Kit and method for detecting PD-1 and PD-L1expression quantity through RNA level

A PD-L1 and PD-1 technology, applied in the field of biomedicine, can solve the problems of poor stability, low specificity, and low sensitivity of immunohistochemical detection, and achieve the effect of wide application range and small sample size

Inactive Publication Date: 2019-04-16
ZHENYUE BIOTECHNOLOGY JIANGSU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims to provide a kit and method for detecting the expression of PD-1 and PD-L1 at the RNA level, so as to solve the technical problems of low sensitivity, low specificity and poor stability of immunohistochemical detection in the prior art

Method used

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  • Kit and method for detecting PD-1 and PD-L1expression quantity through RNA level
  • Kit and method for detecting PD-1 and PD-L1expression quantity through RNA level
  • Kit and method for detecting PD-1 and PD-L1expression quantity through RNA level

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Experimental program
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Embodiment approach

[0023] According to a typical embodiment of the present invention, a kit for detecting the expression of PD-1 and PD-L1 at the RNA level is provided. The kit includes: housekeeping gene amplification primers, PD-1 amplification primers and PD-L1 amplification primers.

[0024] Using the kit of the present invention, the expression level of PD-1 / PD-L1 can be detected from the RNA level. For the tissue samples of cancer patients that cannot be detected by conventional IHC (immunohistochemistry, IHC), especially the tissue preservation solution For soaked tissue samples or puncture samples, the present invention can detect the expression level of PD-1 / PD-L1 in these samples, mainly because the present invention has a wide range of application and requires a small amount of samples. As long as it can extract Both FFPE samples and tissue samples for RNA can be tested.

[0025] Preferably, the housekeeping genes amplified by the housekeeping gene amplification primers include GUSB,...

Embodiment 1

[0102] 1. Design and preparation of the panel

[0103] 1. Panel Design Principles

[0104] This panel selected 12 genes, including PD-1, PD-L1 and 10 House Keeping genes. The specific genes are shown in Table 7.

[0105] In this embodiment, 3 PCR regions are designed for each gene, and the average value of the three regions is taken as the expression level, and normalized to obtain the NRPM value. The principle of primer design is to design spanning exon primers to exclude DNA interference. Through multiplex PCR amplification, the amplicon length is about 110bp. The results of PD-1 and PD-L1 were standardized according to the median of multiples of 10 HK genes = (RPM of HK) / (RPM of HK standard value) to obtain the NRPM value.

[0106] Table 7

[0107]

[0108]

[0109] 2. Primer resuspension and pooling

[0110] 2.1 Dilution of primers

[0111] The synthesized primers contained 0.4 nmol primers in each well, and were diluted to 50 μM (0.05 nmol / μL), that is, 8 μL ID...

Embodiment 2

[0170] FFPE samples from 10 tumor patients with positive PD-1 or PD-L1 detected by NGS were taken for verification.

[0171] The FFPE samples of 10 tumor patients whose PD-1 / PD-L1 was detected by NGS were selected, followed by reverse transcription of RNA into cDNA, specific amplification of amplicons, amplicon purification, library amplification, library purification and For data analysis, etc., the specific operation steps are consistent with the steps in "2. Determination of baseline and interpretation of results" in Example 1.

[0172] Result judgment

[0173] The sequencing results of 10 FFPE samples are shown in Table 8 below:

[0174] Table 8

[0175]

[0176]

[0177] Quality control standard: When HK>=6, On_Target_Ratio>=0.6, Mapped_Reads>200K, the sample sequencing result has passed the quality control, and the expression of PD-1 / PD-L1 can be further determined.

[0178] It is determined that the cutoff value of PD-L1 in the negative sample is NRPM=50, if the ...

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Abstract

The invention discloses a kit and method for detecting the PD-1 and PD-L1 expression quantity through the RNA level. The kit comprises a house-keeping-geneamplification primer, a PD-l amplification primer and a PD-L1amplification primer. By using the kit or method, the PD-1/PD-L1 expression level can be detected through the RNA level; for tissue samples which cannot be detected with a conventionalimmunohistochemistry (IHC) of cancer patients, particularlyfor tissue samples or puncture samples soaked intissue preservation fluid, as the application range of the kit and the method is wide, a small quantity of samples are needed, FFPE samples and tissue samples of the RNA can be extracted, detection can be carried out, the kit or the method can detect the PD-1/PD-L1 expression level of the samples.

Description

technical field [0001] The invention relates to the field of biomedical technology, in particular to a kit and method for detecting the expression of PD-1 and PD-L1 at the RNA level. Background technique [0002] With the official approval of the China Food and Drug Administration (CFDA) for the launch of the first PD-1 inhibitor nivolumab in China, it indicates that my country has entered a new era of immunotherapy. Tumor immunotherapy is to mobilize the body's own immune system and enhance anti-tumor immunity, thereby inhibiting and killing tumor cells. Its appearance has changed the pattern of traditional surgical treatment, radiotherapy and chemotherapy, and is the most promising in the current field of tumor treatment. one of the research directions. In the interaction between tumor cells and immune effector cells, the ligand of programmed cell death-ligand 1 (PD-L1) on the surface of tumor cells and the programmed death on the surface of immune effector cells (T lymph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/106C12Q2600/158C12Q2600/16C12Q2600/166
Inventor 林小静陈敏浚颜林林侯军艳何骥杜波陈维之
Owner ZHENYUE BIOTECHNOLOGY JIANGSU CO LTD
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