Saliva collection, pretreatment and cell sorting method for telomere length detection
Through the pretreatment of saliva samples and the sorting method of neutrophils, the problems of nucleic acid degradation and instability of detection results caused by improper preservation of saliva samples are solved, and efficient and accurate telomere length detection is achieved.
Patent Information
- Application Number
- CN202510283158.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-11
- Publication Date
- 2025-06-06
AI Technical Summary
In the prior art, improper preservation of saliva samples leads to nucleic acid degradation and reduction in yield, and the complex composition of saliva samples affects the reproducibility of subsequent nucleic acid extraction and telomer quantitative detection.
A saliva collection, pretreatment and cell sorting method for telomerular length detection is proposed. Oral cells and impurities are removed by pretreatment of saliva samples, and neutrophil selection is performed using CD66b biotinylated antibodies and avidin magnetic beads, and then nucleic acid extraction is performed for fluorescence quantitative telomerular length detection.
It effectively solves the problems of nucleic acid degradation and reduction in yield caused by improper preservation of saliva samples, improves the completeness of subsequent nucleic acid extraction and the reliability of quantitative detection, and ensures the stability and accuracy of telomere length detection results.
Smart Images
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Abstract
Description
Technical Field
[0001] The present application relates to the field of molecular detection and biological sample processing, and more specifically, to a method for collecting, pre-treating and sorting saliva for telomere length detection. Background Art
[0002] At present, the main method for detecting telomere length is quantitative PCR, and the commonly used test sample types are venous blood, fingertip blood and oral cells. When DNA is extracted from white blood cells in whole blood to detect telomere length, telomere length shows a predictable trend of decreasing with age. However, venipuncture is a relatively invasive, expensive and labor-intensive method of sample collection, which is somewhat traumatic to the person being tested. Fingertip blood sampling is also invasive, and the amount of DNA obtained is insufficient. Oral cells are mainly composed of oral epithelial cells, which are not representative of the overall telomere length of the sample. In addition, unlike the telomere length of white blood cells, the telomere length of oral epithelial cells does not show a trend of decreasing with age. Therefore, there is no correlation between the telomere length measured in samples mainly composed of oral cells and samples mainly composed of white blood cells. Saliva samples have a low risk of infection and are easy to collect in non-clinical settings. There are two main types of cells in saliva samples, oral cells and white blood cells. There are many bacteria in the mouth, and the main function of white blood cells in the mouth is to kill pathogenic microorganisms. Among them, neutrophils mainly play a role in inflammatory response. The neutrophils in the mouth and the neutrophils in the blood have the same source, so in this study, neutrophils were selected as the target white blood cells for sorting. A large number of literatures show that oral saliva can be used as a sample for detecting telomere length, such as Boroumand M, Olianas A, Cabras T, et al. Saliva, a bodily fluid with recognized and potential diagnostic applications [J]. J Sep Sci, 2021, 44 (19): 3677-3690.
[0003] When using whole saliva samples to detect telomere length, the components of saliva samples in the mouth are easily affected by the oral microenvironment, and there are many impurities, which leads to unstable results of quantitative detection of telomere length by whole saliva samples. In addition, improper storage of the samples to be tested or different DNA extraction methods will seriously affect the repeatability of the results of real-time fluorescence quantitative PCR technology for detecting telomere length, which brings great challenges to the accurate detection of telomere length. At present, many articles have proved that the repeatability of this technology for detecting telomere length is low, mainly due to improper sample storage or differences in DNA integrity caused by different DNA extraction methods. For example, Method Specific Calibration Corrects for DNA Extraction Method Effects on Relative Telomere Length Measurements by Quantitative PCR. PLoS One. 2016 Oct 10; 11(10): e0164046 and Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood. PLoS One. 2015 Dec 4; 10(12): e0143889. found that the coefficient of variation of the test results is large due to the different integrity of genomic DNA in the same sample.
[0004] Therefore, it is necessary to propose a saliva collection, pretreatment and cell sorting method for telomere length detection to solve the technical problems in the prior art such as nucleic acid degradation and yield reduction caused by improper storage of the test samples, as well as the impact of the complex composition of saliva samples on subsequent nucleic acid extraction and telomere quantitative detection. Summary of the invention
[0005] In order to solve the above technical problems, the present invention proposes a new method for saliva collection, pretreatment and leukocyte sorting for telomere length detection, selects a saliva sample as the best sample for human telomere length detection, removes oral cells and other impurities in the saliva sample through saliva sample pretreatment, and then uses biotin antibodies corresponding to neutrophil surface specific antigen CD66b to bind to neutrophils, and then uses avidin magnetic beads to specifically bind to the biotin antibodies marked with CD66b, and through the magnetic attraction of a magnetic frame, the neutrophils are positively selected, and the obtained neutrophils can be effectively used for fluorescent quantitative telomere length detection after nucleic acid extraction, avoiding the invasiveness of sampling to the human body, and solving the technical problems in the collection, transportation and storage of saliva samples.
[0006] In order to achieve the above-mentioned purpose of the invention, the present application provides a method for detecting telomere length, wherein saliva is selected as a sample and the method comprises the following steps:
[0007] Step 1: Pretreatment of saliva samples;
[0008] Step 2: Magnetic bead sorting is used to obtain neutrophils in saliva samples. After nucleic acid extraction, the obtained neutrophils are used for fluorescent quantitative telomere length detection.
[0009] Furthermore, the process of step 1 is: collecting a saliva sample, diluting the saliva sample with a preservation solution, filtering the sample with a cell sieve, and collecting the filtrate containing white blood cells.
[0010] Furthermore, in step 1, the cell preservation solution dilutes the saliva sample at a multiple of 1:(1-3).
[0011] Furthermore, in step 1, the cell preservation solution dilutes the saliva sample at a multiple of 1:2.
[0012] Furthermore, in step 1, the preservation solution is any one of physiological saline, PBS, and cell preservation solution.
[0013] Furthermore, in step 1, the preservation solution is a cell preservation solution.
[0014] Furthermore, the process of step 2 is: adding CD66b-biotinylated antibody to the pretreated saliva sample for incubation, centrifuging, resuspending, adding Anti-Botin positive sorting magnetic beads for incubation, sorting and collecting neutrophils, and extracting nucleic acids for fluorescent quantitative telomere length detection.
[0015] Furthermore, the process of step 2 is: adding CD66b-biotinylated antibody to the pretreated saliva sample, mixing, incubating at room temperature for 10 minutes, adding PBS for washing, centrifuging, discarding the supernatant, adding PBS again to resuspend the cells, adding Anti-Botin positive sorting magnetic beads to the cell resuspension, adding cell sorting fluid, mixing, incubating at room temperature for 10 minutes, after the incubation is completed, placing the centrifuge tube on a strong magnetic magnetic stand, removing the supernatant after the magnetic beads are adsorbed, washing twice with cell sorting fluid, removing the supernatant by magnetic adsorption, and then adding cell sorting fluid to suspend and store for use, and after subsequent nucleic acid extraction, it is used for fluorescent quantitative telomere length detection.
[0016] In summary, this application has the following beneficial effects:
[0017] The present invention establishes a complete saliva sample collection, storage and transportation scheme, compares different sample storage methods, and successfully solves the problems of nucleic acid degradation and yield reduction caused by improper storage. In view of the problem that the complex composition of saliva samples will affect the subsequent nucleic acid extraction and telomere quantitative detection, a set of effective saliva sample pretreatment and cell sorting schemes are proposed, which can efficiently and completely sort out neutrophils in saliva, improve the integrity of the subsequent cell nucleic acid extraction genome, and ensure that the subsequent quantitative detection of telomeres is stable and reliable. The method for detecting human telomere length established in this study is of great significance in clinical diagnosis and biomedicine.
[0018] The present invention selects to collect saliva as a sample for telomere detection. The saliva sample is non-invasive to the human body, and a complete set of schemes including saliva collection, transportation, storage, saliva pretreatment and cell sorting are proposed. The present invention effectively solves the problems that the current sample collection is unqualified, the transportation is improper, and the saliva sample is placed for too long, resulting in unqualified quality, affecting the subsequent processing sorting and genome extraction, and causing great interference to the correct detection of telomeres. In addition, the pretreatment and magnetic bead sorting of the saliva sample can achieve the collection of target cells, reduce the interference of saliva impurities, and the genomic DNA obtained by extraction has high integrity and high concentration of the target genome, which is conducive to subsequent fluorescence quantitative detection, so that the detection result is more accurate. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 : Flow chart of the telomere length detection method in the embodiment of the present application;
[0020] Figure 2 :Saliva sample filtered through 30μm cell sieve;
[0021] Figure 3 : Results of salivary leukocyte sorting (A: before sorting; B: after sorting). DETAILED DESCRIPTION
[0022] The technical scheme and effects of the present application are further described in detail below in conjunction with the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention.
[0023] 1. Experimental Materials
[0024] Cell preservation medium (fixed type) (VRCPS) was purchased from Suzhou Weishan Biotechnology Co., Ltd.;
[0025] Phosphate buffered saline (PBS) (E607008-0500) was purchased from Sangon Biotechnology (Shanghai) Co., Ltd.;
[0026] Ezup column animal genomic DNA extraction kit (B518251-0050) was purchased from Sangon Biotech (Shanghai) Co., Ltd.;
[0027] ImunoSep Buffer / cell sorting solution (604050) was purchased from Beijing Novi Biotechnology Co., Ltd.;
[0028] Anti-Biotin positive selection kit (603110) was purchased from Beijing Novi Biotechnology Co., Ltd.;
[0029] Biotin anti-human CD66b (305120) was purchased from Beijing Novi Biotechnology Co., Ltd.;
[0030] FITC-anti-humanCD45 (304006) was purchased from Beijing Novi Biotechnology Co., Ltd.;
[0031] APC-anti-human CD16 (302011) was purchased from Beijing Novi Biotechnology Co., Ltd.;
[0032] 2. Experimental Methods
[0033] 1. Sample collection, storage and transportation:
[0034] 1) Sample collection time and method: Subjects need to collect 2 ml of saliva samples by rubbing both cheeks with their hands before brushing their teeth after getting up in the morning;
[0035] 2) Sample preservation method: Spit the saliva into a 15ml centrifuge tube pre-filled with 4ml of cell preservation solution, and mix by inverting;
[0036] 3) Sample transportation: Place the samples in a foam box with ice packs in advance and seal them for transportation.
[0037] 2. Separation of salivary leukocytes (screening):
[0038] 1) Use a 4 ml pipette to draw half of the saliva mixture and add it dropwise to a 30 um disposable cell sieve for filtration. Centrifuge the filtered liquid at 1500 rpm for 3 minutes and discard the supernatant.
[0039] 2) Add 200 μL PBS to wash, centrifuge at 1500 rpm for 3 minutes, discard the supernatant, and add 100 μL PBS to resuspend.
[0040] 3) Add 2 μL of CD66b-biotinylated antibody, pipette up and down 5 times or vortex to mix, and incubate at room temperature for 10 minutes.
[0041] 4) Add 300 μL PBS to wash, centrifuge at 1500 rpm for 5 min at room temperature, discard the supernatant, and add 100 μL PBS again to resuspend the cells.
[0042] 3. Separation of saliva samples (magnetic bead sorting):
[0043] 1) Add 10 μL of Anti-Botin positive sorting magnetic beads to every 100 μL of cell resuspension, and then add cell sorting solution to 300 μL. Mix well by blowing 3 times with a gun, vortex mix, and incubate at room temperature for 10 minutes.
[0044] 2) After incubation, place the 1.5 mL centrifuge tube on a strong magnetic stand and remove the supernatant after the magnetic beads are adsorbed.
[0045] 3) Wash twice with 300 μL cell sorting solution, remove the supernatant by magnetic adsorption, and then add 100 μL cell sorting solution to suspend and store for later use.
[0046] Example 1
[0047] Test different sample preservation methods and compare to select the best saliva sample preservation method.
[0048] 1. Sample preservation method test selection:
[0049] First, collect 2 mL of saliva samples in the morning before brushing teeth, and mix them with three prepared preservation solutions: physiological saline, PBS, and cell preservation solution. Mix them by inversion. One sample was collected on the same day and filtered and sorted (see the technical solution section for the specific sorting process). The Ezup column animal genomic DNA extraction kit was used for nucleic acid extraction and the operation was performed according to the instructions. The other sample was placed in a 4°C refrigerator for 48 hours before genomic DNA extraction. The concentration of the obtained genomic DNA was measured using a NanoDrop2000 ultra-micro spectrophotometer. The nucleic acid yield is shown in Table 1.
[0050] Table 1 Different saliva sample storage methods
[0051] Sample storage method Normal saline PBS Cell preservation medium Saliva collected on the same day 45 ng / μL 52 ng / μL 87 ng / μL Saliva extracted after 48 hours 17 ng / μL 19 ng / μL 75ng / μL
[0052] 2. Optimal dilution multiple of saliva samples:
[0053] Since the saliva samples are viscous, it is necessary to test the optimal dilution ratio between the saliva samples and the diluent and select the best dilution multiple for the preparation of the preservation solution. Therefore, three saliva samples of 2 mL each were collected on the same day, and the optimal dilution ratio of the saliva samples was tested. The nucleic acid yield is shown in Table 2.
[0054] Table 2 Dilution multiples of saliva samples
[0055] Saliva sample dilution factor 1:1 1：2 1：3 Sample 1 6ng / μL 38 ng / μL 39 ng / μL Sample 2 5ng / μL 39 ng / μL 37 ng / μL Sample 3 5ng / μL 45 ng / μL 39 ng / μL
[0056] Example 2
[0057] A saliva sample pretreatment and cell sorting scheme was established, and neutrophils were successfully extracted from saliva.
[0058] 1. Saliva sample pretreatment (screening):
[0059] After collecting the sample, dilute the saliva sample with cell preservation solution at a ratio of 1:2, filter it with a 30μm disposable cell sieve, collect the filtrate and stain it with Giemsa stain to observe whether the white blood cells are successfully screened. The results are shown in Figure 1 .
[0060] 2. Separation of leukocytes from saliva samples (magnetic bead sorting):
[0061] Neutrophils were selected as the target cells for saliva sample sorting. A saliva sample was collected and divided into two parts after pretreatment. One part was sorted with biotin-avidin immunomagnetic beads, and the other part was not sorted. The two samples were stained with flow cytometry antibodies and tested on a flow cytometer to detect whether the target white blood cells in the saliva were successfully sorted. The test results were as follows: Figure 2 Before the biotin-avidin magnetic bead sorting, the neutrophil content of the sample was about 50%, and after the sorting, the neutrophil content reached 90%.
[0062] The beneficial effect of the present invention is that saliva is selected as a sample for telomere detection, and the saliva sample is non-invasive to the human body, and a complete set of saliva collection, transportation, storage, saliva pretreatment and cell sorting schemes are proposed. The present invention effectively solves the problem that the quality of saliva samples is unqualified due to unqualified sample collection, improper transportation and too long storage, which affects the subsequent processing sorting and genome extraction, and greatly interferes with the correct detection of telomeres. In addition, the pretreatment and magnetic bead sorting of saliva samples can achieve the collection of target cells, reduce the interference of saliva impurities, extract the obtained genomic DNA with high integrity, and the target genome concentration is high, which is conducive to subsequent fluorescence quantitative detection, so that the test results are more accurate.
[0063] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.
Claims
1. A method for detecting telomere length, characterized in that: The method selects saliva as a sample and comprises the following steps: Step 1: Pretreatment of saliva samples; Step 2: Magnetic bead sorting is used to obtain neutrophils in saliva samples. After nucleic acid extraction, the obtained neutrophils are used for fluorescent quantitative telomere length detection.
2. The method according to claim 1, characterized in that The process of step 1 is: collecting a saliva sample, diluting the saliva sample with a preservation solution, filtering the sample with a cell sieve, and collecting the filtrate containing white blood cells.
3. The method according to claim 2, characterized in that In the step 1, the cell preservation solution dilutes the saliva sample at a multiple of 1:(1-3).
4. The method according to claim 3, characterized in that: In the step 1, the cell preservation solution dilutes the saliva sample at a multiple of 1:
2.
5. The method according to claim 2, characterized in that: In the step 1, the storage solution is any one of physiological saline, PBS, and cell storage solution.
6. The method according to claim 5, characterized in that In the step 1, the storage medium is a cell storage medium.
7. The method according to claim 1, characterized in that The process of step 2 is: adding CD66b-biotinylated antibody to the pretreated saliva sample for incubation, centrifuging, resuspending, adding Anti-Botin positive sorting magnetic beads for incubation, sorting and collecting neutrophils, and extracting nucleic acids for fluorescent quantitative telomere length detection.
8. The method according to claim 7, characterized in that The process of step 2 is: adding CD66b-biotinylated antibody to the pretreated saliva sample, mixing, incubating at room temperature for 10 minutes, adding PBS for washing, centrifuging, discarding the supernatant, adding PBS again to resuspend the cells, adding Anti-Botin positive sorting magnetic beads to the cell resuspension, adding cell sorting fluid, mixing, incubating at room temperature for 10 minutes, after the incubation is completed, placing the centrifuge tube on a strong magnetic magnetic rack, removing the supernatant after the magnetic beads are adsorbed, washing twice with cell sorting fluid, removing the supernatant by magnetic adsorption, and then adding cell sorting fluid to suspend and store for use, and after subsequent nucleic acid extraction, it is used for fluorescent quantitative telomere length detection.