CN113322304A - Clinical sample processing method applied to second-generation infection metagenome detection - Google Patents
Clinical sample processing method applied to second-generation infection metagenome detection Download PDFInfo
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Abstract
The invention provides a clinical sample processing method applied to second-generation infection metagenome detection, which is used for sample processing by matching a Lysing Matrix E tube with lysozyme, GB lysate and the like.
Description
Technical Field
The invention relates to the field of gene sequencing, in particular to a clinical sample processing method applied to detection of second-generation infection metagenome.
Background
Over the last 20 years, approximately 1500 million people die worldwide each year from infectious diseases (fudge and morens). The world health organization issued ten major threats to global health in 2019, of which 6 were associated with infectious diseases. Emerging and emerging lethal pathogens, including the transmission of classical pathogens as well as multidrug resistant pathogens (e.g., multidrug resistant klebsiella pneumoniae, neisseria gonorrhoeae, mycobacterium tuberculosis, etc.), emphasize the increasingly serious challenges facing accurate diagnosis and effective treatment of infectious diseases.
Traditional culture diagnostic techniques (e.g., culture, nucleic acid amplification assays, immunoassays, etc.) and relatively new techniques such as 16S rRNA (16S rRNA) gene sequencing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) relied upon for pathogen identification in clinical laboratories have significant advantages and limitations, which often expose weaknesses in the management of complex infections, such as being cumbersome, time consuming, and of limited diagnostic accuracy, resulting in delays or missed diagnoses. The etiology of 40% of intestinal infections (Thomas et al, 2015), about 50% of blood infections (BSI) (Fenollar and Rao μ Lt, 2007) and more than 50% of Central Nervous System (CNS) infections (Glaser et al 2006) has been reported to remain unknown. These undiagnosed cases force physicians to prescribe an empirical broad-spectrum antibiotic treatment that will greatly increase the risk of multidrug-resistant bacteria and potentially increase the mortality rate in severely infected patients.
Next Generation Sequencing (NGS) technology opens another window for diagnosing infectious diseases. The next generation of metagenomic sequencing (mNGS) has become a promising single, universal pathogen detection method for infectious disease diagnosis. This method allows identification and genomic characterization of bacteria, fungi, parasites and viruses without the need to obtain culture results from clinical specimens beforehand. NGS studies of clinical samples or clinical laboratories involve many steps including nucleic acid extraction, DNA and/or RNA concentration, library preparation, PCR amplification, sequencing and bioinformatic analysis. The type of nucleic acid extraction method used in the laboratory is one of the major factors affecting the results, and the amount of nucleic acid released may vary from pathogen to pathogen. For example, mycobacteria require vigorous cell disruption to effectively lyse the cell wall to release nucleic acids, different specimen types require different extraction methods, and efficient extraction methods are key steps to achieve true unbiased sequencing of a sample.
The cell wall of fungi is composed of a variety of polysaccharides, proteins and glycoproteins. Polysaccharides account for approximately 80% of the wall, while proteins account for 20% and there are also small amounts of lipids and waxes, the presence of which prevents drying. The fungal cell wall generally has three layers: the outermost layer consists of glycoproteins and glucans or mannans, the second layer is β -1,3 glucans, and the innermost layer consists of chitin. The structure of the cell wall differs between different groups: the above listed ratios of the components may vary. Their composition also varies depending on the state of the cells within the same species (e.g.spore vs. hyphae). This variation affects the extraction efficiency of a given DNA extraction procedure in different species. The bacteria can be classified into 2 types, gram-positive bacteria and gram-negative bacteria, by gram staining. The cell lysis step is also particularly critical because of the wall thickness of gram-positive bacteria cells, which form a permeability barrier by peptidoglycan network molecules. Cell lysis can be achieved by chemical treatment (with detergents, acids or alkaline reagents), enzymatic degradation of cell wall components (with enzymes such as proteinase K, lytic enzymes and chitinase) or using mechanical/physical methods (e.g.ultrasound, bead beating, high temperature, etc.) (Alessandra Frau et al 2019), the use of either method alone may present some risk of omission.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The core technical problem to be solved by the invention is the phenomenon of detection omission of the second-generation infection metagenome detection caused by insufficient release of the nucleic acid of the pathogenic microorganism of the clinical infection sample, and in order to solve the problems, the technical scheme adopted by the invention is as follows:
the invention firstly provides a method for processing clinical sample pathogens applied to detection of second-generation infection metagenome, which comprises the following steps
1) Sample pretreatment: taking an infection sample, carrying out centrifugal treatment, and reserving part of supernatant and sediment;
2) and (3) sample bacteriolysis and wall breaking:
adding lysozyme with the final concentration of more than 1mg/mL into the pretreated sample for incubation;
adding the incubated sample into a Lysing matrix E tube, and adding GB lysate for lysis; and (5) oscillating and crushing the MP wall breaking instrument, and centrifuging at low temperature.
Further, adding lysozyme with the final concentration of more than 1mg/mL into the pretreated sample in the step 2), and incubating for 10-15min at 35-37 ℃;
further, the sample after incubation in the step 2) is added into an MP Lysing matrix E tube, and a GB lysate is lysed; FastPrep-24 on MP wall breaking instrumentTMShaking for 1-2 times at 5-6m/s for 60-120s at 5G; centrifuging the MP lysine matrix E tube at 12,000-14,000g for 3-8min at low temperature, and storing for later use;
in some embodiments, the sample after incubation in step 2) is added to an MP Lysing matrix E tube and lysed with 200 μ L of GB lysate; FastPrep-24 on MP wall breaking instrumentTMShaking for 120s at 6m/s on 5G for 1 time; centrifuging 14,000g of MP lysine matrix E tube at low temperature for 5min, and storing for later use;
further, the centrifugation in the step 1) is 12,000-14,000g centrifugation for 3-5 min;
further, the step 1) of sample pretreatment comprises the following steps:
for a non-viscous infection sample, taking 1-2mL of the sample, centrifuging for 3-5min at 12,000-14,000g, sucking the supernatant, and reserving part of the supernatant and precipitates in a centrifuge tube for later use;
for viscous infection samples, 100-.
In some embodiments, the step 1) of sample pretreatment comprises:
taking 1-2mL of non-viscous infection sample, centrifuging at 14,000g for 5min, sucking out the supernatant, and reserving part of the supernatant and precipitates in a centrifuge tube for later use;
for viscous infection samples, 200. mu.L of the sample is added with a viscosity breaking agent or PBS, vortexed and shaken at 42 ℃ and 400rpm for 10min, then centrifuged at 14,000g for 5min, the supernatant is aspirated, and part of the supernatant and the precipitate are left in a centrifuge tube for standby.
Further, the non-viscous infection-like sample includes but is not limited to pleural effusion, alveolar lavage fluid, cerebrospinal fluid, urine, pus, non-viscous sputum-like sample and the like; the viscous infection-like sample comprises a sputum sample.
Further, the Lysing matrix E tube is MP BiomedicalstmLysine Matrix E tube.
The invention also provides a second generation infection metagenome library construction method, which is characterized by comprising any one of the treatment methods of the clinical sample pathogen, and further comprising the steps of nucleic acid extraction and library construction.
The invention also provides a clinical sample pathogen treatment kit applied to second-generation infection metagenome detection, which is characterized by comprising lysozyme, a Lysing Matrix E tube, GB lysate and a de-thickening agent.
Furthermore, the kit also comprises a nucleic acid extracting solution and the like.
The invention also provides application of the Lysing Matrix E tube in the treatment of second-generation clinical sample pathogens for infection metagenome detection.
Preferably, the lysine Matrix E is MP Biomedicalstm Lysing Matrix E。
The invention has the beneficial technical effects that:
(1) the clinical sample cell lysis method provided by the invention has low demand on samples and is beneficial to samples which are not easy to collect, such as cerebrospinal fluid and the like.
(2) The sample pretreatment method established by screening optimization has few flows, greatly shortens the treatment time, shortens the whole sequencing detection period, is less than one fourth of the period of the traditional method, and accelerates the report, thereby having significant significance in the industry and being particularly important for clinical severe infection patients.
(3) The method provided by the invention can be used for treating various clinical sample types, including hydrothorax, ascites, cerebrospinal fluid, pus, alveolar lavage fluid and peripheral blood, and is widely applicable.
(4) The clinical sample cell lysis method provided by the invention is suitable for detecting the second-generation sequencing metagenome and can be used for detecting DNA and RNA pathogenic microorganisms.
(5) The method provided by the invention has higher detection rate on pathogens with higher clinical attention such as fungi, gram-positive bacteria, intracellular bacteria and the like, and is more favorable for auxiliary clinical diagnosis.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 11087 sample shows the pre-detection 9 of fungus;
FIG. 2511 shows the rank of fungus detection in the sample;
FIG. 31087 is a schematic representation of sample before detection of bacteria;
fig. 4511 sample bacterial detection ranking.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention, and that the examples are a part of, but not all of the examples of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Definition of partial terms
Unless defined otherwise below, all technical and scientific terms used in the detailed description of the present invention are intended to have the same meaning as commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.
As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If in the following a certain group is defined to comprise at least a certain number of embodiments, this should also be understood as disclosing a group which preferably only consists of these embodiments.
The terms "about" and "substantially" in the present invention denote an interval of accuracy that can be understood by a person skilled in the art, which still guarantees the technical effect of the feature in question. The term generally denotes a deviation of ± 10%, preferably ± 5%, from the indicated value.
Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun.
Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
The invention relates to a method for treating clinical sample pathogens applied to second-generation infection metagenome detection, which comprises the following steps
1) Sample pretreatment:
centrifuging the infected sample, and reserving part of supernatant and sediment in a centrifuge tube for later use;
in some embodiments, the centrifugation is 12,000-14,000g centrifugation for 3-5 min;
in some embodiments, the sample pre-treatment step is:
for a non-viscous infection sample, taking 1-2mL of the sample, centrifuging for 3-5min at 12,000-14,000g, sucking the supernatant, and reserving part of the supernatant and precipitates in a centrifuge tube for later use;
for viscous infection samples, 100-.
In some embodiments, the sample pre-treatment step is:
taking 1-2mL of non-viscous infection sample, centrifuging at 14,000g for 5min, sucking out the supernatant, and reserving part of the supernatant and precipitates in a centrifuge tube for later use;
for viscous infection samples, 200. mu.L of the sample is added with a viscosity breaking agent or PBS, vortexed and shaken at 42 ℃ and 400rpm for 10min, then centrifuged at 14,000g for 5min, the supernatant is aspirated, and part of the supernatant and the precipitate are left in a centrifuge tube for standby.
2) And (3) sample bacteriolysis and wall breaking:
adding lysozyme with the final concentration of more than 1mg/mL into the pretreated sample for incubation;
adding the incubated sample into an MP Lysing matrix E tube, and Lysing GB lysate; oscillating and crushing by using an MP wall breaking instrument; centrifuging at low temperature for later use;
in some embodiments, the sample disruption step is specifically as follows:
adding lysozyme with the final concentration of more than 1mg/mL into the pretreated sample, and incubating at 35-37 ℃;
adding the incubated sample into an MP Lysing matrix E tube, and Lysing GB lysate; FastPrep-24 on MP wall breaking instrumentTMShaking for 60-120s at 6m/s on 5G for 1-2 times; centrifuging the MP lysine matrix E tube at 12,000-14,000g for 3-8min at low temperature, and storing for later use;
in some preferred embodiments, the sample wall breaking step is specifically as follows:
adding lysozyme with the final concentration of more than 1mg/mL into the pretreated sample, and incubating for 15min at 37 ℃;
adding the incubated sample into an MP Lysing matrix E tube, and adding 200 mu L of GB lysate; FastPrep-24 on MP wall breaking instrumentTMShaking for 120s at 6m/s on 5G for 1 time; centrifuging 14,000g of MP lysine matrix E tube at low temperature for 5min, and storing for later use;
in some embodiments, the non-viscous infection-like sample includes, but is not significantly affected by, pleural effusion, alveolar lavage, cerebrospinal fluid, urine, pus, non-viscous sputum-like samples, and the like. The viscous infection-like sample comprises a sputum sample.
The main reagent information used in the examples of the present invention is as follows:
the main instrument information used in the embodiment of the invention is as follows:
example 1 lysis Matrix E lysis tube lysis time optimization
The invention establishes MP Biomedicals by the early optimization of the cracking modetmThe Lysing Matrix E is the most suitable lysis means for next generation sequencing of metagenomic samples.
Further, the wall breaking conditions of the Lysing Matrix E lysis tube are tested and optimized, 3 conditions are selected for testing, and the conditions are as follows, wherein the conditions are as follows: shaking for 40s 2 times at 6 m/s; condition 2: shaking for 60s 2 times at 6 m/s; condition 3: shake 120s 1 times at 6 m/s. Comparing the advantages and disadvantages of the three conditions, repeating the conditions for 3 times, wherein the used sample is a spike in sample, commercial bacteria and virus stock solution is purchased, and the sample is mixed by using a simulated sample mechanism and then treated, and the specific operation steps are as follows:
1 sample pretreatment
1.1 prepared spike in samples were taken from 9 tubes, and centrifuged at 14,000g for 5min at 1 mL/tube. The supernatant was carefully aspirated, and 500. mu.L of the supernatant and pellet were kept in a centrifuge tube for use.
1.2 Add 5. mu.L of 20mg/mL lysozyme prepared (ready for use), incubate for 15min at 37 ℃.
1.3 the incubated sample was added to MP Lysing matrix E tube and 200. mu.L of day root GB lysate was added.
FastPrep-24 on 1.4 MP wall breaking instrumentTMSamples were treated on condition 1, condition 2, and condition 3, respectively, on 5G, with 3 samples per experimental group.
1.5 centrifuging 14,000g of MP lysine matrix E tube for 5min at low temperature, taking all supernatants after centrifugation, adding into a new 2mL EP tube for later use, and performing steps of nucleic acid extraction, library construction, on-machine sequencing and biogenesis analysis;
according to the test, it can be seen from the total amount of nucleic acid extraction that condition 2 ≈ condition 3 is more significant than condition 1, and this point is also consistent with the number of Reads detected by the microorganism, and as can be seen from table 2, the number of Listeria monocytogens Reads detected by conditions 2 and 3 is 2-3 times of that of condition 1, so that the nucleic acid release of conditions 2 and 3 is more significant, that is: shaking at 6m/s for 60s, 2 times or shaking at 6m/s for 120s, 1 time; however, the determination condition 3 is finally selected for subsequent test optimization in consideration of the portability of the operation.
TABLE 1 difference of total amount extracted under different MP wall breaking conditions
TABLE 2 difference of total amount extracted under different MP wall breaking conditions
Example 2: compared with the traditional wall breaking machine
Selecting two media of glass grinding beads (the diameter of the beads is 0.8-1 mm) and a lysine Matrix E cracking tube of an MP manufacturer as a mechanism for mechanically breaking the walls of cells of a clinical sample, taking the mechanism as a single variable, performing wall breaking, nucleic acid extraction, library construction, on-machine sequencing and signal generation analysis according to the process of the invention, comparing the detected advantages and disadvantages of the two media, performing repeated treatment on 2 samples by each wall breaking Matrix, wherein the used samples are spike in samples, purchasing commercial bacteria and virus stock solutions, mixing the samples by using a simulation sample mechanism, and performing treatment, wherein the treatment process of the lysine Matrix E cracking tube is as follows according to the condition 3 in the example 1:
1. spike in samples were taken 1mL and centrifuged at 14,000g for 5 min. The supernatant was carefully aspirated, and 500. mu.L of the supernatant and pellet were kept in a centrifuge tube for use.
2. mu.L of 20mg/mL lysozyme (ready for use) was added and incubated at 37 ℃ for 15 min.
3. The incubated sample was loaded with 0.5mL of prepared beads (beads were sterilized by moist heat (121 ℃,20 min), dried in an oven at 65 ℃ and used), followed by 200. mu.L of GB lysis buffer.
FastPrep-24 on MP wall breaking instrumentTMShake 120s 1 times at 6m/s on 5G.
5. Centrifuging 14,000g of the mixture subjected to wall breaking at low temperature for 5min, taking all supernatants after centrifugation, adding the supernatants into a new 2mL EP tube for later use, and performing nucleic acid extraction, library construction, on-machine sequencing and letter generation analysis;
as can be seen from Table 3, the two samples obtained by breaking the walls with grinding beads showed only the Reads numbers 1/2-1/3 for the MP wall-broken tube, and the other method A showed significantly lower Reads than the MP wall-broken tube of the present invention. In conclusion, the MP wall-breaking pipe has better wall-breaking effect, and the absolute copy number of target pathogens finally detected by the MP wall-breaking pipe is 2.8-3.1 times of that of glass beads.
TABLE 3 comparison of cell disruption effects of two different organisms
Example 3: test of influence of different lysozyme volumes on detection
The invention dilutes the lysozyme to 20mg/mL, then tests the influence of 5 muL, 10 muL, 15 muL and 20 muL of lysozyme on the detection of microorganisms, and uses the volume of the lysozyme as a single variable and adopts the lysine Matrix E as a wall breaking medium for comparative analysis. The used sample is one example of a sputum clinical sample with a nano-pore metagenome detection result, and each treatment group carries out two biological repeated detections. The lysozyme is diluted as follows: the Lysozyme enzyme activity of the invention is more than or equal to 5,000U/mg dw, solid powder, Lysozyme Buffer solution Lysozyme Buffer of 20mM Tris & Cl, pH 8.0; 2mM sodium EDTA; 1.2% Triton X-100 dilution, specifically: 20mg of lysozyme was dissolved in 1ml of Lysozyme Buffer to prepare a 20mg/ml lysozyme solution.
The specific processing operation steps are as follows:
1 sample pretreatment
1.1 respectively taking 200. mu.L of the sputum into 4 different 2mL round-bottom centrifuge tubes, respectively adding 1mL of the de-thickening agent, carrying out vortex oscillation, incubating at 42 ℃ and 400rpm for 10min, then centrifuging at 14,000g for 5min, carefully sucking off the supernatant, and leaving 500. mu.L of the supernatant and precipitating in the centrifuge tubes for later use.
1.2 two tubes of the de-thickened sample are added with 5 mul of 20mg/mL lysozyme (used as prepared), and the other two tubes of the de-thickened sample are added with 10 mul of 20mg/mL lysozyme (used as prepared), and incubated for 15min at 37 ℃.
1.3 the incubated sample was added to an MP Lysing matrix E tube, and 200. mu.L of GB lysate was added.
FastPrep-24 on 1.4 MP wall breaking instrumentTMShake 120s 1 times at 6m/s on 5G.
1.5 centrifuging 14,000g of MP lysine matrix E tube for 5min at low temperature, taking all supernatants after centrifugation, adding into a new 2mL EP tube for later use, and performing steps of nucleic acid extraction, library construction, on-machine sequencing and biogenesis analysis;
as can be seen from the following Table 4, when the volume of lysozyme is 5 μ L, there is a case that the pathogenic microorganism is missed, and when the volume of lysozyme is more than or equal to 10 μ L (the corresponding final concentration is more than 1mg/mL), all the pathogenic microorganisms can be detected normally, which is most suitable for detecting the pathogenic microorganism infected with the sample.
TABLE 4 comparison of different lysozyme systems
Example 4: verification of consistency of different methodologies
20 clinical samples with the detection result of the Nanopore metagenome are selected, 11 cases of alveolar lavage fluid, 4 cases of pus sample, 4 cases of sputum and 1 case of oral mucosa are selected, the storage time of the samples is within 1 month, and the storage condition is-15 to-25 ℃. All samples were processed against the protocol of the present invention and other method A, using the Nanopore detection as a standard.
The process of the invention is as follows:
1.1 sample pretreatment
a pretreatment of alveolar lavage fluid and pus sample
1mL of alveolar lavage fluid and 1mL of pus were collected in a 2mL centrifuge tube and centrifuged at 14,000g for 5 min. Carefully aspirate the supernatant and leave 500. mu.L of supernatant and pellet in the centrifuge tube
b sputum sample pretreatment
Adding 200 μ L of the sputum into a 2mL round-bottom centrifuge tube, adding 1mL of the viscosity breaking agent, performing vortex oscillation, incubating at 42 ℃ and 400rpm for 10min, then centrifuging at 14,000g for 5min, carefully sucking off the supernatant, and leaving 500 μ L of the supernatant and precipitating in the centrifuge tube for later use.
1.2 Add 10. mu.L of 20mg/mL lysozyme prepared (final concentration 1mg/mL) and incubate at 37 ℃ for 15 min.
1.3 the incubated sample was added to an MP Lysing matrix E tube, and 200. mu.L of GB lysate was added.
FastPrep-24 on 1.4 MP wall breaking instrumentTMShake 120s 1 times at 6m/s on 5G.
1.5 centrifuging 14,000g of MP lysine matrix E tube for 5min at low temperature, taking all supernatants after centrifugation, adding into a new 2mL EP tube for later use, and performing steps of nucleic acid extraction, library construction, on-machine sequencing and biogenesis analysis;
the other method A comprises the following detection processes:
1 sample pretreatment
1.1 pretreatment of alveolar lavage fluid, pus and sputum samples, wherein the process is the same as the process 1.1 of the invention;
1.2 Add prepared beads to the incubated sample [ beads need to be sterilized by moist heat (121 ℃,20 min), dried in oven at 65 ℃ and then used ] 0.5mL, then add 200. mu.L of GB lysis solution.
1.3 vortex mix on vortex mixer for 20 min.
1.4 centrifuging the mixture, adding all supernatants into a new 2mL EP tube for later use, and performing nucleic acid extraction, library construction, on-machine sequencing and letter generation analysis;
table 5 shows the comparison of the detection results in this example, it can be seen that the detection of 5 samples in total is superior to that of the other method a in the process of the present invention, and the detection rate is increased by 25%, and 5 samples in total have the detection of deep infectious fungi such as candida, pseudomonas aeruginosa, burkholderia cepacia, alcaligenes faecalis, and conditionally pathogenic bacteria.
TABLE 5 examination of target nanopores for the inventive and Enterprise Process A
In conclusion, the detection of the process of the invention is superior to other methods A. The metagenome clinical sample processing flow disclosed by the invention can ensure the effective second-generation sequencing detection rate, simplify the flow and shorten the processing time, and is particularly important for detecting severe clinical infection patients. Therefore, the invention has significant clinical value.
| Detection method | Experiment time | Detection rate | Convenience of operation |
| NGS-invention | 0.6h | Is higher than | Is higher than |
| NGS-other Process A | 2.5h | In general | In general |
Example 5 clinical multiple sample assay
The method of the invention is used for detecting clinical samples of alveolar lavage fluid, sputum, cerebrospinal fluid, blood and the like of 1087 clinically suspected infected patients. The results showed that the first four fungal species were detected as candida, aspergillus, sporothrix and cryptococcus, and the detection sequence was consistent with that described in the literature (Qing Miao et al 2018), that 16 types of samples suspected of acute or chronic infection, such as sputum, alveolar lavage fluid and blood, were detected in total in 511 clinical patients, and that the detected fungal species were ranked first three as aspergillus, candida and cryptococcus, and were consistent with that of the present invention. Of 1087 samples tested according to the present invention, the bacteria listed at the top 11 were Streptococcus pneumoniae, Haemophilus parainfluenza, Streptococcus pseudopneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Staphylococcus aureus, Corynebacterium wenshuni, Acinetobacter baumannii, Klebsiella pneumoniae, enterococcus faecium, stenotrophomonas maltophilia, enterococcus faecalis, and Pseudomonas putida, respectively, and the bacteria reported in the literature were found to be completely identical to 7 bacteria among the bacteria listed at the top 13, which were Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, enterococcus, Staphylococcus aureus, Haemophilus influenzae, and stenotrophomonas maltophilia.
The detection conditions of fungi in 1087 samples are shown in FIG. 1, which is consistent with that described in the literature, and the ranking of fungi detected in 511 samples in the literature is shown in FIG. 2; the detection of bacteria in 1087 samples is shown in FIG. 3, which is consistent with the detection described in the literature, and the ranking of bacteria detected in 511 samples in the literature is shown in FIG. 4. Therefore, the method can be applied to various clinical samples including but not limited to alveolar lavage fluid, sputum, cerebrospinal fluid, blood and the like, has real and reliable detection results, and is suitable for popularization and industrial application.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The method for treating the clinical sample pathogen applied to the detection of the second generation infection metagenome is characterized by comprising the following steps
1) Sample pretreatment: taking an infection sample, carrying out centrifugal treatment, and reserving part of supernatant and sediment;
2) and (3) sample bacteriolysis and wall breaking:
adding lysozyme with the final concentration of more than 1mg/mL into the pretreated sample for incubation;
adding the incubated sample into a Lysing matrix E tube, and adding GB lysate for lysis; and (5) oscillating and crushing the MP wall breaking instrument, and centrifuging at low temperature.
2. The method according to claim 1, wherein the sample is incubated at 35-37 ℃ for 10-15min after the pretreatment in step 2) with lysozyme added to a final concentration of greater than 1 mg/mL.
3. The process of any one of claims 1 to 2, wherein the sample after incubation in step 2) is added to an MP Lysing matrix E tube and lysed with GB lysate; FastPrep-24 on MP wall breaking instrumentTMShaking for 1-2 times at 5-6m/s for 60-120s at 5G; preferably, the MP Lysing matrix E tube 12,000-14,000g is centrifuged at low temperature for 3-8min and stored for further use.
4. The process according to any one of claims 1 to 3, wherein the centrifugation in step 1) is carried out at 14,000g for 3 to 5min at 12,000.
5. The treatment method as claimed in any one of claims 1 to 4, wherein the step of sample pretreatment in step 1) comprises:
for a non-viscous infection sample, taking 1-2mL of the sample, centrifuging for 3-5min at 12,000-14,000g, sucking the supernatant, and reserving part of the supernatant and precipitates in a centrifuge tube for later use;
for viscous infection samples, 100-.
6. The method of any one of claims 1-5, wherein the non-viscous infection-like sample includes, but is not limited to, pleural effusion, alveolar lavage, cerebrospinal fluid, urine, pus, non-viscous sputum-like samples; the viscous infection-like sample comprises a sputum sample.
7. A second generation infection metagenome library-building method comprising the processing method of any one of claims 1 to 6, and further comprising nucleic acid extraction and library-building steps.
8. A kit for treating clinical sample pathogens applied to second-generation infection metagenome detection, which is characterized in that,
including lysozyme, Lysing Matrix E tube, GB lysate and de-densifier.
9. The kit for the pathogen treatment of clinical samples for the detection of second-generation infection metagenome according to claim 8, wherein the kit further comprises a nucleic acid extracting solution.
Use of the Lysing Matrix E-tube in the treatment of secondary infection metagenomic detected clinical specimen pathogens.
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