Reagent for detecting cross contamination of PDX-model related with human and mouse, the kit comprising the same, and the method for the cross contamination detection
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Publication Number
US-10472678-B2
Publication Date
2019-11-12
Expiration Date
Abstract
The present application relates to a detection kit for genotypes capable of confirming cross contamination that may occur in a banking process of a patient-derived xenograft model or cell-derived xenograft model and a method for determining cross contamination using the same. According to the present invention, it is possible to determine all of cross contamination of mouse related genes, have high detection sensitivity and specificity to be close to 100%, rapidly examine the contamination, and be very useful in predicting mouse contamination.Therefore, according to the present invention, cross contamination of genes related with the human and the mouse is predicted in advance to be applied to evaluation of anticancer drug efficacy using a patient-derived xenograft model or cell-derived xenograft model and contribute to cell banks using the patient-derived xenograft model or cell-derived xenograft model, and as a result, the present invention is very useful in a medical industry.
Core Innovation
The invention relates to a detection kit for determining patient-derived xenograft cells. The kit includes oligonucleotide primers consisting of base sequences of SEQ ID NOS: 9 and 10 and oligonucleotide probes consisting of base sequences of SEQ ID NOS: 9 and 10, with an attached label, and the primers and probes determine a ratio of genes included in xenograft cells of a human and a mouse.
The kit is further defined by measuring a ratio of a human papola gene and a mouse papola gene included in xenograft cells of a human and a mouse. It provides an oligonucleotide probe concentration condition in which the concentration of the oligonucleotide probes is 1 pmol or more, and the labeled probes enable detection based on binding of the probes and resulting gene ratio analysis.
The invention also includes a method for determining cross contamination of mouse genes for human cells in a method for determining patient-derived xenograft cells. The method amplifies a human albumin gene and a mouse albumin gene using oligonucleotide primers consisting of base sequences of SEQ ID NOS: 1 to 4 by a PCR method, hydrolyzes oligonucleotide probes consisting of base sequences of SEQ ID NOS: 9 and 10, and detects binding of the probes to determine a ratio of the human albumin gene and the mouse albumin gene to determine cross contamination.
Claims Coverage
The document contains two independent claims: one directed to a detection kit and one directed to a method for determining cross contamination by determining a human-to-mouse gene ratio. Each independent claim centers on determining a ratio between human and mouse genes in patient-derived xenograft cells, with specific human/mouse gene targets in each claim.
Human-to-mouse gene ratio detection with labeled primers and probes
A detection kit that determines patient-derived xenograft cells comprising oligonucleotide primers consisting of base sequences of SEQ ID NOS: 9 and 10, oligonucleotide probes consisting of base sequences of SEQ ID NOS: 9 and 10, and an attached label, wherein the primers and the probes determine a ratio of genes included in xenograft cells of a human and a mouse.
PCR amplification, probe hydrolysis, and binding-based ratio for cross contamination
A method for determining cross contamination of mouse genes for human cells comprising amplifying a human albumin gene and a mouse albumin gene using oligonucleotide primers consisting of base sequences of SEQ ID NOS: 1 to 4 by a PCR method; hydrolyzing oligonucleotide probes consisting of base sequences of SEQ ID NOS: 9 and 10; and detecting binding of the probes and determining a ratio of the human albumin gene and the mouse albumin gene to determine cross contamination.
Across the independent claims, the coverage is based on determining a human-to-mouse gene ratio in patient-derived xenograft cells, using a kit with labeled oligonucleotide primers/probes (SEQ ID NOS: 9 and 10) or using a method that amplifies human albumin and mouse albumin genes (SEQ ID NOS: 1 to 4 primers) followed by hydrolysis of probes (SEQ ID NOS: 9 and 10) and binding detection to derive the ratio used for cross contamination determination.
Stated Advantages
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