Eukaryotic single cell and detection method and application of eukaryotic single cell and bacteria carried by

Abstract

The invention discloses a detection method of eukaryotic single cells and bacteria carried by the eukaryotic single cells and application of the detection method. The detection method comprises the following steps: providing pretreated eukaryotic single cells and bacteria carried by the eukaryotic single cells; providing a multiple reverse transcription primer system, and carrying out reverse transcription reaction on the multiple reverse transcription primer system, the pretreated eukaryotic single cell and the bacteria carried by the eukaryotic single cell to obtain a reverse transcription product; providing a microbead which is provided with a cell label, mixing the microbead with the reverse transcription product, and carrying out a ligation reaction so as to label the cell label on the reverse transcription product, thereby obtaining a ligation product; performing library construction on the connection product to obtain a single cell transcriptome library and a 16S rRNA library, and sequencing to obtain interaction information between eukaryotic single cells and bacteria carried by the eukaryotic single cells; host cell RNA and intracellular bacteria rRNA are subjected to efficient reverse transcription through a multiple reverse transcription primer system, and a host single cell transcriptome library and a 16S variable region library are constructed respectively, so that simultaneous detection of host cells and intracellular bacteria is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene sequencing and tissue cell sample analysis, in particular to a eukaryotic single cell and a detection method of the eukaryotic single cell carrying bacteria and application thereof. BACKGROUND

[0002] In the tumor of a cancer patient, tumor-associated bacteria play a role in cancer development, metastasis, immune surveillance and drug resistance, and there is strong molecular evidence that bacteria exist in tumors in at least 33 major cancer types, and imaging data shows co-localization of pan-bacterial markers with immune and epithelial cell targets, so it is important to identify which host cells carry bacteria, bacterial species and their regulatory effects on host transcription programs at single-cell resolution, for example, how bacterial communities in the tumor microenvironment affect immune suppression or malignant cell proliferation.

[0003] However, due to the special cell wall of bacteria and the absence of a polyA tail in their 16S rRNA, traditional eukaryotic single-cell transcriptome sequencing scRNA-seq methods based on oligo-dT transcriptome primers cannot capture the bacterial species inside the cell, so simultaneous detection of host cells and intracellular bacteria cannot be achieved. SUMMARY

[0004] The main purpose of the present application is to provide a eukaryotic single cell and a detection method of the eukaryotic single cell carrying bacteria and application thereof, aiming to solve the problems that simultaneous detection of host cells and intracellular bacteria cannot be achieved in the existing eukaryotic single-cell transcriptome sequencing method and the proportion of effective data in the prepared 16S library is extremely small.

[0005] To achieve the above-mentioned purpose, the present application provides a detection method of a eukaryotic single cell and the eukaryotic single cell carrying bacteria, comprising the following steps:

[0006] S10, providing a pretreated eukaryotic single cell and the eukaryotic single cell carrying bacteria;

[0007] S20, providing a multiplex reverse transcription primer system, which is subjected to a reverse transcription reaction with the pretreated eukaryotic single cell and the eukaryotic single cell carrying bacteria to obtain a reverse transcription product;

[0008] S30, providing a microbead having a cell tag, which is mixed with the reverse transcription product to perform a ligation reaction, so as to label the cell tag on the reverse transcription product to obtain a ligation product;

[0009] S40, constructing a library for the ligation product to obtain a single-cell transcriptome library and a 16S rRNA library, and sequencing to obtain interaction information between the eukaryotic single cell and the eukaryotic single cell carrying bacteria.

[0010] In an embodiment, before step S10, further comprising:

[0011] fixing, permeabilizing the eukaryotic single cells to obtain a eukaryotic single cell suspension;

[0012] lysozyme and lysostaphin are added to digest the bacterial cell wall in the cells to obtain pre-processed eukaryotic single cells and bacteria carried by the eukaryotic single cells.

[0013] In an embodiment, the mass ratio of lysozyme to lysostaphin is 1:0.1-4.

[0014] In an embodiment, in step S20, the multiple reverse transcription primer system comprises an oligo-dT primer, a semi-random primer, and a 16S rRNA constant region specific primer.

[0015] In an embodiment, the semi-random primer comprises a first semi-random primer or a second semi-random primer, and the 16S rRNA constant region specific primer comprises a first 16S rRNA constant region specific primer or a second 16S rRNA constant region specific primer, wherein:

[0016] the sequence of the oligo-dT primer is shown in SEQ ID NO. 1; and / or,

[0017] the sequence of the first semi-random primer is shown in SEQ ID NO. 2; and / or,

[0018] the sequence of the second semi-random primer is shown in SEQ ID NO. 3; and / or,

[0019] the sequence of the first 16S rRNA constant region specific primer is shown in SEQ ID NO. 4; and / or,

[0020] the sequence of the second 16S rRNA constant region specific primer is shown in SEQ ID NO. 5.

[0021] In an embodiment, in step S40, the constructing step of the single cell 16S rRNA library comprises:

[0022] using a Read1 Sequencing Primer to perform stepwise PCR amplification with different 16S constant region amplification primers to obtain a single cell 16S rRNA library.

[0023] In an embodiment, the stepwise PCR amplification comprises a first round of PCR amplification and a second round of PCR amplification; wherein the sequence of the 789F primer used in the first round of PCR amplification is shown in SEQ ID NO. 6, and the sequence of the Rd2P-967F primer used in the second round of PCR amplification is shown in SEQ ID NO. 7.

[0024] The present application also provides a sequencing library constructed by the high-throughput single cell and the method for detecting bacteria carried thereby as described above.

[0025] The present application also provides an application of the eukaryotic single cell and the sequencing library constructed by the method for detecting bacteria carried thereby as described above in the field of host-microorganism interaction.

[0026] In the technical solution of the present application, the host cell RNA and intracellular bacterial rRNA can be efficiently reverse transcribed by the multiplex reverse transcription primer system, and the microbeads and the reverse transcription products are mixed by microdroplets, so that the reverse transcription products from the host cell and the bacterial 16S rRNA can be effectively labeled with cell tags in the microdroplets, and then the host single cell transcriptome library and the 16S variable region library are constructed, respectively, so as to realize the simultaneous detection of the host cell and the intracellular bacteria, and the present application is suitable for the host-microorganism interaction research of complex biological systems such as tumor microenvironment and infectious diseases. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the drawings shown.

[0028] Figure 1 . The single cell transcriptome and intracellular 16S detection technical flowchart of the present application;

[0029] Figure 2 . The schematic diagram of the proportion of UMIs matched to positive bacteria (Mycobacterium) to all bacterial 16S UMIs in different experimental schemes;

[0030] Figure 3 . Comparison diagram of the average number of UMIs detected in the top ten Raw cells according to Mycobacterium UMI detection in four experimental schemes;

[0031] Figure 4 . Schematic diagram of the distribution of Mycobacterium UMI in different cell clusters;

[0032] Figure 5 . Comparison of the average UMI detection number of the top ten Raw cells detected by M. smegmatis UMI in the process of this application and INVADEseq technology.

[0033] The purposes, functional features and advantages of the present application will be further described with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION

[0034] In order to make the purposes, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be clearly and completely described below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturers are adopted. If the reagents or instruments used are not specified by the manufacturers, they are all conventional products that can be purchased on the market. In addition, the meaning of "and / or" appearing in the full text includes three parallel solutions. Taking "A and / or B" as an example, it includes the A solution, or the B solution, or the solution of A and B at the same time. In addition, the technical solutions of each embodiment can be combined with each other, but it must be based on the fact that a person skilled in the art can realize it. When the combination of technical solutions appears contradictory or unachievable, it should be considered that the combination of technical solutions does not exist and is not within the scope of protection required by the present application. Based on the embodiments in the present application, all other embodiments obtained by a person skilled in the art without creative labor are within the scope of protection of the present application.

[0035] In the tumors of cancer patients, tumor-associated bacteria play a role in cancer development, metastasis, immune surveillance and drug resistance. There is strong molecular evidence that bacteria exist in tumors in at least 33 major cancer types, and imaging data shows the co-localization of pan-bacterial markers with immune and epithelial cell targets. It is important to identify which host cells carry bacteria, bacterial species and their regulatory effects on host transcriptional programs at single-cell resolution, such as how bacterial communities in the tumor microenvironment affect immune suppression or malignant cell proliferation.

[0036] However, due to the special cell wall of bacteria and the absence of polyA tail in their 16S rRNA, traditional eukaryotic single-cell transcriptome sequencing scRNA-seq methods based on oligo-dT transcriptome primers cannot capture the bacterial species inside the cells, so simultaneous detection of host cells and intracellular bacteria cannot be achieved.

[0037] In view of this, the present application provides a method for detecting eukaryotic single cells and bacteria carried thereby, comprising the following steps:

[0038] S10, providing pre-processed eukaryotic single cells and bacteria carried thereby;

[0039] S20, providing a multiplex reverse transcription primer system, which is used for reverse transcription reaction with the pretreated eukaryotic single cell and the bacteria carried by the eukaryotic single cell, to obtain a reverse transcription product;

[0040] S30, providing microbeads with a cell tag, and mixing the microbeads with the reverse transcription product to perform a ligation reaction, so as to label the cell tag on the reverse transcription product, to obtain a ligation product;

[0041] S40, performing library construction on the ligation product, to obtain a single cell transcriptome library and a 16S rRNA library, and performing sequencing, to obtain interaction information between the eukaryotic single cell and the bacteria carried by the eukaryotic single cell.

[0042] In the present application, the host cell RNA and the intracellular bacterial rRNA can be efficiently reverse transcribed by using a multiplex reverse transcription primer system, and the cDNA from the host cell and the bacterial 16S in the reverse transcription product can be effectively labeled with a cell tag in a microdroplet by mixing the microbeads with the reverse transcription product, and then the host single cell transcriptome library and the 16S rRNA variable region library are constructed respectively, so that the host cell and the intracellular bacteria can be simultaneously detected, and the present application is suitable for host-microorganism interaction research in complex biological systems such as tumor microenvironment and infectious diseases.

[0043] It should be noted that in the present application, the microdroplets are formed by using a microfluidic system, each droplet contains only one cell and the bacteria carried by the cell and one microbead containing the same cell tag, and the cDNA is labeled with a cell tag in the droplet, so that the eukaryotic cDNA from the same cell source and the bacterial 16S rRNA carried by the eukaryotic cDNA have the same cell tag, and the eukaryotic cDNA from different cell sources and the bacterial 16S rRNA carried by the eukaryotic cDNA have different cell tags, so that the original cell source can be accurately traced in subsequent data analysis.

[0044] Specifically, as shown in Figure 1 The detection step of the eukaryotic single cell and the bacteria carried by the eukaryotic single cell is as follows:

[0045] Step 1: fixing the eukaryotic single cell, permeabilizing the host cell membrane, and then using lysozyme and lysostaphin to further permeabilize the intracellular bacterial cell wall;

[0046] Step 2: in situ reverse transcription (intracellular RT) to generate cDNA: using specially designed oligo-dT primers, semi-random primers and specific primers for 16S constant region to reverse transcribe the RNA in the host cell and the bacterial 16S rRNA, respectively;

[0047] Step 3: providing microdroplets, and performing cell tag labeling on the cDNA in the microdroplets, wherein the microfluidic device encapsulates the tens of thousands of single cells and the bacteria carried by the single cells in different microdroplets respectively, and each droplet also encapsulates microbeads carrying cell tags;

[0048] Step 4: constructing a host single cell transcriptome library and a 16S variable region library respectively after oil breaking, wherein the single cell 16S variable region library adopts a ladder PCR enrichment scheme, and relatively pure 16S sequences can be obtained through two rounds of specific incremental PCR amplification.

[0049] In an embodiment, before step S10, the method further comprises:

[0050] Fixing and permeabilizing the eukaryotic single cells to obtain an eukaryotic single cell suspension;

[0051] Adding lysozyme and lysostaphin to digest the bacterial cell wall in the cells to obtain pretreated eukaryotic single cells and bacteria carried by the eukaryotic single cells.

[0052] In this embodiment, the fixation is to prevent the cells from breaking or losing contents in subsequent operations, and to maximize the preservation of cell morphology and subcellular structures. The permeabilization is to allow the subsequent added enzymes (reverse transcriptase, lysozyme, etc.) to enter the cell interior, so as to facilitate the action on the targets such as bacterial cell wall, mRNA, 16S rRNA, etc. The present application performs double permeabilization on the host cells and the bacteria carried by the host cells, so that the subsequent multiple reverse transcription primer system can efficiently reverse transcribe the host cell RNA and intracellular bacterial rRNA.

[0053] Further, the mass ratio of lysozyme to lysostaphin is 1:0.1-4. The lysozyme and lysostaphin are added to the permeabilized cells to destroy the bacterial cell wall, so as to facilitate the efficient performance of the subsequent reverse transcription reaction and ligation reaction. The use of multiple enzymes in cooperation can effectively lyse the cell walls of different types of bacteria. The mass ratio of the two enzymes is 1:0.1-4, and the lysis effect is better within this range.

[0054] In an embodiment, the multiple reverse transcription primer system comprises an oligo-dT primer, a semi-random primer, and a 16S rRNA constant region specific primer. The oligo-dT primer, the semi-random primer, and the 16S rRNA constant region specific primer are added to the reverse transcription reaction system at the same time, and the specific description is as follows:

[0055] Oligo-dT primer: binds to mRNA containing a polyA tail to synthesize a first strand of cDNA (i.e., a classic transcriptome);

[0056] Semi-random primer: binding to any position of RNA, used for non-selective capture of all RNA (including some eukaryotic RNA without polyA, viral RNA, certain IncRNA, rRNA, etc.);

[0057] 16S rRNA constant region specific primer: specifically recognizing the conserved region of bacterial 16S rRNA, used for specifically reverse transcribing bacterial RNA.

[0058] Further, the semi-random primer comprises a first semi-random primer or a second semi-random primer, and the 16S rRNA constant region specific primer comprises a first 16S rRNA constant region specific primer or a second 16S rRNA constant region specific primer, wherein:

[0059] the sequence of the oligo-dT primer is shown in SEQ ID NO. 1; and / or,

[0060] the sequence of the first semi-random primer is shown in SEQ ID NO. 2; and / or,

[0061] the sequence of the second semi-random primer is shown in SEQ ID NO. 3; and / or,

[0062] the sequence of the first 16S rRNA constant region specific primer is shown in SEQ ID NO. 4; and / or,

[0063] the sequence of the second 16S rRNA constant region specific primer is shown in SEQ ID NO. 5.

[0064] Specifically, the sequence of the oligo-dT primer is shown in SEQ ID NO. 1, and the specific sequence of the oligo-dT primer is as follows:

[0065] CGTCCGTCGTTGCTCGT-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN;

[0066] the sequence of the first semi-random primer is shown in SEQ ID NO. 2, and the specific sequence of the first semi-random primer is as follows: CGTCCGTCGTTGCTCGT-NNNNNNNNNNGGGG;

[0067] the sequence of the second semi-random primer is shown in SEQ ID NO. 3, and the specific sequence of the second semi-random primer is as follows: CGTCCGTCGTTGCTCGT-NNNNNNNNNNTTTT;

[0068] The sequence of the first 16S rRNA constant region specific primer is shown as SEQ ID NO. 4, and the specific sequence of the first 16S rRNA constant region specific primer is as follows:

[0069] CGTCCGTCGTTGCTCGT-TCACGRCACGAGCTGACGAC;

[0070] The sequence of the second 16S rRNA constant region specific primer is shown as SEQ ID NO. 5, and the specific sequence is as follows:

[0071] CGTCCGTCGTTGCTCGT-GGGTTGCGCTCGTTG.

[0072] In an embodiment, in step S40, the constructing step of the single-cell 16S rRNA library comprises:

[0073] The single-cell 16S library is obtained by using Read1 SeqPrimer to perform stepwise PCR amplification with different 16S constant region amplification primers, and the eukaryotic single-cell transcriptome library and the single-cell 16S rRNA library are constructed respectively.

[0074] It can be understood that the obtained ligation product is subjected to PCR amplification, and different primer combinations are used for enrichment, the eukaryotic transcriptome library uses Read1 SeqPrimer and template switching sequence primer TSO, and the bacterial 16S rRNA library uses Read1 SeqPrimer and different 16S constant region amplification primers. The use of different 16S constant region amplification primers for multi-step stepwise PCR can improve the capture efficiency of 16S rRNA, avoid the eukaryotic transcriptome data from covering the bacterial signal, allow the use of more sensitive 16S special primers for amplification, and at the same time retain the cell tag information. Finally, the bacterial species and the corresponding host cells can be paired and analyzed by a calculation method, thereby expanding the effective data in the 16S library and improving the sensitivity of the bacterial 16S variable region.

[0075] In an embodiment, the stepwise PCR amplification comprises a first round of PCR amplification and a second round of PCR amplification; wherein the sequence of the 789F primer used in the first round of PCR amplification is shown as SEQ ID NO. 6, and the sequence of the Rd2P-967F primer used in the second round of PCR amplification is shown as SEQ ID NO. 7.

[0076] Specifically, the sequence of the 789F primer is shown as SEQ ID NO. 6, and the specific sequence is as follows:

[0077] TAGATACCCSSGTAGTCC;

[0078] The sequence of the Rd2P-967F primer is shown in SEQ ID NO.7, and its specific sequence is as follows:

[0079] GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-CAACGCGAAGAAC CTTACC.

[0080] Understandably, the single-cell 16S variable region library uses a stepwise PCR enrichment scheme. Through two rounds of specific incremental PCR amplification, a relatively pure 16S sequence can be obtained, which improves the effective data in the prepared 16S library and thus enhances the sensitivity of the bacterial 16S variable region.

[0081] This invention provides a sequencing library, comprising a sequencing library constructed using the high-throughput single-cell and bacteria-carrying detection method described above. This sequencing library possesses all the technical solutions of the detection method for the sequencing library constructed using the high-throughput single-cell and bacteria-carrying detection method, and therefore has all the beneficial effects of the detection method for the sequencing library constructed using the high-throughput single-cell and bacteria-carrying detection method. These will not be elaborated upon further here.

[0082] The present invention also provides an application of the sequencing library described above in the field of host-microbe interaction.

[0083] Example 1: Effects of cell wall permeabilizing enzyme treatment and different primer schemes on the detection of 16S cells in eukaryotic single cells

[0084] 1.1 Cell fixation treatment

[0085] One × 10^6 sterile human peripheral blood mononuclear cells (PBMCs) and mouse RAW264.7 macrophage cell line (ATCCTIB-71) infected with Mycobacterium smegmatis (ATCC 23037) for 48 hours were fixed with PBS buffer (pH 7.4) containing 1% paraformaldehyde at room temperature for 10 minutes. After terminating fixation, the two cell lines were mixed in equal volumes and washed three times with PBS (containing 0.05% Tween-20) pre-cooled at 4°C for 5 minutes each time. The cell pellet was collected by centrifugation at 800g.

[0086] 1.2 Permeabilization and Enzymatic Hydrolysis Treatment

[0087] Step 1.1 The fixed sample is set as T1, except that the sample T1 directly mixes two kinds of cells, the rest of the fixed cells are resuspended in 0.2% Triton X-100 permeabilization solution (containing 5mM EDTA, 10mM Tris-HCl, pH8.0), and permeabilized at room temperature for 7 minutes. After permeabilization, preheated enzyme solution [containing 200U / ml lysozyme, 60U / ml lysoglycerolase in PBS, placed in a 37℃ constant temperature oscillator (300rpm) for 15 minutes, and immediately placed on ice after enzyme digestion to terminate the reaction, divided into 3 samples, labeled as T2, T3 and T4 respectively.

[0088] 1.3 Multi-primer reverse transcription reaction

[0089] In a 0.2ml PCR tube, 20μl reverse transcription system was established: containing 10,000 mixed cells in step 2, multiplex reverse transcription primer (0.5μM 17L-dT30VN, 0.3μM first half random primer 17L-10N4G and 0.3μM second half random primer 17L-10N4T, 1×RT buffer, 10U / μl SuperScript IV reverse transcriptase (Invitrogen). Reaction program: 25℃ for 30min (primer annealing)→42℃ for 60min (reverse transcription), wherein 0.2μM 16S rRNA constant region specific primer (17L-1100R) needs to be added additionally in T3 and T4 samples.

[0090] Among them, the transcriptome primer sequence is as follows:

[0091] 17L-dT30VN (also known as oligo-dT):

[0092] p-CGTCCGTCGTTGCTCGT-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN;

[0093] 17L-10N4G (also known as the first half random primer):

[0094] p-CGTCCGTCGTTGCTCGT-NNNNNNNNNNGGGG;

[0095] 17L-10N4T (also known as the second half random primer):

[0096] p-CGTCCGTCGTTGCTCGT-NNNNNNNNNNTTTT;

[0097] 17L-1061R (first 16S rRNA constant region specific primer):

[0098] p-CGTCCGTCGTTGCTCGT-TCACGRCACGAGCTGACGAC;

[0099] or 17L-1100R (second 16S rRNA constant region specific primer):

[0100] p-CGTCCGTCGTTGCTCGT-GGGTTGCGCTCGTTG;

[0101] It should be noted that N here can be any base.

[0102] 1.4 Water-in-oil ligation and product purification

[0103] Using The water-in-oil ligation system was constructed using the FFPE single cell transcriptome library construction kit (Beijing Xingyin Biotechnology Co., Ltd., K02101), and a single droplet contained 1 hydrogel microbead (containing a cell tag that can be ligated with a reverse transcription primer) and at most 1 cell and the bacteria carried by the cell. After the ligation reaction, demulsification was performed, and the aqueous phase product was collected by magnetic bead purification according to the instructions, and eluted with 21 μl of double distilled water.

[0104] 1.5 Pre-amplification PCR: The PCR system was configured according to the instructions in the kit, except for the Read1 SeqPrimer and template switching sequence primer TSO, and in addition, amplification primers for 16S reverse transcription products were added, wherein T1 and T2 samples need to add amplification primer Rd2P-789F as shown in SEQ ID NO. 8, and T3 and T4 samples need to add amplification primer 789F as shown in SEQ ID NO. 6;

[0105] Rd2P-789F sequence:

[0106] GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-TAGATACCCSSGTA GTCC;

[0107] 789F sequence:

[0108] TAGATACCCSSGTAGTCC;

[0109] 1.6 Construction of transcriptome library: The single cell transcriptome product was enriched from the pre-amplification library using the Read1 SeqPrimer and template switching sequence primer TSO in the kit, and then broken down for library construction;

[0110] 1.7 Construction of intracellular 16S library:

[0111] 1.7.1 Second round of enrichment: sample T3 uses the amplification primer Rd2P-789F combined with Read1 SeqPrimer for PCR enrichment, and sample T4 uses Rd2P-967F primer combined with Read1 SeqPrimer for 15 cycles of amplification (the annealing temperature is increased to 62°C)

[0112] Rd2P-789F sequence:

[0113] GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-TAGATACCCSSGTA GTCC;

[0114] Rd2P-967F sequence:

[0115] GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-CAACGCGAAGAAC CTTACC;

[0116] 1.7.2 Index PCR is constructed to complete the intracellular 16S library.

[0117] 1.8. Sequencing analysis

[0118] Illumina NovaSeq 6000 platform is used for double-end PE150 sequencing, and Table 1 shows the specific experimental condition settings:

[0119] Table 1

[0120] Sample / processing conditions T1 T2 T3 T4 Wall-breaking permeabilization - + + + 16S reverse transcription primer - - + + Stepwise amplification enrichment - - - +

[0121] It should be noted that condition T1 and T2 compare the effects of permeabilization on bacterial detection; condition T2 and T3 compare the effects of one round of amplification and two rounds of amplification on bacterial detection; condition T3 and T4 compare the effects of stepwise amplification enrichment on bacterial detection, wherein the PCR amplification of T3 and T4 includes first round PCR amplification and second round PCR amplification; wherein the sequence of the 789F primer used in the first round PCR amplification is shown in SEQ ID NO. 6, the sequence of the Rd2P-789F primer used in the second round PCR amplification of T3 is shown in SEQ ID NO. 8, and the amplification is at the same position, while the sequence of the Rd2P-967F primer used in the second round PCR amplification of T4 is shown in SEQ ID NO. 7, which is stepwise amplification at different positions.

[0122] According to the above, samples T1, T2, T3 and T4 are obtained, and the sequencing results are shown in Table 2 and Figures 2-3

[0123] ​Table 2. The proportion of total read pairs that can be aligned to 16S in different experimental protocols.

[0124] Sample / processing conditions T1 T2 T3 T4 (16S / total reads) % 15.07％ 51.83％ 63.61％ 84.49％

[0125] From Table 2, we can see that: whether it is the proportion of reads that can be aligned to 16S in the sequencing data (Table 2), or the proportion of positive bacterial UMIs in all bacterial UMIs ( Figure 2 ), or the average number of positive bacterial UMIs detected in host cells ( Figure 3 ), the three optimization treatments of double non-permeabilization of host cells and bacteria (T2 vs T1), addition of bacterial 16S specific reverse transcription primers (T3 vs T2), and ladder PCR enrichment (T4 vs T3) can all obtain more ideal results.

[0126] It should be noted that positive bacteria refer to those bacteria that can be successfully detected by experimental methods (such as reverse transcription, PCR amplification and sequencing). In other words, the 16S rRNA gene fragments of these bacteria can be aligned in the sequencing data, and there is enough supporting evidence (such as UMI count) to show that they indeed exist in the sample; UMI (Unique Molecular Identifier) is a molecular tagging technology used to label each original cDNA molecule to help distinguish between true biological variation and technical noise introduced during PCR amplification or sequencing. For positive bacteria, the number of UMIs can reflect the relative abundance of the bacteria in the sample. The proportion of positive bacterial UMIs in all bacterial UMIs indicates the proportion of UMIs belonging to positive bacteria (i.e. confirmed existing bacteria) in all detected bacterial UMIs. A higher proportion indicates that the experimental design effectively enriches the information of target bacteria and reduces non-specific background signals.

[0127] Example Two: The proportion of UMIs aligned to positive bacteria (Mycobacterium smegmatis) in all bacterial 16S UMIs in different experimental protocols

[0128] 2.1 Cell fixation treatment

[0129] Take 1 x 10^6 sterile human peripheral blood mononuclear cells (PBMCs) and mouse RAW264.7 macrophage cell lines infected with Mycobacterium smegmatis for 48 hours, and fix them with 1% paraformaldehyde in PBS buffer (pH 7.4) at room temperature for 10 minutes. After stopping the fixation, mix the two cells in equal amounts, wash them with pre-cooled PBS (containing 0.05% Tween-20) at 4°C for 3 times, 5 minutes each time, and collect the cell precipitate by centrifugation at 800g.

[0130] 2.2 Construct single-cell transcriptome library and intracellular bacteria 16S library from cells in step 1 according to the optimal protocol T4 flow (i.e. T4-2) and INVADEseq technology flow in Example 1, respectively;

[0131] 2.3 Sequencing and data analysis. First, UMAP cell clustering was performed on the single-cell transcriptome data after human-mouse genome mixed alignment, and the source of each cell cluster (RAW cell or PBMC cell) was determined according to whether the RNA in each cell cluster was aligned to the human or mouse genome. Then, according to the cell label shared in the single-cell transcriptome and intracellular bacteria library, the UMI aligned to M. smegmatis was mapped to the cells, as shown in Figure 4 Most mouse-derived reads (mm10) were distributed in the red box, representing mouse RAW cell lines, and most human-derived reads (GRCh38) were distributed in the green box, representing human PBMC cell clusters. The UMI of M. smegmatis detected by the intracellular bacteria process (T4) of the present application were concentrated in the infected mouse RAW cells, and few UMI were detected in the human PBMC as a negative control. Correspondingly, the INVADEseq process had no significant detection in the mouse RAW cells.

[0132] To further quantify and compare, as shown in Figure 5 The present application compared the average UMI detection number in the top ten Raw cells of M. smegmatis UMI detection according to the process T4-2 and INVADEseq technology of the present application, and found that the UMI detection of the technical process of the present application was more than 10 times that of INVADE-seq.

[0133] The above is only a preferred embodiment of the present application, and does not limit the patent scope of the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the patent protection scope of the present application.

Claims

1. A eukaryotic unicellular cell and a method for detecting a bacterium carried thereby, characterized by, The method comprises the following steps: S10, providing pre-processed eukaryotic single cells and bacteria carried by the eukaryotic single cells; S20, providing a multiplex reverse transcription primer system, and performing a reverse transcription reaction on the pre-processed eukaryotic single cells and the bacteria carried by the eukaryotic single cells to obtain a reverse transcription product; S30, providing microbeads, the microbeads having cell tags, and mixing the microbeads with the reverse transcription product to perform a ligation reaction, so as to label the cell tags on the reverse transcription product to obtain a ligation product; S40, constructing a library for the ligation product to obtain a single cell transcriptome library and a 16S rRNA library, and performing sequencing to obtain interaction information between the eukaryotic single cells and the bacteria carried by the eukaryotic single cells.

2. The eukaryotic unicellular cell and the method for detecting the same according to claim 1, wherein the eukaryotic unicellular cell is a yeast cell. Before step S10, the method further comprises: fixing and permeabilizing the eukaryotic single cells to obtain a eukaryotic single cell suspension; adding lysozyme and lysostaphin to digest bacterial cell walls in the cells to obtain pre-processed eukaryotic single cells and bacteria carried by the eukaryotic single cells.

3. The eukaryotic unicellular cell and the method for detecting the same carrying bacteria according to claim 2, wherein the eukaryotic unicellular cell is a yeast cell. The mass ratio of the lysozyme to the lysostaphin is 1:0.1-4.

4. The eukaryotic unicellular cell and the method for detecting the same according to claim 1, wherein the eukaryotic unicellular cell is a yeast cell. In step S20, the multiplex reverse transcription primer system comprises an oligo-dT primer, a semi-random primer and a 16S rRNA constant region specific primer.

5. The eukaryotic unicellular cell and the method for detecting the same carrying bacteria according to claim 4, wherein, The semi-random primer comprises a first semi-random primer or a second semi-random primer, and the 16S rRNA constant region specific primer comprises a first 16S rRNA constant region specific primer or a second 16S rRNA constant region specific primer, wherein: the sequence of the oligo-dT primer is shown in SEQ ID NO. 1; and / or the sequence of the first semi-random primer is shown in SEQ ID NO. 2; and / or the sequence of the second semi-random primer is shown in SEQ ID NO. 3; and / or the sequence of the first 16S rRNA constant region specific primer is shown in SEQ ID NO. 4; and / or the sequence of the second 16S rRNA constant region specific primer is shown in SEQ ID NO.

5.

6. The eukaryotic unicellular cell and the method for detecting the same according to claim 1, wherein the eukaryotic unicellular cell is a yeast cell. In step S40, the construction step of the single cell 16S rRNA library comprises: using a Read1 Sequencing Primer to perform ladder PCR amplification with different 16S constant region amplification primers to obtain a single cell 16S rRNA library.

7. The eukaryotic unicellular cell and the method for detecting the same according to claim 5, wherein the eukaryotic unicellular cell is a yeast cell. The ladder PCR amplification comprises a first round of PCR amplification and a second round of PCR amplification; wherein the sequence of a 789F primer used in the first round of PCR amplification is shown in SEQ ID NO. 6, and the sequence of a Rd2P-967F primer used in the second round of PCR amplification is shown in SEQ ID NO.

7.

8. A sequencing library, characterized in that, The sequencing library constructed by the high-throughput single cell and bacteria carried by the single cell detection method.

9. Use of the sequencing library of claim 7 in the field of host-microorganism interaction.

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