TWI899908B - Kit for detecting cancer and application thereof - Google Patents

Abstract

The present invention provides a detection kit for detecting whether an individual suffers from cancer, which comprises a plurality of nucleotide sequence molecules, a reverse transcription reagent kit, and a polymerase chain reaction reagent kit. The detection kit for detecting whether an individual suffers from cancer has the advantages of high sensitivity, high accuracy, easy sampling, low invasiveness, low risk, and could be used for efficacy tracking.

Description

Cancer detection kits and their applications

The present invention provides a detection kit for detecting whether an individual has cancer, particularly providing a kit comprising a plurality of nucleotide sequence molecules, a reverse transcription reagent kit, and a polymerase chain reaction reagent kit, and the detection kit is used to detect whether an individual has cancer.

Gene fusions play a crucial role in the initial stages of many cancers. Specifically, certain genes in the body can form fusion genes due to chromosomal translocation and recombination, leading to abnormal cell growth regulation and the development of cancer. The earliest fusion gene discovered was CTNNB1-PLAG1 in salivary gland tumors, which are typically benign tumors. Subsequently, other fusion genes have been found in solid tumors and malignancies, such as glioblastoma, melanoma, prostate cancer, breast cancer, ovarian cancer, lung cancer, colorectal cancer, and head and neck cancer (Taniue and Akimitsu, 2021). Lung cancer is one of the most common malignancies worldwide, with non-small cell lung cancer (NSCLC) accounting for approximately 80%. In 2007, Soda et al. first discovered an inversion on chromosome 2 (p21:p23) in NSCLC tumor specimens, forming a fusion gene between the echinoderm microtubule-associated protein-like 4 (EML4) and the anaplastic lymphoma kinase (ALK). Fusion of the EML4 gene with the ALK gene at different breakpoints results in different EML4-ALK fusion gene variants, including E13, A20, E20, A20, E6, and A20, totaling 11 different fusion gene variants (Han Wei, 2013). More research has found that patients with diseases such as NSCLC (Non-Small Cell Lung Cancer), breast cancer, colorectal cancer, leukemia, lymphoma, thyroid cancer, osteocarcinoma, pancreatic cancer, carcinoma, and soft tissue sarcoma may also harbor other fusion genes besides the EML4-ALK fusion gene. Currently, there are four main types of cancer-related fusion genes:

1. ALK fusion gene: mainly includes (1) EML4-ALK fusion gene, (2) KIF5B-ALK fusion gene, (3) TFG-ALK fusion gene, and (4) HIP1-ALK fusion gene, etc.

2. ROS1 fusion gene: mainly including (1) CD74-ROS1 fusion gene, (2) SDC4-ROS1 fusion gene, (3) SLC34A2-ROS1 fusion gene, (4) GOPC-ROS1 fusion gene, (5) EZR-ROS1 fusion gene, (6) LRIG3-ROS1 fusion gene, and (7) TPM3-ROS1 fusion gene, etc.

3. RET fusion gene: mainly including (1) KIF5B-RET fusion gene, (2) NCOA4-RET fusion gene, (3) CCDC6-RET fusion gene, and (4) TRIM33-RET fusion gene, etc.; and

4. MET gene with skipping of exon 14: mainly includes MET exon 14 skipping fusion genes.

Therefore, the presence of these cancer-associated fusion genes in individual specimens is currently widely used as a marker for determining whether an individual has cancer. For example, in the case of non-small cell lung cancer (NSCLC), the current standard clinical testing method involves obtaining lung tissue from an individual and slicing it. Immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), or real-time polymerase chain reaction (RT-PCR) is then performed to detect the presence of, for example, the EML4-ALK fusion protein or the presence of the EML4-ALK fusion RNA in the lung tissue sections to determine whether an individual has NSCLC. However, all three of the aforementioned testing methods require patient tissue biopsies, making them highly invasive and risky. Furthermore, the real-time polymerase chain reaction (PCR) method, due to limitations such as sensitivity and the instrument's cycle threshold value (Ct value) of 40, requires a large amount of lung tissue. Only when the trace amount of fusion RNA is replicated 40 times or less (due to the upper limit of the Ct value of 40) by the real-time polymerase chain reaction (PCR) can the system produce sufficient fusion RNA to detect and thus determine a positive result.

Generally speaking, the experimental results of real-time polymerase chain reaction are presented as a cycle threshold value (Ct value). When the cycle threshold value is less than or equal to the upper limit of the instrument (for example, 40), it indicates that the sample contains the target nucleic acid, and the test result is positive. When the cycle threshold value exceeds the upper limit of the instrument (for example, 40), it indicates that the sample does not contain the target nucleic acid, and the test result is negative. (A cycle threshold value greater than 40 will result in a serious false positive, so the cycle threshold value cannot exceed 40; a value exceeding 40 is considered a negative test result.) For example, using real-time polymerase chain reaction (RT-PCR) in Figure 1, a commercially available EML4-ALK fusion RNA standard (Horizon™ ALK-RET-ROS1 targeted FFPE RNA Fusion Reference Standards, Cat ID: HD784) was found to have a cycle threshold of 31.84. Further real-time PCR assays in samples confirmed to contain EML4-ALK fusion RNA and saline solution revealed cycle thresholds of 24.68 and 45, respectively. Because the saline solution cycle threshold was 45, exceeding the upper limit of the instrument, the saline solution cycle threshold in Figure 1 is marked with an X, indicating that no EML4-ALK fusion RNA was detected.

Therefore, the market urgently needs a testing method that can overcome current technological limitations to detect whether an individual has cancer. This testing method must have high sensitivity, high accuracy, convenient sampling, low invasiveness, low risk, and be able to facilitate continuous sample collection for treatment efficacy tracking.

In light of the limitations of conventional technology, the present invention provides a test kit for detecting whether an individual has cancer. Specifically, the kit comprises a plurality of nucleotide sequence molecules, a reverse transcription reagent kit, and a polymerase chain reaction reagent kit. The kit is used to detect whether an individual has cancer. The test kit for detecting whether an individual has cancer has advantages such as high sensitivity, high accuracy, convenient sampling, low invasiveness, low risk, and the ability to continuously obtain samples for treatment efficacy tracking.

In this invention, normal messenger RNA (normal mRNA) refers to a single-stranded molecule that is transcribed from DNA in normal human cells and carries the genetic information corresponding to the DNA.

In this invention, the "cycle threshold value (Ct value)" refers to the experimental results of polymerase chain reaction, real-time polymerase chain reaction, or reverse transcription-polymerase chain reaction. This cycle threshold indicates the number of cycles of replication required for the target DNA in the test solution to reach a sufficient level for detection by the instrument. The cycle threshold reflects the amount of target DNA in the test solution. A higher target DNA amount indicates a lower cycle threshold. Conversely, a lower target DNA amount indicates a higher cycle threshold. If the test solution contains no target DNA, the cycle threshold is greater than 40.

In the present invention, a "lipid bilayer membrane structure" refers to a bilayer structure composed of phospholipid molecules. The phospholipid has a hydrophilic end and a lipophilic end. Preferably, the lipid bilayer membrane structure is an exosome.

In the present invention, "binding" or "bonding" refers to the binding or formation of hydrogen bonds, covalent bonds, or ionic bonds between molecules due to interaction forces. For example, thymine and adenine in deoxyribonucleic acid bind or form hydrogen bonds due to interaction forces such as pairing.

In the present invention, "magnetic beads" refer to a magnetic three-dimensional object containing multiple thymines on its surface, which are used to bind or bond with nucleotide sequence molecules.

In this invention, "Basic Local Alignment Search Tool (BLAST)" refers to a tool used in bioinformatics for aligning biological sequences, such as the BLAST tool from the National Center for Biotechnology Information (NCBI).

In this invention, the "E value" refers to the similarity evaluation result obtained by the Basic Local Alignment Search Tool (BLAST) when comparing the nucleotide sequence molecules (capture probes) of the present invention with nucleic acid molecules in a database. A smaller E value indicates a higher degree of similarity. An optimal E value is less than or equal to 0.01. In this invention, the "Query Cover value" refers to the similarity analysis result obtained by the Basic Local Alignment Search Tool (BLAST) when comparing the nucleotide sequence molecules (capture probes) of the present invention with nucleic acid molecules in a database. A larger Query Cover value indicates a higher degree of similarity. An optimal Query Cover value is greater than or equal to 80%.

The present invention provides a detection kit for detecting whether an individual has cancer, comprising: a plurality of nucleotide sequence molecules for binding to (linking to) a plurality of target messenger RNAs (targeted mRNAs) in an in vitro sample to capture the target messenger RNAs; the target messenger RNAs are normal messenger RNAs (normal mRNAs), cancer-associated messenger RNAs (cancer-associated mRNAs), or a combination thereof; wherein the cancer-associated messenger RNA is divided into a first sequence segment and a second sequence segment by an abnormal coding sequence segment, and the nucleotide sequence molecules bind to the first sequence segment of the cancer-associated messenger RNA; furthermore, the normal messenger RNA includes a third sequence segment, and the nucleotide sequence molecules bind to the third sequence segment of the normal messenger RNA;

A reverse transcription reagent kit for reversely transcribing captured normal messenger RNA into normal messenger complementary DNA (normal cDNA), and reversely transcribing captured cancer-associated messenger RNA into cancer-associated messenger complementary DNA (cancer-associated cDNA), wherein the cancer-associated messenger complementary DNA is divided into a first reverse transcribed sequence region and a second reverse transcribed sequence region by an abnormal region reverse transcribed sequence corresponding to the abnormal coding sequence segment; and

A polymerase chain reaction reagent kit includes a first primer and a second primer; In a PCR reaction (PCR), when the cancer-associated complementary DNA denatures and separates into a first abnormal strand and a second abnormal strand, the first primer is used to anneal to the first reverse transcriptase region on the first abnormal strand, and the second primer is used to anneal to the second reverse transcriptase region on the second abnormal strand; at least one of the first primer and the second primer is unable to anneal to either strand of the normal complementary DNA; and, after performing a PCR reaction using the polymerase chain reaction reagent kit, if the cycle threshold value is less than or equal to 40, the individual is determined to have cancer.

In a preferred embodiment, the test kit, wherein the in vitro sample is at least one of a serum sample, a plasma sample, and a blood sample of the individual, or any combination thereof.

In a preferred embodiment, the cancer-associated mRNA is an ALK fusion mRNA, a ROS1 fusion mRNA, a RET fusion mRNA, or a MET mRNA with exon 14 skipped.

In a preferred embodiment, the following features are further included:

(1) The cancer-associated RNA is ALK fusion RNA, and the first sequence segment and the third sequence segment both contain an ALK mRNA sequence segment, and the second sequence segment contains an EML4 mRNA sequence segment, a KIF5B mRNA sequence segment, a TFG mRNA sequence segment, or a HIP1 mRNA sequence segment;

(2) The cancer-associated mRNA is ROS1 fusion mRNA, and the first sequence segment and the third sequence segment both comprise a ROS1 mRNA sequence segment, and the second sequence segment comprises a CD74 mRNA sequence segment, an SDC4 mRNA sequence segment, an SLC34A2 mRNA sequence segment, a GOPC mRNA sequence segment, an EZR mRNA sequence segment, a LRIG3 mRNA sequence segment, or a TPM3 mRNA sequence segment;

(3) The cancer-associated mRNA is RET fusion mRNA, and both the first sequence segment and the third sequence segment include a RET mRNA sequence segment, and the second sequence segment includes a KIF5B mRNA sequence segment, an NCOA4 mRNA sequence segment, a CCDC6 mRNA sequence segment, or a TRIM33 mRNA sequence segment;

(4) The cancer-associated information RNA is the MET information RNA with skipping of exon 14 in the MET mRNA, and both the first sequence segment and the third sequence segment include a sequence segment upstream of the 14th exon of the MET information RNA (MET mRNA), and the second sequence segment includes a sequence segment downstream of the 14th exon of the MET information RNA (MET mRNA); or

(5) The cancer-associated mRNA is a MET mRNA skipping exon 14, and both the first sequence segment and the third sequence segment comprise a sequence segment downstream of exon 14 of the MET mRNA, and the second sequence segment comprises a sequence segment upstream of exon 14 of the MET mRNA.

In a preferred embodiment, the abnormal coding sequence segment is an informational RNA sequence segment transcribed from a chromosomal recombination segment of an ALK fusion gene, a ROS1 fusion gene, or a RET fusion gene resulting from chromosomal recombination; or, the abnormal coding sequence segment is an informational RNA sequence segment transcribed from a segment of exon 14 skipping in the MET gene.

In a preferred embodiment, the aberrant region reverse transcribed sequence is a complementary deoxyribonucleic acid (cDNA) obtained by reverse transcription of the aberrant coding sequence segment, and the cancer-associated information complementary deoxyribonucleic acid is divided into the first reverse transcribed sequence region and the second reverse transcribed sequence region by the aberrant region reverse transcribed sequence.

In a preferred embodiment, the in vitro sample comprises a plurality of lipid bilayer membrane structures, and the target RNAs are encapsulated in the lipid bilayer membrane structures, or are not encapsulated in the lipid bilayer membrane structures, or a combination thereof.

In a preferred embodiment, the method further comprises a solution for mixing with the in vitro sample to release the target RNAs encapsulated in the lipid bilayer membrane. Preferably, the solution is sample preservation solution (Shanghai Rendu Biotechnology Co., Ltd.; Shanghai Puxi Medical Equipment No. 20210018; Sample Preservation Solution), a phenol-chloroform-isoamyl alcohol mixture (Sigmaaldrich 77619), or RNAdvance Blood - Blood RNA Extraction and Purification Reagent Kit (BECKMAN COULTER A35603).

In a preferred embodiment, the lipid bilayer membrane structure is an exosome.

In a preferred embodiment, the nucleotide sequence molecules are bound to or linked to the surface of a plurality of magnetic beads.

In a preferred embodiment, (1) the nucleotide sequence molecules are ALK capture probes, and the sequence of the ALK capture probe is at least one of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or a combination thereof; or, the sequence of the ALK capture probes is at least 80% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; or, a search tool based on a local alignment algorithm (Basic Local Alignment Search Tool) is used to analyze the similarity between the ALK capture probe and SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, and the E value obtained is less than 0.01. Preferably, the sequences of the ALK capture probes are at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. Preferably, the similarity between the ALK capture probes and SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is analyzed using a Basic Local Alignment Search Tool, and the resulting E value is less than 0.01, 0.005, or 0.001. Preferably, the similarity between the ALK capture probe and SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 is analyzed using a Basic Local Alignment Search Tool, and the resulting query coverage value is 80% to 100%, or alternatively, the resulting query coverage value is 80%, 85%, 90%, 95%, or 100%.

(2) The nucleotide sequence molecules are ROS1 capture probes, and the sequence of the ROS1 capture probe is at least one of SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, or a combination thereof; or the sequence of the ROS1 capture probe is at least 80% identical to SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31; or the similarity between the ROS1 capture probe and SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31 is analyzed using a basic local alignment search tool, and the resulting E value is less than 0.01. Preferably, the sequences of the ROS1 capture probes are at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31. Preferably, the similarity between the ROS1 capture probes and SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31 is analyzed using a Basic Local Alignment Search Tool, and the resulting E value is less than 0.01, 0.005, or 0.001. Preferably, the similarity between the ROS1 capture probe and SEQ ID NO: 29, SEQ ID NO: 30, or SEQ ID NO: 31 is analyzed using a Basic Local Alignment Search Tool, and the resulting query coverage value is 80% to 100%, or alternatively, the resulting query coverage value is 80%, 85%, 90%, 95%, or 100%.

(3) The nucleotide sequence molecules are MET capture probes, and the sequence of the MET capture probe is at least one of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, or a combination thereof; or the sequence of the MET capture probe is at least 80% identical to SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44; or the similarity between the MET capture probe and SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44 analyzed using a basic local alignment search tool has an E value of less than 0.01. Preferably, the sequences of the MET capture probes are at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44. Preferably, the similarity between the MET capture probes and SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, and SEQ ID NO:44 is analyzed using a Basic Local Alignment Search Tool, and the resulting E-value is less than 0.01, 0.005, or 0.001. Preferably, a Basic Local Alignment Search Tool is used to analyze the similarity between the MET capture probe and SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, and the resulting query coverage value is 80% to 100%, or the resulting query coverage value is 80%, 85%, 90%, 95%, or 100%.

In a preferred embodiment, (1) the ALK capture probes comprise a first ALK capture probe and a second ALK capture probe; the sequence of the first ALK capture probe is at least 80% identical to SEQ ID NO: 1, and the sequence of the second ALK capture probe is at least 80% identical to SEQ ID NO: 2. Preferably, the sequence of the first ALK capture probe is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 1, and the sequence of the second ALK capture probe is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 2. Preferably, the similarity between the first ALK capture probe and SEQ ID NO: 1, analyzed using a Basic Local Alignment Search Tool, results in an E-value of less than 0.01, 0.005, or 0.001. Preferably, the similarity between the first ALK capture probe and SEQ ID NO: 1, analyzed using a Basic Local Alignment Search Tool, results in a Query Coverage value of 80% to 100%, or a Query Coverage value of 80%, 85%, 90%, 95%, or 100%. Preferably, the similarity between the second ALK capture probe and SEQ ID NO: 2, as analyzed using a Basic Local Alignment Search Tool, results in an E-value of less than 0.01, 0.005, or 0.001. Preferably, the similarity between the second ALK capture probe and SEQ ID NO: 2, as analyzed using a Basic Local Alignment Search Tool, results in a Query Coverage value of 80% to 100%, or alternatively, a Query Coverage value of 80%, 85%, 90%, 95%, or 100%.

(2) The ROS1 capture probes comprise a first ROS1 capture probe and a second ROS1 capture probe; the sequence of the first ROS1 capture probe is at least 80% identical to SEQ ID NO: 29, and the sequence of the second ROS1 capture probe is at least 80% identical to SEQ ID NO: 31. Preferably, the sequence of the first ROS1 capture probe is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 29, and the sequence of the second ROS1 capture probe is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 31. Preferably, the similarity between the first ROS1 capture probe and SEQ ID NO: 29, analyzed using a Basic Local Alignment Search Tool, results in an E-value of less than 0.01, 0.005, or 0.001. Preferably, the similarity between the first ROS1 capture probe and SEQ ID NO: 29, analyzed using a Basic Local Alignment Search Tool, results in a Query Coverage value of 80% to 100%, or a Query Coverage value of 80%, 85%, 90%, 95%, or 100%. Preferably, the similarity between the second ROS1 capture probe and SEQ ID NO: 31, analyzed using a Basic Local Alignment Search Tool, results in an E-value of less than 0.01, 0.005, or 0.001. Preferably, the similarity between the second ROS1 capture probe and SEQ ID NO: 31, analyzed using a Basic Local Alignment Search Tool, results in a Query Coverage value of 80% to 100%, or a Query Coverage value of 80%, 85%, 90%, 95%, or 100%.

(3) The MET capture probes comprise a first MET capture probe and a second MET capture probe; the sequence of the first MET capture probe is at least 80% identical to SEQ ID NO: 42, and the sequence of the second MET capture probe is at least 80% identical to SEQ ID NO: 44. Preferably, the sequence of the first MET capture probe is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 42, and the sequence of the second MET capture probe is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 44. Preferably, the similarity between the first MET capture probe and SEQ ID NO: 42 analyzed using a Basic Local Alignment Search Tool results in an E-value of less than 0.01, 0.005, or 0.001. Preferably, the similarity between the first MET capture probe and SEQ ID NO: 42 analyzed using a Basic Local Alignment Search Tool results in a Query Coverage value of 80% to 100%, or a Query Coverage value of 80%, 85%, 90%, 95%, or 100%. Preferably, the similarity between the second MET capture probe and SEQ ID NO: 44, as analyzed using a Basic Local Alignment Search Tool, results in an E value of less than 0.01, 0.005, or 0.001. Preferably, the similarity between the second MET capture probe and SEQ ID NO: 44, as analyzed using a Basic Local Alignment Search Tool, results in a Query Coverage value of 80% to 100%, or a Query Coverage value of 80%, 85%, 90%, 95%, or 100%.

In a preferred embodiment, (1) the nucleotide sequence molecules comprise a third ALK capture probe, and the sequence of the third ALK capture probe is at least 80% identical to SEQ ID NO: 3; or, the sequence of the third ALK capture probe is at least 80% identical to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; or, the sequence of the third ALK capture probe is analyzed using a search tool based on a local alignment algorithm (Basic Local Alignment Search Tool) to identify the third ALK capture probe and SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17, and the resulting E value is less than 0.01. Preferably, the sequence of the third ALK capture probe is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO:3. Preferably, the sequence of the third ALK capture probe is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17. Preferably, the similarity between the third ALK capture probe and SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17 is analyzed using a Basic Local Alignment Search Tool, and the resulting E value is less than 0.01, 0.005, or 0.001. Preferably, a Basic Local Alignment Search Tool is used to analyze the similarity between the third ALK capture probe and SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, and the resulting Query Coverage value is 80% to 100%, or the resulting Query Coverage value is 80%, 85%, 90%, 95%, or 100%.

(2) The nucleotide sequence molecules comprise a third ROS1 capture probe, and the sequence of the third ROS1 capture probe is at least 80% identical to SEQ ID NO: 30; or the sequence of the third ROS1 capture probe is at least 80% identical to SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28; or the similarity between the third ROS1 capture probe and SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28 is analyzed using a Basic Local Alignment Search Tool, and the resulting E value is less than 0.01. Preferably, the sequence of the third ROS1 capture probe is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 30. Preferably, the sequence of the third ROS1 capture probe is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. Preferably, the similarity between the third ROS1 capture probe and SEQ ID NO: 30, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28, analyzed using a Basic Local Alignment Search Tool, results in an E-value of less than 0.01, 0.005, or 0.001. Preferably, a Basic Local Alignment Search Tool is used to analyze the similarity between the third ROS1 capture probe and SEQ ID NO: 30, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28, and the resulting query coverage value is 80% to 100%, or the resulting query coverage value is 80%, 85%, 90%, 95%, or 100%.

(3) The nucleotide sequence molecules comprise a third MET capture probe, and the sequence of the third MET capture probe is at least 80% identical to SEQ ID NO: 45; or the sequence of the third MET capture probe is at least 80% identical to SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45; or the similarity between the third MET capture probe and SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45 analyzed using a Basic Local Alignment Search Tool (BLSAT) has an E value of less than 0.01. Preferably, the sequence of the third MET capture probe is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 45. Preferably, the sequence of the third MET capture probe is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45. Preferably, the similarity between the third MET capture probe and SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45, analyzed using a Basic Local Alignment Search Tool, results in an E-value of less than 0.01, 0.005, or 0.001. Preferably, a Basic Local Alignment Search Tool is used to analyze the similarity between the third MET capture probe and SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45, and the resulting query coverage value is 80% to 100%, or the resulting query coverage value is 80%, 85%, 90%, 95%, or 100%.

In a preferred embodiment, the cancer is non-small cell lung cancer, breast cancer, colorectal cancer, leukemia, lymphoma, thyroid cancer, osteocarcinoma, pancreatic cancer, carcinoma, or soft tissue sarcoma; or the individual is a human.

In a preferred embodiment, the nucleotide sequence molecules are composed of a plurality of deoxyribonucleotides.

The present invention further provides a nucleotide sequence molecule composition comprising a plurality of nucleotide sequence molecules, each of which is composed of 30 to 60 deoxyribonucleotides and has the following characteristics:

(1) The sequences of the nucleotide sequence molecules are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44; or

(2) The sequences of the nucleotide sequence molecules are at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, or a combination thereof.

In a preferred embodiment, the nucleotide sequence molecules comprise a first nucleotide sequence molecule, a second nucleotide sequence molecule, and a third nucleotide sequence molecule; wherein the sequence of the first nucleotide sequence molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1, the sequence of the second nucleotide sequence molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, and the sequence of the third nucleotide sequence molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2, and the sequence of the third nucleotide sequence molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. NO:17 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical; or the first nucleotide sequence molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:29, the second nucleotide sequence molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:31, and the third nucleotide sequence molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO: NO:28 is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical; the sequence of the first nucleotide sequence molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:42; the sequence of the second nucleotide sequence molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:44; and the sequence of the third nucleotide sequence molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:43, or SEQ ID NO:45.

In a preferred embodiment, the binding affinity between the nucleotide sequence molecules and a first sequence segment of a cancer-associated messenger RNA (cancer-associated mRNA) is greater than a first binding affinity, allowing the nucleotide sequence molecules to bind to the first sequence segment and capture the cancer-associated messenger RNA. Furthermore, the binding affinity between the nucleotide sequence molecules and a third sequence segment of a normal messenger RNA (normal mRNA) is greater than the first binding affinity; wherein the first binding affinity is the binding affinity between the aberrant coding sequence segment and phosphate-buffered saline (PBS).

In a preferred embodiment, the cancer-associated RNA further comprises an abnormal coding sequence segment, and the binding affinity between the nucleotide sequence molecules and the abnormal coding sequence segment is less than the first binding affinity.

The present invention further provides a method for detecting whether an individual has cancer, comprising the following steps:

(A) mixing a plurality of nucleotide sequence molecules with an in vitro sample so that the nucleotide sequence molecules bind to a plurality of target messenger RNAs (targeted messenger RNAs) in the in vitro sample to capture the target messenger RNAs, wherein the target messenger RNAs are normal messenger RNA (normal mRNA), cancer-associated messenger RNA (cancer-associated mRNA), or a combination thereof; wherein the cancer-associated messenger RNA is divided into a first sequence segment and a second sequence segment by an abnormal coding sequence segment, and the nucleotide sequence molecules bind to the first sequence segment of the cancer-associated messenger RNA; and wherein the normal messenger RNA includes a third sequence segment, and the nucleotide sequence molecules bind to the third sequence segment of the normal messenger RNA;

(B) performing a reverse transcription step to reverse transcribe the captured normal information RNA into normal information complementary DNA (normal cDNA), and reverse transcribe the captured cancer-associated information RNA into a cancer-associated information complementary DNA (cancer-associated cDNA); wherein the cancer-associated information complementary DNA is divided into a first reverse transcribed sequence region and a second reverse transcribed sequence region by an abnormal region reverse transcribed sequence corresponding to the abnormal coding sequence segment;

(C) Perform a polymerase chain reaction comprising the following steps:

(c1) performing a denaturation step to denature the cancer-associated complementary DNA and separate it into a first abnormal single strand and a second abnormal single strand, and to separate the normal complementary DNA into a first normal single strand and a second normal single strand;

(c2) performing a primer annealing step, causing a first primer to anneal to the first reverse transcribed sequence region on the first abnormal strand, and causing a second primer to anneal to the second reverse transcribed sequence region on the second abnormal strand; furthermore, at least one of the first primer and the second primer is unable to anneal to the first normal strand or the second normal strand;

Furthermore, after performing the polymerase chain reaction, if the cycle threshold value is less than or equal to 40, the individual is determined to have cancer.

In a preferred embodiment, the in vitro sample is at least one of a serum sample, a plasma sample, and a blood sample of the individual, or any combination thereof.

In a preferred embodiment, in step (A), the nucleotide sequence molecules are dispersed in a solution capable of disrupting the lipid bilayer membrane. Furthermore, the in vitro sample contains a plurality of lipid bilayer membrane structures, and the target information RNA is encapsulated in the lipid bilayer membrane structures, or is not encapsulated in the lipid bilayer membrane structures, or a combination thereof. Step (A) includes:

The solution containing the nucleotide sequence molecules is mixed with the in vitro sample to release the target messenger RNA encapsulated in the lipid bilayer membrane in the in vitro sample and capture the target messenger RNA in the in vitro sample. Preferably, the solution is sample preservation solution (Shanghai Rendu Biotechnology Co., Ltd.; Shanghai Puxi Medical Equipment No. 20210018; Sample Preservation Solution), a phenol-chloroform-isoamyl alcohol mixture (Sigmaaldrich 77619), or RNAdvance Blood - Blood RNA Extraction and Purification Reagent Kit (BECKMAN COULTER A35603).

In a preferred embodiment, the sequence of the first primer is at least 80% identical to SEQ ID NO: 18, and the sequence of the second primer is at least 80% identical to SEQ ID NO: 19. Preferably, the sequence of the first primer is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 18. Preferably, the sequence of the second primer is at least 85%, 90%, 95%, or 100% identical to SEQ ID NO: 19.

The present invention further provides a use of the nucleotide sequence molecular composition described in paragraph [0031] in an in vitro detection method for detecting whether an individual has cancer.

11: Normal information RNA

12: Cancer-related information RNA

121: First sequence segment

122: Abnormal coding sequence segment

123: Second sequence segment

13, 14: Normal information RNA

15: Nucleotide sequence molecule

16: Magnetic beads

21: Normal information complement DNA

211, 212: Single-strand normal RNA

22: Cancer-related information complementary DNA

221: The First Abnormal Single Stock

2211, 2213, 2221, 2223: Single-strand normal RNA

2212: The first single strand of abnormal information RNA

222: Second Abnormal Single Strand

2222: Second strand of abnormal information RNA

31: First Introduction

32: Second Introduction

A: Abnormal region reverse transcriptase sequence

B: First reverse transcriptase region

C: Second reverse transcriptase region

S11-S14: Steps

Figure 1: Bar graph showing the Ct values of the EML4-ALK fusion RNA standard detected by real-time polymerase chain reaction.

Figure 2A and Figure 2B: Schematic diagrams illustrating the detection design of the capture probe (nucleotide sequence molecule in this case).

Figure 3: Flowchart for using the capture probes (nucleotide sequence molecules) in this case to detect whether an individual has cancer.

Figure 4: A bar graph showing the results of experiments using various extraction methods to detect the presence of EML4-ALK fusion RNA in plasma samples from patients with non-small cell lung cancer.

Figure 5: A bar graph showing the efficiency of various ALK capture probes in capturing ALK fusion RNA.

Figure 6: A bar graph showing the efficiency of various ROS1 capture probes in capturing ROS1 fusion RNA.

Figure 7: A bar graph showing the performance of various MET capture probes in capturing MET fusion RNA with exon 14 skipped.

The following is a detailed description of an embodiment of the present invention, as well as the techniques and features of the present invention. However, this embodiment is not intended to limit the present invention. Any modifications and improvements made by anyone skilled in the art that do not depart from the spirit and scope of the present invention should be included in the scope of the patent application of the present invention.

To address the shortcomings of conventional testing techniques, the present invention discloses a detection kit for detecting whether an individual has cancer. Figures 2A and 2B illustrate the general operation of the detection kit.

The detection kit disclosed in the present invention comprises at least a plurality of nucleotide sequence molecules 15, a reverse transcription reagent kit, and a polymerase chain reaction reagent kit.

Taking Figure 2A as an example, the nucleotide sequence molecules 15 are designed to bind to multiple target messenger RNAs (targeted mRNAs) in an in vitro sample and capture these target messenger RNAs.

As shown in the upper left corner of Figure 2A, the target messenger RNAs include at least a normal messenger RNA (normal mRNA) 11 and a cancer-associated messenger RNA (cancer-associated mRNA) 12 (or a combination of the two). The cancer-associated messenger RNA 12 is divided into a first sequence segment 121 and a second sequence segment 123 by an abnormal coding sequence segment 122. It should be noted that when a cancer-associated fusion gene is formed due to, for example, chromosome translocation or recombination, the fusion gene will transcribe cancer-associated information RNA 12, and the cancer-associated information RNA 12 carries (1) a fragment of normal information RNA 11 (i.e., the first sequence segment 121), (2) a fragment of another normal information RNA 13 shown in FIG2A (i.e., the second sequence segment 123), and (3) a breakpoint fusion sequence segment of normal information RNA 11 and normal information RNA 13 (i.e., the abnormal coding sequence segment 122); otherwise, under normal circumstances, normal information RNA 11 and normal information RNA 13 should be independent of each other and not fused. In addition, normal information RNA 14 represents other information RNAs that are unrelated to normal information RNA 11 and normal information RNA 13.

Next, the present invention captures the target RNA using the nucleotide sequence molecule 15. This is achieved by exploiting the complementary relationship between the nucleotides in the nucleotide sequence molecule 15 and specific sequence segments on the target RNA (the third segment on the normal RNA 11 and the first segment 121 on the cancer-associated RNA 12). This is illustrated in Figure 2A, where the process transitions from the upper left step to the upper right step (the first half of number I).

It should be noted that magnetic beads 16 can be further attached to the ends of the nucleotide sequence molecules 15 to separate the captured target RNA (normal RNA 11 and cancer-related RNA 12) from other uncaptured RNA. Therefore, as shown in Figure 2A (from the upper right step to the lower right step (the second half of number I), the present invention utilizes a magnetic adsorption separation process to act on the magnetic beads 16 through magnetic force, thereby retaining only the normal RNA 11 and cancer-related RNA 12 captured by the nucleotide sequence molecules 15, further improving detection accuracy.

Steps from the upper left corner to the upper right corner of Figure 2B (numbered II) are steps of the present invention using a reverse transcription reagent kit to reverse transcribe the retained normal messenger RNA 11 and cancer-associated messenger RNA 12, thereby reverse-transcribing the normal messenger RNA 11 into normal cDNA 21 and the cancer-associated messenger RNA 12 into cancer-associated cDNA 22. As for the reverse transcription of the cancer-associated information RNA 12, the abnormal coding sequence segment 122 is reverse transcribed into an abnormal region reverse transcript sequence A, the first sequence segment 121 is reverse transcribed into a first reverse transcript sequence region B, and the second sequence segment 123 is reverse transcribed into a second reverse transcript sequence region C. The first reverse transcript sequence region B and the second reverse transcript sequence region C also correspond to the coding method of the cancer-associated information RNA 12, with the abnormal region reverse transcript sequence A serving as the boundary. It should be noted that because reverse transcription is a process that converts an unstable single strand into a stable double strand, the single strand of normal information RNA 11 is reverse transcribed into a double strand comprising a first normal single strand 211 and a second normal single strand 212 , and the single strand of cancer-associated information RNA 12 is reverse transcribed into a double strand comprising a first abnormal single strand 221 and a second abnormal single strand 222 . The first sequence segment 121 of the single-stranded cancer-associated information RNA 12 is reverse transcribed into the first reverse transcribed sequence region B (including 2211 on the first abnormal strand 221 and 2221 on the second abnormal strand 222). The second sequence segment 123 of the single-stranded cancer-associated information RNA 12 is reverse transcribed into the second reverse transcribed sequence region C (including 2213 on the first abnormal strand 221 and 2223 on the second abnormal strand 222). The abnormal coding sequence segment 122 of the single-stranded cancer-associated information RNA 12 is reverse transcribed into the abnormal region reverse transcribed sequence A (including 2212 on the first abnormal strand 221 and 2222 on the second abnormal strand 222).

Steps from the lower left corner to the lower right corner of Figure 2B (numbered III) are polymerase chain reactions (PCR) performed by the present invention using a PCR reagent kit, wherein the PCR reagent kit includes a first primer 31 and a second primer 32. During the PCR reaction, the first primer 31 anneals to the first reverse transcriptase region B (2211) on the first aberrant strand 221, and the second primer 32 anneals to the second reverse transcriptase region C (2223) on the second aberrant strand 222. At least one of the first primer 31 and the second primer 32 is unable to anneal to either strand of the normal complementary DNA 21 (the first normal strand 211 and the second normal strand 212). After performing a polymerase chain reaction using the polymerase chain reaction reagent kit, if the corresponding detected cycle threshold value is less than or equal to 40, the individual is determined to have cancer.

Figure 3 is a flowchart of the present invention for detecting whether an individual has cancer using a capture probe (i.e., a nucleotide sequence molecule 15). The flowchart includes the following steps:

Step S11: A plurality of nucleotide sequence molecules 15 are mixed with an in vitro sample, so that the nucleotide sequence molecules 15 bind to a plurality of target RNAs 11 and 12 in the in vitro sample. The target RNAs are normal RNA 11, cancer-related RNA 12, or a combination thereof. The cancer-related RNA 12 is divided into a first sequence segment 121 and a second sequence segment 123 by an abnormally encoded sequence segment 122, and the nucleotide sequence molecules 15 bind to the first sequence segment 121 of the cancer-related RNA 12. The normal RNA 11 includes a third sequence segment, and the nucleotide sequence molecules 15 bind to the third sequence segment of the normal RNA 11.

Step S12: Perform a reverse transcription step to reverse transcribe the captured normal information RNA 11 into normal information complementary DNA 21, and reverse transcribe the captured cancer-related information RNA 12 into cancer-related information complementary DNA 22.

Step S13: Perform a polymerase chain reaction using the first primer 31 and the second primer 32. The first primer 31 and the second primer 32 complement each other with a first abnormal strand 221 and a second abnormal strand 222 of the cancer-associated complementary DNA 22, respectively. At least one of the first primer 31 and the second primer 32 cannot bind to either strand 211 or 212 of the normal complementary DNA.

Step S14: Determine whether the in vitro sample contains cancer-related information RNA 12, and then determine whether the individual has cancer.

The following formally introduces various embodiments of the detection kit disclosed in this invention.

Experiment 1: Using different methods to extract RNA from plasma samples to evaluate the impact of various extraction methods on cancer detection.

It is known that fusion genes in cancer patient tissues transcribe into fusion mRNA. Consequently, large amounts of fusion mRNA are present in the patient's tissues, but only trace amounts are released into the blood. However, due to the difficulty in accurately detecting these trace amounts of fusion mRNA in the blood, there is currently no method for determining whether an individual has cancer by testing for fusion genes in blood samples. To address this issue, the present invention utilizes the presence of trace amounts of fusion genes in the blood of cancer patients to develop a low-invasive, convenient, and highly accurate cancer detection method. This study evaluated the effects of different extraction methods on the detection of the EML4-ALK fusion gene in plasma samples from three individuals using different extraction methods. The extraction methods used included column filtration, exosome isolation and purification, and the target RNA capture probe method provided by the present invention. The three individuals had been diagnosed with non-small cell lung cancer using standard hospital immunohistochemical staining, and their tissue sections contained high levels of the EML4-ALK fusion protein.

Column filtration method: The QIAamp Circulating Nucleic Acid kit (Qiagen, Cat. No. 55114) was used and the protocol provided was followed. Specifically, 0.125 mL of plasma sample was mixed evenly with the QIAGEN Proteinase K reagent and ACL buffer provided in the kit. The plasma was heated at 60°C for 30 minutes to lyse the sample, forming a plasma lysate. The plasma lysate was then mixed with ACB buffer to form a plasma lysate mixture, which was then incubated on ice for 5 minutes. The plasma lysate mixture was passed through the QIAamp Mini column provided in the kit. The QIAamp Mini column was then placed in an elution tube. AVE buffer was then added to the QIAamp Mini column. The solution collected in the elution tube was the total RNA solution extracted by column filtration. Plasma samples from three different sources were prepared using the above steps, yielding total RNA solutions extracted by column filtration from three different individuals.

Exosome purification: The Plasma/Serum Exosome Purification and RNA Isolation Kit (Norgen, Cat. No. 58300) was used, and the protocol provided in the kit was followed. Specifically, 0.125 mL of plasma sample was added to nuclease-free water, ExoC buffer provided in the kit, and Slurry E. The mixture was mixed thoroughly, centrifuged, and the supernatant removed to prepare a ground precipitate. Add ExoR buffer to the ground precipitate, incubate at room temperature for 5 minutes, centrifuge, and pass the supernatant through the filter column provided in the reagent kit. Collect the filtered fraction, which is the exosome solution. Add Lysis Buffer A and Lysis Additive B to the exosome solution, mix thoroughly, and incubate at room temperature for 10 minutes to lyse the exosomes. Then, add 100% ethanol and mix thoroughly. This is the exosome lysis solution. Transfer the exosome lysate to the filter column provided in the reagent kit and centrifuge to remove the liquid that has passed through the filter column. Then, add Wash Solution A, centrifuge, and remove the liquid that has passed through the filter column. Repeat this step twice. Transfer the filter column to a new elution tube, add Elution Solution A, and centrifuge to obtain the total RNA solution extracted from exosomes. Plasma samples from three different sources were prepared using the above steps to prepare total RNA solutions extracted from three different individuals.

Target RNA capture probe method: Using the ALK gene sequence data [ALK receptor tyrosine kinase (homo sapiens (human)); Gene ID: 238] provided by the National Center for Biotechnology Information (NCBI), and evaluating the breakpoints and fusion sites of ALK fusion genes (EML4-ALK fusion gene, KIF5B-ALK fusion gene, TFG-ALK fusion gene, HIP1-ALK fusion gene), target RNA capture probes (i.e., nucleotide sequence molecules) that complement the ALK sequence segment of the ALK fusion RNA were designed. Each target RNA capture probe sequence was tethered to the 3' end with 25-30 adenine residues, namely, the T69 capture probe (sequence: SEQ ID NO: 1) and the T71 capture probe (sequence: SEQ ID NO: 2). NO: 2). The commissioned T69 and T71 capture probes were reconstituted in nuclease-free water and then mixed in a 1:1 ratio to prepare a capture probe solution with a total concentration of 75 nM. Commercially available magnetic beads containing oligo dT were then added to allow the 25-30 adenine residues at the 3 ends of the capture probes to complementarily bind to the oligo dT residues on the beads, thus preparing a capture probe solution containing magnetic beads.

A 0.125 mL plasma sample was added to a high-salt nucleic acid extraction solution (Shanghai Rendu Biotechnology Co., Ltd.; Shanghai Puxi Medical Equipment No. 20190131; Nucleic Acid Extraction Reagent) and mixed thoroughly to lyse the plasma sample and release the total RNA. A capture probe with magnetic beads was then added and heated at 60°C for 10 minutes. The solution was then left at room temperature for 10 minutes to allow the capture probe to bind to the target RNA (including normal ALK RNA and ALK fusion RNA) in the total RNA solution, forming a solution containing the target RNA-capture probe complex. The solution containing the target RNA-capture probe complex was placed in a magnetic bead separation device (DynaMag ^™ -2 Magnet; Cat. No. 12321D) for 10 minutes. Once the target RNA-capture probe complex was completely adsorbed to the tube wall, the remaining liquid was removed, leaving only the target RNA-capture probe complex. Plasma samples from three different sources were prepared using the above steps, yielding target RNA solutions extracted from three different individuals using the capture probe.

The messenger RNA molecules (mRNA) in the above-mentioned (1) total RNA solution extracted by column filtration, (2) total RNA solution extracted by exosome separation and purification, and (3) target RNA solution extracted by capture probe are reverse transcribed into complementary deoxyribonucleic acid (cDNA) using a commercially available reverse transcription reagent kit, thereby obtaining column filtration group cDNA, exosome separation and purification group cDNA, and capture probe group cDNA, respectively. Next, a first primer complementary to the ALK sequence region (SEQ ID: 18), a second primer complementary to the EML4 sequence region (SEQ ID: 19), and a first fluorescent probe (SEQ ID: 20) were mixed with the column-filtered cDNA, the exosome-purified cDNA, or the capture probe cDNA. EML4-ALK complementary DNA was then amplified using a real-time polymerase chain reaction. The first fluorescent probe was used to detect the number of cycles completed in the polymerase chain reaction. The experimental results are shown in Figure 4 and Table 1.

As shown in Figure 4, the capture probe set achieved cycle threshold values (Ct values) of 34.77, 38.92, and 36.32 for target RNA solutions from three patients, respectively. This indicates that the capture probe was able to capture trace amounts of EML4-ALK fusion RNA in the patients' blood samples, yielding positive results. These results were consistent with those obtained using conventional immunohistochemical staining methods used in hospitals (see Table 1). However, the cycle thresholds for total RNA solutions from three patients in both the column filtration group and the exosome isolation and purification group were above 50, indicating that these two extraction methods were unable to extract the trace amounts of EML4-ALK fusion RNA in the patient blood samples, resulting in false-negative test results (see Table 1). This demonstrates that the target RNA capture probe method invented in this case can effectively extract trace amounts of fusion RNA in plasma samples, and the test results are consistent with those of conventional immunohistochemical staining tests in hospitals, with an accuracy rate of 100%. This demonstrates an unexpectedly high efficiency compared to the low extraction rates and high error rates of other RNA extraction methods.

Table 1

Experiment 2: Capture rates of different ALK capture probes

Experiment 2.1: Preparation of Different ALK Capture Probes - Oligo dT Magnetic Beads

Using ALK gene sequence data provided by the National Center for Biotechnology Information (NCBI) [ALK receptor tyrosine kinase (homo sapiens (human)); Gene ID: 238] and the estimated breakpoints and fusion sites of ALK fusion RNAs (EML4-ALK fusion gene, KIF5B-ALK fusion gene, TFG-ALK fusion gene, and HIP1-ALK fusion gene), 17 ALK capture probes (i.e., nucleotide sequence molecules) were designed that complement the ALK sequence segments of the ALK fusion genes. Each ALK capture probe sequence has 25-30 adenine residues at its 3' end.

The ALK capture probe sequences are as follows: T69 (SEQ ID: 1), T71 (SEQ ID: 2), T70 (SEQ ID: 3), T68 (SEQ ID: 4), T47 (SEQ ID: 5), T87 (SEQ ID: 6), T50 (SEQ ID: 7), T49 (SEQ ID: 8), T86 (SEQ ID: 9), T74 (SEQ ID: 10), T72 (SEQ ID: 11), T75 (SEQ ID: 12), T51 (SEQ ID: 13), T22 (SEQ ID: 14), T46 (SEQ ID: 15), T48 (SEQ ID: 16), and T73 (SEQ ID: 17).

The 17 ALK capture probes produced on request were reconstituted in nuclease-free water and divided into groups. The first group consisted of 17 subgroups: T69, T71, T70, T68, T47, T87, T50, T49, T86, T74, T72, T75, T51, T22, T46, T48, and T73 capture probe solutions, all at a concentration of 75 nM. The 17 different ALK capture probes were combined to form the second group of 14 subgroups: T86/T87, T72/T74, T71/T72, T49/T50, and T73. The capture probe solutions were T48/T49, T51/T73, T46/T71, T51/T69, T46/T48, T69/T70, T70/T71, T69/T71, T69/T70/T71, and T48/T69/T70/T71. The total concentration of each group was 75 nM. The two ALK capture probes in the first 12 groups were mixed at a 1:1 ratio; the three ALK capture probes in the 13th group were mixed at a 1:1:1 ratio; and the four ALK capture probes in the 14th group were mixed at a 1:1:1:1 ratio.

Add each capture probe solution to magnetic beads containing oligo dT. This allows the 25-30 adenine residues at the 3 ends of the ALK capture probe to complementarily bind to the oligo dT on the beads. This creates a magnetic bead-attached ALK capture probe solution, such as the T68 ALK capture probe solution and the T69/T70/T71 ALK capture probe solution.

Experiment 2.2: Capture efficiency of EML4-ALK fusion RNA by various ALK capture probes

To prepare the EML4-ALK fusion gene target RNA solution, the sequence data of the EML4-ALK fusion gene variant 1 (Homo sapiens mRNA for fusion protein EML4-ALK variant 1; GenBank: AB274722.1) provided by the National Center for Biotechnology Information (NCBI) was used to align with the ALK gene sequence (gene ID: 238) to confirm the breakpoint location of the EML4-ALK fusion gene. Based on the sequence segments of 200-300 nucleotides toward the 5' end and 200-300 nucleotides toward the 3' end of the breakpoint, an artificial partial fusion RNA molecule of the EML4-ALK fusion gene was prepared. The sequence is shown in SEQ ID: 21. The artificial partial fusion RNA molecules of the commissioned EML4-ALK fusion gene were dissolved in nuclease-free water to prepare the EML4-ALK fusion gene target RNA solution.

The 31 sets of ALK capture probe solutions with magnetic beads prepared in Experiment 2.1 were tested using the following method. For example, using the T68 ALK capture probe solution with magnetic beads, 0.125 mL of the EML4-ALK fusion gene target RNA solution was added to the T68 ALK capture probe solution with magnetic beads. The mixture was mixed thoroughly, heated at 60°C for 10 minutes, and then allowed to stand at room temperature for 10 minutes. This allowed the ALK capture probe to bind to the artificial partial fusion RNA molecule of the EML4-ALK fusion gene, forming a complex solution containing the EML4-ALK fusion RNA and capture probe. Place the EML4-ALK fusion RNA-capture probe complex solution on a magnetic bead separation device for 10 minutes. Once the EML4-ALK fusion RNA-capture probe complex is completely adsorbed to the tube wall, remove the remaining extract, leaving only the EML4-ALK fusion RNA-capture probe complex.

Artificial partial fusion RNA molecules representing the EML4-ALK fusion gene from 31 groups were reverse-transcribed into EML4-ALK complementary DNA (EML4-ALK cDNA) using a commercially available reverse transcription kit. A first primer (SEQ ID: 18) complementary to the ALK sequence, a second primer (SEQ ID: 19) complementary to the EML4 sequence, and a first fluorescent probe (SEQ ID: 20) were mixed with the EML4-ALK cDNA from each of the 31 groups. The EML4-ALK cDNA was then amplified using a real-time polymerase chain reaction. The cycle threshold of a standard solution containing a known amount of complementary DNA (Ct _standard) was used. The cycle threshold obtained for each experimental group (Ct _group) was used to calculate the capture efficiency of each ALK capture probe for the EML4-ALK fusion RNA (i.e., the artificial partial fusion RNA molecule of the EML4-ALK fusion gene).

The capture rate was calculated as follows: Capture rate = (1/2 ^{Ct group - Ct standard} ) x 100%. See Figure 5 for experimental results.

FIG5 is a bar graph showing the efficiency of capturing EML4-ALK fusion RNA using different ALK capture probes. As shown in Figure 5, 28 groups, including T68, T47, T87, T50, T49, T86, T74, T72, T75, T51, T22, T46, T48, T73, T69, T70, T71, T86/T87, T72/T74, T71/T72, T49/T50, T48/T49, T51/T73, T46/T71, T51/T69, T46/T48, T69/T70, and T70/T71, all showed a certain capture rate. Among the single ALK capture probes, T69, T70, and T71 had the best capture rates, demonstrating unexpected results. Furthermore, the capture rates of the EML4-ALK fusion RNA for the three capture probe combinations—T69/T71, T69/T70/T71, and T48/T69/T70/T71—were all greater than or equal to 50%. Furthermore, the T69, T70, T71, and T48 capture probes all exhibited synergistic effects, resulting in unexpected results. The T69/T70/T71 combination achieved the highest capture rate, reaching 65%. This demonstrates that the ALK capture probes and their combinations, such as T69/T70/T71, used in this study, can effectively capture the EML4-ALK fusion RNA in samples. Based on the same ALK capture principle, the ALK capture probes and their combinations in this application can also effectively capture other ALK fusion RNAs, including but not limited to KIF5B-ALK fusion RNAs and TFG-ALK fusion RNAs.

Experiment 3: Capture rates of different ROS1 capture probes

Experiment 3.1: Preparation of Different ROS1 Capture Probes - Oligo dT Magnetic Beads

Using ROS1 gene sequence data provided by the National Center for Biotechnology Information (NCBI) [proto-oncogene 1, receptor tyrosine kinase (Homo sapiens (human)); Gene ID: 6098] and the breakpoints and fusion sites of several ROS1 fusion genes (CD74-ROS1, SDC4-ROS1, SLC34A2-ROS1, GOPC-ROS1, EZR-ROS1, LRIG3-ROS1, and TPM3-ROS1), we designed ten ROS1 capture probes (nucleotide sequences) complementary to the ROS1 sequence of the ROS1 fusion RNA. Each ROS1 capture probe sequence was terminated with 25 to 30 adenine residues at its three termini. The ROS1 capture probes are as follows: T117 (SEQ ID: 22), T112 (SEQ ID: 23), T108 (SEQ ID: 24), T111 (SEQ ID: 25), T116 (SEQ ID: 26), T109 (SEQ ID: 27), T110 (SEQ ID: 28), T114 (SEQ ID: 29), T113 (SEQ ID: 30), and T115 (SEQ ID: 31).

The 10 ROS1 capture probes were dissolved in nuclease-free water and divided into groups. The first group consisted of 10 groups: T117, T112, T108, T111, T116, T109, T110, T114, T113, and T115 capture probe solutions. The concentration of each group was 75 nM. The 10 different ROS1 capture probes were combined to form the second group of 16 groups: T108/T109, T110/T112, T116/T117, T109/T110, T111/T113, T109/T115, T11 The capture probe solutions for groups 3/T114, T114/T115, T108/T109/T110, T110/T112/T114, T109/T110/T113, T110/T113/T114, T109/T113/T114, T110/T113/T115, T109/T113/T115, and T113/T114/T115 were all at a total concentration of 75 nM. The two ROS1 capture probes in the first eight groups were mixed at a 1:1 ratio, while the three ROS1 capture probes in groups 9 to 16 were mixed at a 1:1:1 ratio.

Add each capture probe solution to oligo dT-containing magnetic beads. This allows the 25-30 adenine residues at the 3-terminus of the ROS1 capture probe to complementarily bind to the oligo dT on the beads. This results in the preparation of magnetic bead-containing ROS1 capture probe solutions, such as the T117 ROS1 capture probe solution and the T113/T114/T115 capture probe solution.

Experiment 3.2: Capture efficiency of CD47-ROS1 fusion RNA by various ROS1 capture probes

To prepare the CD74-ROS1 fusion gene target RNA solution, the sequence data of the CD74-ROS1 fusion gene (Homo sapiens mRNA for fusion protein CD74-ROS1; COSMIC: COSF1200) provided by the National Center for Biotechnology Information (NCBI) was used to align with the ROS1 gene sequence data (Gene ID: 6098) to confirm the breakpoint location of the CD74-ROS1 fusion gene. Based on the sequence segments of 200-300 nucleotides toward the 5' end and 200-300 nucleotides toward the 3' end of the breakpoint, an artificial partial fusion RNA molecule of the CD74-ROS1 fusion gene was prepared. The sequence is shown in SEQ ID: 32. The artificial partial fusion RNA molecules of the commissioned CD74-ROS1 fusion gene were dissolved in nuclease-free water to prepare a CD74-ROS1 fusion gene target RNA solution.

The 26 sets of ROS1 capture probe solutions with magnetic beads prepared in Experiment 3.1 were tested using the following method. For example, 0.125 mL of CD74-ROS1 fusion gene target RNA solution was added to the T117 ROS1 capture probe solution with magnetic beads. The mixture was mixed thoroughly, heated at 60°C for 10 minutes, and then allowed to stand at room temperature for 10 minutes. This allowed the ROS1 capture probe to bind to the artificial partial fusion RNA molecules of the CD74-ROS1 fusion gene, forming a complex solution containing the CD74-ROS1 fusion RNA and capture probe. The solution containing the CD74-ROS1 fusion RNA-capture probe complex was placed on a magnetic bead separation device for 10 minutes. Once the CD74-ROS1 fusion RNA-capture probe complex was completely adsorbed to the tube wall, the remaining extract was removed, leaving only the CD74-ROS1 fusion RNA-capture probe complex. Other groups were tested in the same manner.

Using a commercially available reverse transcription reagent kit, artificial partial fusion RNA molecules representing the CD74-ROS1 fusion gene from 26 groups were reverse-transcribed into CD74-ROS1 complementary DNA (CD74-ROS1 cDNA). A third primer (SEQ ID: 33) complementary to the ROS1 sequence region, a fourth primer (SEQ ID: 34) complementary to the CD74 sequence region, and a second fluorescent probe (SEQ ID: 35) were mixed with the CD74-ROS1 cDNA from each of the 26 groups. The CD74-ROS1 cDNA was then amplified using a real-time polymerase chain reaction. See Figure 6 for the experimental results.

Figure 6 is a bar chart showing the capture rates of CD74-ROS1 fusion RNA using different ROS1 capture probes. As shown in Figure 6, 15 groups—T117, T112, T108, T111, T116, T109, T110, T114, T113, T115, T108/T109, T110/T112, T116/T117, T109/T110, and T108/T109/T110—all showed a certain capture rate. Among the individual ROS1 capture probes, T114, T113, and T115 exhibited the highest capture rates, demonstrating unexpected results. In addition, T111/T113, T109/T115, T113/T114, T114/T115, T110/T112/T114, T109/T110/T113, T110/T113/T114, T109/T113/T114, T110/T113/T115, T109/T113/T114 The capture efficiencies of the CD74-ROS1 fusion RNAs in 11 groups, including T114/T115, T109/T113/T114, and T113/T114/T115, were all greater than or close to 30%. Furthermore, the T114, T113, and T115 capture probes were able to form synergistic effects with other capture probes (e.g., T109, T111, T110, and T112), demonstrating unexpected benefits. The highest capture efficiencies were achieved by the groups T114/T115, T109/T113/T114, T110/T113/T115, T109/T113/T115, and T113/T114/T115, reaching 50%-65%. As can be seen from this, the ROS1 capture probes and their combinations in this application can effectively capture the CD74-ROS1 fusion RNA in the specimen. Based on the same ROS1 capture principle, the ROS1 capture probes and their combinations in this application can also effectively capture other ROS1 fusion RNAs, including but not limited to SDC4-ROS1 fusion RNAs, SLC34A2-ROS1 fusion RNAs, GOPC-ROS1 fusion RNAs, EZR-ROS1 fusion RNAs, LRIG3-ROS1 fusion RNAs, and TPM3-ROS1 fusion RNAs.

Experiment 4: Capture rates of different MET capture probes

Experiment 4.1: Preparation of Different MET Capture Probes - Oligo dT Magnetic Beads

Using MET gene sequence data provided by the National Center for Biotechnology Information (NCBI) [MET proto-oncogene, receptor tyrosine kinase ( Homo sapiens (human)); Gene ID: 4233] and the estimated breakpoints of the MET fusion gene (MET exon 14 skipping fusion gene), 10 MET capture probes (nucleotide sequences) were designed that complement the sequence segments upstream or downstream of the exon 14 skipping site of the MET information RNA. Each MET capture probe sequence was ligated with 25 to 30 adenine residues at its 3′ end.

The MET capture probes are as follows: T144 (SEQ ID: 36), T142 (SEQ ID: 37), T141 (SEQ ID: 38), T145 (SEQ ID: 39), T147 (SEQ ID: 40), T139 (SEQ ID: 41), T143 (SEQ ID: 42), T148 (SEQ ID: 43), T140 (SEQ ID: 44), and T146 (SEQ ID: 45).

The 10 MET capture probes produced on request were dissolved in nuclease-free water and divided into groups. The first group consisted of 10 subgroups: T144, T142, T141, T145, T147, T139, T143, T148, T140, and T146 capture probe solutions, each with a concentration of 75 nM. The 10 different MET capture probes were combined to form the second group of 8 subgroups: T143/T146 The capture probe solutions consisted of T146/T148, T143/T148, T140/T148, T140/T146, T140/T143, T140/T146/T148, and T140/T143/T146. The total concentration of each solution was 75 nM. The two MET capture probes in the first six groups were mixed at a 1:1 ratio, while the three MET capture probes in groups 7 and 8 were mixed at a 1:1:1 ratio.

Add each capture probe solution to oligo dT-containing magnetic beads, allowing the 25-30 adenine residues at the 3 ends of the MET capture probe to complementarily bind to the oligo dT on the magnetic beads. This creates a MET capture probe solution with magnetic beads, such as the T144 MET capture probe solution and the T140/T143/T146 MET capture probe solution.

Experiment 4.2: Capture efficiency of various MET capture probes for MET fusion RNA with exon 14 skipped

To prepare the target RNA solution for the MET gene with exon 14 skipped, the sequence data of the MET fusion gene with exon 14 skipped (COSMIC: COSM13245) provided by the National Center for Biotechnology Information (NCBI) was used and compared with the sequence data of the normal MET gene (Gene ID: 4233). The breakpoint location of the MET fusion gene with exon 14 skipped was confirmed. Based on the sequence segments of 200-300 nucleotides toward the 5' end and 200-300 nucleotides toward the 3' end of the breakpoint, an artificial partial fusion RNA molecule of the MET fusion gene with exon 14 skipped was prepared. The sequence is SEQ ID: 46. The artificial partial fusion RNA molecules of the commissioned MET fusion gene with exon 14 skipped were dissolved in nuclease-free water to prepare a MET gene target RNA solution with exon 14 skipped.

The 18 sets of MET capture probe solutions with magnetic beads prepared in Experiment 4.1 were each tested according to the following method. Using the T144 MET capture probe solution with magnetic beads as an example, 0.125 mL of the target RNA solution containing the MET fusion gene with exon 14 skipped was added to the T144 MET capture probe solution with magnetic beads. The mixture was mixed thoroughly, heated at 60°C for 10 minutes, and then allowed to stand at room temperature for 10 minutes. This allowed the MET capture probe solution to bind to the artificial partial fusion RNA molecule of the MET fusion gene with exon 14 skipped, forming a complex solution containing the MET fusion RNA with exon 14 skipped and the capture probe. Place the solution containing the MET exon 14 skipped fusion RNA-capture probe complex on a magnetic bead separation device for 10 minutes. Once the MET exon 14 skipped fusion RNA-capture probe complex is completely adsorbed to the tube wall, remove the remaining extract, leaving only the MET exon 14 skipped fusion RNA-capture probe complex.

Using a commercially available reverse transcription reagent kit, artificial partial fusion RNA molecules of the MET fusion gene with exon 14 skipping in 18 groups were reverse-transcribed into MET exon 14 skipping cDNAs. A seventh primer (SEQ ID: 47), complementary to the sequence region upstream of the MET exon 14 skipping site, an eighth primer (SEQ ID: 48), complementary to the sequence region downstream of the MET exon 14 skipping site, and a fourth fluorescent probe (SEQ ID: 49) were mixed with the MET exon 14 skipping cDNAs in each of the 18 groups. The MET exon 14 skipping cDNAs were then amplified using real-time polymerase chain reactions. Please see Figure 8 for the experimental results.

Figure 7 is a bar chart showing the capture rates of MET fusion RNAs with exon 14 skipped using different MET capture probes. As shown in Figure 7, T144, T142, T141, T145, T147, T139, T143, T148, T140, and T146 all have a certain capture rate, with T139, T143, T148, T140, and T146 achieving the highest capture rates, demonstrating an unexpected effect. Furthermore, the capture rates of MET fusion RNAs with exon 14 skipped in eight groups—T143/T146, T146/T148, T143/T148, T140/T148, T140/T146, T140/T143, T140/T146/T148, and T140/T143/T146—were all greater than or close to 20%. Furthermore, the capture probes T139, T143, T148, T140, and T146 exhibited unexpected synergistic effects. The highest capture rate, reaching 60%, was achieved in the T140/T143/T146 group. This indicates that the MET capture probe and its combination in this case can effectively capture trace amounts of MET fusion RNA with exon 14 skipped in the specimen.

The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the patent application for this invention. Therefore, any other modifications or improvements that do not deviate from the spirit disclosed in this invention should be included in the scope of the patent application for this case.

References

113110877-A0101-15-0001.xml

S11-S14: Steps

Claims (14)

A fusion RNA capture probe set for non-small cell lung cancer-associated fusion information for use in blood or plasma samples, wherein the sequences of the plurality of complementary probes in the capture probe set are at least one of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or any combination thereof; or the capture probe set comprises a first ALK capture probe, a second ALK capture probe, and a third ALK capture probe, and the sequence of the first ALK capture probe is SEQ ID NO: 1, the sequence of the second ALK capture probe is SEQ ID NO: 2, and the sequence of the third ALK capture probe is SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 At least one of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, or any combination thereof. A test kit for detecting whether an individual has non-small cell lung cancer (NSCLC), comprising: a plurality of nucleotide sequence molecules for binding to a plurality of target messenger RNAs (targeted mRNAs) in a blood or plasma sample, thereby directly capturing the target messenger RNAs in the blood or plasma sample; the target messenger RNAs are normal messenger RNAs (normal mRNAs) or cancer-associated messenger RNAs (cancer-associated mRNAs) associated with NSCLC. mRNA), or a combination thereof; wherein the cancer-associated information RNA is divided into a first sequence segment and a second sequence segment by an abnormal coding sequence segment, and the nucleotide sequence molecules are bound to the first sequence segment on the cancer-associated information RNA; and the normal information RNA comprises a third sequence segment, and the nucleotide sequence molecules are bound to the third sequence segment on the normal information RNA; wherein the nucleotide sequence molecules are a plurality of ALK capture probes, and the sequences of the ALK capture probes are at least one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or any combination thereof; or, the nucleotide sequence molecules are a plurality of ROS1 capture probes, and the sequences of the ROS1 capture probes are SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: NO: 31 or any combination thereof; or, the nucleotide sequence molecules are a plurality of MET capture probes, and the MET capture probes include a first MET capture probe, a second MET capture probe, and a third MET capture probe, and the sequences of the first MET capture probe, the second MET capture probe, and the third MET capture probe are SEQ ID NO: 42, SEQ ID NO: 44, and SEQ ID NO: 45, respectively; a reverse transcription reagent kit for reversely transcribing captured normal messenger RNA into a normal messenger complementary DNA (normal cDNA) and reversely transcribing captured cancer-associated messenger RNA into a cancer-associated messenger complementary DNA (cancer-associated cDNA). cDNA), wherein the cancer-associated complementary DNA is divided into a first reverse transcriptase region and a second reverse transcriptase region by an abnormal region reverse transcriptase sequence corresponding to the abnormal coding sequence segment; and a polymerase chain reaction reagent kit comprising a first primer, a second primer, and a fluorescent probe for detecting the number of cycles of the polymerase chain reaction; in the polymerase chain reaction (polymerase chain reaction) reaction; PCR), when the cancer-associated complementary DNA is denatured and separated into a first abnormal strand and a second abnormal strand, the first primer is used to anneal to the first reverse transcriptase region on the first abnormal strand, and the second primer is used to anneal to the second reverse transcriptase region on the second abnormal strand; at least one of the first primer and the second primer is unable to anneal to any strand of the normal complementary DNA; and, after performing a polymerase chain reaction using the polymerase chain reaction reagent kit, if the cycle threshold value is less than or equal to 40, the individual is determined to have non-small cell lung cancer; wherein, (1) The first primer and the second primer are an ALK fusion gene primer pair, and the sequences of the ALK fusion gene primer pair are SEQ ID NO: 18 and SEQ ID NO: 19, respectively, or the ALK fusion gene primer pair can form annealing with the SEQ ID NO: 21 sequence and its complementary sequence in the polymerase chain reaction; and the sequence of the fluorescent probe is SEQ ID NO: 20, or the fluorescent probe can be used to detect the number of cycles that the cancer-related information complementary DNA has undergone in the polymerase chain reaction; or (2) the first primer and the second primer are a ROS1 fusion gene primer pair, and the sequences of the ROS1 fusion gene primer pair are SEQ ID NO: 33 and SEQ ID NO: 34, respectively, or the ROS1 fusion gene primer pair can form annealing with the SEQ ID NO: 21 sequence and its complementary sequence in the polymerase chain reaction; NO: 32 sequence and its complementary sequence form an annealing; and the sequence of the fluorescent probe is SEQ ID NO: 35, or the fluorescent probe can be used to detect the number of cycles that the cancer-related information complementary DNA has undergone in a polymerase chain reaction; or (3) the first primer and the second primer are a MET fusion gene primer pair, and the sequences of the MET fusion gene primer pair are SEQ ID NO: 47 and SEQ ID NO: 48, respectively, or the MET fusion gene primer pair can form an annealing with the sequence of SEQ ID NO: 46 and its complementary sequence in a polymerase chain reaction; and the sequence of the fluorescent probe is SEQ ID NO: 49, or the fluorescent probe can be used to detect the number of cycles that the cancer-associated complementary DNA has undergone in the polymerase chain reaction. The detection kit as described in claim 2, wherein the cancer-associated messenger RNA is an ALK fusion messenger RNA (ALK fusion mRNA), a ROS1 fusion messenger RNA (ROS1 fusion mRNA), or a MET messenger RNA with skipping of exon 14 in the MET mRNA. The detection kit as described in item 2 of the patent application, wherein the abnormal coding sequence segment is an informational RNA sequence segment transcribed from a chromosomal recombination segment of an ALK fusion gene or a ROS1 fusion gene resulting from chromosomal recombination; or, the abnormal coding sequence segment is an informational RNA sequence segment transcribed from a segment of exon 14 skipping in the MET gene. As described in item 2 of the patent application, the detection kit, wherein the abnormal region reverse transcribed sequence is a complementary deoxyribonucleic acid (cDNA) obtained after reverse transcription of the abnormal coding sequence segment, and the cancer-associated information complementary deoxyribonucleic acid is divided into the first reverse transcribed sequence region and the second reverse transcribed sequence region with the abnormal region reverse transcribed sequence as the boundary. In the detection kit described in claim 2, the blood or plasma sample contains a plurality of lipid bilayer membrane structures, and the target information RNA is encapsulated in the lipid bilayer membrane structures, or is not encapsulated in the lipid bilayer membrane structures, or a combination thereof. The detection kit described in claim 6 further comprises a solution for mixing with the blood or plasma sample to release the target RNA encapsulated in the lipid bilayer membrane structure in the blood or plasma sample, wherein the solution is a phenol-chloroform-isoamyl alcohol mixture or other reagent used to extract RNA from blood. The detection kit as described in claim 6, wherein the lipid bilayer membrane structure is an exosome. The detection kit as described in claim 2, wherein the nucleotide sequence molecules are bound to or bonded to the surface of a plurality of magnetic beads. The detection kit as described in item 2 of the patent application, wherein: (1) the nucleotide sequence molecules are a plurality of ALK capture probes, and the ALK capture probes include a first ALK capture probe and a second ALK capture probe; the sequence of the first ALK capture probe is SEQ ID NO: 1, and the sequence of the second ALK capture probe is SEQ ID NO: 2; or (2) the nucleotide sequence molecules are a plurality of ROS1 capture probes, and the ROS1 capture probes include a first ROS1 capture probe and a second ROS1 capture probe; the sequence of the first ROS1 capture probe is SEQ ID NO: 29, and the sequence of the second ROS1 capture probe is SEQ ID NO: 31. The detection kit as described in claim 10, wherein: (1) the nucleotide sequence molecules are a plurality of ALK capture probes, and the ALK capture probes further include a third ALK capture probe, and the sequence of the third ALK capture probe is SEQ ID NO: 3; or the sequence of the third ALK capture probe is SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17; or (2) The nucleotide sequence molecules are a plurality of ROS1 capture probes, and the ROS1 capture probes further include a third ROS1 capture probe, and the sequence of the third ROS1 capture probe is SEQ ID NO: 30; or, the sequence of the third ROS1 capture probe is SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, or SEQ ID NO: 28. The detection kit as described in claim 2, wherein the nucleotide sequence molecules are composed of a plurality of deoxyribonucleotides. The detection kit as described in claim 2, wherein the nucleotide sequence molecules are composed of 30 to 60 deoxyribonucleotides. A detection kit as described in claim 2 is used to detect whether a blood or plasma sample of an individual contains a cancer-associated messenger RNA (cancer-associated mRNA) associated with non-small cell lung cancer, thereby determining whether the individual has non-small cell lung cancer.