CN219709496U - RNA kit for detecting PD-1/PD-L1 expression quantity - Google Patents

RNA kit for detecting PD-1/PD-L1 expression quantity Download PDF

Info

Publication number
CN219709496U
CN219709496U CN202320291427.6U CN202320291427U CN219709496U CN 219709496 U CN219709496 U CN 219709496U CN 202320291427 U CN202320291427 U CN 202320291427U CN 219709496 U CN219709496 U CN 219709496U
Authority
CN
China
Prior art keywords
detecting
expression level
reagent tube
kit
forward primer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202320291427.6U
Other languages
Chinese (zh)
Inventor
童云广
唐剑
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hangzhou Aoming Ruikang Medical Laboratory Co ltd
Original Assignee
Hangzhou Aoming Ruikang Medical Laboratory Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hangzhou Aoming Ruikang Medical Laboratory Co ltd filed Critical Hangzhou Aoming Ruikang Medical Laboratory Co ltd
Priority to CN202320291427.6U priority Critical patent/CN219709496U/en
Application granted granted Critical
Publication of CN219709496U publication Critical patent/CN219709496U/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Landscapes

  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The utility model discloses an RNA kit for detecting the expression quantity of PD-1/PD-L1. The kit comprises a kit body and a kit cover, wherein a reaction liquid and a quality control product for detecting the PD-1/PD-L1 expression quantity are arranged in the kit body, the reaction liquid contains specific forward and reverse primers for detecting the PD-1/PD-L1 expression quantity, the quality control product comprises a negative quality control product, a positive quality control product and an internal reference, the negative quality control product is sterilized nuclease-free water, the positive quality control product is a gene editing cell line PD-L1 expression detection cell strain, and the internal reference is antibodies of housekeeping genes TKT, GP1, RPL-11, USP11 and HADHAI. In addition, hold chamber top and inboard and be provided with cap and the protection sponge of antibacterial and antibiotic function respectively, the supporting use of cap and protection sponge can prevent that the reagent pipe that is equipped with reaction liquid and quality control article from moving at will in holding the intracavity, guarantees transportation safety. The kit has the characteristics of high PD-1/PD-L1 expression detection specificity, good stability, high accuracy, high sensitivity and low cost.

Description

RNA kit for detecting PD-1/PD-L1 expression quantity
Technical Field
The utility model belongs to the technical field of biomedicine, and relates to an RNA kit for detecting PD-1/PD-L1 expression quantity.
Background
Along with the gradual application of the immunotherapeutic drugs in the tumor treatment process, the accurate detection of the highly expressed PD-1/PD-L1 expression level of tumor tissues or cells of cancer patients becomes the clinical key of personalized immunotherapy. Since PD-1/PD-L1 is found to be significantly up-regulated in a variety of tumor tissues, it is currently widely accepted as one of the broad biomarkers for tumor immunotherapy.
Currently, the PD-1/PD-L1 detection methods mainly comprise various methods such as Immunohistochemical (IHC), enzyme-linked immunosorbent assay (ELISA), quantitative Immunofluorescence (IF) and Flow Cytometry (FC). IHC is the most widely used detection method due to its high efficiency and rapidity. However, due to the objective factors such as dyeing technology and conditions, antibody specificity and pathological evaluation criteria, the sensitivity, specificity and stability of the existing PD-1/PD-L1 detection kit are not high. In other words, the clinical detection value of PD-1/PD-L1 is low. Therefore, it is very necessary to develop a detection kit which is easy to unify and quantifiable clinically and accurately detects cancer patients with high expression of PD-1/PD-L1, so as to provide guidance and basis for personalized immunotherapy.
In view of this, the applicant of the present utility model discloses an RNA kit for detecting the expression level of PD-1/PD-L1. The application of the utility model not only enriches the technical means of the existing PD-1/PD-L1 detection, but also provides a trampling basis for improving the clinical benefit of the immunotherapy of cancer patients.
Disclosure of Invention
The technical problem to be solved by the utility model is to provide a kit for detecting the PD-1/PD-L1 expression level of a cancer patient and providing a trampling basis for improving the immunotherapy, which can overcome the problems or partially solve the problems, thereby improving the immunotherapy quality and clinical benefit of the cancer patient.
In order to achieve the above purpose, the utility model adopts the basic conception of the technical proposal that: the sensitivity, the specificity and the stability of PD-1/PD-L1 detection are improved through screening housekeeping genes, designing a specificity amplification primer of the housekeeping genes and designing a specificity amplification primer of the PD-1/PD-L1.
The technical scheme of the utility model is as follows:
the RNA kit for detecting the PD-1/PD-L1 expression quantity comprises a kit body and a kit cover, wherein a reaction liquid and a quality control product for detecting the PD-1/PD-L1 expression quantity are arranged in the kit body, the reaction liquid contains specific forward and reverse primers for detecting the PD-1/PD-L1 expression quantity, the quality control product comprises a negative quality control product, a positive quality control product and an internal reference, the negative quality control product is sterilized nuclease-free water, the positive quality control product is a gene editing cell line PD-L1 expression detection cell strain, and the internal reference is antibodies of housekeeping genes TKT, GP1, RPL-11, USP11 and HADHAI.
Further, the box cover and the box body of the kit are connected in a buckle mode.
Further, a plurality of cubic column-shaped accommodating cavities are arranged in the box body, and the accommodating cavities are arranged to accommodate reagent tubes filled with reaction liquid, wherein the reagent tubes comprise a specific forward primer reagent tube for detecting the PD-1/PD-L1 expression quantity, a specific reverse primer reagent tube for detecting the PD-1/PD-L1 expression quantity, a sterilized nuclease-free water negative quality control product reagent tube for detecting the PD-1/PD-L1 expression quantity, a gene editing cell line PD-L1 expression detection cell line positive quality control product reagent tube for detecting the PD-1/PD-L1 expression quantity, an internal reference housekeeping gene TKT antibody reagent tube for detecting the PD-1/PD-L1 expression quantity, an internal reference housekeeping gene GP1 antibody reagent tube for detecting the PD-1/PD-L1 expression quantity, an internal reference housekeeping gene RPL-11 antibody reagent tube for detecting the PD-1/PD-L1 expression quantity, an internal reference housekeeping gene USP11 antibody reagent tube for detecting the PD-1/PD-L1 expression quantity and an internal reference gene HAAI reagent tube for detecting the PD-1/PD-L1 expression quantity.
Further, a protective sponge is arranged between the inner wall of the accommodating cavity and the reagent tube, the protective sponge is made of gel, and the gel contains 20-35% of bamboo rollers by mass percent.
Further, the accommodating cavity comprises a cap, the cap is made of gel, and the gel contains 20-35% of bamboo by mass percent.
Further, the RNA kit for detecting the PD-1/PD-L1 expression level comprises specific forward and reverse primers for detecting the PD-1/PD-L1 expression level, wherein the specific forward and reverse primers are respectively a housekeeping gene TKT, a housekeeping gene GP1, a housekeeping gene RPL-11, a housekeeping gene USP11, a housekeeping gene HADHAI, a PD-1 gene PDCD-1 and a PD-L1 gene CD 274.
Further, the specific forward primer reagent tube for detecting the PD-1/PD-L1 expression level of the RNA kit comprises a specific forward primer reagent tube of a housekeeping gene TKT, a specific forward primer reagent tube of a housekeeping gene GP1, a specific forward primer reagent tube of a housekeeping gene RPL-11, a specific forward primer reagent tube of a housekeeping gene USP11, a specific forward primer reagent tube of a housekeeping gene HADHAI, a specific forward primer reagent tube of a PD-1 gene PDCD-1, a specific forward primer reagent tube of a PD-L1 gene CD274, and a specific reverse primer reagent tube for detecting the PD-1/PD-L1 expression level comprises a specific reverse primer reagent tube of a housekeeping gene TKT, a specific reverse primer reagent tube of a housekeeping gene GP1, a specific reverse primer reagent tube of a housekeeping gene RPL-11, a specific reverse primer reagent tube of a housekeeping gene USP11, a specific reverse primer reagent tube of a housekeeping gene HADHAI, a specific reverse primer reagent tube of a housekeeping gene PD-1, and a specific reverse primer reagent tube of a PD-L1 gene PDCP-1.
Further, the forward primer sequence of the housekeeping gene TKT is shown as SEQ ID 1, and the reverse primer sequence is shown as SEQ ID 2:
forward primer: 5 '-TCATGTCTGGCGACGTCGA-3' -SEQ ID 1,
reverse primer: 5 '-GGACTGTGCGATCTCGTAT-3' -SEQ ID 2;
the forward primer sequence of the housekeeping gene GP1 is shown as SEQ ID 3, and the reverse primer sequence is shown as SEQ ID 4:
forward primer: 5 '-CACTCGTCGGGTCGACGCA-3' -SEQ ID 3,
reverse primer: 5 '-CTTTCTGTTGTCGTAGTTT-3' -SEQ ID 4;
the forward primer sequence of the housekeeping gene RPL-11 is shown as SEQ ID 5, and the reverse primer sequence is shown as SEQ ID 6:
forward primer: 5 '-ATGAACCACCCGGAGCATG-3' -SEQ ID 5,
reverse primer: 5 '-CTGGAAGTAGCACCCGAAA-3' -SEQ ID 6;
the forward primer sequence of the housekeeping gene USP11 is shown as SEQ ID 7, and the reverse primer sequence is shown as SEQ ID 8:
forward primer: 5 '-GGCGCCTTCTTCCTACAAG-3' -SEQ ID 7,
reverse primer: 5 '-CCAAGAGAAGAAGGGTCCT-3' -SEQ ID 8;
the forward primer sequence of the housekeeping gene USP11 is shown as SEQ ID 9, and the reverse primer sequence is shown as SEQ ID 10:
forward primer: 5 '-CCATCCTCTCGACACGGACT-3' -SEQ ID 9,
reverse primer: 5 '-GTTGAGATGTACATCCTATT-3' -SEQ ID 10;
The forward primer sequence of the gene PDCD-1 is shown as SEQ ID 11, and the reverse primer sequence is shown as SEQ ID 12:
forward primer: 5 '-GCCCTGAGACAAGCGAGTGT-3' -SEQ ID 11,
reverse primer: 5 '-CCTCTCTACGCAGGTCGAAT-3' -SEQ ID 12;
the forward primer sequence of the gene CD274 is shown as SEQ ID 13, and the reverse primer sequence is shown as SEQ ID 14:
forward primer: 5 '-GCGACACCTCTAACTGTGA-3' -SEQ ID 13
Reverse primer: 5 '-GAACGTCGGACAAGAGTAG-3' -SEQ ID 14.
Further, the RNA kit for detecting the PD-1/PD-L1 expression quantity also comprises reverse transcription pre-mixed solution, library amplification mixed solution A, library amplification mixed solution B, 100% ethanol, PCR grade water, magnetic beads and buffer solution;
preferably, the reverse transcription premix comprises LabScript M-MuLV RTase, RNase Inhibitor, random primers, oligo dT Primer, dNTP mix, and tris buffer;
preferably, pool amplification mix a: tris buffer, dNTP mix and AmpliTaq Gold DNA polymerase;
preferably, pool amplification mix B: tris buffer, dNTP mix and Taq DNA polymerase.
Further, an RNA kit for detecting the expression level of PD-1/PD-L1, wherein the use method of the RNA kit comprises the following steps: firstly, extracting RNA in a sample to be detected and reversely transcribing the RNA into cDNA; secondly, specifically amplifying PD-1 genes, PD-L1 genes, housekeeping genes TKT, housekeeping genes GP1, housekeeping genes RPL-11, housekeeping genes USP11 and housekeeping genes HADHAI by taking cDNA as a template; thirdly, constructing a sequencing library and sequencing; fourthly, analyzing the sequencing data, calculating the multiple of the sequencing data quantity of the housekeeping genes TKT, the housekeeping genes GP1, the housekeeping genes RPL-11, the housekeeping genes USP11 and the housekeeping genes HADHAI relative to the standard value, acquiring the median, and simultaneously normalizing the PD-1/PD-L1 expression quantity to obtain an RPM-PDL1 value; fifth step, result report: if RPM-PDL1 is more than or equal to 5, the sample to be tested is a positive sample; if RPM-PDL1 is less than 1, the sample to be tested is a negative sample; if 1 is less than or equal to RPM-PDL1 is less than 5, the sample is a weak positive sample.
Further, the cancer sample includes cells, tissues, serum, plasma and culture supernatant; the tissue includes a tissue sample or a puncture sample of a cancer patient that cannot be soaked with a tissue preservation solution for conventional IHC detection.
Furthermore, the utility model provides an application technical scheme for monitoring immune response of the RNA kit in the cancer immune treatment process, wherein housekeeping genes in the application technical scheme comprise TKT, GP1, RPL-11, USP11 and HADHAI.
Furthermore, the utility model provides an application technical scheme of the RNA kit in predicting the curative effect of tumor immunity drugs, wherein the tumor immunity drugs comprise PD-1 inhibitor, PD-L1 inhibitor, CTLA-4 inhibitor, mRNA drug and annular RNA drug.
Further, a preferred application technical scheme of the RNA kit for detecting the PD-1/PD-L1 expression quantity is tumor immune medicine curative effect prediction application, and the method specifically comprises the following steps:
the tumor tissue or peripheral blood is extracted on the 7 th day after the patient is treated by the tumor immune medicine in a large dose for 4 days, the PD-1/PD-L1 expression amount is detected on the tumor tissue or the extracted serum, a report result is obtained, and if the reduction ratio of the PD-1/PD-L1 expression amount is more than or equal to 25%, the tumor immune medicine treatment can be judged to be effective. In addition, if the test sample reports positive results, it is recommended to consider the conventional tumor immune drug treatment such as PD-1 inhibitor and PD-L1 inhibitor.
Furthermore, the preferred application technical scheme of the RNA kit for detecting the PD-1/PD-L1 expression quantity is the personalized customization of the programmable annular RNA tumor immunity medicament, and the method comprises the following steps of:
after the patient receives the high-dose tumor immune medicine treatment for 4 days, tumor tissues or peripheral blood is extracted on the 7 th day, the PD-1/PD-L1 expression level of the tumor tissues or the extracted serum is detected, a report result is obtained, and if the reduction ratio of the PD-1/PD-L1 expression level is lower than 25%, the treatment effect of the tumor immune medicine is limited, and early stage entering into two-line treatment or individual customized treatment of the programmable annular RNA tumor immune medicine is recommended. Furthermore, if the test sample reports a negative or weak positive result, it is recommended to enter two-line therapy early or to conduct personalized treatment of the programmable circular RNA tumor immunity drug.
The beneficial effects of the utility model are as follows:
firstly, an RNA kit for detecting the PD-1/PD-L1 expression quantity is provided, and the problems of low sensitivity, low specificity and poor stability of the existing PD-1/PD-L1 detection kit are solved, so that a doctor can be accurately assisted to formulate an immunotherapy strategy of a cancer patient, and the clinical treatment benefit of the cancer patient is practically improved;
Secondly, providing the application of the RNA kit in predicting the curative effect of the annular RNA medicine, and predicting the immunotherapy effect of a patient in early stage according to the change of the PD-1/PD-L1 expression quantity, and enabling the patient who is not effective in treatment to enter second-line treatment or personalized immunotherapy annular RNA medicine customization in early stage, so that unnecessary immunotherapy is avoided, the adverse effect of the annular RNA is avoided, and the problem that the existing annular RNA tumor type immune medicine curative effect prediction index is lacking is solved;
thirdly, the containing cavity of the kit is provided with the protective sponge and the cap, and the matching use of the sponge and the cap can prevent the reagent tube from moving randomly in the containing cavity, so that the transportation safety is ensured; the antibacterial and antibacterial functions of the sponge and the cap are beneficial to guaranteeing the physical health and life safety of detection staff.
Drawings
FIG. 1 is a top view of the RNA kit body and cover for PD-1/PD-L1 expression level detection according to the present utility model.
FIG. 2 is a perspective view of an RNA kit body for detecting the expression level of PD-1/PD-L1 according to the present utility model.
FIG. 3 is a cross-sectional view of one of the accommodation cavities 3 in the case of the RNA kit for detecting the expression level of PD-1/PD-L1 according to the present utility model.
Description of the embodiments
The utility model will be described in detail below with reference to the drawings in connection with embodiments. It should be noted that, without conflict, the embodiments of the present utility model and features of the embodiments may be combined with each other. For convenience of description, the words "upper", "lower", "left" and "right" are used hereinafter to denote only the directions corresponding to the upper, lower, left, and right directions of the drawings, and do not limit the structure. The examples of the present utility model are merely for the purpose of explaining the present utility model and are not intended to limit the present utility model, and the examples of the present utility model are not limited to the examples given in the specification. The specific experimental or operating conditions were not noted in the examples and were made under conventional conditions or under conditions recommended by the material suppliers. Furthermore, it is to be understood that the reference to one or more method steps in this disclosure does not exclude the presence of other method steps before or after the combination step or the insertion of other method steps between these explicitly mentioned steps, unless otherwise indicated; it is also to be understood that the use of a combination connection in accordance with the utility model does not exclude the presence of other connections before or after the combination or the insertion of other connections between the two explicitly mentioned connections, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the method steps is merely a convenient tool for identifying the method steps and is not intended to limit the order of arrangement of the method steps or to limit the scope of the utility model in which the utility model may be practiced, as such changes or modifications in their relative relationships may be regarded as within the scope of the utility model without substantial modification to the technical matter. In the examples described below, reagents, materials and apparatus used are commercially available unless otherwise specified.
The components of the box body 1, the box cover 2, the accommodating cavities 3-30, the protective sponge 31 and the cap 32 are all universal standard components or components known to a person skilled in the art, and the structure and the principle of the components are all known to the person skilled in the art through technical manuals or through routine experimental methods.
Example 1
As shown in the accompanying drawings 1 and 2, the RNA kit for detecting the PD-1/PD-L1 expression quantity comprises a kit body 1 and a kit cover 2, the kit body 1 is internally provided with a containing cavity 3 for placing a reverse transcription premix reagent tube, a containing cavity 4 for placing a library amplification mixed liquor A reagent tube, a containing cavity 5 for placing a library amplification mixed liquor B reagent tube, a containing cavity 6 for placing a 100% ethanol reagent tube, a containing cavity 7 for placing a PCR-grade water reagent tube, a containing cavity 8 for placing a magnetic bead reagent tube, a containing cavity 9 for placing a buffer solution, a containing cavity 10 for placing a housekeeping gene TKT specific forward primer reagent tube, a containing cavity 11 for placing a housekeeping gene TKT specific reverse primer reagent tube, a containing cavity 12 for placing a housekeeping gene GP1 specific forward primer reagent tube, a containing cavity 13 for placing a housekeeping gene GP1 specific reverse primer reagent tube, a containing cavity 14 for placing a housekeeping gene RPL-11 specific forward primer reagent tube the method comprises the steps of placing a containing cavity 15 of a housekeeping gene RPL-11 specific reverse primer reagent tube, placing a containing cavity 16 of a housekeeping gene USP11 specific forward primer reagent tube, placing a containing cavity 17 of a housekeeping gene USP11 specific reverse primer reagent tube, placing a containing cavity 18 of a housekeeping gene HADHAI specific forward primer reagent tube, placing a containing cavity 19 of a housekeeping gene HADHAI specific reverse primer reagent tube, placing a containing cavity 20 of a PDCD-1 gene specific forward primer reagent tube, placing a containing cavity 21 of a PDCD-1 gene specific reverse primer reagent tube, placing a containing cavity 22 of a P5 primer reagent tube, placing a containing cavity 23 of a P7 primer reagent tube, placing a containing cavity 24 of a negative quality control sterile water reagent tube, and, A containing cavity 25 for placing a positive quality control product of a gene editing cell line PD-L1 expression detection cell strain, a containing cavity 26 for placing an internal reference housekeeping gene TKT antibody, a containing cavity 27 for placing an internal reference housekeeping gene GP1 antibody, a containing cavity 28 for placing an internal reference housekeeping gene RPL-11 antibody, a containing cavity 29 for placing an internal reference housekeeping gene USP11 antibody and a containing cavity 30 for placing an internal reference housekeeping gene HADHAI antibody;
The forward primer sequence of the housekeeping gene TKT is shown as SEQ ID 1, and the reverse primer sequence is shown as SEQ ID 2:
forward primer: 5 '-TCATGTCTGGCGACGTCGA-3' -SEQ ID 1,
reverse primer: 5 '-GGACTGTGCGATCTCGTAT-3' -SEQ ID 2;
the forward primer sequence of the housekeeping gene GP1 is shown as SEQ ID 3, and the reverse primer sequence is shown as SEQ ID 4:
forward primer: 5 '-CACTCGTCGGGTCGACGCA-3' -SEQ ID 3,
reverse primer: 5 '-CTTTCTGTTGTCGTAGTTT-3' -SEQ ID 4;
the forward primer sequence of the housekeeping gene RPL-11 is shown as SEQ ID 5, and the reverse primer sequence is shown as SEQ ID 6:
forward primer: 5 '-ATGAACCACCCGGAGCATG-3' -SEQ ID 5,
reverse primer: 5 '-CTGGAAGTAGCACCCGAAA-3' -SEQ ID 6;
the forward primer sequence of the housekeeping gene USP11 is shown as SEQ ID 7, and the reverse primer sequence is shown as SEQ ID 8:
forward primer: 5 '-GGCGCCTTCTTCCTACAAG-3' -SEQ ID 7,
reverse primer: 5 '-CCAAGAGAAGAAGGGTCCT-3' -SEQ ID 8;
the forward primer sequence of the housekeeping gene USP11 is shown as SEQ ID 9, and the reverse primer sequence is shown as SEQ ID 10:
forward primer: 5 '-CCATCCTCTCGACACGGACT-3' -SEQ ID 9,
reverse primer: 5 '-GTTGAGATGTACATCCTATT-3' -SEQ ID 10;
The forward primer sequence of the gene PDCD-1 is shown as SEQ ID 11, and the reverse primer sequence is shown as SEQ ID 12:
forward primer: 5 '-GCCCTGAGACAAGCGAGTGT-3' -SEQ ID 11,
reverse primer: 5 '-CCTCTCTACGCAGGTCGAAT-3' -SEQ ID 12;
the forward primer sequence of the gene CD274 is shown as SEQ ID 13, and the reverse primer sequence is shown as SEQ ID 14:
forward primer: 5 '-GCGACACCTCTAACTGTGA-3' -SEQ ID 13
Reverse primer: 5 '-GAACGTCGGACAAGAGTAG-3' -SEQ ID 14;
further, as shown in fig. 3, a protective sponge shown as 31 is arranged between all reagent tubes and the inner walls of all accommodating cavities in the kit, and caps shown as 32 are arranged in all accommodating cavities, so that each accommodating cavity can limit the movement of the placed reagent tube, and the transportation safety is ensured; the antibacterial function of the protective sponge and the cap is used for guaranteeing the physical health and life safety of detection staff.
Example 2 an example of a detection procedure of a tumor tissue sample by an RNA kit for detecting the expression level of PD-1/PD-L1
Firstly, checking whether the tissue sample meets the experimental condition requirement. If the tissue sample is a fresh tissue sample within 30 minutes after operation, the tissue sample can be placed in a preservation solution for normal temperature transportation, and then sent to a laboratory within 48 hours or placed in a freezing storage tube after relevant treatment, dry ice transportation is carried out, and the tissue sample is sent to the laboratory within 36 hours, and the tumor tissue sample is more than or equal to 50 mg, the normal tissue beside cancer is more than or equal to 25 mg, the tumor tissue ratio is more than 20%, and the necrotic tumor tissue ratio is less than 30%; if the tissue is fresh, the tissue can be placed in a preservation solution for normal temperature transportation, and then delivered to a laboratory within 48 hours or placed in a freezing tube after relevant treatment, and dry ice is delivered to the laboratory within 36 hours, wherein the number of the tissue is 2-3, the length is more than 1 cm, the tumor tissue ratio is more than 20%, and the necrotic tumor tissue ratio is less than 30%; determining the type of preservation solution, and recording in time, wherein the preservation solution is 10 times of 10% neutral formalin solution; if the sample is a wax block sample, 25 mg of postoperative tissue or 2-3 biopsies with the length of more than 1 cm are required to be placed in an embedding box, the content of tumor cells is more than 20 percent, the tumor cells are not more than 18 months, HE (human immunodeficiency virus) stained sections are arranged, the thickness is between 5 and 10 microns, and the tissue is transported at normal temperature; if the sample is a postoperative/puncture wax roll sample, the slice thickness is more than or equal to 5 microns, the number of postoperative samples is 10-15 rolls, the number of puncture samples is 20-25 rolls, the sample is placed in a 1.5 ml sterile EP tube, the sealing film is sealed, the tumor cell content is more than 20%, the sample is not more than 18 months, HE (high-density polyethylene) dyed slices are available, the thickness is 5-10 microns, and the sample is transported at normal temperature; if the sample is a postoperative/puncture slice sample, the slice with the thickness of more than or equal to 5 micrometers is placed in the center of an adhesive glass slide subjected to anti-drop treatment, the sample is continuous, intact, flat and wrinkle-free, bubble-free or obvious knife mark-free, the number of the postoperative sample is 10-15 rolls, the number of the puncture sample is 20-25 rolls, the content of tumor cells is more than 20 percent, the tumor cell content is not more than 18 months, HE (high-intensity) stained slices are arranged, and the samples are transported at normal temperature;
Secondly, processing samples meeting the requirements according to a corresponding processing method: if the sample preservation solution is tissue preservation solution, 1 XPBS is added after the protective solution in the tube is absorbed completely, the mixture is inverted, mixed and washed for two times, then the mixture is placed in a fume hood, 10-30 mg of tissue sample is cut after the inner wall of the freezing preservation tube is dried, and if the extraction operation is not immediately carried out, the tissue sample is placed in a corresponding freezing preservation box for preservation at the temperature of minus 80 ℃; if the sample preservation solution is formalin solution, a proper amount of tissue samples are taken, washed twice with 1 ml of 70% medical alcohol, washed twice with 1 ml of PBS solution, and then slightly air-dried to perform nucleic acid extraction operation, if the extraction operation is not performed immediately, the tissue samples are placed in corresponding freezing boxes (the sample preservation solution is not discarded) and preserved at the temperature of minus 20 ℃; if the sample is a wax block sample, a postoperative/puncture wax roll sample and a postoperative/puncture section sample, the nucleic acid extraction operation can be directly carried out, if the extraction operation is not carried out immediately, the sample is stored in a corresponding sample storage box at normal temperature;
thirdly, adding TRNzol Universal reagent into the tissue sample at room temperature; homogenizing with a homogenizer, wherein the volume of the TRNzol Universal reagent is 10 times that of the sample, standing the homogenized sample at room temperature for 5 minutes, adding chloroform with the volume of the TRNzol Universal reagent being 20% of that of the TRNzol Universal reagent, standing for 3 minutes at room temperature after mixing, centrifuging at 12000rpm for 15 minutes at 4 ℃, and transferring the upper water phase into a new centrifuge tube;
Fourthly, adding equal volume of isopropanol into the aqueous phase solution obtained in the third step, uniformly mixing, standing for 10 minutes at room temperature, centrifuging for 10 minutes at 12000rpm at 4 ℃, removing supernatant, adding 75% ethanol solution prepared by using coreless water, washing precipitate, centrifuging for 5 minutes at 10000rpm at 4 ℃, pouring out liquid, centrifuging the rest liquid for a short time, sucking out by using a gun head, airing RNA precipitate at room temperature for 2-3 minutes, adding 30-100 microliters of coreless water, repeatedly blowing and uniformly mixing until the RNA precipitate is fully dissolved; the OD value was then measured to quantify the RNA concentration and the integrity of the RNA was checked using a 1% agarose gel:
fifthly, fully and uniformly mixing 3 microliters of reverse transcription mixed solution and 10 nanograms of RNA obtained in the fourth step, diluting to 10 microliters with coreless water, placing into a PCR instrument, sequentially reacting at 25 ℃ for 12.5 minutes, 45 ℃ for 45 minutes, 90 ℃ for 2 minutes, and finally placing at 4 ℃ to reverse the RNA into cDNA;
sixth, diluting 10 nanograms of cDNA obtained in the fifth step to 10 microliters by using Tris-HCl buffer solution, sequentially adding 2 microliters of library amplification solution A, 5 x forward amplification primers and 5 x reverse amplification primers, thoroughly mixing and centrifuging, then placing a PCR plate filled with the mixed solution in a PCR instrument at a thermal cover of 105 ℃, placing for 15 minutes at 94 ℃, placing for 20 seconds at 94 ℃, amplifying for 12 times at 60 ℃ for 5 minutes each time, placing for 20 minutes at 99 ℃ and then maintaining at 4 ℃;
Seventhly, adding 25 microlitres of magnetic beads thoroughly vortex-mixed into the PCR reaction liquid obtained in the sixth reaction, blowing and mixing the mixture up and down by a pipettor, incubating the mixture for 10 minutes at room temperature, standing the mixture on a magnetic rack for 5 minutes, collecting the magnetic beads until the liquid is clear, discarding the supernatant, adding 175 microlitres of 80% ethanol, incubating the mixture at room temperature for 1 minute, discarding the supernatant, adding 200 microlitres of 80% ethanol again, performing second washing, discarding all ethanol residues, airing the mixture at room temperature for 3 minutes, taking off a PCR plate from the magnetic rack, adding 15 microlitres of Tris-HCl buffer solution into the residue, blowing and mixing the mixture up and down, incubating the mixture at room temperature for 3 minutes, eluting the sample from the magnetic beads, putting the PCR plate on the magnetic rack for 3 minutes, and collecting the magnetic beads;
step eight, taking 10 microliters of the PCR reaction liquid sample eluted by the magnetic beads in the seventh reaction, sequentially adding 5 microliters of library amplification liquid B,2 microliters of primer P5 and 2 microliters of primer P7, diluting to 20 microliters by using Tris-HCl buffer solution, thoroughly mixing and centrifuging, placing the PCR plate in a PCR instrument at a 105 ℃ heat cover, placing at 94 ℃ for 5 minutes, then placing at 94 ℃ for 20 seconds, placing at 60 ℃ for 30 seconds, placing at 70 ℃ for 45 seconds and circulating for 20 times, finally extending at 70 ℃ for 90 seconds, and maintaining at 4 ℃ after extension;
A ninth step of adding 25 microliters of magnetic beads thoroughly vortex-mixed into the PCR reaction solution obtained in the eighth step, uniformly mixing the mixture by blowing up and down with a pipettor, incubating the mixture at room temperature for 10 minutes, standing the mixture on a magnetic rack for 5 minutes, collecting the magnetic beads until the liquid is clear, discarding the supernatant, then adding 175 microliters of 80% ethanol, incubating the mixture at room temperature for 1 minute, discarding the supernatant, adding 175 microliters of 80% ethanol again, performing a second washing, discarding all ethanol residues, airing the mixture at room temperature for 3 minutes, removing a PCR plate from the magnetic rack, adding 20 microliters of Tris-HCl buffer solution into the residue, uniformly blowing up and down, incubating the mixture at room temperature for 3 minutes, eluting the sample from the magnetic beads, placing the PCR plate on the magnetic rack for 3 minutes, and collecting the magnetic beads;
and a tenth step of taking 20 microliters of the PCR reaction liquid sample eluted by the separation magnetic beads in the ninth step of reaction, putting the sample into a low straw, and quantifying and sequencing the library by an upper sequencing platform according to a standardized flow. The quality control standard of the sample sequencing result is HK not less than 10, on_target_ratio not less than 1, and mapped_Reads >400K. And after the sample sequencing result reaches the quality control requirement, carrying out analysis on the expression condition of PD-1/PD-L1. And (3) determining the result: the threshold value of PD-L1 negative in the sample is RPM-PDL1=1, if the RPM-PDL1 of the target gene PD-L1 of the tested sample is less than 1, the sample is negative; the threshold value of positive of PD-L1 in the sample is RPM-PDL1=5, if the RPM-PDL1 of the target gene PD-L1 of the tested sample is more than 5, the sample is positive; if the RPM-PDL1 of the target gene PD-L1 of the sample is 1 < RPM-PDL 1.ltoreq.5, the sample is a weak positive sample.
Example 3 an example of a detection procedure of a tumor blood sample by an RNA kit for detecting the expression level of PD-1/PD-L1
Firstly, taking 150 microliters of peripheral blood which meets the requirement and is thawed on ice after being frozen in a refrigerator at the temperature of minus 80 ℃ or freshly collected peripheral blood, putting the peripheral blood into a 1.5 milliliter EP tube, then adding 1 milliliter of Trizol, blowing and mixing the peripheral blood with a liquid-transferer, placing the peripheral blood on ice for 5 minutes, adding 200 microliters of chloroform, fully blowing and mixing the peripheral blood with the liquid-transferer, and placing the peripheral blood on ice for 15 minutes;
secondly, adding chloroform into the mixed solution after being placed on ice in the first step at 4 ℃, centrifuging at 15000g for 10 minutes, then gently sucking the upper water phase into a new 1.5 ml EP tube, simultaneously adding 500 microliters of isopropanol, uniformly mixing, placing on ice for 10 minutes, centrifuging at 12000g for 10 minutes at 4 ℃, discarding the supernatant, and depositing RNA at the bottom of the tube;
thirdly, adding 1 ml of 75% ethanol to the RNA which is above the bottom of the tube, gently oscillating the EP tube to enable the precipitate to be fully dissolved and suspended, centrifuging at 10000g for 5 minutes at 4 ℃, removing all the supernatant as much as possible, airing at room temperature for 10 minutes until the alcohol is not found, adding 20 microlitres of sterilized DEPC water to dissolve the RNA sample, then standing at 4 ℃ for 15 minutes to measure the OD value and quantify the concentration of the RNA, and detecting the integrity of the RNA by adopting 1% agar gel:
Fourthly, adding equal volume of isopropanol into the aqueous phase solution obtained in the third step, uniformly mixing, standing for 10 minutes at room temperature, centrifuging for 10 minutes at 12000rpm at 4 ℃, removing supernatant, adding 75% ethanol solution prepared by using coreless water, washing precipitate, centrifuging for 5 minutes at 10000rpm at 4 ℃, pouring out liquid, centrifuging the rest liquid for a short time, sucking out by using a gun head, airing RNA precipitate at room temperature for 2-3 minutes, adding 30-100 microliters of coreless water, repeatedly blowing and uniformly mixing until the RNA precipitate is fully dissolved; the OD value was then measured to quantify the RNA concentration and the integrity of the RNA was checked using a 1% agarose gel:
fifthly, fully and uniformly mixing 3 microliters of reverse transcription mixed solution and 10 nanograms of RNA obtained in the fourth step, diluting to 10 microliters with coreless water, placing into a PCR instrument, sequentially reacting at 25 ℃ for 12.5 minutes, 45 ℃ for 45 minutes, 90 ℃ for 2 minutes, and finally placing at 4 ℃ to reverse the RNA into cDNA;
sixth, diluting 10 nanograms of cDNA obtained in the fifth step to 10 microliters by using Tris-HCl buffer solution, sequentially adding 2 microliters of library amplification solution A, 10 x forward amplification primers and 10 x reverse amplification primers, thoroughly mixing and centrifuging, then placing a PCR plate filled with the mixed solution in a PCR instrument at a 105 ℃ heat cover, placing at 94 ℃ for 15 minutes, placing at 94 ℃ for 20 seconds, amplifying at 60 ℃ for 12 times for 5 minutes each time, placing at 99 ℃ for 20 minutes, and then maintaining at 4 ℃;
Seventhly, adding 25 microlitres of magnetic beads thoroughly vortex-mixed into the PCR reaction liquid obtained in the sixth reaction, blowing and mixing the mixture up and down by a pipettor, incubating the mixture for 10 minutes at room temperature, standing the mixture on a magnetic rack for 5 minutes, collecting the magnetic beads until the liquid is clear, discarding the supernatant, adding 175 microlitres of 80% ethanol, incubating the mixture at room temperature for 1 minute, discarding the supernatant, adding 200 microlitres of 80% ethanol again, performing second washing, discarding all ethanol residues, airing the mixture at room temperature for 3 minutes, taking off a PCR plate from the magnetic rack, adding 15 microlitres of Tris-HCl buffer solution into the residue, blowing and mixing the mixture up and down, incubating the mixture at room temperature for 3 minutes, eluting the sample from the magnetic beads, putting the PCR plate on the magnetic rack for 3 minutes, and collecting the magnetic beads;
step eight, taking 10 microliters of the PCR reaction liquid sample eluted by the magnetic beads in the seventh reaction, sequentially adding 5 microliters of library amplification liquid B,2 microliters of primer P5 and 2 microliters of primer P7, diluting to 20 microliters by using Tris-HCl buffer solution, thoroughly mixing and centrifuging, placing the PCR plate in a PCR instrument at a 105 ℃ heat cover, placing at 94 ℃ for 5 minutes, then placing at 94 ℃ for 20 seconds, placing at 60 ℃ for 30 seconds, placing at 70 ℃ for 45 seconds and circulating for 20 times, finally extending at 70 ℃ for 90 seconds, and maintaining at 4 ℃ after extension;
A ninth step of adding 25 microliters of magnetic beads thoroughly vortex-mixed into the PCR reaction solution obtained in the eighth step, uniformly mixing the mixture by blowing up and down with a pipettor, incubating the mixture at room temperature for 10 minutes, standing the mixture on a magnetic rack for 5 minutes, collecting the magnetic beads until the liquid is clear, discarding the supernatant, then adding 175 microliters of 80% ethanol, incubating the mixture at room temperature for 1 minute, discarding the supernatant, adding 175 microliters of 80% ethanol again, performing a second washing, discarding all ethanol residues, airing the mixture at room temperature for 3 minutes, removing a PCR plate from the magnetic rack, adding 20 microliters of Tris-HCl buffer solution into the residue, uniformly blowing up and down, incubating the mixture at room temperature for 3 minutes, eluting the sample from the magnetic beads, placing the PCR plate on the magnetic rack for 3 minutes, and collecting the magnetic beads;
and a tenth step of taking 20 microliters of the PCR reaction liquid sample eluted by the separation magnetic beads in the ninth step of reaction, putting the sample into a low straw, and quantifying and sequencing the library by an upper sequencing platform according to a standardized flow. The quality control standard of the sample sequencing result is HK not less than 10, on_target_ratio not less than 1, and mapped_Reads >400K. And after the sample sequencing result reaches the quality control requirement, carrying out analysis on the expression condition of PD-1/PD-L1. And (3) determining the result: the threshold value of PD-L1 negative in the sample is RPM-PDL1=1, if the RPM-PDL1 of the target gene PD-L1 of the tested sample is less than 1, the sample is negative; the threshold value of positive of PD-L1 in the sample is RPM-PDL1=5, if the RPM-PDL1 of the target gene PD-L1 of the tested sample is more than 5, the sample is positive; if the RPM-PDL1 of the target gene PD-L1 of the sample is 1 < RPM-PDL 1.ltoreq.5, the sample is a weak positive sample.
Example 4 detection and application examples of RNA kit for detecting tumor tissue PD-1/PD-L1 expression level of cancer patient
The detection flow of the embodiment 1 is adopted to detect the PD-1/PD-L1 expression quantity of the tumor tissues of 65 cancer patients, and the RPM-PDL1 of the tumor tissues of 24 cancer patients which are effectively treated by the PD-1 inhibitor through immunotherapy is found to be reduced after the immunotherapy, the reduction amplitude is more than 30 percent, and the reduction amplitude is consistent with the judgment of the immunohistochemical clinical detection result; whereas 41 cases of RPM-PDL1 of tumor tissues of cancer patients judged to have limited or no effect by immunotherapy with PD-1 inhibitors have the conditions of rising, unchanged or slightly falling after the immunotherapy, wherein the falling amplitude is less than 7.5%, and the falling amplitude is consistent with the judgment of immunohistochemical clinical detection results. The embodiment proves that the PD-1/PD-L1 expression quantity can be used for judging the curative effect of the PD-1 inhibitor immunotherapy, and meanwhile, the detection depth and the detection reliability of the RNA kit for detecting the PD-1/PD-L1 expression quantity provided by the utility model on the level detection of the PD-1/PD-L1 expression quantity are good, so that the curative effect condition of a cancer patient in the immunotherapy can be better judged;
at the same time, 41 cancer patients who were judged to be either limited or ineffective with PD-1 inhibitor immunotherapy were very promising to be treated with personalized, custom-made circular RNA programmable circular RNA tumor immunopharmaceuticals encoding cytokines IL-7, IL-12sc, IL-21, IL-23, IL-36 γ, GM-CSF, IFN- α and chemokines CCL3, CCL5, CCL13, CCL21, CXCL9, CXCL10, CXCL11, CX3CL 1. Because the tumor immune curative effect of the subject reaches 100% in 100 cases of the pre-clinical test of the personalized and customized annular RNA programmable tumor immune medicine, which is judged to be limited or ineffective by the PD-1 inhibitor immune treatment.
Example 5 detection and application example of PD-1/PD-L1 expression in peripheral blood of cancer patient
The detection flow of the embodiment 2 is adopted for detecting the PD-1/PD-L1 expression quantity of peripheral blood of 141 cancer patients, and 67 cancer patients which are effectively treated by the PD-L1 inhibitor through immunotherapy are found that the PD-1/PD-L1 expression quantity of the peripheral blood of the cancer patients is reduced after the immunotherapy, and the reduction amplitude is more than 30 percent and is consistent with the judgment of an immunohistochemical clinical detection result; whereas, in 74 cases of cancer patients whose RPM-PDL1 was judged to be either limited or ineffective by immunotherapy with PD-L1 inhibitors, there was an increase, a constant or a slight decrease in RPM-PDL1 following immunotherapy, with the decrease in magnitude being less than 7.5%, consistent with the immunohistochemical clinical test results. The embodiment proves that the PD-1/PD-L1 expression quantity can be used for judging the curative effect of the PD-L1 inhibitor on immunotherapy, and meanwhile, the detection depth and the detection reliability of the RNA kit for detecting the PD-1/PD-L1 expression quantity provided by the utility model on the level detection of the PD-1/PD-L1 expression quantity are good, so that the curative effect condition of a cancer patient on the immunotherapy can be better judged;
at the same time, 74 cancer patients treated with PD-L1 inhibitor immunotherapy, which were judged to be either limited or ineffective, are very promising to be treated with personalized, custom-made circular RNA programmable circular RNA tumor immunopharmaceuticals encoding cytokines IL-7, IL-12sc, IL-21, IL-23, IL-36 γ, GM-CSF, IFN- α and chemokines CCL3, CCL5, CCL13, CCL21, CXCL9, CXCL10, CXCL11, CX3CL 1. Because the tumor immune curative effect effective rate of the subjects reaches 100% in 100 cases of the pre-clinical test of the personalized and customized annular RNA programmable tumor immune medicine, wherein the PD-L1 inhibitor immune treatment is judged to be limited or ineffective.
Example 6 detection and application example of PD-1/PD-L1 expression in peripheral blood of cancer patient
The detection flow of the embodiment 2 is adopted for detecting the PD-1/PD-L1 expression level of peripheral blood of 169 cancer patients, and the fact that the PD-1/PD-L1 expression level in the peripheral blood of 85 cancer patients which are effectively treated by adopting PD-1/PD-L1 combined CTLA-4 inhibitor through immunotherapy is reduced by more than 25% after the immunotherapy, and the reduction amplitude is consistent with the judgment of an immunohistochemical clinical detection result; and 84 cases of cancer patients who are judged to have limited or invalid effects by adopting PD-1/PD-L1 combined CTLA-4 inhibitor immunotherapy have the conditions that the PD-1/PD-L1 expression level in peripheral blood is increased, unchanged or slightly reduced after the immunotherapy, wherein the reduction degree is less than 5%, and the reduction degree is consistent with the judgment of an immunohistochemical clinical detection result. The embodiment proves that the PD-1/PD-L1 expression quantity can be used for judging the curative effect of the PD-1/PD-L1 combined CTLA-4 inhibitor immunotherapy, and meanwhile, the detection depth and the detection reliability of the RNA detection kit for the PD-1/PD-L1 expression quantity provided by the utility model on the PD-1/PD-L1 expression quantity are good, so that the curative effect condition of a cancer patient in the immunotherapy can be better judged;
meanwhile, 84 cancer patients treated with PD-1/PD-L1 combined CTLA-4 inhibitor immunotherapy, which were judged to be either limited or ineffective, are expected to be treated with personalized, customized, circular RNA programmable, circular RNA tumor immunopharmaceuticals encoding cytokines IL-7, IL-12sc, IL-21, IL-23, IL-36 gamma, GM-CSF, IFN-alpha, and chemokines CCL3, CCL5, CCL13, CCL21, CXCL9, CXCL10, CXCL11, CX3CL 1. Because in 100 cases of personalized custom circular RNA programmable tumor immunity drug preclinical experiments in which the PD-1/PD-L1 combined CTLA-4 inhibitor immunotherapy is judged to be limited or ineffective, the tumor immunity efficacy efficiency of the subject reaches 100%.
Example 7 detection and application example of PD-1/PD-L1 expression amount in tumor tissue of cancer patient
The detection flow of the embodiment 1 is adopted to detect the PD-1/PD-L1 expression quantity of the tumor tissues of 59 cancer patients, and the fact that the PD-1/PD-L1 expression quantity in the tumor tissues of 33 cancer patients which are effectively treated by adopting PD-1/PD-L1 combined CTLA-4 inhibitor for immunotherapy is reduced after the immunotherapy, the reduction amplitude is more than 27%, and the reduction amplitude is consistent with the judgment of an immunohistochemical clinical detection result; and 26 cases of cancer patients who are judged to have limited or invalid effects by adopting PD-1/PD-L1 combined CTLA-4 inhibitor immunotherapy have the conditions that the PD-1/PD-L1 expression level in peripheral blood is increased, unchanged or slightly reduced after the immunotherapy, wherein the reduction degree is less than 7%, and the reduction degree is consistent with the judgment of an immunohistochemical clinical detection result. The embodiment proves that the PD-1/PD-L1 expression quantity can be used for judging the curative effect of the PD-1/PD-L1 combined CTLA-4 inhibitor immunotherapy, and meanwhile, the detection depth and the detection reliability of the RNA detection kit for the PD-1/PD-L1 expression quantity provided by the utility model on the PD-1/PD-L1 expression quantity are good, so that the curative effect condition of a cancer patient in the immunotherapy can be better judged;
meanwhile, 26 cancer patients treated with PD-1/PD-L1 in combination with CTLA-4 inhibitors are expected to be treated with personalized, customized, circular RNA programmable, circular RNA tumor immunopharmaceuticals encoding cytokines IL-7, IL-12sc, IL-21, IL-23, IL-36 gamma, GM-CSF, IFN-alpha, and chemokines CCL3, CCL5, CCL13, CCL21, CXCL9, CXCL10, CXCL11, CX3CL 1. Because in 100 cases of personalized custom circular RNA programmable tumor immunity drug preclinical experiments in which the PD-1/PD-L1 combined CTLA-4 inhibitor immunotherapy is judged to be limited or ineffective, the tumor immunity efficacy efficiency of the subject reaches 100%.
Example 8 detection and application example of PD-1/PD-L1 expression in peripheral blood of cancer patient
The detection flow of the embodiment 2 is adopted for detecting the PD-1/PD-L1 expression quantity of peripheral blood of 106 cancer patients, and the fact that the PD-1/PD-L1 expression quantity in the peripheral blood of 56 cancer patients which are effectively treated by the PD-1 inhibitor after immunotherapy is reduced, wherein the reduction amplitude is more than 30 percent, and is consistent with the judgment of an immunohistochemical clinical detection result; and 50 cases of the PD-1 inhibitor immunotherapy, which is judged to have limited or invalid effect, show the condition that the expression level of PD-1/PD-L1 in the peripheral blood of the cancer patient is increased, unchanged or slightly reduced, wherein the reduction amplitude is less than 4%, and the result is consistent with the judgment of the immunohistochemical clinical detection result. The embodiment proves that the PD-1 expression quantity can be used for judging the curative effect of the PD-1 inhibitor on immunotherapy, and meanwhile, the RNA detection kit for the PD-1/PD-L1 expression quantity provided by the utility model has good detection depth and detection reliability for detecting the PD-1/PD-L1 expression quantity, and can better judge the curative effect of a cancer patient on the immunotherapy;
meanwhile, 50 cancer patients judged to be limited or ineffective by immunotherapy with PD-1 inhibitors are expected to be treated by personalized-circular RNA programmable-circular RNA tumor immunopharmaceuticals encoding cytokines IL-7, IL-12sc, IL-21, IL-23, IL-36 gamma, GM-CSF, IFN-alpha, and chemokines CCL3, CCL5, CCL13, CCL21, CXCL9, CXCL10, CXCL11, CX3CL 1. Because the tumor immune curative effect of the subject reaches 100% in 100 cases of the pre-clinical test of the personalized and customized annular RNA programmable tumor immune medicine, which is judged to be limited or ineffective by the PD-1 inhibitor immune treatment.
Example 9 detection and application example of tumor tissue PD-1/PD-L1 expression level of cancer patient
The detection flow of the embodiment 1 is adopted to detect the PD-1/PD-L1 expression quantity of the tumor tissues of 52 cancer patients, and 34 cancer patients with effective PD-L1 inhibitor immunotherapy are found that the PD-1/PD-L1 expression quantity in the tumor tissues of the cancer patients is reduced after the immunotherapy, the reduction amplitude is more than 30 percent, and the reduction amplitude is consistent with the judgment of the immunohistochemical clinical detection result; and 18 cases of cancer patients who are judged to have limited or invalid effects by adopting PD-L1 inhibitor immunotherapy have the conditions that the PD-1/PD-L1 expression level is increased, unchanged or slightly reduced after the immunotherapy, wherein the reduction amplitude is less than 5%, and the reduction amplitude is consistent with the judgment of immunohistochemical clinical detection results. The embodiment proves that the PD-1/PD-L1 expression quantity can be used for judging the curative effect of the PD-L1 inhibitor on immunotherapy, and meanwhile, the detection depth and the detection reliability of the RNA detection kit for the PD-1/PD-L1 expression quantity provided by the utility model on the PD-1/PD-L1 expression quantity are good, so that the curative effect condition of a cancer patient on the immunotherapy can be better judged;
meanwhile, 18 cancer patients judged to be either limited or ineffective by immunotherapy with PD-L1 inhibitors are expected to be treated with personalized, custom circular RNA programmable RNA tumor immunopharmaceuticals encoding cytokines IL-7, IL-12sc, IL-21, IL-23, IL-36 gamma, GM-CSF, IFN-alpha, and chemokines CCL3, CCL5, CCL13, CCL21, CXCL9, CXCL10, CXCL11, CX3CL 1. Because the tumor immune curative effect effective rate of the subjects reaches 100% in 100 cases of the pre-clinical test of the personalized and customized annular RNA programmable tumor immune medicine, wherein the PD-L1 inhibitor immune treatment is judged to be limited or ineffective.
Example 10
The specificity evaluation of the RNA kit for detecting the PD-1/PD-L1 expression quantity is specifically as follows:
the kit is used for detecting the PD-1/PD-L1 expression level of cells, tissues, serum, plasma or culture supernatant of patients suffering from lung cancer, ovarian cancer, breast cancer, bladder cancer, head and neck cancer, hodgkin's lymphoma, melanoma, renal cell carcinoma and merkel cell carcinoma, and the specificity of the kit for detecting the PD-1/PD-L1 expression level is verified by taking sterile water as a blank control. The results showed that no positives were observed in the sterile water, indicating that the system was not contaminated. Further, the experimental results show that the cells, tissues, serum, plasma or culture supernatants of patients with the immune combination determined PD-1/PD-L1 positive lung cancer, ovarian cancer, breast cancer, bladder cancer, head and neck cancer, hodgkin's lymphoma, melanoma, renal cell carcinoma, merkel cell carcinoma have obvious positive expression of PD-1/PD-L1, while the cells, tissues, serum, plasma or culture supernatants of the patients with the immune combination determined PD-1/PD-L1 negative lung cancer, ovarian cancer, breast cancer, bladder cancer, head and neck cancer, hodgkin's lymphoma, melanoma, renal cell carcinoma, merkel cell carcinoma do not have any expression of PD-1/PD-L1. These demonstrate that the primers of the kit of the utility model have good specificity and can be used for distinguishing PD-1/PD-L1 positive ones with good specificity.
Example 11
The sensitivity evaluation of the RNA kit for detecting the PD-1/PD-L1 expression quantity is specifically as follows:
PD-1/PD-L1 positive quality control was controlled according to 1:1 to prepare 10 nanograms per microliter of the mixed solution. The mixed solution is subjected to ten-fold concentration gradient dilution to be respectively diluted into 10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 And 10 -8 A total of 7 gradients were used as negative control with sterile water and the RNA kit of the utility model was used for PD-1/PD-L1 expression level detection and sensitivity experiments. Experimental results show that positive mixed liquor and negative mixed liquor can be obviously distinguished. When the concentration of the mixed solution is as low as 10 -8 Positive can still be detected, which means that the sensitivity of the RNA kit for detecting the PD-1/PD-L1 expression amount is 1 femtogram per microliter.
Example 12
The stability evaluation of the RNA kit for detecting the PD-1/PD-L1 expression quantity is carried out, and the stability evaluation is specifically as follows: the RNA kit for detecting the PD-1/PD-L1 expression level in the examples 1-3 of the utility model compares the detection result of the sample with the detection result of the commercial RNA kit for 100 times of the detection results of the PD-1/PD-L1 expression level and the PD-1/PD-L1 immunohistochemical detection result, and the results show that the detection result of the RNA kit is consistent with the detection result of the commercial RNA kit and the detection result of the commercial RNA kit respectively, so that the stability is good. In addition, compared with the operation time required by detecting samples by using the commercial RNA kit, the utility model shortens 25 minutes and greatly improves the efficiency of the detection process. In addition, compared with the commercial RNA kit, the accuracy of the PD-1/PD-L1 positive detection is improved by 7.2%, and the accuracy is up to 100%.
Example 13
According to the latest published industry recommended standard WS/T650-2019 antibacterial and bacteriostatic effect evaluation method of the national health Committee of the people's republic of China, the antibacterial and bacteriostatic functions of the cap of the RNA kit accommodating cavity for detecting the PD-1/PD-L1 expression amount and the protective sponge placed in the cap are detected:
s1, selecting a carrier soaking quantitative antibacterial test, wherein the test bacteria are staphylococcus aureus (ATCC 6538), escherichia coli (8099) and candida albicans (ATCC 10231) respectively. Fresh slant cultures of test bacteria were washed with PBS for 24 hours and diluted to about 5.0X10 s with PBS 6 CFU per milliliter-5.0X10 7 CFU was prepared as bacterial suspension per ml for use. And (3) dripping 10 microliter of bacterial suspension on a sterilization carrier by using a micropipette, and drying at 36+/-1 ℃ or airing at room temperature for later use. The samples were weighed in an amount of 5 g/piece in a sterile dish, placed in a 20+ -1deg.C water bath for 5 minutes, and the carriers were taken with sterile forceps to completely immerse the carriers in the samples and immediately timed. And (3) respectively taking the carriers to be stained, adding the carriers to a 5.0 ml PBS test tube, uniformly mixing, oscillating, washing test bacteria, respectively sucking 1.0 ml sample liquid, measuring the number of viable bacteria according to a viable bacteria culture counting method, and inoculating 2 plates to each sample liquid. If the number of colonies growing on the plate is large, the plate may be serially diluted 10 times with PBS and then subjected to viable bacteria culture counting. And immersing 2 pieces of bacteria-infected carrier in 10.0 g of control sample which contains no antibacterial component and is made of the same material as the test sample, and performing parallel test to obtain a positive control. The amount of the recovered positive control bacteria was 1.0X10 4 CFU/sheet-9.0x10 4 CFU/slice. The same batch of PBS and medium was used as negative control. All test samples and control samples were cultured at 36±1 ℃ and bacterial propagules were cultured for 48 hours for observation; the results were observed after culturing Candida albicans for 72 hours. The test was repeated 3 times and the bacteriostatic rate was calculated. The bacteriostasis rate is more than or equal to 50-90 percent, and the bacteriostasis effect is judged; the bacteriostasis rate is more than or equal to 90 percent, and the strong bacteriostasis effect is judged. The result shows that the antibacterial rate of the protective sponge and the accommodating cavity cap provided by the utility model on all test bacteria is 100%;
s2, selecting a carrier soaking quantitative antibacterial test, wherein the test bacteria are staphylococcus aureus respectivelyCocci (ATCC 6538), E.coli (8099) and Candida albicans (ATCC 10231). Weighing antibacterial sample according to the amount of 5 g/piece, placing in a sterile plate, placing in a water bath at 20+ -1deg.C for 5 min, taking the carrier for bacteria infection by sterile forceps, completely immersing the carrier in the antibacterial sample, and immediately timing. The carriers to be dyed are interacted with the antibacterial agent for 1 hour, the carriers to be dyed are respectively taken and added into a 5.0 ml neutralizer test tube, and the carriers are evenly mixed. After the neutralization agent is acted for 10 minutes, the test bacteria are washed, 1.0 milliliter of sample liquid is respectively absorbed, the number of viable bacteria is measured according to a viable bacteria culture counting method, and each tube of sample liquid is inoculated with 2 dishes. If the number of colonies growing on the plate is large, serial 10-fold dilutions with PBS may be performed before viable bacteria culture counting. And (3) taking a control sample which is the same as the test sample and does not contain antibacterial components to replace the antibacterial sample to soak 2 pieces of bacteria-staining carriers, and performing parallel test to obtain a positive control. The amount of the recovered positive control bacteria was 1.0X10 4 CFU/sheet-9.0x10 4 CFU/slice. The same batch of diluent, neutralizer and culture medium were used as negative controls. All test samples and control samples were cultured at 36±1 ℃ and bacterial propagules were cultured for 48 hours for observation; the results were observed after culturing Candida albicans for 72 hours. The test was repeated 3 times, and the sterilization rate was calculated. The sterilization rate is more than or equal to 90 percent within 1 hour, and the antibacterial effect is judged; the sterilization rate is more than or equal to 99 percent, and the antibacterial effect is strong. The results show that the antibacterial rate of the protective sponge and the accommodating cavity cap provided by the utility model on all test bacteria is 100%.
Example 14
The RNA kit for detecting the PD-1/PD-L1 expression in the examples 1-3 provided by the utility model can be used for detecting samples such as collected cells, tissues, serum, plasma or culture supernatant after the samples are processed according to the operation flow of clinical test detection standards, and the results show that the detection results of the RNA kit for detecting the PD-1/PD-L1 expression in the examples 2 and 3 can accurately reflect the actual situation of the samples. Compared with the commercial RNA kit, the RNA kit for detecting the PD-1/PD-L1 expression quantity in the embodiment 1-3 has the advantages that the detection accuracy is up to 100%, and the detection time is short. The cost price is calculated, the detection cost of each sample is averagely saved by 27 yuan, the purchase and use of medical insurance in hospitals are facilitated, and one detection cost can be saved for a detector.
The foregoing examples are set forth in order to provide a more thorough description of the present utility model, and are not intended to limit the scope of the utility model, since modifications of the present utility model, in which equivalents thereof will occur to persons skilled in the art upon reading the present utility model, are intended to fall within the scope of the utility model as defined by the appended claims.

Claims (3)

1. The RNA kit for detecting the PD-1/PD-L1 expression quantity is characterized by comprising a kit body and a kit cover, wherein the kit cover is connected with the kit body in a buckle type, a plurality of cubic columnar accommodating cavities are arranged in the kit body, the accommodating cavity is arranged to accommodate a specific forward primer reagent tube for detecting the PD-1/PD-L1 expression level, a specific reverse primer reagent tube for detecting the PD-1/PD-L1 expression level, a sterilized nuclease-free water negative quality control reagent tube for detecting the PD-1/PD-L1 expression level, a positive quality control reagent tube for detecting cell line PD-L1 expression level of a gene editing cell line PD-L1 for detecting the PD-1/PD-L1 expression level, an internal reference housekeeping gene TKT antibody reagent tube for detecting the PD-1/PD-L1 expression level, an internal reference housekeeping gene GP1 antibody reagent tube for detecting the PD-1/PD-L1 expression level, an internal reference housekeeping gene RPL-11 antibody reagent tube for detecting the PD-1/PD-L1 expression level, an internal reference housekeeping gene USP11 antibody reagent tube for detecting the PD-1/PD-L1 expression level, and an internal reference gene HAAI reagent for detecting the PD-1/PD-L1 expression level.
2. The RNA kit for detecting the PD-1/PD-L1 expression level according to claim 1, wherein a protective sponge is arranged between the inner wall of a containing cavity of the RNA kit and the reagent tube, the containing cavity comprises a cap, and the protective sponge and the cap are made of gel.
3. The RNA kit for detecting the expression level of PD-1/PD-L1 according to claim 1, wherein the specific forward primer reagent tube for detecting the expression level of PD-1/PD-L1 of the RNA kit comprises a specific forward primer reagent tube for detecting the expression level of the housekeeping gene TKT, a specific forward primer reagent tube for detecting the expression level of the housekeeping gene GP1, a specific forward primer reagent tube for detecting the expression level of the housekeeping gene RPL-11, a specific forward primer reagent tube for detecting the expression level of the housekeeping gene USP11, a specific forward primer reagent tube for detecting the expression level of the housekeeping gene hadhali, a specific forward primer reagent tube for detecting the expression level of the PD-1/PD-L1 gene CD274, and a specific forward primer reagent tube for detecting the expression level of the PD-1/PD-L1 gene CD274, wherein the specific reverse primer reagent tube for detecting the expression level of the housekeeping gene TKT, the specific reverse primer reagent tube for detecting the housekeeping gene GP1, the specific reverse primer tube for detecting the housekeeping gene RPL 11, the specific forward primer tube for detecting the gene RPL 11, the specific forward primer tube for the housekeeping gene USP11, the specific forward primer tube for the housekeeping gene, and specific forward primer tube for the gene USP11, and specific forward primer tube for the gene-1.
CN202320291427.6U 2023-02-23 2023-02-23 RNA kit for detecting PD-1/PD-L1 expression quantity Active CN219709496U (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202320291427.6U CN219709496U (en) 2023-02-23 2023-02-23 RNA kit for detecting PD-1/PD-L1 expression quantity

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202320291427.6U CN219709496U (en) 2023-02-23 2023-02-23 RNA kit for detecting PD-1/PD-L1 expression quantity

Publications (1)

Publication Number Publication Date
CN219709496U true CN219709496U (en) 2023-09-19

Family

ID=87998802

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202320291427.6U Active CN219709496U (en) 2023-02-23 2023-02-23 RNA kit for detecting PD-1/PD-L1 expression quantity

Country Status (1)

Country Link
CN (1) CN219709496U (en)

Similar Documents

Publication Publication Date Title
EP1356302B1 (en) Method and device for collecting and stabilizing a biological sample
US20200232015A1 (en) Method for detecting brucella infection and application thereof
Wilson et al. Laboratory diagnosis of bone, joint, soft-tissue, and skin infections
Sykes et al. Isolation and identification of aerobic and anaerobic bacteria
CN110016504A (en) Application, the product of neural tube malformation Prenatal Screening and method of the CDR1as in neural tube malformation Prenatal Screening
CN219709496U (en) RNA kit for detecting PD-1/PD-L1 expression quantity
CN105441541A (en) Lung cancer detection quality control product and preparation method thereof
CN116287257A (en) RNA kit for detecting PD-1/PD-L1 expression quantity
CN106048051B (en) A kind of candida krusei fluorescence PCR detection reagent kit
CN105483246A (en) Application of differential expression of gene in oral cancer diagnosis
Clancy et al. Diagnostic methods for detection of blood-borne candidiasis
CN105838799A (en) New application of KCNK2 gene
CN101760518A (en) Method for extracting live bacteria RNA in Mycobacterium tuberculosis and detection kit thereof
CN106399485A (en) Genes highly expressed in tongue squamous carcinoma para-carcinoma tissue and applications of genes
CN112626246A (en) Detection method for identifying three bacteria in milk through multiple PCR
CN113755614A (en) Rapid high-sensitivity differential diagnosis kit for Brucella vaccine strain and wild strain and use method thereof
Alzuguren et al. Infectious abortions in small domestic ruminants in the Iberian Peninsula: Optimization of sampling procedures for molecular diagnostics
CN117165580B (en) Composition for stabilizing nucleic acid in sample, preparation method and application thereof
CN114032301B (en) Application of Rgs12 as gamma-ray radiation biomarker
AU2021101105A4 (en) Application of USP4 as a biomarker of autoimmune liver disease
Kelly-Goss et al. 1181 Prevalence of Celiac Disease-Compatible Human Leukocyte Antigen Haplotypes Across Ethnicities and Regions in the United States
RU2751244C1 (en) Method for extraction of nucleic acids from nail plates
CN112675201B (en) Application of macrophage subgroup and regulator thereof in acute graft-versus-host disease
CN114796524B (en) Application of BMS1P4 gene in preparation of medicines for inhibiting bladder cancer cells
CN107746878B (en) Free gene fixing solution

Legal Events

Date Code Title Description
GR01 Patent grant
GR01 Patent grant