Probe composition for nasopharynx cancer MRD detection, kit and application

Abstract

The invention belongs to the technical field of tumor detection, and particularly relates to a probe composition for nasopharyngeal carcinoma MRD detection, a kit and application, the probe composition for nasopharyngeal carcinoma MRD detection comprises the following probes, the sequences of the probes are SEQ IN NO.1-106; according to the targeted probe capture technology, the probe is designed according to the whole genome sequence of the EB virus, so that all EB virus sequences in a sample are captured, and the detection sensitivity and reliability are improved.

Description

Technical Field

[0001] This invention belongs to the field of tumor detection technology, specifically relating to a probe composition, kit, and application for nasopharyngeal carcinoma MRD detection. Background Technology

[0002] Nasopharyngeal carcinoma (NPC) MRD (minimum residual disease) detection is a highly sensitive monitoring technology based on circulating tumor DNA (ctDNA), which can detect recurrence risk earlier than imaging. Its core applications and key points include: target selection. Probe combinations need to cover core driver mutations in NPC, especially Epstein-Barr virus (EBV)-related gene variants (such as EBNA1, LMP1, etc.). Multi-gene panels (hundreds to thousands of sites) are used to improve detection sensitivity and reduce the risk of missed detection due to tumor heterogeneity. The detection sensitivity of current probe combinations is not high enough and needs further improvement. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a probe composition, kit and application for MRD detection of nasopharyngeal carcinoma, thereby improving the detection sensitivity.

[0004] This invention provides a probe composition for nasopharyngeal carcinoma MRD detection, comprising the following probes, the sequences of which are SEQ IN NO.1-106.

[0005] This invention provides a kit for nasopharyngeal carcinoma MRD detection, comprising the aforementioned probe composition.

[0006] This invention provides an application of the probe composition or the kit described above, wherein the probe composition or kit is used to prepare a substance for nasopharyngeal carcinoma MRD detection.

[0007] The beneficial effects of this invention are that it is a nasopharyngeal carcinoma MRD detection based on EB virus detection. It uses targeted probe capture and high-throughput sequencing (NGS) technology to detect EB virus in the sample. The targeted probe capture technology designs probes targeting the whole genome sequence of EB virus to achieve complete capture of the EB virus sequence in the sample, thereby improving the sensitivity and reliability of detection. Detailed Implementation

[0008] Example 1 The nasopharyngeal carcinoma MRD detection method based on EB virus detection includes the following steps: (1) Sample collection and transportation: 10 ml of peripheral blood was collected using Streck blood collection tubes and transported within the range of 6-37℃. The blood was delivered to the testing laboratory within 5 days after collection.

[0009] (2) cfDNA extraction: Plasma was separated by centrifugation, and then cfDNA was extracted from the plasma sample using the MagMAX™ Cell-Free DNAIsolation Kit.

[0010] (3) Library construction: DNA end repair and dA tail addition were completed in one step, followed by the addition of a Y-shaped adapter using the TA ligation method. Finally, the library was amplified by PCR and purified to obtain the sequencing library. The cfDNA library was constructed using the Hieff NGS® UltimaPro DNA Library Prep Kit for Illumina®.

[0011] (4) Capture: The intermediate library is hybridized with a specific probe designed for EB virus (see SEQ IN NO.1-106). After hybridization incubation, streptomycin-labeled magnetic beads are added to capture the target sequence to obtain a normal library.

[0012] Probe capture offers the following advantages over traditional post-isolation and culture WGS sequencing and ultramultiplex tNGS methods: 1. Compared to the WGS method, the probe capture method does not require viral classification and culture before sequencing to obtain the whole genome sequence. The probe capture is performed in clinical samples (such as BALF, swabs, blood, etc.). With high coverage probes, most of the EB virus genome can be captured directly with high sensitivity. Subsequent studies can be carried out by splicing together a portion of the genome.

[0013] 2. For 172k length EB virus, ultra-multiplex tNGS is very difficult to achieve high coverage due to primer design and amplification length limitations, so its sensitivity is lower than that of probe methods. In addition, PCR-based ultra-multiplex tNGS may not be able to amplify for EB virus with local mutations, further reducing its detection sensitivity. In contrast, probe capture only requires 90% probe fragment consistency to capture the corresponding fragment, and has better sensitivity for highly variable species such as viruses.

[0014] In terms of probe design, the probes used in this method have undergone more targeted adjustments: First, to ensure detection sensitivity, the probe coverage is >50% of the genome to guarantee the possibility of the target fragment being present; second, the probe design is based on clinical viral genome sequences. The inventors merged the EB virus genomes found in domestic clinical trials, while also considering base degeneracy, and designed probes based on their specific sequences that distinguish them from other pathogens; third, in the wet experiment, we used a high concentration of probes to ensure the probes were in a supersaturated state to further improve the sensitivity of capture and enrichment; finally, to ensure the quality of each probe, the probes used in this study are synthesized individually. In addition to basic indicators such as concentration and length, the synthesized probes are also directly sequenced by WGS to check the correctness of each base on the probe.

[0015] (5) Sequencing: The library is circularized and amplified in one step to obtain DNB nanospheres, which can be loaded onto a microarray chip for sequencing. Using optimized combined probe-anchor synthesis (cPAS), sequencing primer anchoring molecules and fluorescent probes are polymerized on DNA nanospheres. A high-resolution imaging system is then used to acquire, read, and identify the light signals to obtain individual base sequence information. The next cycle is then performed to obtain the next base sequence information. After 50-150 cycles of single-end or paired-end sequencing, the base sequence information is finally combined into a complete DNA sequence using an algorithm. The sequencing process is based on the DNBSEQ-G99 sequencing platform for metagenomic SE100 sequencing.

[0016] (6) Bioinformatics analysis and interpretation: The sequencing data after the machine is compared with the pathogen database to obtain the detection status of EB virus in the sample. After manual review, the report is issued.

[0017] This invention improves the detection LOD of EB virus to 5-25 copies / mL, enabling efficient detection of EB virus with lower viral loads. The innovation of this method is reflected in the following three aspects: 1. Probe capture stage: EB virus whole genome capture probe set, a probe set designed for the whole genome sequence of EB virus, can capture all EB virus sequences in the sample library.

[0018] 2. Sequencing process: The latest DNBSEQ-G99 sequencer was used, which accelerated the detection time.

[0019] 3. Analysis and Interpretation: A specially customized database for EB virus greatly speeds up the analysis time and improves accuracy.

[0020] Based on the innovations in these three aspects, we improve the LOD (Level of Detection) of EBV in terms of coverage and sensitivity. The full length of EBV is 172,764 bp. Currently, commonly used qPCR methods can only detect EBV fragments within 200 bp, typically detecting fragments from EBV positions 33052 to 33127, which may result in missed detection of other EBV fragments (PMID: 39059389). NGS can detect trace amounts of EBV signals in plasma. The typical EBV detection load range is 25-150,000 copies / mL (PMID: 10096545, PMID: 38320164). Our method can significantly improve the LOD to 5-25 copies / mL.

[0021] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; within the framework of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of one or more embodiments of this application as described above, which are not provided in detail for the sake of brevity.

[0022] One or more embodiments in this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of one or more embodiments in this application should be included within the protection scope of this application.

Claims

1. A probe composition for MRD detection of nasopharyngeal carcinoma, characterized in that, The probes include the following probes, whose sequences are SEQ IN NO.1-106.

2. A kit for MRD detection of nasopharyngeal carcinoma, characterized in that, Includes the probe composition as described in claim 1.

3. The application of the probe composition as described in claim 1 or the kit as described in claim 2, characterized in that, The probe composition or kit is used to prepare substances for the MRD detection of nasopharyngeal carcinoma.