Trace short tandem repeat detection method

Abstract

The invention relates to the field of gene detection, in particular to a trace short tandem repeat sequence detection method, which comprises the following steps: separating stem cells from a biological sample, combining the stem cells into a cell sample, cracking the cell sample, carrying out polymerase chain reaction and electrophoresis detection, and analyzing obtained data. When the method is used for trace short tandem repeat detection, a high-accuracy sequencing result can be obtained with a small sample size, and detection and analysis of trace samples are facilitated.

Description

Technical Field

[0001] The present invention relates to the field of gene detection, and particularly to a method for detecting trace short tandem repeat sequences. Background Art

[0002] Short tandem repeat (STR) sequences are genetic markers used for individual identification and paternity testing, which are formed by multiple repetitions of short nucleotide sequence units on the DNA strand. STR sequencing analysis is widely used in the fields of forensic medicine, anthropology, genetics, etc., to distinguish individuals by analyzing short tandem repeat sequences at specific loci.

[0003] The field of forensic medicine is an important application scenario for STR sequencing. Usually, after the sampling carrier is fully lysed by soaking it in a lysis solution, it is purified by means such as magnetic beads or separation columns to obtain DNA with relatively high purity and concentration. Then, it is amplified by polymerase chain reaction (PCR) and detected and analyzed by capillary electrophoresis. However, the biological samples collected on-site often have extremely small sample volumes, which leads to significant information loss during the detection process, and a large number of fragments are difficult to detect. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for detecting trace short tandem repeat sequences applicable to the case of a relatively small sample volume.

[0005] To achieve the above purpose, the present invention provides the following technical solutions:

[0006] A method for detecting trace short tandem repeat sequences, comprising:

[0007] Isolating a number of cells from a biological sample to form a cell sample;

[0008] Lysing the cells in the cell sample and performing polymerase chain reaction to obtain a reaction solution;

[0009] Taking the reaction solution for electrophoresis detection and performing data analysis.

[0010] Optionally, taking intact cells from a biological sample, obtaining a cell sample includes:

[0011] Soaking the biological sample in a buffer solution to release the cells in the biological sample into the buffer solution;

[0012] Picking and combining a number of cells from the buffer solution through a capillary to obtain the cell sample.

[0013] Optionally, the total volume of the cell sample is less than or equal to 20% of the reaction system of the polymerase chain reaction, and / or the pipetting volume each time when picking cells is less than or equal to 150 nL.

[0014] Optionally, the time from soaking the biological sample in the buffer to starting the polymerase chain reaction is less than or equal to 2 h.

[0015] Optionally, the buffer is tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid buffer.

[0016] Optionally, the number of cells in the cell sample is any value from 3 to 10.

[0017] Optionally, the number of cells in the cell sample is less than or equal to 50% of the number of intact cells in the biological sample.

[0018] Optionally, each cell in the cell sample has a complete morphology.

[0019] Optionally, lysing the cells in the cell sample and performing polymerase chain reaction to obtain a reaction solution, including:

[0020] Adding a premix and a primer mixture to the cell sample, and making up the volume with a lysis solution, wherein the lysis temperature of the lysis solution is coordinated with the denaturation temperature of the polymerase chain reaction;

[0021] Adding the mixed system to a polymerase chain reaction analyzer, adjusting the temperature to the denaturation temperature, and then standing and incubating until the cells are lysed;

[0022] Performing polymerase chain reaction after the incubation is completed to obtain the reaction solution.

[0023] The beneficial effects of the present invention are as follows: The cells in the sample are separately extracted and combined to obtain a cell sample, thereby removing impurities in the biological sample. After lysing the cell sample, complete STR information is obtained, and after PCR amplification, capillary electrophoresis detection and analysis are performed, which helps to retain complete genetic information, so that relatively accurate data can be detected with a small amount of biological sample.

[0024] Furthermore, soaking the biological sample in a buffer solution to fully release the cells in the biological sample into the buffer solution, and then picking single cells by means of capillary picking and combining them to obtain a cell sample. This method helps to reduce contaminants in the cells and can eliminate non-homologous cells such as bacteria that can be observed under a microscope, which helps to improve the purity of the sample, thereby reducing miscellaneous peaks and obtaining accurate detection data.

[0025] The above description is only an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and be able to implement it according to the content of the specification, the following describes in detail with reference to the preferred embodiments of the present invention and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a flowchart of the trace short tandem repeat sequence detection method shown in the first embodiment of the present invention;

[0027] Figure 2 It is an electrophoresis pattern corresponding to a cell sample containing 1 cell shown in the first embodiment of the present invention;

[0028] Figure 3 It is an electrophoresis pattern corresponding to a cell sample containing 5 cells shown in the first embodiment of the present invention;

[0029] Figure 4 It is an electrophoresis pattern corresponding to a cell sample containing 10 cells shown in the first embodiment of the present invention;

[0030] Figure 5 It is an electrophoresis pattern corresponding to a cell sample containing 50 cells shown in the first embodiment of the present invention;

[0031] Figure 6 It is an electrophoresis pattern obtained by detecting a biological sample shown in Comparative Example 1 of the present invention;

[0032] Figure 7 It is an electrophoresis pattern corresponding to the cell sample shown in Comparative Example 2 of the present invention;

[0033] Figure 8 It is an electrophoresis pattern obtained by detecting the reaction solution shown in Comparative Example 3 of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0035] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be construed as a limitation on the present invention. In addition, the terms "first", "second", "third" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0036] In the description of the present invention, it should be noted that unless otherwise clearly specified and limited, the terms "installed", "connected", "connected" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0037] In addition, the technical features involved in different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0038] Please refer to Figure 1 , the trace short tandem repeat sequence detection method protected by the present invention application includes:

[0039] S100. Isolate several cells from a biological sample to form a cell sample.

[0040] S200. Lyse the cells in the cell sample and perform polymerase chain reaction to obtain a reaction solution.

[0041] S300. Take the reaction solution for electrophoresis detection and data analysis.

[0042] Extract and combine the cells in the sample separately to obtain a cell sample, thereby removing impurities in the biological sample. After lysing the cell sample, complete STR information is obtained, and after PCR amplification, capillary electrophoresis detection and analysis are carried out, which helps to retain complete genetic information, so that relatively accurate data can be detected with a small amount of biological sample.

[0043] In some embodiments, step S100 includes:

[0044] S110. Immerse the biological sample in a buffer solution to release the cells in the biological sample into the buffer solution.

[0045] S120. Pick and combine several cells from the buffer solution through a capillary to obtain a cell sample.

[0046] Immerse the biological sample in a buffer solution. After the cells in the biological sample are fully released into the buffer solution, pick single cells by means of capillary picking, and combine them to obtain a cell sample. This method helps to reduce the contaminants in the cells and can eliminate non-homologous cells such as bacteria that can be observed under a microscope, which helps to improve the purity of the sample, thereby reducing spurious peaks and obtaining accurate detection data.

[0047] In some embodiments, the total volume of the cell sample is less than or equal to 20% of the reaction system of the polymerase chain reaction. For example, it can be any value among 5%, 10%, 13%, 15%, and 20%, which helps to reduce the concentration of impurities doped in the cell sample in the reaction system of the polymerase chain reaction and prevent it from affecting the polymerase chain reaction. And / or the pipetting volume each time when picking cells is less than or equal to 150 nL. For example, it can be any value among 10 nL, 30 nL, 60 nL, 90 nL, 120 nL, and 150 nL, to prevent excessive transfer of the buffer solution during cell separation, resulting in a large amount of impurities being mixed into the cell sample with the buffer solution, thereby affecting the accuracy of the detection data.

[0048] In some embodiments, the time from immersing the biological sample in the buffer solution to starting the polymerase chain reaction is less than or equal to 2 h. For example, it can be any value among 0.5 h, 1 h, 1.5 h, and 2 h, to prevent the degradation of nucleic acids in the cells resulting in fragment deletion and the situation of more spurious peaks caused by non-specific amplification.

[0049] In some embodiments, the buffer solution is tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid (TE) buffer solution, which helps to protect genetic information and prevent nucleic acid degradation.

[0050] In some embodiments, the number of cells in the cell sample is any value from 3 to 10. For example, it can be any value among 3, 5, 7, 8, and 10. Too few cells will result in too little nucleic acid, making it difficult to detect some fragments, and thus resulting in incomplete detection data. Too many cells will cause waste of the sample and is not conducive to the full utilization of trace samples.

[0051] In some embodiments, the number of cells in the cell sample is less than or equal to 50% of the number of intact cells in the biological sample. For example, it can be any value among 20%, 30%, 40%, 45%, and 50%, which is convenient for the full utilization of trace samples.

[0052] In some embodiments, each cell in the cell sample has a complete morphology to prevent the decline of data accuracy caused by incomplete genetic information.

[0053] In some embodiments, step S200 includes:

[0054] S210. Add a premixed solution and a primer mixture to the cell sample, and make up the volume with a lysis solution to obtain a mixed system. The lysis temperature of the lysis solution is coordinated with the denaturation temperature of the polymerase chain reaction.

[0055] S220. Add the mixed system to a polymerase chain reaction analyzer. After adjusting the temperature to the denaturation temperature, let it stand and incubate until the cells are lysed.

[0056] S230. After the incubation is completed, perform a polymerase chain reaction to obtain a reaction solution.

[0057] Integrating lysis and PCR into the same step helps simplify the experimental procedure.

[0058] For details, please refer to the following examples.

[0059] Example 1:

[0060] Please refer to Figure 1 , the trace short tandem repeat sequence detection method shown in a preferred embodiment of this application includes:

[0061] S100. Isolate several cells from a biological sample to form a cell sample.

[0062] S200. Lyse the cells in the cell sample and perform a polymerase chain reaction to obtain a reaction solution.

[0063] S300. Take the reaction solution for electrophoresis detection and data analysis.

[0064] Step S100 includes:

[0065] S110. Immerse the biological sample in a buffer solution to release the cells in the biological sample into the buffer solution.

[0066] S120. Pick and combine several cells from the buffer solution with a capillary tube to obtain a cell sample.

[0067] In this example, step S110 includes:

[0068] S111. Wipe the inner side of the oral mucosa with a cotton swab to obtain a biological sample attached with oral epithelial cells, and store the biological sample in an ice bath.

[0069] S112. Transfer 500 μL of TE buffer pre-cooled at 4°C to a microcentrifuge tube (EP tube) and store it in an ice bath.

[0070] S113. Insert the head of the cotton swab into the EP tube to fully immerse the head of the cotton swab in the TE buffer, and squeeze the head of the cotton swab against the tube wall multiple times to fully release the cells in the biological sample into the TE buffer.

[0071] S114. Transfer the liquid in the EP tube to a 96-well plate pre-cooled in an ice bath sufficiently.

[0072] In step S120, find the cells without contaminants around under a microscope and pick them with a capillary. The contaminants include impurity particles and distinguishable bacteria, etc. The pipetting volume for each pick is 100 nL. Combine several picked cells to obtain multiple cell samples, and ensure the integrity of the cells during the picking process. The number of cells in each cell sample is 1, 5, 10, and 50 respectively, and they are labeled and then parallel experiments are carried out.

[0073] Step S200 includes:

[0074] S201. Mix each cell sample with the lysis buffer pre-cooled at 4 °C sufficiently to lyse the cells. In this embodiment, 3 μL of lysis buffer is added, and it is incubated in a water bath at 95 °C for 10 min to obtain the lysis buffer.

[0075] S202. Take the STR kit and configure the amplification system according to the instructions in the manual. In this embodiment, weigh 1 ng of the DNA template provided in the STR kit as the control sample, add 4 μL of 2.5× premix and 1 μL of 10× primer mixture to the lysis buffer corresponding to each cell sample and the control sample respectively, and make up the volume with the lysis buffer to make the total volume of the amplification system 10 μL.

[0076] S203. Amplify each amplification system with a PCR instrument to obtain the reaction solution. The amplification conditions in this embodiment are shown in Table 1 below.

[0077] Table 1:

[0078]

[0079] In this embodiment, step S113, step S114, step S201, step S202, and step S203 are carried out continuously. From the start of step S113 to the start of step S203, the elapsed time is about 50 min.

[0080] In step S300, capillary electrophoresis is used for detection, and the detection results are shown in Figure 2 、 Figure 3 、 Figure 4 and Figure 5 , and multiple spectra are detected. For the sake of simplicity, only a representative part is shown in this embodiment. By comparison, Figures 3 to 5 has high repeatability. Therefore, it can be proved that picking 5 cells and combining them to form a cell sample can obtain detection data with relatively high accuracy, and the method in this embodiment is applicable to the detection of trace samples. Figure 2Some gene loci have no peaks or the peaks are not obvious, indicating that when detecting with a cell sample formed by 1 cell, the nucleic acid content is too low to be detected.

[0081] In this embodiment, a monomer sampling method is used to obtain a biological sample, so the detected nucleic acid information is the nucleic acid information of a single individual. In other embodiments, there may be multiple biological sources in the obtained biological sample. Therefore, when the number of cells in step S110 is greater than 10, the number of picked cells can be less than or equal to 50% of the total number of cells. When there are 3 or more peaks at any gene locus in the map, the detection method further includes:

[0082] S4. Pick another cell sample by picking the same number of cells and repeat step S3.

[0083] S5. Compare the maps obtained in S3 and S4 and analyze the biological sources of each gene fragment.

[0084] Since the compositions of different biological samples in S2 and S4 are likely to be different, by analyzing the changes in the peak areas of different peaks at the same gene locus, the peaks formed by cells from different biological sources can be separated. When the cell amount is sufficient, the maps obtained by multiple repeated detections can be analyzed and verified.

[0085] In the prior art, when performing various nucleic acid sequencing, it is necessary to immerse the sampling carrier carrying the biological sample in a lysis solution for lysis, and then purify it with magnetic beads or chromatography columns. The obtained purified solution is detected and analyzed by PCR and capillary electrophoresis. Purifying the cell lysate before PCR helps to exclude the influence of substances such as proteins, RNA, cell debris, and other substances that may interfere with the PCR reaction on PCR, thereby preventing amplification failure or the generation of non-specific amplification products, and helping to improve the effective detection rate of the detection. Therefore, in the field of gene sequencing, there is a technical prejudice that "it is necessary to fully purify the cell lysate before PCR". However, although this detection can avoid the influence of other substances in the cell on PCR through good purification effects, it consumes a large amount of nucleic acid in the case of trace samples. Therefore, it is difficult to apply when the sample volume is small, and it is difficult to distinguish nucleic acid information from different biological sources. In the present invention, the above technical prejudice is overcome. By the method of capillary picking, cells and extracellular impurities are physically separated, and the lysate is directly subjected to PCR and electrophoresis detection, greatly retaining nucleic acids and helping to detect trace samples. In the present invention, when performing PCR, the volume of the added sample is greatly reduced, and the sample addition volume is reduced to the nanoliter level or lower. And a small amount of cells means that the content of substances in the lysate that affect PCR is low. Therefore, the final concentration of substances in the PCR reaction system that affect PCR is low, and nucleic acids can still be effectively amplified even without a purification step. In addition, in the method of the present invention, since the amount of detected cells is determined, the sources of nucleic acid fragments of different source cells can be distinguished by detecting different cells multiple times, which helps to analyze the sample source.

[0086] Comparative Example 1:

[0087] The difference between this comparative example and Example 1 is only that a homologous oral epithelial cell sample is taken again by the same method and lysed, and the total number of cells far exceeds one thousand. After lysing the cells, magnetic beads are used to purify the nucleic acid to obtain a purified solution, and PCR and capillary electrophoresis are performed with the purified solution. The detected data are shown in Figure 6 , and comparing Figure 6 and Figure 3 it can be seen that the spectra have a high degree of similarity, thus proving that the data obtained in Example 1 of the present invention have a high degree of accuracy.

[0088] Comparative Example 2:

[0089] The difference between this comparative example and Example 1 is only that the cell sample contains 5 cells and the total pipetting volume exceeds 1 μL. The detected data are shown in Figure 7 . Comparing Figure 7 and Figure 3 it can be seen that a large pipetting volume will result in more impurities, and there are a large number of miscellaneous peaks and partial peak absences in the spectrum.

[0090] Comparative Example 3:

[0091] The difference between this comparative example and Example 1 is only that the cell sample contains 5 cells, and steps S113, S114, S201, S202, and S203 are carried out intermittently. From the start of step S113 to the start of step S203, the elapsed time is about 3.5 h. The detected data are shown in Figure 8 . By comparison Figure 8 and Figure 3 it can be seen that there are many miscellaneous peaks in the spectrum, and some peaks are not clearly shown or even missing, indicating that long-term storage will cause nucleic acid degradation and result in the loss of detection information.

[0092] Example 2:

[0093] The difference between this example and Example 1 is only that step S200 in this example includes:

[0094] S210. Add the premixed solution and primer mixture to the cell sample, and make up the volume with the lysis solution to obtain a mixed system. The lysis temperature of the lysis solution is coordinated with the denaturation temperature of the polymerase chain reaction.

[0095] S220. Add the mixed system to the polymerase chain reaction analyzer, adjust the temperature to the denaturation temperature, and then incubate statically until the cells are lysed.

[0096] S230. After the incubation is completed, carry out the polymerase chain reaction to obtain the reaction solution.

[0097] The spectrum detected in this example is basically the same as the spectrum obtained in Example 1.

[0098] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as these combinations of technical features do not conflict, they should be considered as the scope described in this specification.

[0099] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A method for detecting trace short tandem repeat sequences, characterized in that: include: Isolating a number of cells from a biological sample to form a cell sample; Lysing cells in the cell sample and performing polymerase chain reaction to obtain a reaction solution; The reaction solution is taken for electrophoresis detection and data analysis.

2. The method according to claim 1, characterized in that Take intact cells from biological samples, and obtain cell samples including: Soaking the biological sample in a buffer solution to release cells in the biological sample into the buffer solution; A number of cells are picked up from the buffer solution through a capillary tube and combined to obtain the cell sample.

3. The method according to claim 2, characterized in that The total volume of the cell sample is less than or equal to 20% of the reaction system of the polymerase chain reaction, and / or the pipetting volume when picking cells each time is less than or equal to 150 nL.

4. The method according to claim 2, characterized in that The time from immersing the biological sample in the buffer to starting the polymerase chain reaction is less than or equal to 2 hours.

5. The method according to claim 2, characterized in that The buffer is tris(hydroxymethylaminomethane)-ethylenediaminetetraacetic acid buffer.

6. The method according to claim 1, characterized in that The number of cells in the cell sample is any value between 3 and 10.

7. The method according to claim 1, characterized in that The number of cells in the cell sample is less than or equal to 50% of the number of intact cells in the biological sample.

8. The method according to claim 6 or 7, characterized in that Each cell in the cell sample has a complete morphology.

9. The method according to claim 1, characterized in that Lysing cells in the cell sample and performing polymerase chain reaction to obtain a reaction solution comprising: Adding a premix and a primer mixture to the cell sample, and making up the volume with a lysis solution to obtain a mixed system, wherein the lysis temperature of the lysis solution matches the denaturation temperature of the polymerase chain reaction; Adding the mixed system to a polymerase chain reaction analyzer, adjusting the temperature to the denaturation temperature and then incubating until the cells are lysed; After the incubation is completed, polymerase chain reaction is performed to obtain the reaction solution.

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