Construction method of multiple RT-qPCR Taq Man probe absolute quantitative detection system

Abstract

The invention discloses a construction method of a multiple RT-qPCRTaqMan probe absolute quantitative detection system. The construction method comprises the following steps: (1) constructing a pACYC-MS2 recombinant plasmid; (2), pACYC-MS2-lncRNAs (deoxyribonucleic acids) are selected from the group consisting (3) verifying the accuracy of the pACYC-MS2-lncRNAs recombinant plasmid by sequencing: converting the pACYC-MS2-lncRNAs into BL21 (DE3) escherichia coli, performing induced expression, performing PEG enrichment, performing molecular sieve chromatography purification on MS2VLPs, and performing verification and performance evaluation; (4) constructing a one-step method multiple RT-qPCRTaqMan probe detection system; and (5) establishing a multiple RT-qPCRTaqMan probe absolute quantitative detection system. On the basis of the purpose of absolute quantification of circulating lncRNA in plasma, a novel detection method is established by adopting one-step RT-qPCR, reverse transcription and PCR are completed in the same reaction tube, operations such as uncovering, pipetting and sample adding do not exist in the midway, the experimental efficiency is greatly improved, and the pollution risk of the reaction is reduced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for constructing a multiplex RT-qPCR TaqMan probe absolute quantitative detection system. 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] At present, there are still many problems in the clinical application research of circulating lncRNA, which needs to be continuously improved and perfected. The reported literature basically uses the SYBR dye method, but the content of circulating lncRNA in plasma is very small. Although the SYBR dye method is simple in design and low in cost, its specificity is easily affected, which in turn affects the accuracy of the results. In addition, the SYBR dye method requires the target gene and the internal reference to be detected separately, so it is difficult for the internal reference to truly monitor the detection process of the target gene, and the standardization and accuracy of the results are easily affected, which is not conducive to clinical application.

[0006] Another commonly used fluorescent labeling method is the TaqMan probe method, which can add multiple probes and corresponding primers to the same reaction system to achieve the joint detection of multiple targets, facilitating the establishment of multiplex PCR detection methods. However, due to the high cost of probe labeling, it is currently less used in nucleic acid detection. Tao et al. used two RT-qPCR methods based on TaqMan probes and SYBRGreen dye to detect SARS-CoV-2 respectively, and found that SYBRGreen fluorescent dye is easy to use and has low detection cost, but because it can bind to any DNA double strand, it has no selectivity, which makes its specificity easily affected.

[0007] The results of RT-qPCR experiments are often calculated using two methods: relative quantification and absolute quantification. The relative quantification method often calculates the relative expression level of each target gene relative to the internal reference gene. The absolute quantification method is to accurately quantify the unknown concentration of the test sample through the standard curve. At the same time, in order to monitor the accuracy of the quantitative results, it is necessary to simultaneously amplify endogenous internal reference genes with good stability, or introduce exogenous genes to reduce experimental errors. In the relative quantification process, the use of appropriate internal reference genes is crucial to obtaining meaningful RT-qPCR results. In the absolute quantitative detection process, the use of internal reference genes with good stability can achieve the same tube detection as the target gene, monitor the entire RT-qPCR operation process, and minimize unnecessary errors.

[0008] In order to establish an absolute quantitative detection system for candidate lncRNA TaqMan probes and get rid of the limitations of internal references, the preparation of standards can adopt the armored RNA technology. The basic principle is to obtain the coding sequence of the capsid protein and mature enzyme protein in the MS2 phage genome, insert it into a multiple cloning site of the expression vector, insert the target sequence to be packaged into another multiple cloning site of the vector, and then transcribe the target gene into RNA, wrap it in the capsid protein, and finally assemble it into a spherical RNA-protein complex, generally called MS2 virus-like particle (MS2VLP) or "armored RNA". At present, this technology has been successfully used as a standard and quality control product for nucleic acid detection of RNA viruses such as H7N9, SARS-CoV-2, HCV, HDV, HIV, rubella virus and Zika virus. There are also studies and developments of quantitative standards for p210 and p190BCR-ABL1FG transcripts based on armored RNA. In addition, digital PCR (digitalPCR, dPCR) is an emerging technology for absolute quantitative nucleic acid. There are currently two main types of dPCR systems, namely microfluidic chip detection technology and microdroplet system. Its quantitative process mainly includes two stages: PCR amplification and fluorescence signal acquisition. First, the template is diluted and dispersed into each reaction unit to make it a large number of independent PCR reaction units. Then, the template undergoes single-molecule independent PCR amplification in each unit. Currently, many studies have used digital PCR technology to quantify circulating miRNAs through droplet digital PCR. The application of droplet dPCR has high sensitivity for absolute quantification of hepatitis E virus. The chip-based platform QuantStudio3D microfluidic chip digital PCR technology has also been used to analyze circulating miRNAs in plasma samples of lung cancer patients

[44] . As a newly developed nucleic acid absolute quantification technology, dPCR faces many opportunities and challenges, and it is difficult to completely replace RT-qPCR at present.

[0009] Currently, there is a lack of a method for constructing a multiplex RT-qPCR TaqMan probe absolute quantitative detection system with high experimental efficiency. Summary of the invention

[0010] 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 method for constructing a multiplex RT-qPCR TaqMan probe absolute quantitative detection system with high experimental efficiency.

[0011] To achieve the above object, the present invention provides the following technical solution: A multiplex RT-qPCR TaqMan probe absolute quantitative detection system of the present invention comprises the following steps:

[0012] (1) Using armored RNA technology, BL21 (DE3) expression Escherichia coli was infected with MS2 phage, and the capsid protein and mature enzyme protein coding sequences were reverse transcribed after extracting the MS2 phage genomic RNA, and inserted into the MCS1 site of the pACYCDuet-1 prokaryotic expression vector. The MCS1 site is located between the two restriction sites of BamHI and Not I in the MCS1 site of the pACYCDuet-1 plasmid to construct the pACYC-MS2 recombinant plasmid;

[0013] (2) Total RNA was extracted from lung cancer tissue specimens 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. Finally, a recombinant plasmid was constructed, in which MCS1 contained the mature enzyme protein and capsid protein coding sequences of MS2, and MCS2 contained the MS2 packaging site sequence + three tandem candidate lncRNA target amplification sequences, and was named pACYC-MS2-lncRNAs; the MCS2 site was located between the Fesl and Pac I restriction sites of the MCS1 site of the pACYCDuet-1 plasmid;

[0014] (3) Sequencing to verify the accuracy of the pACYC-MS2-lncRNAs recombinant plasmid: pACYC-MS2-lncRNAs was transformed into BL21 (DE3) Escherichia coli and induced for expression, PEG enrichment, molecular sieve chromatography purification of MS2 VLPs, and verification and performance evaluation;

[0015] (4) Construction of a one-step multiplex RT-qPCR TaqMan probe detection system;

[0016] (5) Establish a multiplex RT-qPCR TaqMan probe absolute quantitative detection system.

[0017] Further, in step (1), the lncRNA target amplification sequences are Linc-PIK3C2G-2, Linc-ABCA5-3, and Linc-MRPL33-1;

[0018] 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;

[0019] 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 shown in SEQ ID No.6.

[0020] The nucleotide sequence of the upstream primer of Linc-MRPL33-1 is shown in SEQ ID No.7, the nucleotide sequence of the downstream primer of Linc-MRPL33-1 is shown in SEQ ID No.8, and the nucleotide sequence of the Probe probe of Linc-MRPL33-1 is shown in SEQ ID No.9;

[0021] In step (4), the nucleotide sequence of the upstream primer of the internal reference gene RPLPO is shown in SEQ ID No.10, the nucleotide sequence of the downstream primer of the internal reference gene RPLPO is shown in SEQ ID No.11, and the nucleotide sequence of the Probe probe of the internal reference gene RPLPO is the nucleotide sequence shown in SEQ ID No.12.

[0022] Furthermore, in step (1), the construction of the MS2 phage prokaryotic expression vector containing three lncRNAs The pACYCDuet-1 expression vector developed by Novagen contains an epitope tag, which is commonly used for gene expression in prokaryotic cells;

[0023] pACYCDuet-1 has two multiple cloning sites (MCS), each MCS has a lactose operator, a T7 promoter and a ribosome binding site, and can express two genes together; the vector also carries a chloramphenicol resistance gene, which can be used for positive colony screening; the capsid protein and mature enzyme protein coding sequences of the MS2 phage genome are inserted into the MCS1 site of pACYCDuet-1 to construct the pACYC-MS2 recombinant plasmid.

[0024] Furthermore, in step (1), step 1 synthesizes the target fragment: referring to the three target genes and the genome sequence of MS2 phage in the GenBank database, artificially synthesizes the nucleic acid fragment of the MS2 phage packaging site and the three target lncRNA target amplification sequences in series.

[0025] Further, in step (1), step 2 target fragment and vector connection: primers of MS2 phage maturase protein and capsid protein are amplified by PCR method to obtain target fragment PCR product, which is cloned into the MCS1 site (BamHI-NotI) of pACYCDuet-1 plasmid to obtain pACYC-MS2 recombinant plasmid.

[0026] Furthermore, in step (2), the pACYC-MS2-lncRNAs recombinant plasmid was constructed:

[0027] The pACYC-MS2 recombinant plasmid was extracted, and the artificially synthesized MS2 phage packaging site and three target lncRNA target amplification sequences were cloned into the pACYC-MS2 recombinant plasmid to obtain the pACYC-MS2-lncRNAs recombinant plasmid, which was then connected, transformed, and verified.

[0028] Furthermore, in step (3), the pACYC-MS2-lncRNAs recombinant plasmid is transferred into Escherichia coli BL21 (DE3) to induce expression, and MS2 bacteriophage virus-like particles (MS2VLPs) are obtained after extraction, enrichment and purification. MS2VLPs are identified by agarose gel electrophoresis, SDS-PAGE electrophoresis, RT-qPCR experiment and transmission scanning electron microscopy, and then the plasmid residues in the MS2VLPs solution are detected, and digital PCR (Digital PCR, dPCR) is used to determine the value as a standard and quality control product, and then the enzyme resistance and stability of MS2VLPs are evaluated.

[0029] Furthermore, in step (4), the internal reference gene RPLPO with good stability was screened out to monitor the lncRNA detection process, and specific primers and TaqMan probes were designed for RPLPO and the three target lncRNAs, respectively, to establish a one-step RT-qPCRTaqMan probe detection single system, and the single system of each gene was optimized from the aspects of annealing temperature, template concentration, primer concentration and probe concentration. First, the optimal single-plex detection system of each gene was determined, and then a one-step multiplex RT-qPCRTaqMan probe detection system of the three target lncRNAs and the internal reference was constructed.

[0030] Furthermore, in step (5), based on the constructed multiplex RT-qPCR TaqMan probe detection method, a multiplex RT-qPCR TaqMan probe absolute quantitative detection system is established using digital PCR-valued MS2 VLPs as standard. The analytical performance is confirmed by analyzing and evaluating the performance indicators of the detection system, such as precision, correctness, linear range, detection limit, and quantification limit.

[0031] Beneficial effects: 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 system, so that reverse transcription and PCR reactions can be completed in the same reaction tube without opening the cover, pipetting and adding samples, which greatly improves the experimental efficiency and reduces the risk of contamination of the reaction.

[0032] Compared with the prior art, the present invention has the following advantages: (1) Compared with the digital PCR method, the clinical test methodological indicators of the multiple RT-qPCR TaqMan 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 are under control, indicating that the precision of the experimental method is good, and its repeatability and the precision of the instrument / method meet the performance requirements.

[0033] (2) When the accuracy performance of the present invention is confirmed, the experimental results pass 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 of the accuracy is less than the allowable bias, and the experimental method passes the accuracy test. In addition, the experimental method has a linear range of 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. By inventing 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.

[0034] (3) 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 this method have good analytical performance.

[0035] (4) The present invention aims to construct a multiplex RT-qPCR TaqMan probe absolute quantitative detection method for these three lncRNAs, evaluate the analytical performance of this method, and preliminarily verify the expression and diagnostic efficacy of the three target lncRNAs in the plasma of NSCLC patients, providing a reference for the subsequent construction of a diagnostic model. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a map of the pACYCDuet-1 vector of the present invention;

[0037] Figure 2 It is a partial sequencing map of the pACYC-MS2 plasmid of the present invention;

[0038] Figure 3 It is a partial sequencing diagram of the pACYC-MS2-lncRNAs plasmid of the present invention;

[0039] Figure 4 This is the peak diagram of the MS2VLPs molecular sieve chromatography purification of the present invention;

[0040] Figure 5 2% agarose gel electrophoresis diagram of the present invention; M: Marker; 1-2 is Linc-MRPL33-1 (94 bp); 4-5 is linc-ABCA5-3 (90 bp); 7-8 is linc-PIK3C2G-2 (162 bp); 3, 6, 9 are negative controls;

[0041] Figure 6 It is the SDS-PAGE electrophoresis diagram of MS2 VLPs of the present invention; M: pre-stained protein marker; 1-6 are the eluates of tubes 1-6, i.e., MS2 VLPs;

[0042] Figure 7 RT-qPCR validation of the RNA packaged in MS2 VLPs of the present invention; linc-MRPL33-1 (red); linc-PIK3C2G-2 (green); linc-ABCA5-3 (blue);

[0043] Figure 8 This is a schematic diagram of the electron microscopy results of MS2VLPs of the present invention;

[0044] Fig. 9The electrophoresis diagram of the enzyme resistance verification result of MS2VLPs of the present invention; A.linc-MRPL33-3; B.linc-PIK3C2G-2; C.linc-ABCA5-3; M: 600bpMarker; 1-2 are MS2VLPs; 3-4 are pACYC-MS2-lncRNAs; 5-6 are RNA extracted from MS2VLPs; 1, 3, 5 are not treated with enzymes; 2, 4, 6 are treated with enzymes;

[0045] Fig.10 This is a diagram showing the results of fluorescence quantitative PCR detection of residual plasmids in MS2 VLPs of the present invention; Fig.10 A is linc-MRPL33-1 of the present invention (red); Fig.10 B is linc-PIK3C2G-2 of the present invention (green); Fig.10 C is linc-ABCA5-3 of the present invention (blue); 1: positive control; 2: reverse transcribed sample; 3: non-reverse transcribed sample; 4. negative control;

[0046] Fig.11 2D scatter plot of dPCR detection of the gene linc-MRPL33-1 of the present invention; green: linc-MRPL33-1 positive droplets; gray: negative droplets;

[0047] Fig.12 This is a 2D scatter plot of dPCR detection of the gene linc-PIK3C2G-2 of the present invention; green: linc-PIK3C2G-2 positive droplets; gray: negative droplets.

[0048] Fig.13 This is a 2D scatter plot of dPCR detection of the gene linc-ABCA5-3 of the present invention; blue: linc-ABCA5-3 positive droplets; gray: negative droplets.

[0049] FIG14 is a graph showing the stability evaluation results of three target genes in MS2 VLPs of the present invention; Fig.14A The stability evaluation result diagram of linc-MRPL33-1 of the present invention; Fig. 14B This is a diagram showing the stability evaluation results of linc-PIK3C2G-2 of the present invention; Fig. 14C This is a diagram showing the stability evaluation results of linc-ABCA5-3 of the present invention.

[0050] Fig.15 It is the gradient PCR electrophoresis diagram of the present invention; Fig.15 A is the linc-MRPL33-1 gradient PCR electrophoresis diagram of the present invention; Fig.15 B is the linc-PIK3C2G-2 gradient PCR electrophoresis diagram of the present invention; Fig.15C is the linc-ABCA5-3 gradient PCR electrophoresis D. RPLPO gradient PCR electrophoresis diagram of the present invention;

[0051] Fig.16 is a template concentration optimization diagram of the present invention; Fig.16 A is the linc-MRPL33-1 template concentration optimization diagram of the present invention; Fig.16 B. linc-PIK3C2G-2 template concentration optimization diagram; Fig.16 C. linc-ABCA5-3 template concentration optimization D. RPLPO template concentration optimization diagram;

[0052] Fig.17 It is the primer concentration optimization diagram of the present invention; Fig.17 A is the linc-MRPL33-1 primer concentration optimization diagram of the present invention; Fig.17 B is a graph showing the optimization of the linc-PIK3C2G-2 primer concentration of the present invention; Fig.17 C is the concentration optimization diagram of linc-ABCA5-3 primers of the present invention; D. RPLPO primer concentration optimization diagram;

[0053] Fig.18 It is a probe concentration exploration diagram of the present invention; Fig.18 A. linc-MRPL33-1 probe concentration exploration diagram; Fig.18 B is a concentration exploration diagram of the linc-PIK3C2G-2 probe of the present invention; Fig.18 C is a concentration exploration diagram of the linc-ABCA5-3 probe of the present invention; Fig.18 D is the RPLPO probe concentration exploration diagram of the present invention;

[0054] Fig.19 This is a probe low concentration optimization diagram of the present invention; Fig.19 A. Optimization of low concentration of linc-MRPL33-1 probe Fig.19 B is a low concentration optimization diagram of the linc-PIK3C2G-2 probe of the present invention; Fig.19 C is a low concentration optimization diagram of the linc-ABCA5-3 probe of the present invention; Fig.19 D is the low concentration optimization diagram of the RPLPO probe of the present invention;

[0055] Fig. 20 It is a graph of the amplification curve of the plasma specimen multiplex real-time fluorescence quantitative PCR of the present invention; linc-MRPL33-1 (red); linc-PIK3C2G-2 (green); linc-ABCA5-3 (blue); RPLPO (orange). DETAILED DESCRIPTION

[0056] 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.

[0057] Example 1

[0058] Preparation of MS2 bacteriophage virus-like particle standards containing linc-MRPL33-1, linc-PIK3C2G-2 and linc-ABCA5-3 target amplification sequences

[0059] 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.

[0060] 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.

[0061] Materials and Methods

[0062] 1 Experimental Materials

[0063] 1.1 Plasmids and strains

[0064] The expression host Escherichia coli BL21 (DE3) and the prokaryotic expression plasmid pACYCDuet-1 were both from General Biosystems (Anhui) Co., Ltd.

[0065] 1.2 The main experimental instruments are shown in Table 1:

[0066] Table 1

[0067]

[0068]

[0069] 1.3 Main experimental consumables

[0070] Table 2

[0071]

[0072] 1.4 Main experimental reagents

[0073] Table 3

[0074]

[0075]

[0076]

[0077] 1.5 Reagent preparation

[0078] (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.

[0079] (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.

[0080] (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.

[0081] (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.

[0082] (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.

[0083] (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.

[0084] 2 Experimental methods

[0085] 2.1 Primer and probe design of target LncRNA

[0086] 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.

[0087] Table 4

[0088]

[0089] 2.2 Construction of MS2 phage prokaryotic expression vector containing three lncRNAs

[0090] The pACYCDuet-1 expression vector developed by Novagen contains an epitope tag and is commonly used for gene expression in prokaryotic cells. pACYCDuet-1 has two multiple cloning sites (MCS). Each MCS has a lactose operon, a T7 promoter, and a ribosome binding site, which can express two genes together. The vector also carries a chloramphenicol resistance gene, which can be used for positive colony screening. The capsid protein and mature enzyme protein coding sequences of the MS2 phage genome are inserted into the MCS1 site of pACYCDuet-1 to construct the pACYC-MS2 recombinant plasmid; the MS2 packaging site sequence + the 3 target lncRNA target amplification sequences in series are inserted into the MCS2 of pACYC-MS2 to construct the pACYC-MS2-lncRNAs recombinant plasmid. PACYCDuet-1 vector see Figure 1 .

[0091] 2.2.1 Construction of pACYC-MS2 plasmid

[0092] (1) Synthesize the target fragment:

[0093] With reference to the genome sequence of the three target genes and MS2 phage (NC_001417) in the GenBank database, General Biosystems (Anhui) Co., Ltd. was commissioned to artificially synthesize a nucleic acid fragment concatenated with the MS2 phage packaging site and the three target lncRNA target amplification sequences.

[0094] (2) Connection between target fragment and vector:

[0095] The primers of MS2 phage mature enzyme protein and capsid protein were amplified by PCR to obtain the target fragment PCR product, which was cloned into the MCS1 site (BamHI-NotI) of pACYCDuet-1 plasmid to obtain pACYC-MS2 recombinant plasmid. The ligation system is as follows: prepare the ligation solution according to Table 2, and ligate at 52°C for 30 minutes.

[0096] The target fragment and vector connection system are shown in Table 5:

[0097] Table 5

[0098]

[0099]

[0100] (3) Transformation of recombinant plasmid:

[0101] 1) Take 2 μl of plasmid with a concentration of about 100 ng / μl and add it to 100 μl Top10 competent cells, shake gently to mix, and place on ice for 3 minutes.

[0102] 2) Place in a 42°C water bath for 90 seconds (the centrifuge tube cannot be moved during the water bath).

[0103] 3) After taking out the centrifuge tube, quickly place it on ice for 3 minutes.

[0104] 4) Add 500 μl of LB liquid culture medium at about 37°C and culture with shaking at 37°C, 200 rpm for 40 min.

[0105] (4) Verification of recombinant plasmid

[0106] 1) Take 100 μl of bacterial solution and spread it evenly on a chloramphenicol resistance (34 μg / ml) LB plate, invert the plate, and culture it in a 37°C incubator overnight.

[0107] 2) Positive colonies were picked and expanded in LB liquid medium (chloramphenicol resistance) at 37°C, 250 rpm, on a constant temperature shaker overnight, and the recombinant plasmid was verified by bacterial liquid sequencing.

[0108] 2.2.2 Construction of pACYC-MS2-lncRNAs recombinant plasmid

[0109] Extract the pACYC-MS2 recombinant plasmid, clone the artificially synthesized MS2 phage packaging site and three target lncRNA target amplification sequences into the pACYC-MS2 recombinant plasmid (Fesl-PacI restriction vector) to obtain the pACYC-MS2-lncRNAs recombinant plasmid. The ligation, transformation, and verification are the same as 2.2.1.

[0110] 2.3 Expression and transformation of recombinant virus-like particles

[0111] 2.3.1 Transformation of BL21(DE3) Escherichia coli with pACYC-MS2-lncRNAs

[0112] (1) Before opening the plasmid powder, centrifuge at 12,000 rpm for 1 min to prevent the plasmid from being lost. Add 50 μl of DEPC water to dissolve it and shake to mix.

[0113] (2) Take a tube of BL21(DE3) competent cells, thaw it and set aside.

[0114] (3) Add 2 μl of pACYC-MS2-lncRNAs plasmid, shake gently to mix, and place on ice for 30 min.

[0115] (4) Take out the centrifuge tube and place it in a 40°C water bath for heat shock for 90 seconds.

[0116] (5) Quickly transfer the centrifuge tube to an ice bath and cool for 2 min. Keep the centrifuge tube still during this process.

[0117] (6) Add 500 μl of LB liquid medium without resistance and pre-culture in a constant temperature incubator at 37°C and 175 rpm for 40 min.

[0118] (7) Centrifuge at 3000 rpm for 4 min.

[0119] (8) Discard the supernatant and spread an appropriate amount of the product evenly on a chloramphenicol-resistant LB plate.

[0120] (9) Invert the plate and incubate at 37°C overnight.

[0121] 2.3.2 Inducible expression of recombinant virus-like particles

[0122] (1) Take out the overnight culture plate, pick a single positive colony and place it in LB liquid medium containing 34 μg / ml chloramphenicol resistance, and culture it at 37°C with shaking at 220 rpm overnight.

[0123] (2) The overnight culture was added to two conical flasks containing 250 ml of chloramphenicol-resistant LB liquid medium (ratio 1:100) for expansion culture. The culture was incubated at 37°C with shaking at 220 rpm for about 4 h until the OD value was about 0.6.

[0124] (3) Add IPTG inducer (final concentration: 1 mmol / L) and culture at 37°C, 200 rpm for 12 h.

[0125] (4) Collect the bacterial solution in a 50 ml centrifuge tube and centrifuge at 8000 rpm for 5 min. Discard the supernatant and collect the bacterial precipitate.

[0126] (5) Wash the precipitate with 1× PBS, centrifuge at 8000 rpm for 5 min, discard the supernatant, collect the bacterial precipitate, and repeat the process twice.

[0127] 2.3.3 Ultrasonic fragmentation of recombinant virus-like particles

[0128] (1) Resuspend the bacteria obtained in the previous step in 30 ml of 1× PBS.

[0129] (2) Use an ultrasonic cell disruptor to disrupt the bacteria. Set the ultrasonic power to 20%, and disrupt for 2 seconds and pause for 3 seconds each time. The total disruption time is 45 minutes. During this process, the bacteria must be kept on ice to avoid temperature rise that may cause protein denaturation.

[0130] (3) After the fragmentation is completed, centrifuge at 8000 rpm for 30 min and collect the supernatant, which is the expression product.

[0131] 2.3.4 Enrichment of recombinant virus-like particles by PEG20000

[0132] Polyethylene glycol (PEG) is a nonionic water-soluble polymer that can cause nonspecific precipitation of proteins, and the process is reversible and will not cause protein denaturation. Different concentrations of PEG are selected according to the molecular weight of the protein, and the molecular weight of the protein is inversely proportional to the concentration of PEG used for precipitation. The present invention uses PEG6000 with good protein selectivity to perform preliminary purification and enrichment of recombinant virus-like particles. The role of solid NaCl is to promote the rapid separation of bacterial fragments from virus-like particles, while chloroform is to remove polyethylene glycol and bacterial fragments in the suspension.

[0133] (1) Add solid NaCl to the supernatant collected after ultrasonic disruption to a final concentration of 1 mol / L and shake to dissolve the NaCl.

[0134] (2) Add PEG6000 to a final concentration of 1 mmol / L and shake vigorously in a 37°C constant temperature shaker for 15 min to completely dissolve it.

[0135] (3) Place in an ice bath at 4°C overnight.

[0136] (4) Centrifuge at 8000 rpm for 45 min and discard the supernatant.

[0137] (5) Add 5 ml of PBS to suspend the precipitate, add DNaseI and RNaseA to the final concentrations of 200 U / ml and 10 mg / ml, respectively, and digest in a 37°C incubator for 2 h (the bacterial RNA is completely digested, while the virus-like particles are enzyme-resistant and the RNA encapsulated in the shell is not digested).

[0138] (6) Add an equal volume of chloroform to the centrifuge tube and shake at 220 rpm in a shaker at 37°C for 10 min.

[0139] (7) Centrifuge at 8000 rpm for 15 min to separate the organic phase and the aqueous phase, and recover the upper aqueous phase containing MS2 VLPs.

[0140] (8) The collected aqueous phase is added to a dialysis bag with a molecular weight cutoff of 8000-14000Da and embedded with PEG20000 (when clamping the dialysis bag with a dialysis clamp, remove as many bubbles as possible) to achieve the purpose of concentration, so as to facilitate the next step of propylene glycol gel chromatography purification (the dialysis bag is pre-treated with 2% sodium bicarbonate and 1mmol / L ETA pH 8.0).

[0141] 2.3.5 Gel chromatography purification of recombinant virus-like particles

[0142] Gel chromatography can show different flow rates according to the molecular weight of the protein in the sample solution. Proteins with large molecular weight are eluted first due to vertical movement, while proteins with small molecular weight have a long movement distance and are more obstructed, so elution takes longer, thereby achieving the purpose of separating proteins of different molecular weights. MS2VLPs are further separated and purified by gel chromatography after enrichment with PEG6000. The steps of gel chromatography experiment are as follows:

[0143] (1) Before connecting the column to the chromatographic system, start the pump to purge all air in the system, especially the air in the pipes and valves, and stop the pump after the air is purged.

[0144] (2) Install the Sephacryl S-200 High Resolution Hi Prep 26 / 60 chromatography column in a vertical position into the ClearFirst protein purification system, remove the bottom stopper, and connect the inlet tubing to the purification system drop-by-drop.

[0145] (3) Turn on the instrument and computer, enter the software operation interface, create a new experiment, and determine the correlation coefficient in the purification method setting. The purification procedure is shown in Table 3.

[0146] (4) First, equilibrate the chromatography column with deionized water filtered through a 0.22 μm filter membrane and PBS buffer at a flow rate of 1.3 ml / min.

[0147] (5) Draw the sample into the syringe, push out excess bubbles, filter the sample through a 0.22 μm filter membrane, and slowly push it into the sample injection port.

[0148] (6) Elute the target protein with PBS buffer at a flow rate of 1.3 ml / min. Observe the protein peak on the elution spectrum and manually collect the corresponding fractions, collecting 1 ml in each tube. Equilibrate the column with the eluent.

[0149] (7) The prepacked column contains 20% ethanol. If the column is stored for more than 2 days after use, clean the column with four column volumes of distilled water and then with four column volumes of 20% ethanol. For more stringent cleaning, select the appropriate cleaning steps depending on the specific situation.

[0150] (8) To avoid the formation of bubbles in the column, use a delivery device. Connect the delivery device to the capillary at the column outlet, start the pump, and fill the column with 20% ethanol to approximately 50% of the column volume.

[0151] (9) The same component solutions were mixed and placed into a dialysis bag, embedded with PEG20000, and finally placed at 4°C for concentration.

[0152] (10) After concentration, the mixture was stored at -20°C for future use and named as MS2 VLPs. The propylene glycol gel chromatography method is shown in Table 6:

[0153] Table 6

[0154]

[0155] Note: Buffer A and Buffer B are deionized water and PBS filtered through a 0.22 μm filter membrane, respectively.

[0156] 2.4 Identification of recombinant virus-like particles

[0157] 2.4.1 Identification of MS2 VLPs by agarose gel electrophoresis

[0158] The purified MS2 VLPs were identified by 2% agarose gel electrophoresis.

[0159] 2.4.2 SDS-PAGE identification of recombinant virus-like particles

[0160] (1) Sample treatment: Take an appropriate amount of MS2 VLPs sample and 2× SDS loading buffer in a 1:1 volume ratio and mix them. Heat them in a constant temperature metal bath at 100°C for 10 min to fully denature the protein.

[0161] (2) Install and fix the thick and thin glass panels in pairs.

[0162] (3) Determine the required volume of gel solution and prepare 15% separation gel according to Table 4.

[0163] (4) Add TEMED last, mix quickly to avoid agglomeration, and immediately pour into the gaps between the fixed plastic sheets.

[0164] (5) Add isopropanol (about 1 ml, isolate oxygen and flatten the gel surface) above the separation gel surface. After the separation gel is completely polymerized (about 40 minutes), pour out the isopropanol above the separation gel and absorb it with filter paper.

[0165] (6) According to the required volume of concentrated gel, prepare 5% upper concentrated gel according to Table 4.

[0166] (7) Add TEMED last, mix quickly and pour it onto the upper layer of the separation gel. Carefully insert a clean loading comb into the concentrated gel to avoid bubbles. Leave it at room temperature for about 40 min until the concentrated gel is completely solidified before proceeding with subsequent operations.

[0167] (8) After the concentrated gel is completely polymerized, pull out the loading comb vertically.

[0168] (9) Remove the gel and fix it on the electrophoresis device. Press down the glass plate firmly to prevent the inner tank from leaking. Add 1× protein electrophoresis solution to the inner tank and observe whether there is any leakage of electrophoresis solution. Adjust the tank until there is no leakage. Then add buffer solution to the outer tank before electrophoresis can be performed.

[0169] (10) Loading: Add samples to the loading wells in sequence, with a loading volume of 10 μl per well.

[0170] (11) Connect the power supply to 80 V. After the dye enters the lower separation gel, increase the voltage to 120 V. Stop electrophoresis when bromophenol blue moves to about 1 cm from the bottom of the separation gel.

[0171] (12) SDS-PAGE staining: Remove the glass plate from the electrophoresis mold, take out the separation gel, and observe the bands by Coomassie Brilliant Blue staining (refer to the reagent instructions for staining steps). 1) Put the separation gel into a container, add water and heat it in a microwave oven. Stop after boiling, place it on a shaker and shake for 5-10 minutes, and discard the aqueous solution. 2) Add rapid staining solution to completely cover the gel surface, heat it in a microwave oven, keep it in a boiling state for 30 seconds, then stop heating, shake it on a shaker for 10 minutes, and the Coomassie Brilliant Blue staining solution can be recycled. 3) Add decolorizing solution to immerse the gel, heat it in a microwave oven, repeat the above steps, replace the decolorizing solution until the target protein band is clearly visible, and observe the results. The preparation table of SDS-PAGE separation gel and concentrated gel is shown in Table 7:

[0172] Table 7

[0173]

[0174] 2.4.3 RT-qPCR identification of target RNA sequences contained in MS2 VLPs

[0175] RNA extraction and one-step RT-qPCR amplification were performed on the purified recombinant virus-like particles to identify whether the three target lncRNAs were successfully packaged in the prepared MS2VLPs. The reagent used was TaKaRa One Step Prime Script III RT-qPCR Mix, and the PCR system and amplification were referred to the instructions.

[0176] 2.4.3.1 Extraction of recombinant virus-like particles

[0177] The extraction reagent uses Tianlong Technology Ex-DNA / RNA virus (4.0) nucleic acid extraction or purification reagent. Add 20μl proteinase K and 200μl sample to the 1st or 7th column of the pre-packaged reagent respectively. Place stirring bars in the 2nd and 8th columns of the deep-well plate before running the program. Use GeneRotex96 nucleic acid extractor to perform automatic extraction according to the program in Table 8: The nucleic acid extraction program is shown in Table 9:

[0178] Table 8

[0179]

[0180]

[0181] 7 Washing 350:300:000:002000800120

[0182] Table 9

[0183]

[0184] After the nucleic acid extraction procedure is completed, transfer the eluate from columns 6 and 12 to clean enzyme-free tubes.

[0185] 2.4.3.2 Real-Time PCR

[0186] The one-step reaction system was configured according to the instruction manual of TAKARA One Step Prime Script III RT-qPCR Mix. The real-time fluorescence quantitative PCR reaction system is shown in Table 10.

[0187] Table 10 (Linc-MRPL33-1, linc-PIK3C2G-2, linc-ABCA5-3)

[0188]

[0189] Table 11

[0190]

[0191] 2.4.4 Identification of recombinant virus-like particles by transmission scanning electron microscopy

[0192] The morphology of virus-like particles was observed using a JEM-1400 Flash transmission electron microscope with an accelerating voltage of 80 kV and 2% phosphotungstic acid negative staining.

[0193] 2.5 Enzyme resistance test of MS2VLPs

[0194] DNaseI (200 U / ml) and RNaseA (10 mg / ml) were added to the purified recombinant virus-like particles and digested in a 37°C constant temperature incubator for 2 h. At the same time, RNA extracted from MS2VLPs and pACYC-MS2-lncRNAs recombinant plasmid were used as parallel controls.

[0195] 2.6 Detection of residual plasmid in MS2 VLPs

[0196] The RNA extracted from MS2 VLPs was used as a template. The extraction steps were as described in 2.4.3.1. The samples that needed to be reverse transcribed were transcribed according to TAKARA PrimeScript TM The configuration of RTreagentKitwithgDNAEraser (PerfectRealTime) is detailed in Table 10 and Table 11. It is set up into two groups, one for reverse transcription and the other for non-reverse transcription, and the pACYC-MS2-lncRNAs recombinant plasmid is set as the positive control and ddH2O as the negative control. The primer and probe sequences are shown in Table 1, and RT-qPCR is used to verify whether there is plasmid DNA residue in the recombinant virus-like particle solution.

[0197] (1) Genomic DNA removal: The genomic DNA removal system is shown in Table 12:

[0198] Table 12

[0199]

[0200] The probe-based qPCR analysis method can use a maximum of 2 μg of Total RNA; 42°C for 2 min, followed by cooling at 4°C.

[0201] (2) Reverse transcription reaction, probe RT-qPCR analysis method reverse transcription system configuration is shown in Table 13:

[0202] Table 13

[0203]

[0204]

[0205] The reverse transcription procedure was 37°C for 15 min; 85°C for 5 s; and cooling at 4°C.

[0206] (3) The two-step RT-qPCR reaction system is shown in Table 14:

[0207] Table 14

[0208]

[0209] (4) The two-step RT-qPCR reaction procedure is shown in Table 15.

[0210] Table 15

[0211]

[0212] 2.7 Digital PCR determination

[0213] Digital PCR (dPCR) is a newly developed absolute quantitative detection technology for nucleic acid samples. Its experimental steps mainly include PCR amplification and fluorescence signal detection. The reaction program will first dilute the template and distribute the nucleic acid to each independent reaction unit. After the PCR reaction of the template molecule in the unit is completed. The copy number of the sample is calculated by endpoint detection, which does not rely on real-time monitoring of the amplification curve, so there is no need to use internal reference and standard curve for relative quantification and absolute quantification.

[0214] Currently, there are two main dPCR analysis systems: microfluidics and microdroplets. Microfluidics chip technology divides the sample fluid into tens of thousands of independent reaction units through valves on the chip, and then separates the sample to be tested and performs PCR amplification. Microdroplet digital PCR forms oil-in-water microdroplets containing nucleic acid molecules, each droplet represents an independently dispersed sample carrier. After PCR amplification, the fluorescent signal contained in each oil-in-water droplet is detected by a microdroplet analyzer.

[0215] The TD-1 digital PCR system used in the present invention is the droplet dPCR technology of the above two dPCR methods. The channels for detecting target genes using the probe method in this system only contain two channels, FAM and VIC. Therefore, probes that meet the channel detection requirements must be selected for the three target genes.

[0216] The channels used for the three target genes linc-MRPL33-1, linc-PIK3C2G-2, and linc-ABCA5-3 in the digital PCR determination of the present invention are VIC, VIC, and FAM, respectively. The three target genes are tested three times, the dPCR scatter plot is observed, and finally the data is exported for analysis. The specific operation steps are as follows:

[0217] (1) After diluting the purified MS2 VLPs 10,000-fold with PBS buffer, RNA in the MS2 VLPs was extracted according to 2.4.3.1.

[0218] (2) Use the TD-1 digital PCR system to determine the value. The specific steps are as follows:

[0219] 1) Reagent preparation: fully dissolve premix A and premix B, primers, probes and DEPC water (negative wells need to be set up, DEPC water is used for negative well templates, and the rest of the system remains unchanged). Prepare the dPCR system as shown in Table 16:

[0220] Table 16

[0221]

[0222] 2) Microdroplet generation: Place the microdroplet generation chip into the microdroplet generation chip adapter, cover the adapter cover, add 30 μl of the aqueous phase system and 160 μl of the generation oil to the aqueous phase solution well and the oil phase solution well of the chip in sequence (add the aqueous phase reaction system first, then the microdroplet generation oil), and cover the microdroplet chip sealing gasket.

[0223] 3) Turn on the computer and the DropMarker sample preparation instrument, put the 8-tube strip into the machine, and put the adapter into the corresponding position of the sample preparation instrument, press down the handle, cover the machine, and click the DropMarker software on the computer desktop to run the instrument.

[0224] 4) PCR amplification: After the microdroplet generation program is completed, the 8-tube strip is capped and taken out and placed in a common PCR amplification instrument. There is no need to collect fluorescence. The dPCR amplification program conditions are set as shown in Table 17:

[0225] Table 17

[0226]

[0227] 5) Turn on the ChipReader biochip analyzer and place the 8-tube strip containing the sample to be tested after PCR amplification into the micro-droplet detection adapter.

[0228] 6) Install the micro-droplet detection chip into the detection adapter and press the chip to ensure that the chip punctures the 8-row tube cover.

[0229] 7) Close the adapter, press the buckle, add 430μl and 500μl of detection oil to the first and second rows of chip holes respectively, cover the 8-strip sealing gasket cover on the micro-droplet chip, put it into the instrument, press the handle, and close the instrument cover.

[0230] 8) Click the ChipReader software on the computer desktop to start the detection program;

[0231] 9) After the chip detection is completed, perform data analysis, calculate the copy number, check the 1D scatter plot and 2D scatter plot of each sample and negative well, and export the data.

[0232] 2.8 Evaluation of long-term stability of recombinant virus-like particles

[0233] (1) Sample dilution: MS2 VLPs were diluted with PBS at three different dilutions (approximately 108 copies / ml, 106 copies / ml, and 104 copies / ml).

[0234] 1) Extract RNA from MS2 VLPs. Refer to 2.4.3.1 for extraction steps.

[0235] 2) Digital PCR determination, see step 2.7 for details.

[0236] 3) Based on the RNA copy number, MS2VLPs were diluted to three different concentrations to comprehensively evaluate the stability of MS2VLPs as standards and quality controls.

[0237] (2) Sample packaging: Wipe and disinfect the biosafety cabinet in advance and expose it to ultraviolet light for a sufficient period of time before starting the operation. Since samples with higher concentrations are very likely to contaminate samples with lower concentrations, samples with lower concentrations should be packaged first.

[0238] 1) Use centrifuge tubes without DNaseI and RNaseA to dispense different concentrations of MS2VLPs in a biosafety cabinet, 1 ml per tube, first dispense the lower concentration group, then the higher concentration group, to avoid high concentration samples contaminating low concentration samples.

[0239] 2) Storage conditions: Three groups of concentration samples, the temperature was set to -20℃, 4℃, 25℃ and 37℃, a total of 4 temperatures, and the storage time period was 1 day, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 8 weeks, a total of 7 time periods (10 4 The samples with less than 10 copies / ml were only placed for 4 weeks), and the specific conditions are shown in Table 16. The stability study of MS2 VLPs is shown in Table 18:

[0240] Table 18

[0241]

[0242] 3) After the storage time is reached, the samples are collected and placed in a -80°C refrigerator. Each sample is subsequently tested and compared with the original sample placed in the refrigerator in advance.

[0243] 4) The extraction steps are detailed in 2.4.3.1, and the detection is performed using Takara One Step Prime Script III RT-qPCR Mix reagent.

[0244] result

[0245] 1 Construction of prokaryotic expression vector of MS2 bacteriophage virus-like particles containing three LncRNAs

[0246] pACYC-MS2 and pACYC-MS2-lncRNAs recombinant plasmids were constructed and synthesized by General Biosystems (Anhui) Co., Ltd. pACYC-MS2 was identified by double restriction digestion with BamHI / NotI, while pACYC-MS2-lncRNAs recombinant plasmid was identified by double restriction digestion with FseI / PacI. The band size detected by agarose gel electrophoresis was consistent with the expected value. Partial sequencing of pACYC-MS2 recombinant plasmid is shown in Figure 2 , pACYC-MS2-lncRNAs recombinant plasmid was partially sequenced as shown in Figure 3 , sequencing was correct and the recombinant plasmid was constructed successfully. Figure 2 It is a partial sequencing map of the pACYC-MS2 plasmid of the present invention. Figure 3 This is a partial sequencing diagram of the pACYC-MS2-lncRNAs plasmid of the present invention.

[0247] 2 Expression and purification of recombinant virus-like particles

[0248] After the PACYC-MS2-lncRNAs recombinant plasmid was transferred into E. coli BL21 (DE3) for induction of expression, the supernatant containing MS2VLPs was subjected to ultrasonic disruption and residual nucleic acids were digested with high concentrations of DNaseI and RNaseA. The MS2VLPs were purified by a combination of PEG enrichment and concentration and molecular sieve chromatography. A purification peak diagram was made based on the absorbance and volume during the purification process, such as Figure 4 As shown, the eluate corresponding to the first peak was collected. At this time, the absorbance at 280 nm and 215 nm were both maximum values, indicating that the content of the target molecule and the protein shell was the highest here, which was the recombinant virus-like particle prepared this time. Figure 4 MS2VLPs molecular sieve chromatography purification peak diagram;

[0249] 3. Identification of recombinant virus-like particles

[0250] 3.1 Identification of RT-PCR amplification products of MS2 VLPs by agarose gel electrophoresis

[0251] MS2 VLPs were amplified by RT-PCR and then identified by 2% agarose gel electrophoresis. With reference to DNA Marker I, bright bands were observed at 94 bp, 90 bp, and 162 bp. Figure 5 , which was consistent with the expected amplified fragment size of the three target genes Linc-MRPL33-1, linc-ABCA5-3 and linc-PIK3C2G-2, and the results were in line with expectations. Figure 5It is a 2% agarose gel electrophoresis diagram of the present invention. Note: M: Marker; 2 is Linc-MRPL33-1 (94 bp); 4-5 are linc-ABCA5-3 (90 bp); 7-8 are linc-PIK3C2G-2 (162 bp); 3, 6, 9 are negative control diagrams.

[0252] 3.2 Identification of recombinant virus-like particles by SDS-PAGE electrophoresis

[0253] During the purification process, 6 tubes of eluate were collected from the components corresponding to the first elution peak. All 6 tubes of eluate were subjected to SDS-PAGE electrophoresis. The results showed that there was an obvious band at 14kDa in 6 tubes without interference from other bands, which was the purified MS2VLPs. The MS2VLPs bands in the second and third tubes were darker, indicating that their concentration was the highest. Figure 6 , the results are consistent with expectations.

[0254] Figure 6 The figure is the SDS-PAGE electrophoresis diagram of the MS2 VLPs of the present invention; M: pre-stained protein marker; 6 is the 6th tube of eluate, i.e., MS2 VLPs.

[0255] 3.3 RT-qPCR identification of target RNA sequences contained in MS2 VLPs

[0256] Based on the previous research of the research group, the RNA packaged in MS2VLPs was extracted and purified according to step 2.4.3.1, and identified by one-step RT-qPCR. Figure 7 ,RT-qPCR results showed that the prepared recombinant virus-like particles were able to amplify three target lncRNA sequences after extraction and amplification, and the Ct value was small, indicating a high concentration, proving that the target sequence was correctly packaged in the recombinant virus-like particles.

[0257] Figure 7 This is a diagram of RNA packaged in MS2 VLPs verified by RT-qPCR of the present invention; Note: linc-MRPL33-1 (red); linc-PIK3C2G-2 (green); linc-ABCA5-3 (blue).

[0258] 3.4 Identification of recombinant virus-like particles by transmission scanning electron microscopy

[0259] The morphology of MS2 VLPs was observed using a JEM-1400 Flash transmission electron microscope at an accelerating voltage of 80 kV and negative staining with 2% phosphotungstic acid at a magnification of 120,000×. Figure 8As shown, MS2VLPs are spherical under the microscope, with uniform size, complete morphological structure and a diameter of about 26nm. Observation under an electron microscope shows that MS2VLPs are densely distributed, and too high concentration causes a small number of them to aggregate into clusters. The results are consistent with expectations. Figure 8 Schematic diagram of MS2VLPs electron microscopy results.

[0260] In summary, the results of the four identification methods, namely agarose gel electrophoresis, SDS-PAGE, RT-qPCR and transmission scanning electron microscopy, all proved that MS2VLPs were successfully prepared and three specific target genes were correctly packaged.

[0261] 4. Enzyme resistance test of recombinant virus-like particles

[0262] Armor RNA technology uses the characteristics of MS2 bacteriophage to package itself into a pseudovirus structure, and encapsulates the three specific target lncRNA fragments involved in this experiment into the protein shell. The virus-like particles made in this way can monitor the entire process and can withstand digestion by DNaseI and RNaseA. After long-term treatment with these two enzymes, the exposed DNA and RNA are degraded, while the RNA encapsulated by VLPs is not affected. Fig. 9 shown. Fig. 9 The electrophoresis diagram of the enzyme resistance verification result of MS2VLPs of the present invention. Note: A.linc-MRPL33-3; B.linc-PIK3C2G-2; C.linc-ABCA5-3; M: 600bpMarker; 2 is MS2VLPs; 3-4 are pACYC-MS2-lncRNAs; 5-6 are RNA extracted from MS2VLPs; 1, 3, 5 are not treated with enzymes; 2, 4, 6 are treated with enzymes.

[0263] 5. Detection of residual plasmid in recombinant virus-like particles

[0264] Using RNA extracted from recombinant virus-like particles as template, the reverse transcribed and non-reverse transcribed templates were detected simultaneously to amplify three target lncRNAs. The pACYC-MS2-lncRNAs recombinant plasmid was set as a positive control, and ddH2O was set as a blank control. Fig.10 The Ct values ​​of genes linc-MRPL33-1, linc-PIK3C2G-2, and linc-ABCA5-3 in the reverse transcribed samples were 24.78, 24.15, and 22.62, respectively, while the Ct values ​​of fluorescence quantitative PCR without reverse transcription were 36.82, 37.98, and 36.97, respectively, indicating that there was almost no plasmid DNA residue in MS2VLPs, and the results were consistent with expectations.

[0265] Fig.10The figure is a fluorescence quantitative PCR detection result of residual plasmids in MS2VLPs of the present invention; A. linc-MRPL33-1 (red); B. linc-PIK3C2G-2 (green); C. linc-ABCA5-3 (blue); 1: positive control; 2: reverse transcribed sample; 3: non-reverse transcribed sample; 4. negative control.

[0266] 6 Digital PCR quantification results of MS2VLPs

[0267] The purified MS2VLPs were diluted 10,000 times with PBS buffer and used as the standard for determination. After determination, they were used in subsequent experiments. RNA was extracted from MS2VLPs, and the TD-1 digital PCR system was used to determine the value of the three target genes three times, and the results were averaged.

[0268] 6.1 Scatter plot and results of dPCR detection of Linc-MRPL33-1 gene in MS2 VLPs standard products Scatter plot and results of dPCR detection of Linc-MRPL33-1 gene in MS2 VLPs standard products Fig.11 and Table 17. The 2D scatter plots of the three tests showed that the positive droplets (green) containing the Linc-MRPL33-1 gene were clearly distinguished from the negative droplets (gray), and the results were consistent with expectations.

[0269] Fig.11 This is a 2D scatter plot of the dPCR detection of the gene linc-MRPL33-1 of the present invention; green: linc-MRPL33-1 positive droplets; gray: negative droplets. The results of the dPCR detection of the gene Linc-MRPL33-1 are shown in Table 19:

[0270] Table 19

[0271]

[0272] 6.2 Scatter plot and results of linc-PIK3C2G-2dPCR detection in MS2VLPs standard

[0273] The scatter plot and results of dPCR detection of gene linc-PIK3C2G-2 in MS2VLPs standard are shown in Fig.12 and Table 18. The 2D scatter plots of the three tests showed that the positive droplets (green) and negative droplets (gray) containing the linc-PIK3C2G-2 gene were clearly distinguished, which was consistent with expectations.

[0274] Fig.12This is a 2D scatter plot of the linc-PIK3C2G-2dPCR detection of the gene of the present invention; green: linc-PIK3C2G-2 positive droplets; gray: negative droplets. The results of the linc-PIK3C2G-2dPCR detection of the gene are shown in Table 20:

[0275] Table 20

[0276]

[0277]

[0278] 6.3 Scatter plot and results of 3dPCR detection of gene linc-ABCA5 in MS2 VLPs standard

[0279] The scatter plot and results of 3dPCR detection of gene linc-ABCA5 in MS2VLPs standard are shown in Fig.13 and Table 19. The 2D scatter plots of the three tests showed that the positive droplets (blue) containing the linc-ABCA5-3 gene were clearly distinguished from the negative droplets (gray), which was consistent with expectations. Fig.13 This is a 2D scatter plot of the linc-ABCA5-3dPCR detection of the gene of the present invention; blue: linc-ABCA5-3 positive droplets; gray: negative droplets. The results of the linc-ABCA5-3dPCR detection of the gene are shown in Table 21:

[0280] Table 21

[0281]

[0282] 7 Long-term stability experiment of recombinant virus-like particles

[0283] The standard was diluted into three concentrations (about 108 copies / ml, 106 copies / ml and 104 copies / ml), and the three concentrations of MS2VLP were placed at four temperatures of -20℃, 4℃, 25℃ and 37℃ for 1d, 3d, 1w, 2w, 3w, 4w and 8w respectively (104 copies / ml samples were only placed for 4 weeks). After the placement time, the samples were stored at -80℃, and all samples were tested uniformly after the placement was completed. The stability results of Linc-MRPL33-1, linc-PIK3C2G-2 and linc-ABCA5-3 in MS2VLPs are shown in Figure 14. The high-concentration samples have better stability and are more conducive to long-term storage. The MS2VLPs prepared this time can be stably stored at -20℃ for two months, meeting the stability requirements of quality control products.

[0284] FIG. 14 shows the stability evaluation results of three target genes (linc-MRPL33-1, linc-PIK3C2G-2 and linc-ABCA5-3) in the MS2 VLPs of the present invention.

[0285] Establishment of a multiplex RT-qPCR TaqMan probe quantitative detection system for Linc-MRPL33-1, linc-PIK3C2G-2 and linc-ABCA5-3

[0286] The research team evaluated the expression stability of 7 commonly used internal reference genes (RPLP0, β-actin, GAPDH, B2M, CYC, GUSB, 18SrRNA) in plasma in the early stage, and finally determined that the internal reference gene with better stability in plasma was human large ribosomal protein (RPLPO). Therefore, on this basis, the present invention uses RPLPO as an endogenous gene with better stability, and uses multiple RT-qPCR to detect lncRNA and endogenous internal reference in the same tube, so as to monitor the lncRNA extraction and amplification processes, thereby making up for the deficiency that the test sample and the standard cannot monitor the detection process in the lncRNA tube due to the separate detection of the test sample and the standard.

[0287] Therefore, in this experiment, specific primers and oligonucleotide probes were designed for the internal reference RPLPO and the three target lncRNAs, and a TaqMan probe RT-qPCR one-step single detection system was established for each gene; each single system was then optimized in terms of annealing temperature, template concentration, primer concentration and probe concentration to construct a TaqMan probe one-step multiplex fluorescence quantitative PCR detection method. The constructed multiplex RT-qPCR TaqMan probe detection method was applied, and MS2VLPs were used as standard and quality control products. The copy numbers of the three lncRNAs in the plasma of lung cancer patients, the plasma of patients with benign lung diseases, and the plasma of apparently healthy people were calculated by drawing standard curves. Taking clinical diagnosis as the gold standard, the diagnostic efficacy of the three target lncRNAs detected separately and in combination based on the absolute quantitative system was evaluated by drawing ROC curves to understand the clinical application value of the absolute quantitative detection system.

[0288] Establishment of a multiplex RT-qPCR TaqMan probe quantitative detection system for Linc-MRPL33-1, linc-PIK3C2G-2 and linc-ABCA5-3

[0289] Materials and Methods

[0290] 1. Experimental Materials

[0291] 1.1 Main experimental instruments

[0292] Table 22

[0293]

[0294]

[0295] 1.2 Main experimental reagents

[0296] Table 23

[0297]

[0298] 2. Experimental Methods

[0299] 2.1 Ethical issues and specimen collection and processing

[0300] The sample collection and testing involved in this invention have been approved by the Ethics Review Committee of this unit, Ethics No.: (2022) Lunshen L No. 222. The subjects are kept confidential and the relevant national laws and regulations are strictly observed.

[0301] The plasma specimens used in the present invention were collected from the Department of Thoracic Surgery and the Department of Respiratory Medicine of this hospital from April 2022 to November 2022. The plasma samples of the patients before surgical treatment were collected, transferred to RNase-free EP tubes, and stored in a -80°C refrigerator for future use. The inclusion criteria are: (1) Lung cancer group: The patient's pathological biopsy diagnosis was NSCLC; no treatment (including surgery, immunotherapy, radiotherapy, chemotherapy, etc.) before surgery; no other malignant tumors, chronic wasting diseases (such as diabetes) and immune diseases (including systemic lupus erythematosus, rheumatoid arthritis, hyperthyroidism, etc.) were combined.

[0302] (2) Benign lung disease group: patients with pathological diagnosis of benign lung tumors; or diagnosed with other benign lung diseases (such as emphysema, pneumonia, etc.), without other malignant tumors, other immune diseases and wasting diseases. (3) Apparently healthy group: chest and lung CT and routine laboratory test results showed no obvious abnormalities, without other malignant tumors, wasting diseases (such as diabetes) and immune diseases (such as rheumatoid arthritis).

[0303] 2.2 RNA extraction from MS2 VLPs standards and plasma samples

[0304] The specimens were taken out of the -80℃ freezer and thawed at room temperature. The extraction reagent used was Tianlong Technology Ex-DNA / RNA virus (4.0) nucleic acid extraction or purification reagent. 20μl proteinase K and 200μl sample were added to the 1st or 7th column of the pre-packaged reagent respectively. Stirring bars were placed in the 2nd and 8th columns of the deep-well plate before the program was run. The GeneRotex96 nucleic acid extractor automated program extraction steps were used for automated extraction according to the program in Table 24:

[0305] Table 24

[0306]

[0307] After the nucleic acid extraction procedure is completed, transfer the RNA from column 6 or column 12 (pay attention to the effective well position) to an enzyme-free tube.

[0308] 2.3 Optimization of experimental conditions for target lncRNA fluorescence quantitative PCR

[0309] 2.3.1 Primers and probes for target lncRNA

[0310] To facilitate the construction of a multiplex RT-qPCR detection system, the reporter groups of the probes were labeled with four fluorescent dyes to distinguish them, and the quencher groups were labeled with BHQ (Black Hole Quencher) to improve the detection sensitivity. The primer and probe powders were centrifuged before opening the lid, carefully opened, and the corresponding TE buffer was added to a concentration of 100 μM. The aliquots were stored at -20°C (the fluorescent probes needed to be stored away from light), and before use, they were taken out and diluted to a concentration of 10 μM to avoid repeated freezing and thawing. The primer and probe sequences of the target lncRNA and internal reference are shown in Table 25:

[0311] Table 25

[0312]

[0313] 2.3.2 Annealing temperature optimization

[0314] Annealing temperature has a great influence on the sensitivity and specificity of PCR reaction. Within a certain temperature range, when the annealing temperature is higher, the specificity of PCR reaction can be improved, while the sensitivity is reduced. In order to obtain the best annealing temperature, this experiment performs gradient PCR amplification within 5°C range according to the Tm value of each gene primer. After the reaction is completed, the product is verified by 2% agarose gel electrophoresis to find the most suitable annealing temperature for constructing multiple PCR.

[0315] (1) Sample extraction refers to step 2.2.

[0316] (2) cDNA synthesis: FastKing cDNA first strand synthesis kit was used, the configuration system is shown in Table 25, and the reaction was carried out at 42°C for 3 min. The g DNA removal reaction system is shown in Table 26:

[0317] Table 26

[0318] Element volume 5×gDNABuffer 2μl Total RNA <![CDATA[RNase-FreeddH2O]]> up to 10 μl

[0319] (3) The reverse transcription mixture preparation system is shown in Table 27.

[0320] Table 27

[0321]

[0322] (4) Add the mixed solution prepared in Table 23 to the reaction system after gDNA removal and mix thoroughly.

[0323] (5) Maintain at 42°C for 15 min; incubate at 95°C for 3 min. After reverse transcription, cDNA is obtained and can be used for subsequent experiments or stored at -20°C for future use.

[0324] (6) Ordinary PCR reaction: Thaw the 2×TaqPCR premix reagent at room temperature in advance, mix well, and prepare the reaction system according to Table 27. Note that the mixed solution should be prepared first, and then the template should be added after it is dispensed into eight tubes. The ordinary PCR reaction system is shown in Table 28:

[0325] Table 28

[0326]

[0327] (7) The amplification procedure is as follows:

[0328] The general PCR reaction program is shown in Table 29:

[0329] Table 29

[0330]

[0331] (8) 2% agarose gel electrophoresis:

[0332] 1) Add 1g agarose and 50ml 1×TAE to a conical flask and heat in a microwave oven to dissolve it. Cool to about 60℃ and add 3μl nucleic acid dye. Mix immediately and pour into the electrophoresis mold. Wait for about 20 minutes for it to completely solidify.

[0333] 2) Place the gel in a horizontal electrophoresis tank with the sample well facing the negative electrode, and pour TAE buffer over the gel surface.

[0334] 3) Sample loading: Take 5 μl of nucleic acid sample and add 1 μl of 6× loading buffer, mix well, and add all to the loading well.

[0335] 4) Set the voltage to 120 V and run the electrophoresis for about 30 min. Observe the results using a gel imaging system and take photos for preservation.

[0336] 2.3.3 Template concentration optimization

[0337] Select an example with a concentration of about 10 4For samples with 100 copies / ml, extract RNA according to step 2.2, and add 0.5μl, 1.0μl, 1.5μl, 2.0μl, 2.5μl, and 3.0μl to the 25μl system. Record the template concentration corresponding to the minimum Ct value. Configure the reaction system according to Table 30. The RT-qPCR template concentration optimization reaction system is shown in Table 31:

[0338] Table 30

[0339]

[0340] The one-step RT-qPCR reaction procedure is shown in Table 31:

[0341] Table 31

[0342]

[0343]

[0344] 2.3.4 Primer concentration optimization

[0345] Set 6 primer concentration gradients to observe the Ct value: 0.25μl, 0.5μl, 0.75μl, 1.0μl, 1.25μl, 1.5μl, and add the corresponding unidirectional primers to the 25μl system, that is, the final reaction concentrations of the primers are 0.1μM, 0.2μM, 0.3μM, 0.4μM, 0.5μM, and 0.6μM, respectively. The other conditions are the same, and the primer concentration corresponding to the minimum Ct value is recorded. Configure the reaction system according to Table 31, and the amplification program is shown in Table 31. The RT-qPCR primer concentration optimization reaction system is shown in Table 32:

[0346] Table 32

[0347]

[0348] 2.3.5 Probe concentration optimization

[0349] First, set up 6 probe concentration gradients to observe the Ct value. Add 0.25μl, 0.5μl, 0.75μl, 1.0μl, 1.25μl, and 1.5μl of probe into the 25μl system, respectively. That is, the final reaction concentrations of the probes are 0.1μM, 0.2μM, 0.3μM, 0.4μM, 0.5μM, and 0.6μM, respectively. The configuration of the RT-qPCR reaction system is shown in Table 200. The amount of probe corresponding to the minimum Ct value is recorded. Since the content of lncRNA in plasma is very small, the Ct value fluctuation caused by the change of probe concentration is more sensitive, so it is necessary to further reduce the probe concentration to optimize the system. Add 0.05μl, 0.1μl, 0.15μl, 0.2μl, and 0.25μl to the 25μl system respectively. The final reaction concentrations of the probe are 0.02μM, 0.04μM, 0.06μM, 0.08μM, and 0.1μM respectively. The other conditions are the same as before. The amplification program is shown in Table 33:.

[0350] Table 33

[0351]

[0352]

[0353] 2.3.6 Determination of the multiplex RT-qPCR TaqMan probe absolute quantitative detection system

[0354] Based on the constructed one-step single amplification detection system, a multiplex PCR system was established according to the optimized conditions, so that three target lncRNAs and the endogenous gene RPLPO can be detected simultaneously in one amplification tube, and a TaqMan probe one-step multiplex fluorescence quantitative PCR detection method is constructed.

[0355] 1. Optimization of lncRNA fluorescence quantitative PCR experimental conditions

[0356] result

[0357] 1.1 Annealing temperature optimization results

[0358] The gradient PCR function of the PCR instrument was used to set the annealing temperature of each gene primer within the range of 5°C above and below the respective Tm values, and 2% agarose gel electrophoresis was performed to verify the completion of PCR amplification. In order to construct a multiplex PCR reaction system, all genes are required to have good amplification effects. When determining the most suitable annealing temperature, a common temperature with obvious band brightness, no dimer formation, and good amplification effects of each gene was selected. The results showed that the amplification products of linc-MRPL33-1, linc-PIK3C2G-2, linc-ABCA5-3, and RPLPO were around 94bp, 162bp, 90bp, and 122bp, respectively, which met the expected product lengths, proving that the primer design was successful. Each gene can obtain a good amplification effect within the set temperature range. Considering the sensitivity and specificity of amplification, 60°C was selected as the common optimal annealing temperature for constructing one-step multiplex RT-qPCR. The annealing temperature optimization electrophoresis results of the PCR products of each gene are shown in Figure 14.

[0359] Figure 14 is a gradient PCR electrophoresis diagram of the present invention; A. linc-MRPL33-1 gradient PCR electrophoresis B. linc-PIK3C2G-2 gradient PCR electrophoresis; C. linc-ABCA5-3 gradient PCR electrophoresis D. RPLPO gradient PCR electrophoresis.

[0360] 1.2 Template concentration optimization results

[0361] 0.5μl, 1μl, 1.5μl, 2μl, 2.5μl, and 3μl of RNA samples were added to the 25μl system, respectively. The results showed that the three target lncRNAs and the internal reference first showed fluorescent signals (the smaller the Ct value) when the template concentration was higher. According to the instructions of the kit, the amount of RNA sample added should be less than 1 / 10 of the total reaction solution (that is, a maximum of 2.5μl RNA template can be added to the 25μl system), and the ideal addition range is 10pg-1μg. If the amount used exceeds 1 / 10 of the total reaction solution due to the low concentration of the target RNA, it may have an inhibitory effect on the RT-qPCR reaction. In addition, the one-step multiplex RT-qPCR system requires a large number of components such as primers and probes to be added, and the total system should not exceed 25μl. After comprehensive consideration, it was finally decided to add an RNA template volume of 2.5μl. See Fig.15 . Fig.15 is a template concentration optimization diagram of the present invention; Fig.15 A is the linc-MRPL33-1 template concentration optimization diagram of the present invention; 15B is the linc-PIK3C2G-2 template concentration optimization diagram of the present invention; 15C is the linc-ABCA5-3 template concentration optimization diagram of the present invention; 15D is the RPLPO template concentration optimization diagram of the present invention.

[0362] 1.3 Primer concentration optimization results

[0363] When optimizing the primer concentration for each gene, 0.25 μl, 0.5 μl, 0.75 μl, 1.0 μl, 1.25 μl, and 1.5 μl of forward primer and reverse primer were added to the 25 μl system, respectively, that is, the final reaction concentrations of the primers were 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, and 0.6 μM, respectively. The results showed that the three target genes linc-MRPL33-1, linc-PIK3C2G-2, linc-ABCA5-3, and the internal reference RPLPO all first showed fluorescent signals when the primer concentration was 0.1 μM, and the Ct value was the lowest, that is, 0.25 μl of the unidirectional primer of each gene was added to the 25 μl reaction system. The PCR amplification curve results are shown in Fig.16 . Fig.16 This is a primer concentration optimization diagram of the present invention; 16A. linc-MRPL33-1 primer concentration optimization B. linc-PIK3C2G-2 primer concentration optimization, C. linc-ABCA5-3 primer concentration optimization D. RPLPO primer concentration optimization.

[0364] 1.4 Probe concentration optimization results

[0365] When 0.25 μl, 0.5 μl, 0.75 μl, 1.0 μl, 1.25 μl, and 1.5 μl of the probe were added to the 25 μl system, that is, when the final reaction concentrations of the probe were 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, and 0.6 μM, respectively, the three target lncRNAs and the internal reference RPLPO first showed fluorescence signals when the probe concentration was 0.1 μM, that is, 0.25 μl of the probe was added to the reaction system, as shown in Fig.17 . Further, 0.05μl, 0.1μl, 0.15μl, 0.2μl, and 0.25μl of probe were added to the 25μl system, that is, the final reaction concentrations of the probes were 0.02μM, 0.04μM, 0.06μM, 0.08μM, and 0.1μM, respectively. The results showed that the three target lncRNAs and the internal reference RPLPO first showed fluorescence signals when the probe concentration was 0.02μM, and the Ct value was the smallest. That is, it was finally determined that 0.05μl of probe was added to the reaction system, see Fig.18 .

[0366] Fig.17 It is a probe concentration exploration diagram of the present invention; Fig.17 A is a concentration exploration diagram of the linc-MRPL33-1 probe of the present invention; 17B is a concentration exploration diagram of the linc-PIK3C2G-2 probe of the present invention; 17C is a concentration exploration diagram of the linc-ABCA5-3 probe of the present invention; 17D is a concentration exploration diagram of the RPLPO probe of the present invention.

[0367] Fig.18 This is a probe low concentration optimization diagram of the present invention; Fig.18 A is a low concentration optimization diagram of the linc-MRPL33-1 probe of the present invention; Fig.18 B is the low-concentration optimization diagram of the linc-PIK3C2G-2 probe of the present invention; 18C is the low-concentration optimization diagram of the linc-ABCA5-3 probe of the present invention; 18D is the low-concentration optimization diagram of the RPLPO probe of the present invention.

[0368] 1.5 Determination of fluorescence quantitative PCR reaction system

[0369] By optimizing the reaction condition parameters, a one-step multiplex real-time fluorescence quantitative PCR amplification reaction system for lung cancer plasma specimen detection was determined. The constructed system was used to perform RT-qPCR experiments on plasma specimens. The amplification curves were as follows: Fig.19 .

[0370] Fig.19 It is a plasma sample multiple real-time fluorescence quantitative PCR amplification curve diagram of the present invention; Note: linc-MRPL33-1 (red); linc-PIK3C2G-2 (green); linc-ABCA5-3 (blue); RPLPO (orange), the optimized reaction system of plasma sample multiple real-time fluorescence quantitative PCR is shown in Table 34:

[0371] Table 34

[0372]

[0373] The MS2VLPs standard and plasma samples were extracted and amplified simultaneously using the constructed one-step multiplex RT-qPCR TaqMan probe absolute quantitative detection system. The expression levels of the three target lncRNAs in the plasma of lung cancer group, benign lung disease group and healthy group were determined by drawing standard curves, and the internal reference genes were monitored in the same tube for plasma samples. The results showed that compared with the healthy group, linc-PIK3C2G-2, linc-ABCA5-3 and Linc-MRPL33-1 were highly expressed in the lung cancer group (P<0.0001) and the benign lung disease group (P<0.001). Compared with the benign lung disease group, the three target genes were also highly expressed in the lung cancer group (P<0.05). Subsequently, the ROC curves of independent detection and combined detection were drawn according to the expression levels of the three lncRNAs. The results showed that the three lncRNAs had the potential to distinguish NSCLC patients from healthy people, and the diagnostic efficiency of combined detection was better than that of single detection.

[0374] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for constructing a multiplex RT-qPCR TaqMan probe absolute quantitative detection system, characterized in that The steps include: (1) Using armor RNA technology, BL21(DE3) expression Escherichia coli was infected with MS2 phage, and the capsid protein and mature enzyme protein coding sequences were reverse transcribed after extracting the MS2 phage genomic RNA, and inserted into the MCS1 site of the pACYCDuet-1 prokaryotic expression vector to construct the pACYC-MS2 recombinant plasmid; (2) Total RNA was extracted from lung cancer tissue specimens 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. Finally, a recombinant plasmid was constructed, which contained the mature enzyme protein and capsid protein coding sequences of MS2 in MCS1, the packaging site sequence of MS2, and the three candidate lncRNA target amplification sequences in tandem, and was named pACYC-MS2-lncRNAs. The target amplification sequences of the three candidate lncRNAs were connected in sequence and cloned into the pACYCDuet-1 plasmid. (3) Sequencing to verify the accuracy of the pACYC-MS2-lncRNAs recombinant plasmid: pACYC-MS2-lncRNAs was transformed into BL21 (DE3) Escherichia coli and induced for expression, PEG enrichment, molecular sieve chromatography purification of MS2 VLPs, and verification and performance evaluation; (4) The internal reference gene RPLPO was used to monitor the lncRNA detection process and to construct a one-step multiplex RT-qPCR TaqMan probe detection system; (5) Establish a multiplex RT-qPCR TaqMan probe absolute quantitative detection system.

2. The method for constructing the multiplex RT-qPCR TaqMan probe absolute quantitative detection system according to claim 1, characterized in that: In step (1), the lncRNA target amplification sequences are Linc-PIK3C2G-2, Linc-ABCA5-3, and Linc-MRPL33-1; 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; 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 shown in SEQ ID No.

6. The nucleotide sequence of the upstream primer of Linc-MRPL33-1 is shown in SEQ ID No.7, the nucleotide sequence of the downstream primer of Linc-MRPL33-1 is shown in SEQ ID No.8, and the nucleotide sequence of the Probe probe of Linc-MRPL33-1 is shown in SEQ ID No.9; In step (4), the nucleotide sequence of the upstream primer of the internal reference gene RPLPO is shown in SEQ ID No.10, the nucleotide sequence of the downstream primer of the internal reference gene RPLPO is shown in SEQ ID No.11, and the nucleotide sequence of the Probe probe of the internal reference gene RPLPO is the nucleotide sequence shown in SEQ ID No.

12.

3. The method for constructing the multiplex RT-qPCR TaqMan probe absolute quantitative detection system according to claim 1, characterized in that: In step (1), the MS2 phage prokaryotic expression vector containing three lncRNAs was constructed. The pACYCDuet-1 expression vector developed by Novagen contains an epitope tag and is commonly used for gene expression in prokaryotic cells; pACYCDuet-1 has two multiple cloning sites, each MCS has a lactose operator, a T7 promoter and a ribosome binding site, and can express two genes together; the vector also carries a chloramphenicol resistance gene, which can be used for positive colony screening; the capsid protein and mature enzyme protein coding sequences of the MS2 phage genome are inserted into the MCS1 site of pACYCDuet-1 to construct the pACYC-MS2 recombinant plasmid.

4. The method for constructing the multiplex RT-qPCR TaqMan probe absolute quantitative detection system according to claim 1, characterized in that: In step (1), step 1 synthesizes the target fragment: referring to the three target genes and the genome sequence of MS2 phage in the GenBank database, artificially synthesizes the nucleic acid fragment of the MS2 phage packaging site and the three target lncRNA target amplification sequences in series.

5. The method for constructing the multiplex RT-qPCR TaqMan probe absolute quantitative detection system according to claim 1, characterized in that: In step (1), step 2 target fragment and vector connection: primers of MS2 phage maturase protein and capsid protein are amplified by PCR to obtain the target fragment PCR product, which is cloned into the MCS1 site (BamHI-NotI) of the pACYCDuet-1 plasmid to obtain the pACYC-MS2 recombinant plasmid.

6. The method for constructing the multiplex RT-qPCR TaqMan probe absolute quantitative detection system according to claim 1, characterized in that: In step (2), construct the pACYC-MS2-lncRNAs recombinant plasmid: The pACYC-MS2 recombinant plasmid was extracted, and the artificially synthesized MS2 phage packaging site and three target lncRNA target amplification sequences were cloned into the pACYC-MS2 recombinant plasmid to obtain the pACYC-MS2-lncRNAs recombinant plasmid, which was then connected, transformed, and verified.

7. The method for constructing the multiplex RT-qPCR TaqMan probe absolute quantitative detection system according to claim 1, characterized in that: In step (3), the pACYC-MS2-lncRNAs recombinant plasmid is transferred into Escherichia coli BL21 (DE3) to induce expression, and MS2 bacteriophage virus-like particles (MS2VLPs) are obtained after extraction, enrichment and purification. MS2VLPs are identified by agarose gel electrophoresis, SDS-PAGE electrophoresis, RT-qPCR experiment and transmission scanning electron microscopy. Then, the plasmid residues in the MS2VLPs solution are detected, and the residues are determined by digital PCR and used as standards and quality control products. The enzyme resistance and stability of MS2VLPs are evaluated.

8. The method for constructing the multiplex RT-qPCR TaqMan probe absolute quantitative detection system according to claim 1, characterized in that: In step (4), the internal reference gene RPLPO monitors the lncRNA detection process, and specific primers and TaqMan probes are designed for RPLPO and the three target lncRNAs, respectively, to establish a one-step RT-qPCR TaqMan probe detection single system. The single system of each gene is optimized in terms of annealing temperature, template concentration, primer concentration and probe concentration. First, the optimal single-plex detection system of each gene is determined, and then a one-step multiplex RT-qPCR TaqMan probe detection system combining the three target lncRNAs with the internal reference is constructed.

9. The method for constructing the multiplex RT-qPCR TaqMan probe absolute quantitative detection system according to claim 1, characterized in that: In step (5), based on the constructed multiplex RT-qPCR TaqMan probe detection method, a multiplex RT-qPCR TaqMan probe absolute quantitative detection system is established using digital PCR-valued MS2 VLPs as standards.

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