A lung adenocarcinoma detection kit, method and application

Abstract

The present application relates to the field of disease detection, in particular to a lung adenocarcinoma detection kit, method and application. The present application provides a lung adenocarcinoma detection kit, which comprises detection reagents for detecting the relative expression amount of LRPPRC gene and EGLN3 gene in a biological sample, wherein the relative expression amount is the ratio of a first expression amount and a second expression amount, the first expression amount is the ratio of the expression amount of LRPPRC gene and the expression amount of PPIB gene, and the second expression amount is the ratio of the expression amount of EGLN3 gene and the expression amount of PPIB gene. The present application can to some extent avoid the influence of sample amount on the detection result, and can specifically distinguish lung adenocarcinoma patients from other populations, thereby achieving auxiliary diagnosis of early diagnosis of lung adenocarcinoma.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of disease detection, in particular to a lung adenocarcinoma detection kit, method and application. BACKGROUND

[0002] Lung cancer (LC), i.e. primary bronchogenic lung cancer, is a malignant tumor originating from the bronchial mucosa or glands of the lung. According to the world cancer statistics published by the International Agency for Research on Cancer (IARC) in 2020, there are about 1930 million new cases and 1000 million deaths worldwide, with 220.6 million new cases of lung cancer and 179.6 million deaths. Lung adenocarcinoma (LUAD) is the pathological type with the highest incidence and mortality rate among lung cancers. Previous studies (DOI: 10.3390 / ijms241713302) have shown that lung adenocarcinoma is insidious and progresses rapidly, and due to the lack of reliable diagnostic markers, about 75% of patients are in the advanced stage at the time of diagnosis, and the five-year survival rate is only 15%. If lung adenocarcinoma can be diagnosed and treated in the early stage, the five-year survival rate can be increased to 45%.

[0003] Although Chinese patent application CN112522409A discloses that the EGLN3 gene can be used as a biomarker for lung adenocarcinoma detection, previous studies (DOI: 10.1016 / j.heliyon.2024.e33206) have shown that the EGLN3 (Egl-9 family hypoxia-inducible factor 3) gene not only has a high expression in lung adenocarcinoma, but also has a higher expression level in breast cancer (BRCA), cervical squamous cell carcinoma and cervical adenocarcinoma (CESC), and cholangiocarcinoma (CHOL) than in normal tissues. Therefore, the EGLN3 gene alone is not sufficient as a biomarker for lung adenocarcinoma. SUMMARY

[0004] In a first aspect, the present application provides a lung adenocarcinoma detection kit, which comprises a detection reagent for detecting the relative expression amount of the LRPPRC gene (leucine rich ppr motif containing protein, PPR motif protein rich in leucine) and the EGLN3 gene (Egl-9 family hypoxia-inducible factor 3, EGL-9 family hypoxia-inducible factor 3) in a biological sample.

[0005] In some embodiments, the relative expression amount is the ratio of the first expression amount to the second expression amount.

[0006] In some embodiments, the first expression level is the ratio of the expression level of the LRPPRC gene to the expression level of the PPIB (Peptidylprolyl Isomerase B) gene.

[0007] In some embodiments, the second expression level is the ratio of the expression level of the EGLN3 gene to the expression level of the PPIB gene.

[0008] Gene expression level generally refers to the relative or absolute amount of mRNA transcribed from a certain gene, which is used to measure the expression activity of the gene in a particular tissue, cell or experimental condition. The expression level can be detected and quantified by various techniques, such as PCR, etc.

[0009] In some embodiments, the detection reagent comprises:

[0010] an upstream primer for detecting the LRPPRC gene, the nucleotide sequence of which is shown as SEQ ID NO: 1; a downstream primer for detecting the LRPPRC gene, the nucleotide sequence of which is shown as SEQ ID NO: 3; a probe for detecting the LRPPRC gene, the nucleotide sequence of which is shown as SEQ ID NO: 2;

[0011] an upstream primer for detecting the EGLN3 gene, the nucleotide sequence of which is shown as SEQ ID NO: 4; a downstream primer for detecting the EGLN3 gene, the nucleotide sequence of which is shown as SEQ ID NO: 6; a probe for detecting the EGLN3 gene, the nucleotide sequence of which is shown as SEQ ID NO: 5;

[0012] an upstream primer for detecting the PPIB gene, the nucleotide sequence of which is shown as SEQ ID NO: 7; a downstream primer for detecting the PPIB gene, the nucleotide sequence of which is shown as SEQ ID NO: 9; a probe for detecting the PPIB gene, the nucleotide sequence of which is shown as SEQ ID NO: 8.

[0013] In some embodiments, the biological sample is derived from the lung tissue or blood of the subject.

[0014] In some embodiments, the biological sample comprises lung adenocarcinoma cells and / or lung adenocarcinoma tissue.

[0015] In some embodiments, the lung adenocarcinoma detection kit is configured for detecting RNA extracted from the biological sample.

[0016] In some embodiments, the quality of the RNA comprises 0.5 - 2 μg.

[0017] In some embodiments, the quality of the RNA comprises 0.5 – 1 μg.

[0018] In some embodiments, the lung adenocarcinoma detection kit is configured for detection based on digital PCR.

[0019] Digital PCR (digital PCR) is a molecular biology technique for absolute quantification of target nucleic acid molecules to achieve single-molecule level amplification and detection, thereby obtaining the copy number of target nucleic acid molecules without standard curve.

[0020] In some embodiments, the expression amount of the LRPPRC gene, the expression amount of the PPIB gene and the expression amount of the EGLN3 gene refer to absolute quantification results obtained based on digital PCR technology.

[0021] In some embodiments, the lung adenocarcinoma detection kit is configured for simultaneously detecting the expression amount of the LRPPRC gene, the EGLN3 gene and the PPIB gene in one tube of digital PCR reaction.

[0022] In a second aspect, the present application provides a method for using the above-mentioned kit, which comprises:

[0023] S101 extracting nucleic acid from a biological sample;

[0024] In some embodiments, the biological sample is derived from lung tissue or blood of a subject.

[0025] In some embodiments, the biological sample comprises lung adenocarcinoma cells and / or lung adenocarcinoma tissue.

[0026] In some embodiments, the nucleic acid is RNA.

[0027] In some embodiments, the quality of the RNA comprises 0.5 – 2 μg.

[0028] In some embodiments, the quality of the RNA comprises 0.5 – 1 μg.

[0029] S102 performing PCR amplification on the nucleic acid using the above-mentioned kit;

[0030] In some embodiments, the PCR is digital PCR.

[0031] In some embodiments, the amplification procedure of the digital PCR comprises: 53℃ 25min; 95℃ 5min; 95℃ 15s, 58℃ 25s, 35 cycles.

[0032] S103 obtaining absolute quantification results of the LRPPRC gene, the EGLN3 gene and the PPIB gene in the biological sample.

[0033] In some embodiments, the S103 further comprises obtaining the relative expression amount of the LRPPRC gene and the EGLN3 gene in the biological sample.

[0034] In some embodiments, the relative expression amount is a ratio of a first expression amount and a second expression amount.

[0035] In some embodiments, the first expression amount is a ratio of the expression amount of the LRPPRC gene and the expression amount of the PPIB gene.

[0036] In some embodiments, the second expression amount is a ratio of the expression amount of the EGLN3 gene and the expression amount of the PPIB gene.

[0037] In some embodiments, the method further comprises S104 when the relative expression amount in the biological sample is ≤ a preset positive threshold, the subject corresponding to the biological sample is evaluated as a lung adenocarcinoma patient.

[0038] In some embodiments, when the relative expression amount in the biological sample is ≥ a preset negative threshold, the subject corresponding to the biological sample is evaluated as a non-lung adenocarcinoma patient.

[0039] In some embodiments, when the relative expression amount in the biological sample is between the preset positive threshold and the preset negative threshold (i.e. positive threshold < relative expression amount < negative threshold), the subject corresponding to the biological sample is recommended for follow-up for further observation and evaluation.

[0040] In a third aspect, the present application provides a use of a detection reagent in the preparation of a lung adenocarcinoma detection kit, wherein the detection reagent is used for detecting the relative expression amount of the LRPPRC gene and the EGLN3 gene in a biological sample.

[0041] In some embodiments, the biological sample is derived from the lung tissue or blood of a subject.

[0042] In some embodiments, the biological sample comprises lung adenocarcinoma cells and / or lung adenocarcinoma tissue.

[0043] In some embodiments, the lung adenocarcinoma detection kit is used for detecting RNA extracted from the biological sample.

[0044] In some embodiments, the quality of the RNA comprises 0.5 – 2 μg.

[0045] In some embodiments, the quality of the RNA comprises 0.5 – 1 μg.

[0046] In some embodiments, the lung adenocarcinoma detection kit is used for detection based on digital PCR.

[0047] In some embodiments, the LRPPRC gene expression amount, the PPIB gene expression amount and the EGLN3 gene expression amount refer to absolute quantitative results obtained based on digital PCR technology.

[0048] In some embodiments, the lung adenocarcinoma detection kit is used for simultaneously detecting the expression amounts of the LRPPRC gene, the EGLN3 gene and the PPIB gene in one tube of digital PCR reaction.

[0049] In some embodiments, the relative expression amount is a ratio of the first expression amount to the second expression amount.

[0050] In some embodiments, the first expression amount is a ratio of the LRPPRC gene expression amount to the PPIB gene expression amount.

[0051] In some embodiments, the second expression amount is a ratio of the EGLN3 gene expression amount to the PPIB gene expression amount.

[0052] In some embodiments, the detection kit comprises:

[0053] an upstream primer for detecting the LRPPRC gene, the nucleotide sequence of which is shown in SEQ ID NO: 1; a downstream primer for detecting the LRPPRC gene, the nucleotide sequence of which is shown in SEQ ID NO: 3; a probe for detecting the LRPPRC gene, the nucleotide sequence of which is shown in SEQ ID NO: 2;

[0054] an upstream primer for detecting the EGLN3 gene, the nucleotide sequence of which is shown in SEQ ID NO: 4; a downstream primer for detecting the EGLN3 gene, the nucleotide sequence of which is shown in SEQ ID NO: 6; a probe for detecting the EGLN3 gene, the nucleotide sequence of which is shown in SEQ ID NO: 5;

[0055] an upstream primer for detecting the PPIB gene, the nucleotide sequence of which is shown in SEQ ID NO: 7; a downstream primer for detecting the PPIB gene, the nucleotide sequence of which is shown in SEQ ID NO: 9; a probe for detecting the PPIB gene, the nucleotide sequence of which is shown in SEQ ID NO: 8.

[0056] In some embodiments, the application further comprises:

[0057] when the relative expression amount in the biological sample is ≤ a preset positive threshold, the subject corresponding to the biological sample is evaluated as a lung adenocarcinoma patient;

[0058] When the relative expression amount in the biological sample is greater than or equal to the preset negative threshold, the subject corresponding to the biological sample is evaluated as a non-lung adenocarcinoma patient.

[0059] In some embodiments, when the relative expression amount in the biological sample is between the preset positive threshold and the preset negative threshold (i.e., positive threshold < relative expression amount < negative threshold), the subject is recommended to be followed up for further observation and evaluation.

[0060] In some embodiments, the positive threshold can be determined according to the average value of the relative expression amount of lung adenocarcinoma positive samples minus 2 times the standard deviation.

[0061] In some embodiments, the negative threshold can be determined according to the average value of the relative expression amount of lung adenocarcinoma negative samples plus 2 times the standard deviation.

[0062] In some embodiments, the positive threshold can be 5.0156.

[0063] In some embodiments, the negative threshold can be 24.6983.

[0064] Compared with the prior art, the present application has at least the following advantages:

[0065] The experimental results of the present application show that the EGLN3 gene and the LRPPRC gene in different biological samples have a certain degree of expression. Therefore, detecting the expression level (expression amount) of the EGLN3 gene or the LRPPRC gene in the biological sample alone is insufficient to achieve precise detection of lung adenocarcinoma.

[0066] The previous research results of the present application show that the EGLN3 gene and the LRPPRC gene in lung adenocarcinoma cells have a specific mutual regulation effect, which further affects the occurrence and development of lung adenocarcinoma. However, the present application found that the absolute quantitative results of the expression amount of the EGLN3 gene and the LRPPRC gene in the biological sample are directly related to the sample amount (e.g., the amount of RNA extracted from the biological sample added) of the biological sample.

[0067] Based on this, the application creatively uses the PPIB gene as an internal reference gene in the quantitative detection of digital PCR with the LRPPRC gene and the EGLN3 gene as detection target genes. The application finds that using the PPIB gene as an internal reference gene to standardize the data (i.e., the relative expression amount of the LRPPRC gene and the EGLN3 gene set by the application) can monitor whether the nucleic acid extraction and digital PCR amplification process of the biological sample is abnormal. In addition, the application also finds that the expression amount of the PPIB gene in different subject groups (including normal people, lung adenocarcinoma people and other tumor people) is relatively stable, so when the PPIB gene is abnormal, it can prompt that re-detection is needed.

[0068] Further, the application also finds that the relative expression amount of the LRPPRC gene and the EGLN3 gene obtained by using the PPIB gene as an internal reference gene can to some extent avoid the influence of sample amount on the detection result, and can specifically distinguish lung adenocarcinoma patients from other groups (for example, according to the "double threshold" set by the application, i.e., the positive threshold and the negative threshold), thereby achieving auxiliary diagnosis for early diagnosis of lung adenocarcinoma.

[0069] Specifically, when the relative expression amount in the biological sample is ≤ the preset positive threshold, the subject is a lung adenocarcinoma patient (i.e., a more certain lung adenocarcinoma positive); when the relative expression amount in the biological sample is ≥ the preset negative threshold, the subject is a non-lung adenocarcinoma patient (i.e., a more certain lung adenocarcinoma negative); and when the relative expression amount in the biological sample is between the positive threshold and the negative threshold, the subject is recommended to be followed up for further observation and evaluation, which is helpful for timely diagnosis and treatment of lung adenocarcinoma. For such subjects, comprehensive judgment can be made in combination with other clinical symptoms and monitoring can be performed in the form of follow-up. In other words, the "double threshold" set by the application conforms to the progressive evolution characteristics of lung adenocarcinoma, which not only can provide more meaningful results for clinical auxiliary diagnosis, but also is helpful for early discovery and early diagnosis of lung adenocarcinoma. BRIEF DESCRIPTION OF DRAWINGS

[0070] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, hereinafter, a brief introduction will be given to the drawings needed to be used in the embodiments or the prior art description. In all the drawings, similar elements or parts are generally identified by similar reference signs. In the drawings, the elements or parts are not necessarily drawn according to the actual proportions. Obviously, the drawings described below are some embodiments of the application, and those skilled in the art can obtain other drawings according to these drawings without paying creative labor.

[0071] Figure 1Figure 1 is a graph of the quantification results of each gene in lung adenocarcinoma positive samples based on digital PCR (repeat 1);

[0072] Figure 2 Figure 2 is a graph of the quantification results of each gene in lung adenocarcinoma positive samples based on digital PCR (repeat 2);

[0073] Figure 3 Figure 3 is a graph of the quantification results of each gene in lung adenocarcinoma positive samples based on digital PCR (repeat 3). DETAILED DESCRIPTION

[0074] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of the present application.

[0075] As used herein, "and / or" includes any and all combinations of one or more of the associated items.

[0076] As used herein, "plurality" means two or more, i.e., it includes two, three, four, five, etc.

[0077] It should be noted that, in this document, the terms "comprising", "containing", or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or apparatus that includes a list of elements not only includes those elements, but also includes other elements not explicitly listed, or further includes elements inherent in such process, method, article, or apparatus. Without more limitations, the element defined by the statement "comprising a" does not exclude the presence of additional identical elements in the process, method, article, or apparatus that includes the element.

[0078] As used in this specification, the term "about", typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, even more typically + / - 0.5% of the stated value.

[0079] In this specification, certain embodiments can be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and that one of skill in the art would understand that the range format is applicable to each and every specification, example, and embodiment of the present application. For example, a range of "1 to 6" is intended to include individually each number in the range, i.e. 1, 2, 3, 4, 5, and 6. The same applies to ranges of "1 to 3", "5 to 10", and 10 to 100. In addition, it should be understood that all sub-ranges and ranges of sub-ranges within a broader range are intended to be specifically disclosed. For example, a range of "1 to 6" should be specifically understood to include the ranges of "1 to 3", "1 to 4", "1 to 5", "2 to 4", "2 to 6", "3 to 6", and "5 to 6", as well as the individual numbers 1, 2, 3, 4, 5, and 6. The same applies to the ranges of "1 to 3" and "5 to 10".

[0080] Example 1

[0081] Primer and probe design

[0082] Conserved regions of the LRPPRC gene, the EGLN3 gene, and the PPIB gene are selected to design primers and probes. The sequences of the primers and probes involved in the present application are shown in Table 1.

[0083] Table 1

[0084]

[0085] Note: LRPPRC gene-P(643): 5' 6-FAM, 3' BHQ1; EGLN3 gene-P(185): 5' ROX, 3' BHQ2; PPIB gene-P(2741): 5' HEX, 3' BHQ1.

[0086] Example 2

[0087] RNA extraction, nucleic acid content, and digital PCR detection

[0088] 1. RNA extraction

[0089] RNA in the sample is extracted by taking the chloroform-free universal total RNA extraction kit as an example. Other RNA extraction kits can also be used in the present application.

[0090] The sample conditions are as follows: positive samples of lung adenocarcinoma (lung adenocarcinoma tissues from lung adenocarcinoma patients), negative samples of lung adenocarcinoma (other tumor samples and samples from normal people).

[0091] The extraction method is as follows:

[0092] (1) The sample is pretreated according to the instructions, and then repeatedly punched 5 times with a pipette.

[0093] (2) The homogenate sample is placed at room temperature for 5 min at 15-30°C to completely separate the nucleic acid protein complex.

[0094] (3) 12000 rpm centrifuge 5 min, transfer the supernatant to a new centrifuge tube.

[0095] (4) Add an equal volume of absolute ethanol to the lysate, mix well, and transfer the solution and precipitate into the RNA adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and if not transferred completely at one time, transfer it in several times.

[0096] (5) Add 500 μL of deproteinizing solution RE to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube.

[0097] (6) Add 500 μL of rinse solution RW to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube.

[0098] (7) Optional step: Repeat step (6) once.

[0099] (8) Place the RNA adsorption column back into the empty collection tube, centrifuge at 12000 rpm for 2 min, and dry the residual liquid.

[0100] (9) Place the centrifuged adsorption column in a new 1.5 ml plastic centrifuge tube, add 50 ~ 100 μL of RNase-Free ddH2O elution, and place it at room temperature for 2 min. 12,000 rpm centrifuge for 1 min, and the centrifuge tube solution is RNA. The collected RNA solution should be stored at -70°C to prevent degradation.

[0101] 2. RNA concentration determination (ultraviolet spectrophotometry method)

[0102] (1) Material preparation

[0103] Reagent: DEPC water or RNase Free dH2O

[0104] Article: Enzyme-free gun head

[0105] Instrument: Nanoprop ultraviolet spectrophotometer

[0106] (2) Operation process

[0107] a. Open the Nanoprop 2000 software

[0108] b. Select Nucieic Acid ↓

[0109] c. Select RNA, add 2 μL of DEPC water or RNase Free dH2O, and wash the sample hole twice

[0110] d. Add 2 μL of DEPC water or RNase-free dH2O and click “Blank”

[0111] e. Change "Type" to "RNA", add 1 μL of sample, and measure the concentration (Measure)

[0112] f. The Nanoprop 2000 software displays a final concentration of XX ng / µL. Based on the sample volume, the nucleic acid amount can be calculated as XX µg.

[0113] If the RNA extraction kit is operated correctly according to the instructions, the RNA purity can meet the detection standards. The specific requirements are: 1.8 <A260 / A280<2.2,A260 / A230比值高于1.8。如果纯度不满足要求,需要重新提取。

[0114] 3. Digital PCR Detection

[0115] Using the AccuONE digital PCR instrument (GE041-B / AccuONE-L100 / AccuONE-R200) ​​and its matching reaction system and detection chip, the digital PCR amplification system is as follows:

[0116]

[0117] Note: The final concentration of primers for all genes is 333 nM, and the final concentrations of probes are: LRPPRC gene: 100 nM, PPIB gene: 67 nM, EGLN3 gene: 167 nM.

[0118] Digital PCR amplification procedure:

[0119]

[0120] Example 3

[0121] Detection of EGLN3 gene expression in lung adenocarcinoma tissue samples using digital PCR

[0122] First, in a preliminary experiment, different downstream primers (i.e., EGLN3 gene-R(263) and EGLN3 gene-R(231)) were used to amplify the EGLN3 gene. The experimental results showed that the amplification efficiency was low when EGLN3 gene-R(263) was used as the downstream primer to amplify the EGLN3 gene. Therefore, in this example, a combination of EGLN3 gene-F(164), EGLN3 gene-P(185), and EGLN3 gene-R(231) (combination 1) was used to amplify the EGLN3 gene.

[0123] The results are shown in Table 2. Under the premise of using lung adenocarcinoma tissues of the same source as samples, the present application finds that, when the expression amount of the EGLN3 gene in the lung adenocarcinoma tissue samples is detected by using combination 1, the expression amount of the EGLN3 gene is easily affected by factors such as sample amount, the expression amount of the EGLN3 gene in lung adenocarcinoma tissues of different sample amounts fluctuates greatly, and does not have a good linear relationship with the sample amount. This specifically embodies that although the intra-group repeatability of the 0.5 μg group, the 1.0 μg group and the 2.0 μg group is good, and the results are reliable, however, when the sample amount is increased from 0.5 μg to 1.0 μg (i.e. the sample amount is increased by 2 times), the copy number is increased by about 1.49 times, which is lower than the expected 2 times; when the sample amount is increased from 0.5 μg to 2.0 μg (i.e. the sample amount is increased by 4 times), the copy number is increased by about 5.84 times, which is higher than the expected 4 times; when the sample amount is increased from 1.0 μg to 2.0 μg (i.e. the sample amount is increased by 2 times), the copy number is increased by about 3.92 times, which is higher than the expected 2 times. That is to say, the detected copy number of the EGLN3 gene does not have a good linear proportional relationship with the amount of RNA input.

[0124] Table 2

[0125]

[0126] In other words, although the prior art discloses that the EGLN3 gene can be used as a biomarker for detecting lung adenocarcinoma, the expression level of the EGLN3 gene is greatly affected by the sample amount, therefore, only based on the expression level of the EGLN3 gene is insufficient for accurately diagnosing lung adenocarcinoma. On this basis, the present application proposes that the detection of the expression amount of the EGLN3 gene needs to control the amount of nucleic acid before the experiment so as to control the amount of the EGLN3 gene within the range of the quantification limit, and then the accuracy of the detection result is realized.

[0127] Example Four

[0128] Detection of the expression amount of the LRPPRC gene in lung adenocarcinoma tissue samples by using digital PCR

[0129] Firstly, in the pre-experiment, different downstream primers (i.e. LRPPRC gene-R(769), LRPPRC gene-R(720), LRPPRC gene-F(617)) are used to amplify the LRPPRC gene. The experimental results show that, when the LRPPRC gene-R(769) and the LRPPRC gene-R(720) are used as the downstream primers to amplify the LRPPRC gene, the amplification efficiency is low, and it is difficult to be applied to the absolute quantification detection of digital PCR. Therefore, in the present embodiment, the combination (combination 2) of the LRPPRC gene-F(617), the LRPPRC gene-P(643) and the LRPPRC gene-R(692) is used to amplify the LRPPRC gene.

[0130] As shown in Table 3, under the premise of using the same lung adenocarcinoma tissue in the same state as the sample, the present application found that, when detecting the expression amount of the LRPPRC gene in lung adenocarcinoma tissue by using combination 2, the absolute quantification result of digital PCR showed that the added amount was the main influencing factor of the quantitative result, and the added amount was extremely susceptible to the accuracy of the range of the pipette and the influence of the experimental operator, so it was difficult to use the absolute expression amount of the LRPPRC gene single gene as a diagnostic index.

[0131] Table 3

[0132]

[0133] In other words, although the prior art shows that the LRPPRC gene can be used as a therapeutic target for lung adenocarcinoma, the expression level of the LRPPRC gene is greatly affected by the sample amount. Therefore, the expression level of the LRPPRC gene alone is not sufficient for accurate diagnosis of lung adenocarcinoma.

[0134] Similarly to the case of the EGLN3 gene, the present application found that the nucleic acid amount of the LRPPRC gene also needs to be controlled within the quantitative limit range to ensure the accuracy of the detection result.

[0135] Example Five

[0136] Based on Examples Three and Four, the present application found that the PPIB gene (Peptidylprolyl Isomerase B) encodes cyclophilin B, and the expression amount in different sample types of the human body is relatively stable, and is suitable for being used as an internal standard gene. Therefore, this example uses the PPIB gene as an internal standard gene to standardize the LRPPRC gene and the EGLN3 gene, thereby to a certain extent, avoiding the influence caused by different added amounts due to different samples when detecting all genes.

[0137] The expression amounts of the LRPPRC gene, the EGLN3 gene, and the PPIB gene were detected simultaneously using samples that had been definitely positive for lung adenocarcinoma and negative for lung adenocarcinoma, and the digital PCR reaction system of the above-mentioned examples (using combination 1, combination 2, and combination 3) was used. The specific results are shown below.

[0138] First, the present application uses different downstream primers (i.e. PPIB gene-R(2801), PPIB gene-R(2760)) to amplify the PPIB gene in the pre-experiment. The experimental results show that when PPIB gene-R(2760) is used as a downstream primer to amplify the PPIB gene, the amplification efficiency is low, and it is difficult to apply to the absolute quantitative detection of digital PCR. Therefore, the present embodiment uses the combination of PPIB gene-F(2695), PPIB gene-P(2741) and PPIB gene-R(2801) (combination 3) to amplify the PPIB gene.

[0139] The present application creatively uses the ratio of LRPPRC gene / PPIB gene and EGLN3 gene / PPIB gene (i.e. (LRPPRC gene / PPIB gene) / (EGLN3 gene / PPIB gene)) to analyze the above results. The results are shown in Tables 4 and 5, and it is appropriate to keep the sample amount between 0.5 μg-1.0 μg, preferably 0.5 μg.

[0140] Table 4 LRPPRC gene / PPIB gene results

[0141]

[0142] The results of Table 5 further confirm that it is appropriate to keep the sample amount between 0.5 μg-1.0 μg, preferably 0.5 μg.

[0143] Table 5 EGLN3 / PPIB gene results

[0144]

[0145] In particular for the application scenario of physical examination, the healthy population accounts for a large proportion. Insufficient specificity can easily lead to false positives and cause psychological burden to the subjects. In order to determine the threshold of lung adenocarcinoma positive and lung adenocarcinoma negative, and to a certain extent, avoid the problem of insufficient specificity in tumor biomarker detection, the present embodiment attempts to adjust the threshold as follows: positive threshold ≤ mean-2SD, the purpose is to provide clear positive auxiliary diagnosis results for the clinic; negative threshold ≥ mean+2SD, the purpose is to provide clear negative auxiliary diagnosis results for the clinic. The gray area between the positive threshold and the negative threshold is recommended to be comprehensively judged in combination with other clinical symptoms, and regular follow-up is recommended.

[0146] As shown in Table 6, the present embodiment finds that when the above results are analyzed using the ratio of LRPPRC gene / PPIB gene to EGLN3 gene / PPIB gene, the positive threshold of LRPPRC gene / PPIB gene to EGLN3 gene / PPIB gene of lung adenocarcinoma samples is determined to be 5.0156. That is, using the ratio of (LRPPRC gene / PPIB gene) to (EGLN3 gene / PPIB gene) (i.e., (LRPPRC gene / PPIB gene) / (EGLN3 gene / PPIB gene)) to analyze the quantitative results of each gene in lung adenocarcinoma samples based on digital PCR (Table 7, Figure 1 , Figure 2 and Figure 3 ), can to some extent avoid the problem of inaccurate quantitative results caused by sample size changes.

[0147] Table 6 Results of the ratio of (LRPPRC gene / PPIB gene) to (EGLN3 gene / PPIB gene)

[0148]

[0149] Table 7

[0150]

[0151] Note: In this table, 2 μL, 4 μL and 8 μL represent the addition amount of RNA samples diluted by 4000 times with RNA concentration of 624 ng / μL, which correspond to 0.5 μg, 1 μg, 2 μg of RNA sample amount (i.e., the copy number corresponding to 0.5 μg, 1 μg, 2 μg sample amount is calculated based on the copy number corresponding to 2 μL, 4 μL and 8 μL). The results of Tables 2-6 are calculated based on this table.

[0152] The present embodiment also tests lung adenocarcinoma negative samples with a sample size of 1.0 μg, and calculates the results of (LRPPRC gene / PPIB gene) / (EGLN3 gene / PPIB gene), as shown in Tables 8-1, 8-2 and 8-3.

[0153] Table 8-1 LRPPRC gene / PPIB gene results

[0154]

[0155] Table 8-2 EGLN3 gene / PPIB gene results

[0156]

[0157] Table 8-3 Results of the ratio of (LRPPRC gene / PPIB gene) / (EGLN3 gene / PPIB gene)

[0158]

[0159] The above results show that the negative threshold value determined based on the results of (LRPPRC gene / PPIB gene) / (EGLN3 gene / PPIB gene) in lung adenocarcinoma negative samples is 24.6983, which is significantly different from lung adenocarcinoma positive samples. This can be due to the research results of the inventors of the present application that both the EGLN3 gene and the LRPPRC gene have specific mutual regulation in lung adenocarcinoma cells, thereby affecting the occurrence and development of lung adenocarcinoma. It should be noted that the reason why the above negative threshold value is higher than the positive threshold value can be that, compared with lung adenocarcinoma positive samples, the EGLN3 gene and the LRPPRC gene can not have specific mutual regulation in lung adenocarcinoma negative samples, and the expression amount of the EGLN3 gene is lower than that of the LRPPRC gene.

[0160] Therefore, the kit provided by the present application finally determines 5.0156 as the positive threshold value and 24.6983 as the negative threshold value to specifically detect lung adenocarcinoma through the above results. Specifically, when the detection result of (LRPPRC gene / PPIB gene) / (EGLN3 gene / PPIB gene) in a biological sample is less than or equal to the positive threshold value, the corresponding subject is evaluated as lung adenocarcinoma positive; when the detection result of (LRPPRC gene / PPIB gene) / (EGLN3 gene / PPIB gene) in a biological sample is greater than or equal to the negative threshold value, the corresponding subject is evaluated as lung adenocarcinoma negative; and when the detection result of (LRPPRC gene / PPIB gene) / (EGLN3 gene / PPIB gene) in a biological sample is between the negative threshold value and the positive threshold value, the corresponding subject is suggested to be further examined and / or prevented for further observation and evaluation.

[0161] The embodiments of the present application are described above in combination with the drawings, but the present application is not limited to the above specific embodiments, and the above specific embodiments are only illustrative and not restrictive. Those skilled in the art can make many forms under the inspiration of the present application without departing from the purpose of the present application and the scope protected by the claims, and these all belong to the protection of the present application.

Claims

1. A lung adenocarcinoma detection kit, characterized in that: The kit includes a detection reagent for detecting the relative expression levels of the LRPPRC gene and the EGLN3 gene in a biological sample, wherein the relative expression level is a ratio of a first expression level to a second expression level, the first expression level is a ratio of the LRPPRC gene expression level to the PPIB gene expression level, and the second expression level is a ratio of the EGLN3 gene expression level to the PPIB gene expression level, and the detection reagent includes: The upstream primer for detecting the LRPPRC gene has a nucleotide sequence as shown in SEQ ID NO: 1; the downstream primer for detecting the LRPPRC gene has a nucleotide sequence as shown in SEQ ID NO: 3; and the probe for detecting the LRPPRC gene has a nucleotide sequence as shown in SEQ ID NO:

2. The upstream primer for detecting the EGLN3 gene has a nucleotide sequence shown in SEQ ID NO:4; the downstream primer for detecting the EGLN3 gene has a nucleotide sequence shown in SEQ ID NO:6; and the probe for detecting the EGLN3 gene has a nucleotide sequence shown in SEQ ID NO:

5. The upstream primer for detecting the PPIB gene has a nucleotide sequence as shown in SEQ ID NO:7; the downstream primer for detecting the PPIB gene has a nucleotide sequence as shown in SEQ ID NO:9; and the probe for detecting the PPIB gene has a nucleotide sequence as shown in SEQ ID NO:

8. The lung adenocarcinoma detection kit is configured to detect RNA extracted from the biological sample; the lung adenocarcinoma detection kit is configured to perform detection based on digital PCR; the mass of the RNA ranges from 0.5 to 1 μg.

2. The kit according to claim 1, wherein The biological sample is derived from lung tissue or blood of the subject.

3. The kit according to claim 1, wherein The biological sample includes lung adenocarcinoma cells and / or lung adenocarcinoma tissue.

4. The kit according to claim 1, wherein The digital PCR amplification program includes: 53°C for 25 min; 95°C for 5 min; 95°C for 15 s, 58°C for 25 s, for 35 cycles.

5. Use of a detection reagent in preparing a lung adenocarcinoma detection kit, characterized in that: The detection reagent is used to detect the relative expression levels of the LRPPRC gene and the EGLN3 gene in a biological sample, wherein the relative expression level is a ratio of a first expression level to a second expression level, the first expression level is a ratio of the LRPPRC gene expression level to the PPIB gene expression level, and the second expression level is a ratio of the EGLN3 gene expression level to the PPIB gene expression level, and the detection reagent comprises: The upstream primer for detecting the LRPPRC gene has a nucleotide sequence as shown in SEQ ID NO: 1; the downstream primer for detecting the LRPPRC gene has a nucleotide sequence as shown in SEQ ID NO: 3; and the probe for detecting the LRPPRC gene has a nucleotide sequence as shown in SEQ ID NO:

2. The upstream primer for detecting the EGLN3 gene has a nucleotide sequence shown in SEQ ID NO:4; the downstream primer for detecting the EGLN3 gene has a nucleotide sequence shown in SEQ ID NO:6; and the probe for detecting the EGLN3 gene has a nucleotide sequence shown in SEQ ID NO:

5. The upstream primer for detecting the PPIB gene has a nucleotide sequence as shown in SEQ ID NO:7; the downstream primer for detecting the PPIB gene has a nucleotide sequence as shown in SEQ ID NO:9; the probe for detecting the PPIB gene has a nucleotide sequence as shown in SEQ ID NO:8; the lung adenocarcinoma detection kit is used to detect RNA extracted from the biological sample; the lung adenocarcinoma detection kit is used for detection based on digital PCR; the mass of the RNA ranges from 0.5 to 1 μg; the application includes: when the relative expression amount in the biological sample is ≤ a preset positive threshold, the subject corresponding to the biological sample is evaluated as a lung adenocarcinoma patient; when the relative expression amount in the biological sample is ≥ a preset negative threshold, the subject is evaluated as a non-lung adenocarcinoma patient; the positive threshold is determined based on the average value of the relative expression amount of the lung adenocarcinoma positive sample minus 2 times the standard deviation; the negative threshold is determined based on the average value of the relative expression amount of the lung adenocarcinoma negative sample plus 2 times the standard deviation.

6. The use according to claim 5, characterized in that When the relative expression level in the biological sample is between the preset positive threshold and the preset negative threshold, the subject is recommended to follow up for further observation and evaluation.

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