IncRNA diagnosis marker combination and application thereof

Abstract

The invention discloses an lncRNA (long non-coding ribonucleic acid) diagnostic marker combination and application thereof, the lncRNA diagnostic marker combination comprises a specific primer, a TaqMan probe and a standard positive template, and is characterized in that the lncRNA diagnostic marker comprises Linc-PIK3C2G-2, Linc-ABCA5-3 or Linc-MRPL33-1, and the specific primer, the TaqMan probe and the standard positive template are used for detecting the lncRNA diagnostic marker combination. The invention also discloses the application of the lncRNA diagnosis marker combination in lung cancer diagnosis molecular markers. The circulating linc-MRPL33-1, the linc-PIK3C2G-2 and the linc-ABCA5-3 are highly expressed in plasma of a patient with the lung cancer, the linc-PIK3C2G-2 has the highest sensitivity in diagnosis of the lung cancer, the linc-ABCA5-3 has the highest specificity in diagnosis of the lung cancer, and the value of combined diagnosis of the lung cancer by the three lncRNAs is superior to that of single diagnosis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a lncRNA diagnostic marker combination and application thereof. Background Art

[0002] According to the data registered by the National Cancer Center, lung cancer is one of the important causes of cancer death. It is the malignant tumor with the highest incidence and mortality rate in China. It is also the most common cancer among Chinese men. It is prevalent in all regions and poses a huge threat to human life. There are two types of lung cancer: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC is further divided into squamous cell carcinoma (LSCC), large cell carcinoma and adenocarcinoma (LAD). NSCLC accounts for more than 85% of the total. Most patients are diagnosed with advanced lung cancer, with a 5-year survival rate of only 10-15%. Therefore, identifying new targets may guide future lung cancer treatment.

[0003] At present, bronchoscopy combined with pathological examination is the gold standard for diagnosing lung cancer, but the sampling process is somewhat traumatic and patient compliance is poor. In addition, due to problems such as the size of the sampling specimen, the non-specific sampling location, and tumor heterogeneity, a single test may not be able to diagnose, and it is even more difficult to perform dynamic monitoring by taking multiple samples. Although imaging examinations have improved the detection rate of lung cancer, they still have disadvantages such as radiation damage and difficulty in positioning. The most widely used tumor markers in clinical practice, such as carcinoembryonic antigen, have low diagnostic efficacy. Therefore, the development of minimally invasive or even non-invasive markers with high specificity and sensitivity will provide new insights into the early diagnosis and treatment of lung cancer, so as to improve and supplement the current early screening methods for lung cancer.

[0004] The mammalian genome expresses two main types of genes through RNA polymerase II-mediated transcription: protein-coding transcription units and non-coding RNA transcription units. Non-coding RNA is further divided into relatively abundant structural RNA, such as small RNA, and long non-coding RNA (lncRNA). LncRNA is longer than 200 nt and is RNA with no protein coding function or limited coding function. It is worth noting that the transcription, processing and metabolism of lncRNA are different from the regulation of protein-coding genes. LncRNA has been tried for the diagnosis, prognosis and targeted treatment of some diseases. Studies have found that a large number of lncRNAs are abnormally expressed in lung cancer compared with adjacent tissues. The upregulation of AQP3 expression in lung cancer is associated with the proliferation, invasion and metastasis of NSCLC; the abnormal expression of SOX4 protein is associated with the proliferation, migration and invasion and metastasis of lung cancer, and indicates a poor prognosis; high expression of CDK2 protein and low expression of p27kip1 protein are associated with the malignant progression of lung cancer. LncRNAs such as BANCR, GHRLOS, PANDAR, MEG3, GAS6-AS1, etc. are downregulated in lung cancer, while CCAT2, FOXD2-AS1, MALAT1, HOTAIR, UCC, etc. are upregulated. These lncRNAs can regulate the occurrence and development of lung cancer by promoting or inhibiting the biological behaviors of lung cancer cell proliferation, migration, apoptosis, etc. In addition, studies have found multiple circulating lncRNA markers with potential application value from various tumor types. For example, PAC3, a urine marker for early diagnosis and prognosis evaluation of prostate malignancies. HULC is highly upregulated in human breast cancer and hepatocellular carcinoma, and HULC is used for the diagnosis of hepatocellular carcinoma. Xie et al. found that low expression of SOX2OT and ANRI was associated with a higher survival rate, and SOX2OT could be used as an independent prognostic factor. After combining the two lncRNAs SOX2OT and ANRIL with three traditional tumor markers CEA, CYFRA21-1 and SCCA, it was found that the diagnostic value of this model was better than any of the biomarkers alone.

[0005] Currently, there is a lack of a diagnostic marker combination and its application. Summary of the invention

[0006] In view of the problems of the prior art, the purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a lncRNA diagnostic marker combination and application thereof.

[0007] To achieve the above object, the present invention provides the following technical solution: a lncRNA diagnostic marker combination of the present invention, wherein the lncRNA diagnostic marker combination includes one or a combination of several of Linc-PIK3C2G-2, Linc-ABCA5-3 or Linc-MRPL33-1.

[0008] Furthermore, the lncRNA diagnostic marker combination includes specific primers, TaqMan probes and standard positive templates.

[0009] The nucleotide sequence of the upstream primer of the Linc-PIK3C2G-2 is shown in SEQ ID No.1, the nucleotide sequence of the downstream primer of the Linc-PIK3C2G-2 is shown in SEQ ID No.2, and the nucleotide sequence of the Probe probe of the Linc-PIK3C2G-2 is the nucleotide sequence shown in SEQ ID No.3;

[0010] Or the nucleotide sequence of the upstream primer of Linc-ABCA5-3 is shown in SEQ ID No.4, the nucleotide sequence of the downstream primer of Linc-ABCA5-3 is shown in SEQ ID No.5, and the nucleotide sequence of the Probe probe of Linc-ABCA5-3 is the nucleotide sequence shown in SEQ ID No.6;

[0011] Or the nucleotide sequence of the upstream primer of the Linc-MRPL33-1 is shown in SEQ ID No.7, the nucleotide sequence of the downstream primer of the Linc-MRPL33-1 is shown in SEQ ID No.8, and the nucleotide sequence of the Probe probe of the Linc-MRPL33-1 is the nucleotide sequence shown in SEQ ID No.9.

[0012] The present invention provides a kit containing the lncRNA diagnostic marker combination, wherein the kit includes reagents used to detect the expression level of the marker combination.

[0013] Furthermore, the kit includes: PCR primers, TaqMan probes, PCR buffer, hot start enzyme, dNTP, standard positive template, and reference substance; the reference substance is ultrapure water.

[0014] Furthermore, the reagent for detecting the expression amount includes a reagent for detecting the protein expression amount and / or the mRNA expression amount.

[0015] Application of the lncRNA diagnostic marker combination of the present invention in lung cancer diagnostic molecular markers.

[0016] Beneficial effects: Application of the lncRNA diagnostic marker combination of the present invention in lung cancer diagnostic molecular markers. Circulating linc-MRPL33-1, linc-PIK3C2G-2, and linc-ABCA5-3 of the present invention are highly expressed in the plasma of lung cancer patients, linc-PIK3C2G-2 has the highest sensitivity in diagnosing lung cancer, and linc-ABCA5-3 has the highest specificity in diagnosing lung cancer. The value of the combined diagnosis of lung cancer by the three lncRNAs is better than that of a single diagnosis.

[0017] Compared with the prior art, the present invention has the following advantages: (1) The present invention prepares a triple armor RNA containing linc-MRPL33-1, linc-PIK3C2G-2 and linc-ABCA5-3 as a stable standard and quality control product.

[0018] (2) A one-step multiplex RT-qPCR TaqMan probe absolute quantitative detection method combining three target lncRNAs and internal reference was established. The analytical performance of the multiplex RT-qPCR TaqMan probe absolute quantitative detection method for linc-MRPL33-1, linc-PIK3C2G-2 and linc-ABCA5-3 was good. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the data distribution diagram of the high concentration standard of the present invention; Note: Mean (X): 7.80; Standard deviation (S): 0.18; Coefficient of variation (CV): 2.4%

[0020] Figure 2 This is the data distribution diagram of the low concentration standard of the present invention; Note: Mean (-X): 3.67; Standard deviation (S): 0.15; Coefficient of variation (CV): 4.2%.

[0021] Figure 3 The amplification curve and standard curve of the target lncRNA contained in the MS2VLPs of the present invention; Note: linc-MRPL33-1 (red), linc-PIK3C2G-2 (green), linc-ABCA5-3 (blue).

[0022] Figure 4 The dot graphs are the expression levels of the three target lncRNAs in the three groups of plasma samples of the present invention; (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0023] Figure 5 It is the ROC curve diagram of independent detection and combined detection of three lncRNAs of the present invention. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Example 1

[0026] In a first aspect, an embodiment of the present application provides a lncRNA diagnostic marker combination, wherein the lncRNA diagnostic marker combination includes one or a combination of several of Linc-PIK3C2G-2, Linc-ABCA5-3 or Linc-MRPL33-1.

[0027] The lncRNA diagnostic marker combination includes specific primers, TaqMan probes and standard positive templates.

[0028] The nucleotide sequence of the upstream primer of the Linc-PIK3C2G-2 is shown in SEQ ID No.1, the nucleotide sequence of the downstream primer of the Linc-PIK3C2G-2 is shown in SEQ ID No.2, and the nucleotide sequence of the Probe probe of the Linc-PIK3C2G-2 is the nucleotide sequence shown in SEQ ID No.3;

[0029] Or the nucleotide sequence of the upstream primer of Linc-ABCA5-3 is shown in SEQ ID No.4, the nucleotide sequence of the downstream primer of Linc-ABCA5-3 is shown in SEQ ID No.5, and the nucleotide sequence of the Probe probe of Linc-ABCA5-3 is the nucleotide sequence shown in SEQ ID No.6;

[0030] Or the nucleotide sequence of the upstream primer of Linc-MRPL33-1 is shown in SEQ ID No.7, and the nucleotide sequence of the downstream primer of Linc-MRPL33-1 is shown in SEQ ID No.8,

[0031] The nucleotide sequence of the Probe probe of Linc-MRPL33-1 is the nucleotide sequence shown in SEQ ID No.9.

[0032] A second aspect of an embodiment of the present application provides a kit containing the lncRNA diagnostic marker combination, wherein the kit includes reagents used to detect the expression level of the marker combination.

[0033] The kit comprises: PCR primers, TaqMan probes, PCR buffer, hot start enzyme, dNTP, standard positive template, and reference substance; the reference substance is ultrapure water.

[0034] Reagents for detecting expression levels include reagents for detecting protein expression levels and / or mRNA expression levels.

[0035] Application of the lncRNA diagnostic marker combination of the present invention in lung cancer diagnostic molecular markers.

[0036] Test Example 1

[0037] Preparation of MS2 bacteriophage virus-like particle standards containing linc-MRPL33-1, linc-PIK3C2G-2 and linc-ABCA5-3 target amplification sequences

[0038] In this chapter, based on the armor RNA technology, BL21 (DE3) expression Escherichia coli was infected with MS2 phage, the MS2 phage genomic RNA was extracted, and the capsid protein and mature enzyme protein coding sequences were reverse transcribed and inserted into the MCS1 site of the pACYCDuet-1 prokaryotic expression vector to construct the pACYC-MS2 recombinant plasmid; total RNA from lung cancer tissue specimens was extracted and reverse transcribed to obtain cDNA fragments containing candidate lncRNA target amplification sequences, which were inserted into the second multiple cloning site MCS2 of pACYC-MS2 in a tandem manner, and finally a recombinant plasmid was constructed with MCS1 containing MS2 mature enzyme protein and capsid protein coding sequences and MCS2 containing MS2 packaging site sequence + 3 tandem candidate lncRNA target amplification sequences, named pACYC-MS2-lncRNAs, and sequencing was used to verify the accuracy of the pACYC-MS2-lncRNAs recombinant plasmid.

[0039] pACYC-MS2-lncRNAs were transformed into BL21(DE3) Escherichia coli and induced for expression, PEG enrichment, molecular sieve chromatography purification of MS2VLPs, and verification and performance evaluation were performed.

[0040] Materials and Methods

[0041] 1 Experimental Materials

[0042] 1.1 Plasmids and strains

[0043] The expression host Escherichia coli BL21 (DE3) and the prokaryotic expression plasmid pACYCDuet-1 were both from General Biosystems (Anhui) Co., Ltd.

[0044] 1.2 The main experimental instruments are shown in Table 1:

[0045] Table 1

[0046]

[0047] 1.3 Main experimental consumables

[0049] Table 2

[0050]

[0051] 1.4 Main experimental reagents

[0052] Table 3

[0053]

[0054]

[0055] 1.5 Reagent preparation

[0056] (1) Luria-Bertani (LB) broth medium: 25 g LB powder, dilute to 1000 ml with deionized water, and sterilize with high pressure steam at 121°C for 20 min.

[0057] (2) LB liquid medium containing chloramphenicol: Take 250 ml of LB liquid medium with a temperature below 60°C, add 170 μl of 50 mg / ml chloramphenicol solution to a final concentration of 34 μg / ml, mix well, and store at 4°C.

[0058] (3) 1.5% agar LB solid medium containing 34 μg / ml chloramphenicol: 3.75 g agar powder was diluted to 250 ml with LB medium. The solution was sterilized by high pressure sterilization at 121°C for 20 min. The temperature was lowered to about 60°C. 170 μl of 50 mg / ml chloramphenicol solution was added. After mixing, the solution was quickly poured into a 90 mm culture dish. After solidification, the solution was packaged and sealed at 4°C for later use.

[0059] (4) SDS loading buffer (2×): 0.01 g bromophenol blue, 0.2 g SDS, 1.25 ml 0.5 M Tris-HCl (pH 6.8), 0.2 ml β-mercaptoethanol, 1 ml glycerol, and dilute to 5 ml with deionized water. Aliquot and store at -20°C.

[0060] (5) 5× Tris-glycine buffer: 15.1 g Tris base, 94 g glycine, 5 g SDS powder, add deionized water to make up to 1 L, mix well, and store at room temperature.

[0061] (6) Agarose gel (2%): Weigh 1 g of agarose and dissolve it in 50 ml of 1×TAE. Heat in a microwave oven to completely melt it. When the temperature drops to about 60°C, add 2.5 μl of nucleic acid dye and mix thoroughly. Pour the mixture into a mold and wait for about 25 min. After the gel solidifies, pull up the comb vertically and place it in an electrophoresis apparatus for subsequent operations.

[0062] 2 Experimental methods

[0063] 2.1 Primer and probe design of target LncRNA

[0064] The UCSC database was used to search for three lncRNA sequences, and primers and probes were designed to avoid the sequence overlap with mRNA. The lncRNA sequences were entered into the Shanghai Bioengineering website, and Bioengineering (Shanghai) Co., Ltd. was commissioned to synthesize the three lncRNA primer sequences, synthesize the probe sequences, and label with fluorescent groups. The three lncRNA primer and probe sequences are shown in Table 4.

[0065] Table 4

[0066]

[0067] Test Example 2

[0068] Multiplex RT-qPCR validation of the expression of three target lncRNAs in lung cancer plasma

[0069] After confirming the analytical performance of the detection method, multiple RT-qPCR was used to detect lncRNA and internal reference in the same tube, and MS2VLPs were used as standard and quality control products. The copy numbers of the three lncRNAs in the plasma of 141 lung cancer patients, 137 benign lung disease patients and 145 apparently healthy people were calculated by drawing standard curves, and ROC curves were drawn to evaluate the diagnostic efficacy of the three lncRNAs detected alone and in combination. Multivariate logistic regression was used to construct a clinical prediction model.

[0070] Statistical methods

[0071] GraphPad Prism 9.3.0 software was used for statistical analysis and dot plots of differentially expressed lncRNAs were drawn. Figure 4 And ROC curve Figure 5 , the potential clinical application value of the target lncRNA as a diagnostic marker for NSCLC was evaluated, and P < 0.05 was considered statistically significant (* represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and **** represents P < 0.0001).

[0072] Test Example 2

[0073] Methodological evaluation of multiplex RT-qPCR TaqMan probe assay

[0074] Precision performance evaluation

[0075] Using -X±2SD as the warning limit and -X±3SD as the out-of-control limit, quality control was performed on 80 test data of two concentrations. Figure 1 and Figure 2 , 80 data of samples with two concentrations are valid. Figure 1 Data distribution diagram of high concentration standard; Figure 2 Data distribution of low concentration standard; Note: Mean (-X): 3.67; Standard deviation (S): 0.15; Coefficient of variation (CV): 4.2%

[0076] Precision evaluation experimental data

[0077] The data are expressed as mean ± standard deviation, see Table 5. Precision acceptability pre-experimental data statistics, see Table 6. Statistics of acceptable data target values ​​and standard deviations every 5 days, see Table 7. The precision confirmation results of high-value MS2VLPs and low-value MS2VLPs standards are shown in Tables 3-5. The intra-batch CVs of the standards at the two concentration levels were 2.2% and 2.4%, respectively; the inter-batch CVs were 0.93% and 1.30%, respectively; the inter-day precision CV of the high-value MS2VLPs standard was 0, indicating that the inter-day variation was almost entirely caused by intra-batch variation, so the value was taken as 0; the total indoor CVs were 2.4% and 4.2%, respectively. The precision experiment result data (-X ± S) are shown in Table 5:

[0078] Table 5

[0079]

[0080] The statistics of the precision acceptability pre-experimental data are shown in Table 6:

[0081] Table 6

[0082]

[0083] The precision data of high and low level quality control products are shown in Table 8:

[0084]

[0085] Linear range

[0086] The standard curves of Linc-MRPL33-1, 9 and Linc-ABCA5-3 are y=-3.3254x+47.067, y=-3.3243x+46.685, y=-3.366+46.525, R 2The linear ranges of linc-MRPL33-1, linc-PIK3C2G-2 and linc-ABCA5-3 in the detected MS2 VLPs were 8.73×10 8 -8.73×10 2 copies / ml, 7.73×10 8 -7.73×10 2 copies / ml, 9.78×10 8 -9.78×10 2 copies / ml. Figure 3 The amplification curve and standard curve of the target lncRNA contained in the MS2VLPs of the present invention; Note: linc-MRPL33-1 (red), linc-PIK3C2G-2 (green), linc-ABCA5-3 (blue).

[0087] Limit of Detection and Limit of Quantitation

[0088] The total number of tests for each concentration and the number of times that can be detected are shown in Table 9. When the copy number of the sample is 750 copies / ml, the number of times that can be detected in 20 tests is 18 times, which exceeds 90% of the total number of tests, so the detection limit is 750 copies / ml. In addition, when the copy number of the sample is 800 copies / ml, the Log initial copy number bias between the measured value and the target value of 20 tests is less than 0.4, so the quantitative limit of this method is 800 copies / ml. Table 9 Probability analysis for determining the detection limit and quantitative limit MS2VLPs (copies / ml)

[0089] Table 9

[0090]

[0091] Verify the expression of three target lncRNAs in the plasma of lung cancer patients

[0092] After the performance of the detection system was confirmed, the differential expression of linc-MRPL33-1, linc-PIK3C2G-2 and lnc-ABCA5-3 in the plasma of 141 lung cancer patients, 137 benign lung disease patients and 145 apparently healthy subjects was detected by constructing a one-step absolute quantitative multiplex PCR amplification system. The experimental data were statistically analyzed using GraphPadPrism9.0.0 software and a dot plot of differentially expressed lncRNA was drawn. Figure 4 . Figure 4The expression levels of the three target lncRNAs in the three groups of plasma samples of the present invention (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0093] The absolute quantitative results of one-step multiplex real-time fluorescence quantitative PCR in plasma showed that the expression levels of the three target genes linc-MRPL33-1, linc-PIK3C2G-2 and linc-ABCA5-3 were statistically different between the lung cancer group and the benign lung disease group (P<0.05), the lung cancer group and the apparently healthy population group (P<0.0001), and the benign lung disease group and the apparently healthy population healthy group (P<0.001). They were highly expressed, which was consistent with the trend of the previous tissue verification results and chip detection results of the research team.

[0094] Preliminary application of three target lncRNAs as diagnostic markers for lung cancer

[0095] The ROC curve was used to draw the ROC curves of independent detection and combined detection of the expression data of the three lncRNAs in the plasma of the lung cancer group and the control group (including the benign lung disease group and the healthy group) to observe the diagnostic efficacy of the model. Figure 5 and Table 10. The analysis showed that compared with the control group, the three target genes were upregulated in the lung cancer group, and the differences were statistically significant (P<0.0001). The three lncRNAs have the potential to distinguish patients with non-small cell lung cancer from patients with benign lung diseases and healthy people.

[0096] The AUC of linc-MRPL33-1 was 0.638 (95% CI: 0.584-0.692), with a sensitivity of 54.6% and a specificity of 68.8%; the AUC of linc-PIK3C2G-2 was 0.631 (95% CI: 0.577-0.685), with a sensitivity of 90.1% and a specificity of 30.5%; the AUC of linc-ABCA5-3 was 0.622 (95% CI: 0.567-0.678), with a sensitivity of 46.8% and a specificity of 70.9%. In addition, the AUC of the three target lncRNAs combined for diagnosis of lung cancer was 0.647 (95% CI: 0.593-0.700), the sensitivity was 94.3%, and the specificity was 30.1%. The combined diagnostic efficiency was better than that of individual diagnosis, indicating that the combined detection of the three target lncRNAs has a relatively greater diagnostic value for distinguishing NSCLC patients from benign lung disease groups and apparently healthy people. Figure 5 1 is the ROC curve diagram of the independent detection and combined detection of the three lncRNAs of the present invention; Table 10 is the ROC curve statistical results of the independent detection and combined detection of the three lncRNAs of the present invention;

[0097] Table 10

[0098]

[0099]

[0100] The expression levels of the three candidate lncRNAs were included to construct a multivariate logistic regression equation, and the results are shown in Table 11. Binary multivariate logistic regression was used to analyze the risk factors for lung cancer progression in the control group (including the benign lung disease group and the healthy group). The results showed that Linc-MRPL33-1 (OR = 46.619, 95% CI: 1.093-1988.512, P = 0.045) and Linc-PIK3C2G-2 (OR = 7.033, 95% CI: 1.311-37.745, P = 0.023) were independent risk factors for lung cancer, while Linc-ABCA5-3 (OR = 0.048, 95% CI: 0.001-0.860, P = 0.146) was a protective factor. The prediction equation is: P = ex / (1 + ex), X = -12.469 + 3.842 * linc-MRPL33-1 + 1.951 * linc-PIK3C2G-2 - 3.030 * linc-ABCA5-3. Wherein, e represents the natural logarithm. The influencing factors of lung cancer were analyzed by multivariate logistic regression as shown in Table 11:

[0101] Table 11

[0102]

[0103] 4.7 Relationship between the expression levels of three lncRNAs in plasma samples and clinical data and pathological characteristics

[0104] The relationship between the expression levels of the three lncRNAs in plasma and the clinical pathological parameters of lung cancer is shown in Table 12:

[0105] Table 12

[0106]

[0107]

[0108] In summary, the expression levels of linc-MRPL33-1, linc-PIK3C2G-2 and linc-ABCA5-3 in plasma had no correlation with the patient's age, tissue typing, and lymph node metastasis (P>0.05); the expression levels of linc-MRPL33-1 and linc-ABCA5-3 had no correlation with TNM stage, but the expression level of linc-PIK3C2G-2 was correlated with TNM stage (P<0.05).

[0109] Based on the purpose of absolute quantification of circulating lncRNA in plasma, the present invention adopts a one-step RT-qPCR to establish a new detection method, so that reverse transcription and PCR reactions are completed in the same reaction tube, without opening the cover, pipetting and adding samples in the middle, which greatly improves the experimental efficiency and reduces the risk of contamination of the reaction. Using dPCR as a comparison method, the methodological performance of the constructed RT-qPCR experimental method is confirmed. RT-qPCR collects fluorescence signals for each cycle and measures the fluorescence cumulative signal in real time, while digital PCR first performs PCR amplification. This process does not require fluorescence collection, only PCR products need to be amplified, and finally the fluorescence signal is collected through the detection system. The reaction unit with nucleic acid template can collect the fluorescence signal. In general, the original copy number of the target DNA molecule is equal to the number of units with fluorescence signals, but in fact, there is not necessarily only a single target molecule in each unit, and the initial copy number of the sample needs to be calculated according to the Poisson distribution. dPCR has the advantages of high sensitivity, high accuracy and good repeatability for the detection of target DNA molecules, and has low sample requirements. It can be used to determine the accurate sample copy number and achieve data reproducibility, which can be used as an important supplement to RT-qPCR.

[0110] Compared with the digital PCR method, the clinical test methodological indicators of the multiplex RT-qPCRTaqMan probe absolute quantitative detection method constructed by the present invention have good analytical performance. For the precision performance confirmation, the detection data of the two concentration samples were under control, indicating that the precision of the experimental method was good, and its repeatability and the precision of the instrument / method met the performance requirements. When the accuracy performance was confirmed, the experimental results passed the intra-method and inter-method outlier tests. The scatter plot shows that the experimental method's measured values ​​fit the comparison method very well, and the bias plot shows that the bias between the two methods is small, and no outliers appear. The upper limit of the expected bias for accuracy is less than the allowable bias, and the experimental method passes the accuracy test. In addition, the detection linear ranges of the experimental method for circulating Linc-MRPL33-1, linc-PIK3C2G-2 and linc-ABCA5-3 are 8.73×10 8 -8.73×10 2 copies / ml, 7.73×10 8 -7.73×10 2 copies / ml, 9.78×10 8 -9.78×10 2 The method has a wide linear range and can fully achieve the purpose of quantitative detection of trace lncRNA in plasma. Through the study of the detection limit of the constructed experimental method, it was found that the detection limit and quantification limit of the method were both 800 copies / ml, which is suitable for quantitative detection of low-copy plasma samples.

[0111] At present, most studies only focus on the diagnostic efficacy and prognostic evaluation of a single lncRNA. Compared with the combination of multiple lncRNAs, the sensitivity and specificity of a single lncRNA in diagnosing lung cancer are often not high, and the clinical diagnostic significance is not great. Studies have shown that lncRNATUG1 is lowly expressed in the plasma of NSCLC patients, while lncRNAUCA1 is highly expressed. The diagnostic efficacy of the combined detection of the two for lung cancer is better than that of a single detection. Studies have shown that the application value of the combined diagnosis of NSCLC by lncRNATNILA, lncRNAAGAP2-AS1 and soluble fragment of cytokeratin 19 (Cyfra21-1) is also better than that of a single diagnosis. Although multiple lncRNA combinations with potential for early diagnosis of lung cancer have been reported, they are still in the research stage and there is still no recognized effective combination.

[0112] The present invention prepares stable standards and quality control products that can monitor and quantify processes such as sample extraction and amplification, and constructs a multiple TaqMan probe absolute quantitative detection method based on the standards, which can achieve the amplification of three target lncRNAs in the same reaction system to meet the needs of multiple detection. In addition, the methodological indicators such as precision, correctness, analytical measurement range, and detection limit of the method have good analytical performance. Due to the versatility of the method, the evaluation scheme in the present invention will provide a reference for other newly established detection systems or methods.

[0113] The present invention prepares a triple armor RNA containing linc-MRPL33-1, linc-PIK3C2G-2 and linc-ABCA5-3 as a standard and quality control product. The analytical performance of the multiple RT-qPCR TaqMan probe absolute quantitative detection method of linc-MRPL33-1, linc-PIK3C2G-2 and linc-ABCA5-3 is determined. Linc-MRPL33-1, linc-PIK3C2G-2, and linc-ABCA5-3 are highly expressed in the lung cancer group, and the application value of the combined detection of the three lncRNAs is better than that of individual detection.

[0114] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and the scope of protection of the present invention is defined by the attached claims, description and their equivalents.

Claims

1. A combination of lncRNA diagnostic markers, Features: The lncRNA diagnostic marker combination includes one or a combination of several of Linc-PIK3C2G-2, Linc-ABCA5-3 or Linc-MRPL33-1.

2. The lncRNA diagnostic marker combination according to claim 1, Features: The lncRNA diagnostic marker combination includes specific primers, TaqMan probes and standard positive templates. The nucleotide sequence of the upstream primer of the Linc-PIK3C2G-2 is shown in SEQ ID No.1, the nucleotide sequence of the downstream primer of the Linc-PIK3C2G-2 is shown in SEQ ID No.2, and the nucleotide sequence of the Probe probe of the Linc-PIK3C2G-2 is the nucleotide sequence shown in SEQ ID No.3; Or the nucleotide sequence of the upstream primer of Linc-ABCA5-3 is shown in SEQ ID No.4, the nucleotide sequence of the downstream primer of Linc-ABCA5-3 is shown in SEQ ID No.5, and the nucleotide sequence of the Probe probe of Linc-ABCA5-3 is the nucleotide sequence shown in SEQ ID No.6; Or the nucleotide sequence of the upstream primer of the Linc-MRPL33-1 is shown in SEQ ID No.7, the nucleotide sequence of the downstream primer of the Linc-MRPL33-1 is shown in SEQ ID No.8, and the nucleotide sequence of the Probe probe of the Linc-MRPL33-1 is the nucleotide sequence shown in SEQ ID No.

9.

3. A kit containing the lncRNA diagnostic marker combination according to claim 1, Features: The kit comprises reagents used to detect the expression level of the marker combination according to claim 1.

4. The kit according to claim 3, Features: The kit comprises: PCR primers, TaqMan probes, PCR buffer, hot start enzyme, dNTP, standard positive template, and reference substance; the reference substance is ultrapure water.

5. The kit according to claim 3, Features: Reagents for detecting expression levels include reagents for detecting protein expression levels and / or mRNA expression levels.

6. Use of the lncRNA diagnostic marker combination according to any one of claims 1 to 5 in a molecular marker for lung cancer diagnosis.

Images