CN114182011A - Primer pair, kit and method for detecting stability of microsatellite BAT25 locus - Google Patents
Primer pair, kit and method for detecting stability of microsatellite BAT25 locus Download PDFInfo
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Abstract
The invention provides a primer pair, a kit and a method for detecting the stability of a microsatellite BAT25 locus. The primer pair has a base sequence shown as SEQ ID NO. 1 and a base sequence shown as SEQ ID NO. 2. The primer pair disclosed by the invention is high in detection sensitivity and specificity, and accurate, rapid and low in cost when the primer is used for detecting the stability of the BAT25 locus of the microsatellite by adopting the HRM method, so that the clinical requirements can be met, and the application prospect is wide.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a primer pair, a kit and a method for detecting the stability of a microsatellite BAT25 locus.
Background
Colorectal cancer (CRC) is one of the most common malignant tumors, and the incidence rate of the CRC is 3 rd, the mortality rate of the CRC is 2 nd in the malignant tumor ranks, and the CRC accounts for the first global incidence and death in the gastrointestinal malignant tumors, thereby seriously threatening the health and survival of human beings. In recent years, with the change of life style and dietary structure of Chinese people, it has become a second-grade malignant tumor with the first-grade morbidity of digestive systems in China. Microsatellite instability (MSI) is an important cause of colorectal cancer, plays an important role in predicting prognosis and chemotherapy response of intestinal cancer patients, and has important significance in diagnosing Lynch syndrome.
In recent years, the relevant clinical and fundamental aspects of MSI have been well studied, and the relationship between MSI and CRC has been deeply understood. In 2016, the united states national cancer complex treatment consortium (NCCN) in the "clinical practice guidelines for colorectal cancer" (2016.V2) states that Lynch syndrome should be tested for MSI or mismatch repair (MMR): 1) diagnosis of patients with colorectal cancer at age 70 and below, and patients greater than 70 who meet the Bethesda guidelines, should be tested for Lynch syndrome tumor; 2) all stage II patients should be tested for MSI or MMR, since stage II patients with MSI-H have a better prognosis and do not benefit from 5-fluorouracil monotherapy. For stage II colon cancers with MSI-H characteristics, poor tissue differentiation is no longer considered a high risk factor. In addition, the NCCN guidelines indicate that MSI or MMR detection is required for patients with locally advanced, recurrent or metastatic gastric cancer prior to treatment with a PD-1 inhibitor of interest. FDA approved the anti-PD-1 drug pembrolizumab for use in second-line or subsequent treatment options for solid tumors that are unresectable or metastatic MSI-H or dMMR. BAT25 is one of the important sites for microsatellite instability detection in colorectal cancer, and changes in the number of mononucleotide repeats occur when instability occurs.
Currently, methods for detecting microsatellite instability include: an MMR protein immunohistochemical method, a DNA sequencing method, a fragment analysis method, a high performance liquid chromatography technology, a high-resolution melting (HRM) method and the like. Wherein: fragment analysis is currently recognized as the gold standard; however, this method is costly, requires a long Detection time, and requires analysis on an expensive sequencer, and most laboratories cannot perform Detection (V Descholmester et al, Detection of microbial activity in a biological laboratory using an alternative multiplex assay of quantitative-monomeric monoclonal antibodies markers, J Mol Diagn, 2008,10(2): 154-9). The HRM method is based on the physical property of nucleic acid for analysis, only some saturated dyes are added on the basis of conventional PCR, the shape of a melting curve depends on GC content, fragment length and a product sequence, and the melting curve can be distinguished through the difference of the melting curve; the sample is directly subjected to HRM analysis after PCR amplification, PCR products are directly analyzed in the same PCR tube without transferring to other analysis devices, the closed tube operation is realized, and the method has the advantages of high speed, low cost, high sensitivity and the like, and can be used for analyzing on a fluorescence quantitative PCR instrument. However, the HRM method requires a high level of primer design: the primer has good specificity, and the amplified fragment is required to be short enough to obtain good detection sensitivity. Based on this, the HRM method has not been successfully applied to the detection of BAT25 site stability.
Disclosure of Invention
Based on the above, the main objective of the invention is to provide a primer pair, a kit and a method for detecting the stability of the BAT25 locus of the microsatellite. The primer provided by the invention is used for detecting the stability of the BAT25 locus of the satellite, and has high sensitivity and good specificity.
The purpose of the invention is realized by the following technical scheme:
a primer pair for detecting the stability of a microsatellite BAT25 locus is provided, wherein the primer has a base sequence shown as SEQ ID NO. 1 and a base sequence shown as SEQ ID NO. 2.
A kit for detecting the stability of a microsatellite BAT25 locus comprises a primer pair with base sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2.
In one embodiment, the kit further comprises at least one of an amplification buffer, dntps, a DNA polymerase, a saturating fluorescent dye, and water.
In one embodiment, the DNA polymerase is Taq enzyme.
In one embodiment, the saturated fluorescent dye is Eva Green.
A method for detecting microsatellite BAT25 site stability based on a high resolution melting curve, the method comprising the steps of:
respectively taking DNA of a sample to be detected and DNA of a reference substance as templates, carrying out PCR reaction by using primer pairs with base sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2, collecting fluorescent signals, and drawing a melting curve;
and (4) analyzing a high-resolution melting curve, and comparing the high-resolution melting curve of the sample to be detected with the high-resolution melting curve of a reference substance to identify the stability of the microsatellite BAT25 locus of the sample to be detected.
In one embodiment, the PCR reaction system comprises:
in one embodiment, the procedure of the PCR reaction is: 93-97 ℃ for 9-11 min; 93-97 ℃, 18-22 s, 56-58 ℃, 28-32 s, 71-73 ℃, 28-32 s, 38-42 cycles; the melting temperature is 75-78 ℃; 38-42 ℃ for 28-32 s.
In one embodiment, the frequency of the fluorescence signal is from 11 times/deg.C to 13 times/deg.C.
In one embodiment, the frequency of the fluorescence signal is 12 times/deg.C.
In one embodiment, the concentration of the template is 50 ng/. mu.L to 100 ng/. mu.L.
In one embodiment, the template is at a concentration of 75 ng/. mu.L.
The invention has the following beneficial effects:
the primer pair disclosed by the invention is high in detection sensitivity and specificity, and when the stability of the BAT25 locus of the microsatellite is detected by adopting the primer disclosed by the invention to carry out an HRM method, the sensitivity is high (for example, up to 91.20%) and the specificity is good (for example, 97.01%), and the clinical requirements can be met. In addition, when the primers are used for HRM method detection of the stability of the BAT25 locus of the microsatellite, the requirements on equipment are greatly reduced, a gene analyzer is not needed, and only a fluorescence quantitative PCR instrument with HRM function is needed. Meanwhile, the primers are adopted to detect the stability of the locus of the microsatellite BAT25 by the HRM method, the operation procedure is greatly simplified, the time required for detecting one sample is shortened by about 1 hour compared with the gold standard method, and the cost is reduced by 80 percent; therefore, the method has good application prospect in detecting the stability of the locus of the microsatellite BAT 25. Overall, the present invention provides an accurate, rapid and inexpensive method for detecting the stability of BAT25 locus.
Drawings
FIG. 1 shows the melting curve of the primer set 1 for the fluorescent quantitative PCR detection of the sample 1.
FIG. 2 shows the melting curve of the primer set 1 for the fluorescent quantitative PCR detection of the sample 2.
FIG. 3 is a melting curve of the primer set 2 for the fluorescent quantitative PCR detection of sample 2.
FIG. 4 shows the melting curve of the primer set 3 for the fluorescent quantitative PCR detection of sample 2.
FIG. 5 is a fluorescent quantitative PCR amplification curve of primer pair 4 versus sample 2.
FIG. 6 is a fluorescent quantitative PCR amplification curve of primer pair 5 versus sample 2.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The embodiment of the invention provides a primer pair for detecting the stability of a microsatellite BAT25 locus, wherein the primer has a base sequence shown as SEQ ID NO. 1 and a base sequence shown as SEQ ID NO. 2.
The primer pair disclosed by the invention is high in detection sensitivity and specificity, and when the stability of the BAT25 locus of the microsatellite is detected by adopting the primer disclosed by the invention to carry out an HRM method, the sensitivity is high (for example, up to 91.20%) and the specificity is good (for example, 97.01%), and the clinical requirements can be met. In addition, when the primers are used for HRM method detection of the stability of the BAT25 locus of the microsatellite, the requirements on equipment are greatly reduced, a gene analyzer is not needed, and only a fluorescence quantitative PCR instrument with HRM function is needed. Meanwhile, the primers are adopted to detect the stability of the locus of the microsatellite BAT25 by the HRM method, the operation procedure is greatly simplified, the time required for detecting one sample is shortened by about 1 hour compared with the gold standard method, and the cost is reduced by 80 percent; therefore, the method has good application prospect in detecting the stability of the locus of the microsatellite BAT 25. Overall, the present invention provides an accurate, rapid and inexpensive method for detecting the stability of BAT25 locus.
The embodiment of the invention also provides a kit for detecting the stability of the BAT25 locus of the microsatellite, which comprises a primer pair with the base sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2.
Preferably, the kit further comprises at least one of an amplification buffer, dntps, a DNA polymerase, a saturated fluorescent dye, and water.
Preferably, the DNA polymerase is Taq enzyme.
It is understood that the embodiment of the present invention does not specifically limit the kinds of the saturated fluorescent dye, including but not limited to Eva Green. The following embodiment explains the technical solution of the present invention by taking Eva Green as an example.
The embodiment of the invention also relates to a method for detecting the stability of the BAT25 locus of the microsatellite based on the high-resolution melting curve, which comprises the following steps:
respectively taking DNA of a sample to be detected and DNA of a reference substance as templates, carrying out PCR reaction by using primer pairs with base sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2, collecting fluorescent signals, and drawing a melting curve;
and (4) analyzing a high-resolution melting curve, and comparing the high-resolution melting curve of the sample to be detected with the high-resolution melting curve of a reference substance to identify the stability of the microsatellite BAT25 locus of the sample to be detected.
It is understood that the "sample to be tested" in the embodiments of the present invention may be tumor tissue, and the "control" in the embodiments of the present invention refers to healthy normal tissue, where the tumor tissue and the healthy normal tissue are both derived from the same tumor patient. In the case of colorectal cancer, the "sample to be tested" is tumor tissue of a patient suffering from colorectal cancer, and the "control" is paracancerous normal tissue or blood from the patient suffering from colorectal cancer. Taking colorectal cancer as an example, if the melting curve of the tumor tissue of a colorectal cancer patient shows two or more melting peaks, and the melting curve of the normal tissue shows only one melting peak, judging that the two melting peaks are inconsistent, and indicating that the colorectal cancer patient is a patient with unstable microsatellite BAT25 locus; otherwise, the colorectal cancer patient is the microsatellite BAT25 site stable patient. It will be appreciated that the above-described methods provided by embodiments of the present invention may, for example, be used for detection for non-diagnostic purposes.
Preferably, the system of the PCR reaction comprises:
for example: the system of the PCR reaction comprises: 10 XBuffer 2.5. mu.L, dNTP 2.5. mu.L, template 5. mu.L, fluorescent dye 1.25. mu.L, primer 1.25. mu.L shown in SEQ ID NO:1, primer 1.25. mu.L shown in SEQ ID NO:2, Taq enzyme 0.25. mu.L, and water to 25. mu.L. It is understood that the water described in the examples of the present invention is enzyme-free water.
Preferably, the procedure of the PCR reaction is: 93-97 ℃ for 9-11 min; 93-97 ℃, 18-22 s, 56-58 ℃, 28-32 s, 71-73 ℃, 28-32 s, 38-42 cycles; the melting temperature is 75-78 ℃; 38-42 ℃ for 28-32 s. For example: the procedure of PCR reaction is 95 ℃ for 10 min; 40cycles of 95 ℃ 20s, 57 ℃ 30s, 72 ℃ 30 s; the melting temperature is 76 ℃ and 40 ℃ for 30 s.
Preferably, the frequency of the fluorescence signal is from 11 times/DEG C to 13 times/DEG C. For example, the frequency of the fluorescence signal is 12 times/. degree.C
Preferably, the concentration of the template is 50 ng/. mu.L to 100 ng/. mu.L. For example, the template concentration is 75 ng/. mu.L.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 establishment of a method for detecting the stability of the microsatellite BAT25 site
1. Primer and method for producing the same
This example provides a primer pair for detecting the stability of BAT25 locus, the nucleotide sequence of the primer pair is shown in SEQ ID NO. 1 and SEQ ID NO. 2.
Upstream primer (SEQ ID NO: 1): 5'-cctccaagaatgtaagtggga-3', respectively;
downstream primer (SEQ ID NO: 2): 5'-ggctctaaaatgctctgttctca-3' are provided.
2. Reaction system
Using the above primer set, a fluorescent quantitative PCR reaction system as shown in Table 1 was prepared using Blend Taq Plus enzyme (Toyobo, CAT NO. BTQ-201):
TABLE 1 fluorescent quantitative PCR reaction System
| Name (R) | Dosage range (mu L) | Specific dosage (mu L) |
| 10×Buffer | 2μL～2.5μL | 2.5μL |
| dNTP | 2μL～2.5μL | 2.5μL |
| Form panel | 1μL～5μL | 1μL |
| Fluorescent dye Eva Green | 1μL～1.25μL | 1.25μL |
| Upstream primer | 1μL～1.25μL | 1.25μL |
| Downstream primer | 1μL～1.25μL | 1.25μL |
| Taq enzyme | 0.2μL～0.25μL | 0.25μL |
| Enzyme-free water | 15μL～17μL | Adding water to 25 μ L |
3. Detection method
(1) Sample source and genomic DNA extraction: all tumor and normal samples of colorectal cancer patients were obtained from the pathology department of the sixth hospital affiliated to the university of Zhongshan, tissues were collected, and genomic DNA was extracted using a paraffin sample extraction kit. Adjusting the DNA of tumor tissues and normal tissues of colorectal cancer patients to 75 ng/mu L, respectively adding 1 mu L of DNA into the reaction system, uniformly mixing by vortex, performing fluorescent quantitative PCR reaction by using a Roche LightCycler 480 fluorescent quantitative PCR instrument according to the following program, collecting fluorescent signals at the frequency of 12 times/DEG C, and drawing a melting curve;
the procedure for the fluorescent quantitative PCR reaction was: 10min at 95 ℃; 40cycles of 95 ℃ 20s, 57 ℃ 30s, 72 ℃ 30 s; the melting temperature is 76 ℃ and 40 ℃ for 30 s.
(2) And (3) comparing the melting curves of the tumor tissue and the normal tissue of the colorectal cancer patient, and judging whether the colorectal cancer patient is a microsatellite BAT25 site stable patient according to the following method:
if the melting curve of the tumor tissue of the colorectal cancer patient shows two or more melting peaks, and the melting curve of the normal tissue only shows one melting peak, judging that the peak types of the two are inconsistent, and indicating that the colorectal cancer patient is a patient with unstable microsatellite BAT25 site; otherwise, the colorectal cancer patient is the microsatellite BAT25 site stable patient.
Example 2 construction of a kit for detecting microsatellite BAT25 site stability
The embodiment provides a kit for detecting the stability of a microsatellite BAT25 locus, which comprises the following components: the nucleotide sequence is shown as an upstream primer and a downstream primer shown as SEQ ID NO. 1 and SEQ ID NO. 2, Eva Green, Buffer, dNTP and Taq enzyme.
Upstream primer (SEQ ID NO: 1): 5'-cctccaagaatgtaagtggga-3', respectively;
downstream primer (SEQ ID NO: 2): 5'-ggctctaaaatgctctgttctca-3' are provided.
Test example 1 comparison of results of detecting the stability of the BAT25 site of microsatellite with different primer pairs
1. Experimental methods
In this test example, the method for detecting the stability of microsatellite BAT25 site, which was constructed in example 1, was used to investigate the influence of 5 primer pairs (Table 2) on the experimental results for detecting the stability of tumor microsatellite BAT25 site in patients with colorectal cancer, and to find out the results of the method (HRM analysis) of the present invention and the commercial kit (Beijing GenBank technology Co., Ltd.) for the conventional fragment analysis method
MSI detection kit) were compared and the working procedures of the commercial kit were referred to the product instructions. Sample 1 and sample 2 of this test example were both from the sixth hospital pathology department affiliated with the university of zhongshan.
2. Results of the experiment
The comparison of 5 sets of primer pairs and their peak patterns and interpretation results are shown in table 2, the melting curve of the primer pair 1 for the fluorescence quantitative PCR detection of the sample 1 is shown in fig. 1, the melting curve of the primer pair 1 for the fluorescence quantitative PCR detection of the sample 2 is shown in fig. 2, the melting curve of the primer pair 2 for the fluorescence quantitative PCR detection of the sample 2 is shown in fig. 3, the melting curve of the primer pair 3 for the fluorescence quantitative PCR detection of the sample 2 is shown in fig. 4, the fluorescence quantitative PCR amplification curve of the primer pair 4 for the sample 2 is shown in fig. 5, the fluorescence quantitative PCR amplification curve of the primer pair 5 for the sample 2 is shown in fig. 6, and it can be seen from table 2 and fig. 1 to 6 that the sample 2 is detected and interpreted as unstable microsatellite BAT25 site by using a commercial kit; however, the method for detecting the stability of the BAT25 locus of the microsatellite, which is constructed in example 1, by using the 5 primer pairs, is found that only the primer pair 1 (the nucleotide sequences of which are shown as SEQ ID NO:1 and SEQ ID NO:2) can accurately interpret the instability of the BAT25 locus of the microsatellite of the sample 2, and the rest 4 primer pairs (the primer pairs 2 to 5) cannot be accurately interpreted. Thus, primer pair 1 was used as a primer pair for detecting the stability of the BAT25 site of the microsatellite.
Table 2, 5 primer pairs and comparison of HRM peak patterns and interpretation results of amplification products thereof
Note: the CT value of the sample amplification is preferably 18-25, and if the CT value of the sample amplification is not in the range, the amplification effect of the fluorescence quantitative PCR reaction is poor or the amplification cannot be performed, and finally the quantity of the amplification product and the subsequent HRM analysis are possibly influenced.
Test example 2, detection Rate, detection time and cost of the method of the present invention and the existing fragment analysis commercialized kit
Comparison
1. Experimental methods
192 pairs (namely tumor tissues and corresponding normal tissues, numbered 1-192) of colorectal cancer patient samples are all from the pathology department of the sixth Hospital affiliated to Zhongshan university, sample genome DNAs are respectively extracted according to the method in example 1, the primer pair 1 (nucleotide sequences are shown as SEQ ID NO:1 and SEQ ID NO:2) obtained in the test example 1 is adopted, and the method for detecting the stability of the BAT25 locus of the microsatellite, which is constructed in example 1, is used for respectively detecting 192 pairs of samples.
Upstream primer (SEQ ID NO: 1): 5'-cctccaagaatgtaagtggga-3', respectively;
downstream primer (SEQ ID NO: 2): 5'-ggctctaaaatgctctgttctca-3' are provided.
Meanwhile, the existing fragment analysis method is utilized to commercialize the kit (Beijing Microgene technology Co., Ltd.)
MSI detection kit) (the working principle of the kit is: detection of microsatellite instability based on fluorescent quantitative PCR coupled with capillary electrophoresis) the 192 pairs were tested.
In addition, the detection time and cost used in the above 2 detection methods were accounted and compared.
2. Results of the experiment
The detection rate comparison results of the method and the existing commercial kit by the fragment analysis method are shown in table 3, and it can be seen that the method and the existing commercial kit by the fragment analysis method have 179 cases with consistent results, and 13 cases with inconsistent results; the sensitivity of the method of the invention is 114/(114+11) × 100%: 91.20%, and the specificity is 65/(65+2) × 100%: 97.01%.
Therefore, compared with the existing commercial kit (gold standard method) of the fragment analysis method, the specificity of the method is 97.01%, the sensitivity is up to 91.20%, and the clinical requirement can be met.
TABLE 3 detection Rate comparison of commercial kits for the method of the invention and the existing fragment analysis
The results of comparing the detection time and cost of the method of the invention with those of the commercial kit of the existing fragment analysis method are shown in Table 4, and it can be seen that the detection time of each sample is shortened by 33.3% and the detection cost of each sample is reduced by 80% when the method of the invention is used for detecting the stability of the BAT25 locus of the microsatellite compared with the detection time of the commercial kit of the existing fragment analysis method.
Therefore, the method for detecting the stability of the locus of the microsatellite BAT25 can greatly save the detection time and cost and better serve clinical patients.
TABLE 4 comparison of detection time and cost of the commercial kits of the present invention and the existing fragment analysis
| The method of the invention | Commercial kit for existing fragment analysis method | |
| Time of detection | 2 hours/sample | 3 hours/sample |
| Cost of experiment | 10 yuan/sample | 50 yuan/sample |
In conclusion, the primer pair provided by the embodiment of the invention has high detection sensitivity and specificity, and when the primer provided by the embodiment of the invention is used for detecting the stability of the BAT25 locus of the microsatellite by using the HRM method, the sensitivity is high (for example, up to 91.20%) and the specificity is good (for example, 97.01%), so that the clinical requirements can be met. In addition, when the primers are used for HRM method detection of the stability of the BAT25 locus of the microsatellite, the requirements on equipment are greatly reduced, a gene analyzer is not needed, and only a fluorescence quantitative PCR instrument with HRM function is needed. Meanwhile, the primers are adopted to detect the stability of the locus of the microsatellite BAT25 by the HRM method, the operation procedure is greatly simplified, the time required for detecting one sample is shortened by about 1 hour compared with the gold standard method, and the cost is reduced by 80 percent; therefore, the method has good application prospect in detecting the stability of the locus of the microsatellite BAT 25. In general, the embodiments of the present invention provide an accurate, fast, and inexpensive method for detecting the stability of BAT25 locus.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Sequence listing
<110> secondary sixth Hospital of Zhongshan university
Sun Yat-Sen University
<120> primer pair, kit and method for detecting stability of microsatellite BAT25 locus
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Claims (10)
1. A primer pair for detecting the stability of a microsatellite BAT25 locus is characterized by having a base sequence shown as SEQ ID NO. 1 and a base sequence shown as SEQ ID NO. 2.
2. A kit for detecting the stability of a microsatellite BAT25 locus is characterized by comprising a primer pair with base sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2.
3. The kit for detecting microsatellite BAT25 site stability according to claim 2 further comprising at least one of an amplification buffer, dNTPs, DNA polymerase, a saturating fluorescent dye, and water.
4. The kit for detecting the stability of BAT25 as a microsatellite according to claim 3 wherein said DNA polymerase is Taq enzyme.
5. The kit for detecting the stability of a microsatellite BAT25 locus as claimed in claim 3 wherein said fluorescent dye is Eva Green.
6. A method for detecting microsatellite BAT25 site stability based on a high resolution melting curve, the method comprising the steps of:
respectively taking DNA of a sample to be detected and DNA of a reference substance as templates, carrying out PCR reaction by using primer pairs with base sequences shown as SEQ ID NO. 1 and SEQ ID NO. 2, collecting fluorescent signals, and drawing a melting curve;
and (4) analyzing a high-resolution melting curve, and comparing the high-resolution melting curve of the sample to be detected with the high-resolution melting curve of a reference substance to identify the stability of the microsatellite BAT25 locus of the sample to be detected.
7. The method for detecting the stability of BAT25 locus, according to claim 6, wherein the PCR reaction system comprises:
8. the method for detecting the stability of BAT25 locus as defined in claim 6, wherein the PCR reaction is performed by: 93-97 ℃ for 9-11 min; 93-97 ℃, 18-22 s, 56-58 ℃, 28-32 s, 71-73 ℃, 28-32 s, 38-42 cycles; the melting temperature is 75-78 ℃; 38-42 ℃ for 28-32 s.
9. The method for detecting the stability of BAT25 locus as claimed in any one of claims 6 to 8, wherein the frequency of the fluorescence signal is from 11 times/° C to 13 times/° C.
10. The method for detecting the stability of BAT25 locus as claimed in any one of claims 6 to 8, wherein the concentration of the template is 50ng/μ L to 100ng/μ L.
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| WO2013153130A1 (en) * | 2012-04-10 | 2013-10-17 | Vib Vzw | Novel markers for detecting microsatellite instability in cancer and determining synthetic lethality with inhibition of the dna base excision repair pathway |
| US20150045369A1 (en) * | 2012-04-10 | 2015-02-12 | Vib Vzw | Novel markers for detecting microsatellite instability in cancer and determining synthetic lethality with inhibition of the dna base excision repair pathway |
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| WO2013153130A1 (en) * | 2012-04-10 | 2013-10-17 | Vib Vzw | Novel markers for detecting microsatellite instability in cancer and determining synthetic lethality with inhibition of the dna base excision repair pathway |
| US20150045369A1 (en) * | 2012-04-10 | 2015-02-12 | Vib Vzw | Novel markers for detecting microsatellite instability in cancer and determining synthetic lethality with inhibition of the dna base excision repair pathway |
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