Protective solution for protecting cell-free supernatant free RNA (Ribonucleic Acid) of cytological specimen

Abstract

The invention discloses a protective solution for protecting cell-free supernatant free RNA (Ribonucleic Acid) of a cytology specimen, the protective solution is a guanidine isothiocyanate solution prepared by taking 1 * TEbuffer (10mMTrisHCL + 1mMEDTA (Potential of Hydrogen) = 8.0) as a solvent, and the concentration of the guanidine isothiocyanate solution is 1M-4M. The protective liquid is suitable for all cytological specimens, including pleural effusion, alveolar lavage fluid, sputum and puncture cytological suspension. By protecting cfRNA in a cytological specimen supernatant, the problems of low cfRNA concentration, low yield and easiness in degradation at present are solved, so that the liquid biopsy specimen cfDNAamp is realized; and the possibility of double-extraction and double-detection of cfRNA is reduced. By protecting the cfRNA of the cytological sample, the gene fusion detection sensitivity in NSCLC multi-gene detection is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a formula of cell free RNA (cfRNA) protection solution, which is used to extract cfRNA from cell-free supernatant of cytological specimens of patients with non-small cell lung cancer (NSCLC). The obtained cfRNA can be used to detect driver genes with gene fusion changes in NSCLC patients. Background Art

[0002] About 70% of NSCLC patients in my country have driver gene mutations that can be treated with targeted therapies. Genetic testing can be used to identify driver mutations and select targeted therapeutic drugs, which is the so-called "precision medicine". Domestic and foreign NSCLC diagnosis and treatment guidelines recommend that patients routinely undergo a group of gene change tests, including EGFR, KRAS, ALK, ROS1, BRAF, RET, Her-2, C-MET, NTRK1 / 2 / 3 and other genes, in order to select corresponding targeted drugs.

[0003] The specimens for relevant driver gene testing of NSCLC patients are tumor tissue or tumor cytology specimens, but 2 / 3 of NSCLC are discovered in the late stage. Clinical testing faces the problem that some patients cannot obtain tumor specimens or the tumor specimens are not suitable for genetic testing. Therefore, liquid biopsy technology, that is, the use of peripheral blood for driver gene testing, came into being. However, blood sample testing has many problems such as low sensitivity and inability to perform quality control before testing.

[0004] Cytology is an important specimen type for pathological diagnosis of NSCLC patients. There are various types of cytological specimens that can be used for pathological diagnosis, including pleural effusion, sputum, alveolar lavage fluid, cerebrospinal fluid, puncture cytological suspension, etc. Such specimens can be divided into three types: high tumor cell content, low tumor cell content and no tumor. Our previous research and testing work showed that specimens with high tumor cell content can be directly used for driver gene testing using tumor cells through preparation or cytological wax blocks, while specimens with low tumor or no tumor can try to use the cell-free supernatant of cytological specimens to extract cfDNA and complete the test using liquid biopsy technology.

[0005] The use of cell-free supernatant cfDNA from cytological specimens for driver gene testing has its advantages: 1) The concentration of cfDNA obtained is high, which can meet the requirements of PCR-based and next generation sequencing (NGS) technology testing; 2) cfDNA quality is stable, not easily degraded, and can be stored for a long time. However, the problems of cfDNA in multi-gene testing are also prominent. The detection sensitivity of fusion-type altered genes is not high (about 50%), and the detection accuracy needs to be improved, which affects its clinical application value.

[0006] Among the driver genes carried by NSCLC patients, ALK, ROS1, RET, and NTRK1 / 2 / 3 are all fusion-type gene changes. The application of RNA level detection for fusion-type changes can greatly improve its detection sensitivity, but there are two problems with the cell-free RNA in the supernatant of cytological specimens: 1) The concentration and amount of cfRNA extracted are low and cannot be detected; 2) cfRNA is unstable and easily degraded, so it cannot be repeatedly detected. cfRNA needs to be efficiently protected after obtaining the supernatant of cytological specimens to protect the concentration and quality of cfRNA, so as to improve the success rate of RNA level detection and the accuracy of fusion-type gene change detection. Summary of the invention

[0007] As mentioned above, currently liquid biopsy of cytological specimens mostly uses cell-free supernatant cfDNA for driver gene detection. Although it has its advantages, there are also prominent problems in multi-gene detection, which is mainly reflected in the problem of low detection sensitivity of fusion gene changes (about 50%). However, more than half of the driver genes that need to be detected in NSCLC patients are fusion gene changes. The application of RNA level detection of fusion changes can greatly improve its detection sensitivity.

[0008] Existing technologies directly extract and detect cell free RNA (cfRNA) from the supernatant of cytological specimens. There are urgent technical problems that need to be solved, including: 1) The amount of cfRNA obtained by extracting nucleic acids from unprotected cytological supernatant is low, and subsequent PCR-based detection cannot be completed; 2) The extracted cfRNA is unstable and easily degraded; 3) There is no cfRNA protection solution for cytological supernatant and applied to driver gene detection; 4) There is no cfRNA protection solution suitable for the supernatant of all cytological specimens (pleural effusion, sputum, alveolar lavage fluid, cerebrospinal fluid, puncture cytological suspension, etc.); 5) The specific formula of the protection solution, the application scheme of the protection solution and the protection efficiency need to be verified by clinical specimens; 6) The value of improving the accuracy of detection of NSCLC fusion gene changes needs to be determined.

[0009] In order to solve the above problems, the present invention provides a protective solution for protecting cfRNA in the supernatant of cytological specimens, which has high protection efficiency for cfRNA and improves the accuracy of NSCLC fusion driver gene detection.

[0010] The protective solution for protecting cfRNA in the supernatant of cytological specimens provided by the present invention is: a guanidine thiocyanate solution prepared with 1×TE buffer (10mM Tris HCL+1mM EDTA pH=8.0) as a solvent;

[0011] 100ml 1×TE buffer is prepared as follows: 1ml 1M Tris HCI + 0.02ml 0.5M EDTA + pure water, dilute to 100ml;

[0012] The concentration of the guanidine thiocyanate solution may be 1M-4M, specifically 4M;

[0013] The 4M guanidine thiocyanate solution is prepared by the following method: 1×TE buffer is added to 47.2 g guanidine thiocyanate and the volume is fixed to 100 ml.

[0014] The protective solution for protecting cfRNA in the supernatant of cytological specimens is applicable to all cytological specimens;

[0015] The cytological specimens include pleural effusion, bronchoalveolar lavage fluid, sputum and puncture cytological suspension, etc.

[0016] The present invention also provides a reagent for pretreatment of cytological specimen supernatant, comprising the protective solution and cytological specimen supernatant fixative.

[0017] Wherein, the supernatant fixative of the cytological specimen is 50% ethanol + 2% polyethylene glycol 400.

[0018] The pleural effusion supernatant is prepared by the following method: centrifuging the collected pleural effusion specimen at 2000 rpm for 5 min, and retaining the supernatant;

[0019] The alveolar lavage fluid supernatant is prepared by the following method: 10 ml of the alveolar lavage fluid specimen is added to the cytology specimen supernatant fixative at a ratio of 1:1, mixed, and then 1 ml of sputum-resolving fluid is added to perform sputum-resolving treatment, shaken and mixed, centrifuged at 2000 rpm for 5 min, and the supernatant is retained to obtain the obtained product;

[0020] The sputum supernatant is prepared by the following method: collecting sputum specimens ≥ 3 ml, adding cytology specimen supernatant fixative at a ratio of 1:1 to 3, adding 2 ml of sputum clearing solution, performing sputum clearing treatment, oscillating and mixing, centrifuging at 2000 rpm for 5 min, and retaining the supernatant to obtain the sputum supernatant;

[0021] The cytological specimen supernatant fixative is: 50% ethanol + 2% polyethylene glycol 400, and 100 ml is prepared by adding 2 ml polyethylene glycol 400 + 50 ml anhydrous ethanol + pure water to a fixed volume of 100 ml;

[0022] The expectorant solution is a 0.5M dithiothreitol aqueous solution, which is prepared by adding 7.71g dithiothreitol + pure water and diluting the volume to 100ml to obtain 100ml;

[0023] The puncture cytology suspension supernatant is prepared by the following method: the puncture specimen (fine needle puncture specimen or coarse needle puncture specimen) is injected into 20 ml of cytology specimen supernatant fixative to prepare a puncture cell / tissue suspension, the suspension is centrifuged at 2000 rpm for 5 min, and the supernatant is retained to obtain the suspension.

[0024] The present invention also provides a method for protecting cfRNA in the supernatant of cytological specimens.

[0025] The method for protecting cfRNA in the supernatant of cytological specimens provided by the present invention comprises: mixing the above-mentioned protection solution for protecting cfRNA in the supernatant of cytological specimens with the prepared supernatant of cytological specimens, and mixing them evenly.

[0026] The volume ratio of the protective solution used to protect cfRNA in the supernatant of the cytological specimen to the supernatant of the cytological specimen is 1:1-3, specifically 1:1, 1:2, 1:3;

[0027] The cytological specimen supernatant includes pleural effusion supernatant, bronchoalveolar lavage fluid supernatant, sputum supernatant and puncture cytological suspension supernatant;

[0028] The pleural effusion supernatant is prepared by the following method: centrifuging the collected pleural effusion specimen at 2000 rpm for 5 min, and retaining the supernatant;

[0029] The alveolar lavage fluid supernatant is prepared by the following method: 10 ml of the alveolar lavage fluid specimen is added to the cytology specimen supernatant fixative at a ratio of 1:1, mixed, and then 1 ml of sputum-resolving fluid is added to perform sputum-resolving treatment, shaken and mixed, centrifuged at 2000 rpm for 5 min, and the supernatant is retained to obtain the obtained product;

[0030] The sputum supernatant is prepared by the following method: collecting sputum specimens ≥ 3 ml, adding cytology specimen supernatant fixative at a ratio of 1:1 to 3, adding 2 ml of sputum clearing solution, performing sputum clearing treatment, oscillating and mixing, centrifuging at 2000 rpm for 5 min, and retaining the supernatant to obtain the sputum supernatant;

[0031] The cytological specimen supernatant fixative is: 50% ethanol + 2% polyethylene glycol 400, and 100 ml is prepared by adding 2 ml polyethylene glycol 400 + 50 ml anhydrous ethanol + pure water to a fixed volume of 100 ml;

[0032] The expectorant solution is a 0.5M dithiothreitol aqueous solution, which is prepared by adding 7.71g dithiothreitol + pure water and diluting the volume to 100ml to obtain 100ml;

[0033] The puncture cytology suspension supernatant is prepared by the following method: the puncture specimen (fine needle puncture specimen or coarse needle puncture specimen) is injected into 20 ml of cytology specimen supernatant fixative to prepare a puncture cell / tissue suspension, the suspension is centrifuged at 2000 rpm for 5 min, and the supernatant is retained to obtain the suspension.

[0034] The cytological specimen supernatant fixative is: 50% ethanol + 2% polyethylene glycol 400, and 100 ml is prepared by adding 2 ml polyethylene glycol 400 + 50 ml anhydrous ethanol + pure water to a fixed volume of 100 ml;

[0035] The expectorant solution is a 0.5M dithiothreitol aqueous solution, which is prepared by adding 7.71g dithiothreitol to pure water and adjusting the volume to 100ml to obtain 100ml.

[0036] The above method further comprises storing the obtained mixed solution at -20°C.

[0037] The use of the above-mentioned protective solution for protecting cfRNA in the supernatant of cytological specimens or the reagent for pretreatment of the supernatant of cytological specimens in the preparation of RNA detection reagents also falls within the protection scope of the present invention.

[0038] In the application, the RNA detection can specifically be NSCLC fusion driver gene detection.

[0039] The present invention designs a protective solution formula for protecting cfRNA in the cell-free supernatant of cytological specimens. The concentration, quality and fusion driver gene detection results of cfRNA obtained from protected and unprotected specimens are compared, showing that the cfRNA protective solution formula designed by the present invention has advantages in the following aspects:

[0040] (1) The clinical specimen validation results conducted by the present invention show that by protecting cfRNA in the supernatant of cytological specimens, the current problems of low cfRNA concentration, low yield and easy degradation are solved, thereby realizing the possibility of dual extraction and dual testing of cfRNA and cfRNA in liquid biopsy specimens, which is a breakthrough progress.

[0041] (2) By protecting cfRNA of cytological samples according to the present invention, the detection sensitivity of fusion gene changes in multi-gene detection of NSCLC is improved.

[0042] (3) Verification of the detection method: After protection by the protective solution of the present invention, the cfRNA in the cell-free supernatant of the cytological specimen obtained is suitable for RT-qPCR method (Xiamen Aide Biopharmaceutical Technology Co., Ltd.) and digital PCR method (Beijing Xinyi Biotechnology Co., Ltd.), both of which can complete NSCLC multi-gene joint detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The RT-qPCR method in Example 1 of the present invention detects the difference in the protective effect of different cfRNA protection solution concentrations on cfRNA. From left to right, the first curve is 1:1 (protective solution: supernatant); the second curve is 1:2 (protective solution: supernatant); the third curve is 0:1 (no protective solution); and the fourth curve is 1:3 (protective solution: supernatant).

[0044] Figure 2 The difference in the results of the RT-qPCR method in Example 1 of the present invention between the 1:1 group (protective solution: supernatant) and the 0:1 group (no protective solution) after cfRNA extraction for 30 days (-80°C freezing). The rising curve is 1:1 (protective solution: supernatant), and the CT is 20.80. The unprotected specimen 0:1 (no protective solution) did not detect the rising curve, indicating that the cfRNA in the supernatant of the unprotected cytological specimen has been degraded.

[0045] Figure 3 The RT-qPCR method was used to detect cfRNA in the pleural effusion supernatant of a non-small cell lung cancer patient in Example 1 of the present invention, and the results showed ALK gene fusion. From left to right, the first curve is the FAM signal as the RNA internal control, and the second curve is the HEX signal, showing ALK gene fusion.

[0046] Figure 4 In order to verify the RT-qPCR test results by using the digital PCR method in Example 1 of the present invention, cfRNA was extracted from the protected pleural effusion supernatant specimen to confirm ALK gene fusion. The scattered points in the lower right corner of the figure are VIC signals, which are positive droplets for the internal reference gene, and the scattered points in the upper left corner are FAM signals, which are positive droplets for the ALK gene.

[0047] Figure 5 The RT-qPCR method was used in Example 1 of the present invention to detect cfRNA in the pleural effusion supernatant of a patient with non-small cell lung cancer, and the results showed RET gene fusion. From left to right, the first curve is the FAM signal as the RNA internal control, and the second curve is the HEX signal, showing RET gene fusion.

[0048] Figure 6 In order to verify the RT-qPCR test results by using the digital PCR method in Example 1 of the present invention, cfRNA was extracted from the protected pleural effusion supernatant specimen to confirm RET gene fusion. The scattered points in the lower right corner of the figure are droplets with VIC signals as the internal reference gene, and the scattered points in the upper left corner are droplets with FAM signals as the RET gene positive. DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0050] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0051] Example

[0052] (1) Preparation of Solution A: Cytology supernatant fixative formula:

[0053] 50v / v% ethanol + 2v / v% polyethylene glycol 400

[0054] For example, to prepare 100ml: 2ml polyethylene glycol 400 + 50ml anhydrous ethanol + pure water, dilute to 100ml

[0055] Solution storage: -20℃

[0056] Solution B: Cytology supernatant cfRNA protection solution formula:

[0057] Solvent: 1×TE buffer (10mM Tris HCL+1mM EDTA PH=8.0)

[0058] For preparing 100ml: 1ml 1M Tris HCI + 0.02ml 0.5M EDTA + pure water, make up to 100ml

[0059] Use 1×TE buffer to prepare 4M guanidine thiocyanate

[0060] Weigh 47.2g of guanidine thiocyanate, add 1×TE buffer, and adjust the volume to 100ml

[0061] Solution storage: 4°C

[0062] Solution C (sputum solution): 0.5 M dithiothreitol

[0063] 7.71g dithiothreitol + pure water, dilute to 100ml

[0064] Solution storage: room temperature

[0065] (2) Different methods of collecting cytological specimens

[0066] Pleural effusion: Collect pleural effusion in a clean container with a lid, with a specimen volume of 50 ml [Note: if the specimen is bloody, add heparin for anticoagulation (add 2500u heparin for every 100ml of pleural effusion)], send it to the cell room of the pathology department, and store it at 4°C.

[0067] Alveolar lavage fluid: Collect the alveolar lavage fluid in a 50 ml clean centrifuge tube with a cap. Collect 10 ml of the specimen, send it to the cell room of the pathology department, add solution A in a 1:1 ratio, and store at 4°C.

[0068] Sputum: Collect the sputum in a clean sputum box with a lid. The collected specimen volume should be ≥ 3 ml. After sending it to the cell room of the pathology department, add solution A in a ratio of 1:3 and store it at 4°C.

[0069] Cytological suspension: Place 20 ml of solution A in a 50 ml clean centrifuge tube with a cap, inject the puncture tissue or cytological specimen directly into solution A, mix gently and send to the pathology department.

[0070] (3) Preparation of supernatants from different cytological specimens and pathological evaluation

[0071] Pleural effusion: Centrifuge the collected pleural effusion specimens at 2000rpm for 5 minutes and keep the supernatant. Resuspend the centrifugal precipitate to prepare liquid-based cytology slides or cytology wax blocks.

[0072] Alveolar lavage fluid: Add 1 ml of solution C to the collected alveolar lavage fluid specimen, treat with expectoration, oscillate and mix, centrifuge at 2000 rpm for 5 min, and keep the supernatant. Resuspend the centrifugal precipitate to make liquid-based cytology slides or cytology wax blocks.

[0073] Sputum: Add 2 ml of solution C to the collected sputum specimens, treat with expectoration, oscillate and mix, centrifuge at 2000 rpm for 5 min, and keep the supernatant. Resuspend the centrifugal precipitate to make liquid-based cytology slides or cytology wax blocks.

[0074] Cytological suspension: The collected puncture tissue specimens are taken out to make formalin-fixed paraffin-embedded (FFPE) histological wax blocks; the collected fine needle aspiration specimens are shaken and mixed, centrifuged at 2000rpm for 5min, the supernatant is collected, the fine needle aspiration specimens are centrifuged and precipitated and resuspended to make liquid-based cytological slides, or cytological wax blocks are made.

[0075] Pathological evaluation content: whether cancer cells are found, the proportion of cancer cells, and the source and typing of cancer cells. If cancer cell specimens are found and the tumor proportion is less than 20%, it is recommended to use the supernatant of cytological specimens for driver gene detection.

[0076] (4) Protection of cfRNA in supernatant of cytological specimens

[0077] Prepare the supernatant of pleural effusion, bronchoalveolar lavage fluid, sputum and puncture cytology suspension collected after centrifugation, mix solution B with the supernatant, and mix according to four concentration gradients of 0:1 (no protective solution), 1:1, 1:2, and 1:3. The total volume of the mixed solution of each concentration is 4 ml, including 4 ml supernatant (4 ml supernatant without solution B), 1:1 (2 ml solution B + 2 ml supernatant), 1:2 (1.3 ml solution B + 2.7 ml supernatant), and 1:3 (1 ml solution B + 3 ml supernatant). Oscillate and mix by four concentration gradients and store at -20°C.

[0078] (5) Extraction of free nucleic acid from the supernatant of cytological specimens

[0079] Circulating nucleic acids were extracted using a nucleic acid extraction kit (Xiamen Aide Biopharmaceutical Technology Co., Ltd.). The free nucleic acids extracted from the unprotected supernatant were used to detect gene mutations at the DNA level, and the free nucleic acids extracted from the protected supernatant were used to detect RNA levels. Double extraction was used to determine the success rate of cfRNA protection in the free nucleic acids after protection (evaluated by the CT value obtained by RT-qPCR detection).

[0080] (6) Detection of cfRNA quality in cytological supernatant

[0081] The free nucleic acid extracted from the protection supernatant was reverse transcribed, and the cfRNA quality (CT value) was detected by RT-qPCR. At the same time, the free nucleic acid extracted from the protection supernatant was stored at -80°C, and the cfRNA quantity and quality were detected for 30 days to determine the stability of the protection solution on cfRNA protection.

[0082] (7) Detection of multi-gene mutation status in cfDNA and cfRNA in supernatant of cytological specimens in NSCLC patients

[0083] Cytological specimens of NSCLC patients were selected, and the supernatant of cytological specimens was used for multi-gene mutation status detection. Inclusion criteria: (1) NSCLC patients; (2) cancer cells were found in cytological specimens; (3) known driver gene detection results were detected by tumor specimens. The supernatant of cytological specimens was tested for EGFR, KRAS, BRAF, Her2 (cfDNA level detection); ALK, ROS1, RET, NTRK1 / 2 / 3, MET (cfRNA level detection) gene mutation status by qPCR and RT-qPCR using the NSCLC 11 Mutation Gene Kit (Xiamen Aide Biopharmaceutical Technology Co., Ltd.) of NSCLC cytological supernatant samples cfDNA and cfRNA (samples protected by cfRNA protection solution).

[0084] At the same time, the digital PCR method was used to detect 13 genes of human lung cancer (Beijing Xinyi Biotechnology Co., Ltd.), including EGFR, KRAS, NRAS, PIKCA, BRAF, Her2 (DNA level detection); ALK, ROS1, RET, NTRK1 / 2 / 3, MET (RNA level detection), to verify the detection results of qPCR and RT-qPCR methods.

[0085] (8) Experimental results

[0086] The protection efficiency of cfRNA protection solution in supernatant of cytological specimens

[0087] The protection efficiency of different protection solution concentrations in the supernatants of 21 cytological specimens was compared: the cfRNA quality within and among the four groups of 0:1 (no protection solution) / 1:1 (protection solution: supernatant); 0:1 (no protection solution) / 1:2 (protection solution: supernatant); 0:1 (no protection solution) / 1:3 (protection solution: supernatant) was compared, and the CT value differences were detected by RT-qPCR method.

[0088] Figure 1 The RT-qPCR method was used to detect the difference in cfRNA protection effects of different cfRNA protection solution concentrations. From left to right, the first curve is 1:1 (protection solution: supernatant); the second curve is 1:2 (protection solution: supernatant); the third curve is 0:1 (no protection); and the fourth curve is 1:3 (protection solution: supernatant).

[0089] 1) 0:1 (no protection solution) vs. 1:1 (protection solution: supernatant): RT-qPCR results showed that there were differences in CT values ​​between the two groups. The median CT value of the 0:1 no protection group was 24.18, and the median CT value of the 1:1 (protection solution: supernatant) group was 21.68. Statistical analysis showed that the difference between the two groups was statistically significant (p < 0.05, Wilcoxon rank sum test);

[0090] 2) 0:1 (no protection solution) vs. 1:2 (protection solution: supernatant): RT-qPCR results showed that there were differences in CT values ​​between the two groups. The median CT value of the 0:1 no protection group was 24.18, and the median CT value of the 1:2 (protection solution: supernatant) group was 21.79. Statistical analysis showed that the difference between the two groups was statistically significant (p < 0.05, Wilcoxon rank sum test);

[0091] 3) 0:1 (no protective solution) vs. 1:3 (protective solution: supernatant): The median CT value of the 0:1 no protective solution group was 24.18, and the median CT value of the 1:3 (protective solution: supernatant) group was 24.14. There was no significant difference between the two groups in statistical analysis (p>0.05, Wilcoxon rank sum test);

[0092] 4) 1:1 (protective solution: supernatant) vs. 1:2 (protective solution: supernatant): Statistical analysis showed that there was no significant difference in CT values ​​between the two groups in RT-qPCR results (p>0.05, chi-square test).

[0093] Furthermore, the successful protection of cfRNA protective solution was defined as: CT value ≤ 23.5, 1:1 (protective solution: supernatant) protection success rate was 90.5% (19 / 21), 1:2 (protective solution: supernatant) protection success rate was 76.2% (16 / 21), 0:1 (no protective solution) CT value ≤ 23.5% ratio was 42.9% (9 / 21).

[0094] The results showed that the protection efficiency was best with a ratio of 1:1 (protection solution: supernatant) (CT value was 19.02).

[0095] Based on the above results, this study recommends the application of a 1:1 (protection solution: supernatant) scheme for the protection of cytological sample supernatants and the next step of clinical specimen verification.

[0096] Clinical specimen validation results

[0097] The sample size was expanded to further verify the protective effect of cfRNA protective solution. The supernatants of 89 cytological samples were collected to compare the protection efficiency of 0:1 (no protective solution) and 1:1 (protective solution: supernatant).

[0098] RT-qPCR results showed that there were differences in CT values ​​between the two groups. The median CT value of the 0:1 unprotected group was 25.25, and the median CT value of the 1:1 (protective solution: supernatant) group was 23.09. Statistical analysis showed that the difference between the two groups was statistically significant (p < 0.05, Wilcoxon rank sum test).

[0099] "1:1 protection group" protection efficiency

[0100] (1) The protective effects of 0:1 (no protective solution) and 1:1 (protective solution: supernatant) were compared. When the CT value of 0:1 (no protective solution) was greater than the CT value of 1:1 (protective solution: supernatant), it was defined as effective protection. The effective protection rate in this study was 92.1% (82 / 89).

[0101] (2) Successful protection with cfRNA protective solution was defined as: CT value ≤ 23.5. The protection success rate of 1:1 (protective solution: supernatant) was 73.0% (65 / 89), and the ratio of CT value ≤ 23.5 in the 0:1 (no protection) group was 38.2% (34 / 89).

[0102] (3) Samples frozen for more than 30 days were subjected to RT-qPCR again to detect the quality of cfRNA. When the CT value of 0:1 (no protective solution) was greater than the CT value of 1:1 (protective solution: supernatant), it was defined as effective protection, with a protection efficiency of 96.4% (27 / 28). Among the 28 tested samples, 3 cases (10.7%, 3 / 28) showed no increase in the cfRNA curve, indicating cfRNA degradation.

[0103] Figure 2 The RT-qPCR method was used to detect the difference in results between the 1:1 group (protective solution: supernatant) and the 0:1 group (no protective solution) after cfRNA extraction and cryopreservation for 30 days (-80℃ cryopreservation). The rising curve was 1:1 (protective solution: supernatant), and the CT was 20.80. No rising curve was detected in the unprotected specimen 0:1 (no protective solution). The results showed that the cfRNA quality of the 1:1 group (protective solution: supernatant) was intact after cfRNA extraction and cryopreservation for 30 days, while the cfRNA of the unprotected specimen was degraded.

[0104] Results of multi-gene joint detection in supernatant of NSCLC lung cancer cytology specimens

[0105] Thirty-three cytological specimens that met the selection criteria were selected, including 18 pleural effusions, 12 bronchoalveolar lavage fluids, 4 sputums, and 1 puncture cytological suspension. 4 ml of the supernatant of the specimens at 0:1 (without protective solution) and 1:1 (protective solution: supernatant) were aspirated to extract free nucleic acids for gene detection. The NSCLC 11 Mutation Gene Kit (Xiamen Aide Biopharmaceutical Technology Co., Ltd.) was used to detect the mutation status of EGFR, KRAS, BRAF, Her2 (DNA level detection) and ALK, ROS1, RET, NTRK1 / 2 / 3, MET (RNA level detection) in the supernatant samples of NSCLC cytology by RT-qPCR method.

[0106] Four cases of ALK gene fusion were detected based on cfRNA levels (see Figure 3 , Figure 4 ), 1 case of ROS1 gene fusion, 1 case of RET mutation (see Figure 5 , Figure 6), 1 case of C-MET gene exon 14 skipping deletion mutation, which was consistent with the tumor specimen detection results (cfRNA fusion gene detection sensitivity 100%). cfDNA levels detected 7 cases of EGFR exon 21 L858R mutation, 8 cases of EGFR exon 19 deletion mutation, 3 cases of EGFR exon 20 mutation, 1 case of EGFR exon 18 G719X mutation, 4 cases of KRAS mutation, and 1 case of BRAF mutation. Compared with the tumor specimen detection results, the overall detection sensitivity was 93.9% (31 / 33). The digital PCR detection results were consistent with the qPCR and RT-qPCR results.

[0107] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A protective solution for protecting cfRNA in the supernatant of cytological specimens, comprising: a guanidine thiocyanate solution prepared with 1×TE buffer as a solvent; in, 1×TE buffer is 10mM TrisHCL+1mM EDTA, pH=8.

0.

2. The protective liquid according to claim 1, characterized in that: The concentration of the guanidine isothiocyanate solution is 1M-4M.

3. A reagent for pretreatment of cytological specimen supernatant, comprising the protective solution according to claim 1 and a cytological specimen supernatant fixative, in, The cytological specimen supernatant fixative is 50% ethanol+2% polyethylene glycol 400.

4. A method for protecting cfRNA in the supernatant of a cytological specimen, comprising: mixing the protective solution for protecting cfRNA in the supernatant of a cytological specimen according to claim 1 or 2 with the supernatant of the cytological specimen, and mixing them evenly.

5. The method according to claim 4, characterized in that: The cytological specimen supernatant includes pleural effusion supernatant, alveolar lavage fluid supernatant, sputum supernatant and puncture cytological suspension supernatant.

6. The method according to claim 5, characterized in that: The pleural effusion supernatant is prepared by the following method: centrifuging the pleural effusion specimen at 2000 rpm for 5 min and retaining the supernatant; The alveolar lavage fluid supernatant is prepared by the following method: 10 ml of the alveolar lavage fluid specimen is added to the cytology specimen supernatant fixative at a ratio of 1:1, mixed, and then 1 ml of sputum-resolving fluid is added to perform sputum-resolving treatment, shaken and mixed, centrifuged at 2000 rpm for 5 min, and the supernatant is retained to obtain the obtained product; The sputum supernatant is prepared by the following method: collecting sputum specimens ≥ 3 ml, adding cytology specimen supernatant fixative at a ratio of 1:1 to 3, adding 2 ml of sputum clearing solution, performing sputum clearing treatment, oscillating and mixing, centrifuging at 2000 rpm for 5 min, and retaining the supernatant to obtain the sputum supernatant; Wherein, the supernatant fixative of the cytological specimen is 50% ethanol + 2% polyethylene glycol 400, and the expectorant is 0.5M dithiothreitol aqueous solution; The puncture cytology suspension supernatant is prepared by the following method: a fine needle puncture specimen is injected into 20 ml of cytology specimen supernatant fixative to prepare a puncture cell suspension, the suspension is centrifuged at 2000 rpm for 5 min, and the supernatant is retained to obtain the suspension; or a coarse needle puncture specimen is injected into 20 ml of cytology specimen supernatant fixative, mixed, centrifuged at 2000 rpm for 5 min, and the supernatant is retained to obtain the suspension.

7. The method according to any one of claims 4 to 6, characterized in that: The volume ratio of the protective solution used to protect cfRNA in the supernatant of cytological specimens to the supernatant of cytological specimens is 1:1-2.

8. Use of the protective solution for protecting cfRNA in the supernatant of cytological specimens according to claim 1 or 2 or the reagent for pretreatment of the supernatant of cytological specimens according to claim 3 in the preparation of RNA detection reagents.

9. The use according to claim 8, characterized in that: The RNA detection is for NSCLC fusion driver gene detection.

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