CN113897438A - CD74-ROS1 rearranged DNA standard substance and RNA standard substance for molecular diagnosis and application thereof - Google Patents
CD74-ROS1 rearranged DNA standard substance and RNA standard substance for molecular diagnosis and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of gene rearrangement standard products, and provides a CD74-ROS1 rearranged DNA standard product, an RNA standard product and application thereof for molecular diagnosis. The method comprises the steps of constructing sgRNA aiming at the intron No.6 of the CD74 gene on a vector pX330 to obtain a plasmid C4, constructing sgRNA aiming at the intron No.33 of the ROS1 gene on the vector pX330 to obtain a plasmid R3, transfecting host cells with the plasmid C4 and the plasmid R3 together to obtain recombinant cells, performing monoclonation on the recombinant cells to obtain cells for preparing a DNA standard product or an RNA standard product, and further obtaining the DNA and RNA standard product. The CD74-ROS1 rearrangement standard substance sequence and the cell provided by the invention can stably provide a sample for a long time, and are very suitable for performance evaluation and long-term quality control requirements of LDT or IVD development.
Description
Technical Field
The invention relates to the technical field of gene rearrangement standard products, in particular to a CD74-ROS1 rearranged DNA standard product, an RNA standard product and application thereof for molecular diagnosis.
Background
ROS protooncogene 1(ROS1), encoded by the ROS1 gene located on chromosome 6q22.1, belongs to the tyrosine kinase insulin receptor subfamily and was first discovered in 1986 in studies involving chicken sarcoma RNA UR2 oncovirus. The physiological role of ROS1 remains controversial; its expression is observed mainly in lung tissue, followed by the cervix and colon. It is envisioned that the tyrosine kinase insulin receptor plays a key role in embryonic development. The ROS1 protein shows a great deal of homology to ALK (both belonging to the insulin receptor superfamily), particularly in the ATP binding site (84% homology) and the kinase domain (64% homology). Fusion mutations of ROS1 are well known and often result in gene fusions with several fusion partners, and the resulting fusion proteins are powerful oncogenic drivers. Thus, ROS1 kinase activity is continuously activated, resulting in increased cell proliferation, survival and migration due to upregulation of JAK/STAT, PI3K/AKT and MAPK/ERK signaling pathways. ROS1 has shown tumorigenic potential in vitro and in vivo, and glioblastoma was the first human cancer shown to have ROS1 rearrangements. ROS1 fusions were subsequently observed in other malignancies, including NSCLC, melanoma, and occasionally cholangiocarcinoma, angiosarcoma, ovarian cancer, gastric cancer, and colorectal cancer. ROS1 changes are neither inherited nor obtained as genetic changes.
The ROS1 rearrangement accounts for approximately 2% of NSCLC patients, and the fusion partners common in NSCLC are shown in table 1, with the highest of these being the CD74 gene.
TABLE 1Main ROS1 fusion partners in non-small cell lung cancer (NSCLC)
CD74 is located on chromosome 5q33.1, and the protein encoded by the gene is related to the Major Histocompatibility Complex (MHC) class II and is an important molecular chaperone for regulating antigen presentation in immune response. It also acts as a cell surface receptor for the cytokine macrophage Migration Inhibitory Factor (MIF), which when bound to the encoded protein, initiates survival pathways and cell proliferation. This protein also interacts with Amyloid Precursor Protein (APP) and inhibits the production of amyloid beta (Abeta).
In the diagnosis of non-small cell lung cancer, the NCCN guidelines explicitly describe "ROS 1 a receptor type kinase that can distinguish between in NSCLC, and that can detect in both differentiation and inhibition of the formation of the kinase domain". The NCCN guidelines explicitly describe "ROS 1 kinase domain (ROS1 is a receptor tyrosine kinase, which causes gene rearrangement in non-small cell lung cancer, resulting in deregulation and abnormal signaling of the kinase domain." emphasize the meaning of molecular diagnosis of ROS1, a molecular marker that must be detected, and the recommended detection method is described as "FISH fragment-antibody reagent detected"; host computer, detection under-detection of FIG-1 variant. IHC assay reagent attached IHC 2 reagent, detection of ROS 387 nucleic acid molecules, ROS 387 detection of ROS nucleic acid molecules, ROS nucleic acid reagent attached nucleic acid molecule, ROS 2 nucleic acid molecules, coding nucleic acid molecules, ROS 387, and coding nucleic acid molecules, ROS 3875, the method of this invention is based on the discovery of the presence of a single probe, and the detection of multiple transcripts of FIG-ROS1, and the detection of IHC (immunohistochemistry) with the disadvantage of low specificity, requiring subsequent detection methods to confirm as a supplementary verification.
In recent years, many companies develop kits for detecting CD74-ROS1 in China, in the development of the kits, standard substances are required to perform performance evaluation on the whole detection system, the performance evaluation is described according to the guiding principle of the development of the third type of medical instrument tumor detection kit, the corresponding standard substances are required for experimental performance evaluation, and the standard substances cannot be plasmids and are preferably clinical samples. However, in the actual detection process, clinical samples are often not available and need to be exactly met with such patients, and the samples are not reproducible and small in quantity, so that the samples are not suitable for being used as standard substances and quality control substances for developing IVD kits for a long time.
How to solve the contradiction is two methods, the first method is to prepare a clinical sample containing the target CD74-ROS1 rearrangement into an immortalized cell line, but the preparation process usually needs half a year to a year, the success rate is not high, and one patient corresponds to a rearrangement mode and has no diversity, so the method is basically excluded and is not preferred; the second method is to rearrange and introduce CD74-ROS1 into a cell line by means of gene recombination and gene editing on an immortalized cell line to form a stable genome structure, so that the genome (DNA and RNA) containing CD74-ROS1 rearrangement can be continuously and stably supplied, the requirement of diversity is met, and the requirements of standard products and quality control products in the whole process of performance evaluation are met. However, little research has been currently conducted on the development of CD74-ROS1 rearrangement standards.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a CD74-ROS1 rearranged DNA standard substance, an RNA standard substance and application thereof for molecular diagnosis.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a CD74-ROS1 rearranged DNA standard substance for molecular diagnosis, and the nucleotide sequence of the DNA standard substance is shown as SEQ ID NO. 35.
The invention also provides a CD74-ROS1 rearranged RNA standard for molecular diagnosis, and the nucleotide sequence of the RNA standard is shown as SEQ ID NO. 42.
The invention also provides a cell for preparing the DNA standard substance or the RNA standard substance, and the genome of the cell is recombined with the nucleotide sequence of the DNA standard substance.
Preferably, the cell is a host cell in a human immortalized cell line;
the host cell is selected from one of HEK293, HCT116, DLD-1, RKO and SW 48.
The invention also provides a preparation method of the cell for preparing the DNA standard substance or the RNA standard substance, which comprises the following steps:
(1) the sgRNA of intron 6 of the CD74 gene is constructed on a vector pX330 to obtain a plasmid C4;
(2) constructing sgRNA aiming at intron 33 of ROS1 gene on a vector pX330 to obtain a plasmid R3;
(3) co-transfecting host cells with the plasmid C4 and the plasmid R3 to obtain recombinant cells;
(4) and (3) performing monoclonality on the recombinant cells to obtain cells for preparing a DNA standard substance or an RNA standard substance.
Preferably, the sequence of sgRNA directed to intron 6 of the CD74 gene in step (1) is shown in SEQ ID No. 18.
Preferably, the sequence of sgRNA directed against intron 33 of ROS1 gene in step (2) is shown in SEQ ID No. 27.
Preferably, the single cloning in step (4) is carried out by limiting dilution.
The invention also provides application of the DNA standard substance or RNA standard substance of CD74-ROS1 rearrangement in the preparation of a kit for detecting CD74-ROS1 rearrangement.
The invention also provides application of the CD74-ROS1 rearranged DNA standard substance or RNA standard substance in performance evaluation of an LDT or IVD development system.
Compared with the prior art, the invention has the following beneficial effects:
the CD74-ROS1 rearranged DNA standard or RNA standard provided by the invention is suitable for being used as a CD74-ROS1 rearranged standard for molecular diagnosis, samples can be stably provided for a long time, the obtained numerous clones present the diversity of intron breakpoints, are correctly transcribed into RNA products, have complete and accurate breakpoints, contain the genome with the full length of human, are the best standard for simulating clinical samples, and are very suitable for performance evaluation and long-term quality control requirements for LDT or IVD development.
Drawings
FIG. 1 shows ddPCR detection of the copy number of ROS1 in DNA in HCT 116;
FIG. 2 is a graph of ddPCR detecting the copy number of ROS1 in mRNA in HCT 116;
FIG. 3 shows the rearrangement of CD74-ROS1 at the DNA level (note: partial sequence of intron 6 of CD74 to the left of the vertical line and partial sequence of intron 33 of ROS1 to the right of the vertical line);
FIG. 4 shows the rearrangement frequency of clone No.23 of CD74-ROS1 detected by ddPCR;
FIG. 5 shows RNA sequences of clone No.23 of CD74-ROS1 detected by sanger sequencing (note: the tail sequence of exon No.6 of CD74 on the left side of the arrow, and the head sequence of exon No.34 of ROS1 on the right side of the arrow);
FIG. 6 shows the copy number of CD74(E6) -ROS1(E34) at RNA level detected by ddPCR.
Detailed Description
The invention provides a CD74-ROS1 rearranged DNA standard substance for molecular diagnosis, and the nucleotide sequence of the DNA standard substance is shown as SEQ ID NO. 35.
The invention also provides a CD74-ROS1 rearranged RNA standard for molecular diagnosis, and the nucleotide sequence of the RNA standard is shown as SEQ ID NO. 42.
The invention also provides a cell for preparing the DNA standard substance or the RNA standard substance, and the genome of the cell is recombined with the nucleotide sequence of the DNA standard substance.
In the present invention, the cell is preferably a host cell in the human immortalized cell line;
the host cell is preferably one of HEK293, HCT116, DLD-1, RKO and SW48, and is more preferably HCT 116.
The invention also provides a preparation method of the cell for preparing the DNA standard substance or the RNA standard substance, which comprises the following steps:
(1) the sgRNA of intron 6 of the CD74 gene is constructed on a vector pX330 to obtain a plasmid C4;
(2) constructing sgRNA aiming at intron 33 of ROS1 gene on a vector pX330 to obtain a plasmid R3;
(3) co-transfecting host cells with the plasmid C4 and the plasmid R3 to obtain recombinant cells;
(4) and (3) performing monoclonality on the recombinant cells to obtain cells for preparing a DNA standard substance or an RNA standard substance.
In the present invention, the sequence of sgRNA directed to intron 6 of CD74 gene in step (1) is preferably as shown in SEQ ID No. 18.
In the present invention, the sequence of sgRNA directed to intron 33 of ROS1 gene in step (2) is preferably as shown in SEQ ID No. 27.
In the present invention, the single cloning in step (4) is preferably carried out by a limiting dilution method, and the limiting dilution method is preferably carried out by: cells were collected at 1x10e6, configured at the following densities: respectively paving the cells with the densities to a 96-well plate, 100 mu l/hole, namely 1 cell/hole of the cells with the densities of 10cells/ml, 2 cells/hole of the cells with the densities of 20cells/ml and 5 cells/hole of the cells with the densities of 50cells/ml, paving the line A, the line B and the line C of the 96-well plate with the densities of 1 cell/hole, paving the line D, the line E and the line F of the 96-well plate with the densities of 2 cells/hole, paving the line G and the line H of the 96-well plate with the densities of 5 cells/hole, and adding the 10 blocks 96 of the plates according to the mode to continue cell culture;
on the 2 nd day, after the cells adhere to the wall, each hole is observed under a microscope, one cell exists in a single hole, marking is carried out, and no cell or more than 1cell hole is left;
observing the marked holes in 96 holes under a microscope every day within 5-10 days later, after a clone island is formed, carrying out trypsinization, and paving the wells into a 24-hole plate;
after the 24-hole plate grows to 70% confluence degree, digesting by pancreatin, and paving into a 6-hole plate;
when the cells in the 6-well plate grow to the confluence of more than 70%, the cells are digested by pancreatin and collected to form the monoclonalized cells.
The invention also provides application of the CD74-ROS1 rearranged DNA standard or RNA standard in preparation of a kit for detecting CD74-ROS1 rearrangement, and the application specifically refers to that the CD74-ROS1 rearranged DNA standard or RNA standard is used as a standard or control for detecting CD74-ROS1 rearrangement to replace a clinical sample.
The invention also provides application of the CD74-ROS1 rearranged DNA standard substance or RNA standard substance in performance evaluation of an LDT or IVD development system.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Experimental example 1
The mother cells with higher plasmid transfection efficiency were selected as alternatively infected host cells as shown in table 2:
TABLE 2 sources and media for different host cell lines
| Cell lines | Source | Culture medium |
| HEK293 | ATCC;CRL-1573 | DMEM+10%FBS |
| HCT116 | ATCC;CCL-247 | McCoy's 5a+10%FBS |
| DLD-1 | ATCC;CRL-2577 | RPMI-1640+10%FBS |
| RKO | ATCC;CCL-221 | MEM+10%FBS |
| SW48 | ATCC;CCL-231 | DMEM+10%FBS |
Culturing 5cells according to the ATCC published specifications, adjusting to exponential growth, plating the cells into a 6-well plate according to the density of 1x10e6cell/well, and culturing overnight;
observing the cells under a microscope on the next day, when the cell confluence reaches 80%, transfecting 5 host cells with plasmid PX458 with GFP (phosphoprotein) by 1.5 mu g/hole, continuously culturing the cells according to the instructions of lipo3000 by a transfection method, observing the expression of GFP of the cells by a fluorescence microscope for 48 hours, observing the cells again for 72 hours, and selecting the cells with the best infection efficiency according to the results of the two observations. The results showed that the positive rate of GFP expression in HEK293 was 70%, the positive rate of GFP expression in HCT116 was 90%, the positive rate of GFP expression in DLD-1 was 50%, the positive rate of GFP expression in RKO was 80%, and the positive rate of GFP expression in SW48 was 70%. Thus, HCT116 was selected as the host cell for later experiments.
HCT116 cells were cultured further and 1 × 10e6 cells were collected at the following densities: 10cells/ml, 20cells/ml, 50cells/ml, corresponding cells were plated in 96-well plates, 100 μ l/well, corresponding to 1 cell/well, 2 cells/well, 5 cells/well, wherein 1 cell/well was plated in line a, line B, and line C of 96-well plates, 2 cells/well was plated in line D, line E, and line F of 96-well plates, and 5 cells/well was plated in line G and line H of 96-well plates. In the same protocol, 10 plates of 96-well plates were plated in total and cell culture was continued.
The next day, after the cells were attached, each well was observed under a microscope, one cell was present in a single well, marked, wells with no cells or greater than 1cell were discarded altogether. Within 5-10 days thereafter, the labeled wells in the 96 wells were observed under a microscope every day, and after the formation of cloned islands, they were trypsinized and plated into 24-well plates, and after the 24-well plates were grown to 70% confluence, they were trypsinized and plated into 6-well plates.
When the cells in the 6-well plate grow to be more than 70% of confluence, pancreatin is used for digestion, the cells are collected, the gDNA is extracted by using Quick-DNA Miniprep of Zymo, the concentration is detected by using qubit 4.0, the OD260/280 (between 1.8 and 2.0) is detected by using Nano, the integrity of the gDNA (a single band of more than 15000 bp) is observed by gel electrophoresis, and the quality of the gDNA is ensured.
Designing a Copy number ddPCR system to detect ROS1 for detecting the background Copy number of ROS1 in each clone; ddPCR system to detect ROS1 expression was designed to test whether ROS1 was transcribed normally in each clone. The design is as follows:
the copy number of ROS1 was designed as follows:
selecting a target sequence shown as SEQ ID NO. 1;
primers and probes were designed by Primer3, and the sequences were:
ROS1-T-23-F is shown in SEQ ID NO.2, ROS1-T-23-R is shown in SEQ ID NO.3, and ROS1-T-23-P is shown in SEQ ID NO. 4.
The copy number of the reference gene EIF2C1 was designed as follows:
selecting a target sequence hg19| chr1:36359339-36359461 as shown in SEQ ID NO. 5;
primers and probes were designed by Primer3, and the sequences were:
EIF2C1-CNV-F is shown as SEQ ID NO.6, EIF2C1-CNV-R is shown as SEQ ID NO.7, and EIF2C1-CNV-P is shown as SEQ ID NO. 8.
The Expression of ROS1 is designed as follows:
selecting a target sequence hg19| chr6:117650560-117658407 as shown in SEQ ID NO. 9;
primers and probes were designed by Primer3, and the sequences were:
ROS1-mRNA-F is shown as SEQ ID NO.10, ROS1-mRNA-R is shown as SEQ ID NO.11, and ROS1-mRNA-P is shown as SEQ ID NO. 12.
Using ddPCR assay, monoclonal data No.27 was obtained, as shown in fig. 1 and 2. Copy number 2 (found 2.07), mRNA copy number 98 copies/ng, normal transcription, indicating that monoclonal No.27 is structurally complete and normal in the genome of ROS1, and is suitable for further gene editing.
The most common rearrangement of CD74-ROS1 is the splicing of exon 6 of CD74 with exon 34 of ROS1, so that the breakpoint in the DNA rearrangement is intron 6 of CD74 and intron 33 of ROS 1. Wherein, the intron sequence No.6 of CD74 is shown in SEQ ID NO.13, and the intron sequence No.33 of ROS1 is shown in SEQ ID NO. 14.
Sgrnas were designed using the online criprpr cas9 design software of zhang lab, and the design results are shown in tables 3 and 4.
Table 3 sgRNA design results for intron 6 of CD74
| No. | Orientation | sgRNA Sequence | PAM Sequence | On-Target Efficacy Score |
| C1 | antisense | GAGAGGTCACCTGTACCCTG (shown in SEQ ID NO. 15) | TGG | 0.7676 |
| C2 | sense | GGGTTCCTAAGGGCCCACAG (shown as SEQ ID NO. 16) | AGG | 0.7576 |
| C3 | antisense | TCACACGGATGGGAAAACTG (shown in SEQ ID NO. 17) | AGG | 0.7555 |
| C4 | antisense | GGAACCCTTAGACAAGATGG (shown in SEQ ID NO. 18) | GGG | 0.7457 |
| C5 | antisense | AGTGAGTGAGCTCTGAACCA (shown in SEQ ID NO. 19) | GGG | 0.7187 |
| C6 | sense | ACTCCCTGGTCCCAGCACCG (shown as SEQ ID NO. 20) | CGG | 0.7023 |
| C7 | antisense | TGTACCATCCTCTATGAGAG (shown as SEQ ID NO. 21) | AGG | 0.6887 |
| C8 | antisense | GCAGCTTCAGGAGAGCCCCG (shown in SEQ ID NO. 22) | AGG | 0.6886 |
| C9 | antisense | TTCAGGACCCTGAATCCTAG (shown in SEQ ID NO. 23) | AGG | 0.6795 |
| C10 | antisense | AACTGAGGCCTAGACCAACA (shown in SEQ ID NO. 24) | AGG | 0.6782 |
Table 4 sgRNA design results for intron 33 of ROS1
| No. | Orientation | sgRNA Sequence | PAM Sequence | On-Target Efficacy Score |
| R1 | sense | TGTGTGCTTAGGTAGAGCTG (shown in SEQ ID NO. 25) | GGG | 0.745 |
| R2 | antisense | AACTAAAATAAAATACACAG (shown as SEQ ID NO. 26) | TGG | 0.7356 |
| R3 | antisense | TCTAGGCTGAGGCAAACACA (shown in SEQ ID NO. 27) | GGG | 0.7326 |
| R4 | antisense | CCATTGTGGGTATTTCAGAG (shown in SEQ ID NO. 28) | AGG | 0.6939 |
| R5 | sense | GGATGCTTTCAAGCTACCAA (shown as SEQ ID NO. 29) | CGG | 0.6899 |
| R6 | antisense | TGTAGAGGAAACAAACACGT (shown as SEQ ID NO. 30) | AGG | 0.6882 |
| R7 | antisense | TGATGTGGTAGGAATTAAGG (shown as SEQ ID NO. 31) | GGG | 0.6852 |
| R8 | sense | ATTGAGTGGATATATTCCAG (shown in SEQ ID NO. 32) | TGG | 0.6747 |
| R9 | sense | ACTTATTCATAGCTAATAGG (shown in SEQ ID NO. 33) | GGG | 0.6737 |
| R10 | antisense | TTAAATTAGGAAAGCCAAGG (shown in SEQ ID NO. 34) | TGG | 0.6716 |
10 sgRNAs of CD74 are constructed on a vector pX330, 10 sgRNAs of ROS1 are also constructed on the vector pX330, 20 plasmids are summed up, 100 mu g is extracted, HCT116 cells cloned in No.27 are transfected in a 24-well plate, after the cells grow to 70-80% confluence, the cells are digested and plated in a 6-well plate, after the cells grow to 70-80% confluence in the 6-well plate, the cells are trypsinized and collected, gDNA is extracted by using Quick-DNA Miniprep of Zymo, the concentration is detected by using qubit 4.0, the OD260/280 (between 1.8 and 2.0) is detected by Nano, the integrity of the gDNA (a single band of more than 15000 bp) is observed by gel electrophoresis, and the quality of the gDNA is ensured.
A total of 20 gDNAs were sent to a third party for sanger sequencing (primers were designed according to the sequences on both sides of each breakpoint, and any cleavage inconsistent with the wild type was considered to have occurred by sequencing), and according to the sequencing results, C2, C4 and C5 were found to be the first 3 of CD74 with the highest cleavage efficiency for intron 6, and R2, R3 and R6 were found to be the first 3 of ROS1 with the highest cleavage efficiency for intron 33, and were used in further downstream experiments.
The pX330 plasmids corresponding to C2, C4, C5, R2, R3 and R6 are co-transferred into a 24-well plate in a pairwise combination mode (3 x3=9 combinations in total), HCT116 cells cloned in number 27 are co-transferred in the 24-well plate, after the cells grow to 70-80% confluence, the cells are digested and plated into a 6-well plate, after the cells grow to 70-80% confluence in the 6-well plate, the cells are digested by pancreatin and collected, gDNA is extracted by using Quick-DNA Miniprep of Zymo, the concentration is detected by using qubit 4.0, the OD260/280 (between 1.8 and 2.0) is detected by Nano, the integrity of the gDNA (a single band larger than 15000 bp) is observed by gel electrophoresis, and the quality of the gDNA is ensured.
And sending the total 9 gDNAs to a third party for sanger sequencing (according to two breakpoints of CD74-ROS1, an F directional primer is designed on the left side of the breakpoint of CD74, an R directional primer is designed on the right side of the breakpoint of ROS1, and all PCR products are used for sequencing as long as the PCR products exist), and according to the electrophoresis result and the sequencing result of the PCR products, finding that obvious PCR products appear in C4-R3, thereby indicating that various CD74-ROS1 rearrangements occur.
The cell of HCT116-C4-R3 is subjected to monoclonality, and the specific operation is as follows: cells were collected at 1x10e6, at the following densities: 10cells/ml, 20cells/ml, 50cells/ml, corresponding cells were plated in 96-well plates, 100 μ l/well, corresponding to 1 cell/well, 2 cells/well, 5 cells/well, wherein 1 cell/well was plated in line a, line B, and line C of 96-well plates, 2 cells/well was plated in line D, line E, and line F of 96-well plates, and 5 cells/well was plated in line G and line H of 96-well plates. In the same protocol, 10 plates of 96-well plates were plated in total and cell culture was continued.
The next day, after the cells were attached, each well was observed under a microscope, one cell was present in a single well, marked, wells with no cells or greater than 1cell were discarded altogether. Within 5-10 days thereafter, the labeled wells in the 96 wells were observed under a microscope every day, and after the formation of cloned islands, they were trypsinized and plated into 24-well plates, and after the 24-well plates were grown to 70% confluence, they were trypsinized and plated into 6-well plates.
When the cells in the 6-well plate grow to be more than 70% of confluence, pancreatin is used for digestion, the cells are collected, the gDNA is extracted by using Quick-DNA Miniprep of Zymo, the concentration is detected by using qubit 4.0, the OD260/280 (between 1.8 and 2.0) is detected by using Nano, the integrity of the gDNA (a single band of more than 15000 bp) is observed by gel electrophoresis, and the quality of the gDNA is ensured.
All HCT116-C4-R3 monoclonals were sent to a third party for sanger sequencing (based on two breakpoints of CD74-ROS1, F primers were designed to the left of the breakpoints of CD74, R primers were designed to the right of the breakpoints of ROS1, and all were used for sequencing as long as PCR products were available), and positive rearrangement was found in C4-R3 based on the electrophoresis results and sequencing results of the PCR products. As shown in particular in figure 3. As can be seen in FIG. 3, the partial sequence of intron 6 of CD74 is to the left of the vertical line, and the partial sequence of intron 33 of ROS1 is to the right of the vertical line, indicating that a DNA-level rearrangement of CD74-ROS1 was successfully achieved.
Experimental example 2
ddPCR detection of copy number at DNA level of rearranged Standard cells of CD74-ROS 1:
clone No.23 of CD74-ROS1 rearrangement was selected, and ddPCR was designed to detect the frequency of the desired rearrangement, and the sequence of interest is shown in SEQ ID NO. 35.
Design ddPCR for the sequence of interest:
CD74-ROS1-23-F is shown as SEQ ID NO. 36;
CD74-ROS1-23-R is shown as SEQ ID NO. 37;
CD74-ROS1-23-P is shown as SEQ ID NO. 38;
ROS1-T-23-F is shown in SEQ ID NO. 39;
ROS1-T-23-R is shown in SEQ ID NO. 40;
ROS1-T-23-P is shown in SEQ ID NO. 41.
The ddPCR assay revealed that the rearrangement frequency of clone No.23, CD74-ROS1, was 50%, i.e., there were 2 copies of ROS1 in HCT116, 1 copy rearranged, and the other 1 copy did not. As shown in fig. 4. FAM probe detected the number of copies of CD74-ROS1 rearrangement, VIC probe detected the number of copies of ROS1 as a whole, and as can be seen from FIG. 4, the frequency of CD74-ROS1 rearrangement was 50%.
Experimental example 3
ddPCR detection of copy number at RNA level of rearranged Standard cells of CD74-ROS 1:
RNA of clone No.23 of CD74-ROS1 was extracted by using the Tissue RNA Miniprep Kit from Biomiga and sent to a third party sanger for sequencing, and the detection data shows that the sample is correctly extracted mRNA, and after reverse transcription, the sequencing data is correct and is CD74(E6) -ROS1(E34) fusion. The results are shown in FIG. 5.
Aiming at the RNA splicing sequence of CD74(E6) -ROS1(E34), ddPCR is developed, and the copy number of the corresponding RNA is detected, wherein the corresponding splicing sequence is shown as SEQ ID NO. 42.
Design ddPCR for the sequence of interest:
CD74-ROS1-RNA-F is shown as SEQ ID NO. 43;
CD74-ROS1-RNA-R is shown as SEQ ID NO. 44;
CD74-ROS1-RNA-P is shown as SEQ ID NO. 45.
The RNA-level copy number of CD74(E6) -ROS1(E34) rearranged clone No.23 was determined by ddPCR at 691copies/ng, as shown in FIG. 6.
In conclusion, the invention obtains clone No.23 by gene editing on HCT116 cells, the clone has 50% of rearrangement of CD74-ROS1 (verified by sanger sequencing and ddPCR) at the DNA level, and the breakpoint is in an intron; rearrangement of CD74(E6) -ROS1(E34) occurred at the RNA level (detected by sanger sequencing and ddPCR) with breakpoints at exons.
The CD74-ROS1 rearranged DNA standard or RNA standard provided by the invention is suitable for being used as a CD74-ROS1 rearranged standard for molecular diagnosis, samples can be stably provided for a long time, the obtained numerous clones present the diversity of intron breakpoints, are correctly transcribed into RNA products, have complete and accurate breakpoints, contain the genome with the full length of human, are the best standard for simulating clinical samples, and are very suitable for performance evaluation and long-term quality control requirements for LDT or IVD development.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Nanjing Kebai Biotech Co., Ltd
<120> CD74-ROS1 rearranged DNA standard substance, RNA standard substance for molecular diagnosis and application thereof
<160> 45
<170> SIPOSequenceListing 1.0
<210> 1
<211> 208
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
aaaaaaaagc aaaaacacct tgcttttgat ttcacatggc ataaacactg tctgtatgga 60
tgctttcaag ctaccaacgg tctaacaact ggcttgcaaa aatccagtag tagctagctc 120
tgctatatta ctctgtgtgc ttaggtagag ctggggcaac ttagctttta tctatgaatt 180
aatttctttt tctgatttat attattag 208
<210> 2
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
caccttgctt ttgatttcac atg 23
<210> 3
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gttgttagac cgttggtagc ttga 24
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ataaacactg tctgtatgga tg 22
<210> 5
<211> 123
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gggcgcgagg tctggttcgg ctttcaccag tctgtgcgcc ctgccatgtg gaagatgatg 60
ctcaacattg atggtgagtg gggagagcta tggagccagg ggcaccccaa gtccagtgac 120
cac 123
<210> 6
<211> 16
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
gtgcgccctg ccatgt 16
<210> 7
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
catagctctc cccactcacc at 22
<210> 8
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
aagatgatgc tcaacatt 18
<210> 9
<211> 123
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
tttactccct tctagtaatt tgggaatgcc tggtttattt gggactccag cttttgtctt 60
aaagctttct ggaagtgagg tgctattttc tcccgtctta taaaccacca ctactctgac 120
att 123
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gggaatgcct ggtttatttg g 21
<210> 11
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
aatagcacct cacttccaga aagc 24
<210> 12
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
actccagctt ttgtcttaa 19
<210> 13
<211> 1367
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
gtacagggag tgggagcttt agcgtgccag ggcttctgga ccctcggggc tctcctgaag 60
ctgctgaggc cggggcctcc agcactccct ggtcccagca ccgcggaatc tcccatcctc 120
tcagctctca cttctttctc acttctctct ctctcctgtc tgttccttct tggtttggct 180
gtccccctcc cctgacccac ccccatcttg tctaagggtt cctaagggcc cacagaggcc 240
tgtcaccaca gggtacaggt gacctctctc atagaggatg gtacagcaca gggtgcctgg 300
ggtagaacct gcccgaaaca ctgcagaaag gaatccttgt aatgaccttg ccccagtgct 360
gcccgcaatc cagtgagggc gccaaggtca cagctgctgc caagagagcc ttgggcgttt 420
cccacctcat ggacaatgca gactaggatg tttttagacc caaagacaga gtgctgtttc 480
tatcccggtg ctgtctctaa tttgctatgt aactttggac aagtcccctt ccctctagga 540
ttcagggtcc tgaagtagaa ggtcaaaggg ccaccctgcc tggggcctca gtttctgcat 600
cagattcata gaaggcacct tacatgctat ctccaactcc tagctgatgc ttaaacccct 660
ctaagacatc tccaacaaac ggtaaacccc atttctactt gagaactccc agtaacagga 720
agcttagact taccaaggtg cccattgctt ttctctagaa tcagaatcgc aagtgcaatt 780
ccaaactgta atggtgtttt tttgtttgat tgtttgtttt tgagatagag tctggctctg 840
tcgcccaggc tggagtgcag tggtgcgatc tcagctcact gcaacctcag cctcccgggt 900
tcaagcaatt ctcctgcctc ggcctcccga gtagctggga ttataggcat gtgccaccac 960
gcccagctaa tttttgtatt tttagtagag acagggtttc accatgttgg ccaggctggt 1020
cttgaactcc tgacctcaag tgatccgccc acctcggcct cccaaagtgc taagattaca 1080
ggcatgagcc actgtgcctg gcccaaaact gtaatgggct ttgagtgtca gaagaaacca 1140
tcaacttctg aggtgaattg gacacatggc cattcacttc ctttttgatc tcagaccttg 1200
ttggtctagg cctcagtttt cccatccgtg tgatggctgg agtgagtaaa gccacttggg 1260
aagaaggcat taagcccaca gcagtggtgt gtgggtcttt agctctgctc agaccctggt 1320
tcagagctca ctcactcact gtgtcctcat catgcctgtc gcttcag 1367
<210> 14
<211> 1808
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gtatgttacc atgtctgtct acacactagc ttattaccta aaggttcagt aataaatatc 60
agtacattct taacattagc aatagataat ggtgactata tatatatata tatatatata 120
tatatatata taatcttatt atccaaacta ttaaactatt ccattcatgt gatgtgatct 180
ttctaaaaca tcattatcat catgtcattt ttctccccaa taataatcag ccaccccctt 240
aattcctacc acatcaattc catgctccta attaggcttc caaggctgtt cacaatctgt 300
tctatccagc catataattc tattcacatt ataccagcct tatgaccact cctacattat 360
acataacagg cctaagacct ttgtttcttc cccaatatct acctcttcca ctgtgtattt 420
tattttagtt tatgatatat tgtctttcta atcacatagg caggaaatct cagtgccata 480
tgtgattttt ccctctactc agtaatcagg tcctgttgat tttaccatct tgatttttct 540
ctgatgagtt ctcatcttcc tctttccacc ttggctttcc taatttaagg tatcatttct 600
tgcctgtgcc cttgacttac gcatactgct gacagttaaa tttagttgaa gcacaggctg 660
gattacttaa tccctctctg aaatacccac aatggctctc catttactgc tttcagaatc 720
agctacaaac ttctttgtgg catgtgaggt cttctgtaat ttatcctcca attgtggttt 780
atcttttcca atatttatgt ctgtgctgta gccatatcag accagtaaaa agtttttatg 840
tcacttagtc tttggccctg tgtttgcctc agcctagaat acaggtcccc actacctacc 900
ctgtgcccct tagctgtgat ttcctattat ttattttctg ccaattgaaa ttcttctcat 960
ccttcaagac ttaaatgaac tcatcataat gcttacctga tgctccttag tcaaatgaat 1020
tattgcattt tatacactca catgctatat aaaacttaca tgattcttgt ttatattata 1080
gttagttatc tagttagttg tgtacagaag tttgctaccc agctcagcaa actttttctg 1140
aaaagaacca aatagtaaat atgttagact ttgcaggcta catgtgatct ctgtagcatg 1200
ttcttctttt cttctttttc tgttctcttt ttccttctcc ttctttttaa aaataacact 1260
ttacaaaggt aaaacttatt catagctaat agggggtaca aaatcaggct atagccagat 1320
ttggtcctca tgccatagtt tgccagactc tggcctacgt gtttgtttcc tctacacaac 1380
tgaaactacc taagagaaat taccatgttt attcctcagt ttaatatcca tgaaattaaa 1440
tatgtatgaa gatattatac aaaataataa tgccaactat ttagtatcca aagactgaga 1500
tttcttggtc ctaaatttat taaaaagata tatatgtttc ctaagtcatt ttaaagtaga 1560
agattgagtg gatatattcc agtggtttgt tgctctctgc aaaaaaaagc aaaaacacct 1620
tgcttttgat ttcacatggc ataaacactg tctgtatgga tgctttcaag ctaccaacgg 1680
tctaacaact ggcttgcaaa aatccagtag tagctagctc tgctatatta ctctgtgtgc 1740
ttaggtagag ctggggcaac ttagctttta tctatgaatt aatttctttt tctgatttat 1800
attattag 1808
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
gagaggtcac ctgtaccctg 20
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gggttcctaa gggcccacag 20
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
tcacacggat gggaaaactg 20
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
ggaaccctta gacaagatgg 20
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
agtgagtgag ctctgaacca 20
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
actccctggt cccagcaccg 20
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
tgtaccatcc tctatgagag 20
<210> 22
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
gcagcttcag gagagccccg 20
<210> 23
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
ttcaggaccc tgaatcctag 20
<210> 24
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
aactgaggcc tagaccaaca 20
<210> 25
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
tgtgtgctta ggtagagctg 20
<210> 26
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
aactaaaata aaatacacag 20
<210> 27
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
tctaggctga ggcaaacaca 20
<210> 28
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
ccattgtggg tatttcagag 20
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
ggatgctttc aagctaccaa 20
<210> 30
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
tgtagaggaa acaaacacgt 20
<210> 31
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
tgatgtggta ggaattaagg 20
<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
attgagtgga tatattccag 20
<210> 33
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
acttattcat agctaatagg 20
<210> 34
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
ttaaattagg aaagccaagg 20
<210> 35
<211> 658
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
gttcctaagg gcccacagag gcctgtcacc acagggtaca ggtgacctct ctcatagagg 60
atggtacagc acagggtgcc tggggtagaa cctgcccgaa acactgcaga aaggaatcct 120
tgtaatgacc ttgccccagt gctgcccgca atccagtgag ggcgccaagg tcacagctgc 180
tgccaagaga gccttgggcg tttcccacct catggacaat gcagactagg atgtttttag 240
acccaaagac agagtgctgt ttctatcccg gtgctgtctc taatttgcta tgtaactttg 300
gacaagtccc cttccctcta ggattcaggg tcctgaagta gaaggctgga ttacttaatc 360
cctctctgaa atacccacaa tggctctcca tttactgctt tcagaatcag ctacaaactt 420
ctttgtggca tgtgaggtct tctgtaattt atcctccaat tgtggtttat cttttccaat 480
atttatgtct gtgctgtagc catatcagac cagtaaaaag tttttatgtc acttagtctt 540
tggccctgtg tttgcctcag cctagaatac aggtccccac tacctaccct gtgcccctta 600
gctgtgattt cctattattt attttctgcc aattgaaatt cttctcatcc ttcaagac 658
<210> 36
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
tccccttccc tctaggattc a 21
<210> 37
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
tgtgggtatt tcagagaggg attaa 25
<210> 38
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
ctgaagtaga aggctggatt 20
<210> 39
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
caccttgctt ttgatttcac atg 23
<210> 40
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
gttgttagac cgttggtagc ttga 24
<210> 41
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
ataaacactg tctgtatgga tg 22
<210> 42
<211> 416
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
cgccccaggg gcccagcaga agccaccaag aggcaacaga cagaggacca ggagcaccgc 60
ccagaagcga cccccgaagg gacccgccac gaaggggagc cccggagaac cgagacacca 120
agaacaccag gagaccaaga cggaaggcga gagcggagca ccaggcccgg aaagagcagg 180
cacccggagc aaaagcccac gacgcccacc gaaagagagg aaccagaaac aagcaacaca 240
aagggaaacg ggacaaccca cgaccgcggc aagaagaaaa gaacaaaaaa ggccaaggaa 300
gggggacagg caaaacgaag acaaagaggg cgagcgcgag gcggcagccg gagaggccgg 360
caagccgcag caaacaaccc caacccaaga ggagagaaaa cccgcccccc gggaaa 416
<210> 43
<211> 17
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
gcacccggag caaaagc 17
<210> 44
<211> 20
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
gggagaacaa ccagaaaacc 20
<210> 45
<211> 13
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
ccaccgaaag aga 13
Claims (10)
1. A CD74-ROS1 rearranged DNA standard for molecular diagnosis, which is characterized in that the nucleotide sequence of the DNA standard is shown as SEQ ID NO. 35.
2. A CD74-ROS1 rearranged RNA standard for molecular diagnosis, which is characterized in that the nucleotide sequence of the RNA standard is shown as SEQ ID NO. 42.
3. A cell for producing the DNA standard according to claim 1 or the RNA standard according to claim 2, wherein the genome of the cell is recombined with the nucleotide sequence of the DNA standard according to claim 1.
4. The cell of claim 3, wherein the cell is a host cell of a human immortalized cell line;
the host cell is selected from one of HEK293, HCT116, DLD-1, RKO and SW 48.
5. The method for producing a cell for producing a DNA standard or an RNA standard according to claim 3 or 4, comprising the steps of:
(1) the sgRNA of intron 6 of the CD74 gene is constructed on a vector pX330 to obtain a plasmid C4;
(2) constructing sgRNA aiming at intron 33 of ROS1 gene on a vector pX330 to obtain a plasmid R3;
(3) co-transfecting host cells with the plasmid C4 and the plasmid R3 to obtain recombinant cells;
(4) and (3) performing monoclonality on the recombinant cells to obtain cells for preparing a DNA standard substance or an RNA standard substance.
6. The method for preparing a cell that is a DNA or RNA standard according to claim 5, wherein the sgRNA for intron 6 of CD74 in step (1) has a sequence shown in SEQ ID NO. 18.
7. The method for preparing a cell for a DNA or RNA standard according to claim 6, wherein the sequence of sgRNA directed to intron 33 of ROS1 gene in step (2) is shown in SEQ ID NO. 27.
8. The method for producing a cell for producing a DNA or RNA standard according to claim 7, wherein the single cloning in the step (4) is performed by a limiting dilution method.
9. Use of the CD74-ROS1 rearranged DNA standard of claim 1 or the CD74-ROS1 rearranged RNA standard of claim 2 in the preparation of a kit for detecting CD74-ROS1 rearrangement.
10. Use of the CD74-ROS1 rearranged DNA standard of claim 1 or the CD74-ROS1 rearranged RNA standard of claim 2 for performance evaluation in LDT or IVD development systems.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114381523A (en) * | 2022-01-11 | 2022-04-22 | 仁宽(上海)生物科技有限公司 | EGFR vIII rearrangement DNA standard substance and RNA standard substance for molecular diagnosis and application thereof |
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