CN117721202A - Capture probe pool and reagent kit for lung cancer MRD detection - Google Patents

Abstract

The invention provides a capture probe pool for lung cancer MRD detection and a detection kit. The capture probe pool comprises positive chain capture probes and negative chain capture probes which are designed for 16 lung cancer related genes and are distributed in a staggered manner, and can cover all target gene sequences, so that the problems of low probe utilization rate, low capture efficiency, high background noise and the like caused by the mutual hybridization among the probes can be effectively avoided, and the positive chain capture probes and the negative chain capture probes of target genes in a library can be captured at the same time, so that library abundance is improved, and hybridization capture efficiency is improved. The invention also provides a lung cancer MRD detection kit comprising the capture probe pool for lung cancer MRD detection and hybridization solution, which has good detection sensitivity and specificity, low background and short hybridization capture time, can effectively detect various variation types of 16 lung cancer related genes, and can well realize lung cancer MRD detection.

Description

Capture probe pool and reagent kit for lung cancer MRD detection

Technical Field

The invention belongs to the technical field of biology, relates to molecular biology detection, and in particular relates to a capture probe pool and a kit for lung cancer MRD detection.

Background

The minimal residual lesions (Minimal residual disease, MRD) are also referred to as measurable residual lesions (Measurable residual disease), molecular residual lesions (Molecular residual disease), meaning that very few cancer cell survival or cancer cell-derived molecular abnormalities are detectable by liquid biopsy, which are undetectable by traditional imaging or experimental methods after treatment of cancer patients (Wang Y et al Biomedical Transformation,2021, 2:49-59). Included in MRD are micro recurrent residual foci (M-REC), micro non recurrent residual foci (MN-REC), micro residual foci precursors (MRPs), micro Residual Nucleic Acids (MRNAs) and Micro Residual Metabolites (MRMs) (Luskin MR et al, nat Rev Cancer,2018, 18:255-263). In addition to MN-REC, M-REC and MRPs are both one of the possibilities for distant recurrence of MRD in the hidden stage of tumor development, and MRNAs and MRMs may also be associated with recurrence of tumors. Therefore, MRD is considered to be the main cause of cancer recurrence/metastasis.

In lung cancer, MRD is a potential source of distant metastasis and recurrence in patients, and monitoring of MRD during lung cancer is important. According to the expert consensus of residual focus of non-small cell lung cancer molecules, the application of MRD detection in lung cancer has three main aspects. In a first aspect, use of an operable early stage non-small cell lung cancer (NSCLC) MRD, comprising: (1) After radical excision of an early NSCLC patient, the MRD positive prompt has high recurrence risk, close follow-up management is needed, and one MRD detection is recommended every 3-6 months; (2) It is suggested to develop perioperative clinical trials of operable NSCLC based on MRD, providing as accurate a treatment regimen of perioperative period as possible; (3) It is suggested to explore the role of MRD in both types of patients, driving gene positive and driving gene negative, respectively. In a second aspect, use of a locally advanced NSCLC MRD, comprising: (1) Complete remission of patients after radical chemoradiotherapy of locally advanced NSCLC suggests detection of MRD, which is helpful for judging prognosis and formulating further treatment strategies; (2) It is recommended to develop clinical trials of MRD-based chemoradiotherapy consolidation therapy, providing as accurate a consolidation therapy regimen as possible. In a third aspect, use of an advanced NSCLC MRD, comprising: (1) Patients are completely relieved after treatment of the advanced NSCLC system, and MRD detection is recommended, so that prognosis judgment and further treatment strategies are facilitated; (2) It is suggested to develop MRD-based treatment strategy studies in fully-palliative patients, extending the time of full-palliative as much as possible, allowing the patients to benefit most.

Therefore, the MRD detection of the lung cancer has huge clinical application prospect, not only can early find the patients with high risk of recurrence, but also can early intervene in treatment, strives for curing more patients, reduces recurrence and metastasis, improves prognosis, can also screen the patients with low risk of recurrence after lung cancer operation/auxiliary treatment, avoids related risks caused by excessive treatment, and provides a real treatment-free holiday for the patients. In general, the MRD detection of lung cancer is used for predicting the recurrence risk of lung cancer, guiding adjuvant therapy, predicting curative effect, judging whether a patient can enter a non-therapeutic holiday, judging tumor load in the patient, and the like, and finally assisting accurate diagnosis and treatment of lung cancer.

Currently, lung cancer MRD detection is mainly achieved through cfDNA-MRD detection, and a common detection method is based on hybrid capture targeted high throughput sequencing (NGS). The method is a method for obtaining and analyzing the sequence of a target region through hybridization of a capture probe and a specific region in a genome, and can detect point mutation, insertion deletion and copy number variation of small fragments and gene rearrangement at a DNA level, and can find some unknown fusion, wherein the detection sensitivity can reach more than 95%. However, due to the fact that the method has a plurality of operation steps and a relatively complex process, some background noise is inevitably introduced, so that low-frequency mutation in cfDNA-MRD detection is often submerged in the background noise, a false negative or false positive result is caused, and the sensitivity and the specificity of cfDNA-MRD detection are affected. Therefore, how to solve the background interference problem of the targeted NGS method based on hybrid capture becomes the challenge for the current lung cancer MRD detection.

Disclosure of Invention

Based on the above, the invention aims to provide a capture probe pool for lung cancer MRD detection and a detection kit, wherein the detection kit adopts a capture probe pool which is carefully designed for 16 lung cancer related genes and an optimized hybridization solution formula, and performs hybridization capture-based targeting NGS on lung cancer patient plasma sample cfDNA, so that the background interference can be effectively reduced, the detection sensitivity and the detection kit have good detection specificity, and lung cancer MRD detection can be well realized.

The technical scheme for achieving the purpose comprises the following steps.

According to a first aspect of the invention, a capture probe pool for lung cancer MRD detection is provided, wherein the capture probe pool consists of a positive strand capture probe and a negative strand capture probe aiming at least one target gene, and the capture probes of each target gene are sequentially designed and staggered according to the positive strand nucleotide sequence and the negative strand nucleotide sequence of the target gene, so that the capture probes can cover all target gene sequences; among the positive strand capture probes and the negative strand capture probes, two adjacent probes are positive strand capture probes, one is a positive strand capture probe, the other is a negative strand capture probe, and the aimed target gene sequences are not overlapped; the target gene is selected from at least one of the following 16 types: ALK, BRAF, EGFR, HER2, KRAS, MET, NRAS, MEK1, PIK3CA, RET, ROS1, TP53, NTRK1, NTRK2, NTRK3 and PD-L1.

In some embodiments, the 5' ends of the capture probes in the capture probe well are each modified by Biotin (Biotin) labeling.

In some embodiments, the length of the capture probes in the capture probe pool is 80-130 bp; preferably, the length of the capture probes in the capture probe pool is 100-110 bp.

In some of these embodiments, the positive strand capture probes and the negative strand capture probes of the target gene in the capture probe pool are selected from at least one of the following groups:

a capture probe for ALK gene with the sequence shown in SEQ ID NO. 1-SEQ ID NO. 30;

a capture probe for the BRAF gene, the sequence of which is shown as SEQ ID NO. 31-SEQ ID NO. 40;

a capture probe for EGFR gene with the sequence shown as SEQ ID NO. 41-SEQ ID NO. 64;

a capture probe for the HER2 gene with the sequence shown as SEQ ID NO. 65-SEQ ID NO. 80;

a capture probe for KRAS genes, the sequence of which is shown as SEQ ID NO. 81-SEQ ID NO. 86;

a capture probe for MET gene with the sequence shown as SEQ ID NO. 87-SEQ ID NO. 110;

a capture probe for NRAS genes, the sequence of which is shown as SEQ ID NO. 111-SEQ ID NO. 116;

a capture probe for MEK1 gene with the sequence shown as SEQ ID NO. 117-SEQ ID NO. 128;

A capture probe for PIK3CA gene with the sequence shown in SEQ ID NO. 129-SEQ ID NO. 140;

a capture probe for RET gene with the sequence shown as SEQ ID NO. 141-SEQ ID NO. 160;

a capture probe for ROS1 gene with the sequence shown as SEQ ID NO. 161-SEQ ID NO. 180;

a capture probe for TP53 gene with the sequence shown as SEQ ID NO. 181-SEQ ID NO. 192;

a capture probe for NTRK1 gene with a sequence shown as SEQ ID NO. 193-SEQ ID NO. 212;

a capture probe for NTRK2 gene with a sequence shown as SEQ ID NO. 213-SEQ ID NO. 224;

a capture probe for NTRK3 gene with a sequence shown as SEQ ID NO. 225-SEQ ID NO. 238;

the sequence is shown as SEQ ID NO. 239-SEQ ID NO.248 and aims at the capture probe of the PD-L1 gene.

In a second aspect, the present invention provides a hybridization solution for hybridization capture of the capture probe pool, wherein the hybridization solution comprises 300-600 mM sodium phosphate buffer solution (pH 7.0), 10-50% (v/v) of 2 XSSC (pH 7.0), 0.5-1.5% (w/v) of SDS, 1-3% (v/v) of Denhardt solution, 1-5% (w/v) of dextran sulfate, 1-10% (w/v) of trehalose, and 0.1-0.5% (v/v) of Tween-20.

In some of these embodiments, the hybridization solution comprises 400-500 mM sodium phosphate buffer (pH 7.0), 20-40% (v/v) 2 XSSC (pH 7.0), 0.8-1.2% (w/v) SDS, 1.5-2.5% (v/v) Denhardt's solution, 2-4% (w/v) dextran sulfate, 4-7% (w/v) trehalose, 0.2-0.4% (v/v) Tween-20.

In some preferred embodiments, the hybridization solution comprises 450mM sodium phosphate buffer (pH 7.0), 30% (v/v) 2 XSSC (pH 7.0), 1% (w/v) SDS, 2% (v/v) Denhardt's solution, 3% (w/v) dextran sulfate, 5.5% (w/v) trehalose, 0.3% (v/v) Tween-20.

In some of these embodiments, the hybridization solution has a pH of 6.8 to 7.2.

In some preferred embodiments, the hybridization solution has a pH of 7.0.

In a third aspect of the invention, there is provided a lung cancer MRD detection kit comprising a hybridization solution as described above and a capture probe pool; wherein the capture probe pool is formed by mixing and diluting each capture probe in the capture probe pool for lung cancer MRD detection to the use concentration according to the same molar ratio.

In some of these embodiments, the concentration of each capture probe used is from 0.1pM to 6pM.

In some preferred embodiments, the concentration of each capture probe used is 1pM to 2pM; more preferably 1.4pM to 1.6pM.

In a fourth aspect of the present invention, there is provided a lung cancer MRD detection method, mainly comprising the steps of:

s1, extracting cfDNA of plasma: collecting a blood sample of a subject, centrifugally separating plasma, and extracting the plasma cfDNA sample according to the product specification by using a commercial plasma cfDNA extraction kit;

s2, constructing a library: library construction of the extracted plasma cfDNA samples using a commercially available library construction kit according to its product instructions;

s3, hybridization capture: hybridization is carried out by using the lung cancer MRD detection kit provided by the invention, and target fragments are captured;

s4, on-machine sequencing and data analysis.

In some of these embodiments, the hybridization capture step, hybridization time is 1 to 16 hours; preferably, the hybridization time is 1.5 to 2.5 hours.

The invention provides a capture probe pool for lung cancer MRD detection, which consists of a positive strand capture probe and a negative strand capture probe aiming at least one target gene, wherein the target gene is selected from at least one of the following 16 types: ALK, BRAF, EGFR, HER2, KRAS, MET, NRAS, MEK, PIK3CA, RET, ROS1, TP53, NTRK1, NTRK2, NTRK3 and PD-L1, wherein the capture probes are designed by staggered arrangement of positive and negative chain probes, the capture probes of each target gene are sequentially designed according to the positive and negative chain nucleotide sequences of the target gene, are staggered and arranged, cover all target gene sequences, not only avoid the problems of low probe utilization rate, low capture efficiency, high background noise and the like caused by mutual hybridization among the probes, but also simultaneously capture the positive and negative chain of the target gene in the library, improve library abundance, increase the opportunity of hybridization between the capture probes and the target gene sequences in the library, and further improve hybridization capture efficiency.

The invention also provides a hybridization solution, which can not only effectively shorten the hybridization capture time and save the time cost, but also reduce the nonspecific background noise while enhancing the hybridization signal of the target gene, thereby well controlling the background value generated during hybridization and improving the hybridization capture effect.

The invention provides a lung cancer MRD detection kit and a detection method thereof, wherein the kit has good detection sensitivity and specificity, low background and short hybridization capture time, can effectively detect a plurality of mutation types such as ALK, BRAF, EGFR, HER2, KRAS, MET, NRAS, MEK1, PIK3CA, RET, ROS1, TP53, NTRK1, NTRK2, NTRK3 and PD-L1 total 16 lung cancer related genes, such as point mutation/insertion/deletion, gene fusion, gene copy number mutation and the like, and can well realize lung cancer MRD detection.

Drawings

FIG. 1 is a schematic flow chart of the lung cancer MRD detection method of the invention.

Detailed Description

The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Experimental methods, in which specific conditions are not noted in the examples below, are generally carried out according to conventional conditions, for example, green and Sambrook-s.A.fourth edition, molecular cloning, A.laboratory Manual (Molecular Cloning: A Laboratory Manual), published in 2013, or according to the conditions recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

Furthermore, as used herein, the term "or" is an inclusive "or" symbol and is equivalent to the term "and/or" unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. Furthermore, throughout the specification, the meaning of "a", "an", and "the" include plural referents. The meaning of "in" is included "in" and "on".

In some embodiments, the target gene capture probes are synthesized by the division of biological engineering (Shanghai) and are prepared into mother solutions of 100 mu M respectively for storage, and the mother solutions of the capture probe pools are mixed according to the same molar ratio according to requirements for standby.

In the extraction of the plasma cfDNA, cfDNA was extracted using a commercially available plasma cfDNA extraction kit QIAamp Circulating Nucleic Acid Kit according to the product instructions, and the obtained plasma cfDNA samples were quantified using Qubit 4.0.

The above extracted plasma cfDNA samples were library constructed using a commercially available library construction kit xGen Prism DNA Library Prep Kit according to their product instructions. The cfDNA library constructed was quantified using Qubit 4.0 and fragment distribution analysis was performed using Bioanalyzer (Agilent).

The present invention will be described in further detail with reference to specific examples.

Example 1 preparation of Capture Probe pool, hybridization solution and kit for Lung cancer MRD detection

The capture probe pool consists of positive and negative strand capture probes which are designed for the target gene positive and negative strand nucleotide sequences in sequence and are arranged alternately, and can cover all target gene sequences. The target genes are finely selected according to NCCN guidelines, FDA/CFDA guidelines and combined with a plurality of authority databases, and comprise 16 lung cancer related genes including ALK, BRAF, EGFR, HER2, KRAS, MET, NRAS, MEK1, PIK3CA, RET, ROS1, TP53, NTRK1, NTRK2, NTRK3 and PD-L1; in the positive strand capture probes and the negative strand capture probes, two adjacent probes, one is a positive strand probe and the other is a negative strand probe, and the targeted target gene sequences are not overlapped; the 5' end of the probe is modified by Biotin (Biotin) marking; the probe length is 80 to 130bp, and the preferred length is 100 to 110bp in this example (refer to example 4). The base sequences of the probes are shown in Table 1.

TABLE 1 base sequence of target Gene Capture Probe

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Description

The target gene capture probe is synthesized by the general biological engineering (Shanghai) Co., ltd, respectively prepared into mother liquor of 100 mu M for storage, and mixed into capture probe pool mother liquor according to the same molar ratio as required for standby.

2. Preparation of hybridization solution

The hybridization solution is used for hybridization capture of the capture probe pool and comprises 300-600 mM sodium phosphate buffer solution (pH value 7.0), 10-50% (v/v) of 2 XSSC (pH value 7.0), 0.5-1.5% (w/v) of SDS, 1-3% (v/v) of Denhardt solution, 1-5% (w/v) of dextran sulfate, 1-10% (w/v) of trehalose and 0.1-0.5% (v/v) of Tween-20. The pH value of the hybridization solution is 6.8-7.2.

Preferably, the hybridization solution comprises 400-500 mM sodium phosphate buffer (pH 7.0), 20-40% (v/v) of 2 XSSC (pH 7.0), 0.8-1.2% (w/v) of SDS, 1.5-2.5% (v/v) of Denhardt solution, 2-4% (w/v) of dextran sulfate, 4-7% (w/v) of trehalose, 0.2-0.4% (v/v) of Tween-20, and the pH of the hybridization solution is 7.0;

in this example, the hybridization solution was used to contain 450mM sodium phosphate buffer (pH 7.0), 30% (v/v) of 2 XSSC (pH 7.0), 1% (w/v) of SDS, 2% (v/v) of Denhardt's solution, 3% (w/v) of dextran sulfate, 5.5% (w/v) of trehalose, and 0.3% (v/v) of Tween-20 (see example 5), and the hybridization solution had a pH of 7.0 (see example 6).

Preparing hybridization solution according to the above hybridization solution formula according to the requirement.

3. Preparation of the kit

The lung cancer MRD detection kit comprises the hybridization solution and the capture probe pool solution. The capture probe pool solution is formed by diluting the capture probe pool mother solution to the use concentration. Each capture probe of the capture probe pool solution is used at a concentration of 0.1pM to 6pM. The concentration of each capture probe used in the capture probe pool solution described in this example is preferably 1.5pM (refer to example 8). The hybridization solution and the capture probe pool solution are packaged and assembled according to the specification of the kit.

The embodiment also relates to a detection method for lung cancer MRD detection by using the kit, which mainly comprises the following steps:

s1, extracting cfDNA of plasma: collecting a blood sample of a subject, centrifugally separating plasma, and extracting the plasma cfDNA sample according to the product specification by using a commercial plasma cfDNA extraction kit;

s2, constructing a library: library construction of the extracted plasma cfDNA samples using a commercially available library construction kit according to its product instructions;

s3, hybridization capture: hybridization is carried out by using the lung cancer MRD detection kit provided by the invention, and target fragments are captured;

S4, on-machine sequencing and data analysis.

In the hybridization capturing step, the hybridization time is 1-16 hours; preferably, the hybridization time in this example is 2 hours (see example 9).

Example 2 detection of samples Using the kit and detection method of example 1

The various reagents are shown in Table 2.

List of reagents described in Table 2

Reagent name	Source
		QIAamp Circulating Nucleic Acid Kit	From QIAGEN company, cat No.: 55114
xGen Prism DNA Library Prep Kit	From IDT company, product number: 10006203
		Human Cot DNA	From IDT company, product number: 1080769
Universal Blockers-TS Mis	From IDT company, product number: 1075475
		Hybridization solution	Homemade, from the inventive kit described in example 1
Capture probes Chi Rongye	Homemade, from the inventive kit described in example 1
		xGen Hybridization and Wash Kit	From IDT company, product number: 1080584
2×KAPA HiFi PCR Master Mix	From Kapa Biosystems, cat: KK2601
		xGen Library Amplification Primer Mix	From IDT company, product number: 1077677
Nuclease-free water	From Thermo Fisher Scientific company, cat No.: AM9938

In this example, a blood sample of a lung cancer patient was collected and plasma cfDNA was detected in the sample, wherein both the hybridization solution and the capture probe pool solution used the corresponding components in the kit described in example 1.

1. Extraction of blood plasma cfDNA

A blood sample of the subject was collected using a asian blood collection tube, the plasma was centrifuged, cfDNA was extracted using a commercial plasma cfDNA extraction kit QIAamp Circulating Nucleic Acid Kit according to its product instructions, and the resulting plasma cfDNA sample was quantified using Qubit 4.0.

2. Construction of libraries

The above extracted plasma cfDNA samples were library constructed using a commercially available library construction kit xGen Prism DNA Library Prep Kit according to their product instructions. The cfDNA library constructed was quantified using Qubit 4.0 and fragment distribution analysis was performed using Bioanalyzer (Agilent).

3. Hybrid capture

Hybridization was performed using the lung cancer MRD detection kit described in example 1, and the target fragment was captured. The method comprises the following specific steps:

(1) Library blocking and concentration: a blocking solution was prepared from 5. Mu.L of Human Cot DNA and 2. Mu. L Universal Blockers-TS Mis, 500ng of the cfDNA library was added thereto, and after mixing, it was dried by vacuum concentration.

(2) Library rapid hybridization: 16. Mu.L of the hybridization solution described in example 1 and 4. Mu.L of the capture probe Chi Rongye described in example 1 were prepared as a hybridization reaction solution, which was added to the above-mentioned extraction tube, mixed well and allowed to stand for 10 minutes, and the hybridization reaction program as shown in Table 3 was run on a PCR instrument.

TABLE 3 hybridization reaction procedure

(3) Library capture and washing: after the hybridization reaction procedure was completed, library capture and washing was performed using the reagent components in the commercial library capture and wash kit xGen Hybridization and Wash Kit according to the product instructions thereof, to obtain 20 μl of the captured cfDNA library.

(4) PCR amplification of the capture library: a PCR reaction system was prepared according to Table 4, and the obtained library was subjected to PCR amplification by running the PCR reaction program shown in Table 5 on a PCR instrument.

TABLE 4 PCR reaction System for PCR amplification Capture library

Reagent name	Volume (mu L)
		2×KAPA HiFi PCR Master Mix	25
xGen Library Amplification Primer Mix	1.25
		Nuclease-free water	3.75
Captured cfDNA library	20
		Total volume of	50

TABLE 5 PCR reaction procedure for PCR amplification of Capture library

(5) And (3) purifying a PCR product: the PCR product was purified using 75. Mu. L Agencourt AMPure XP beads (1.5X), and the purified PCR product was quantified using Qubit 4.0 and analyzed for fragment distribution using Bioanalyzer (Agilent).

4. On-machine sequencing and data analysis

And (3) sequencing the purified PCR product on a machine, and analyzing a sequencing result. Sequencing was performed using the HiSeq X Ten NGS platform from Illumina, inc., using a 2X 150bp double-ended sequencing mode. The positive judgment standard is as follows: when mutation abundance is detected to be more than or equal to 0.02%, determining that MRD is positive; otherwise, it is judged that the MRD is negative.

Blood samples (sample numbers 1 to 6) of 6 healthy subjects and blood samples (sample numbers 7 to 16) of 10 lung cancer patients, whose positive genetic variation was confirmed by Sanger sequencing/FISH method/digital PCR method, were tested according to the test method described above using the kit described in example 1. The specific results are shown in Table 6.

TABLE 6 sample detection results

The detection shows that the detection data Q30 of all samples is more than 85%, the average sequencing depth is more than 8000×, the coverage is high, the capturing efficiency is high, the uniformity is good, and the kit and the detection method can be used for constructing a high-quality sequencing library. Meanwhile, according to the detection results, the blood samples of healthy people are detected, the lung cancer related gene mutation is not found, and the blood samples are MRD negative and accord with the expectation; detecting a blood sample of a lung cancer patient, wherein the detected mutation types have 100% of coincidence rate with clinical detection results, and are all MRD positive; the kit and the detection method thereof have high accuracy.

EXAMPLE 3 Capture Effect verification of Capture probes of the invention

1. Design for verification of capture effect of capture probe

In order to verify the capture effect of the capture probes in the capture probe pool for lung cancer MRD detection, experimental groups 1-4 were designed, and each experimental group was identical except for the types of the capture probes, and the detection effects were compared, and specific designs are shown in Table 7.

TABLE 7 design of capture probes

Experimental group	Type of capture probe
		Group 1	The capture probes of the present invention comprise staggered positive and negative strand probes, the sequences of which are described in example 1
Group 2	Positive strand probe having the same probe design position as the capture probe of the present invention described in example 1
		Group 3	The negative strand probe was designed to have the same position as the capture probe of the present invention described in example 1
Group 4	Double-stranded probes having the same probe design position as the capture probes of the present invention described in example 1

2. Sample detection

In this example, 6 samples (sample numbers 1 to 6, mutation types were EGFR p.T790M, KRAS p.Q61H, PIK3CA p.E545K, BRAF p.V600E, NRAS p.G12S, ALK p.L1196M) were selected and tested by the above-described kit according to the method of example 2. The specific results are shown in Table 8.

TABLE 8 Capture Probe Capture Effect verification detection results

From the above detection results, the four groups can realize accurate detection, and all the mutation types of the samples can be detected correctly. However, experimental group 1 was comparable to experimental group 4, but higher than experimental groups 2 and 3, in terms of the amount of captured library, Q30, and average sequencing depth; in terms of repetition rate, experimental group 1 was comparable to experimental group 4, but lower than experimental groups 2 and 3; in terms of capture efficiency, uniformity, mutation rate, experimental group 1 was higher than experimental groups 2, 3, and 4. The method shows that the capture probes have better capture effect, can simultaneously realize the technical effects of large library capturing quantity, high capture efficiency, low data repetition rate and high sequencing depth, and avoid the problem of high background noise caused by the mutual hybridization among the probes, thereby avoiding the problem of low mutation rate caused by high background noise.

Example 4 determination of the Length of Capture probes of the invention

1. Design of capture probe length

In order to determine the length of the capture probe in the capture probe pool for lung cancer MRD detection, experimental groups 1-5 were designed, and the detection effect of each experimental group was compared with the detection effect of the other experimental groups except for the different length of the capture probe, and the specific design is shown in Table 9.

TABLE 9 design of Capture Probe Length

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2. Sample detection

In this example, 6 samples (sample numbers 1 to 6, mutation types were KRAS p.g12v, BRAF p.v600e, EGFR p.l8611 q, MEK1 p.q56p, HER2 amplification, PIK3CA p.h1047r) were selected and verified by Sanger sequencing/FISH method, and the kit prepared by the above design was used to perform detection according to the method of example 2. The specific results are shown in Table 10.

TABLE 10 comparison of detection results for capture probes of different lengths

From the detection results, the experimental groups 2, 3 and 4 can realize accurate detection, and the mutation types of all samples can be detected correctly, wherein the Q30, the average sequencing depth, the capturing efficiency and the uniformity of the experimental group 3 are slightly better than those of the experimental groups 2 and 4, and the capturing effect is better; while the experimental groups 1 and 5 cannot realize accurate detection, the detection results of the experimental groups cannot respectively have false positive and false negative results (see the group 1 sample 2 and the group 5 sample 4 in the table 10), and the Q30, the average sequencing depth, the capturing efficiency and the uniformity of the experimental groups are lower than those of the experimental groups 2, 3 and 4, presumably because the capturing probe length is too short to increase nonspecific hybridization, the capturing probe length is too long to increase hybridization difficulty, so that the hybridization effect of hybridization between the target gene sequence and the capturing probe is poor, the capturing effect is poor, and even the false positive and/or false negative results are caused. Therefore, the length of the capture probe in the capture probe pool for lung cancer MRD detection is 80-130 bp, preferably 100-110 bp.

EXAMPLE 5 determination of the concentration of the constituents of the hybridization solution of the present invention

1. Design of concentration of hybridization solution composition

In order to determine the concentrations of the components of the hybridization solution of the present invention, experimental groups 1 to 7 were designed, and each experimental group was identical except for the concentrations of the components of the hybridization solution, and the detection effects thereof were compared, and specific designs are shown in Table 11.

TABLE 11 design of hybridization fluid compositions

2. Sample detection

In this example, 6 samples with different mutation types (sample numbers 1 to 6, mutation types were in turn BRAF p.V600E, KRAS p.G12D, TP53 p.P72R, EGFR p.G7199S, PIK3CA p.E542K, and MET amplification) were selected by Sanger sequencing/FISH method, and the kit prepared by the above design was used for detection according to the method of example 2. The specific results are shown in Table 12.

TABLE 12 comparison of detection results of hybridization solutions of different component concentrations

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From the above detection results, all of the experimental groups 2, 3, 4, 5 and 6 can realize accurate detection, and all of the variation types of the samples can be detected correctly, wherein the Q30, the average sequencing depth, the capturing efficiency and the uniformity of the experimental group 4 are better than those of the experimental groups 2, 3, 5 and 6, and the detection effect is better; the experimental groups 1 and 7 cannot realize accurate detection, the detection results of the experimental groups 1 and 7 have false negative results (see the sample 3 of the group 1 and the sample 6 of the group 7 in the table 12), and the Q30, the average sequencing depth, the capturing efficiency and the uniformity of the experimental groups are lower than those of the experimental groups 2, 3, 4, 5 and 6, presumably because the concentration of the components of the hybridization solution is too low or too high, the using effect of the hybridization solution is reduced, the hybridization effect of hybridization between the target gene sequence and the capturing probe is poor, the detection effect is poor, and even the false negative result is caused.

Thus, the hybridization solution of the present invention comprises 300 to 600mM sodium phosphate buffer (pH 7.0), 10 to 50% (v/v) of 2 XSSC (pH 7.0), 0.5 to 1.5% (w/v) of SDS, 1 to 3% (v/v) of Denhardt's solution, 1 to 5% (w/v) of dextran sulfate, 1 to 10% (w/v) of trehalose, 0.1 to 0.5% (v/v) of Tween-20; preferably, the hybridization solution of the present invention comprises 400 to 500mM sodium phosphate buffer (pH 7.0), 20 to 40% (v/v) of 2 XSSC (pH 7.0), 0.8 to 1.2% (w/v) of SDS, 1.5 to 2.5% (v/v) of Denhardt's solution, 2 to 4% (w/v) of dextran sulfate, 4 to 7% (w/v) of trehalose, 0.2 to 0.4% (v/v) of Tween-20; more preferably, the hybridization solution of the present invention comprises 450mM sodium phosphate buffer (pH 7.0), 30% (v/v) of 2 XSSC (pH 7.0), 1% (w/v) of SDS, 2% (v/v) of Denhardt's solution, 3% (w/v) of dextran sulfate, 5.5% (w/v) of trehalose, 0.3% (v/v) of Tween-20.

EXAMPLE 6 determination of the pH of the hybridization solution of the present invention

1. Design of hybridization solution pH value

In order to determine the pH value of the hybridization solution of the invention, experimental groups 1 to 5 were designed, and each experimental group was identical except for the pH value of the hybridization solution, and the detection effect was compared, and the specific design is shown in Table 13.

TABLE 13 design of hybridization solution pH

Experimental group	pH value of hybridization solution
		Group 1	The hybridization solution composition of the present invention was as described in example 1, pH 6.5
Group 2	The hybridization solution composition was as described in example 1, with a pH of 6.8
		Group 3	The hybridization solution composition was as described in example 1, with a pH of 7.0
Group 4	The hybridization solution composition of the present invention was as described in example 1, pH 7.2
		Group 5	The hybridization solution composition was as described in example 1, with a pH of 7.5

2. Sample detection

In this example, 6 samples with different mutation types (sample numbers 1 to 6, mutation types of PIK3CA p.h1047r, EGFR p.e746_a750del, BRAF p.v600e, ALK fusion, NRAS p.q61k, KRAS p.g13d) were selected and verified by Sanger sequencing/FISH method, and the kit prepared by the above design was used to perform detection according to the method of example 2. The specific results are shown in Table 14.

TABLE 14 comparison of detection results of hybridization solutions at different pH values

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From the detection results, the experimental groups 2, 3 and 4 can realize accurate detection, and the mutation types of all samples can be detected correctly, wherein the Q30, the average sequencing depth, the capturing efficiency and the uniformity of the experimental group 3 are better than those of the experimental groups 2 and 4, and the detection effect is better; the experimental groups 1 and 5 cannot realize accurate detection, the detection results of the experimental groups 1 and 5 have false negative results (see the samples 4 in the groups 1 and 5 in the table 14), and the Q30, the average sequencing depth, the capturing efficiency and the uniformity of the experimental groups are lower than those of the experimental groups 2, 3 and 4, presumably due to the fact that the pH value of the hybridization solution is too low or too high, the using effect of the hybridization solution is reduced, the hybridization effect of hybridization between the target gene sequence and the capturing probe is poor, the detection effect is poor, and even the false negative result is caused. Therefore, the pH of the hybridization solution of the present invention is 6.8 to 7.2, preferably 7.0.

EXAMPLE 7 verification of the Effect of Using the hybridization solution of the present invention

1. Design for verifying using effect of hybridization solution

In order to verify the use effect of the hybridization solution of the present invention, experimental group 1-2 was designed, the hybridization solution of the present invention was selected for experimental group 1, the hybridization solution component of commercial kit xGen Hybridization and Wash Kit (from IDT company, cat# 1080584) was selected for experimental group 2, and the other components were the same except for the hybridization solution. In hybridization, test group 1 was hybridized as in example 2, test group 2 was hybridized as in the kit instructions, and the detection results were compared, and the specific designs are shown in Table 15.

TABLE 15 design for verification of hybridization fluid usage effect

2. Sample detection

In this example, 6 samples with different mutation types (sample numbers 1 to 6, mutation types were EGFR p.L858R, PIK3CA p.H1047L, RET fusion, KRAS p.G12C, BRAF p.V600E, HER2 p.A775_G776 insYVMA) were selected and verified by Sanger sequencing/FISH method, hybridization was performed according to the above method, and other steps were performed according to the method of example 2. The specific results are shown in Table 16.

Table 16 shows the results of the test for verifying the effect of hybridization

From the above detection results, both groups can realize accurate detection, and all the mutation types of samples can be detected correctly, but the Q30, average sequencing depth, capturing efficiency and uniformity of the experimental group 1 are better than those of the experimental group 2, and the use effect is better. The possible reason is that the hybridization solution of the invention can reduce nonspecific background noise while enhancing hybridization signals of target genes, thereby well controlling background values generated during hybridization and improving hybridization capturing effect. In addition, comparing the hybridization reaction program of the hybridization solution of the present invention with the hybridization reaction program of the hybridization solution component in the commercially available kit, it was found that the hybridization time period of the hybridization solution of the present invention when used for hybridization was 2 hours, whereas the hybridization time period of the hybridization solution component in the commercially available kit when used for hybridization was at least 4 to 16 hours. This shows that the hybridization solution of the invention can effectively shorten hybridization capture time and save time cost under the condition of ensuring better capture efficiency and uniformity of captured library.

Example 8 determination of the use concentration of Capture Probe pool solution in the kit of the invention

1. Design of use concentration of capture probe pool solution

In order to verify the use concentration of each capture probe of the capture probe pool solution in the kit of the present invention, experimental groups 1 to 5 were designed, each of which was identical except for the use concentration of each capture probe of the capture probe pool solution, and the detection effect was compared, and the specific design is shown in table 17.

TABLE 17 design of capture probe pool solution use concentration

Experimental group	Design of use concentration of capture probe pool solution
		Group 1	Each capture probe was used at a concentration of 0.05pM, the probe sequences are as described in example 1
Group 2	Each capture probe was used at a concentration of 0.1pM, the probe sequences are as described in example 1
		Group 3	The concentration of each capture probe used was 1.5pM, as described in example 1 of the present invention for capture probe Chi Rongye
Group 4	Each capture probe was used at a concentration of 6pM, the probe sequences are as described in example 1
		Group 5	Each capture probe was used at a concentration of 6.5pM, the probe sequences are as described in example 1

2. Sample detection

In this example, 6 samples with different mutation types (sample numbers 1 to 6, mutation types were KRAS p.G12A, MEK1 p.K57N, EGFR p.G719A, PIK3CA p.H1047R, NTRK1 fusion, BRAF p.V600E) were selected and verified by Sanger sequencing/FISH method, and the kit prepared by the above design was used for detection according to the method of example 2. The specific results are shown in Table 18.

TABLE 18 comparison of detection results of Capture Probe pool solutions of different use concentrations

From the detection results, the experimental groups 2, 3 and 4 can realize accurate detection, and the mutation types of all samples can be detected correctly, wherein the Q30, the average sequencing depth, the capturing efficiency and the uniformity of the experimental group 3 are better than those of the experimental groups 2 and 4, and the detection effect is better; while the experimental groups 1 and 5 cannot realize accurate detection, the detection results of the experimental groups 1 and 5 have false negative results (see the samples 5 of the groups 1 and 5 in the table 18), and the Q30, the average sequencing depth, the capturing efficiency and the uniformity of the experimental groups are lower than those of the experimental groups 2, 3 and 4, presumably because the too low concentration of the capturing probe pool solution can lead to insufficient hybridization between the target gene sequence and the capturing probe, and the too high concentration of the capturing probe pool solution can lead to increased background noise and nonspecific hybridization, so that the hybridization effect of the hybridization between the target gene sequence and the capturing probe is poor, the capturing effect is poor, and even the false negative result is caused. Thus, the concentration of each capture probe used in the capture probe pool solution of the present invention is 0.1pM to 6pM, preferably 1.5pM.

Example 9 determination of hybridization time in the detection method of the present invention

1. Design of hybridization time

In order to determine the hybridization time in the detection method of the present invention, experimental groups 1 to 5 were designed, each of which was identical except for the hybridization time, and the detection effect was compared, and specific designs are shown in Table 19.

TABLE 19 design of hybridization time

Experimental group	Hybridization time ofDesign of
		Group 1	Hybridization time was 30 minutes, otherwise as described in examples 1 and 2
Group 2	Hybridization time was 1 hour, otherwise as described in examples 1 and 2
		Group 3	Hybridization time was 2 hours as described in examples 1 and 2 of the present invention
Group 4	Hybridization time was 16 hours, otherwise as described in examples 1 and 2
		Group 5	Hybridization time was 20 hours, otherwise as described in examples 1 and 2

2. Sample detection

In this example, 6 samples with different mutation types (sample numbers 1 to 6, mutation types were ROS1 fusion, BRAF p.V600E, KRAS p.G12R, TP53 p.R273C, PIK3CA p.E542Q, EGFR p.L8611Q in this order) were selected and verified by Sanger sequencing/FISH method, and the hybridization time designed as described above was measured using the kit described in example 1 and the method described in example 2. The specific results are shown in Table 20.

TABLE 20 comparison of the detection results for different hybridization times

From the detection results, the experimental groups 2, 3 and 4 can realize accurate detection, and the mutation types of all samples can be detected correctly, wherein the Q30, the average sequencing depth, the capturing efficiency and the uniformity of the experimental group 3 are better than those of the experimental groups 2 and 4, and the detection effect is better; the experimental groups 1 and 5 cannot realize accurate detection, the detection results of the experimental groups cannot have false negative results (see the table 20, the group 1 and the group 5, the sample 1), and the Q30, the average sequencing depth, the capturing efficiency and the uniformity of the experimental groups are lower than those of the experimental groups 2, 3 and 4, presumably, the reason is that the hybridization time is too short to enable the target gene sequence and the capturing probe to be hybridized insufficiently, the hybridization time is too long to enable the background noise and the nonspecific hybridization to be increased, so that the hybridization effect of the hybridization of the target gene sequence and the capturing probe is poor, the capturing effect is poor, and even the false negative result is caused. Therefore, in the hybridization capturing step of the detection method of the present invention, the hybridization time is 1 to 16 hours, preferably 2 hours.

Example 10 sensitivity verification of the kit and the detection method thereof

1. Design for sensitivity verification

In order to verify the sensitivity of the kit and the detection method thereof, experiments are designed to detect samples with different mutation types and mutation rates.

2. Sample detection

In this example, 16 samples (sample numbers 1 to 16, mutation types of ALK fusion, BRAF p.v600e, EGFR p.l858r, HER2 p.a775_g776insvma, KRAS p.g12d, MET p.d1010h, NRAS p.g12s, MEK1 p.q56p, PIK3CA p.e545k, RET fusion, ROS1 fusion, TP53 p.r175h, NTRK1 fusion, NTRK2 fusion, NTRK3 fusion, PD-L1 amplification) were selected and used for mutation rate determination by digital PCR, and wild type samples were mixed to give samples with mutation rates of 0.002%, 0.02%, 0.2% using the kit described in example 1, and were tested according to the method of example 2. The specific results are shown in Table 21.

Table 21 sensitivity verification test results

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From the detection results, the kit and the detection method thereof can accurately detect the corresponding mutation type with the theoretical mutation rate of 0.02% -0.2%, and the detected mutation rate accords with the theoretical value. This shows that the kit and the detection method thereof can stably detect 0.02% of ultra-low frequency mutation, namely the detection sensitivity of the kit and the detection method thereof is 0.02%.

Example 11 accuracy of the detection method of the present invention for detecting MRD status of lung cancer

1. Design for detecting accuracy of lung cancer MRD state

In order to examine the accuracy of the reagent kit and the detection method for detecting the MRD state of lung cancer, an experiment is designed to detect clinical samples.

2. Sample detection

In this example, 53 clinical plasma samples were selected, 21 of which were from patients with completely-relieved pathology and 32 of which were from patients with incompletely-relieved pathology, and tested according to the method of example 2 using the kit described in example 1. The specific results are shown in Table 22.

Table 22 results of 53 cases of clinical plasma sample tests

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From the detection results, the kit and the detection method thereof can detect 28 MRD positive results and 4 MRD negative results in the clinical samples of the plasma of the patients with the lung cancer in the incomplete pathological remission, and detect 1 MRD positive result and 20 MRD negative results in the clinical samples of the plasma of the patients with the lung cancer in the complete pathological remission, and the sensitivity of detecting the MRD state of the lung cancer is 87.5%, the specificity is 95.2% and the accuracy is 90.6%. The above description shows that the kit and the detection method thereof can accurately judge the MRD state of lung cancer, and can provide auxiliary information for lung cancer condition monitoring.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (11)

1. The capture probe pool for the MRD detection of the lung cancer is characterized by comprising a positive strand capture probe and a negative strand capture probe aiming at least one target gene, wherein the capture probes of each target gene are sequentially designed and staggered according to the nucleotide sequences of the positive strand and the negative strand of the target gene, and can cover the whole sequence of the target gene; the two adjacent capture probes in the positive chain capture probe and the negative chain capture probe are positive chain capture probes, the other capture probe is negative chain capture probe, the target gene sequences are not overlapped, and the length of each capture probe in the capture probe pool is 80-130 bp; the target gene is selected from at least one of the following 16 types: ALK, BRAF, EGFR, HER2, KRAS, MET, NRAS, MEK1, PIK3CA, RET, ROS1, TP53, NTRK1, NTRK2, NTRK3 and PD-L1.

2. The capture probe pool for MRD detection of lung cancer according to claim 1, wherein the capture probe is 100-110 bp in length; and/or the 5' end of the capture probe in the capture probe pool is modified by a marker; preferably, the label is modified to be a biotin label.

3. The capture probe pool for MRD detection of lung cancer according to claim 1, wherein the positive and negative strand capture probes of the target gene are selected from at least one group of:

a capture probe for ALK gene with the sequence shown in SEQ ID NO. 1-SEQ ID NO. 30;

a capture probe for the BRAF gene, the sequence of which is shown as SEQ ID NO. 31-SEQ ID NO. 40;

a capture probe for EGFR gene with the sequence shown as SEQ ID NO. 41-SEQ ID NO. 64;

a capture probe for the HER2 gene with the sequence shown as SEQ ID NO. 65-SEQ ID NO. 80;

a capture probe for KRAS genes, the sequence of which is shown as SEQ ID NO. 81-SEQ ID NO. 86;

a capture probe for MET gene with the sequence shown as SEQ ID NO. 87-SEQ ID NO. 110;

a capture probe for NRAS genes, the sequence of which is shown as SEQ ID NO. 111-SEQ ID NO. 116;

a capture probe for MEK1 gene with the sequence shown as SEQ ID NO. 117-SEQ ID NO. 128;

A capture probe for PIK3CA gene with the sequence shown in SEQ ID NO. 129-SEQ ID NO. 140;

a capture probe for RET gene with the sequence shown as SEQ ID NO. 141-SEQ ID NO. 160;

a capture probe for ROS1 gene with the sequence shown as SEQ ID NO. 161-SEQ ID NO. 180;

a capture probe for TP53 gene with the sequence shown as SEQ ID NO. 181-SEQ ID NO. 192;

a capture probe for NTRK1 gene with a sequence shown as SEQ ID NO. 193-SEQ ID NO. 212;

a capture probe for NTRK2 gene with a sequence shown as SEQ ID NO. 213-SEQ ID NO. 224;

the sequence is shown as SEQ ID NO. 225-SEQ ID NO.238 and aims at a capture probe of the NTRK3 gene.

4. The capture probe pool for MRD detection of lung cancer according to claim 3, wherein the capture probe pool comprises capture probes for the following 16 genes ALK, BRAF, EGFR, HER2, KRAS, MET, NRAS, MEK1, PIK3CA, RET, ROS1, TP53, NTRK1, NTRK2, NTRK3 and PD-L1.

5. A lung cancer MRD detection kit, characterized in that it comprises the capture probe pool for lung cancer MRD detection according to any one of claims 1-4.

6. The MRD detection kit for lung cancer according to claim 5, further comprising a hybridization solution comprising 300-600 mM sodium phosphate buffer, 10-50% (v/v) 2 x SSC, 0.5-1.5% (w/v) SDS, 1-3% (v/v) Denhardt solution, 1-5% (w/v) dextran sulfate, 1-10% (w/v) trehalose, 0.1-0.5% (v/v) Tween-20.

7. The MRD detection kit for lung cancer according to claim 6, wherein the hybridization solution comprises 400-500 mM sodium phosphate buffer, 20-40% (v/v) of 2 x SSC, 0.8-1.2% (w/v) of SDS, 1.5-2.5% (v/v) of Denhardt solution, 2-4% (w/v) of dextran sulfate, 4-7% (w/v) of trehalose, and 0.2-0.4% (v/v) of Tween-20.

8. The MRD detection kit for lung cancer according to claim 7, wherein the hybridization solution is Tween-20 comprising 450mM sodium phosphate buffer, 30% (v/v) 2 x SSC, 1% (w/v) SDS, 2% (v/v) Denhardt solution, 3% (w/v) dextran sulfate, 5.5% (w/v) trehalose, 0.3% (v/v).

9. The lung cancer MRD detection kit according to claims 6-8, wherein the pH of the hybridization solution is 6.8-7.2; preferably, the pH of the hybridization solution is 7.0.

10. The lung cancer MRD detection kit according to claims 5-8, wherein each capture probe in the capture probe pool for lung cancer MRD detection is mixed and configured in the same molar ratio; and/or preferably, each capture probe is used at a concentration of 0.1pM to 6pM, preferably 1pM to 2pM, more preferably 1.4 to 1.6pM.

11. The lung cancer MRD detection kit according to claims 5-8, characterized in that the kit employs a detection method comprising the steps of:

s1, extracting cfDNA of plasma: collecting a blood sample of a subject, centrifugally separating plasma, and extracting the plasma cfDNA sample according to the product specification by using a commercial plasma cfDNA extraction kit;

s2, constructing a library: library construction of the extracted plasma cfDNA samples using a commercially available library construction kit according to its product instructions;

s3, hybridization capture: hybridization is carried out by using the lung cancer MRD detection kit provided by the invention, and target fragments are captured;

s4, sequencing and data analysis on the machine;

in the step S3, the hybridization time is 1-16 hours; preferably, in the step S3, the hybridization time is 1.5 to 2.5 hours.