CN112725418A

CN112725418A - Method and kit for detecting expression level of PD-L1 based on free RNA

Info

Publication number: CN112725418A
Application number: CN202110099183.7A
Authority: CN
Inventors: 陈建国; 陈川; 张瑜巨
Original assignee: Shenzhen Letu Biological Technology Co ltd
Current assignee: Shenzhen Letu Biological Technology Co ltd
Priority date: 2021-01-25
Filing date: 2021-01-25
Publication date: 2021-04-30

Abstract

A method for detecting the expression level of PD-L1 based on free RNA and a kit thereof, wherein the method comprises the following steps: a reverse transcription step, which comprises the step of reversely transcribing the free RNA sample to be detected into cDNA; and the real-time fluorescent quantitative PCR detection step comprises the steps of carrying out real-time fluorescent quantitative PCR detection on the PD-L1 gene and the internal reference gene in the cDNA, and predicting the strength of the PD-L1 expression quantity of the individual to which the sample to be detected belongs according to the Ct values of the PD-L1 gene and the internal reference gene. The invention is used as a curative effect evaluation means which is carried out before an immunotherapy scheme is used for a patient who can not or is difficult to carry out tissue immunohistochemical detection PD-L1, and the benefit of the patient in the immunotherapy is improved.

Description

Method and kit for detecting expression level of PD-L1 based on free RNA

Technical Field

The invention relates to the technical field of biology, in particular to a method for detecting PD-L1 expression quantity based on free RNA and a kit thereof.

Background

Cancer is an important factor affecting human health and life worldwide, and most cancer patients are diagnosed at an advanced stage, lose the chance of surgery and are insensitive to conservative treatment modes such as radiotherapy and chemotherapy, so that prognosis is generally biased. With the continuous development of immunooncology, the immunotherapy of tumors becomes a new hotspot after molecular targeted therapy, wherein the PD-1/PD-L1 signaling pathway is one of the research hotspots of immunotherapy in recent years, and at present, several drugs aiming at the signaling pathway are marketed for the treatment of cancers such as melanoma, lung cancer, colorectal cancer, and the like. However, PD-1/PD-L1 immunotherapy is not effective for every patient, and clinical experimental results show that only less than 30% of patients can benefit from PD-1/PD-L1 immunotherapy, and the treatment cost is high, the treatment period is long, and if the treatment is ineffective, the patients lose the opportunity to receive other treatment methods in time, and more importantly, adverse reactions caused by immunotherapy cannot be ignored. Therefore, it is necessary to perform appropriate tests to predict the efficacy of an immunotherapy prior to the selection of the immunotherapy regimen. In PD-L1-based immunotherapy, the therapeutic efficacy of PD-1/PD-L1 inhibitors is closely related to the expression level of PD-L1.

Currently, the expression level of PD-L1 is detected mainly by an immunohistochemical method. Specifically, the expression level of PD-L1 in a cancer tissue specimen is detected by means of hybridization color development by using specific antibodies (such as 28-8, 22C3, SP263 and SP 142). However, for some patients who have failed to obtain tumor tissue or have difficulty obtaining tumor tissue, the evaluation of PD-L1 becomes difficult. In addition, the current immunohistochemical method also faces some problems, for example, in terms of detection technology, different detection antibodies, platforms and settings of different thresholds have different influences on the detection result, and in terms of biology, the detection result may not truly reflect the expression level of PD-L1 due to intratumoral and intratumoral heterogeneity; tissue origin, there were differences in PD-L1 expression levels in primary sites and metastases for cytological, archival and fresh specimens. In addition, the curative effect of immunotherapy is evaluated by a method for detecting Tumor Mutation Burden (TMB) in ctDNA through NGS large panel, but the method has complex detection process and high cost, and is a heavy economic burden for many patients.

Disclosure of Invention

According to a first aspect, there is provided in one embodiment a method for detecting the expression level of PD-L1 based on free RNA, comprising:

a reverse transcription step, which comprises the step of reversely transcribing the free RNA sample to be detected into cDNA;

and the real-time fluorescent quantitative PCR detection step comprises the steps of carrying out real-time fluorescent quantitative PCR detection on the PD-L1 gene and the internal reference gene in the cDNA, and predicting the strength of the PD-L1 expression quantity of the individual to which the sample to be detected belongs according to the Ct values of the PD-L1 gene and the internal reference gene.

According to a second aspect, an embodiment provides a probe primer combination for real-time fluorescent quantitative PCR detection of PD-L1 gene, wherein the probe comprises a nucleotide sequence as shown in SEQ ID No. 3, and the primer includes but is not limited to at least one of the following nucleotide sequence combinations:

1) the nucleotide sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2;

2) the nucleotide sequences shown as SEQ ID NO. 7 and SEQ ID NO. 8.

According to the third aspect, an embodiment provides a probe primer combination for real-time fluorescence quantitative PCR detection of PD-L1 gene and reference gene, the probe for real-time fluorescence quantitative PCR detection of PD-L1 gene comprises a nucleotide sequence shown as SEQ ID NO. 3, and the primer includes but is not limited to at least one of the following nucleotide sequence combinations:

1) the nucleotide sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2;

2) the nucleotide sequences shown as SEQ ID NO. 7 and SEQ ID NO. 8;

the internal reference gene is selected from beta-Actin, the probe for detecting the internal reference gene by real-time fluorescence quantitative PCR comprises a nucleotide sequence shown in SEQ ID NO. 6, and the primer is selected from at least one of the following nucleotide sequence combinations:

1) the nucleotide sequences shown as SEQ ID NO. 4 and SEQ ID NO. 5;

2) the nucleotide sequences shown as SEQ ID NO. 9 and SEQ ID NO. 10.

According to a fourth aspect, an embodiment provides a kit for detecting the expression level of PD-L1 based on free RNA, which comprises a probe primer combination for real-time fluorescent quantitative PCR detection of the PD-L1 gene.

According to the method for detecting the expression level of PD-L1 based on free RNA and the kit thereof in the embodiment, the method is used as a curative effect evaluation means which is carried out before an immunotherapy scheme is used for a patient who cannot or is difficult to carry out tissue immunohistochemical detection of PD-L1, and the benefit of the patient in the immunotherapy is improved.

Drawings

FIG. 1 is a diagram of immunohistochemical weak expression;

FIG. 2 is an expression profile in immunohistochemistry;

FIG. 3 is a diagram of immunohistochemical strong expression;

FIG. 4 is a graph of fluorescence quantitative weak expression amplification;

FIG. 5 is a graph showing the expression amplification curve in the fluorescence quantification;

FIG. 6 is a graph of fluorescence quantitative strong expression amplification.

Detailed Description

The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. Wherein like elements in different embodiments are numbered with like associated elements. In the following description, numerous details are set forth in order to provide a better understanding of the present application. However, those skilled in the art will readily recognize that some of the features may be omitted or replaced with other elements, materials, methods in different instances. In some instances, certain operations related to the present application have not been shown or described in detail in order to avoid obscuring the core of the present application from excessive description, and it is not necessary for those skilled in the art to describe these operations in detail, so that they may be fully understood from the description in the specification and the general knowledge in the art.

Furthermore, the features, operations, or characteristics described in the specification may be combined in any suitable manner to form various embodiments. Also, the various steps or actions in the method descriptions may be transposed or transposed in order, as will be apparent to one of ordinary skill in the art. Thus, the various sequences in the specification and drawings are for the purpose of describing certain embodiments only and are not intended to imply a required sequence unless otherwise indicated where such sequence must be followed.

The numbering of the components as such, e.g., "first", "second", etc., is used herein only to distinguish the objects as described, and does not have any sequential or technical meaning. The term "connected" and "coupled" when used in this application, unless otherwise indicated, includes both direct and indirect connections (couplings).

Herein, PD-L1 refers to Programmed cell death-ligand 1(Programmed cell death 1ligand 1), also known as cluster of differentiation 274 (CD 274) of surface antigen or B7 homolog (B7 homolog 1, B7-H1), a protein in the human body, encoded by the CD274 gene. PD-L1 is a first type of transmembrane protein of size 40kDa that is believed to be involved in the suppression of the immune system in certain specific situations (e.g. pregnancy, tissue transplantation, autoimmune diseases, and certain diseases such as hepatitis). The immune system will normally respond to foreign antigens that accumulate in the lymph nodes or spleen, triggering antigen-specific cytotoxic T cells (CD8+ Tcell proliferation). The programmed cell death receptor-1 (PD-1) is combined with programmed cell death-ligand 1(PD-L1) to transmit inhibitory signals and reduce the proliferation of lymph node CD8+ T cells, and the PD-1 can control the accumulation of antigen-specific T cells in lymph nodes by regulating Bcl-2 genes.

Herein, PD-1 refers to programmed death receptor 1, also known as CD279, or cluster of differentiation 279.

The results of previous studies showed that the mRNA level of PD-L1 in tumor tissues was consistent with the protein level. Meanwhile, free RNA released into peripheral blood by tumor tissues is positively correlated with the expression level of PD-L1 in the tissues, so that a complementary detection means for evaluating the curative effect of immunotherapy can be provided for patients who cannot carry out tissue immunohistochemical detection through the relative expression level of PD-L1 in the free RNA in the peripheral blood.

In some embodiments, the invention mainly aims to provide a detection method for detecting the expression level of PD-L1 in plasma free RNA, which is used as a curative effect evaluation means before an immunotherapy scheme is used for a patient who cannot or is difficult to carry out tissue immunohistochemical detection of PD-L1, so that the benefit of the patient in the immunotherapy is improved.

In order to achieve the above objects, in some embodiments, the present invention establishes a plasma free RNA quantitative detection method based on RT-qPCR (reverse transcription-real-time fluorescent quantitative PCR), by performing exon-spanning specific primer design for PD-L1 gene and selecting beta-actin with stable relative expression as an internal reference gene. Extracting free RNA from peripheral blood of a cancer patient, performing reverse transcription by a one-step method, performing qPCR detection, and judging the expression level of PD-L1 in a tumor focus of the tumor patient by comparing the Ct value difference of a PD-L1 gene and an internal reference gene so as to guide the immunotherapy of the tumor patient.

Based on the above, in some embodiments, the present invention provides a method for detecting the expression level of PD-L1 in plasma free RNA, which uses one-step RT-qPCR technology for plasma free RNA of tumor patients, performs quantitative detection using specific primers of PD-L1 and internal reference gene, and determines the expression level of PD-L1 of tumor patients by calculating the Ct difference between PD-L1 and internal reference gene.

The one-step RT-qPCR technology refers to: firstly, RNA is reversely transcribed into cDNA under the action of reverse transcriptase, and then the cDNA is quantitatively detected by qPCR under the amplification action of DNA polymerase, all of which are completed in one tube.

According to a first aspect, in some embodiments, there is provided a method for detecting the expression level of PD-L1 based on free RNA, comprising:

The Ct value is the number of cycles corresponding to the inflection point from the baseline to the exponential increase.

The PD-L1 gene refers to a gene capable of expressing PD-L1.

In some embodiments, the free RNA targeted by the invention differs primarily with respect to total RNA in the tissue or cells in the pleural effusion by: firstly, because the total amount of free RNA is lower, the expression amount of RNA used for translating PD-L1 is lower than that of RNA used for translating PD-L1 in total RNA in cells; secondly, the free RNA is a mixture of free RNA released from different cells, and the tissue sample can be regarded as a homogeneous tissue to a certain extent under the condition of ensuring the tumor content.

In some embodiments, the cDNA is double-stranded cDNA.

It should be noted that the expression level of PD-L1 is only an intermediate result, and when the health status of the subject is evaluated, the final diagnosis result can be obtained by combining the tumor type, stage, or the Microsatellite instability (MSI) of the patient. In addition, the invention can also be used for evaluating the curative effect of a medicament, for example, screening of new drug candidates, and when the curative effect of the medicament is evaluated, the final curative effect evaluation result can be obtained by combining the tumor type, the stage or the Microsatellite instability (MSI) and other indexes of a patient in addition to the expression quantity of PD-L1. Thus, the present invention is not a diagnostic method of disease, much less a therapeutic method.

In some embodiments, the method comprises: adding a free RNA sample to be detected, a probe primer combination for performing real-time fluorescent quantitative PCR detection on the PD-L1 gene and a probe primer combination for performing real-time fluorescent quantitative PCR detection on the reference gene into a one-step reverse transcription-real-time fluorescent quantitative PCR reaction system, sequentially completing reverse transcription and real-time fluorescent quantitative PCR detection, and predicting the strength of the PD-L1 expression of an individual to which the sample to be detected belongs according to Ct values of the PD-L1 gene and the reference gene. The reverse transcription and real-time fluorescent quantitative PCR detection of the PD-L1 gene and the reference gene can be carried out in the same reaction system or different reaction systems.

In some embodiments, a part of the free RNA sample to be detected and a probe primer combination for real-time fluorescent quantitative PCR detection of the PD-L1 gene are added to one-step reverse transcription-real-time fluorescent quantitative PCR reaction system, and another part of the free RNA sample to be detected and a probe primer combination for real-time fluorescent quantitative PCR detection of the reference gene are added to another one-step reverse transcription-real-time fluorescent quantitative PCR reaction system, so that reverse transcription of the PD-L1 gene and the reference gene and real-time fluorescent quantitative PCR detection are performed in different reaction systems.

In some embodiments, the degree of the expression level of PD-L1 of the individual to which the test sample belongs is predicted according to the absolute value of the difference value of the Ct values of the PD-L1 gene and the reference gene.

In some embodiments, the method for predicting the degree of the PD-L1 expression level of the individual to which the test sample belongs according to the Ct values of the PD-L1 gene and the reference gene is as follows:

when the absolute value of the Ct difference value between the PD-L1 gene and the reference gene is more than or equal to a first threshold value, the weak expression of PD-L1 is predicted; when the second threshold value is less than or equal to the absolute value of the Ct difference value between the PD-L1 gene and the reference gene is less than the first threshold value, the expression in PD-L1 is predicted; and when the absolute value of the Ct difference value of the PD-L1 gene and the reference gene is less than a second threshold value, strong expression of PD-L1 is predicted.

In some embodiments, the first threshold is 15.

In some embodiments, the second threshold is 10.

The setting method of the threshold value is as follows: according to the absolute value of the Ct value difference detected in the sample of the embodiment and the tissue immunohistochemical result of the corresponding sample, a threshold value is defined through statistics and comparison.

In some embodiments, the probe for real-time fluorescent quantitative PCR detection of PD-L1 gene includes but is not limited to the nucleotide sequence shown in SEQ ID NO. 3.

In some embodiments, the primers useful for amplifying the PD-L1 gene include, but are not limited to, at least one of the following combinations of nucleotide sequences:

1) the nucleotide sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2;

2) the nucleotide sequences shown as SEQ ID NO. 7 and SEQ ID NO. 8. Usually, one set of primers is selected as a front primer and a back primer for amplifying the PD-L1 gene. Two groups of sequences can also be used as primers, and reverse transcription and real-time fluorescence quantitative PCR are respectively carried out in two reaction systems.

In some embodiments, the reference gene is selected from at least one of β -Actin, GAPDH, U6, HMBS, B2M, TUBB, SDHA, 18S rRNA, ACTB, RPL4, PPIA, HPRT1, YWHAZ, RPP30, ERG. Real-time fluorescent quantitative PCR detection can also be performed using a plurality of reference genes.

The reference gene refers to a gene whose expression is relatively constant in body fluid such as blood.

In some embodiments, the reference gene is selected from β -Actin, and the probe for real-time fluorescent quantitative PCR detection of the reference gene includes, but is not limited to, the nucleotide sequence shown in SEQ ID NO. 6.

In some embodiments, the reference gene is selected from β -Actin, and the primers used for real-time fluorescent quantitative PCR detection of the reference gene include, but are not limited to, at least one of the following combinations of nucleotide sequences:

1) the nucleotide sequences shown as SEQ ID NO. 4 and SEQ ID NO. 5;

2) the nucleotide sequences shown as SEQ ID NO. 9 and SEQ ID NO. 10. Usually, one set of primers is selected as a front primer and a back primer for amplifying the reference gene. Two groups of sequences can also be used as primers, and reverse transcription and real-time fluorescence quantitative PCR are respectively carried out in two reaction systems.

In some embodiments, the number of cycles of the real-time fluorescent quantitative PCR reaction is 40-50, and fluorescence is collected upon cycling the reaction.

In some embodiments, the real-time fluorescent quantitative PCR reaction, each cycle of the reaction is as follows: 94-98 deg.C for 10-20 s; 56-62 ℃ for 15-35 s.

In some embodiments, the procedure prior to cycling the reaction is as follows in order for real-time fluorescent quantitative PCR reaction: at 40-45 deg.C for 94-98 min; 94-98 deg.C, 1-5 min.

In some embodiments, the 5 'end of the probe for performing real-time fluorescence quantitative PCR detection on PD-L1 gene is modified with a first label molecule, the 3' end of the probe capable of binding to PD-L1 gene is modified with a second label molecule, the 5 'end of the probe for performing real-time fluorescence quantitative PCR detection on reference gene is modified with a third label molecule, the 3' end of the probe for performing real-time fluorescence quantitative PCR detection on reference gene is modified with a fourth label molecule, the first label molecule and the second label molecule undergo fluorescence energy resonance transfer when they are physically close to each other, and the third label molecule and the fourth label molecule undergo fluorescence energy resonance transfer when they are physically close to each other.

In some embodiments, the first labeling molecule, the third labeling molecule, and the second labeling molecule, the fourth labeling molecule are fluorescent reporters and fluorescent quenchers.

In some embodiments, the first labeling molecule emits a fluorescence wavelength different from the fluorescence wavelength emitted by the third labeling molecule.

In some embodiments, the fluorescent reporter group includes, but is not limited to, at least one of FAM, HEX, VIC, ROX, Cy 5.

In some embodiments, the second labeling molecule and the fourth labeling molecule are the same or different fluorescence quenchers.

In some embodiments, the fluorescence quenching group includes, but is not limited to, at least one of BHQ1, BHQ2, MGB.

In some embodiments, the real-time fluorescent quantitative PCR detection of the PD-L1 gene and the real-time fluorescent quantitative PCR detection of the reference gene may be performed in the same or different reaction vessels.

In some embodiments, the free RNA sample is derived from a human or animal body. The animal body may be a mammal, and specifically may include, but is not limited to, a mouse, a rat, a rabbit, and the like.

In some embodiments, the free RNA sample is extracted from at least one of the group including, but not limited to, blood, pleural fluid, uterine fluid, urine, and the like.

Blood is a red, opaque, viscous liquid that flows in blood vessels and the heart of the human or animal body. Blood is mainly composed of plasma and blood cells.

In some embodiments, the free RNA sample is extracted from peripheral blood of the tumor patient, which is typically blood other than bone marrow.

In some embodiments, the tumor comprises at least one of a solid tumor, a non-solid tumor. The invention is particularly applicable to patients with solid tumors.

In some embodiments, the tumor includes, but is not limited to, at least one of lung cancer, liver cancer, breast cancer, esophageal cancer, colorectal cancer.

In some embodiments, the free RNA is extracted from plasma.

According to a second aspect, in some embodiments, there is provided a probe primer combination for real-time fluorescent quantitative PCR detection of PD-L1 gene, the probe including but not limited to the nucleotide sequence shown in SEQ ID No. 3, the primer including but not limited to at least one of the following combinations of nucleotide sequences:

1) the nucleotide sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2;

2) the nucleotide sequences shown as SEQ ID NO. 7 and SEQ ID NO. 8.

According to a third aspect, in some embodiments, there is provided a probe primer combination for real-time fluorescent quantitative PCR detection of PD-L1 gene, reference gene, the probe for real-time fluorescent quantitative PCR detection of PD-L1 gene includes but is not limited to the nucleotide sequence shown in SEQ ID No. 3, and the primer includes but is not limited to at least one of the following nucleotide sequence combinations:

1) the nucleotide sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2;

2) the nucleotide sequences shown as SEQ ID NO. 7 and SEQ ID NO. 8;

the internal reference gene is selected from beta-Actin, a probe for detecting the internal reference gene by real-time fluorescence quantitative PCR comprises but is not limited to a nucleotide sequence shown in SEQ ID NO. 6, and a primer comprises but is not limited to at least one of the following nucleotide sequence combinations:

1) the nucleotide sequences shown as SEQ ID NO. 4 and SEQ ID NO. 5;

2) the nucleotide sequences shown as SEQ ID NO. 9 and SEQ ID NO. 10.

According to a fourth aspect, in some embodiments, there is provided a kit comprising a probe primer combination for real-time fluorescent quantitative PCR detection of the PD-L1 gene.

In some embodiments, the kit further comprises a probe primer combination for detecting the reference gene by real-time fluorescent quantitative PCR.

In some embodiments, the kit is a kit for detecting the expression level of PD-L1 based on free RNA.

In some embodiments, the probe sequence for real-time fluorescent quantitative PCR detection of the PD-L1 gene includes, but is not limited to, the nucleotide sequence shown in SEQ ID NO. 3.

In some embodiments, the primers used for real-time fluorescent quantitative PCR detection of PD-L1 gene include, but are not limited to, at least one of the following combinations of nucleotide sequences:

1) the nucleotide sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2;

2) the nucleotide sequences shown as SEQ ID NO. 7 and SEQ ID NO. 8.

In some embodiments, the reference gene is selected from at least one of β -Actin, GAPDH, U6, HMBS, B2M, TUBB, SDHA, 18S rRNA, ACTB, RPL4, PPIA, HPRT1, YWHAZ, RPP30, ERG.

1) the nucleotide sequences shown as SEQ ID NO. 4 and SEQ ID NO. 5;

2) the nucleotide sequences shown as SEQ ID NO. 9 and SEQ ID NO. 10.

In some embodiments, the 5 'end of the probe for real-time fluorescent quantitative PCR detection of the PD-L1 gene is modified with a first marker molecule, and the 3' end is modified with a second marker molecule; the 5 'end of the probe used for carrying out real-time fluorescent quantitative PCR detection on the reference gene is modified with a third marker molecule, and the 3' end is modified with a fourth marker molecule; the first labeling molecule and the second labeling molecule can generate fluorescence energy resonance transfer when being physically close to each other, and the third labeling molecule and the fourth labeling molecule can generate fluorescence energy resonance transfer when being physically close to each other.

In some embodiments, the first labeling molecule emits fluorescence at a wavelength different from the fluorescence emitted by the third labeling molecule.

In some embodiments, the kit further comprises reagents for reverse transcription of free RNA and real-time fluorescent quantitative PCR detection. Reagents for reverse transcription of free RNA and real-time fluorescent quantitative PCR detection are generally commercially available.

In some embodiments, the reagents for reverse transcription of free RNA and real-time fluorescent quantitative PCR detection are one-step reverse transcription-real-time fluorescent quantitative PCR reaction solutions (i.e., RT-qPCR MIX).

In some embodiments, the reagents for reverse transcription of free RNA and real-time fluorescent quantitative PCR detection include, but are not limited to, RT-qPCR MIX.

In some embodiments, the kit further comprises a container for separately storing each probe, primer, reagent. The probes, the primers and the reagents can be independently stored in different containers or different chambers of the same container, and the probes, the primers and the reagents are mixed when in use.

In some embodiments, the kit further comprises instructions for instructing a user to use the kit.

In some embodiments, a qPCR primer probe that specifically binds PD-L1 gene and reference gene β -Actin is provided, the specific primer probe sequences being shown in SEQ ID No. 1 to SEQ ID No. 10.

TABLE 1 primer probes

Serial number	Sequence (5 '-3')	Serial number	Sequence (5 '-3')
				SEQ ID NO:1	TCCCTCTTGGCCATATTCTG	SEQ ID NO:6	GGGCATGGAGTCCTGTGGCA
SEQ ID NO:2	CCAACACCACAAGGAGGAGT	SEQ ID NO:7	GTACCTTGGCTTTGCCACAT
				SEQ ID NO:3	GGCAAGAATTGTGGCTGAGCAAGG	SEQ ID NO:8	CAACACCACAAGGAGGAG
SEQ ID NO:4	GGACTTCGAGCAAGAGATGG	SEQ ID NO:9	CTCTTCCAGCCTTCCTTCCT
				SEQ ID NO:5	AGCACTGTGTTGGCGTACAG	SEQ ID NO:10	GCACTGTGTTGGCGTACA

In Table 1, the nucleotide sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 are front and back amplification primers for amplifying the PD-L1 gene, and the sequence shown in SEQ ID NO. 3 is a probe capable of being specifically bound to the PD-L1 gene; the nucleotide sequences shown in SEQ ID NO. 4 and SEQ ID NO. 5 are front and back amplification primers for amplifying the internal reference gene beta-Actin, and the nucleotide sequence shown in SEQ ID NO. 6 is a probe capable of being specifically combined with the internal reference gene beta-Actin.

The nucleotide sequences shown in SEQ ID NO. 7 and SEQ ID NO. 8 are respectively front and back amplification primers aiming at the PD-L1 gene; the nucleotide sequences shown in SEQ ID NO 9 and SEQ ID NO 10 are respectively front and back amplification primers aiming at an internal reference gene.

In some embodiments, the 5' end of the probe sequence shown in SEQ ID NO. 3 that specifically binds to PD-L1 gene is modified, the modifying group is FAM fluorophore; the 3' end is also modified, and the modified group is a BHQ1 quenching group.

In some embodiments, the 5' end of the probe sequence specifically binding to the beta-Actin reference gene shown in SEQ ID NO. 6 is modified, and the modifying group is a VIC fluorophore; the 3' end is modified, and the modifying group is a BHQ1 quenching group;

in some embodiments, the method for detecting the expression level of PD-L1 in the plasma free RNA of a tumor patient based on RT-qPCR (quantitative reverse transcription PCR) technology comprises the following steps:

a reverse transcription step, adding the extracted plasma free RNA of a sample to be detected into one-step RT-qPCR reaction solution for random reverse transcription to form cDNA;

a PD-L1 gene amplification and quantification step, in a first reaction tube, performing specific amplification and quantification on PD-L1 gene in cDNA by using primer probes shown as SEQ ID NO: 1-SEQ ID NO: 3;

and (3) amplifying and quantifying the internal reference gene beta-Actin, and specifically amplifying and quantifying the internal reference gene beta-Actin in the cDNA by using a primer probe shown in SEQ ID NO. 4-SEQ ID NO. 6 in a second reaction tube.

In some embodiments, the reverse transcription and specific amplification and quantification are performed in the same reaction tube without the need for a lid being opened midway.

In some embodiments, the reverse transcription and quantification reaction procedure is as follows:

TABLE 2

In some embodiments, the expression of PD-L1 in a tumor patient is determined by calculating the Ct difference between PD-L1 and an internal reference gene.

In some embodiments, when the Ct difference between PD-L1 and the reference gene is greater than or equal to 15, then weak expression of PD-L1 is predicted; when the difference between the CT of the PD-L1 and the CT of the reference gene is less than 15 and is more than or equal to 10, the expression is predicted to be in PD-L1; when the CT difference between PD-L1 and the reference gene is less than 10, strong expression of PD-L1 is predicted.

In some embodiments, the invention is used for detecting free RNA in plasma of a tumor patient, a specific primer is designed for carrying out quantitative detection on PD-L1 and an internal reference gene, and the expression condition of PD-L1 of the tumor patient is judged by calculating the Ct value difference between the two genes, so that compared with the existing tissue sampling, the sampling difficulty is obviously reduced, and the detection result deviation caused by the difference of the expression levels of PD-L1 in a primary part and a metastatic focus is effectively avoided.

In some embodiments, the subject to be tested by the present invention is patient peripheral blood free RNA, which has the advantage of being able to be sampled repeatedly, and can be adapted to the needs of immunotherapy efficacy assessment for patients who have no and/or difficult access to tumor tissue.

In some embodiments, the peripheral blood detected by the invention is collected and transported at normal temperature by adopting a cfDNA storage tube, so that free RNA in a sample can be kept stable and is not degraded, and the stability of a detection result is good.

In some embodiments, the invention is based on an RT-qPCR method, and the method is simple and rapid, has good repeatability and low cost, and can provide detection service with extremely high cost performance for patients.

In some embodiments, the present invention has a higher consistency with the results of tumor tissue immunohistochemistry; in the research process, the method and the immunohistochemical method are synchronously adopted, and the results of the two methods are compared and counted, so that the threshold value of the Ct difference value for result judgment is set. The judgment result has higher consistency with the immunohistochemical result.

To better illustrate the problems solved, the technical solutions adopted and the effects achieved by the present invention, the present invention will be further described with reference to specific examples and related materials, and it should be noted that the present invention includes, but is not limited to, the following examples and their combined embodiments.

In the embodiments of the present invention, specific techniques or conditions are not specified, and they can be obtained by conventional means such as market purchase, and the method flow can be described with reference to reagents or equipment.

Example 1

The method of the embodiment comprises the following steps:

collecting peripheral blood of healthy people and tumor patients (particularly lung cancer), wherein blood samples of the healthy people comprise 20 cases, 12 cases of women and 8 cases of men; blood samples from tumor patients were taken in 64 total, plasma free RNA was extracted, and quantitative detection of PD-L1 was performed.

Specifically, after peripheral blood Plasma was isolated, Plasma free RNA was extracted using a commercial Kit miRNeasy Serum/Plasma Advanced Kit (cat # 217204, manufacturer: QIAGEN), and the extraction method was performed with reference to the Kit instructions.

The concentration of extracted free RNA was measured using a Qubit RNA HS detection kit (cat # Q32855, manufacturer: Thermo Fisher), and this concentration was mainly used for preliminary assessment of success of extraction.

Designing a plurality of groups of primer probes aiming at PD-L1 and an internal reference gene beta-Actin, respectively carrying out RT-qPCR detection, evaluating and screening out optimal probe primers, wherein the optimal primer group sequences are nucleotide sequences shown in SEQ ID NO. 1-SEQ ID NO. 6.

In the first reaction tube, the PD-L1 gene in the cDNA is specifically amplified and quantified by using primer probes shown in SEQ ID NO. 1-SEQ ID NO. 3.

Meanwhile, in a second reaction tube, a primer probe shown in SEQ ID NO. 4-SEQ ID NO. 6 is used for carrying out specific amplification and quantification on the internal reference gene beta-Actin in the cDNA.

Adding the extracted plasma free RNA of the sample to be detected into one-step RT-qPCR reaction solution (ABScript II one-step RT-qPCR probe kit, product number: RK20407, manufacturer: ABClonal) for random reverse transcription to form cDNA, and specifically mixing the free RNA, a primer probe working solution and 2 xRT-qPCR MIX according to the following reaction system:

the reaction system for amplifying and quantitatively detecting the PD-L1 gene comprises the following components: mu.L of free RNA, 12.5. mu.L of 2 XTT-qPCR MIX, 1. mu.L each of the pre-primer, the post-primer and the probe of the PD-L1 gene, the volume was made up to 25. mu.L using nuclease-free water. The front primer and the rear primer of the PD-L1 gene are nucleotide sequences shown as SEQ ID NO 1 and SEQ ID NO 2, the probe of the PD-L1 gene is shown as SEQ ID NO 3, the 5' end of the probe sequence is modified, and the modifying group is an FAM fluorescent reporter group; the 3' end is also modified, and the modified group is a BHQ1 fluorescence quenching group. The manufacturers of FAM, VIC, BHQ1 were Invitrogen.

The reaction system for amplifying and quantitatively detecting the reference gene comprises the following components: 2 mul of free RNA, 12.5 mul of 2 xRT-qPCR MIX, and 1 mul of front primer, back primer and probe of internal reference gene respectively; the volume was made up to 25 μ L using nuclease free water. The front primer and the rear primer of the reference gene are shown as SEQ ID NO. 4 and SEQ ID NO. 5, the probe is shown as SEQ ID NO. 6, the 5' end of the probe sequence is modified, and the modifying group is a VIC fluorescent reporter group; the 3' end is also modified, and the modified group is a BHQ1 fluorescence quenching group.

The two reaction systems are prepared in different reaction tubes.

Reverse transcription, specific amplification and quantification are completed in the same reaction tube without opening the cover in the middle.

Optimizing the reaction conditions of the primer probe, and determining the optimal reaction conditions as follows:

TABLE 3

Comparing the interpretation result with an immunohistochemical result (a sample of an immunohistochemical experiment is taken from a tissue of a subject) of a corresponding cancer patient, comparing the consistency and setting an interpretation threshold; the antibody model of immunohistochemistry is 28-8, 22C3, SP142 and SP263, TPS and CPS are stained and calculated to judge the detection result, wherein the detection results of immunohistochemistry with low expression, medium expression and high expression are shown in the figure 1, figure 2 and figure 3 in sequence. The three figures are representative figures selected from the respective groups.

And (3) calculating the Ct values of the PD-L1 gene and the reference gene of each sample according to the amplification curve, and judging the expression quantity of the PD-L1 according to the Ct value difference. When the absolute value of the Ct difference value between the PD-L1 gene and the reference gene is more than or equal to 15, the weak expression of PD-L1 is judged; when the absolute value of the Ct difference value between the PD-L1 gene and the reference gene is more than or equal to 10 and less than 15, the expression in PD-L1 is judged; when the absolute value of the Ct difference value between the PD-L1 gene and the reference gene is less than 10, the strong expression of PD-L1 is judged.

Amplification profiles of the weak expression, the medium expression and the strong expression are shown in FIG. 4, FIG. 5 and FIG. 6 in this order, and the three graphs are representative graphs selected from the respective groups. In the three graphs, the abscissa indicates the cycle number, and the ordinate indicates the Relative Fluorescence Units (RFU), the upper curve in each graph is the amplification curve for the PD-L1 gene, and the lower curve is the amplification curve for the reference gene. As can be seen, in FIG. 4, the difference in Ct between the PD-L1 gene and the reference gene is greater than 15, and it is judged that the expression is weak in PD-L1; in FIG. 5, the difference in Ct between the PD-L1 gene and the reference gene is greater than 10 and less than 15, and it is judged that the gene is expressed in PD-L1; in FIG. 6, the Ct difference between the PD-L1 gene and the reference gene is less than 10, and strong expression of PD-L1 is determined.

Based on the above results, it was found that the degree of expression of PD-L1 could be detected by blood sampling in subjects who could not be subjected to tissue sampling. The invention can also be used as a supplementary test for subjects who can be sampled for tissue.

The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.

SEQUENCE LISTING

<110> Shenzhen Letu Biotech Limited

<120> method for detecting expression level of PD-L1 based on free RNA and kit thereof

<130> 20I31086

<160> 10

<170> PatentIn version 3.3

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Claims

1. A method for detecting the expression level of PD-L1 based on free RNA, which is characterized by comprising the following steps:

2. The method of claim 1, comprising: adding a free RNA sample to be detected, a probe primer combination for performing real-time fluorescent quantitative PCR detection on the PD-L1 gene and a probe primer combination for performing real-time fluorescent quantitative PCR detection on the reference gene into a one-step reverse transcription-real-time fluorescent quantitative PCR reaction system, sequentially performing reverse transcription and real-time fluorescent quantitative PCR detection, and predicting the strength of the PD-L1 expression quantity of an individual to which the sample to be detected belongs according to Ct values of the PD-L1 gene and the reference gene;

and/or predicting the strength degree of the PD-L1 expression quantity of the individual to which the sample to be detected belongs according to the absolute value of the difference value of the Ct values of the PD-L1 gene and the reference gene.

3. The method of claim 1 or 2, wherein the method for predicting the degree of the expression level of PD-L1 of the individual to which the test sample belongs according to the Ct values of the PD-L1 gene and the internal reference gene is as follows:

when the absolute value of the Ct difference value between the PD-L1 gene and the reference gene is more than or equal to a first threshold value, the weak expression of PD-L1 is predicted;

when the second threshold value is less than or equal to the absolute value of the Ct difference value between the PD-L1 gene and the reference gene is less than the first threshold value, the expression in PD-L1 is predicted;

and when the absolute value of the Ct difference value of the PD-L1 gene and the reference gene is less than a second threshold value, strong expression of PD-L1 is predicted.

4. The method of claim 3, wherein the first threshold is 15;

and/or the second threshold is 10.

5. The method as claimed in claim 1 or 2, wherein the probe for real-time fluorescent quantitative PCR detection of PD-L1 gene comprises the nucleotide sequence shown in SEQ ID NO. 3;

and/or, the primer for carrying out real-time fluorescent quantitative PCR detection on the PD-L1 gene is selected from at least one of the following nucleotide sequence combinations:

1) the nucleotide sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2;

2) the nucleotide sequences shown as SEQ ID NO. 7 and SEQ ID NO. 8;

and/or the reference gene is selected from at least one of beta-Actin, GAPDH, U6, HMBS, B2M, TUBB, SDHA, 18S rRNA, ACTB, RPL4, PPIA, HPRT1, YWHAZ, RPP30 and ERG;

and/or the reference gene is selected from beta-Actin, and a probe for carrying out real-time fluorescent quantitative PCR detection on the reference gene comprises a nucleotide sequence shown as SEQ ID NO. 6;

and/or the reference gene is selected from beta-Actin, and the primer for real-time fluorescent quantitative PCR detection of the reference gene is selected from at least one of the following nucleotide sequence combinations:

1) the nucleotide sequences shown as SEQ ID NO. 4 and SEQ ID NO. 5;

2) the nucleotide sequences shown as SEQ ID NO. 9 and SEQ ID NO. 10;

and/or, the cycle number of the real-time fluorescence quantitative PCR reaction is 40-50, and fluorescence is collected during the cycle reaction;

and/or, real-time fluorescent quantitative PCR reaction, each cycle reaction is as follows: 94-98 deg.C for 10-20 s; 56-62 ℃ for 15-35 s;

and/or, in the real-time fluorescence quantitative PCR reaction, the sequence before the cycle reaction is as follows: at 40-45 deg.C for 94-98 min; 94-98 deg.C for 1-5 min;

and/or the 5 'end of the probe used for carrying out real-time fluorescence quantitative PCR detection on the PD-L1 gene is modified with a first marker molecule, and the 3' end is modified with a second marker molecule; the 5 'end of the probe used for carrying out real-time fluorescent quantitative PCR detection on the reference gene is modified with a third marker molecule, and the 3' end is modified with a fourth marker molecule; when the first marker molecule and the second marker molecule are physically close to each other, fluorescence energy resonance transfer can occur, and when the third marker molecule and the fourth marker molecule are physically close to each other, fluorescence energy resonance transfer can occur;

and/or the first marker molecule and the third marker molecule are fluorescence reporters, and the second marker molecule and the fourth marker molecule are fluorescence quenchers;

and/or the wavelength of fluorescence emitted by the first labeling molecule is different from the wavelength of fluorescence emitted by the third labeling molecule;

and/or the fluorescent reporter group is selected from at least one of FAM, HEX, VIC, ROX and Cy 5;

and/or the second marker molecule and the fourth marker molecule are the same or different fluorescence quenching groups;

and/or the fluorescence quenching group is selected from at least one of BHQ1, BHQ2 and MGB.

6. The method of claim 1 or 2, wherein the real-time fluorescent quantitative PCR detection of PD-L1 gene and the real-time fluorescent quantitative PCR detection of reference gene are performed in the same or different reaction vessels;

and/or, the free RNA sample is derived from a human or animal body;

and/or, the free RNA sample is extracted from at least one of blood, hydrothorax and ascites, uterine cavity fluid and urine;

and/or, the free RNA sample is extracted from peripheral blood of a tumor patient;

and/or, the tumor comprises a solid tumor, a non-solid tumor;

and/or, the tumor comprises lung cancer, liver cancer, breast cancer, esophageal cancer, colorectal cancer;

and/or, the free RNA sample is extracted from plasma.

7. A probe primer combination for real-time fluorescent quantitative PCR detection of PD-L1 gene is characterized in that the probe comprises a nucleotide sequence shown as SEQ ID NO. 3, and the primer is selected from at least one of the following nucleotide sequence combinations:

1) the nucleotide sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2;

2) the nucleotide sequences shown as SEQ ID NO. 7 and SEQ ID NO. 8.

8. A probe primer combination for real-time fluorescent quantitative PCR detection of PD-L1 gene and reference gene is characterized in that a probe for real-time fluorescent quantitative PCR detection of PD-L1 gene comprises a nucleotide sequence shown as SEQ ID NO:3, and the primer is selected from at least one of the following nucleotide sequence combinations:

1) the nucleotide sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2;

2) the nucleotide sequences shown as SEQ ID NO. 7 and SEQ ID NO. 8;

1) the nucleotide sequences shown as SEQ ID NO. 4 and SEQ ID NO. 5;

2) the nucleotide sequences shown as SEQ ID NO. 9 and SEQ ID NO. 10.

9. A kit for detecting the expression level of PD-L1 based on free RNA is characterized by comprising a probe primer combination for detecting PD-L1 gene and reference gene by real-time fluorescent quantitative PCR.

10. The kit of claim 9, wherein the probe sequence for real-time fluorescent quantitative PCR detection of PD-L1 gene comprises the nucleotide sequence shown in SEQ ID No. 3;

1) the nucleotide sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2;

2) the nucleotide sequences shown as SEQ ID NO. 7 and SEQ ID NO. 8;

1) the nucleotide sequences shown as SEQ ID NO. 4 and SEQ ID NO. 5;

2) the nucleotide sequences shown as SEQ ID NO. 9 and SEQ ID NO. 10;

and/or, the kit also comprises a reagent for reverse transcription of free RNA and real-time fluorescent quantitative PCR detection;

and/or the kit also comprises a one-step reverse transcription-real-time fluorescence quantitative PCR reaction solution for reverse transcription of free RNA and real-time fluorescence quantitative PCR detection;

and/or, the kit also comprises a container for independently storing each probe, primer and reagent;

and/or, the kit further comprises instructions.