathogenic microorganism nucleic acid was detected by referring to the Aspergillus detection kit provided by Shanghai Jienuo Biotechnology Co., Ltd.
[0143] (3) Sample N was treated with 400 μL of commercial sodium hydroxide treatment reagent for 20 min. After treatment, it was centrifuged at 12000 rpm for 5 min and 500 μL of supernatant was carefully discarded. 0.5 times the sample volume of PBS was added to neutralize the sodium hydroxide. After treatment, the remaining sample was resuspended and nucleic acid extraction was performed according to the instructions of the magnetic bead nucleic acid extraction reagent (Shanghai Jienuo GF1). The pathogen nucleic acid was detected by referring to the Aspergillus detection kit provided by Shanghai Jienuo Biotechnology Co., Ltd.
[0144] (4) Add 0.5 times the sample volume of the first reagent to sample O, and add 30 μL of the second reagent. After mixing evenly, incubate at room temperature for 5 min. After processing, perform relevant nucleic acid extraction directly according to the instructions of the magnetic bead nucleic acid extraction reagent (Shanghai Jienuo GF1), and perform nucleic acid detection according to the Aspergillus detection kit provided by Shanghai Jienuo Biotechnology Co., Ltd. The first reagent includes 150 mM glycine, 25 mM disodium ethylenediaminetetraacetate, 1% saponin, 1.5% ethyl phenyl polyethylene glycol, and pH 8.5; the second reagent includes 1.2 M dithiothreitol.
[0145] 2. Experimental Results
[0146] The time required for sample liquefaction, liquefaction state, and nucleic acid concentration measurement data are shown in Table 1. The detection results for sample M are shown in Table 1. Figure 20 The detection results for sample N are shown in [link to test results]. Figure 21 The detection results for sample O are shown below. Figure 22 The comparison results of samples M, N, and O are shown in [the original text]. Figure 23 .
[0147] Table 1 Performance comparison of different processing methods
[0148]
[0149] The results showed that the detection results using the processing method provided in this embodiment, as well as the dithiothreitol and sodium hydroxide processing methods, were consistent with the clinical detection results. The detected Ct values showed that the processing method provided in this embodiment was superior to the dithiothreitol and sodium hydroxide processing methods. Furthermore, the processing method provided in this embodiment greatly shortened the processing time, enabling faster completion of the pretreatment process for sputum and bronchoalveolar lavage fluid samples and improving detection efficiency.
[0150] Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.
Claims
1. A method for detecting nucleic acid in pathogenic microorganisms, characterized in that, include: (1) The pretreatment reagent is mixed with the sample and incubated at room temperature. The pretreatment reagent includes a first reagent and a second reagent. The first reagent includes a buffer, an organic polycarboxylic acid compound containing multiple carboxyl groups and nitrogen atoms, and a surfactant. The second reagent is a thiol-based reducing agent. (2) Nucleic acid extraction was performed on the pretreated sample using nucleic acid extraction reagents; (3) Amplification is performed using amplification primers and probes for the pathogenic microorganism to determine the presence of the pathogenic microorganism or to quantify it.
2. The method for detecting pathogenic microorganism nucleic acid according to claim 1, characterized in that, The pathogenic microorganisms include at least one of Streptococcus pneumoniae, Mycoplasma pneumoniae, Klebsiella pneumoniae, Mycobacterium tuberculosis, and Aspergillus. Preferably, the primer sequences for detecting Streptococcus pneumoniae are shown in SEQ ID No. 1-2, and the probe sequences are shown in SEQ ID No. 3; the primer sequences for detecting Mycoplasma pneumoniae are shown in SEQ ID No. 4-5, and the probe sequences are shown in SEQ ID No. 6; the primer sequences for detecting Klebsiella pneumoniae are shown in SEQ ID No. 7-8, and the probe sequences are shown in SEQ ID No. 9; the primer sequences for detecting Mycobacterium tuberculosis are shown in SEQ ID No. 10-11, and the probe sequences are shown in SEQ ID No. 12; and the primer sequences for detecting Aspergillus are shown in SEQ ID No. 13-14, and the probe sequences are shown in SEQ ID No.
15.
3. The method for detecting pathogenic microorganism nucleic acid according to claim 1, characterized in that, In step (3), the nucleic acid is subjected to real-time quantitative PCR to obtain the Ct value of the microorganism.
4. The method for detecting pathogenic microorganism nucleic acid according to claim 1, characterized in that, The second reagent includes at least one of sulfur-containing amino acids, amino acid derivatives containing acetyl groups, organic compounds containing thiol functional groups, and organophosphorus compounds.
5. The method for detecting pathogenic microorganism nucleic acid according to claim 1, characterized in that, The first reagent comprises: 50-600 mM of buffer, 1-50 mM of organic polycarboxylic acid compound containing multiple carboxyl groups and nitrogen atoms, and 0.5-5% of surfactant; Preferably, the pH of the first reagent is 6-10.
6. The method for detecting pathogenic microorganism nucleic acid according to claim 1, characterized in that, The buffer comprises at least one of glycine, acetate, phosphate, tris(hydroxymethyl)aminomethane, 3-morpholinopropanesulfonic acid, and 4-hydroxyethylpiperazine ethanesulfonic acid.
7. The method for detecting pathogenic microorganism nucleic acid according to claim 1, characterized in that, The surfactant includes at least one of saponins, surfactants, ethyl phenyl polyethylene glycol, polyoxyethylene sorbitan monolaurate, polyethylene glycol octylphenyl ether, polyoxyethylene ether, and sodium dodecyl sulfate.
8. The method for detecting pathogenic microorganism nucleic acid according to claim 1, characterized in that, In step (1), the incubation temperature is 20-42℃ and the incubation time is 3-30 min; Preferably, the mixture is shaken at 80-1200 rpm during incubation.
9. The method for detecting pathogenic microorganism nucleic acid according to claim 1, characterized in that, The volume ratio of the first reagent to the sample is 0.5:1 to 3:1, preferably 0.5:1 to 1.5:1; Preferably, the volume ratio of the second reagent to the sample is 0.1:1 to 0.5:1, more preferably 0.1:1 to 0.45:1; Preferably, the final concentration of the second reagent is 5-300 mM, and more preferably 5-150 mM.
10. A nucleic acid detection kit for pathogenic microorganisms, characterized in that, include: (1) Pretreatment reagent, wherein the pretreatment reagent includes a first reagent and a second reagent, the first reagent includes: a buffer, an organic polycarboxylic acid compound containing multiple carboxyl groups and nitrogen atoms and a surfactant, and the second reagent is a thiol-based reducing agent; and (2) Amplification primers and / or probes for the pathogenic microorganism.