Method for detecting state of microsatellite site BAT-26

Abstract

The invention discloses a method for detecting the state of a microsatellite site BAT-26, and belongs to the field of biological medicine, the sequences of a primer and a probe are shown in SEQ ID No: 1 to SEQ ID No: 11, the primer and the probe are adopted for detecting the state of the microsatellite site BAT-26 through a quantitative PCR end point fluorescence signal ratio method, the method has the advantages of being easy and rapid to operate and objective in result interpretation, uncovering electrophoresis detection is not needed, the detection time is shortened, and the detection efficiency is improved. Meanwhile, an expensive sequencer does not need to be purchased, and analysis can be carried out only by one fluorescent quantitative PCR instrument, so that the method is convenient to popularize.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a method for detecting the state of a microsatellite locus BAT-26. Background Art

[0002] Microsatellite markers, also known as short tandem repeats (STRs) or simple sequence repeats (SSRs), are simple repeat sequences evenly distributed in the genomes of eukaryotic organisms. They are composed of tandem repeat fragments of 1 to 6 nucleotides. Because the number of repetitions of the repeating units is highly variable among individuals and the number is abundant, microsatellite markers are widely used. There are already 19 million known microsatellite loci in the human genome.

[0003] At present, the methods for detecting the status of microsatellite loci include fluorescent PCR-capillary electrophoresis, fluorescent PCR melting curve method, etc. The fluorescent PCR-capillary electrophoresis method is a widely used detection method, which is to amplify by PCR, analyze the amplified product by electrophoresis and separate alleles according to size for detection, but it has high requirements for detection conditions, complex operation process, high cost, and requires expensive sequencers; the fluorescent PCR melting curve method has low requirements for detection equipment, low detection cost, simpler operation, and shorter detection time, but the method has low resolution and cannot effectively distinguish the difference between wild type and mutant type, insertion type or deletion type, making the result interpretation more subjective.

[0004] The present invention designs primers, probes and amplification systems for detecting the state of microsatellite locus BAT-26 by using quantitative PCR end-point fluorescence signal ratio method to solve the above problems. Summary of the invention

[0005] The main purpose of the present invention is to provide a method for detecting the status of the microsatellite locus BAT-26 by a simple and rapid operation, objective result interpretation, no need to open the cover for electrophoresis detection, and no need to purchase expensive sequencers, and only a fluorescent quantitative PCR instrument is required.

[0006] The purpose of the present invention can be achieved by adopting the following technical solutions:

[0007] A method for detecting the status of microsatellite locus BAT-26 comprises the following steps:

[0008] Step 1: Obtain a sample and extract genomic DNA from the sample;

[0009] Step 2: Prepare a quantitative PCR reaction system, use Roche Light Cycler 480Ⅱ fluorescence quantitative PCR instrument to perform real-time quantitative detection of BAT-26 locus, and collect detection probe and reference probe signals at the same time;

[0010] Step 3: Obtain the fluorescence values ​​of the BAT-26 reference probe and the BAT-26 detection probe at the same cycle number during the amplification plateau phase and calculate the ratio between the two;

[0011] Step 4: Compare the calculated fluorescence ratio with a preset threshold value. If the fluorescence ratio is less than or equal to the threshold value, the microsatellite locus BAT-26 is in a stable state; if the fluorescence ratio is greater than the threshold value, the microsatellite locus BAT-26 is in an unstable state.

[0012] Preferably, the BAT-26 detection probe and the BAT-26 reference probe are in the same amplicon, which includes a BAT-26 site detection primer, a BAT-26 detection probe and a BAT-26 reference probe. The detection probe covers the BAT26 site repeat region, and the reference probe covers the conserved region adjacent to the repeat region. If the BAT-26 site repeat region has not mutated, it is wild type, and the BAT-26 detection probe and the BAT-26 reference probe are completely matched, and the hydrolysis signals of the two are almost the same, and the fluorescence ratio is close to 1 at this time; if the BAT-26 site mutates, the BAT-26 detection probe cannot be completely paired with the template, and the binding force is affected, resulting in a decrease in its hydrolysis signal, but the BAT-26 reference probe can still be completely paired with the template, and the binding force is not affected, so at this time the reference probe signal value divided by the detection probe signal value will be greater than 1, and the ratio is proportional to the mismatch intensity.

[0013] Preferably, the BAT-26 detection probe is an oligonucleotide containing a repeat sequence or a complementary sequence of a wild-type BAT-26 microsatellite locus, and both ends of the oligonucleotide are labeled with a fluorescent group and a corresponding quenching group, respectively;

[0014] The BAT-26 reference probe is an oligonucleotide with an adjacent sequence or a complementary sequence to the BAT-26 detection probe, and the two ends of the oligonucleotide are respectively labeled with a fluorescent group and a corresponding quenching group;

[0015] There can be 6-10 bp overlap between the BAT-26 detection probe and the BAT-26 reference probe, introducing competition to enhance the sensitivity of the detection probe to mutations and improve resolution;

[0016] The fluorescent group is selected from but not limited to the following types: FAM, HEX, VIC, ROX, CY5, CY3, TET, JOE;

[0017] The quenching group is selected from but not limited to the following types: TAMRA, BHQ1, BHQ2, DABCYL, BHQ3, MGB-NFQ.

[0018] Preferably, the quantitative PCR reaction system comprises 1×PCR buffer, 0.1U / μL TaqDNA polymerase, 0.0125U / μL UDG enzyme, 0.2mM dNTPs (dUTP plus), 0.2μM SEQ ID No: 1 primer, 0.2μM SEQ ID No: 2 primer, 0.2μM BAT-26 detection probe and 0.2μM BAT-26 reference probe.

[0019] Preferably, the threshold in step 4 is determined using the ROC curve method.

[0020] Beneficial technical effects of the present invention:

[0021] The present invention provides a method for detecting the state of microsatellite locus BAT-26. The method provided by the present invention greatly simplifies the detection operation procedure and shortens the detection time. At the same time, only the fluorescence signal ratio needs to be obtained to interpret the result, which improves the result.

[0022] Objectivity and simplicity of interpretation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a ROC curve calculated based on the threshold of probe set 1 according to a preferred embodiment of a method for detecting the status of the microsatellite locus BAT-26 of the present invention.

[0024] Figure 2 This is an ROC curve of threshold calculation of probe group 2 to probe group 6 according to a preferred embodiment of a method for detecting the status of the microsatellite locus BAT-26 of the present invention.

[0025] Figure 3-Figure 8 Probe groups 1 to 6 according to a preferred embodiment of a method for detecting the status of the microsatellite locus BAT-26 of the present invention detect the fluorescence ratios of clinical samples S1 to S10.

[0026] Figure 9-10 These are the results of fluorescent PCR-capillary electrophoresis detection of the BAT-26 locus of clinical samples S1-S10.

[0027] Fig.11 The diagram is a probe state diagram of a preferred embodiment of a method for detecting the state of the microsatellite locus BAT-26 according to the present invention. DETAILED DESCRIPTION

[0028] In order to make the technical solution of the present invention more clear and specific to those skilled in the art, the present invention is further described in detail below in conjunction with embodiments and drawings, but the implementation manner of the present invention is not limited thereto.

[0029] Example 1 Primer probe design.

[0030] According to the gene sequence of the microsatellite locus BAT-26, the present invention follows the primer and probe design principles to design multiple primers and probes, the 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group. After a large number of experimental screening tests, the primers and probes shown in Table 1 are selected. The screened detection probes and reference probes are combined, and the preferred probe combination is shown in Table 2.

[0031] Table 1

[0032]

[0033]

[0034] Table 2

[0035]

[0036] Example 2 Fluorescence ratio acquisition.

[0037] This embodiment specifically discloses how to obtain the fluorescence ratio, which specifically includes:

[0038] Obtaining a sample and extracting genomic DNA from the sample;

[0039] Prepare real-time quantitative PCR (qPCR) reaction system:

[0040] Table 3

[0041] Components Dosage (μL) 2×PCR buffer 5 TaqDNA polymerase (5U / μL) 0.2 UDG enzyme (1U / μL) 0.125 dNTPs (dUTP plus) (25 mM each) 0.08 Primer 1 (10 μM) 0.2 Primer 2 (10 μM) 0.2 Detection probe (10 μM) 0.2 Reference probe (10 μM) 0.2 Nuclease-free water 2.795 Template DNA (2-50ng / μL) 1

[0042] Roche Light Cycler 480Ⅱ fluorescence quantitative PCR instrument was used for detection, and the program was as shown in Table 4:

[0043] Table 4

[0044]

[0045]

[0046] The present invention is calculated according to the following fluorescence ratio method:

[0047] Obtain qPCR raw fluorescence value data;

[0048] Select the starting cycle number and the ending cycle number for calculating the baseline fluorescence value, and calculate the average fluorescence value of all cycles between the start and the end as the baseline fluorescence value R-baseline;

[0049] According to the qPCR curve, the nth cycle was selected so that the detection probe and reference probe signals both entered the plateau phase. The ΔR detection probe and ΔR reference probe were equal to the nth cycle fluorescence value minus the corresponding baseline fluorescence value R-baseline. The fluorescence ratio was calculated as follows:

[0050]

[0051] Example 3 Threshold calculation.

[0052] This embodiment provides a method for establishing a microsatellite locus result interpretation threshold, which specifically comprises the following steps:

[0053] Using the sample DNA template detected by fluorescent PCR-capillary electrophoresis, the sample qPCR system was prepared using the preferred primer-probe combination of Example 1. The preparation method was the same as the qPCR system of Example 2. The fluorescence ratio of each sample was obtained according to the fluorescence ratio obtaining method of Example 2, and the ROC curve method was used to analyze and obtain the optimal threshold.

[0054] Attached Figure 1 , 2 The ROC curve of the preferred probe combination of Example 1 is shown.

[0055] The optimal ROC threshold results of the preferred probe combination in Example 1 are shown in Table 5 below:

[0056] Table 5

[0057]

[0058]

[0059] From the above table, it can be seen that the AUC value corresponding to group 1 is 0.979, which means that the value of group 1 for detecting the status of the microsatellite locus BAT-26 is very high, and the corresponding optimal threshold is 2.398 (at this time, the sensitivity is 87.5% and the specificity is 100%).

[0060] The AUC value corresponding to group 2 is 1.000, which means that the value of group 2 for detecting the status of the microsatellite locus BAT-26 is very high, and the corresponding optimal threshold is 2.238 (at this time, the sensitivity is 100% and the specificity is 100%).

[0061] The AUC value corresponding to group 3 is 1.000, which means that the value of group 3 for detecting the status of the microsatellite locus BAT-26 is very high, and the corresponding optimal threshold is 2.062 (at this time, the sensitivity is 100% and the specificity is 100%).

[0062] The AUC value corresponding to group 4 is 1.000, which means that the value of group 4 for detecting the status of the microsatellite locus BAT-26 is very high, and the corresponding optimal threshold is 1.827 (at this time, the sensitivity is 100% and the specificity is 100%).

[0063] The AUC value corresponding to group 5 is 1.000, which means that the value of group 5 for detecting the status of the microsatellite locus BAT-26 is very high, and the corresponding optimal threshold is 1.875 (at this time, the sensitivity is 100% and the specificity is 100%).

[0064] The AUC value corresponding to group 6 is 1.000, which means that group 6 has a very high value for detecting the status of the microsatellite locus BAT-26, and the corresponding optimal threshold is 2.243 (at this time, the sensitivity is 100% and the specificity is 100%).

[0065] In this embodiment, the microsatellite locus and its adjacent sequences are quantitatively amplified in real time in the same amplicon, which includes a detection probe and a reference probe.

[0066] In this embodiment, the detection probe is an oligonucleotide including a repeat sequence or a complementary sequence of a wild microsatellite locus;

[0067] The two ends of the oligonucleotide are respectively labeled with a fluorescent group and a corresponding quenching group;

[0068] The reference probe is an oligonucleotide with a sequence adjacent to or complementary to the detection probe;

[0069] Both ends of the oligonucleotide are labeled with a fluorescent group and a corresponding quenching group respectively.

[0070] In this embodiment, the kit includes a BAT-26 primer pair, a BAT-26 detection probe, a BAT-26 reference probe, Taq DNA polymerase, UDG enzyme, PCR buffer, dNTPs (dUTP plus), and nuclease-free water.

[0071] In this embodiment, the BAT-26 primer pair is used to amplify the BAT-26 site and its adjacent sequences.

[0072] In this example, the 5' end of the BAT-26 detection probe is modified with a FAM fluorescent group, and the 3' end is modified with a BHQ1 or MGB-NFQ quenching group.

[0073] In this example, the 5' end of the BAT-26 reference probe is modified with a VIC fluorescent group, and the 3' end is modified with a BHQ1 or MGB-NFQ quenching group.

[0074] Example 4 Clinical sample detection.

[0075] This embodiment provides a method for detecting the status of the microsatellite locus BAT-26 in clinical samples, which specifically includes the following steps:

[0076] Acquisition of genomic DNA: Extract DNA from tumor tissue FFPE samples S1-S10, dilute the extracted DNA template to 10 ng / μL, use the preferred primer probe set of Example 1 to prepare a sample qPCR system, the preparation method is the same as the qPCR system of Example 2, obtain the fluorescence ratio of each sample according to the fluorescence ratio acquisition method of Example 2, and analyze the microsatellite status of the sample according to the threshold value of Example 3.

[0077] Attached Figure 3-8 The fluorescence ratios of clinical samples S1-S10 are displayed. The fluorescence ratio of S1-S4 is less than the threshold, and the BAT-26 site is stable; the fluorescence ratio of S5-S10 is greater than the threshold, and the BAT-26 site is unstable.

[0078] Furthermore, the fluorescence PCR-capillary electrophoresis method was used to detect the status of the S1-S10 BAT-26 loci. Figure 9-10 The results of fluorescence PCR-capillary electrophoresis detection of the S1-S10 BAT-26 sites in clinical samples were shown. The S1-S4 BAT-26 sites were stable, while the S5-S10 BAT-26 sites were unstable, and the results were consistent.

[0079] exist Fig.11 It can be seen that the first group is in the wild state: the reference probe and the detection probe are in the same amplicon, and the hydrolysis signals of the two are almost the same, and the fluorescence ratio is close to 1.

[0080] The second group is mutation state 1: the template repeat unit is missing or there is a mismatch. The detection probe’s binding force is affected due to the presence of the mismatch, resulting in a decrease in its hydrolysis signal. However, the mismatch does not affect the reference probe, so the fluorescence ratio will be greater than 1 at this time. The size of the fluorescence ratio is proportional to the mismatch intensity.

[0081] The third group is mutation state 2: the template repeating unit is redundant or has mismatches. The binding force of the detection probe is affected due to the presence of mismatches, resulting in a decrease in its hydrolysis signal, but the mismatch does not affect the reference probe. Therefore, the fluorescence ratio will be greater than 1 at this time, and the size of the fluorescence ratio is proportional to the mismatch intensity.

[0082]

[0083] The above are only further embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and concepts of the present invention within the scope disclosed by the present invention, which belong to the protection scope of the present invention.

Claims

1. A method for detecting the status of the microsatellite locus BAT-26, characterized in that: The steps include: Step 1: Obtain a sample and extract genomic DNA from the sample; Step 2: Prepare a quantitative PCR reaction system, use Roche Light Cycler 480Ⅱ fluorescence quantitative PCR instrument to perform real-time quantitative detection of BAT-26 locus, and collect detection probe and reference probe signals at the same time; Step 3: Obtain the fluorescence values ​​of the BAT-26 reference probe and the BAT-26 detection probe at the same cycle number during the amplification plateau phase and calculate the ratio between the two; Step 4: Compare the calculated fluorescence ratio with a preset threshold value. If the fluorescence ratio is less than or equal to the threshold value, the microsatellite locus BAT-26 is in a stable state; if the fluorescence ratio is greater than the threshold value, the microsatellite locus BAT-26 is in an unstable state.

2. A method for detecting the status of the microsatellite locus BAT-26 according to claim 1, characterized in that: The BAT-26 detection probe and the BAT-26 reference probe are in the same amplicon, which includes the BAT-26 site detection primer, the BAT-26 detection probe and the BAT-26 reference probe. The detection probe covers the BAT26 site repeat region, and the reference probe covers the conserved region adjacent to the repeat region. If the BAT-26 site repeat region has not mutated, it is wild type, and the BAT-26 detection probe and the BAT-26 reference probe are completely matched, and the hydrolysis signals of the two are almost the same. At this time, the fluorescence ratio is close to 1; if the BAT-26 site mutates, the BAT-26 detection probe cannot be completely paired with the template, and the binding force is affected, resulting in a decrease in its hydrolysis signal, but the BAT-26 reference probe can still be completely paired with the template, and the binding force is not affected. Therefore, at this time, the reference probe signal value divided by the detection probe signal value will be greater than 1, and the ratio is proportional to the mismatch intensity.

3. A method for detecting the status of microsatellite locus BAT-26 according to claim 2, characterized in that: The sequences of the BAT-26 site detection primers are SEQ ID No: 1 and SEQ ID No:

2.

4. A method for detecting the status of microsatellite locus BAT-26 according to claim 2, characterized in that: The BAT-26 detection probe is an oligonucleotide containing a repeat sequence or a complementary sequence of the wild BAT-26 microsatellite locus, and the two ends of the oligonucleotide are respectively labeled with a fluorescent group and a corresponding quenching group; The BAT-26 reference probe is an oligonucleotide with a sequence adjacent to or complementary to the BAT-26 detection probe, and both ends of the oligonucleotide are labeled with a fluorescent group and a corresponding quenching group, respectively.

5. A method for detecting the status of microsatellite locus BAT-26 according to claim 4, characterized in that: The sequences of the BAT-26 detection probe and the BAT-26 reference probe are SEQ ID No: 3 to SEQ ID No:

11.

6. A method for detecting the status of microsatellite locus BAT-26 according to claim 1, characterized in that: The quantitative PCR reaction system included 1× PCR buffer, 0.1 U / μL Taq DNA polymerase, 0.0125 U / μL UDG enzyme, 0.2 mM dNTPs (dUTP plus), 0.2 μM SEQ ID No: 1 primer, 0.2 μM SEQ ID No: 2 primer, 0.2 μM BAT-26 detection probe and 0.2 μM BAT-26 reference probe.

7. A method for detecting the status of microsatellite locus BAT-26 according to claim 1, characterized in that: The threshold in step 4 is determined using the ROC curve method.

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