CN111850129A - Primer pair, kit and method for detecting stability of NR21 locus of microsatellite - Google Patents
Primer pair, kit and method for detecting stability of NR21 locus of microsatellite Download PDFInfo
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Abstract
The invention discloses a primer pair, a kit and a method for detecting the stability of a microsatellite NR21 locus. The invention firstly provides a primer pair for detecting the stability of the NR21 locus of the microsatellite, the stability of the NR21 locus of the microsatellite can be accurately, quickly and specifically detected by utilizing the primer pair, compared with the gold standard method of the existing fragment analysis commercial kit, the method for detecting the stability of the NR21 locus of the microsatellite or the kit constructed by the invention has the advantages that the detection sensitivity is 95.77 percent, the specificity is 100 percent, the requirement on equipment is greatly reduced, a gene analyzer is not required, and only a fluorescent quantitative PCR instrument with the HRM function is required; in addition, the operation procedure of the method or the kit 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 or the kit has good application prospect in detecting the stability of the locus of the NR21 microsatellite.
Description
Technical Field
The invention belongs to the field of biotechnology. More particularly, relates to a primer pair, a kit and a method for detecting the stability of a microsatellite NR21 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 and first-grade malignant tumor of digestive system incidence 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.
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. Fragment analysis is currently recognized as the gold standard; however, this method is costly, takes a long time to detect, 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 biochemical specimen analyzer, J Mol Diagn, 2008,10(2): 154-9).
The HRM method is completely based on the analysis of the physical properties of nucleic acid, only some saturated dyes are added on the basis of the 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 being transferred to other analysis devices, the tube closing 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 has high requirements on primer design: the primer has good specificity, and the amplified fragment is required to be short enough to obtain good detection sensitivity.
NR21 is one of the important sites for the microsatellite instability detection of colorectal cancer, and the number of single nucleotide repeats changes when instability occurs. Therefore, there is a need to screen an accurate, rapid and inexpensive method for detecting the stability of the NR21 site.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the prior art and provide a primer pair, a kit and a method for detecting the stability of a microsatellite NR21 locus.
The invention aims to provide application of a high-resolution melting curve method in detecting the stability of a microsatellite NR21 site.
Another objective of the invention is to provide a primer pair for detecting the stability of the NR21 locus of the microsatellite.
The invention also aims to provide application of the primer pair in detection of the stability of the NR21 locus of the microsatellite or preparation of a reagent/kit for detecting the stability of the NR21 locus of the microsatellite.
The invention also aims to provide a kit for detecting the stability of the NR21 locus of the microsatellite.
The invention also aims to provide a method for detecting the stability of the NR21 locus of the microsatellite.
The invention also aims to provide the application of the primer pair, the kit or the method in detecting the stability of the site NR21 of the microsatellite.
The above purpose of the invention is realized by the following technical scheme:
the invention provides application of a high-resolution melting curve method in detecting the stability of a locus NR21 of a microsatellite.
The invention provides a primer pair for detecting the stability of a microsatellite NR21 locus, and the nucleotide sequences of the primer pair are shown as SEQ ID NO. 1 and SEQ ID NO. 2.
Upstream primer (SEQ ID NO: 1): 5'-tggcacagttctatttttatattta-3', respectively;
downstream primer (SEQ ID NO: 2): 5'-cactttctggtcactcgcgtttaca-3' are provided.
The application of the primer pair in detecting the stability of the site NR21 of the microsatellite or preparing a reagent/kit for detecting the stability of the site NR21 of the microsatellite also belongs to the protection scope of the invention.
The invention also provides a kit for detecting the stability of the locus NR21 of the microsatellite, which comprises the primer pair.
Preferably, the kit further comprises a saturating fluorescent dye.
More preferably, the saturated fluorescent dye is Eva Green.
Preferably, the kit further comprises Buffer, dNTP and Taq enzyme.
The invention also provides a method for detecting the stability of the NR21 locus of the microsatellite, which utilizes the primer pair to carry out fluorescence quantitative PCR reaction and HRM analysis.
Preferably, the method comprises the steps of:
(1) respectively taking DNA of tumor tissues and DNA of normal tissues of colorectal cancer patients as templates, performing fluorescence quantitative PCR reaction and HRM analysis by using the primer pair, collecting fluorescence signals, and drawing a melting curve;
(2) and (3) comparing the melting curves of the tumor tissue and the normal tissue of the colorectal cancer patient to judge whether the colorectal cancer patient is a microsatellite NR21 site stable patient.
Preferably, the method for determining whether the patient is a microsatellite NR21 site stable patient in the step (2) comprises the following steps: 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 microsatellite NR21 site unstable patient; otherwise, the colorectal cancer patient is indicated to be a microsatellite NR21 site stable patient.
Preferably, the system of the fluorescent quantitative PCR reaction in the step (1) is as follows: 1-5 mu L of DNA, 10 XBuffer 2-2.5 mu L, dNTP 2-2.5 mu L of DNA, 1-1.25 mu L of Eva Green, 1-1.25 mu L of upstream primer, 1-1.25 mu L, Taq mu of downstream primer, 0.2-0.25 mu L of enzyme, and 20-25 mu L of enzyme-free water.
More preferably, the system of the fluorescent quantitative PCR reaction in the step (1) is as follows: DNA 1. mu.L, 10 XBuffer 2.5. mu. L, dNTP 2.5.5. mu.L, Eva Green 1.25. mu.L, forward primer 1.25. mu.L, reverse primer 1.25. mu. L, Taq enzyme 0.25. mu.L, enzyme-free water to 24. mu.L.
Preferably, the procedure of the fluorescent quantitative PCR reaction in the step (1) is as follows: 10min at 95 ℃; 40cycles of 95 ℃ 20s, 62 ℃ 20s, 72 ℃ 20 s; the melting temperature is gradually increased from 73 ℃ to 83 ℃, and the temperature increasing speed is 1 ℃/s and 11 s; 30s at 40 ℃.
Preferably, the frequency of the collected fluorescence signal in the step (1) is 11-13 times/DEG C.
More preferably, the frequency of the collected fluorescence signal of step (1) is 12 times/DEG C.
Preferably, the concentration of the DNA in the step (1) is 50-100 ng/. mu.L.
More preferably, the concentration of the DNA of step (1) is 75 ng/. mu.L.
In addition, the application of the primer pair, the kit or the method in detecting the stability of the NR21 locus of the microsatellite is also within the protection scope of the invention.
The invention has the following beneficial effects:
the invention provides a primer pair, a kit and a method for detecting the stability of a microsatellite NR21 locus. The invention firstly provides a primer pair for detecting the stability of the NR21 locus of the microsatellite, and the stability of the NR21 locus of the microsatellite can be accurately, quickly and specifically detected by utilizing the primer pair and the method for detecting the stability of the NR21 locus of the microsatellite;
compared with the existing gold standard method of the fragment analysis commercial kit, the method has the advantages that the sensitivity is 95.77 percent, the specificity is 100 percent, the clinical requirements can be met, the requirements on equipment are greatly reduced, a gene analyzer is not needed, and only a fluorescence quantitative PCR instrument with the HRM function is needed; in addition, the operation procedure of the method 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 NR21 of the microsatellite.
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
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
EXAMPLE 1 establishment of method for testing stability of NR21 site of microsatellite
1. Primer and method for producing the same
This example provides a primer pair for detecting the stability of the NR21 locus of a microsatellite, the nucleotide sequences of which are shown in SEQ ID NO. 1 and SEQ ID NO. 2.
Upstream primer (SEQ ID NO: 1): 5'-tggcacagttctatttttatattta-3', respectively;
downstream primer (SEQ ID NO: 2): 5'-cactttctggtcactcgcgtttaca-3' are provided.
2. Reaction system
Using the above primer set, a fluorescent quantitative PCR reaction system (24. mu.L) 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 (mu L) |
| 10×Buffer | 2-2.5 |
| dNTP | 2-2.5 |
| Eva Green | 1-1.25 |
| Upstream primer | 1-1.25 |
| Downstream primer | 1-1.25 |
| Taq enzyme | 0.2-0.25 |
| Enzyme-free water | 15-17 |
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, 62 ℃ 20s, 72 ℃ 20 s; the melting temperature is 78 ℃, 40 ℃ and 30 s.
(2) 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 NR21 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 microsatellite NR21 site unstable patient; otherwise, the colorectal cancer patient is the microsatellite NR21 site stable patient.
Example 2 construction of a kit for detecting stability of the site NR21 of microsatellite
A kit for detecting the stability of a microsatellite NR21 locus 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'-tggcacagttctatttttatattta-3', respectively;
downstream primer (SEQ ID NO: 2): 5'-cactttctggtcactcgcgtttaca-3' are provided.
Test example 1 comparison of results of detecting stability of site NR21 of microsatellite by using different primer pairs
1. Experimental methods
In this test example, the method for detecting the stability of the site NR21 of the microsatellite, which was constructed in example 1, was used to investigate the influence of 5 primer pairs (Table 2) on the test results for detecting the stability of the site NR21 of the tumor microsatellite of a patient with colorectal cancer, and the results of the method (HRM analysis) of the present invention and the commercial kit for the conventional fragment analysis (Beijing Microgene technology Co., Ltd.) were investigated
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 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 as can be seen from table 2 and fig. 1-6, the sample 2 is detected and interpreted as unstable microsatellite NR21 site by using a commercial kit; the detection is carried out by using the 5 groups of primer pairs and the method for detecting the stability of the NR21 locus of the microsatellite, which is constructed in the embodiment 1, and the result shows 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 judge the instability of the NR21 locus of the microsatellite in the sample 2, and the rest 4 pairs of primers (the primer pairs 2-5) cannot be accurately judged. Therefore, primer pair 1 is used as a primer pair for detecting the stability of the NR21 locus of the microsatellite.
TABLE 25 comparison of HRM Peak types and interpretation results of primer pairs and 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 commercial kit of the present invention and the existing fragment analysis method were compared
1. Experimental methods
141 pairs (i.e., tumor tissue and normal tissue corresponding thereto, Nos. 1-141) of colorectal cancer patient samples were obtained from the sixth Hospital Pathology department affiliated to Zhongshan university, and genomic DNAs of the samples were extracted, respectively, according to the method of example 1, and 141 were tested, respectively, using the method for testing the stability of the NR21 site of microsatellite, constructed in example 1, using primer pair 1 (nucleotide sequences shown in SEQ ID NO:1 and SEQ ID NO:2) obtained in test example 1.
Upstream primer (SEQ ID NO: 1): 5'-tggcacagttctatttttatattta-3', respectively;
downstream primer (SEQ ID NO: 2): 5'-cactttctggtcactcgcgtttaca-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) was performed on the above 141 samples.
In addition, the detection time and cost used in the above 2 detection methods were accounted and compared.
2. Results of the experiment
The result of the detection rate comparison between the method and the commercial kit of the existing fragment analysis method is shown in table 3, and it can be seen that the method of the invention has 138 cases of consistent results with the commercial kit of the existing fragment analysis method, and the results of 3 cases of samples are not consistent; the sensitivity of the method of the invention is 68/(68+3) × 100%: 95.77%, and the specificity is 70/(70+0) × 100%: 100%.
Therefore, compared with the existing commercial kit (gold standard method) of the fragment analysis method, the specificity of the method is 100%, the sensitivity is as high as 95.77%, and the clinical requirement can be met.
TABLE 3 detection Rate comparison of commercial kits for the methods 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 NR21 locus of the microsatellite compared with that of the commercial kit of the existing fragment analysis method.
Therefore, the method for detecting the stability of the site NR21 of the microsatellite can greatly save the detection time and cost and better serve clinical patients.
TABLE 4 comparison of detection time and cost of commercial kits for the methods of the invention and the existing fragment analysis methods
| The method of the invention | Commercial kit for existing fragment analysis method | |
| Time of |
2 hours/ |
3 hours/sample |
| Cost of experiment | 10 yuan/sample | 50 yuan/sample |
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
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SUN YAT-SEN University
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Claims (10)
1. The application of a high-resolution melting curve method in detecting the stability of the NR21 site of the microsatellite.
2. The primer pair for detecting the stability of the site NR21 of the microsatellite is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1 and SEQ ID NO. 2.
3. Use of the primer pair of claim 2 in detecting the stability of the NR21 locus of a microsatellite or preparing a reagent/kit for detecting the stability of the NR21 locus of a microsatellite.
4. A kit for detecting the stability of a microsatellite NR21 site, comprising the primer set according to claim 1.
5. The kit of claim 4, further comprising a saturating fluorescent dye.
6. A method for detecting the stability of the NR21 locus of a microsatellite, which comprises carrying out a fluorogenic quantitative PCR reaction using the primer set according to claim 2 and carrying out HRM analysis.
7. The method of claim 6, comprising the steps of:
(1) respectively taking DNA of tumor tissues and DNA of normal tissues of colorectal cancer patients as templates, performing fluorescence quantitative PCR reaction and HRM analysis by using the primer pair of claim 2, collecting fluorescence signals, and drawing a melting curve;
(2) and (3) comparing the melting curves of the tumor tissue and the normal tissue of the colorectal cancer patient to judge whether the colorectal cancer patient is a microsatellite NR21 site stable patient.
8. The method of claim 7, wherein the step (2) of determining whether the patient has a stable position for the NR21 microsatellite is performed by: 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 microsatellite NR21 site unstable patient; otherwise, the colorectal cancer patient is indicated to be a microsatellite NR21 site stable patient.
9. The method of claim 7, wherein the fluorescent quantitative PCR reaction in step (1) is performed in a system comprising: 1-5 mu L of DNA, 10 multiplied by 2-2.5 mu L, dNTP 2-2.5 mu L of Buffer, 1-1.25 mu L of Eva Green, 1-1.25 mu L of upstream primer, 1-1.25 mu L, Taq enzyme 0.2-0.25 mu L of downstream primer, and adding enzyme-free water to 20-25 mu L;
the fluorescent quantitative PCR reaction procedure in the step (1) is as follows: 10min at 95 ℃; 40cycles of 95 ℃ 20s, 62 ℃ 20s, 72 ℃ 20 s; the melting temperature is gradually increased from 73 ℃ to 83 ℃, and the temperature increasing speed is 1 ℃/s and 11 s; 30s at 40 ℃.
10. Use of the primer pair according to claim 2, the kit according to any one of claims 4 to 5 or the method according to any one of claims 6 to 9 for detecting the stability of the NR21 locus of a microsatellite.
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