CN116574782A - Lesion tissue sample dissociation host method applied to mNGS pathogen detection - Google Patents
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Abstract
The invention relates to a focus tissue sample dissociation host method applied to mNGS pathogen detection. The method comprises the following steps of S1, preparing a cell dehast enzyme system: dissolving the cell deHost mixed enzyme dry powder to 100mg/mL by adopting deHost buffer, filtering by adopting a 0.22 mu m membrane after dissolving, and storing filtrate in a brown EP tube at the temperature of-20 ℃ to be used as an enzyme stock solution; the cell deHost mixed enzyme dry powder contains casein enzyme, collagenase, trypsin and clostridium protease, and the activity of the mixed enzyme dry powder is more than or equal to 250U/mg; s2, preparing a focus tissue sample; s3, dissociation treatment; s4, removing the host. The invention realizes the cell dispersion of the tissue sample in the mNGS pathogen detection, improves the host removal effect, improves the detection performance of pathogenic bacteria, reduces the pathogen detection omission caused by high human occupation ratio, and improves the accuracy of mNGS sequencing data. The invention has simple operation flow, and can fully digest the tissue blocks into dispersed cell clusters or single cell states by adding the reagent incubation step, thereby efficiently realizing the host dissociation effect.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a focus tissue sample dissociation host method applied to mNGS pathogen detection, in particular to a treatment method for dissociating focus tissue blocks into smaller cell clusters or single cells.
Background
Metagenome sequencing (mNGS) is a novel technology capable of rapidly and deeply identifying infectious pathogens without culturing pathogenic microorganisms, and compared with the traditional culture method, the mNGS has higher sensitivity and is compatible with the concept of accurate diagnosis and treatment, and is an effective important tool in clinical virology. The mNGS can immediately obtain complete viral genetic information, thereby performing epidemiological monitoring of viruses or identifying specific mutations of viral resistance, being able to identify highly diverse viral genomes, rare pathogens, and finding viruses that are missing by targeted PCR, and using mNGS for metagenomic analysis is an effective method to determine the abundance of viral infection and identify the infection in a non-preferential manner.
The high-throughput sequencing does not depend on the separation and culture of traditional pathogens, does not need specific primers, can identify and type the pathogens by single operation, can detect unknown pathogens, realizes qualitative and quantitative description of the pathogens, and has incomparable advantages of the traditional etiology detection technology especially aiming at the aspects of fever caused by unknown reasons for a long time, negative conventional detection, poor empirical anti-infection treatment effect, difficult and serious infection and the like. The present mNGS is widely applied to diagnosis and treatment of various infectious diseases including respiratory system infection, central nervous system infection, blood system infection and the like, although the sensitivity is high compared with that of the traditional pathogen detection technology, in actual use, partial NGS results are negative in positive samples of clinical culture, so that false negative results appear, because most of microbial nucleic acids in the samples are influenced by host source background, usually, human source data can occupy more than 99% of the total data, a small amount of human source nucleated cells in the samples can completely submerge pathogen genetic materials with low abundance, in order to improve the coverage of pathogen genome sequences, sequencing depth is often required to be improved, the detection sensitivity is severely limited, and the data waste is caused. Therefore, elimination of host background nucleic acid interference is one of the most important links to increase pathogen detection sensitivity and further reduce detection costs.
Currently, common methods of decommissioning include: 1. the enzyme digestion method is to directly digest a sample with DNaseI and then carry out metagenome sequencing, and the result shows that the human sequence occupation ratio in the digested sample is slightly reduced, but the number of detected pathogenic microorganisms is not obviously increased; 2. the probe capturing method is characterized in that a reverse specificity probe is designed for capturing a human genome, and then the rest sample is used for detection, so that the method has high cost, long flow and non-ideal detection result; 3. the differential digestion method utilizes the difference of the cell structures of the human cells and the pathogenic microorganisms to selectively lyse host cells, and utilizes nuclease to digest released host DNA, and then performs enrichment and purification of the pathogenic microorganism DNA. The method has obvious effect on most of sputum, alveolar lavage fluid and other samples, but has poor performance in infected tissue samples, compared with liquid clinical samples, the tissue samples cannot completely lyse human cells due to the specificity of the tissue samples by directly using digestive reagents, and the size of human genome is about 3×10 9 While bacterial genomes are about 3 x 10 in size 6 The increase of the host nucleic acid ratio improves the detection of the human host background, greatly reduces the intracellular pathogenic microorganism ratio and the number of reads, and simultaneously improves the host cell ratio due to the invasion of immune cells in the tissue sample taken from the infected part. In addition, some tissues contain connective tissue, contractile proteins, collagen, etc., and are not subjected to special pretreatment, thus bringing about the subsequent nucleic acid extraction of pathogenic microorganismsGreat difficulty is encountered.
Therefore, a method for dispersing cells in a tissue sample with low cost and simple operation is needed, so that the tissue cells can be fully contacted with a lysis reagent, the proportion of human sequences is effectively reduced, and the sensitivity and the detection rate of detecting pathogenic microorganisms are improved.
Disclosure of Invention
The invention provides a focus tissue sample solution host method applied to mNGS pathogen detection for solving the technical problems in the prior art, overcomes the defects of the existing tissue sample solution host technology, is suitable for detecting mNGS pathogen microorganisms by using various focus tissue samples such as puncture tissues, tumor tissues and the like, and can reduce the proportion of host sources and improve the detection performance of the pathogen microorganisms after the samples are treated by adopting the method.
The invention adopts the technical proposal for solving the technical problems in the prior art that: a method for separating host from focus tissue sample for mNGS pathogen detection includes such steps as,
s1, preparing a celldbast enzyme system:
dissolving celldbuffer mixed enzyme dry powder to 100mg/mL, filtering with 0.22 μm membrane, and storing filtrate in brown EP tube at-20deg.C to obtain enzyme stock solution; the celldeHost mixed enzyme dry powder contains caseinase, collagenase, trypsin and clostridium protease, and the activity of the mixed enzyme dry powder is more than or equal to 250U/mg;
s2, preparing a focus tissue sample:
cutting focus tissue from the sampled tissue block in a biosafety cabinet by using a sterile blade, and transferring the cut focus tissue into an EP tube;
s3, dissociation treatment:
diluting the celldbuff enzyme stock solution into low-concentration working solution by adopting deHostbuffer, adding the working solution into an EP tube containing focus tissues, putting the EP tube into a metal bath for digestion for 20min, wherein the temperature condition of the metal bath is 37 ℃ and the rotating speed condition is 1000rpm, and after dissociation, the tissue blocks disappear and a small amount of floccules are present in the liquid;
s4, removing hosts:
200 mu L of liquid obtained by dissociation is subjected to instantaneous centrifugation, 100 mu L of clear liquid on the upper layer is removed, and 100 mu L of liquid containing flocculent components on the lower layer is obtained; and supplementing 106 mu L of PBS buffer solution into 100 mu L of the liquid, namely 206 mu L of the total system, adding a host removing reagent, uniformly mixing, incubating for 10min at 37 ℃ for carrying out the processes of human cell lysis, human gDNA release and digestion, and obtaining a product after host removal.
Preferably: the deHostbuffer contains NaCl 8.00-8.05 mg/mL and Na 2 HPO 4
0.05～0.06mg/mL、KCl0.39～0.42mg/mL、KH 2 PO 4 0.05～0.06mg/mL、MgSO 4 0.09～0.11mg/mL、CaCl 2 0.13-0.14 mg/mL, 0.98-1.03 mg/mL of D-glucose, and NaHCO 3 0.33-0.36 mg/mL, and the pH value of the buffer solution after preparation is 7.2-7.4.
Preferably: the concentration of the diluted working solution in the step S3 is 10-20 mg/mL.
The invention has the advantages and positive effects that:
the invention can realize the cell dispersion of the tissue sample in the mNGS pathogen detection, improve the host removal effect, improve the detection performance of pathogenic bacteria, reduce the pathogen detection omission caused by high human occupation ratio and improve the accuracy of mNGS sequencing data. In the current operation mode of directly cutting out tissue blocks for experiments, pathogenic microorganisms are wrapped in the tissues and cannot be obtained, and the condition that the pathogenic microorganisms are missed after sequencing exists. In the host removing method, tissue dissociation is carried out, so that tissue blocks are digested into single cells to the greatest extent, pathogenic microorganisms attached to the tissue cells are exposed, and a better pathogenic microorganism detection effect is achieved.
The method has simple operation flow, and can efficiently realize the host dissociation effect by adding the reagent incubation step of 20min at 37 ℃ and 1000rpm before the host dissociation operation of the focus tissue sample, so that the tissue blocks are fully digested into dispersed cell clusters or single cell states, and the method has good popularization prospect.
Detailed Description
In order to further understand the summary, features and advantages of the present invention, the following examples are set forth in detail.
Example 1
The embodiment shows that the cell dissociation enzymes with different working solution concentrations have positive effects on tissue extraction and library construction results in the process of applying the cell dissociation enzymes to the dissociation hosts of focus tissue samples in the field of mNGS pathogen detection.
And S1, preparing a celldbast enzyme system.
The celldbuffer was used to dissolve the celldbast mixed enzyme dry powder to a concentration of 100mg/mL, after dissolution, the solution was filtered using a 0.22 μm membrane and the filtrate was kept in a brown EP tube at a temperature of-20 ℃ as enzyme stock.
Wherein the celldeHost mixed enzyme dry powder contains casein enzyme, collagenase, trypsin and clostridium protease, and the activity of the mixed enzyme dry powder is more than or equal to 250U/mg.
Wherein the components and the content of the deHostbuffer are NaCl 8.00-8.05 mg/mL and Na 2 HPO 4 0.05～0.06mg/mL、KCl0.39～0.42mg/mL、KH 2 PO 4 0.05～0.06mg/mL、MgSO 4 0.09～0.11mg/mL、CaCl 2
0.13-0.14 mg/mL, 0.98-1.03 mg/mL of D-glucose, and NaHCO 3 0.33-0.36 mg/mL, and the pH value of the buffer solution after preparation is 7.2-7.4. The preferable scheme is as follows: deHostbuffer contains NaCl8.00mg/mL and Na 2 HPO 4 0.05mg/mL、KCl0.41mg/mL、KH 2 PO 4 0.06mg/mL、MgSO 4 0.10mg/mL、CaCl 2 0.14mg/mL, D-glucose 1.00mg/mL, naHCO 3 The pH of the buffer after preparation was 7.3 at 0.35 mg/mL.
Step S2, preparing a focus tissue sample.
Placing focus tissue sample of clinical infectious disease patient into culture dish, cutting 1.5mm from sampled tissue block on culture dish with aseptic blade in secondary biosafety cabinet 3 Is used for experiments, and is cut with a blade for 3 to 5 times to minimize the volume of the focal tissue mass, and the cut focal tissue is transferred to1.5mL of the EP tube.
And S3, dissociation processing.
Firstly, preparing a celldeHost enzyme working solution: taking celldbehest enzyme stock solution, melting the enzyme stock solution at room temperature, and diluting the enzyme stock solution into working solutions with enzyme concentration of 10mg/mL and 20mg/mL respectively by using deHostbuffer in an ultra-clean workbench.
Adding 200 μL of working solution into 1.5mLEP tube containing focus tissue, and performing metal bath digestion at 37deg.C and 1000rpm for 20min; after dissociation the tissue mass disappeared and a small amount of filiform material was present in the liquid.
S4, removing the host.
200. Mu.L of the liquid obtained by the dissociation was subjected to instantaneous centrifugation, and 100. Mu.L of the supernatant was removed to obtain 100. Mu.L of the liquid containing the flocculent component as the lower layer. In general, the human cells are in a discrete state in a large quantity after the tissue blocks are dissociated, so that only 100 mu L of the human cells are reserved for experiments, and the detection of pathogenic microorganisms is prevented from being influenced by the excessive human cells.
Adding 106 mu L of PBS buffer solution into the obtained 100 mu L of liquid containing flocculent components, namely 206 mu L of total system, adding a host removing reagent, uniformly mixing, incubating for 10min at 37 ℃ for human cell lysis and human gDNA release and digestion processes, and obtaining a host removed product, which is a mixed liquid.
Among them, PBS buffer, phosphate buffered saline, commercial reagent brand is "Life science", product number "F2209".
Wherein, the host removing reagent is selected as a reagent component in a host removing kit, the brand of the commercial kit is gold key medicine, and the product number is 2007-01 (Jinjie preparation 20210385).
And supplementing 1mL of PBS buffer solution into the product mixture after the host removal, uniformly mixing, centrifuging for 3min under the centrifugal condition of 12000rpm, removing supernatant to keep layer precipitation, and completing the collection of pathogenic organisms.
The pathogenic microorganism sediment is supplemented with 800 mu L of PBS buffer solution, the solution is placed into a LysingMatrixE wall breaking tube, and physical wall breaking is carried out by using 'key medical' sample pretreatment system equipment (Jinqianzhi 20210673) to complete the microorganism physical wall breaking operation. After breaking the wall, performing instantaneous centrifugation, taking 600 μl of liquid, and extracting nucleic acid of pathogenic microorganism with magnetic bead method nucleic acid extraction kit (DNA/RNA) (product No. 20210111, product No. 2005-01). The extracted nucleic acid can be used for operations such as library building, sequencing and the like.
Control group settings: the control group does not perform tissue dissociation enzyme action, directly performs extraction and library establishment after the conventional treatment of hosts according to the dissociation of the gold key tissue sample;
the above control group and test group were each provided with 2 technical replicates, and the results are shown in table 1.
TABLE 1 Effect of cell dissociating enzymes on tissue sample extraction and library construction
Note that: library amplification efficiency = (library concentration × 40/initial amount of library construction)/(1/cycle number) -1
As can be seen from table 1, the test group applies cell dissociation enzyme to the pretreatment of tissue sample dissociation host for tissue cell dispersion, so that the total cell number, the ex-warehouse concentration and the amplification efficiency can be improved; the extraction concentration can be obviously reduced: the effect of the subsequent experiment to remove the host can be enhanced by dissociating the tissue into single cells or more clusters, which is manifested by a decrease in the extraction concentration. Overall, the positive effect on the host for dissociation of the tissue sample is demonstrated at a tissue dissociation enzyme concentration of 10mg/mL and 20mg/mL.
Example two
The embodiment illustrates that the cell dissociation enzyme with the same concentration is applied to focus tissue samples in the field of mNGS pathogen detection, different components are selected for extraction, library construction and sequencing after dissociation, and the dissociation has positive effects on pathogen detection and the like.
Focal tissue sample preparation: focal tissue samples were taken from 2 clinically infected patients, and the remaining experimental procedures and conditions remained consistent with the examples.
Firstly, preparing a celldeHost enzyme working solution: taking celldbehest enzyme stock solution, melting the enzyme stock solution at room temperature, and diluting the enzyme stock solution into working solution with the enzyme concentration of 20mg/mL by using deHostbuffer in an ultra-clean workbench;
dissociation treatment: adding 200 μL of working solution into 1.5mLEP tube containing focus tissue, and performing metal bath digestion at 37deg.C and 1000rpm for 20min; transferring the dissociated liquid into a new 1.5mLEP tube, extracting, building a library and sequencing the dissociated liquid and the precipitate which are not dissociated fully according to the conventional treatment of the host for dissociation of the tissue sample of the 'key medical' respectively;
wherein the control group and the test group are not provided with repeated technology, and the rest experimental operation and conditions are consistent with the examples; the results are shown in tables 2 and 3.
TABLE 2 influence of cell dissociating enzymes on tissue sample extraction, pooling and detection
TABLE 3 influence of cell dissociating enzymes on pathogen detection performance of tissue samples
As can be seen from table 2 and table 3, before the test group applies the cell dissociation enzyme to the tissue sample dissociation host experiment, different components are selected for experiment after dissociation, and the reduction of the extraction concentration and the reduction of the human source indicate that the host removal effect is better after the tissue dissociation; the number of detected species, the microorganism ratio, the pathogen detection performance and the like are all superior to those of a control group. In conclusion, the cell dissociation enzyme has positive effect on the dissociation host of the tissue sample, and the pathogen detection performance is obviously improved.
Example III
The embodiment illustrates that the cell dissociation enzyme with the same concentration is applied to focus tissue samples in the field of mNGS pathogen detection, dissociation liquid and sediment mixed components are taken for extraction, library establishment and sequencing after dissociation, and the dissociation enzyme has positive effects on pathogen detection and the like.
Focal tissue sample preparation: taking focus tissue samples of 5 clinical infectious patients, and keeping the rest experimental operation and conditions consistent with those of the examples;
firstly, preparing a celldeHost enzyme working solution: taking celldbehest enzyme stock solution, melting the enzyme stock solution at room temperature, and diluting the enzyme stock solution into working solution with the enzyme concentration of 20mg/mL by using deHostbuffer in an ultra-clean workbench;
dissociation treatment: 200. Mu.L of working solution was added to a 1.5mLEP tube containing focal tissue, and subjected to metal bath digestion at 37℃and 1000rpm for 20min. In the second embodiment, the detection performance of each single component pathogen after dissociation is obviously improved, so the embodiment discards 100 mu L of dissociation liquid, and extracts and builds a warehouse after the dissociation host is conventionally processed according to the 'key medical' tissue sample by using the rest liquid and the sediment mixed components;
wherein, the control group and the test group are provided with two repeated technologies, and the rest experimental operation and conditions are consistent with the examples; the results are shown in tables 4 and 5.
TABLE 4 influence of cell dissociating enzymes on tissue sample extraction, pooling and detection
TABLE 5 influence of cell dissociating enzymes on pathogen detection performance of tissue samples
As can be seen from tables 4 and 5, before the cell dissociation enzyme is applied to the tissue sample to dissociate the host, the test group keeps part of the liquid and the mixed components of the sediment for experiment after dissociation, and the concentration of the extracted liquid is reduced, the reduction of human sources indicates that the host removal effect is better after the tissue dissociation; the number of detected species, the microorganism ratio, the pathogen detection performance and the like are all increased, which indicates that the cell dissociation enzyme has positive action on the tissue sample dissociation host, and the pathogen detection performance is obviously improved.
In summary, the three examples illustrate that positive effects can be obtained with different celldbast enzyme concentrations, and different components after dissociation for the de-hosting experiments.
The above-described embodiments are provided for illustrating the technical idea and features of the present invention, and are intended to enable those skilled in the art to understand the present invention and to implement the same, and the scope of the present invention is not limited to the embodiments, i.e., the disclosed functions and equivalent changes or modifications are still within the scope of the present invention.
Claims (3)
1. A focus tissue sample dissociation host method applied to mNGS pathogen detection is characterized in that: comprises the steps of,
s1, preparing a cell dehast enzyme system:
dissolving the cell deHost mixed enzyme dry powder to 100mg/mL by adopting deHost buffer, filtering by adopting a 0.22 mu m membrane after dissolving, and storing filtrate in a brown EP tube at the temperature of-20 ℃ to be used as an enzyme stock solution; the cell deHost mixed enzyme dry powder contains casein enzyme, collagenase, trypsin and clostridium protease, and the activity of the mixed enzyme dry powder is more than or equal to 250U/mg;
s2, preparing a focus tissue sample:
cutting focus tissue from the sampled tissue block in a biosafety cabinet by using a sterile blade, and transferring the cut focus tissue into an EP tube;
s3, dissociation treatment:
diluting a cell deHost enzyme stock solution into a low-concentration working solution by adopting a deHost buffer, adding the working solution into an EP tube containing focus tissues, putting the EP tube into a metal bath for digestion for 20min, wherein the temperature condition of the metal bath is 37 ℃ and the rotating speed condition is 1000rpm, and after dissociation, the tissue blocks disappear and a small amount of floccules are present in the liquid;
s4, removing hosts:
200 mu L of liquid obtained by dissociation is subjected to instantaneous centrifugation, 100 mu L of clear liquid on the upper layer is removed, and 100 mu L of liquid containing flocculent components on the lower layer is obtained; and supplementing 106 mu L of PBS buffer solution into 100 mu L of the liquid, namely 206 mu L of the total system, adding a host removing reagent, uniformly mixing, incubating for 10min at 37 ℃ for carrying out the processes of human cell lysis, human gDNA release and digestion, and obtaining a product after host removal.
2. The method for solving a host by using a focus tissue sample for detecting mNSS pathogen according to claim 1, wherein: the components and the content of the deHostbuffer are 8.00-8.05 mg/mL NaCl and Na 2 HPO 4 0.05～0.06mg/mL、KCl 0.39～0.42mg/mL、KH 2 PO 4 0.05～0.06mg/mL、MgSO 4 0.09～0.11mg/mL、CaCl 2
0.13-0.14 mg/mL, 0.98-1.03 mg/mL of D-glucose, and NaHCO 3 0.33-0.36 mg/mL, and the pH value of the buffer solution after preparation is 7.2-7.4.
3. A lesion tissue sample dissociation host method for use in mNGS pathogen detection according to claim 2, wherein: the concentration of the diluted working solution in the step S3 is 10-20 mg/mL.
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